relatório de actividades.2006

RELATÓRIO DE ACTIVIDADES
2006
ÍNDICE
PÁG.
1.
INTRODUÇÃO
2.
INVESTIGAÇÃO CIENTÍFICA
3
2a. Cancer Biology
6
2b. Cancer Genetics
16
2c. Carcinogenesis
28
2d. Genetics, Evolution and Pathology
35
2e. Population Genetics
43
2f. Tumour Evolution and Development
53
2g. Genetics Diversity and Bioinformatics
56
2h. Public Health and Cancer
60
3.
EDUCAÇÃO CONTÍNUA E DIFUSÃO CIENTÍFICA
66
4.
SERVIÇO À COMUNIDADE
4a. UPS
69
4b. UPSi
74
4c. UPSs
77
5. RECÉM-DOUTORADOS
81
6. RESUMO DOS PROJECTOS E SEU FINANCIAMENTO
85
7. TRABALHOS PUBLICADOS
89
8. REVISTAS CIENTÍFICAS INTERNACIONAIS COM MEMBROS
DO IPATIMUP NOS SEUS EDITORIAL BOARDS
9. NÚCLEO DE AMIGOS DO IPATIMUP
96
97
2
Introdução
Dando sequência ao procedimento seguido nos últimos anos por sugestão dos avaliadores externos do IPATIMUP,
o relatório de actividades de 2006 é precedido de uma introdução que resume os elementos mais expressivos da
actividade no ano passado.
Os investigadores do IPATIMUP publicaram, em 2006,101 artigos científicos, 99 dos quais em revistas
internacionais e 2 em revistas portuguesas. Noventa e um (91) desses artigos foram publicados em revistas com
Factor de Impacto. Cerca de 50% desses 91 artigos foram publicados em revistas com Factor de Impacto entre 3 e
29,4.
A WEB of KNOWLEDGE (ISI) tem uma lista dos artigos de todas as áreas cientificas mais citados no mundo.
Portugal tinha, em Janeiro de 2007, 250 artigos nesta lista. Destes 250 artigos 13 têm como autores ou co-autores
investigadores do IPATIMUP (mais de 5% do total).
A WEB of KNOWLEDGE (ISI) publica também os artigos mais citados por instituição. O Hospital de S. João tem 6
artigos nessa lista – 5 desses 6 artigos foram publicados por investigadores do IPATIMUP.
Nos quadros seguintes documenta-se a evolução da qualidade da produção científica no IPATIMUP.
Re vis tas
Inte rnacionais
com Factor de
Im pacto
Produção Científica (2000-2006)
100
90
80
Artigos e m
Re vis tas c/FI>1,0
70
60
50
Artigos e m
Re vis tas c/FI>3,0
40
30
20
Artigos e m
Re vis tas c/FI>6,0
10
0
2000
100
2001
2002
2003
2004
2005
2006
Produção Cientifica (2005-2006)
2005
2006
90
80
70
60
50
40
30
20
10
0
Revistas
A rtigos em
A rtigos em
A rtigos em
Internacionais Revistas c/FI>1,0 Revistas c/FI>3,0 Revistas c/FI>6,0
com Factor de
Impacto
3
Nos quadros seguintes resumimos a origem das verbas que sustentaram a actividade do IPATIMUP e as Agências
que financiaram os Projectos em curso.
Projectos de investigação - financiamento
Fonte
FCT
Outros
19%
.000 euros
*
FCT
36%
488,9
F. Gulbenkian
BCP
7%
183,8
SXXI
99,2
Agências internacionais
248,6
BCP
100,0
Outros
255,2
Total
Agências internacionais
18%
F. Gulbenkian
13%
SXXI
7%
1.375,7
* Exclui 402 m. euros recebidos para projectos de reequipamento
Projectos de investigação em curso
Projectos de investigação em curso
(31/12/2006) N = 47
APSA
1
Ministério da Saúde
2
Adi
2
Indústria, bancos, associações
6
F. C. Gulbenkian
4
Agências Internacionais
5
Farmacêuticas
FCT
7
20
20
20
18
16
14
12
10
8
6
4
2
0
6
1
AP
SA
2
4
5
7
2
Ad
M in
isté i
r io
da
FC
F.
F
T
Gu Ag ên a r m
úst
a
lbe
c
ria
n k ias In cêu ti
,b
ia n
ca
an
te r
s
c
n
os,
ac
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io n
e
as
a is
soc
ia ç
õe
s
In d
Sa
O número de doutoramentos foi, em 2006, de 5 (As provas de um desses doutoramentos tiveram de ser adiadas
para 2007)
O apoio mecenático permitiu o estabelecimento, em 2006, das bases do Programa INFOCANCER, um programa
destinado a esclarecer as dúvidas e a estimular a prevenção e o diagnóstico precoce das situações de cancro
familiar, tendo como alvos a população portuguesa e os profissionais de saúde.
Continuaram-se em 2006 os programas de Mestrado e Doutoramento e realizaram-se, na Fundação Gulbenkian e
em Serralves, as sessões do terceiro Ciclo de Colóquios sobre Medicina Preventiva do Cancro para mais de mil
professores de Biologia e Físico-Química. Em Outubro/Novembro, o IPATIMUP organizou e realizou, em
colaboração com a Associação de Estudantes da Faculdade de Medicina da Universidade do Porto, o terceiro
Curso de Introdução à Medicina Molecular do Cancro para 100 alunos dos 1ºs anos de licenciaturas em Medicina e
outras Ciências da Saúde. Em 2006, o IPATIMUP organizou ainda, em conjunto com a Sociedade Portuguesa de
Genética Humana, um Curso de 2 dias, o ABZ da Genética, sobre Identificação Humana. Continuaram-se as
actividades de divulgação científica junto de numerosas escolas básicas e secundárias (Projecto do Autolaboratório, bem como os Programas Ciência Viva em Férias e Universidade Júnior).
4
Mantivemos as actividades de apoio à comunidade e de consultadoria tanto de diagnóstico anatomo-patológico
como de patologia molecular. Os cerca de 170 casos nestas condições foram enviados ao IPATIMUP por hospitais
e institutos de cancro de 21 países.
Os investigadores do IPATIMUP integram os Corpos Editoriais de 23 revistas científicas internacionais.
O IPATIMUP realizou, em 2006, como nos anos anteriores, três reuniões internacionais – uma sobre Genética
Populacional e Genética Forense (Portugaliae Genetica – 9º edição), outra sobre Cancro (Porto Cancer Meeting –
15ª edição) e a terceira sobre Ciência e Cultura (Conferências do Equinócio – 10ª edição).
Em 2006 continuámos o reforço da ligação ao IBMC e INEB e avançámos, dando seguimento à decisão da
Assembleia Geral, no estudo da viabilidade (e bondade) de uma futura associação entre as três instituições.
Nota: O IPATIMUP voltou a contar, em 2006, com o apoio excepcional dos seus Associados Efectivos
(Universidade do Porto, Câmara Municipal do Porto, Fundação Luso-Americana Para o Desenvolvimento, Liga
Portuguesa Contra o Cancro, Comissão de Coordenação e Desenvolvimento da Região Norte, Instituto de
Genética Medica Dr. Jacinto de Magalhães e Cruz Vermelha Portuguesa) e Aderentes (Faculdade de Medicina,
Faculdade de Ciências, Instituto de Ciências Biomédicas Abel Salazar, Faculdade das Ciências da Alimentação e
Nutrição, Faculdade de Medicina Dentária, Faculdade de Farmácia, Hospital de S. João e Instituto Português de
Oncologia – Centro Regional do Norte), assim como de várias instituições públicas e privadas: Fundação Calouste
Gulbenkian, Fundação Millennium-bcp, Fundação Oriente, Fundação para a Ciência e Tecnologia, Fundação
Ciência Viva e Agência Portuguesa de Inovação.
5
Cancer Biology
Coordinator: Paula Soares.
Scientific Consultant: Manuel Sobrinho-Simões
Principal Investigators: Clara Sambade; Helena Vasconcelos; José Manuel Lopes.
Post- Docs: Valdemar Máximo; Ana Preto; Ana Sofia Rocha, Patrícia Castro.
PhD Students: Jorge Lima*, Vitor Trovisco, Raquel Lima, Helena Populo.
MSc Students: Tália Feijão*, Hugo Seca, Noémia Leal, Luís Dias (CS)
BI’s:, Patrícia Pontes, Inês Bento, Joana Gonçalves, Marta Oliveira, Paula Boaventura.
Undergraduate students: André Silva (JL), Liliana Santos (PS), Joana Silva (VT), Ricardo Celestino (PC), Paula
Parreira e Ana Brandão (VM), Teresa Costa (HV)
Clinicians: Manuel Cardoso de Oliveira, José Teixeira Gomes, Duarte Pignatelli.
* - Concluded their thesis in 2006 (see below).
Objectives/Goals of the research activity
1. The main objective is to progress in the understanding of the etiopathogenesis of some types of human cancer,
with an emphasis on thyroid and neuroendocrine tumours. Within this frame a particular attention is paid to: a)
genetic alterations in tyrosine kinase receptors and signal transducing molecules involved in the mitogen-activated
protein kinases (MAPK) pathway; and b) mitochondrial alterations secondary to mitochondrial DNA
mutations/deletions or to mutations in nuclear genes encoding mitochondrial enzymes.
2. Some members of the group are also involved in clinico-pathological studies in other types of human tumours,
and in projects aiming to validate and/or identify molecular targets for cancer treatment, namely via the utilization of
cell signalling inhibitors and the down regulation of gene expression with siRNAs.
Main research topics
Oncobiology of differentiated thyroid tumours
Oncobiology of familial forms of thyroid and neuroendocrine tumours
Role of mitochondrial alterations in the etiopathogenesis of endocrine/neuroendocrine cancers .
Radiation induced thyroid tumourigenesis
Oncobiology of some haematological malignancies
Validation of molecular targets (namely targets in cancer chemotherapy resistance)
Background and major achievements in 2006
1. Oncobiology of differentiated thyroid tumours
Continuing collecting and studying a large series of papillary and follicular carcinomas with detailed clinicopathologic data, appropriately sampled material (paraffin-embedded and, whenever possible, also frozen material),
long follow-up and reliable information on regional and distant metastases, with the following main objectives:
1a) To clarify the influence on the metastatic pattern of the tumours and on the patients’ prognosis of BRAF
mutations, RAS mutations and PAX8/PPAR translocation. In relation to this objective, we have analysed, in
collaboration with clinicians from H.S. João, Santiago de Compostela (Spain) and USP (Brazil) a cohort of PTC
patients in an attempt to find if there is an increase in the frequency or in any particular type of BRAF mutation
associated with nodal metastization (as reported by others and reviewed by us in Paper 6). In 2006 we finished
the study of 35 primary-metastasis paired PTC cases. Our main findings are: a) We found a 90% concordance
in the BRAF status between the primary and the respective metastasis; b) There is no requirement of BRAF
mutations, neither mutation frequency increase, in PTC nodal metastases; c) The BRAF VK600-1E mutation was
not detected in any metastasis sample therefore not being specific of PTC metastases; d) The previously
described phenotype-genotype correlation is maintained and strengthened. In summary, our results do not
confirm the advanced higher propensity for metastization of BRAF mutated tumours (Trovisco et al, manuscript
in preparation).
6
1b) To disclose the molecular characteristics of the follicular variant of PTC which is known to behave differently
from conventional PTC, namely regarding its tendency to give rise to lung and bone metastases. In previous
studies we have shown that this variant displayed a different BRAF mutation (BRAFK601E in about 7-9% of the
cases and no BRAFV600E), and that it shares some of the molecular features of follicular carcinomas: frequent
occurrence of PAX8/PPAR translocation and of N-RAS mutations (Paper 1). In 2006 we start the study of
these characteristics in an independent tumour series (Spanish cases). Furthermore we initiated the study of a
small series of the so-called “mixed” carcinomas, which are papillary thyroid carcinomas with a follicular and
papillary pattern in comparable amounts. We intend to verify if in this context the two components of the tumour
harbour the genetic alterations characteristics of the conventional PTC and of the FVPTC.
1c) To identify molecular alterations underlying the development of thyroid tumours. Overexpression of TKRs
(c-MET, EGFR, ERBB2) is presented as a common feature of PTC. Since no subjacent genetic alterations
could be assigned to these genes, it is assumed that epigenetic regulation can be in the basis of this abnormal
expression. In 2006 having has working hypothesis the idea that the cell membrane composition (caveolae)
may modulate signal transduction and protein expression in thyroid cells, namely in PTC, we have evaluate
TKR (EGFR, ERBB2 and c-MET) and caveolin-1 expression in a series of cPTC, FVPTC and FTC. We verified
that caveolin-1(either by over or under expression) is deregulated in thyroid cancer. The comparison of the
expression of caveolin-1 and the three TKRs did not reveal any strong association between each pair. The
hypothesis that caveolin-1 can modulate signal transduction and protein expression of the three TKRS, does
not seem valid.
Our paper in the expression of p75 neurotrophin receptor in PTC was published in 2006 (Paper 2).
In 2006 we have also conclude a study based on the description of transgenic mice for mutant alleles in thyroid
hormone receptor beta (THRB) gene, which develop spontaneously follicular thyroid carcinomas that
progressed to undifferentiated carcinomas in a similar fashion to human follicular thyroid carcinoma
progression. In a more pathologic based perspective, we evaluate the presence of mutations in THRB gene in a
panel of thyroid carcinomas. We studied several types of thyroid pathologies and no mutations were found
(Paper 7)
1.d) As a a sort of cell biology counterpart of the clinicopathological studies listed in Points 1a), b) and c) and to
progress in the understanding of the role played by BRAF mutations, we have established in vitro systems in
which we can evaluate the biological role (proliferation, apoptosis, motility and invasiveness) and the signalling
pathways activated by the different BRAF mutations in thyroid cancer derived cell lines. We have been using
this system to study the effects of kinase signalling inhibitors (BAY, Gleevec and PD) on the rate of proliferation
and apoptosis. In 2006, we have been evaluating the effect of silencing of BRAF gene by siRNA in the same
thyroid cancer derived cell lines (Post- doc project of Ana Preto). We observed that BAY 43-9006 is able to
inhibit proliferation in thyroid cancer cell lines and to induce apoptosis in cells harboring the BRAFV600E
mutation (8505C and NPA). The mechanisms by which BAY 43-9006 induces apoptosis in cell lines with
BRAFV600E mutation seem to reflect a decrease/increase balance of the anti-apoptotic proteins Mcl-1 and Bcl2. We observed also that the apoptotic effect of BAY 43-9006 is independent of BRAF since specific inhibition
of BRAF by RNAi has no effect in apoptosis. Our results show that BRAF signaling is important in the
proliferation of thyroid cells with different genetic backgrounds regardless of being PTC or ATC derived cell
lines. We have also shown that the growth inhibition of BRAF pathway induced by BAY 43-9006 is different
from that obtained with BRAF siRNA.
Finally, in transfection cell models, we have been comparing the transcription activation patterns of BRAF
mutants with those of RET/PTC; the results obtained to date show that STAT1/STAT3 appears to be
differentially activated by RET/PTC and by BRAF (PhD Project of Vitor Trovisco). During this year we attempted
to verify if the in vitro studies were reproduced in in vivo samples. For that we are studying the
immunohistochemical expression of STAT3, phosphoSTAT3 (activated) and STAT1 in a large series of thyroid
tumours previously characterized for BRAF, RET/PTC and RAS genes alterations. Our preliminary results
suggest that STAT1 and 3 are overexpressed rather than activated in thyroid lesions, namely in PTC.
Furthermore, our results do not evidence striking differences in the expression and activation of those proteins
that can be assigned to different genetic alterations. Another section of the work consisted in the “construction”
of stable clones of thyroid cell lines harbouring both mutated B-RAF and RET/PTC-1. We transfect TPC-1 cell
line (RET/PTC-1 positive) with wild-type and mutated B-RAF (Post-doc project of Ana Sofia Rocha). Although
we were able to isolate viable stable clones from both B-RAF forms we could not detect changes in B-RAF
7
protein expression by western-blot. A similar attempt was performed in PCCL3 cells, using GFP-tagged BRAF
forms; however, no GFP-marked clones could be isolated. When GFP-tagged B-RAF was expressed transiently
in both cell lines, we could see a small increase in B-RAF expression, suggesting that for unknown reasons the
expression of transfect B-RAF is lost during the process of cell culture. This work was done in collaboration with
the group of Luis Teixeira da Costa.
1.e)To identify mechanisms underlying the occurrence of aneuploidy in thyroid tumours.
The two major histotypes of differentiated thyroid carcinoma display different patterns of DNA content: follicular
carcinomas are clearly aneuploid whereas almost every papillary carcinoma is diploid or quasi–diploid. We
found an association between the presence of a polymorphism in H-Ras and the occurrence of aneuploidy in
thyroid tumours. We also detected a link between the presence of H-RAS 81-C allele and significantly higher
amounts of H-RAS p21 mRNA isoform. These findings support the hypothesis that H-RAS 81 T-C may induce
aneuploidy through the overexpression of p21 isoform of H-RAS (Paper 4). In 2006 we continued to study the
association between the presence of the H-RAS 81C polymorphism and the ploidy of thyroid tumors. We
confirm that the presence of the H-RAS 81C polymorphism is associated with patients with aneuploid thyroid
tumors in an independent tumor series (Spanish cases). We also tried to verify if this association is restricted to
thyroid tumors. In order to do that we studied a series of 75 bladder cancers with known ploidy and we found
the same association between the presence of the H-RAS 81C polymorphism and aneuploidy in bladder cancer
patients. We concluded that the association of the H-RAS 81C polymorphism and aneuploidy is not restricted
to thyroid tumors.
2. Oncobiology of familial forms of thyroid and neuroendocrine tumours
The occurrence of nodules in the thyroid is one of the most frequent human diseases. It is estimated that more than
one out of every three persons will develop a benign or malignant thyroid nodule during her/his life time. Papillary
thyroid carcinoma (PTC) is the most common endocrine malignancy in humans. It is particularly frequent in
countries like Portugal, Iceland, Ireland, Norway and others in which there is a high iodine intake due to prominent
costal areas and a close relationship with the ocean. PTCs together with the less frequent follicular carcinoma,
constitute the so-called non-medullary thyroid cancer (NMTC) in general, and familial PTCs (fNMTC) in particular,
are associated with some of the highest cancer risks among all cancer sites. The model of inheritance of fNMTC
appears, mostly, to be autosomal dominant although the majority of families are small (sibs/trios) and therefore a
multigenic inheritance is plausible. Four loci have been identified through genetic linkages: MNG on 14q32, TCO1
on 19p13.2, fPTC on 1p21 and NMTC1 on 2q21.
In the previous years, in collaboration with clinicians, we have collected samples from individuals belonging to
families with thyroid tumours aggregation. We have now 17 families with more than one affected individual and
several non-affected members.Using linkage analysis in this series of families we intend to select regions of the
genome in which genes predisposing for fNMTC can be localized. We started the study in 6 families, 4 in which
multinodular goiter was the main pathologic finding and 2 showing Hürthle cell tumours. We did not found any
linkage association with the previous identified loci, suggesting that in those families a new locus is associated with
the disease. The results are the support of the Master thesis of Teresa Almeida, entitled “Formas Familiares de
Bócio” present in the Medical Faculty of the University of Porto.
We maintain also collaboration with the IPO-Coimbra in this subject (Paper 3).
3. Role of mitochondrial alterations (mitochondrial DNA mutations/deletions or mutations in nuclear genes
encoding mitochondrial enzymes) in the etiopathogenesis of endocrine/neuroendocrine cancers
It is not well understood the role of the mtDNA mutations/variants, nor the role of genetic alterations in nuclear
genes that codify for mitochondrial proteins, on the etiopathogenesis of several types of tumors exhibiting
mitochondrion rich neoplastic cells.
Mitochondrion-rich or oxiphilic tumours constitute an unusual form of neoplasm composed of cells with a
voluminous, granular, eosinophilic cytoplasm due to the huge amount of structurally abnormal mitochondria number
being more often found in tumours of tissues with low turn-over. In a previous study, we analysed the mtDNA in
benign and malignant thyroid tumours and we concluded that mtDNA variants and mtDNA somatic mutations of
Complex I and Complex IV genes appear to be involved in thyroid tumourigenesis. In order to see if this association
exists in oncocitic tumors of other organs we searched, during 2006, for mtDNA mutations (mtDNA large deletions,
D-loop instability and point mutations in ATPase 6 and ATPase 8 genes) in a series of 19 Warthin tumours of the
salivary gland and 14 renal oncocytomas. The data we obtained is similar to those found in Hürthle cell tumours and
8
will be used as support for the Master thesis of Noémia Leal entitled – “Mitochondrial DNA deletions and mutations
in renal oncocytic tumours and Warthin tumours of the salivary gland.”
The existing literature in this subject was reviewed in Paper 5.
GRIM-19 is a cell death regulatory gene that promotes apoptosis, is a negative regulator of cell growth, and is also
involved in mitochondrial metabolism. The dual role of GRIM-19 in apoptosis and mitochondrial biogenesis turned it
a good candidate for being a gene involved in the etiopathogenesis of oncocytic cell tumours. Its localization to the
same region as the previous region containing a gene [thyroid cell oxyphilia (TCO gene)] predisposing to Hürthle
cell familial tumours (chromosome 19p13.2) also suggested that it could be involved in the etiopathogenesis of
familial Hürthle cell tumours. We have searched for GRIM-19 mutations in a series of thyroid tumours. Sequence
determination of the 5 exons of GRIM-19 in 26 apparently sporadic Hürthle cell tumours disclosed the existence of
four mutations. We did not detect mutations in any of the 20 cases of non-Hürthle follicular and papillary
carcinomas, nor in any of the 96 blood donor samples. Since, the major role of GRIM-19 in control of cell growth is
exerted through STAT3 we studied the expression of a downstream STAT3 gene - ICAM1 -, and we have shown
that the cases with GRIM-19 mutations display increased expression of the STAT3 regulated gene ICAM1; this
finding fits with a loss of function of GRIM-19.
Now we intend to progress in the understanding of the functional role of GRIM-19 mutations in thyroid tumours. In
order to address this aim we intend:
A1- To verify the frequency of GRIM-19 mutations in thyroid tumours
We will search for GRIM-19 mutations in a larger series of benign and malignant familial and sporadic tumours of
the thyroid, with and without Hürthle cell features, in order to verify our previous association of GRIM-19 mutations
and Hürthle cell tumours.
A2 - To verify the functional consequence of GRIM-19 down-regulation/mutation
a) To induce GRIM-19 down-regulation by small interfering RNAs (siRNA).
b) To transfect thyroid cell lines with GRIM-19 mutated cDNAs.
Using small interfering RNAs we will study the effect of GRIM-19 down-regulation in human thyroid cell lines,
namely the effect in the OXPHOS system activity, in the expression of STAT3 downstream genes and in cell
apoptosis and proliferation. In order to access the functional role of previous detected (by our group) GRIM-19
mutations in thyroid tumours we will transfect human thyroid cell lines with GRIM-19 mutated cDNAs and we will
study the effect of these mutations in the OXPHOS system activity, in the expression of STAT3 downstream genes
and in cell apoptosis and proliferation.
During 2006 we started the siRNAs assays. We used two different siRNAs and one of them has provided positive
results. We are now confirming the results and starting the study of the effect of GRIM-19 downregulation by the
analysis of the expression of STAT3 downstream genes. Our preliminary results show an increased expression of
CyclinD1 in the cell lines treated with siRNA against GRIM-19.
Concerning the study of the role of GRIM-19 in the etiopathogenesis of oncocytic tumours we checked for the
expression of GRIM-19 expression in a series of 31 Hürthle cell tumours by immunohistochemistry. We found a
decrease in the expression of GRIM-19 in 58.1% of the tumours studied and in 1 case (3.2%) a complete loss of
GRIM-19. No mutations in GRIM-19 gene were found in the case without immunohistochemical expression. In the
cases with decreased GRIM-19 expression and in the case with complete loss of GRIM-19 expression we observed
a low frequency of cells with nuclear AIF expression compared with that found in cases with normal GRIM-19
expression, suggesting a decrease in apoptosis when GRIM-19 is dowregulated, and confirming the role in GRIM19 in apoptosis regulation. Similar results were found in a series of 14 renal cell carcinomas.
The succinate dehydrogenase (SDH) mitochondrial proteins (Complex I of the mitochondrial respiratory chain and
Krebs cycle) were implicated in familial forms of neuroendocrine tumours (paraganglioma, pheocromocytoma), but
their role in C-cell derived tumours was unknown. We identified in 2003, in a familial case of C-cell hyperplasia
without ret mutation, a new SDHD gene variation that segregates with the disease (Lima et al, JCME, 2003). In
2006 we have estimating the frequency of alterations in SDH genes in a series of 20 patients with medullary
carcinomas without ret mutation. No germline alterations where found in SDH genes; however we have identified
polymorphic variants in SDHB and SDHD. The SDHD polymorphisms where more frequent in the carcinoma
patients group than in control group and the patients harbouring them showed a lower age at diagnosis.
9
4. Radiation induced thyroid tumourigenesis
The studies on the role of mitochondrial alterations in tumourigenesis, involves also the study of the effect of
irradiation in mitochondrial DNA and for that we continue in 2006, to have the possibility to use a series of postChernobyl tumours (in these point we will also use in the future samples from the cohort irradiated for the treatment
of Tinea Capitis in the 1950’s., see below).In a series of 49 patients with post-Chernobyl tumours and 10 patients
with sporadic thyroid tumours we found the presence of the common deletion in virtually all the samples. In addition
in 18/48 (38%) of the post-Chernobyl tumours large mtDNA deletions and rearrangements were found. None of the
10 sporadic cases show these abnormal fragments. We advanced that these alterations can act as irradiation
biomarkers (Post-Doc project of Valdemar Máximo and PhD project of Jorge Lima).
In the field of radiation induced carcinogenesis we initiate in 2006 a project (with financial support from Fundação
Calouste Gulbenkian) aiming to evaluate Head and Neck Tumours in a cohort of about 5000 individuals irradiated
for the treatment of Tinea Capitis in the 1950’s. Until now 220 individuals from the cohort have been clinically
observed and part of them showed lesions that can be potentially related with head and neck irradiation (thyroid and
skin). From all the individuals peripheral blood and samples from oral mucosa have been collected. We intend in
2007 continue the clinical observation of the cohort and start the genetic characterization of the cases.
5. Clinico-pathological studies in other types of endocrine/neuroendocrine tumours
a) Our Group will host the National DataBase of GastroIntestinal Stromal Tumours (GIST) and NeuroEndocrine
Tumours (NET), which have been designed as REGIST and REGENE respectively (Responsible: José Manuel
Lopes). The establishment and maintenance of the DataBase will be supported by NOVARTIS ONCOLOGY.
a) During 2006 we have collected from the files of IPATIMUP and H S João 100 cases of gastro-entero-pancreatic
NETS. The clinical revision of the series was performed as well as the histological re-classification. Areas were
selected to perform tissue-arrays of these cases. JM Lopes was involve in the Consensus Conference for
establishment of new guidelines for clinical management of these tumours (Paper 9). In 2007 we intend to start the
biopathologic classification of the series in terms of proliferation (and cell cycle proteins) and production of
neuroendocrine markers.
b) Clearly distinct morphological and biologically tumours derive from the two distinct areas of the adrenal gland, the
cortex and the medulla. In particular a great deal of attention has been given to those from the cortex mostly
because of their clinical characteristics which are frequently rather notorious. However, not many studies at a
cellular level have been done mostly due to their relative rarity. In 2006, organization of the existing histological
material was recognized as an essential step to allow a rapid access to the samples of the different types of
tumours, in their different developmental stages. Simultaneously, with this systematic identification, a correlation of
the pathological with both the biochemical and the clinical data is in the process of completion. In 2007 we intend to
conclude this first phase of the construction of this database introducing the already mentioned data together with
completing the organization of the samples. We will do tissue arrays of the different types of tumours and then start
performing immunohistochemical studies taking special attention to cell cycle molecules. In this study we aim to
verify if there are alterations in the pathways related to cell proliferation as well as modifications in the expression of
apoptosis markers in adrenal cortical tumours.
6. Validation of molecular targets in cancer treatment or chemoresistance.
PI. Helena Vasconcelos
During the current year we have found that downregulation of expression of the antiapoptotic protein XIAP may be a
good approach to enhance sensitivity to Imatinib Mesylate (a drug developed by Novartis for the targeted therapy of
Chronic Myeloid Leukemia) even in the case of existing P-glycoprotein overexpression (Paper B1). On the basis of
these results we have been awarded a prize for best poster –laboratorial work, from “Sociedade Portuguesa de
Hematologia” at the “Reunião Anual da Sociedade Portuguesa de Hematologia”( see below). We have also been
continuing the work on validation of the relevance of XIAP in the resistance of acute myelogenous leukaemia cells to
chemotherapy, financed by ”Associação Portuguesa contra a Leucemia”– APCL.
Future directions will consist of investigating the mechanisms by which downregulation of XIAP expression reverts
the effects of P-gp overexpression. The work on XIAP will also be pursued as collaboration with Belfast, UK, for
which we have been awarded a travelling grant “Acções Integradas Luso-Britânicas” from CRUP/British Council.
Future work will also contribute to the validation of the relevance of viral antiapoptotic proteins in drug resistance, in
EBV infected lymphoma cells. Pending approval of projects submitted to FCT, validation of the antitumour activity of
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small pharmacological molecules modulators of p53 or apoptotic proteins will also be carried out, as collaboration
with the Faculty of Pharmacy of Porto University.
7. Oncobiology of some haematological malignancies
Over 2006 the research activity of the Hematopathology group under the supervision of Clara Sambade was
focused on 2 projects entitled: A) Analysis of “minimal residual disease” in acute lymphoblastic leukaemia by “realtime PCR” using ASO-primers and TaqMan probes (financed by Fundação Calouste Gulbenkian); B) Abnormal
cytokinesis (cleavage furrow regression) in Hodgkin´s lymphoma cells: molecular and cellular analysis of potentially
involved genes (financed by Liga Portuguesa Contra a Leucemia). A brief description of the work performed and the
main results obtained in each project follows:
A) DNA from diagnostic samples of 33 cases of acute lymphoblastic leukaemia has been previously analysed for the
IgH locus according to the following steps: detection of clonal rearrangements by multiplex PCR followed by
Heteroduplex analysis and GeneScan analysis; direct sequencing (with the appropriate primer pairs) of the clonal
products; sequence analysis of the specific rearrangement and design of an ASO-primer; sensitivity tests for the
ASO-primer/TaqMan probe. Whenever the sensitivity obtained was higher than 10-03 new ASO-primers were
designed and tested.
Over 2006 the same general strategy was used to analyse the IgK and the TcRG locus in the samples previously
studied for IgH. The data obtained for each clonality target as well as for the use of more than one target per case
(IgH and IgK; IgH and TcRG; IGH, IgK and TcRG) are similar to the scarce data published in the literature
reinforcing the conclusion that analysis of “minimal residual disease by quantitative real-time PCR” is a robust
strategy with valuable clinical application. From our data, and in keeping with some published discrepancies, the
analysis of IgK is more difficult, requiring additional primer sets for sequencing of clonal products, and also
considerable experience.
This project is considered concluded; the results are under final revision and submission for publication is expected
to be possible (in approximately 2 months).
B) Previous studies by time-lapse videomicroscopy of 3 Hodgkin’s lymphoma derived cell lines have documented
abnormal cytokinesis due to cleavage furrow regression. In an attempt to identify genes potentially involved in the
documented abnormal phenotype 2 strategies were chosen: 1) mutational analysis and (co) immunolocalization
studies of the genes (and respective proteins) shown, when abnormal, to result in cleavage furrow regression; 2)
obtention of purified mononucleated Hodgkin’s cells synchronized in G1 and significantly enriched in G2/M, as well
as appropriately synchronized non-Hodgkin’s lymphoma control cells, to perform a “large-scale” microarray analysis
of gene expression.
Following the molecular and cellular analysis of the chromosomal passenger complex previously performed, over
2006 the components of centralspindlin complex (MKLP1/CHO1 and MgcRacGAP), the Rho-A and its GEF Ect2
were studied. In addition, the mutational analysis of Anillin and Septin-7 was also performed. From the results
obtained we stress the finding of an abnormal mRNA of Ect2 detected in two of the cell lines that display the
cleavage furrow regression phenotype. If translated, the abnormal mRNA may give rise to a dominant-negative form
of Ect2 that would be expected to interfere with the normal localization of Rho-A at the cleavage furrow. In keeping
with this hypothesis, we have observed deficient Rho-A localization in the cleavage furrow in the very same cells
where the abnormal Ect2 mRNA was detected.
To further explore the potential mechanism of multinucleation mediated by the abnormal and eventually dominantnegative Ect2 form, we have obtained a myc-tagged-Ect2-expression vector to transfect Hodgkin’s cells, after
suppression of endogenous Ect2 by RNAi methodology, to verify if such a strategy prevents (or reverts) the
multinucleation phenotype.
Concerning the above mentioned strategy involving “large-scale” microarray analysis of gene expression, the
appropriately purified and synchronized sub-populations of Hodgkin’s cells and controls were prepared by elutriation
(in collaboration with Prof. William Earnshaw, head of the Structure and Chromosome Laboratory at the University
of Edinburgh). The microarray study is currently on-going (in collaboration with Prof. Martin-Leo Hansmann, head of
the Department of Pathology of the University of Frankfurt).
Seminars, Scientific meetings:
Clara Sambade was the local Organiser of the Sixth ESH-UT MD Andersson Cancer Center International
Euroconference “Mechanisms of cell death and disease: advances in therapeutic intervention and drug
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development”. European School of Haematology . Cascais, Portugal. October 2006
II GENE Meeting “Apresentação de casuistica (2000-2006)dos Serviços de Anatomia Patologica Nacionais”
IPATIMUP, November 2006.
PhD Theses:
Jorge Lima. Succinate dehydrogenase in human tumors: Mutational analysis of SDHB, SDHC AND SDHD in
paragangliomas, Phaechromocytomas and medullary thyroid carcinomas. Supervisor Manuel Sobrinho-Simões
(Porto, Portugal); cosupervisor Dillwyn Williams (Cambridge, U.K.). Medical Faculty of University
of Porto, December 2006.
Papers: Jorge Lima, Valdemar Máximo, Paula Soares, Manuel Sobrinho-Simões Comment on: Alterations of the
SDHD gene locus in midgut carcinoids. Genes, Chromosomes & Cancer 2003; April 36(4):424
Jorge Lima, José Teixeira-Gomes, Paula Soares, Valdemar Máximo, Mrinalini
Honavar, Dillwyn Williams, Manuel Sobrinho-Simões Germline SDHD mutation
segregating with familial non-RET C-cell hyperplasia. The Journal of Clinical
Endocrinology & Metabolism 2003; October 88(10) 4932-4937
Jorge Lima, Tália Feijao, Isabel Pereira-Castro, Gregorio FernandezBallester, Valdemar Maximo, Agustin Herrero, Luis Serrano, Manuel SobrinhoSimões, Ginesa Garcia-Rostan High frequency of unreported germline SDHB
mutations in sporadic head and neck paragangliomas from Northern Spain:
Structural consequences and clinico-pathological features. (submitted)
MSc Theses:
Susana Ribeiro. “Molecular and cellular analysis of cytokinesis regulators (MKLP1/CHO1, MgcRacGAP, Ect2 and
Rho-A)“ Supervisor: Clara Sambade. Medical Faculty of Porto. June 2006
Tália Figueiredo. Implications of the expression of cell membrane proteins in Well Differentiated Thyroid
Carcinomas and Thyroid Cell Lines. Supervisor Paula Soares. Medical Faculty of University of Porto, December
2006.
Hugo Seca Teixeira. Estudo da relevância da diminuição da expressão do XIAP por RNA de interferência na
resposta de células de LMC ao Imatinib. Supervisor M. Helena Vasconcelos Meehan; Co-Supervisor: José Eduardo
Guimarães. Medical Faculty of University of Porto, January 2007.
Publications
A. Related with the main research topics
1. Castro P, Rebocho AP, Soares RJ, Magalhaes J, Roque L, Trovisco V, Vieira de Castro I, Cardoso-deOliveira M, Fonseca E, Soares P, Sobrinho-Simoes M. PAX8-PPARgamma rearrangement is frequently
detected in the follicular variant of papillary thyroid carcinoma. J Clin Endocrinol Metab, 2006, 91:213-220.
2. Rocha AS, Risberg B, Magalhães J, Trovisco V, Vieira de Castro I, Lazarovici P, Soares P, Davidson B,
Sobrinho-Simões M. The p75 neurotrophin receptor is widely expressed in conventional papillary thyroid
carcinoma. Hum Pathol, 2006, 37:562-568.
3. Prazeres HJ, Rodrigues F, Figueiredo P, Naidenov P, Soares P, Bugalho, MJ, Lacerda M, Campos B,
Martins TC. Occurrence of the Cys611Tyr mutation and a novel Arg886Trp substitution in the RET protooncogene in multiple endocrine neoplasia type 2 families and sporadic medullary thyroid carcinoma cases
originating from the central region of Portugal. Clin Endocrinol (Oxf), 2006, 64:659-666,.
4. Castro P, Soares P, Gusmão L, Seruca R, Sobrinho-Simões M. H-RAS 81 polymorphism is significantly
associated with aneuploidy in follicular tumors of the thyroid. Oncogene 25:4620-4627, 2006.
5. Sobrinho-Simoes M, Maximo V. Warthin's tumour. Virchows Arch; 448:877-878, 2006.
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6. Trovisco V, Soares P, Sobrinho-Simoes M. B-RAF mutations in the etiopathogenesis, diagnosis, and
prognosis of thyroid carcinomas. Hum Pathol. 37:781-786, 2006.
7. Rocha AS, Marques R, Bento I, Soares R, Magalhaes J, de Castro IV, Soares P. Thyroid hormone
receptor {beta} mutations in the 'hot-spot region' are rare events in thyroid carcinomas. J Endocrinol
192:83-86, 2007.
8. Fonseca E, Soares P, Cardoso-Oliveira M, Sobrinho-Simoes Diagnostic criteria in well-differentiated
thyroid carcinomas. Endocr Pathol 17:109-117, 2006
9. Rindi G, Kloppel G, Alhman H, Caplin M, Couvelard A, de Herder WW, Erikssson B, Falchetti A, Falconi M,
Komminoth P, Korner M, Lopes JM, McNicol AM, Nilsson O, Perren A, Scarpa A, Scoazec JY,
Wiedenmann B; and all other Frascati Consensus Conference participants; European Neuroendocrine
Tumor Society (ENETS). TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including
a grading system. Virchows Arch 449:395-401, 2006.
B. Other topics
B1. Lima RT, Martins LM, Guimarães JE, Sambade C and Vasconcelos MH. Chemosensitization Effects of
XIAP Downregulation in K562 Leukemia Cells. J Chemother, 18, 98-102, 2006.
B2. Cameselle-Teijeiro J, Abdulkader I, Barreiro-Morandeira F, Ruiz-Ponte C, Reyes-Santias R, Chavez E,
Sobrinho-Simoes M. Breast tumor resembling the tall cell variant of papillary thyroid carcinoma: a case report.
Int J Surg Pathol14:79-84, 2006.
B3. Sambade C, Berglund M, Lagercrantz S, Sallstrom J, Reis RM, Enblad G, Glimelius B, Sundstrom C. U2940, a human B-cell line derived from a diffuse large cell lymphoma sequential to Hodgkin lymphoma. Int J
Cancer 118:555-563, 2006.
B4. Bruggemann M, White H, Gaulard P, Garcia-Sanz R, Gameiro P, Oeschger S, Jasani B, Ott M, Delsol G,
Orfao A, Tiemann M, Herbst H, Langerak AW, Spaargaren M, Moreau E, Groenen PJ, Sambade C, Foroni L,
Carter GI, Hummel M, Bastard C, Davi F, Delfau-Larue MH, Kneba M, van Dongen JJ, Beldjord K, Molina TJ.
Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of
the BIOMED-2 Concerted Action BHM4 CT98-3936. Leukemia. 2006 Dec 14; [Epub ahead of print]
B5. Langerak AW, Molina TJ, Lavender FL, Pearson D, Flohr T, Sambade C, Schuuring E, Al Saati T, van
Dongen JJ, van Krieken JH. Polymerase chain reaction-based clonality testing in tissue samples with reactive
lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936.
Leukemia. 2006 Dec 14; [Epub ahead of print]
Communications
Figueiredo J, Lima RT, Seca H, Vasconcelos MH. Downregulation of XIAP expression with siRNAs in K562
cells: effects on cellular sensitivity to doxorubicin. Book of Abstracts XVth National Congress of Biochemistry,
Aveiro, Portugal, 8-10 December 2006, selected communication SH.2, page 42 (Oral communication).
Lima J, Máximo V, Soares P, Thomas GA, Bogdanova T, Williams D, Sobrinho-Simões M. Mitochondrial DNA
large deletions and complex rearrangements in post-Chernobyl thyroid tumors. 6TH ETA-CRN MEETING,
September 2, 2006 NAPLES (Oral communication).
Seca H, Lima RT, Guimarães JE, Vasconcelos MH. Evaluation of XIAP as a Molecular Target to Increase
Sensitivity to Imatinib. 48th ASH (American Society of Hematology) Annual Meeting, Orlando, USA, 9-12
Dezembro 2006. (Abstract published in Blood, Volume 108, issue 11, Abstract 1381), 2006.
Figueiredo C;. Maximo V; Soares P; Machado JC; Seruca R; Carneiro F; Sobrinho-Simoes M. Mitochondrial
DNA (mtDNA) mutations in Helicobacter pylori chronic gastritis and gastric carcinoma. Digestive Diseases
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Week 2006 – American Gastroenterological Association. Los Angeles, California, EUA (Abstract published in
Gastroenterology 130 Suppl. 2: A-54, 2006), 2006.
Seca H, Lima RT, Guimarães JE, Vasconcelos MH.. Modulation of anti-apoptotic XIAP as an approach to
enhance response to Imatinib Mesylate. Book of Abstracts XVth National Congress of Biochemistry, Aveiro,
Portugal, 8-10 December 2006.
Seca H, Lima RT, Guimarães JE, Vasconcelos MH.. Efeitos da diminuição dos níveis de expressão de P-gp ou
de XIAP na resposta ao Mesilato de Imatinib na linha celular K562Dox. Livro de Resumos da Reunião Anual
da Sociedade Portuguesa de Hematologia,16-18 Novembro de 2006, Viseu, 2006.
Pereira M, Pinheiro C, Bento I, Costa S, Soares P, Pardal F, Lopes JM, Reis RM: Association of EGFR Intron 1
CA repeat with glioma risk. Reunião anual da Sociedade Portuguesa de Genética Humana, Coimbra, 2006.
Prazeres H, Rodrigues F, Naidenov P, Figueiredo P, Soares P, Lacerda M, Campos B, Martins TC. Análise de
perda de heterozigotia na região 19p13.2 em tumores não-medulares familiares da tireóide. VII Congresso
Português de Endocrinologia – 57ª Reunião Anual da SPEDM, Vilamoura, Portugal, 2006.
Other communications
Raquel T. Lima, J.E. Guimarães, M. Helena Vasconcelos. Overcoming resistance of a Chronic Myeloid
Leukemia cell line to Imatinib by downregulation of P-glycoprotein using siRNAs. Oral presentation by Raquel
Lima at the Course “Smart Toxicology: Subcellular targets of Anti-cancer Therapy” organized by Centro de
Neurociências e Biologia Celular da Universidade de Coimbra (CNC) and Instituto de Medicina Legal,
Faculdade de Medicina, Coimbra.
Prizes:
Best Poster “Poster Trabalho Laboratorial”, from Sociedade Portuguesa de Hematologia. Efeitos da diminuição
dos níveis de expressão de P-gp ou de XIAP na resposta ao Mesilato de Imatinib na linha celular K562Dox.
Annual Meeting Sociedade Portuguesa de Hematologia, 16-18 November 2006, Viseu, Portugal.
Best Presentation “Prémio de Investigação Básica” from Sociedade Portuguesa de Diabetes Endocrinologia e
Metabolismo. Análise de perda de heterozigotia na região 19p13.2 em tumores não-medulares familiares da
tireóide. VII Congresso Português de Endocrinologia – 57ª Reunião Anual da SPEDM, 26-29 January 2006,
Vilamoura, Portugal.
On going projects:
“Biological role of BRAF oncogene activaction in thyroid carcinogenesis. STI-571/gleeved as an inhibitor agent.”
Principal Investigators: Raquel Seruca and Paula Soares
Duration:1/5/2004 to 1/12/2007;. Budget:78869 Euros(2005: 38752 E; 2006:117647 E)
Funding: NOVARTIS
“Teste para selecção in vitro de compostos com potencial actividade anti-oxidante, obtidos através de plantas
medicinais utilizadas tradicionalmente em Portugal.”
Principal Investigator: Valdemar Máximo
Duration:1/2/2005 to 1/2/2008;. Budget :48468 Euros(2005: 19387 E; 2006/2007:19387 E)
Funding: UNICER (beverage and food company)
“Mitochondrial DNA deletions and mutations in post-Chernobyl thyroid tumors and in the respective normal thyroid
parenchyma.”
Principal Investigators: M Sobrinho-Simões and Valdemar Máximo.
Duration:1/7/2004 to 1/7/2006; Budget: 46305 Euros (2004: 30105; 2005: 10000).
Funding:FCT (Concluded in 2006)
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“Potential mechanisms of tolerance to wild-type p53 in human tumours using differentiated thyroid carcinoma as a
prototype.”
Principal Investigators: Paula Soares and Ana Preto.
Duration: 1/7/2003 to 1/12/2006. Budget: 67825 Euros (2005: 23150 E; 2006:22525 E)
Funding: FCT (Concluded in 2006)
“Role of raft domains in the modulation/alteration of the transducing pathways in normal thyroid and in papillary
thyroid carcinoma.”
Principal Investigator: Paula Soares
Duration:1/1/2004 to 1/7/2007. Budget: 72645 Euros (2005: 24219 E; 2006:24218 E)
Funding: FCT
“Biological role of BRAF oncogene activaction in thyroid carcinogenesis.”
Principal Investigators: Paula Soares
Duration: 1/9/2005 to 1/9/2008. Budget: 95250 Euros (2005: 30950 E; 2006:31900 E)
Funding: FCT
“Mapping of Genes Predisposing to Familial Thyroid Tumours.”
Principal Investigators: Valdemar Máximo
Duration: 1/09/2006 to 1/09/2009. Budget: 86700€
“Risco de cancro em indivíduos irradiados para tratamento da tinha do couro cabeludo (Tinea Capitis) durante a
infância. Estudo de follow-up de uma coorte do norte de Portugal.”
Principal Investigator: Teixeira Gomes
Duration: 1/3/2006 to 1/3/2008. Budget: 91000 Euros.
Funding: Fundação Calouste Gulbenkian
“Overcoming resistance to imatinib mesylate in leukemia cell line models by specific downregulation of MDR1 with
siRNAs”
Principal Investigators: J.E. Guimarães and M. Helena Vasconcelos Meehan
Duration: 15/2/2004 to 15/2/2006. Budget: 51000 Euros.
Funding: NOVARTIS
“Validação do papel do XIAP na resistência da leucemia mielógena aguda à quimioterapia: abrir caminho para uma
possível nova estratégia de tratamento tendo o XIAP como alvo terapêutico”
Principal Investigators: M. Helena Vasconcelos Meehan
Duration: 1/6/2005 to 1/9/2007. Budget: 15000 Euros. (2005: 7500 E; 2006:7500 E)
Funding: "Associação Portuguesa Contra a Leucemia":
“Molecular dissection of the multinucleation phenotype of the neoplastic cells in Hodgkin´s lymphoma.”
Principal investigator: Clara Sambade
Duration: 1/6/2005 to 1/6/2007. Budget: 15000 Euros. (2005: 7500 E; 2006:7500 E)
Funding: "Associação Portuguesa Contra a Leucemia":
“Neuroendocrine tumors: Clinico-pathological and immunohistochemistry characterization and identification of
biological factors of aggressiveness”
Principal Investigator: José Manuel Lopes
Duration: 23/9/2004; to 23/9/2007. Budget: 53150 Euros.
Funding: NOVARTIS
“Is the mTOR pathway relevant in the initiation/progression and/or a putative therapeutic target in melanomas?”
Principal Investigators: José Manuel Lopes and Paula Soares
Duration:1/5/2006 to 1/5/2008;. Budget :54500 Euros(First year: 27250 E; Second year:27250 E)
Funding: NOVARTIS
15
Cancer Genetics
Coordinator: Raquel Seruca
Principal Investigators: Carla Oliveira, Céu Figueiredo, Fátima Carneiro, Fernando Schmitt, Gianpaolo
Suriano, José Carlos Machado.
Post-Doc students: Ana Preto, Cecília Durães, Joana Paredes, Maria José Oliveira.
PhD students: Ana Costa, Ana Machado, André Albergaria, António Carlos Ferreira, Catarina Alves,
Fernanda Milanezi, Gonçalo Regalo, Joana Correia, Paulo Canedo, Rachid Karam, Rita Mateus, Sérgia
Velho, Sílvia Carvalho, Sónia Melo.
The research of our group focuses on the molecular genetics of three common types of epithelial cancer
(gastric, breast, and colorectal carcinoma). We aim at: 1) identifying individuals at risk for the various
forms of these tumours; 2) identifying pathological features and molecular markers occurring in the
setting of familial and sporadic carcinoma; 3) identifying signalling pathways mediated by genetic and
environmental factors in tumour development, in order to find new molecular targets for therapeutic
intervention.
In 2006 the Cancer Genetics Group aimed at answering to the aforementioned questions
1. We aimed at identifying epi/genetic mechanisms leading to E-cadherin dowregulation in the
germline of HDGC patients and second-hit mechanisms leading to E-cadherin silencing in
sporadic and hereditary forms of diffuse gastric carcinoma and to
2. We aimed at further studying the signalling pathways mediated by E-cadherin mutations, that
were shown to have distinct cellular behaviours, and find the pivotal proteins for cell motility (in
vitro) and invasion in diffuse gastric cancer.
3. We aimed at investigating how H. pylori strains with different virulence mediate host cell effects
and interfere with signalling pathways relevant to gastric carcinogenesis.
4. We aimed to unravel the signalling pathways that lead to overexpression of CEBP in gastric
carcinoma and studying its cellular effects.
5. We aimed at further studying the signalling pathways mediated by de novo expression of Pcadherin and find the pivotal proteins for cell invasion in breast cancer.
6. We aimed at evaluating if metaplastic breast carcinomas share molecular and pathological
features previously found in basal like breast carcinomas in order to be able to offer a precise
prognosis and correct therapeutics in this type of breast cancer.
7. We aimed at identifying the cellular effects mediated by BRAF activating in colon cancer cell
lines and to verify its timing of occurrence using colorectal lesion in different stages of
malignancy.
In this report we will describe results obtained in 2006
1) Identifying individuals at risk of gastric cancer
E-cadherin is the only gene identified, to date, with a causative role in Hereditary Diffuse Gastric Cancer.
Germline mutations in the E-cadherin gene CDH1, cause 30% of hereditary diffuse gastric cancer
(HDGC), an autosomal dominant gastric cancer susceptibility syndrome which also predispose to familial
breast and colon cancer. A minor proportion of these families was studied for the inactivation of the
CDH1 wild-type allele in HDGC tumour samples. The mechanisms found to be involved were:
hypermethylation of the CDH1 gene promoter, somatic CDH1 mutations and CDH1 intragenic deletion.
nd
In 2006, we have enlarged the analysis of the 2 hit in tumours (n=8) from HDGC patients and found
that 6/8 (75%) of them display CDH1 promotor methylation (unpublished results). In 60-70% of the
HDGC families and in 90% of families with at least two gastric cancers but with diffuse histotpathology
confirmed in a single case (FDGC families), CDH1 germline inactivation failed to be identified. Although
current techniques of genotyping did not reveal CDH1 mutations, diffuse gastric tumours occurring in
these families display similar morphological features and E-cadherin immunostaining to those harbouring
CDH1 germline mutations (unpublished results). We discarded large deletions of the CDH1 gene, which
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are easily missed by PCR based techniques, by MLPA in 128 gastric cancer affected members from 109
families with aggregation of gastric cancer (HDGC+FDGC) from different geographic backgrounds (data
not published).
HDGC tumours displaying E-cadherin loss of expression and function impairment, which are negative for
CDH1 germline alterations, represent the ideal target to identify mechanisms and target sequences
involved in E-cadherin downregulation. In 2006, we designed a new strategy to analyse, in the germline
of diffuse gastric cancer family members, allele specific CDH1 expression. This analysis, performed in
14 probands, allowed the identification of six (6/14-43%) patients harbouring monoallelic E-cadherin
expression downregulation, who probably harbour a molecular mechanism different from the currently
screened mutations, acting as a first hit. From these six probands, one harboured germline methylation
in the promoter of CDH1, and this epimutation was absent in two other cancer associated gene
promotors hMLH1 and RASSF1 used as negative controls. As for the five germline promoter methylation
negative probands, displaying allelic expression imbalance, we submitted the full CDH1 sequence to
GENSCAN and thirteen putative transcribed sequences within CDH1 introns and untranslated regions
were predicted. Two transcribed regions inside intron 2 were already identified and are expressed in a
RNA pool of different organs: one splicing in-frame (exona) and another splicing out-of-frame (exonb)
with exon 3 of the native form of E-cadherin. Real-time PCR performed in two cancer cell lines and 4
different organs showed differential expression of each of these transcripts in comparison with the CDH1
native form. Although preliminary, these results seem to show that the expression of these transcripts is
tissue specific, variable and independent from the expression of the native form. We have reasons to
believe that one carries an in-frame AUG putative initiation codon, and may be a translated isoform of Ecadherin (isoform a), the other lacks a initiation signal and may work as a non-coding mRNA regulatory
molecule (non-coding sequence b) (data not published).
Besides identifying epi/genetic mechanism leading to E-cadherin dowregulation in hereditary forms of
diffuse gastric carcinoma our team focuses on the understanding the role of E-cadherin germline
missense mutations found in the setting of both hereditary diffuse gastric cancer and early onset diffuse
gastric cancer. As reference laboratory of the International Gastric Cancer Linkage Consortium, we have
been involved in the functional characterization of new HDGC-associated E-cadherin germline missense
mutations, aiming at unrevealing their pathogeneicity, using in vitro assays. In 2006 we have completed
the functional characterization of 6 new HDGC-associated E-cadherin germiline missense mutations (2
from Canada, 2 from New Zeland, 1 from Italy and 1 from England), which proved in vitro to be all
pathogenic. Results were submitted to the respective laboratories, and shared with the clinicians
responsible for the genetic counselling and clinical follow-up of the carrier families. On the other hand,
results were also integral part of research publications in highly recognised medical journals. This work
has been also pivotal for the establishment or strengthening of international collaborations. Taking
advantage of the large panel of E-cadherin germline missense mutations in our hand and in line with
similar approaches used for BRCA1 and BRCA2 germline missense mutations in breast cancer, we have
outlined a multivariate approach to infer the significance of such variants. We reviewed all HDGCassociated E-cadherin germline missense mutations reported to date. The information collected
included: co-segregation of the mutation within pedigrees, frequency in healthy population control,
recurrence in independent families, and functional in vitro and in silico data. We used the neighborjoining method to group mutations according to the collected information and assessed the robustness of
mutation clusters with a bootstrap test. CDH1 germline missense variants were classified according to
the parameters defined in the multivariate analysis. This analysis allowed the distribution of the variants
into two distinct groups: neutral variants versus mutations, thus demonstrating its usefulness in
predicting the deleterious nature of CDH1 germline missense variants of unknown significance.
We have been actively involved in the identification of inflammation-related genetic polymorphisms
associated with risk of development of sporadic GC. Our work culminated with the demonstration that it
is possible to define a specific genetic profile associated with highest risk of developing GC. In 2006, we
extended our analysis to additional polymorphisms in inflammation-related genes such as IL8, TNFA and
IFNGR1. We performed a case-control analysis, including 693 controls, 187 chronic gastritis cases and
333 GC cases, in order to determine the association between the IL8-251 polymorphism and risk of
chronic gastritis and GC in the northern Portugal population. We found no significant association
between the IL8-251 polymorphism and increased risk of chronic gastritis or GC, in agreement with was
has been reported in other populations of Caucasian origin. The retrospective analysis of published data
shows that the association between the IL8-251 polymorphism and risk of GC tends to be reproducible in
populations of Asian origin. The estimated effect of the polymorphism under analysis was not
significantly different in subgroups of GC cases defined by histologic type and anatomic site of the
17
tumours, and by sex and age of the subjects. In conclusion our results indicate that although the IL8-251
polymorphism might be a relevant host susceptibility factor for GC development, this association is likely
to be ethnic-specific.
One of the best studied examples of the association between inflammation-related host polymorphisms
and GC risk is the tumour necrosis factor alpha (TNFA)-308 polymorphism. This association finds further
support in functional evidence showing that the TNFA-308*A allele increases the transcriptional activity
of the TNFA gene. However, in some studies the association between the TNFA-308*G/*A
polymorphism and risk of GC could not be replicated. In the present study, our aims were: 1) to perform
a case-control study, including 713 controls and 508 GC patients, in order to confirm the association
between the TNFA-308*G/*A polymorphism and risk of GC; 2) to extend our analysis to five polymorphic
microsatellite markers (TNFA-a, -b, -c, -d and -e), in 169 controls and 122 GC patients, in order to
establish the haplotypic structure associated with the TNFA-308*G/*A polymorphism in both cases and
controls. Haplotype determination is likely to provide a basis for elucidating whether the association
between the TNFA-308*A allele and increased risk of GC is etiological or secondary to linkage
disequilibrium, with an as yet unidentified locus. Our results confirm the existence of a significant
association between the pro-inflammatory TNFA-308*A*A genotype and increased risk of GC with an
OR of 1.7 (95% CI, 1.3-2.2). Haplotype inference disclosed a higher number of haplotypes associated
with the TNFA-308*G allele than with the TNFA-308*A allele in both cases and controls, providing
support to the hypothesis that the TNFA-308*A allele is the most recent allele. Although all inferred
haplotypes have been observed in both cases and controls, we found significant differences in its
frequencies between the two groups. The most significant difference was that found for the TNFA
a2b3c2-308*Ad2e4 haplotype which was observed in 33.1% of the cases and in 12.5% of the controls.
When microsatellite markers were considered individually, we found higher magnitude associations
between microsatellite markers and increased risk of GC, than that observed with the TNFA-308*A
allele; as example, the TNFd2 allele was significantly associated with development of GC with an OR of
2.8 (95% CI, 1.4-6.4). These results support the hypothesis that the observed association between the
TNFA-308*A allele and increased risk of GC is likely to be secondary to linkage disequilibrium with an as
yet unidentified locus.
Recently polymorphisms in the gene encoding the chain 1 of the interferon gamma receptor (IFNGR1)
were found to be associated with increased susceptibility to H. pylori infection. We aimed to determine
the association between the IFNGR1-611G/A IFNGR1-56C/T IFNGR1+1004*A/*C and
IFNGR1+1400*T/*C polymorphisms in the IFNGR1 gene and risk of chronic non-atrophic gastritis
(CNAG), chronic atrophic gastritis (CAG) and GC. In a case-control study including 733 controls and
393 GC patients, the polymorphism were genotyped. In individuals with early onset GC (defined as
having less than 40 years of age at the time of diagnosis) we found a significant over-representation of
the IFNGR1-56*T/*T homozygous genotype with an odds ratio (OR) of 4.1 (95% confidence interval [CI]
1.6-10.6). The effect of the -56C/T promoter polymorphism in the level of expression of the IFNGR1
gene was evaluated by an IFNGR1-56C/T allele specific luciferase reporter assay. In this assay we
observed a 10-fold increase (P < 0.001) in luciferase expression associated with the IFNGR1-56*T
allele. Our results indicate that the IFNGR1-56C/T polymorphism is a relevant host susceptibility factor
for GC development. Our data also indicate that this genetic polymorphism is functionally relevant and
may be related with early development of GC. The -56*T allele is associated with significantly increased
expression of the IFNGR1 gene and may therefore play an important role in modulating the host
inflammatory response to H. pylori infection.
1) Identifying individuals at risk of breast cancer
The purpose of this study was to evaluate the role of polymorphisms in DNA repair genes as genetic
indicators of susceptibility to familial and sporadic breast cancer. In 2006, we analysed DNA samples
from 285 breast cancer patients and 442 control subjects, for XRCC1 Arg399Gln, XPD Lys751Gln,
RAD51 G135C and XRCC3 Thr241Met polymorphisms, using PCR-RFLP. We observed that women
carriers of XRCC1 399Gln genotypes, and without family history (FH) of breast cancer, have a protective
effect concerning this disease (OR = 0.54 95% CI 0.35-0.84; p = 0.006). Furthermore, we found that
carriers of XRCC3 241Met genotypes without FH have an increased susceptibility for breast cancer (OR
= 2.21 95% CI 1.42-3.44; p < 0.001). Additionally, we verified an increased risk of breast cancer in
women with FH and carrying RAD51 135C genotypes (OR = 2.17 95% CI 1.19-3.98; p = 0.012). Our
results suggest XRCC1 Arg399Gln and XRCC3 Thr241Met DNA repair polymorphisms as important
biomarkers to sporadic breast cancer susceptibility, as well as, RAD51 G135C polymorphism as a real
risk modifier in familial breast cancer cases.
18
2) Identifying pathological features and molecular markers occurring in the setting of familial and
sporadic cancer
A) in Brest Cancer:
Gene expression profiles of invasive breast carcinomas have identified a subgroup of tumours with
worse prognosis, which have been called "basal-like". These are characterized by a specific pattern of
expression, being estrogen receptor (ER) and HER2 negative, and frequently expressing at least one
basal marker such as basal cytokeratins or epidermal growth factor receptor (EGFR). In 2005, our group
characterized basal-like tumours in a series of invasive breast carcinomas using P-cadherin (P-CD), p63
and cytokeratin 5 (CK5). Based on thist study, we hypothesized that those high-grade basal-like invasive
carcinomas might have a pre-invasive counterpart, which could be identified using the same approach.
In 2006, a series of 79 ductal carcinomas in situ (DCIS) were classified into distinct subgroups according
to their ER, HER2 and basal markers expression. Sixty five percent of the cases of DCIS expressed ER,
25% of the cases had HER2 overexpression and did not express ER and 10% of the cases lack the
expression of ER and HER2 and expressed at least one basal marker (P-CD, CK5, CK14, p63, vimentin
and/or EGFR). These basal-like DCIS were mostly high-grade, with comedo-type necrosis, and
consistently showed expression of P-CD and CK5. In conclusion, DCIS with a basal-like phenotype
represent a small percentage in our series, being P-CD and CK5, the most useful adjunct markers to
distinguish this subset of carcinomas in situ of the breast.
B) in Colorectal Cancer:
In sporadic colorectal cancer (CRC), oncogenic mutations affecting KRAS and BRAF occur in about 30
and 10% of the cases, respectively. KRAS mutations have been observed in colorectal tumours
independently of their microsatellite instability (MSI) status. In sporadic MSI CRCs, KRAS mutations are
inversely associated to the oncogenic BRAFV600E mutation. In contrast to sporadic MSI CRC, data on
the presence of both KRAS and BRAF oncogenic mutations in sporadic MSS CRC and their relationship
with tumour progression were scarce.
In 2006, we screened KRAS and BRAF in 250 MSS primary CRC and 45 lymph node (LN) metastases
and analysed the pathological features of the cases to understand the involvement of KRAS–BRAF
activation in progression and metastasis. Forty-five per cent of primary MSS CRCs carried mutations in
at least one of these genes and mutations were associated with wall invasion (p=0.02), presence and
number of LN metastases (p=0.02 and p=0.03, respectively), distant metastases (p=0.004) and
advanced stage (p=0.01). We demonstrated that KRAS and BRAF are alternative events in Tis and
T1MSS CRC and, KRAS rather than BRAF mutations, contributed to the progression of MSS CRC. The
frequency of KRAS and/or BRAF mutations was higher in LN metastases than in primary carcinomas
(p=0.0002). Mutated LN metastases displayed KRAS associated or not with BRAF mutations. BRAF
mutations were never present as a single event. Concomitant KRAS and BRAF mutations increased
along progression of MSS CRCs, suggesting that in this subset of CCR activation of both genes is likely
to harbour a synergistic effect.
3) Identification of signaling pathways mediated by genetic and environmental factors in the
distinct cancer models, and target-specific therapeutic drugs
Signaling pathways mediated by H. pylori
The interaction of H. pylori with the host gastric epithelium is the basis for the development of gastric
disease. In 2006, we have explored the relationship between H. pylori virulence factors and cell invasion.
We have demostrated, using the gastric carcinoma-derived AGS cell line as a model, that H. pylori
induces cell invasion. We have shown that H. pylori-induced cell invasion is dependent on the activation
of the c-Met receptor and on increased MMP-2 and MMP-9 activity. Studies with different H. pylori wildtype strains and their respective mutants for virulence factors such as the cag pathogenicity island (cag
PAI), CagA, and VacA, showed that cell invasion, c-Met receptor activation, and increased MMP-2 and
MMP-9 activity, were all dependent on the presence of a functional bacterial type IV secretion system
(encoded by the cag PAI), but not on VacA cytotoxicity. In order to identify the molecular mechanisms
involved in H. pylori-mediated cell invasion downstream c-Met receptor we will transfect cells with siRNA
for c-Met adaptors, c-Met effectors, and GTPases and perform invasion assays using those cells after
infection with H. pylori. We will also use westernblot and immunoprecipitation analysis for evaluating
protein interactions. Part of this work will be performed in collaboration with Thomas Meyer’s group at
the Max Planck Institute for Infection Biology, in Berlin, Germany.
19
Signaling pathways mediated by P-cadherin
P-cadherin molecule is overexpressed in basal-like breast carcinomas, predicting a worse prognosis for
patients. We showed that this pro-invasive activity of P-cadherin is awarded to its juxtamembrane
domain, and probably also to its interaction partners, like p120-catenin (p120ctn). Analogously to Pcadherin, p120ctn has been implicated in the carcinogenesis process, although there are few studies
about its role in breast cancer.
In 2006 we aimed at was to clarify if there is an effective role of P-cadherin together with p120ctn in
breast cancer. We were particularly interested in studying the influence of P-cadherin expression on
p120ctn subcellular localization in human invasive breast carcinomas. We found that P-cadherin
overexpression in E-cadherin positive breast carcinomas induces p120ctn to move from the membrane
to the cell cytoplasm. p120ctn cytoplasmic localization was significantly correlated with the lack of
estrogen receptor, poorly differentiated carcinomas and poor patient survival in a short-term follow-up.
Hence we can conclude that p120ctn play a relevant role in breast cancer, since it seems to constitute
an important mediator of the oncogenic effects derived from P-cadherin overexpression in these
tumours.
In order to elucidate the role of P-cadherin in cell invasion, we also investigated the effect of this
molecule on cell migration, as well as the expression and activity profile of matrix metalloproteases
(MMPs) using a breast cancer cell line (MCF-7/AZ) retrovirally transduced with human P-cadherin. Using
wound-healing migration assay and time-lapse microscopy, we demonstrated that P-cadherin
overexpression is also able to promote breast cancer cell migration and cell motility. Considering the
pattern of MMPs expression and activity, we showed that P-cadherin overexpressing cells cultured in a
collagen type I matrix, have an induction in activity of MMP-1 and MMP-2. Concerning MMPs secretion
to the medium, an increase in expression of MMP-1, MMP-2 and TIMP-1 was observed in P-cadherin
transduced cells, as well as a decrease in expression of MMP-3 and MMP-9. Concomitantly with the
increase in activity and expression of some MMPs in the conditioned medium of MCF-7/AZ.P-cad cells, it
was also detected a soluble P-cadherin fragment, with 80kDa, although we do not know yet its biological
relevance.
Taken together, all the results show that P-cadherin overexpression promotes cell migration and
increases expression and activity of MMP-1, MMP-2 and TIMP-1 in breast cancer cells, cultured in a
collagen type I matrix.
Furthermore, in 2006 we cloned CDH3 promoter in a luciferase reporter plasmid, in order to identify
which transcription factors can activate or repress P-cadherin expression.
Signaling pathways mediated by E-cadherin
E-cadherin missense mutations represent a unique and subtle research opportunity to understand the
role of E-cadherin deregulation in cancer, and in particular in cell motility and invasion. Using cell lines
stably expressing mutant forms of E-cadherin we have shown that mutations on the extracellular domain
of the protein exhibit enhanced cell motility mediated through RhoA activation. In 2006, we have
challenged the hypothesis of the existence of a bidirectional cross-talk between E-cadherin and EGFR
activity, and characterized in vitro the effect of its subversion on cell behaviour. We built this hypothesis
on the knowledge that EGFR and E-cadherin colocalize at the basolateral areas of polarized epithelial
cells. EGFR activation has been shown to inhibit E-cadherin-dependent adhesion; but also EGFdependent activation of EGFR has been reported to be inhibited in an E-cadherin adhesion-dependent
manner. Furthermore, EGFR has been found in many human tumours and it has been associated to
enhanced cell motility and invasion. Our preliminary data obtained using a subset of E-cadherin germline
missense mutations supported the existence of a physical interaction between E-cadherin and EGFR,
likely taking place at the extracellular side. We further explore this model and showed that E-cadherin
might function as a natural inhibitor of EGFR, through the formation of inactive heterodimers. Loss of
such interactions (i.e. caused by mutations on the extracellular domain of E-cadherin) leads to the
activation of EGFR and consequently to enhanced cell motility. In keeping with our previous observation,
the effect of EGFR activation on cell motility was shown to be transmitted through the activation of the
small GTPase RhoA.
In 2006, among the E-cadherin germline missense mutations that were submitted to us for functional
analysis, a clustering was observed in the juxtamembrane domain of the E-cadherin protein. This
domain is localized within the cytoplasmic tail of the protein, and represents the binding site for p120cat
and Hakai, two molecules that together with Arf6 have recently merged as key regulator of E-cadherin
membrane trafficking and stabilization. In 2006, cell lines stably expressing the above mentioned Ecadherin germline missense mutation were obtained and preliminary data suggest that these mutations
interfere with the stability of the E-cadherin/p120 complex as well as with the pattern of E-cadherin
20
phosphorylation. Characterization of E-cadherin cellular distribution revealed that mutations cause
displacement of the protein from the plasma membrane to the cytoplasm and perinuclear region,
supporting the hypothesis of protein trafficking deregulation. These preliminary observations have paved
the way to a new research project, which aims at exploring the role of E-cadherin membrane trafficking
deregulation in carcinoma progression. A major effort has been put in the preparation for this new task.
Cell lines stably expressing GFP tagged forms of E-cadherin wild-type and mutants were established,
which will allow the characterization of E-cadherin cellular fate in living cells. Yeast-two-hybrid analysis
for the identification of new E-cadherin partner at the level of the juxtamembrane domain was launched
and results are expected shortly. A new important international collaboration was established with Prof.
Frans van Roy in Ghent, and which will be pivotal for a successful accomplishment of the project.
C/EBP transcription factors and gastric carcinogenesis
CCAAT/enhancer-binding protein (C/EBP) family members are transcription factors containing a highly
conserved basic leucine zipper domain (bZIP) that is involved in homo or heterodimerization and DNA
binding. The C/EBP transcription factors have been shown to play a pivotal role in regulating key
biological processes such as cell proliferation, differentiation and apoptosis, which are thought to be
crucial in tumourigenesis. The C/EBPbeta transcription factor has been associated with several cancer
models and was recently found to be overexpressed in gastric carcinoma (GC). In 2006, We analyzed
the expression of C/EBPbeta in a series of 90 GC. CEBPB gene mutations were screened for in a series
of 35 primary GC. The functional activity of C/EBPbeta was analysed in GC cell lines. In normal gastric
mucosa, C/EBPbeta expression was restricted to cells with a proliferative phenotype. In intestinal
metaplasia, dysplasia, and GC of the intestinal and atypical subtypes, C/EBPbeta was overexpressed
(P=0.0005, for the association with histological type). C/EBPbeta and Ki67, a marker of cell proliferation,
were also co-expressed in primary GC. In one GC we found a mutation of the CEBPB gene. Using GC
cell lines we showed that transfected C/EBPbeta is able to regulate the expression of endogenous
cyclooxygenase-2 (COX-2). Back in the series of metaplasia, dysplasia and GC lesions we observed a
very high degree of overlap between expression of C/EBP-beta and COX-2. In GC cell lines we have
also shown that transfected C/EBP-beta is able to modulate proliferation in these cells. These results
suggest that C/EBPbeta may both be a marker of neoplastic transformation and play an active role in
gastric carcinogenesis through its ability to up-regulate COX-2 expression.
Signaling pathways mediated by BRAF in MSI colorectal carcinoma (CRCs)
KRAS and BRAF are members of the MAP kinase (MAPK) pathway. In sporadic MSI CRCs, KRAS
mutations are inversely associated to the oncogenic BRAFV600E mutation, suggesting that each
mutation can induce similar cellular effects and signal through the same pathway. However, the
V600E
biological significance of mutated BRAF
in MSI colorectal carcinomas remains to be elucidated.
In 2006, we studied the cellular effects and the associated signalling pathways mediated by BRAFV600E
mutation in MSI CRCs by using RNA interference (RNAi) to selectively knockdown BRAF in CRCs cell
lines harbouring KRAS or BRAF activating mutations. Inhibition of BRAF in MSI CRCs with BRAFV600E
or KRASG13D mutations significantly inhibits proliferation (by BrdU incorporation). Inhibition of BRAF
induces a significant increase in apoptosis (by TUNEL assay) only in cell lines with BRAFV600E
(CO115, RKO). No significant differences were seen in apoptosis in the cell line with KRASG13D
mutation.
We further analysed the possible signalling pathways that might be implicated in proliferation (pERK1/2,
Cyclin D1, p27), and apoptotic pathways (Bcl-2, Bax, Mcl-1, pAkt, pBad, XIAP). We found a decrease in
ERK1/2 phosphorylation and in Cyclin D1 levels in all cell lines independently of the genetic background.
No alterations were found Bax, Mcl-1, pAkt, pBad, XIAP. In cell lines with mutated BRAF we found an
increase in p27 levels and a decrease in the levels of the antiapoptotic protein Bcl-2. In summary, we
showed that MSI KRAS and BRAF mutant cell lines responded differently to BRAF knockdown. We
demonstrated that BRAFV600E in MSI CRCs provide cell survival signals that involve Bcl-2. We provide
evidences that BRAF is likely to be a good target for therapeutic intervention in a specific subset of MSI
colorectal cancer.
4) Known target-specific therapeutic drugs in breast cancer
As we reported in 2005, metaplastic breast carcinomas harbour epidermal growth factor receptor
(EGFR) overexpression in up to 80% of the cases. In one-third of these overexpressing EGFR
carcinomas, gene amplification is the underlying genetic mechanism. To confirm our previous results, in
2006, we screened EGFR gene amplification and expression in a cohort of 47 metaplastic breast
21
carcinomas. Furthermore, in these series we investigated EGFR mutations in exons 18, 19, 20, and 21.
Thirty-two cases showed EGFR overexpression and of these, 11 (34%) harboured EGFR gene
amplification. The association between amplification and overexpression of EGFR was significantly
associated (p < 0.0094) and was restricted to carcinomas with homologous metaplasia. No activating
EGFR mutations were identified, suggesting that this is unlikely to be a common alternative underlying
genetic mechanism for EGFR expression in metaplastic breast carcinomas. Given that metaplastic
breast carcinomas are resistant to conventional chemotherapy or hormone therapy regimens and that
tumours with EGFR amplification are reported to be sensitive to EGFR tyrosine kinase inhibitors, these
findings indicate that further studies are warranted to explore EGFR tyrosine kinase inhibitors as
potential therapeutic agents for metaplastic breast carcinomas harbouring amplification of 7p11.2.
HER2/neu is another member of the HER family that is overexpressed in 15 to 30% of invasive breast
carcinomas and is associated with poor prognosis and resistance to hormonal therapy. Overexpression
and/or amplification of HER2 is an eligibility requirement for trastuzumab therapy, a target-specific
therapy that acts by blocking the extracellular domain of the receptor. However, almost 50% of breast
cancer cases considered positive for HER2 did not respond to the trastuzumab therapy. Most of the
antibodies used to recognize HER2 are not specific for the extracellular domain (ECD) of the protein that
is the target of trastuzumab. Since it was described cleavage of HER2 with loss of the ECD, these cases
although positive for HER2 can not respond to therapy. One can hypothesize that the ECS-directed
antibodies constitute an excellent tool to predict the availability of the trastuzumab target epitope. In
2006, our group use a novel rabbit monoclonal antibody (SP3) that recognize the ECD of the HER2
receptor in a series of 190 cases of invasive breast carcinomas and compare the results with
amplification of the gene. SP3 staining had a high specificity (99%) and a moderate sensibility (52.3%) to
assess HER2, having CISH as a gold standard. So, we conclude that the assessment of SP3 could
constitute an excellent tool to predict the availability of the trastuzumab target epitope and can explain
some positive cases assessed for other antibodies that do not respond to therapy. Now our group is
aiming to study another molecular mechanisms that can explain trastuzumab resistance in HER2
positive cases.
Financial support of the Cancer Genetics Group
The Cancer Genetics Group got for 2006, after deduction of 20% overheads, grants and missions for lab
running costs- 287.965,27Euros.
PI: F Schmitt
• “DNA repair gene polymorphisms in breast cancer patients from Portuguese origin”, FLAD
(172/2002), 5.692,59 €.
•
“Myoepithelial differentiation in breast carcinomas: pathological characterization and clinical
implications”, FCT (POCTI/CBO/45157/2002), 8.911,85 €.
•
“P-cadherin in Breast Cancer: what regulates its aberrant expression and how it can induce
invasion of neoplastic cells?”, FCT (POCTI/BIA-BCM/59252/2004), 26.757,57 €.
PI: J Paredes
• “Cell signalling mediated by P-cadherin expression in neoplastic invasion modulation using in
vitro assays”, Prémio Gulbenkian de Estímulo à Investigação (FCG 55/05), 5.220,06 €.
PI: G Suriano
• “E-cadherin germline missense mutations and hereditary diffuse gastric cancer: a model for the
identification of the E-cadherin-dependent signaling pathways pivotal for cell invasion”,
FCT(POCI / SAU - OBS / 57670 / 2004), 15.630,00 €
•
“Estudo da heterogeneidade fenotípica patológica usando a proteína ornitina transcarbamilase
(OTC) como modelo”, FCT (POCI / CVT / 58082 / 2004), 15.019,00 €
•
“Mechanisms of cell invasion: possible application to oncobiology”. Programa IDEIA, Agência de
Inovação (INV-ONC-DPN), 24.940,00€
PI: C Oliveira
• Identificação de mecanismos celulares subjacentes ao desenvolvimento de cancro gástrico em
familiares portadores e não por-mutações germinativas da caderina – E, FCT(POCI / SAU OBS / 58111 / 2004), 21.332,00 €
22
PI: R Seruca
• No cancro colorectal com instabilidade de microssatélites são os genes BRAF e KRAS novos
marcadores de prognóstico e novas terapêuticas?, FCT (POCI / SAU - OBS / 56921 / 2004),
18.627,20 €
•
Prevention, diagnosis and molecular characterisation of mismatch repair defect-related
hereditary cancers of the digestive system, FP6 (FP6-2004-LIFESCIHEAL TH-5), 30.000,00 €
PI: JC Machado
• The role of chronic infections in the development of cancer, INCA, FP6, 16.000,00 €
•
Identification of genetic markers for risk of development of gastric carcinoma in the Portuguese
population, SAUDE XXI, 54.203,00 €
•
Teste de susceptibilidade genética para determinação de risco de desenvolvimento de Doença
de Crohn na população Portuguesa. TSG-CROHN, IDEIA-ADI, 33.400,00 €
PI: F Carneiro
• Análise funcional dos repressores da caderina-E (Slug, ZEB1 e E12/E47) em carcinomas do
estômago, FCT (POCI / SAU - OBS / 57275 / 2004), 13.540,00 €
PI: C Figueiredo
• Efeitos da infecção por Helicobacter pylori em células epiteliais gástricas, FCT (POCI / SAU IMI / 56681 / 2004), 14.692,00 €
Supervision
1. Fernando Schmitt – Jorge Sérgio Reis-Filho – PhD thesis – “Breast Cancer Molecular
Pathology: Resolving quandaries”, Escola de Ciências da Saúde, Universidade do Minho,
Braga, 2006.
2. Fernando Schmitt – Ana Sofia Ribeiro – Master thesis - “P-cadherin role in Breast Cancer Cell
Migration and Invasion”, Molecular Oncology Master Course of Medical Faculty, Porto
University, 2006.
3. Fernando Schmitt – Maria de Fátima Duarte – PhD thesis – “Mechanisms utilized by growth
factors and cytokines in angiogenesis: role of thrombin in the cross-talk between the FGF1 and
Notch signaling pathways”, Escola de Ciências da Saúde, Universidade do Minho, Braga, 2006.
4. Joana Paredes – Ana Luísa Correia – Estágio de Licenciatura - “The importance of p120-catenin
in Breast Cancer”, Licenciatura em Biologia Aplicada, Universidade do Minho, Braga, 2006.
5. Céu Figueiredo- Ana Clara Reis Teixeira- Master thesis- “Caracterização molecular das estirpes
de Helicobacter pylori numa população pediátrica”, Molecular Oncology Master Course of
Medical Faculty, Porto University, 2006.
Papers published in 2006:
In 2005, the Cancer Genetics Group planned to publish in 2006 between 15-25 manuscripts in peerreview International Journals with IF above 3 and 2 manuscripts above 6 IF.
In 2006, the Cancer Genetics Group published 44 manuscripts , 40 (91%) with IF, 18 with IF ≥ 3 and < 6
and 10 with IF ≥ 6.
The next Table describes in detail the papers published in 2006
1. Agudo A, Sala N, Pera G, Capella G, Berenguer A, Garcia N, Palli D, Boeing H, Del
Giudice G, Saieva C, Carneiro F, Berrino F, Sacerdote C, Tumino R, Panico S, Berglund
G, Siman H, Stenling R, Hallmans G, Martinez C, Amiano P, Barricarte A, Navarro C,
Quiros JR, Allen N, Key T, Bingham S, Khaw KT, Linseisen J, Nagel G, Overvad K,
Tjonneland A, Olsen A, Bueno-de-Mesquita HB, Boshuizen HC, Peeters PH, Numans
ME, Clavel-Chapelon F, Boutron-Ruault MC, Trichopoulou A, Lund E, Blaker H, Jenab
M, Ferrari P, Norat T, Riboli E, Gonzalez CA. No association between polymorphisms in
CYP2E1, GSTM1, NAT1, NAT2 and the risk of gastric adenocarcinoma in the European
4.5
23
prospective investigation into cancer and nutrition. Cancer Epidemiol Biomarkers Prev
15: 1043-5, 2006.
2. Allazzouzi H, Suriano G, Guerra A, Plaja A, Espin E, Armengol M, Alhopuro P, Velho S,
Shinomura Y, Gonzalez-Aguilera JJ, Yamamoto H, Aaltonen LA, Moreno V, Capella G,
Peinado MA, Seruca R, Arango D, Schwartz Jr S. Tumour selection advantage of nondominant negative p53 mutations in homozygous mdm2-snp309 colorectal cancer cells.
J Med Genet 44: 75-80, 2007.
4.3
3. Azevedo L, Suriano G, van Asch B, Harding RM, Amorim A. Epistatic interactions: how
strong in disease and evolution? Trends Genet 22: 581-5, 2006.
12.0
4. Botelho CH, Magalhaes AV, Mello PA, Schmitt FC, Casulari LA. Expression of p53, Ki-67
and c-erb B2 in growth hormone-and/or prolactin-secreting pituitary adenomas. Arq
Neuropsiquiatr 64: 60-6, 2006.
0.4
5. Cameselle-Teijeiro JF, Cortizo-Torres ME, Schmitt FC. Medical chronobiology:
inheritance and cancer. Rev Clin Esp 206: 60-1; author reply 61, 2006.
0.3
6. Cardoso H, Nunes AC, Carneiro F, Tavarela Veloso F. Successful infliximab therapy for
oral Crohn's disease. Inflamm Bowel Dis 12: 337-8, 2006.
3.0
7. Carneiro F, Chaves P. Pathologic risk factors for therapy for adenocarcinoma of the
gastric cardia and gastroesophageal junction. Surg Oncol Clin N Am 15: 697-714, 2006.
-
8. Carvalho B, Buffart TE, Reis RM, Mons T, Moutinho C, Silva P, van Grieken NC,
Grabsch H, van de Velde CJ, Ylstra B, Meijer GA, Carneiro F. Mixed gastric carcinomas
show similar chromosomal aberrations in both their diffuse and glandular components.
Cell Oncol 28: 283-94, 2006.
4.2
9. Castro P, Soares P, Gusmao L, Seruca R, Sobrinho-Simoes M. H-RAS 81 polymorphism
is significantly associated with aneuploidy in follicular tumors of the thyroid. Oncogene
25: 4620-7, 2006.
6.9
10. Chen C-Y, Chi K-H, George RW, Cox DL, Srivastava A, Silva MR, Carneiro F, Lauwers
GY, Ballard RC. Diagnosis of gastric syphilis by direct immunofluorescence staining and
real-time PCR testing. J Clinical Microbiol 44: 3452-6, 2006.
3.5
11. Costa S, Pinto D, Pereira D, Rodrigues H, Cameselle-Teijeiro J, Medeiros R, Schmitt F.
DNA repair polymorphisms might contribute differentially on familial and sporadic breast
cancer susceptibility: a study on a Portuguese population. Breast Cancer Res Treat,
2006 (DOI: 10.1007/s10549-006-9364-z).
4.6
12. Davalos V, Dopeso H, Velho S, Ferreira AM, Cirnes L, Diaz-Chico N, Bilbao C, Ramirez
R, Rodriguez G, Falcon O, Leon L, Niessen RC, Keller G, Dallenbach-Hellweg G, Espin
E, Armengol M, Plaja A, Perucho M, Imai K, Yamamoto H, Gebert JF, Diaz-Chico JC,
Hofstra RM, Woerner SM, Seruca R, Schwartz S, Arango D. High EPHB2 mutation rate
in gastric but not endometrial tumors with microsatellite instability. Oncogene 26: 308-11,
2007.
6.9
13. Frebourg T, Oliveira C, Hochain P, Karam R, Manouvrier S, Graziadio C, Vekemans M,
Hartmann A, Baert-Desurmont S, Alexandre C, Lejeune Dumoulin S, Marroni C, Martin
C, Castedo S, Lovett M, Winston J, Machado JC, Attie T, Jabs EW, Cai J, Pellerin P,
Triboulet JP, Scotte M, Le Pessot F, Hedouin A, Carneiro F, Blayau M, Seruca R. Cleft
lip/palate and CDH1/E-cadherin mutations in families with hereditary diffuse gastric
cancer. J Med Genet 43:138-42, 2006.
4.3
14. Gonzalez CA, Jakszyn P, Pera G, Agudo A, Bingham S, Palli D, Ferrari P, Boeing H, del
Giudice G, Plebani M, Carneiro F, Nesi G, Berrino F, Sacerdote C, Tumino R, Panico S,
Berglund G, Siman H, Nyren O, Hallmans G, Martinez C, Dorronsoro M, Barricarte A,
Navarro C, Quiros JR, Allen N, Key TJ, Day NE, Linseisen J, Nagel G, Bergmann MM,
Overvad K, Jensen MK, Tjonneland A, Olsen A, Bueno-de-Mesquita HB, Ocke M,
Peeters PH, Numans ME, Clavel-Chapelon F, Boutron-Ruault MC, Trichopoulou A,
Psaltopoulou T, Roukos D, Lund E, Hemon B, Kaaks R, Norat T, Riboli E. Meat intake
and risk of stomach and esophageal adenocarcinoma within the European Prospective
Investigation Into Cancer and Nutrition (EPIC). J Natl Cancer Inst 98: 345-54, 2006.
15.2
24
15. Gonzalez CA, Pera G, Agudo A, Bueno-de-Mesquita HB, Ceroti M, Boeing H, Schulz M,
Del Giudice G, Plebani M, Carneiro F, Berrino F, Sacerdote C, Tumino R, Panico S,
Berglund G, Siman H, Hallmans G, Stenling R, Martinez C, Dorronsoro M, Barricarte A,
Navarro C, Quiros JR, Allen N, Key TJ, Bingham S, Day NE, Linseisen J, Nagel G,
Overvad K, Jensen MK, Olsen A, Tjonneland A, Buchner FL, Peeters PH, Numans ME,
Clavel-Chapelon F, Boutron-Ruault MC, Roukos D, Trichopoulou A, Psaltopoulou T,
Lund E, Casagrande C, Slimani N, Jenab M, Riboli E. Fruit and vegetable intake and the
risk of stomach and oesophagus adenocarcinoma in the European Prospective
Investigation into Cancer and Nutrition (EPIC-EURGAST). Int J Cancer 118: 2559-66,
2006.
4.7
16. Gouvea AP, Milanezi F, Olson SJ, Leitao D, Schmitt FC, Gobbi H. Selecting antibodies
to detect HER2 overexpression by immunohistochemistry in invasive mammary
carcinomas. Appl Immunohistochem Mol Morphol 14: 103-8, 2006
1.4
17. Jakszyn P, Bingham S, Pera G, Agudo A, Luben R, Welch A, Boeing H, Del Giudice G,
Palli D, Saieva C, Krogh V, Sacerdote C, Tumino R, Panico S, Berglund G, Siman H,
Hallmans G, Sanchez MJ, Larranaga N, Barricarte A, Chirlaque MD, Quiros JR, Key TJ,
Allen N, Lund E, Carneiro F, Linseisen J, Nagel G, Overvad K, Tjonneland A, Olsen A,
Bueno-de-Mesquita HB, Ocke MO, Peeters PH, Numans ME, Clavel-Chapelon F,
Trichopoulou A, Fenger C, Stenling R, Ferrari P, Jenab M, Norat T, Riboli E, Gonzalez
CA. Endogenous versus exogenous exposure to N-nitroso compounds and gastric
cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPICEURGAST) study. Carcinogenesis 27: 1497-501, 2006.
5.1
18. Jenab M, Riboli E, Ferrari P, Friesen M, Sabate J, Norat T, Slimani N, Tjonneland A,
Olsen A, Overvad K, Boutron-Ruault MC, Clavel-Chapelon F, Boeing H, Schulz M,
Linseisen J, Nagel G, Trichopoulou A, Naska A, Oikonomou E, Berrino F, Panico S, Palli
D, Sacerdote C, Tumino R, Peeters PH, Numans ME, Bueno-de-Mesquita HB, Buchner
FL, Lund E, Pera G, Chirlaque MD, Sanchez MJ, Arriola L, Barricarte A, Quiros JR,
Johansson I, Johansson A, Berglund G, Bingham S, Khaw KT, Allen N, Key T, Carneiro
F, Save V, Giudice GD, Plebani M, Kaaks R, Gonzalez CA. Plasma and dietary
carotenoid, retinol and tocopherol levels and the risk of gastric adenocarcinomas in the
European prospective investigation into cancer and nutrition. Br J Cancer 95: 406-15,
2006.
4.1
19. Jenab M, Riboli E, Ferrari P, Sabate J, Slimani N, Norat T, Friesen M, Tjonneland A,
Olsen A, Overvad K, Boutron-Ruault MC, Clavel-Chapelon F, Touvier M, Boeing H,
Schulz M, Linseisen J, Nagel G, Trichopoulou A, Naska A, Oikonomou E, Krogh V,
Panico S, Masala G, Sacerdote C, Tumino R, Peeters PH, Numans ME, Bueno-deMesquita HB, Buchner FL, Lund E, Pera G, Sanchez CN, Sanchez MJ, Arriola L,
Barricarte A, Quiros JR, Hallmans G, Stenling R, Berglund G, Bingham S, Khaw KT, Key
T, Allen N, Carneiro F, Mahlke U, Del Giudice G, Palli D, Kaaks R, Gonzalez CA. Plasma
and dietary vitamin C levels and risk of gastric cancer in the European Prospective
Investigation into Cancer and Nutrition (EPIC-EURGAST). Carcinogenesis 27: 2250-7,
2006.
5.1
20. Kocjan G, Feichter G, Hagmar B, Kapila K, Kardum-Skelin I, Kloboves V, Kobayashi TK,
Koutselini H, Majak B, Schenck U, Schmitt F, Tani E, Totch M, Onal B, Vass L, Vielh P,
Weynand B, Herbert A. Fine needle aspiration cytology: a survey of current European
practice. Cytopathology 17: 219-26, 2006.
0.8
21. Lambros MB, Simpson PT, Jones C, Natrajan R, Westbury C, Steele D, Savage K,
Mackay A, Schmitt FC, Ashworth A, Reis-Filho JS. Unlocking pathology archives for
molecular genetic studies: a reliable method to generate probes for chromogenic and
fluorescent in situ hybridization. Lab Invest 86: 398-408, 2006.
3.9
22. Lunet N, Valbuena C, Carneiro F, Lopes C, Barros H. Antioxidant vitamins and risk of
gastric cancer: A case-control study in Portugal. Nutrition and Cancer 55: 71-7, 2006.
2.4
23. Moreira MA, Longato-Filho A, Taromaru E, Queiroz G, Jube LF, Pinto SA, Schmitt FC.
Investigation of human papillomavirus by hybrid capture II in cervical carcinomas
including 113 adenocarcinomas and related lesions. Int J Gynecol Cancer 16: 586-90,
2006.
1.4
25
24. Oliveira C, Seruca R, Carneiro F. Genetics, pathology, and clinics of familial gastric
cancer. Int J Surg Pathol 14: 21-33, 2006.
0.9
25. Oliveira C, Velho S, Moutinho C, Ferreira A, Preto A, Domingo E, Capelinha AF, Duval
A, Hamelin R, Machado JC, Schwartz S Jr, Carneiro F, Seruca R. KRAS and BRAF
oncogenic mutations in MSS colorectal carcinoma progression. Oncogene 26: 158-63,
2007.
6.9
26. Oliveira MJ, Costa AC, Costa AM, Henriques L, Suriano G, Atherton JC, Machado JC,
Carneiro F, Seruca R, Mareel M, Leroy A, Figueiredo C. Helicobacter pylori induces
gastric epithelial cell invasion in a C-Met and type IV secretion system dependent
manner. J Biol Chem 281: 34888-96, 2006.
5.9
27. Paredes J, Albergaria A, Carvalho S, Schmitt FC. "Basal-like" breast carcinomas:
identification by P-cadherin, p63, EGFR and basal cytokeratins expression. Applied
Cancer Research 26: 41-55, 2006.
-
28. Paredes J, Lopes N, Milanezi F, Schmitt FC. P-cadherin and cytokeratin 5: useful adjunct
markers to distinguish basal-like ductal carcinomas in situ. Virchows Arch, 2006 (DOI:
10.1007/s00428-006-0334-y).
2.2
29. Pereira F, Ferreira AC, Tavares M, Canedo P, Costa A, Figueiredo C, Trindade E,
Carneiro F, Machado JC, de Almeida MDV, Amil J: Doença celíaca e aleitamento
materno: evidências epidemiológicas. Alimentação Humana 12:29-35, 2006.
-
30. Pereira PS, Teixeira A, Pinho S, Ferreira P, Fernandes J, Oliveira C, Seruca R, Suriano
G, Casares F. E-cadherin missense mutations, associated with hereditary diffuse gastric
cancer (HDGC) syndrome, display distinct invasive behaviors and genetic interactions
with the Wnt and Notch pathways in Drosophila epithelia. Hum Mol Genet 15: 1704-12,
2006.
7.8
31. Regalo G, Canedo P, Suriano G, Resende C, Campos M, Oliveira M, Figueiredo C,
Rodrigues-Pereira P, Blin N, Seruca R, Carneiro F, Machado J: C/EBPbeta is overexpressed in gastric carcinogenesis and is associated with COX-2 expression. J Pathol
210: 398–404, 2006.
6.2
32. Reis-Filho JS, Milanezi F, Steele D, Savage K, Simpson PT, Nesland JM, Pereira EM,
Lakhani SR, Schmitt FC. Metaplastic breast carcinomas are basal-like tumours.
Histopathology 49: 10-21, 2006.
2.6
33. Reis-Filho JS, Pinheiro C, Lambros MBK, Milanezi F, Carvalho S, Savage K, Simpson
PT, Jones C, Swift S, Mackay A, Reis RM, Hormick JL, Pereira EM, Baltazar F, Fletcher
CDM, Ashworth A, Lakhani SR, Schmitt FC. EGFR amplification and lack of activating
mutations in metaplastic breast carcinomas. J Pathol 209: 445-53, 2006.
6.2
34. Reis-Filho JS, Simpson PT, Turner N, Lambros MB, Jones C, Mackay A, Grigoriadis A,
Sarrio D, Savage K, Dexter T, Iravani M, Fenwick K, Weber B, Hardisson D, Schmitt FC,
Palacios J, Lakhani SR, Ashworth A. FGFR1 emerges as a potential therapeutic target
for lobular breast carcinomas. Clin Cancer Res 12: 6652-62, 2006.
5.7
35. Reis-Filho JS, Steele D, Di Palma S, Jones RL, Savage K, James M, Milanezi F, Schmitt
FC, Ashworth A. Distribution and significance of nerve growth factor receptor
(NGFR/p75NTR) in normal, benign and malignant breast tissue. Mod Pathol 19: 307-19,
2006.
3.4
36. Ricardo S, Milanezi F, Carvalho S, Leitao D, Schmitt. HER2 Evaluation through the novel
rabbit monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast
carcinomas. J Clin Pathol, 2006 (DOI:10.1136/jcp.2006.040287).
2.2
37. Roviello F, Corso G, Pedrazzani C, Marrelli D, De Falco G, Berardi A, Garosi L, Suriano
G, Vindigni C, De Stefano A, Leoncini L, Seruca R, Pinto E. Hereditary diffuse gastric
cancer and E-cadherin: Description of the first germline mutation in an Italian family. Eur
J Surg Oncol. 2006 Nov 23; [Epub ahead of print]
3.2
38. Schmitt FC. Thyroid cytology: FNA is still the best diagnostic approach. Cytopathology
17: 210-6, 2006.
0.8
26
39. Seixas S, Suriano G, Carvalho F, Seruca R, Rocha J, Di Rienzo A. Sequence Diversity
at the Proximal 14q32.1 SERPIN Subcluster: Evidence for Natural Selection Favoring the
Pseudogenization of SERPINA2. Mol Biol Evol. 2006 Nov 29; [Epub ahead of print]
6.2
40. Serpa J, Mesquita P, Mendes N, Oliveira C, Almeida R, Santos-Silva F, Reis CA,
LePendu J, David L. Expression of Lea in gastric cancer cell lines depends on FUT3
expression regulated by promoter methylation. Cancer Lett 242: 191-7, 2006.
3.0
41. Suriano G, Ferreira P, Mateus AR, Correia J, Henriques L, Seruca R. Genetics of
hereditary diffuse gastric cancer: progress and future challenges. Future Oncol 2: 36370, 2006.
-
42. Suriano G, Seixas S, Rocha J, Seruca R. A model to infer the pathogenic significance of
CDH1 germline missense variants. J Mol Med 84: 1023-31, 2006.
4.7
43. Turner NC, Reis-Filho JS, Russell AM, Springall RJ, Ryder K, Steele D, Savage K, Gillett
CE, Schmitt FC, Ashworth A, Tutt AN. BRCA1 dysfunction in sporadic basal-like breast
cancer. Oncogene, 2006 (DOI: 10.1038/sj.onc.1210014).
6.2
44. Utagawa ML, di Loreto C, de Freitas C, Milanezi F, Longatto Filho A, Pereira SM, Maeda
MY, Schmitt FC. Pero Vaz de Caminha: an-interchange program for quality control
between Brazil and Portugal. Acta Cytol 50: 303-8, 2006.
1.0
Research plans for 2007
The group aims at answering to the following specific research questions:
• We expect to identify intermediate genes (like transcription factors, co-activators or co-repressors)
that are able to induce P-cadherin upregulation.
• We aim to determine how P-cadherin expression interferes with E-cadherin function, using breast
cancer cells as a model system.
• We aim to further studying the signalling pathways mediated by de novo expression of P-cadherin
and find the pivotal proteins for cell motility, migration and invasion in breast cancer. Further, we aim
to determine the role P-cadherin in metastatization and angiogenesis by using breast cancer cells
stably expressing this protein in a E-cadherin wild-type background.
• We plan to further explore the E-cadherin mutation-dependent activation of EGFR and its effect on
cell motility and invasion. In particular, a more complex working system has been elaborated as to
take into account the effect of cell-matrix interaction in this process.
• On the basis of the observation in vitro on the existence of a regulatory cross-talk between Ecadherin and EGFR, we are exploring in a subset of sporadic gastric tumours the E-cadherin and
EGFR status both for expression, activation and the presence of mutations.
• Major effort will be put on the E-cadherin membrane trafficking project. This will range from in vitro
characterization mainly through the use of confocal and time-lapse microscopy, as well as through
the identification of new E-cadherin cytoplasmic partners.
• We aim at identifying the molecular mechanisms involved in H. pylori-mediated cell invasion
downstream c-Met receptor.
We aim to publish between 15-20 manuscripts in peer-review International Journals with IF above 3 and
5 manuscripts above 6 IF.
27
Carcinogenesis
Group Leader: Leonor David, MD PhD, Full Professor of Pathology from the Medical
Faculty of the University of Porto and senior Researcher of IPATIMUP
Staff members: Fátima Gartner, PhD, Full Professor at ICBAS; Celso Reis, PhD,
senior Investigator; Raquel Almeida, PhD, senior Investigator; Luis Filipe Santos Silva,
PhD, senior Investigator; Ana Carvalho, post-Doc; Hugo Osório, post-Doc; Nuno
Marcos, PhD student; Paula Paulo, PhD student; Rita Barros, PhD student; Salomé
Pinho, PhD student; César Ribeiro, PhD student (jointly with INEB); Liliana Silva, PhD
student (jointly with the Dental Faculty); Ana Magalhães, BI; Natália Costa, BI; Maria
Manuel Azevedo, BI; Joana Gomes, BI; Nuno Mendes, technician; Joana Oliveira,
Veterinary graduation student.
Objectives/Goals of the research activity - The main objective of the group is to
identify alterations of mucins and mucin glycosylation, associated with gastric
carcinoma and precancerous lesions, that may be relevant for the development of
diagnostic and therapeutic strategies. We are also engaged in understanding the
molecular mechanisms involved in the development of such alterations, including the
identification
of
transcription
factors
responsible
for
cancer/pre-cancer
transdifferentiation, as well as to extend our expertise on other cancer models (ex:
mammary cancer from dogs).
Major achievements during 2006 – The group continued previous studies on the
clarification of the mechanisms involved in the development of intestinal metaplasia
(IM) and on the capacity of this pre-neoplastic lesion to eradicate Helicobacter pylori
infection. We compiled the group and literature contributions in a review paper where
we propose IM is not a cell differentiation disease but a tissue differentiation process
instead (Paper 1). The group progressed in this line of research and succeeded in
showing that matrix derived BMP2/4, through the Smad4 pathway, are responsible for
regulation of the CDX2 homeobox transcription factor in gastric cells (paper submitted
for publication). We are therefore gathering strong and innovative evidence in favor of a
matrix-derived initiation signaling pathway for IM development. In view of a recent
paper showing that the terminal alpha1,4-GlcNAc structure, identified in gastric glands,
has an antibiotic effect against Helicobacter pylori, we hypothesized that this glycan
structure might be involved in eradication of bacterial infection in IM. We tested this
hypothesis by searching for immunohistochemical expression of alpha1,4-GlcNAc in
intestinal metaplasia and we could not support that alpha1,4-GlcNAc is overexpressed
in IM and therefore we can not sustain it ’s role in Helicobacter pylori eradication in IM
(Paper 2).
We continued previous studies on the genotype/phenotype associations in the Secretor
and Lewis systems in an attempt to clarify their relevance for gastrointestinal infectious
diseases – Helicobacter pylori and Calicivirus – and for the generation of cancer
associated Lewis structures. On the first issue we demonstrated, in contradiction to
most published data, that Secretor status, together with ABO histo-blood group
phenotype for some viral strains, is determinant for adhesion of Helicobacter pylori and
Calicivirus. Studies to be carried in 2007 at Thomas Boren Lab in Umea will hopefully
consolidate this observation and, if so, will clarify the existing controversies on the in
vivo relevance of Secretor/Lewis status for these human infections. We will also
28
continue, in 2007, our present collaboration with the group of Jorge Rocha from
IPATIMUP, to elucidate the phenotype/genotype discrepancies in the Secretor and
Lewis systems. On the second line of study, of generation of cancer associated Lewis
structures, we demonstrated that hypomethylation of the FUT3 gene promoter is a key
factor for the aberrant expression of Lewisa in cancer (Paper 3).
We have previously established stable transfectants of gastric cell lines expressing the
sialyltransferase ST6GalNAc-I and demonstrated that it is responsible for the
biosynthesis of the cancer-associated carbohydrate antigen Sialyl-Tn. We have used
these cell line models to demonstrate that Sialyl-Tn per se induces an aggressive
behaviour of cancer cells (Paper 4). The biosynthesis of MUC1 mucin glycoforms with
high occupancy by Tn and Sialyl-Tn allowed the establishment of cancer cell-specific
immune responses and also the development of a glycopeptide-specific monoclonal
antibody with promising diagnostic and immunopreventive potential (Paper 5).
The group progressed in the characterization of the host mucins relevant for
Helicobacter pylori adhesion. Using clones of GP202 gastric cell line expressing MUC1
with different tandem repeat lengths we evaluated the impact of the MUC1 VNTR
variability in the adhesion of H. pylori to gastric cells, using pathogenic (strain 26695)
and non-pathogenic (Tx30a) H. pylori strains. All transfected clones showed higher
adhesion of the pathogenic strain and both H. pylori strains showed increased
adhesion in clones with a higher number of tandem repeats of MUC1 mucin,
consolidating previous evidence for a role of MUC1 VNTR polymorphism in early steps
of gastric carcinogenesis.
The group has evaluated the expression of genes related to glycoconjugate
biosynthesis upon adhesion of Helicobacter pylori using a Glyco-gene Chip array. H.
pylori binding/interaction with host cells is known to alter the expression of host genes,
leading to expression of pro-inflammatory molecules and modification of carbohydrate
structures. Our results showed that H. pylori induced significant expression alterations
in 168 of the 1031 genes tested. The most virulent H. pylori strain led to increased
expression of glycosyltransferases participating in the biosynthesis of glycans such as
Sialyl-Lex antigen, the ligand of H. pylori SabA adhesin. Our results also showed that
virulent H. pylori strains induced a remarkable expression of Syndecan-4, suggesting
that this proteoglycan may be involved in the modulation of H. pylori-associated
gastritis. The group initiated the characterization of the role that alpha2,3Sialyltransferases (ST3Gal-III, ST3Gal-IV, ST3gal-VI) play in building the terminal sialylation
of the carbohydrate chain, leading to the formation of Sialyl-L
Lewisa and Sialyl- Lewisx.
These cellular models are presently being tested and will be used in functional assays.
The group has generated/incorporated interesting models during 2006 that will be used
in projects to develop in 2007: a gastric carcinoma cell line with silencing of the MUC1
mucin by siRNA (Master thesis of Paula Paulo); Transgenic mice obtained from the
Consortium for Functional Glycomics (Celso Reis is a member of this consortium) with
knock-out of one of the following glycosyltransferases - Core2-GlcNAc (controlling
mucin type O-glycosylation braching), FUT2 (controlling terminal H-type 1 terminal
chain), ST3Gal-IV (controlling terminal sialylation forming the Sialyl-Lewis structures),
and the control wild type mice with the C57BL6 background. The knock-out mice, with
29
major modifications in the glycan structures they synthesize, will be used to identify the
in vivo relevance of the different glycans for Helicobacter pylori infection.
During 2006 we finished the field-collection of samples/questionnaires from a study of
follow-up of gastric pathology in a cohort of healthy volunteers from Estaleiros Navais
de Viana do Castelo. A parallel study was developed in Mozambique to access a
population that, despite having a high rate of Helicobacter pylori infection, has a low
rate
of
intestinal
metaplasia
and
gastric
carcinoma.
During
2006
samples/questionnaires from 100 Mozambicans were completed. The initial analysis
from Viana showed that high-virulent H. pylori strains are preferentially associated with
incomplete IM, whereas smoking is associated with complete IM (Paper 6). A first
approach to the clinical data was done and a paper was submitted for publication by
our clinical co-workers. Comparative analysis between the 2 populations, histology,
interleukin polymorphisms and Helicobacter pylori strains genotypes, will be carried
during 2007.
Fátima Gartner has come into close contact with the main research interests of the
group. Sialyl-Lewisx expression was demonstrated in all cases of canine mammary
carcinomas and the levels of expression are significant associated to lymph node
metastases. Moreover, an inverse relationship was observed for E-Cadherin and sLex
expression. A poorly differentiated mammary carcinoma cell line - CMT-U27 generously supplied by Eva Hellmén, Uppsala University, was characterized and
showed positivity for Sialyl-Lewis X, Lewis X and Lewis A as well as a strong
immunoexpression for E-cadherin. Female athymic nude mice were evaluated for its
tumorigenic and metastatic capacity and metastization was observed in the lung, heart,
liver, spleen and ovary. Cell lines from two complex carcinomas and two complex
adenomas, cultured from mammary canine primary tumours, are being established and
characterized.
The group maintains an interest in developing, together with a biotechnology company
from Portugal – ATGC, and a company from Denmark - Dako, a kit for the diagnosis of
intestinal metaplasia based on the identification of alterations of the mucin expression
profile using monoclonal antibodies previously produced by the group. However, little
progress was made during 2006.
The group, under the leadership of Celso Reis and with a strong input from Hugo
Osório, is establishing a Mass Spectrometry Unit. The Unit has a MALDI-TOF mass
spectrometer that was acquired under the Re-equipment Project from Fundação para a
Ciência e a Tecnologia (REEQ/899/SAU/2005). The Unit has the capability of
performing MS and MS/MS analysis. The equipment is already functioning and we are
presently working on the establishment of the standard protocols for protein
identification and analysis of post-translational modifications.
Apart from the mainstream of the group objectives we collaborated in several
publications, mainly clinico-pathologic descriptions (see “Other” publications).
Financing/Projects
The group leaded the acquisition of a MALDI-TOF equipment under a Re-equipment
Project from Fundação para a Ciência e a Tecnologia: “Mass spectrometry (MALDI-
30
TOF) for protein characterization and proteomics in the north of Portugal”.
(REEQ/899/SAU/2005) PI: Celso Reis. Total budget for 2005-2006: 536.690€.
The group won Royalties from monoclonal antibodies sells: 2006 – 576€
• MUC5AC (MAB2011, Clone CLH2). CHEMICON INTERNATIONAL INC.
28835 Single oak drive, Temecula, CA 92590; California, USA.
• MUC5AC (NCL-MUC5). NOVOCASTRA LABORATORIES Ltd. Balliol
Business Park West, Benton Lane, Newcastle upon Tyne; NE12 8EW,
United Kingdom.
• MUC6 (NCL-MUC6). NOVOCASTRA LABORATORIES Ltd. Balliol
Business Park West, Benton Lane, Newcastle upon Tyne; NE12 8EW,
United Kingdom.
The group gathered 301.356€ for funding during 2006 and has approved funding for
2007 (267.544€). The group is waiting for evaluation of 5 projects leaded by the
Investigators in the last FCT contest. The funding for 2006, after deduction of 20%
overheads (60.271,2€), and salaries (102.693,76€: one technician – 14.835€/year; one
pos-doc – 17.940€/year; an informatic technician at the Epidemiology Department in
the Medical Faculty – 9.866,16€/year; a student who performed dietary questionnaires
in Mozambique - 2.500€/year and 57.552,6€ for the 6BIs/variable periods), leaves
138.391,04 € for Lab running costs.
1. “Clarification of the biological role of MUC1 mucin variability in gastric
carcinogenesis”.
Fundação
para
a
Ciência
e
a
Tecnologia
(POCTI/CBO/44812/2002). PI: Luís Filipe Santos Silva. Total budget for 20032007 - 72.000€ (2006 - 24.000€; 2007 - 10.000€).).
2. “Clarification of the relevance of MUC1 mucin polymorphism in Helicobacter
pylori infection”. Fundação para a Ciência e a Tecnologia (POCI/SAUIMI/56895/2004). PI: Luís Filipe Santos Silva. Total budget for 2005-2008 100.000€ (2006 - 33.000€; 2007 - 34.000€).
3. “Molecular mechanisms of biosynthesis of cancer associated mucin
carbohydrate antigens in intestinal metaplasia”. Fundação para a Ciência e a
Tecnologia (POCTI/CBO/44598/2002). PI: Celso Reis. Total budget for 20032006 - 66.600€, extended until April 2007. (2006 – 22.200€; 2007 – 24.079€).
4. “Molecular mechanisms of biosynthesis of cancer associated mucin
carbohydrate antigens in gastric carcinoma”. Association for International
Cancer Research (AICR grant Ref: 05-088). PI: Celso Reis. Total budget for
2005 – 2008 - 112.320€ (2006 – 37.440€; 2007 -37.440€).
5. “Identification of Glycosylation-associated genes induced by H. pylori in gastric
cells: a glycomic approach”. Fundação para a Ciência e a Tecnologia.
POCI/SAU-OBS/56686/2004 PI: Celso Reis. Total budget for 2005-2007:
55.000€. (2006 – 27.500€; 2007 -13.470€).
6. “Genes associated to intestinal metaplasia of the gastric mucosa (MUC2 mucin
and fucosyltransferase FUT3): transcriptional regulation and relevance for
31
Helicobacter pylori adhesion” (Project POCI/SAU-OBS/55549/2004). PI: Leonor
David. Total budget for 2005-2008: 96.680€ (2006 – 33.000€; 2007 – 29.420€).
7. “Lesions of the gastric mucosa in Mozambique and Portugal: the “African
enigma”. Study of biologic factors and lifestyle that contribute to understand the
differences in incidence of gastric carcinoma in two countries with a high
prevalence of Helicobacter pylori infection: Mozambique and Portugal”.
Fundação Calouste Gulbenkian (Project FC-68697). PI: Leonor David. Total
budget for 2005-2008: 100.000€ (2006 - 33.000€; 2007 - 33.000€).
8. “Identification of signalling pathways involved in Cdx2 regulation in two human
models of altered intestinal differentiation: intestinal metaplasia and juvenile
polyposis”. Fundação para a Ciência e a Tecnologia (Project POCTI/SAUOBS/55840/2004). PI: Raquel Almeida. Total budget for 2005-2008: 98.500€
(2006 – 32.833€; 2007 – 28.635€).
9. “Biological characterization of canine mammary mixed tumours: histogenesis,
tumour progression and genetic alterations”. Fundação para a Ciência e a
Tecnologia (POCI/CVT/57795/2004). PI: Fátima Gartner. Total budget for 20052008: 92.225€ (2006 – 30.741€; 2007 – - 50.000€).
10. “Cat mammary tumours-Pathologic, Molecular and Cytogenetic approaches”.
Fundação para a Ciência e a Tecnologia (POCI/CVT/62940/2004). PI: Fátima
Gartner. Total budget for 2005-2008: 82.927€ (2006 – 27.642€; 2007 - 7.500€).
International collaborations
•
Eppley Institute, University of Omaha – Michel Anthony Hollingsworth. This
collaboration was fundamental for the development of the SiRNA for silencing
endogenous MUC1 gene expression.
•
Faculty of Health Sciences of the University of Copenhagen – Ulla Mandel and
Henrik Clausen. This collaboration has been fundamental for characterization of
carbohydrate antigens and glycosyltransferases using unique monoclonal
antibodies.
•
INSERM, Nantes – Jacques Le Pendu. This collaboration has been critical for
the prosecution of the study of Secretor and Lewis phenotypes/genotypes, due
to the unique expertise of Jacques Le Pendu in such complex field.
•
INSERM, Lille – Isabelle Van Seuningen. This collaboration has been
fundamental for the establishment of promoter studies, namely transient
transfection assays with luciferase reporters and electrophoretic mobility shift
assays (EMSA).
•
INSERM, Strasbourg – Jean-Noel Freund. This collaboration started in 2006
with the acceptance of Jean-Noel Freund to be co-supervisor of the PhD thesis
of Rita Barros. Raquel Almeida and Jean-Noel Freund successfully applied for
travel money for one year at the CRUP. This collaboration will be essential for
the study of CDX2 regulation in intestinal metaplasia.
32
•
IMIM, Barcelona – Francisco Real and Carme de Bolos. This collaboration has
allowed completion of in situ hybridization techniques for mucin gene
expression.
•
Universite des Sciences et Technologies de Lille – Anne Harduin-Lepers. This
collaboration has been useful in the cloning and recombinant expression of
sialyltransferases.
•
Consortium for Functional Glycomics – The Scripps Research Institute, CA,
USA - James Paulson. This collaboration has provided the use of resources of
the Consortium, including Microarray analysis and knock-out mice.
Master Thesis
In 2006 one Master student of the group defended her thesis at the Medical Faculty of
Porto.
•
Paula Paulo – The work was focused on the development of in vitro models
with stable silencing of MUC1 expression, mediated by RNA interference.
Silencing was directed to two regions of MUC1 coding sequence in GP202 and
MKN45 gastric carcinoma cell lines. MUC1 down-regulation had an impact on
mucin glycosylation with decreased expression of Tn and Sialyl-Tn antigens in
both cell lines and de novo expression of Sialyl-Lea in MKN45. MUC1 downregulation also affected β-catenin expression and cellular distribution in both cell
lines. Paula further showed that MUC1 down-regulation had a wider impact in
MKN45 gene expression profile, using a gene expression microarray. The
silencing model established by Paula will contribute to understand the relevance
of MUC1 expression in gastric cancer cells, both as a molecule involved in
epithelial protection and as a player in signaling pathways.
Selected publications:
1. Mesquita P, Almeida R, Lunet N, Reis CA, Santos-Silva F, Serpa J, Van Seuningen
I, Barros H, David L: Metaplasia – a transdifferentiation process that facilitates
câncer development. The model of gastric intestinal metaplasia. Critical Reviews
in Oncogenesis 12: 3-26, 2006.
2. Ferreira B, Marcos NT, David L, Nakayama J, Reis CA: Terminal alpha1,4-linked
N-acetylglucosamine in Helicobacter pylori-associated intestinal metaplasia of
the human stomach and gastric carcinoma cell lines. Journal of Histochemistry and
Cytochemistry 54: 585-591, 2006.
3. Serpa J, Mesquita P, Mendes N, Oliveira C, Almeida R, Santos-Silva F, Reis CA, Le
Pendu J, David L: Expression of Lea in gastric cancer cell lines depends on FUT3
expression regulated by promoter methylation. Cancer Letters 242: 191-197, 2006.
4. Pinho S, Marcos NT, Ferreira B, Carvalho AS, Oliveira MJ, Santos-Silva F, Harduin33
Lepers A, Reis CA: Biological significance of cancer-associated silyl-Tn antigen:
modulation of malignant phenotype in gastric carcinoma cells. Cancer Letters (in
press).
5. Sorensen AL, Reis CA, Tarp MA, Mandel U, Ramachandran K, Sankaranarayanan
V, Schwientek T, Graham R, Taylor-Papadimitriou J, Hollingsworth MA, Burchell J,
Clausen H: Chemoenzymatically synthesized Tn/STn MUC1 glycopeptides elicit
cancer-specific anti-MUC1 antibody responses and override tolerance.
Glycobiology 16: 96-107, 2006.
6. Peleteiro B, Lunet N, Figueiredo C, Carneiro F, David L, Barros H: Smoking,
Helicobacter pylori virulence, and type of intestinal metaplasia in Portuguese
males. Cancer Epidemiology, Biomarkers and Prevention (in press).
“Other” publications:
1. Babu SD, Jayanthi V, Devaraj N, Reis CA, Devaraj H: Expression profile of
mucins (MUC2, MUC5AC and MUC6) in Helicobacter pylori infected preneoplastic and neoplastic human gastric epithelium. Molecular Cancer 5: 10-17,
2006.
2. Escrevente C, Machado E, Brito C, Reis CA, Stoeck A, Runz S, Marme A, Altevogt
P, Costa J: Different expression levels of alpha3/4 fucosyltransferases and Lewis
determinants in ovarian carcinoma tissues and cell lines. International Journal of
Oncology 29: 557-566, 2006.
3. Pudelko M, Lindgen A, Tengel T, Reis CA, Elofsson M, Kihlberg J: Formation of
lactones from sialylated MUC1 glycopeptides. Organic & Biomolecular Chemistry 4:
713-720, 2006.
4. Matos AJ, Lopes C, Faustino AM, Carvalheira JG, dos Santos MS, Rutteman GR,
Gartner MF: MIB-1 labelling indices according to clinico-pathological variables in
canine mammary tumours: a multivariate study. Anticancer Research 26: 18211826, 2006.
5. Matos AJ, Faustino AM, Lopes C, Rutteman GR, Gartner F: Detection of lymph
node micrometastases in malignant mammary tumours in dogs by cytokeratin
immunostaining. Veterinary Record 158: 626-630, 2006.
6. Matos AJ, Lopes C, Carvalheira J, Santos M, Rutteman GR, Gartner F: E-cadherin
expression in canine malignant mammary tumours: relationship to other clínicopathological variables. Journal of Comparative Pathology 134: 182-189, 2006.
7. Pinto de Sousa J, David L: Role of immunohistochemical expression of MUC5B
in gastric carcinoma. In: Handbook of immunohistochemistry and in situ hybridization
of human carcinomas (Volume 4). MA Hayat (ed.). Elsevier Academic Press,
Burlington and San Diego, USA, London, UK, 2006, pp 191-194.
34
Genetics, Evolution and Pathology
Information about the activities of 2006
Jorge Rocha
35
1. Team members
Group leader:
Jorge Rocha (BSc, PhD)
Post-doc students
Susana Seixas (BSc, PhD)
Sandra Beleza (BSc, PhD)
PhD students
Margarida Coelho (BSc)
Project grants
João Pedro Oliveira (BSc)
Undergraduate students (from March to September de 2006)
Luís Pedro Resende (Biological Sciences student)
2. Major activities
The major activities developed in 2006 were essentially related with the first year of Margarida
Coelho PhD project, the first year of Sandra Beleza post-doc project , the third and last year of
Susana Seixas post-doc project and the first year of the research project
POCI/BIABDE/56654/2004 entitled “Biocultural Adaptation: human adaptive responses to
changes in subsistence economy.” These activities will be now summarized in accordance with
the major subjects of research in which they are centered.
2.1 Characterization of the genetic structure and of the history of African populations
In agreement with the objectives planned for Margarida Coelho PhD project, we started a
collaboration with Prof. Joanna Mountain from the Laboratory of Anthropological Genetics at
the Stanford University (California, USA), aiming to develop a new kind of genetic markers
designated by UEPSTRs, where a short tandem repeat (STR) with a high mutation rate is linked to
a biallelic polymorphism originated by a unique mutational event (“unique event polymorphism”;
UEP). These markers will be used together with polymorphisms from the Y- chromosome and
mtDNA to characterize samples from 29 populations from Cameroon, Angola and Mozambique, as
part of a detailed study of the Bantu expansions. The samples from Cameroon will be provided by
the “Istituto de Biologia Animale e del Uomo” from the University of Roma (“La Sapienza”). The
samples from Angola and Mozambique were collected by during our own field missions to these
countries in 2005.
The development of the UEPSTRs comprises the following steps: a) Identification in the human
genome of DNA sequences with linked UEPs and STRs markers; b) Establishment of automated
protocols for the simultaneous typing of UEPSTRs by PCR; c) Experimental validation of protocols
and analysis of genetic diversity in large dimension samples (n>100). Until now we have identified
21 regions that include linked UEPs and STRs and presently we have validated protocols for 5
UEPSTRs. Our objective is to define a set of 20-30 independent systems.
36
On the other hand, we started to collect data for the sequence variation of the HVR-1 region from
the mitochondrial DNA molecule in populations from the Namibe desert (southern Angola) whose
genetic diversity still remains undocumented.
The data collected with different types of markers will be used to estimate several parameters of the
history of Bantu populations (migration rates, subdivision ages, size and rate of population
increment) with new analytical and computational approaches that, hopefully, will make our studies
of the population structure progressively less descriptive.
To obtain financial support to this study we have submited to FCT a project entitled On the edge of
the Bantu expansions: inference of recent population history with independently evolving haplotypic
systems. This project awaits approval by the FCT. Meanwhile, we were invited by Prof Joanna
Mountain to participate as collaborators in a similar project, enrolling other African population that
is now under submission to NIH.
In addition, still in the area area of the genetic structure of the African populations, we have
completed and submitted for publication in Current Anthropology journal the work that
summarizes the major results from the research project POCTI/42510/ANT/2001 entitled
Anthropogenetics of São Tomé e Príncipe Archipelago: a case study of human microevolution. This
work, done in collaboration with the Population Genetics group from IPATIMUP, is included in
Margarida Coelho’s PhD and is entitled Human microevolution and the Atlantic slave trade: the
case study of São Tomé. The work was recently sent to us for revision with a positive appreciation
and was considered for publication. The full acceptance of the article is dependent on that review.
The Current Anthropology journal is a prestigious publication in the Anthropology that is quite
restrive in regard to articles that are not trans-disciplinary and are not considered of great interest for
a large audience of anthropologists. For this reason, consider the publication of the work in Current
Anthropology our greatest short term priority. This work was also selected for an oral presentation in
the next congress of the American Association of Physical Anthropology (AAPA), to be held in
Philadelphia (USA) in March 2007.
2.2 Study of the evolutionary history of human adaptation candidate genes
This area of activity may be divided in two parts. The first corresponds to the research project
Biocultural Adaptation: human adaptive responses to changes in subsistence economy where João
Oliveira and Margarida Coelho are involved as a project granted student and as a PhD student,
respectively. Margarida is also enrolled in this project since the study of evolutionary history of
adaptive mutations in Africa is complementary to the analysis of the genetic structure of African
populations. The second part corresponds integrally to the post-doc project of Susana Seixas and is
centered in the analysis of the signatures of natural selection in the SERPIN gene family at
chromosome 14q32.1 in both African and European populations.
In the year 2006, the work developed for the project about the biocultural adaptation was mainly
carried out by João Oliveira and was centered in the collection of data for the intra-allelic variation
linked to the S mutation of the β-globin gene (HBB*S). This gene is well known for causing
falciform anemia in homozigosity and for conferring the most effective genetic protection to malaria
when in heterozygosity. Taking advantage from the sample collections from our field work in Africa
and from our collaboration with the Istituto de Biologia Animale e del Uomo from the University of
Rome, it was possible to gather a large number of HBB*S alleles (>250) from populations of
Angola, Mozambique, São Tomé and Príncipe, Cape Verde, Cameroon and Sudan, which belong to
very different ethnic and geographic contexts. The patterns of inter-allelic variation are being
37
evaluated with microsatellites located at different distances from the β-globin gene and will be used
to test alternative hypotheses regarding the age, origin and major pathways of HBB*S spread in
Africa. Preliminary analyses of results were presented by João Oliveira in a poster communication
at Second Euroconference on Disorders of Iron Homeostasis,erythrocytes and Erythropoiesis
(Cascais) . In order to contextualize the study of the evolutionary history of HBB*S allele in the
scope of the diffusion of mutations that confer resistance to malaria, we are collecting additional
data concerning the phylogeography of other loci, like the α-Thalassemia (João Oliveira), the
Glucose-6-phosphate-desidrogenase and the allele FY*O from the Duffy Blood group (Margarida
Coelho). Our team will make an effort to use the collected data about geographic variation and interallelic diversity to test models of selection and diffusion of adaptive mutations, with more
sophisticated methods of simulation and statistical inference than those we have been using. This
topic will be pursued by the Jorge Rocha during his 2 month visit to Department of Human Genetics
of the University of Chicago (Illinois, USA).
In addition, it is important to mention that our 2005 work about the evolution of the lactose
tolerance (Coelho et al. Hum Genet 117: 329-339), also included in the project of biocultural
evolution, has been cited by the two most important articles of 2006 that were focused on the
evolutionary history of this polymorphism (Ingram et al., Hum Genet,Epub ahead of print; Tishkoff
et al., Nat Genet, Advance Online Publication). The Nature Genetics article, in particular, has been
much commented and presents a strong evidence for the occurrence of several distinct lactose
tolerance mutations, as we had predicated before. This work was cited as well in a large revision of
natural selection in Humans published in Yearbook of Physical Anthropology (Harris and Meyer,
Yearbook Phys Anthropol 49: 89-130; 2006), together with another work of our team about the
evolution of the Duffy Blood Group. These citations shows that our work starts to gain relevance in
the bibliography about the evolution of lactose intolerance and in a more general way, in the
bibliography about natural selection and the genetic variation of Human adaptations.
The second part of the study of the evolutionary history of genes involved in Human adaptation was
developed by Susana Seixas, during her post-doc in collaboration with Prof. Anna Di Rienzo from
the Department of Human Genetics of the University of Chicago. This work ended in 2006 with the
preparation and the acceptance for publication in the Molecular Biology and Evolution of the article
entitled Sequence diversity at the proximal 14q32.1 SERPIN subcluster: evidence for natural
selection favoring the pseudogenization of SERPINA2. This work was also done in collaboration
with the Cancer Genetics group at IPATIMUP. The article is in many aspects, the most complex
work done up to now by our group and builts upon a hypothesis formulated during Susana’s PhD
work that could only be tested thanks to the improvement of her data analysis skills in during her
visits to Prof. Anna Di Rienzo lab (Department of Human Genetics of the University of Chicago).
Besides, the demonstration of the signature of natural selection in a human gene – an increasingly
demeanding exercise - the major interest of this article is the presentation of empirical evidence that
natural selection may be used to pinpoint genomic regions and genetic variants with functional
relevance. In order to develop this concept and to pursue the study of the evolution of genes of
proteolysis, Susana submitted a research project to FCT entitled Looking for evidences of human
adaptation in the proteolysis universe: the case study of serine protease inhibitors. This project will
have the collaboration of the Cancer Genetics group and will search for signatures of natural
selection in carefully selected serine protease inhibitors genes. Meanwhile, Susana will use this
project to apply to a second post-doctoral grant. These activities will have the collaboration Prof.
Anna Di Rienzo from the Department of Human Genetics of the University of Chicago.
38
2.3 Characterization of admixed populations to study the genetic basis of complex
phenotypes
This area of activity corresponds to Sandra Beleza’s post-doc project, entitled “The admixture
structure of Cape Verde: implications for anthropological and biomedical research”, which has as
consultants Professors Esteban Parra (University of Toronto, Canada) and Mark Shriver
(Pennsylvania State University, USA) and which benefits from an ongoing partnership with the
University of Cape Verde. The major aim of the project is to study the genetic structure of Cape
Verde and to apply admixture mapping strategies to study the genetic basis of three complex
traits with anthropological, evolutionary and biomedical relevance: skin and eye pigmentation,
obesity and hypertension.
Sandra and Jorge visited Cape Verde (Cidade da Praia and Mindelo) during the months of March
and April 2006 to promote our research proposal and establish an institutional protocol with the
local University. Besides having a series of conferences and meetings with the local authorities, it
was possible to carry out a pilot study to test the practicality of our field work protocol and
identify the main difficulties that may arise during the bigger sampling. The most important
activities of the field work are: a) to perform sociodemographic questionnaires; b) to collect
biological material for DNA extraction; c) to collect phenotypic data (blood pressure,
anthropometrical measures related with obesity, skin reflectometry and eye pictures). The pilot
study was undertaken with the participation and collaboration of students from the Instituto
Superior de Educação (ISE). This successful interaction with the students led to the addition of an
educational/formative component to the project, which will start with a one-month visit of two
Biology students from ISE to IPATIMUP in February/March 2007. These students will be trained
in several experimental methods such as DNA extraction, PCR and SNP and STR genotyping.
Additionally, training in statistical data analyses will be also provided.
In order to obtain financial support for all these activities, we choose to write two projects: one
focused in the analysis of pigmentation and the other in the study of obesity and hypertension. The
project related with pigmentation and entitled Understanding the genetic architecture and evolution
of human pigmentary traits: admixture mapping studies in Cape Verde was submitted to FCT (a
summary of the project can be found in appendix 1). The project about obesity and hypertension and
entitled Caracterização de Populações Miscigenadas para o estudo da Etiologia da Obesidade e
Hipertensão: O Caso Exemplar de Cabo Verde (“Characterization of admixed populations to
study the genetic basis of obesity and hypertension: the case-study of Cape Verde”) was
submitted to the program Fronteira das Ciências da Vida of Gulbenkian Foundation. If neither of
the projects is accepted, the field work at the scale that it was thought will be irremediably
compromised.
Still in the scope of the project about obesity and hypertension, we started some collaborations with
medical doctors that do research in these areas. These collaborations should be intensified in 2007.
Not being able to perform the field work in Cape Verde during her first year as a post-doc student,
Sandra integrated Mark Shriver’s and Esteban Parra’s teams and started to collaborate in ongoing
studies about pigmentation genes. In this way, she participated in the paper entitled The genetic
architecture of normal variation in human pigmentation: an evolutionary perspective model
(McEvoy et al, Hum Mol Genet 15: R176-R181; 2006; summary in Appendix 2), in which a first
phylogenetic model for skin colour variation is drawn and where the strategy consisted in using
signs of natural selection to identify candidate genes that may have influenced the differences in
pigmentation observed across humans populations, a similar approach to the one used by Susana in
her work. Sandra is also participating in an admixture mapping study with African Americans,
aimed at evaluating the influence of the gene KIT ligand (KITLG) in skin pigmentation and started
39
collaborating in the preliminary analysis of the evolutionary history of two other genes (SLC24A5
and MATP) whose influence in skin colour was previously demonstrated.
Finally, Sandra organized the visit from Mark Shriver to Porto, aiming to perform a field work
related with the study of the genetic structure and skin pigmentation variation across European
populations, and went to Brazil integrated in Mark’s team, to perform a sample collection also in the
scope of admixture mapping studies.
2.4 Other activities
In addition to the activities related with the main research areas of our group, we participated in the
supervising of Luís Pedro Resende, a student from the Science Department of University of Porto
and we collaborated in other groups’ research works.
The work done by Luís Pedro, entitled Estudo da distribuição de polimorfismos humanos do gene
FUT2 e a sua contribuição para o fenótipo secretor (“Distribution of polymorphisms in human
FUT2 gene and its contribution to the secretor phenotype”), co-supervised by Prof. Leonor David
and Jorge Rocha, encompassed the beginning of a collaboration with the Carcinogenesis group that
we intend to proceed with the study of the relationship between the genetic diversity of FUT genes
and susceptibility to Helicobacter pylori. This subject comprised the aim of the project Secretor and
Lewis phenotypes/genotypes in Helicobacter pylori and calicivirus infection submitted by Leonor
David to FCT, in which we participate.
Still within the subject about the relationship between host’s genetic variation and susceptibility to
infectious and carcinogenic agents, we have collaborated in the elaboration of the Work Package
entitled Human genetic markers and risk of infection with Helicobacter pylori, led by Prof. José
Carlos Machado and included in a proposal submitted by the European consortium Parasite and
host genetic diversity in Helicobacter infections (HELDIVNET) to an international competition
promoted by ERA-NET PathoGenoMics. This project was recently approved.
Our collaboration in other research studies resulted in co-authorship of the following papers: Ecadherin germline missensse mutations: a clinical dilemma. How to infer their pathogenic
significance in HDGC (Suriano et al, J Mol Med 84: 1023-1031; 2006; Color and genomic ancestry
in Brazilians: a study with forensic microsatellites (Pimenta et al, Hum Hered 62: 190-195; 2006);
Phylogeography of the human mitochondrial L1c haplogroup: genetic signatures of the prehistory
of Central Africa (Batini et al, Mol Phyl Evol; em publicação); Lobar brain hemorrages and white
matter changes: clinical, radiological and laboratory profiles (Maia et al, Cerebrovas Dis 22: 155161; 2006) (summaries in appendix 2).
In addition, Jorge Rocha was invited by Prof. Leonor Cancela, president of the Portuguese Society
of Biochemistry, to write a paper describing our group activities in order to be published in a special
number of the society’s journal (Canal Bioquímica) dedicated to the subject Science at South, aimed
at thinking about the partnerships and research proposals in Southern countries (South from
Europe). This paper, shown in appendix 4, makes a summary of our recent activities and makes
some considerations about the increasingly central role of the African continent in our research.
Finally, Jorge Rocha took part of the organization committee of the 2º Encontro de Biologia
Evolutiva (“2nd Meeting of Evolutionary Biology”), together with Dr. Jorge Vieira (IBMC) and Prof.
Nuno Ferrand (CIBIO). The purpose of the meeting is to annually join PhD students that are
developing a project in the area of Evolutionary Biology, in order to stimulate the discussion of
themes that are of common interest and to facilitate future collaborations.
40
2.5 Work plan for 2007
During 2007 we intend to: a) start Susana’s work, aimed at extending the study of natural selection
patterns to other protease inhibitor families, in collaboration with Raquel Seruca and Gianpaolo
Suriano; b) to proceed with Margarida’s PhD work plan, namely with the ascertainment of UEPSTR sets and data collection in Bantu populations; c) to interpret the data of intra-allelic variation of
HBB*S mutation an to test formal models about its origin and spread in Africa; d) to proceed with
Sandra’s post-doc work plan and, in particular, to perform the fundamental field work in Cape
Verde; e) to proceed with the educational activities that will be concentrated in hosting two Cape
verdian students and in the supervising of two undergraduate students that will work in the
evolutionary history of skin pigmentation genes; f) to develop the collaborations with Leonor David
and José Carlos regarding the study on the relationship between host’s genetic variation and
susceptibility to infectious and carcinogenic agents.
Regarding the strategy that should be carried out to accomplish these activities, it is our opinion that
we should strengthen our ability to analyse the results. Sometimes, the feeling is that the way we act
to surpass our weakness in this field is to accumulate a great amount of data, when we could have
more significant conclusions and save time (and money) in the lab just by a better analysis and
interpretation of the results. In other words, with a better analytical capability it is possible to extract
more conclusions with less data. Given that, today, there are labs that may produce data in very
short period of time (months) the data that would take us years to obtain, it is essential that we
dedicate more time trying to produce and apply interpretive methodologies of better quality. The
type of analysis done by Susana, or some of the approaches used in the paper in which Sandra
participated, already offer us some encouraging perspectives that we should explore to construct less
descriptive evolutionary genetics studies.
Another aspect related with our activity has to do with its link to medical research and, specifically,
to a more applied clinical research. In many situations, our focus on evolutionary studies will drive
us away from collaborations with doctors, or at least will not allow that these collaborations to
become more strong. Nevertheless, this should not be necessarily contradicted, since, on the other
hand, it will make us closer to other areas of Biology and to other type of collaborations that may
increase our research quality. In some cases, however, our aims are not only compatible but also
dependent on collaborations with doctors. A good example is the section regarding obesity and
hypertension from Cape Verde’s project, which represents an excellent opportunity to reinforce the
links with medical research and which we will try to accomplish in 2007.
2.6 Ongoing projects
“Bio-cultural adaptation: human evolutionary responses to major changes in subsistence economy”
(FCT-project; POCTI/BIA-BDE/56654/2004). PI: Jorge Rocha. Total budget for 2005-2008 = 78
000 €.
2.7 Submitted projects
2.7.1 Projects in which the principal investigator is a group member
“On the edge of the Bantu expansions: inference of recent population history with independently
evolving haplotypic systems” (FCT). PI: Jorge Rocha.
41
“Looking for evidences of human adaptation in the proteolysis universe: the case study of serine
protease inhibitors” (FCT). PI: Susana Seixas.
“Understanding the genetic architecture and evolution of human pigmentary traits: admixture
mapping studies in Cape Verde” (FCT). PI: Sandra Beleza.
“Caracterização de Populações Miscigenadas para o estudo da Etiologia da Obesidade e
Hipertensão: O Caso Exemplar de Cabo Verde” (Fronteira das Ciências da Vida Fundação
Gulbenkian). PI: Sandra Beleza.
2.7.2 Projects in collaboration
“Secretor and Lewis phenotypes/genotypes in Helicobacter pylori and calicivirus infection” (FCT)
PI: Leonor David.
“Human genetic markers and risk of infection with Helicobacter pylori” (ERA-NET
PathoGenoMics). PI: José Carlos Machado.
2.8 Publications
2.8.1 Published papers or in press
Batini C, Coia V, Battagia C, Rocha J, Pilkington MM, Spedini G, Comas D, Destro-Bisol G,
Calafell F. 2006. Phylogeography of the human mitochondrial L1c haplogroup: genetic signatures of
the prehistory of Central Africa. Molecular Phylogenetics and Evolution (in press).
Maia LF, Vasconcelos C, Seixas S, Magalhães R, Correia M. 2006. Lobar brain hemorrages and
white matter changes: clinical, radiological and laboratory profiles. Cerebrovascular Diseases 22:
155-161.
McEvoy B, Beleza S, Shriver M. 2006. The genetic architecture of normal variation in human
pigmentation: an evolutionary perspective and model. Human Molecular Genetics 15: R175-R181.
Pimenta JR, Zuccherato LW, Debes AA, Maselli L, Soares RP, Moura-Neto R, Rocha J, Bydlowski
SP, Pena SDJ. 2006. Color and genomic ancestry in Brazilians: a study with forensic microsatellites.
Human Heredity (in press).
Rocha J. 2006. Genética de populações humanas: o centro está no sul. Canal BQ (in press).
Seixas S, Suriano G, Carvalho F, Seruca R, Rocha J, Di Rienzo A. 2006. Sequence diversity at the
proximal 14q32.1 SERPIN subcluster: evidence for natural selection favoring the pseudogenization
of SERPINA2. Molecular Biology and Evolution (in press).
Suriano G, Seixas S, Rocha J, Seruca R. 2006. E-cadherin germline missense mutations: a clinical
dilemma. How to infer their pathogenic significance in HDGC. Journal of Molecular Medicine 84:
1023-1031.
2.8.2 Papers submitted for publication
Coelho M, Alves C, Coia V, Luiselli D, Uselli A, Hagemeijer T, Amorim A, Destro- Bisol G, Rocha J. 2006.
Human microevolution and the Atlantic slave trade: the case study of São Tomé (Gulf of Guinea). Submitted to
Current Anthropology
42
Population Genetics
Group Leader: António Amorim, PhD
Staff members: Maria João Prata, PhD; Leonor Gusmão, PhD; Luísa Azevedo, PhD (post-Doc);
Alexandra Lopes, PhD (post-Doc); Ana Goios, PhD student; Barbara van Asch, PhD student; Elisabete
Oliveira, PhD student; Filipe Pereira, PhD student; Iva Gomes, PhD student; Sandra Martins, PhD
student; Rita Quental, PhD student; Sofia Quental, PhD student; Rui Pereira, MSc student; Alfredo
Gusmão, MSc student; Inês Soares MSc student; Nádia Pinto, MSc student; Cíntia Alves, chief
technician; Verónica Gomes, technician.
Objectives/Goals of the research activity
The group aims at understanding the origin and evolution of (mainly) human genetic diversity and
their consequences and applications, both normal and pathological (using autosomal, X and Y linked, as
well as mtDNA markers).
This requires the development of descriptive and analytical formal tools and techniques adequate to
specific genomic segments, in order to achieve the genetic characterisation of normal populations, their
origins, phylogeny and evolution, and disease susceptibility profiles.
The applications in which we concentrate our efforts are molecular diagnostics and forensics, but a
line of research involving the history, conservation and management of domesticates and laboratory
animals as well as food quality assessment is now established.
Background and major achievements during 2006
According to the work plan formulated in 2005,
A PCR decaplex for X-chromosome STR typing was successfully developed and the results are
reported in [P11]; the kit proved to be robust and the set of markers very informative, with large
potential application in anthropological studies and forensics [1,16,17,28].
The results of the studies of normal and pathological variation associated with mtDNA include
(a) the assessment of the utility of a new technique suitable for vestigial and/or degraded samples [2];
(b) the evaluation of the informativeness of haplogroup H subtyping in Eurasia [27] the demonstration
of the genetic complexity of the Tunisian populations samples [21]; the establishment/validation of the
maternal lineages history (the microphylogeny) of the lab mouse [15]; the creation of a software tool for
the analysis of circular genomes, particularly useful for mtDNA [14]; the study of the pitfalls in mtDNA
recovery from mixtures [12] and the disclosure of the founder effects of the maternal lineages of the
Ashkenazi Jews.
The extension of the genetic profiling of Portuguese domesticates included the characterisation
of the sheep mtDNA lineages and the discovery of a strong Mediterranean signature on its husbandry
[26]. A collaborative exercise/proficiency test on dog mtDNA has been launched under our coordination
within the Spanish and Portuguese Working Group of the International Society of Forensic Genetics
(www.gep-isfg.org/ISFG/Portugues/Grupos_de_trabalho/Genetica_forense_nao_humana/propuesta_2006_port.php).The progress on the genetic
traceability of processed animal products has been slow due to the need of the previous characterisation
of pig mtDNA variation, which is nearly finished in one of the autochthonous breeds.
The impact of cis- and trans- acting variation on differences in expression levels was addressed
in [20], demonstrating sexual dimorphism of expression for a gene belonging to the protocadherin
family in brain.
A substantial contribution to clarify the origin and dispersion of Machado Joseph Disease [36]
and the evolutionary dynamics of the locus were made, evidencing a dual mutational mechanism on
action at this trinucleotide repeat locus [24].
The importance of TPMT genotype on the management of paediatric acute lymphoblastic
leukaemia was confirmed, revealing that carriers of deficient alleles show a higher rate of relapse [P15]
- The mutational and transcriptional instability of specific genomic regions was studied in [4], where
the mucopolysaccharidosis type II gene was shown to be prone to splicing mutations, and in [5], where
43
it was demonstrated that the knowledge of the linkage disequilibrium between markers at the OTC is
essential to the detection of deletions in carriers.
- The issues of the demographic history and genetic epidemiology of disease causing genes were
addressed in the analysis of the congenital disorder of glycosylation type Ia, where the two most
common mutations at the PMM2 locus were shown to result from just two independent events and one
of them of Iberian origin [29] as well in the unraveling of a distinctive mutational spectrum of the TPMT
locus in the ancient genetic isolate of Sardinia [30].
- The group has also been involved in the population genetics analyses of the polymorphisms involved
in oncogenesis [11] and cardiomyopathies [23].
- The results of anthropological and forensic studies of human populations, with particular emphasis
on Portugal and Timor were: the haplotype distribution of X-chromosome STR markers in Spain [1],
Africa [16], US ethnic groups [17] the extension of the Y-chromosome STR haplotype database in
Portugal [3], Colombia [9, 10], Tunisia [13], Brasil [31] and Timor [33], the microphylogeography and
demographic history of Portuguese [8] and S. Tomé e Príncipe [35] male lineages, the mosaic structure
of the male and female gene pools of Jerba Island, Tunisia [21], the autosomal databases of Colombia
[22] and Argentina [34], and, finally the correlation between linguistic groups and Y chromosome
diversity in East Timor [32].
- We have also participated in the expert group on the use of Y chromosome STRs in forensics, which
has issued a set of recommendations [18, 19] and in the revision and correction of the nomenclature
[25].
- Last, but not least, a review on the role of epistatic interactions in disease and evolution was
published in Trends in Genetics [6].
WORK PLAN FOR 2007
We intend to study
- The dynamics of normal and pathogenic mtDNA diversity, in particular the variation among SAM
(senescent lab mouse lines)
- Nuclearly transferred segments of mtDNA (NUMTs): distinction from genuine mtDNA
- Expression of X and Y chromosome genes in male germ line in humans and in mouse models of
infertility
- Development of molecular tools for genotyping Aspergillus strains
- Variation of PKLR gene in relation to malaria susceptibility
- Timings and mechanisms of triplet repeat instability
- The genetic pools of Portuguese pig, sheep and goat autochthonous breeds in order to allow the
development of molecular tools for the control, certification and traceability of meat products
- Genetic factors in the susceptibility to, and treatment of, paediatric acute lymphoblastic leukaemia
- The newly defined X chromosome markers in terms of population analysis and forensic applications
and the development of new high throughput typing technologies
- The genes involved in congenital disorders of glycosylation, in terms of disease-causing mutations,
establishment of orthologues, and phylogenetic relationships
- The mutational spectrum of Portuguese maple syrup urine disease patients
44
- Meta-patterns of DNA sequence variation: development of detecting algorithms and comparative
genomics
PAPERS
1. ALER M, SÁNCHEZ-DIZ P, GOMES I, GISBERT M, CARRACEDO A, AMORIM A, GUSMÃO L. Genetic
data of 10 X-STRs in a Spanish population sample". Forensic Sci Int (in press).
2. ALONSO A, ALBARRAN C, MARTIN P, GARCIA P, CAPILLA J, GARCIA O, DE LA RUA C, IZAGUIRRE N,
PEREIRA F, PEREIRA L, AMORIM A, SANCHO M (2006) Usefulness of microchip electrophoresis for
the analysis of mitochondrial DNA in forensic and ancient DNA studies. Electrophoresis. 27(24): 51019.
3. ALVES C, GOMES V, PRATA MJ, AMORIM A, GUSMÃO L: Population data for Y-chromosome
haplotypes defined by 17 STRs (AmpFlSTR YFiler) in Portugal. Forensic Sci Int (in press).
4. ALVES S, MANGAS M, PRATA MJ, RIBEIRO G, LOPES L, RIBEIRO H, PINTO-BASTO J, LIMA MR,
LACERDA L (2006) Molecular characterization of Portuguese patients with mucopolysaccharidosis
type II shows evidence that the IDS gene is prone to splicing mutations. J Inherit Metab Dis 29:743754.
5. AZEVEDO L, SOARES PA, QUENTAL R, VILARINHO L, TELES EL, MARTINS E, DIOGO L, GARCIA P,
CENNI B, WERMUTH B, AMORIM A (2006) Mutational Spectrum and Linkage Disequilibrium Patterns
at the Ornithine Transcarbamylase Gene (OTC) Ann Hum Genet 70(Pt 6):797-801.
6. AZEVEDO L, SURIANO G, VAN ASCH B, HARDING RM, AMORIM A (2006) Epistatic interactions: how
strong in disease and evolution? Trends Genet. 22(11): 581-5.
7. BEHAR DM, METSPALU E, KIVISILD T, ACHILLI A, HADID Y, TZUR S, PEREIRA L, AMORIM A,
QUINTANA-MURCI L, MAJAMAA K, HERRNSTADT C, HOWELL N, BALANOVSKY O, KUTUEV I,
PSHENICHNOV A, GURWITZ D, BONNE-TAMIR B, TORRONI A, VILLEMS R, SKORECKI K (2006) The
Matrilineal Ancestry of Ashkenazi Jewry: Portrait of a Recent Founder Event. Am J Hum Genet 78:
487-497
8. BELEZA S, GUSMAO L, LOPES A, ALVES C, GOMES I, GIOUZELI M, CALAFELL F, CARRACEDO A,
AMORIM A (2006) Micro-phylogeographic and demographic history of Portuguese male lineages. Ann
Hum Genet 70: 181-94.
9. BUILES JJ, BRAVO ML, GOMEZ C, ESPINAL C, AGUIRRE D, GOMEZ A, RODRIGUEZ J, CASTANEDA P,
MONTOYA A, MORENO M, AMORIM A, GUSMAO L (2006) Y-chromosome STRs in an Antioquian
(Colombia) population sample. Forensic Sci Int. 164: 79-86
10. BUILES JJ, MARTINEZ B, GOMEZ A, CARABALLO L, ESPINAL C, AGUIRRE D, MONTOYA A, MORENO
M, AMORIM A, GUSMAO L, BRAVO ML Y chromosome STR haplotypes in the Caribbean city of
Cartagena (Colombia). Forensic Sci Int. 2006 Jan 30; [Epub ahead of print]
11. CASTRO P, SOARES P, GUSMÃO L, SERUCA R, SOBRINHO-SIMOES M (2006) H-RAS 81
polymorphism is significantly associated with aneuploidy in follicular tumors of the thyroid. Oncogene
25:4620-4627
12. CRESPILLO M, PAREDES MR, PRIETO L, MONTESINO M, SALAS A, ALBARRAN C, V AI, AMORIM A,
BERNIELL-LEE G, BREHM A, CARRIL JC, CORACH D, CUEVAS N, DI LONARDO AM, DOUTREMEPUICH
C, ESPINHEIRA RM, ESPINOZA M, GOMEZ F, GONZALEZ A, HERNANDEZ A, HIDALGO M, JIMENEZ M,
LEITE FP, LOPEZ AM, LOPEZ-SOTO M, LORENTE JA, PAGANO S, PALACIO AM, PESTANO JJ,
PINHEIRO MF, RAIMONDI E, RAMON MM, TOVAR F, VIDAL-RIOJA L, VIDE MC, WHITTLE MR, YUNIS
JJ, GARCIA-HIRSCHFEL J (2006) Results of the 2003-2004 GEP-ISFG collaborative study on
mitochondrial DNA: Focus on the mtDNA profile of a mixed semen-saliva stain. Forensic Sci Int. 160:
157-167
13. FRIGI S, PEREIRA F, PEREIRA L, YACOUBI B, GUSMAO L, ALVES C, KHIL HK, CHERNI L, AMORIM A,
GAAIED AE (2006) Data for Y-chromosome haplotypes defined by 17 STRs (AmpFLSTRYfilertrade
mark) in two Tunisian Berber communities. Forensic Sci Int 160 (1): 80-83
14. GOIOS A, MEIRINHOS J, ROCHA R, LOPES R, AMORIM A, PEREIRA L. (2006) RepeatAround: A
software tool for finding and visualizing repeats in circular genomes and its application to a human
mtDNA database. Mitochondrion 6(4): 218-24.
15. GOIOS A, PEREIRA L, BOGUE M, MACAULAY V, AMORIM A. MtDNA phylogeny and evolution of
laboratory mouse strains. Genome Reseach (in press)
45
16. GOMES I, ALVES C, MAXZUD K, PEREIRA R, PRATA MJ, SÁNCHEZ-DIZ P, CARRACEDO A, AMORIM
A, GUSMÃO L. Analysis of 10 X-STRs in three African populations". Forensic Sci Int (“DNA in
Forensics” -special edition, in press).
17. GOMES I, PRINZ M, PEREIRA P, MEYERS C, MIKULASOVICH RS, AMORIM A, CARRACEDO A,
GUSMÃO L. Genetic analysis of three US population groups using an X-chromosomal STR decaplex.
Int J Legal Med. (in press) DOI 10.1007/s00414- 006-0146-2.
18. GUSMÃO L, BUTLER JM, CARRACEDO A, GILL P, KAYSER M, MAYR MR, MORLING N, PRINZ M,
ROEWER L, TYLER-SMITH C, SCHNEIDER PM (2006) DNA Commission of the International Society of
Forensic Genetics (ISFG): An update of the recommendations on the use of Y-STRs in forensic
analysis. Int J Legal Med 120:191-200.
19. GUSMÃO L, BUTLER JM, CARRACEDO A, GILL P, KAYSER M, MAYR MR, MORLING N, PRINZ M,
ROEWER L, TYLER-SMITH C, SCHNEIDER PM (2006) DNA Commission of the International Society of
Forensic Genetics (ISFG): An update of the recommendations on the use of Y-STRs in forensic
analysis. Forensic Sci Int 157:187-197.
20. LOPES AM, ROSS N, CLOSE J, DAGNALL A, AMORIM A, CROW TJ (2006) Inactivation status of
PCDH11X: sexual dimorphisms in gene expression levels in brain. Hum Genet. 119(3):267-75
21. LOUESLATI BY, CHERNI L, KHODJET-ELKHIL H, ENNAFAA H, PEREIRA L, AMORIM A, BEN AYED F,
BEN AMMAR ELGAAIED A (2006) Islands inside an island: reproductive isolates on Jerba island. Am J
Hum Biol 18:149-53.
22. MARTINEZ B, CARABALLO L, BARON F, GUSMAO L, AMORIM A, CARRACEDO A (2006) Analysis of
STR loci in Cartagena, a Caribbean city of Colombia. Forensic Sci Int. 160:221-223.
23. MARTINS E, SILVA-CARDOSO J, ALVES C, PEREIRA H, SOARES B, DAMASCENO A, ABREU-LIMA C,
AMORIM A, ROCHA-GONCALVES F (2006) Familial dilated cardiomyopathy with troponin T K210del
mutation. Rev Port Cardiol 25(3): 295-300
24. MARTINS S, CALAFELL F, WONG VC, SEQUEIROS J, AMORIM A (2006) A multistep mutation
mechanism drives the evolution of the CAG repeat at MJD/SCA3 locus. Eur J Hum Genet. 14(8):93240
25. MULERO JJ, BUDOWLE B, BUTLER JM, GUSMAO L (2006) Letter to the editor--nomenclature and
allele repeat structure update for the Y-STR locus GATA H4. J Forensic Sci. 51(3):694.
26. PEREIRA F, DAVIS SJ, PEREIRA L, MCEVOY B, BRADLEY DG, AMORIM A (2006) Genetic Signatures
of a Mediterranean Influence in Iberian Peninsula Sheep Husbandry. Mol Biol Evol. 23(7):1420-1426.
27. PEREIRA L, RICHARDS M, GOIOS A, ALONSO A, ALBARRAN C, GARCIA O, BEHAR DM, GOLGE M,
HATINA J, AL-GAZALI L, BRADLEY DG, MACAULAY V, AMORIM A (2006) Evaluating the forensic
informativeness of mtDNA haplogroup H sub-typing on a Eurasian scale. Forensic Sci Int 159: 43-50
28. PEREIRA R, GOMES I, AMORIM A, GUSMÃO L. Genetic diversity of 10 X-chromosome STRs in
northern Portugal". Int J Legal Med. DOI 10.1007/s00414- 006-0144-4.
29. QUELHAS D, QUENTAL R, VILARINHO L, AMORIM A, AZEVEDO L. (2006) Congenital Disorder of
Glycosylation Type Ia: Searching for the Origin of Common Mutations in PMM2. Ann Hum Genet.
2006 Dec 12; [Epub ahead of print]
30. ROSSINO R, VINCIS C, ALVES S, PRATA MJ, MACIS MD, NUCARO AL, SCHIRRU E, CONGIA M (2006)
Frequency of the Thiopurine S-Methyltransferase alleles in the ancient genetic isolate of Sardinia. J
Clin Pharm Ther 31:283-287.
31. SILVA DA, CARVALHO E, COSTA G, TAVARES L, AMORIM A, GUSMAO L (2006) Y-chromosome
genetic variation in Rio De Janeiro population. Am J Hum Biol 18(6): 829-837
32. SOUTO L, GUSMAO L, AMORIM A, CORTE-REAL F, VIEIRA DN (2006) Y-STR haplotype diversity in
distinct linguistic groups from East Timor. Am J Hum Biol 18(5): 691-701.
33. SOUTO L, GUSMÃO L, FERREIRA E, AMORIM A, CÔRTE-REAL F, VIEIRA DN (2006) Y-chromosome
STR haplotypes in East Timor: Forensic evaluation and population data. Forensic Sci Int 156: 261265.
34. TOSCANINI U, GUSMAO L, BERARDI G, AMORIM A, CARRACEDO A, SALAS A, RAIMONDI E (2007)
Testing for genetic structure in different urban Argentinian populations. Forensic Sci Int. 165(1):3540.
35. TROVOADA MJ, TAVARES L, GUSMÃO L, ALVES C, ABADE A, AMORIM A, PRATA MJ (2007)
Dissecting the genetic history of São Tomé e Príncipe: a new window from Y-chromosome biallelic
markers. Ann Hum Genet 71:77-85.
36. MARTINS S, CALAFELL F, GASPAR C, WONG VCN, SILVEIRA I, NICHOLSON GA, BRUNT E,
TRANEBJAERG L, STEVANIN G, HSIEH M, SOONG B-W, LOUREIRO L, DÜRR A, TSUJI S, WATANABE
M, JARDIM L, GIUNTI P, RIESS O, RANUM LPW, BRICE A, ROULEAU GA, COUTINHO P, AMORIM A,
46
SEQUEIROS J (2007) The worldwide spread mutational event in Machado-Joseph disease has an
Asian origin. Arch Neurol (in press).
Books/ Book Chapters
AMORIM A, CORTE-REAL F, MORLING N, eds. (2006) Progress in Forensic Genetics 11. International
Congress Series, Elsevier.
Full papers in Procceedings
P1
ALVES C, COELHO M, ROCHA J, AMORIM A (2006): The Amelogenin locus displays a high
frequency of X homologue failures in São Tomé island (West Africa). Progress in Forensic Genetics
11. International Congress Series 1288: 271-273.
P2
Alves C, Gusmão L, Meirinhos J, Amorim A (2006) Making the most of Y-STR haplotypes: The
HapYDive. Progress in Forensic Genetics 11. International Congress Series 1288:201-203. Elsevier
Science. Amsterdam.
P3
Amorim A, Alves C, Gusmão L, Pereira L (2006) Extended Northern Portuguese database on 21
autosomal STRs used in genetic identification. Progress in Forensic Genetics 11. International Congress
Series 1288:364-366. Elsevier Science. Amsterdam.
P4
BETTENCOURT C, MONTIEL R, SANTOS C, PRATA MJ, ALUJA MP, LIMA M (2006) Diversity of
maternal and paternal lineages in the geographic extremes of the Azores (Santa Maria and Flores
islands): insights from mtDNA, Y-chromosome and surname data. Progress in Forensic Genetics 11.
International Congress Series 1288:88-90.
P5
Builes JJ, Bravo MLJ, Gómez A, Caraballo L, Espinal C, Aguirre D, Montoya AE, Moreno MA,
Pancorbo M, Gusmão L, Martínez B (2006) Analysis of 16 Y-chromosome STRs in a Cartagena
(Colombia) population sample. Progress in Forensic Genetics 11. International Congress Series
1288:358-360. Elsevier Science. Amsterdam.
P6
Builes JJ, Castañeda SP, Espinal C, Aguirre D, Gómez MV, Villamarin D, Pancorbo M, Gusmão L,
Moreno MA, Bravo MLJ (2006) Analysis of 16 Y-chromosomal STRs in a Valle (Colombia) population
sample. Progress in Forensic Genetics 11. International Congress Series1288:219-221. Elsevier Science.
Amsterdam.
P7
Builes JJ, Castañeda SP, Espinal C, Aguirre D, Rodríguez JR, Gómez MV, Villamarin D, Moreno M,
Pancorbo M, Gusmão L, Caraballo L, Martínez B, Bravo MLJ (2006) Analysis of 16 Y-chromosomal STRs
in a Córdoba (Colombia) population sample. Progress in Forensic Genetics 11. International Congress
Series 1288:174-176. Elsevier Science. Amsterdam.
P8
Builes JJ, Hau J, Rodríguez JI, Montoya AE, Izarra F, Ochoa O, Pérez L, Pancorbo M, Gusmão L,
Bravo MLJ (2006) Peruvian population study with 16 Y-STR loci. Progress in Forensic Genetics 11.
International Congress Series 1288:216-218. Elsevier Science. Amsterdam.
P9
Frigi S, Yacoubi B, Pereira F, Pereira L, Cherni L, Amorim A, Elgaaied A, mtDNA lineages in
two Tunisian Berber communities: comparing diversities between villages and towns, Progress in
Forensic Genetics 11. 1288, 121– 123.
P10
Goios A, Amorim A, Pereira L. (2006) Mitochondrial DNA pseudogenes in the nuclear
genome as possible sources of contamination. Progress in Forensic Genetics 11.International
Congress Series 1288:697-699.
P11
Gomes I, Carracedo A, Amorim A, Gusmão L (2006) A multiplex PCR design for simultaneous
genotyping of X chromosome short tandem repeat markers. Progress in Forensic Genetics 11.
International Congress Series 1288:313-315. Elsevier Science. Amsterdam.
P12
Gusmão L, Sánchez-Diz P, Gomes I, Alves C, Carracedo A, Prata MJ, Amorim A (2006) Genetic
analysis of autosomal and Y-specific STRs in the Karimojong population from Uganda. Progress in
Forensic Genetics 11. International Congress Series 1288:213-215. Elsevier Science. Amsterdam.
P13
López-Parra AM, Tavares L, Gusmão L, Mesa MS, Prata MJ, Amorim A, Arroyo-Pardo E (2006) YSTR polymorphisms from Basque-speaking region of Cinco Villas (Navarra) in the context of the
Pyrenean genetic landscape. Progress in Forensic Genetics 11. International Congress Series 1288:198200. Elsevier Science. Amsterdam.
P14
Moreno MA, Builes JJ, Jaramillo P, Espinal C, Aguirre D, Pancorbo M, Gusmão L, Bravo MLJ
(2006) Validation of five X-chromosome STRs DXS6800, DXS6807, DXS6798, DXS8377 and DXS7423 in
an Antioquian population sample. Progress in Forensic Genetics 11. International Congress Series
1288:295-297. Elsevier Science. Amsterdam.
47
P15
OLIVEIRA E, ALVES S, QUENTAL S, FERREIRA F, NORTON L, COSTA V,
AMORIM A, PRATA MJ (2006) Outcome in acute lymphoblastic leukaemia: influence of thiopurine
methyltransferase genetic polymorphism. Progress in Forensic Genetics 11. International Congress
Series 1288:789-791.
P16
Pereira F, Meirinhos J, Amorim A, Pereira L (2006) Analysis of inter-specific mitochondrial
DNA diversity for accurate species identification. Progress in Forensic Genetics 11.1288, 103– 105.
P17
PEREIRA L, GOIOS A, AMORIM A (2006) Sampling efficiency for Amerindian female lineages.
Progress in Forensic Genetics 11. International Congress Series 1288:322-324.
P18
PEREIRA L, MORALES AC, GOIOS A, DUARTE R, RODRIGUES C, ENDICOTT P, ALONSO A,
MARTIN P, TORRES C, AMORIM A (2006) The Islamization of Iberian Peninsula: a demographic shift
or a cultural change? Search for an answer using extant and ancient DNA from Mértola (Southeast
Portugal). Progress in Forensic Genetics 11. International Congress Series 1288:828-830.
P19
Prata MJ, Tavares L, Trovoada MJ, Gusmão L, Beleza S, Alves C, Amorim A (2006) Highresolution analysis of Y-biallelic markers in three populations from São Tomé e Príncipe. Progress in
Forensic Genetics 11. International Congress Series 1288:28-30. Elsevier Science. Amsterdam.
P20
SANTOS C, MONTIEL R, BETTENCOURT C, PRATA MJ, ABADE A, ALUJA MP, LIMA M (2006)
Peopling, demographic history and genetic structure of the Azores Islands: Integrating data from
mtDNA and Y-chromosome. Progress in Forensic Genetics 11. International Congress Series
1288:85-87.
P21
Soares PA, Pereira F, Brion M, Alves C, Richards M, Carracedo A, Amorim A, Gusmão L (2006)
Relative Y-STR mutation rates estimated from the variance inside SNP defined lineages. Progress in
Forensic Genetics 11. International Congress Series 1288:82-84. Elsevier Science. Amsterdam.
P22
Souto L, Gusmão L, Ferreira E, Pires A, Rocha AM, Amorim A, Côrte-Real F, Vieira DN (2006) Ychromosome haplotypes in East Timor (Timor-Leste): Evidences of population differentiation. Progress in
Forensic Genetics 11. International Congress Series 1288:256-258. Elsevier Science. Amsterdam.
P23
Toscanini U, Berardi G, Amorim A, Carracedo A, Salas A, Gusmão L, Raimondi E (2006) Forensic
considerations on STR databases in Argentina. Progress in Forensic Genetics 11. International Congress
Series 1288:337-339. Elsevier Science. Amsterdam.
P24
Toscanini U, Gusmão L, Berardi G, Amorim A, Carracedo A, Salas A, Raimondi E (2006) Genetic
variability of 17 Y chromosome STRs in two Native American populations from Argentina. Progress in
Forensic Genetics 11. International Congress Series 1288:154-155. Elsevier Science. Amsterdam.
Prizes
Best oral presentation
Quental S, Matos R, Vilarinho L, Martins E, Teles E L, Rodrigues E, Diogo L, Garcia P, Eusébio F, Gaspar
A, Sequeira S, Furtado F, Lança I, Amorim A, Prata MJ “Maple Syrup Urine Disease – Molecular Spectrum
in Portugal”. 4º Simpósio Internacional da Sociedade Portuguesa de Doenças Metabólicas, Funchal,
24/25 Nov.
PhDs
-
Finished
Lopes A Gene evolution and regulation within the Xq21.3/Yp11.2 hominid-specific homology block.
(FCUP,14Jul; supervisor: A Amorim)
-
-
-
Submitted
Martins S “Evolutionary and epidemiological genetics of Machado-Joseph disease”, Faculty of
Sciences, University of Porto, IPATIMUP and Univ. Pompeu Fabra, Barcelona
Ongoing
Pereira F “Development of uniparentally transmitted genetic markers for the characterization of male
and female gene pools of Portuguese small ruminants autochthonous breeds” Faculty of Sciences,
University of Porto, IPATIMUP, and Department of Genetics, Smurfit Institute, Trinity College, Dublin
2, Ireland. Fundação para a Ciência e Tecnologia (SFRH/BD/19585/2004). Since October 2004
Oliveira E “Study of Genetic Polimorphisms in Pediatric Acute Lymphoblastic Leukemia Pharmacogenetic Role in the Treatment and Relationship with susceptibility to the leukemogenic
48
-
-
-
-
-
process” Faculty of Sciences, University of Porto, IPATIMUP, and School of Medicine , Washington
University in St. Louis. Fundação para a Ciência e Tecnologia (SFRH/BD/17124/2004). Since
November 2004.
Goios A “Dynamics of the mitochondrial genome – the moving boundary between normal and
pathogenic diversity” Faculty of Sciences, University of Porto, IPATIMUP and Department of
Statistics, University of Glasgow. Fundação para a Ciência e Tecnologia (SFRH/BD/16518/2004).
Since November 2004.
Gomes I “X chromosome markers: genetic characterization, population analysis and forensic
applications” University Santiago de Compostela, IPATIMUP and CEGEN (National Genotyping Center
of Spain). Fundação para a Ciência e Tecnologia (SFRH/BD/21647/2005). Since November 2004.
Quental R “Congenital Disorders of Glycosylation (CDG): Enzymatic, Biochemical and Molecular
Features” Faculty of Sciences, University of Porto, IPATIMUP and Department of Human Genetics,
Catholic University of Leuven, Belgium. Fundação para a Ciência e Tecnologia
(SFRH/BD/23657/2005). Since February 2006.
Quental S “Functional, Expression and Structural investigation of the mutational spectrum of
Portuguese Maple Syrup Urine Disease patients” Faculty of Sciences, University of Porto, IPATIMUP
and Department of Human Genetics, School of Medicine, Emory University, Atlanta, USA. Fundação
para a Ciência e Tecnologia (SFRH/BD/22685/2005). Since January 2006.
Van Asch B “Development of uniparentally transmitted genetic markers for the characterization of
male and female gene pools of Portuguese pig (Sus scrofa) autochthonous breeds: applications for
the control, certification and traceability of meat products” Faculty of Sciences, University of Porto,
IPATIMUP and Modelo Continente Hipermercados SA. Fundação para a Ciência e Tecnologia
(SFRH/BDE/15581/2006). Since July 2006.
MScs
Finished
- PINTO N ” Construção de Algoritmos de Reconstrução de Genealogias” ENGENHARIA MATEMÁTICA,
FCUP, Oct, 2006; co-supervision António Amorim, Pedro Silva, Dep.Matem.Pura, FCUP
- SOARES I “Avaliação da dependência/independência de marcadores do cromossoma Y”. ENGENHARIA
MATEMÁTICA, FCUP, 24Nov2006; co-supervision Leonor Gusmão, Pedro Silva, Dep.Matem.Pura,
FCUP
OngoingSubmitted
- PEREIRA R “Marcadores do cromossoma X: Perfil genético da população do norte de Portugal e
aplicações forenses” CIÊNCIAS FORENSES. Faculdade de Medicina. Universidade do Porto. Submitted
Oct 2006
- GUSMÃO A “A comunidade cigana em Portugal: padrão de diversidade no gene pool masculino
baseado em marcadores do cromossoma Y” CIÊNCIAS DA TERRA E DA VIDA PARA O ENSINO.
Universidade de Lisboa. Faculdade de Ciências. Departamento de Geologia. Submitted Jan 2007
NATIONAL AND INTERNATIONAL COOPERATIONS
The group is currently engaged in various collaborative projects with
-
CEPROCOR (Centro de Excelencia en Productos y Procesos), Complejo Hospitalario Santa María de
Punilla, Córdoba, Argentina (population and forensic genetics), Nidia Modesti
Dep. Statistics, Univ. Glasgow (mtDNA), Vincent Macaulay
Department of Pathology, Cambridge University (sex chromosomes biology), Nabeel A Affara
Faculdade de Medicina, UP (Aspergillus genotyping), Cidália Pina-Vaz
Hôpital Nôtre Dame, Montreal, Canada (Machado-Joseph disease), Guy Rouleau
Inst. Med. Legal, Univ. Santiago Compostela (forensic genetics), Angel Carracedo
Instituto de Genética Médica Jacinto de Magalhães (human genetic diseases), Laura Vilarinho
Instituto Nacional de Medicina Legal (forensic genetics and databases), Francisco Corte-Real
Instituto Nacional de Toxicologia y Ciencias Forenses, Madrid (forensic genetics), Antonio Alonso
Instituto Português de Arqueologia, Lisboa, Portugal (domesticates), Simon J.M. Davis
Laboratorio de Genetica Forense, Universidad de Valencia (forensic genetics), Mercedes Aler
New York City Office of Chief Medical Examiner, forensic genetics), Mechthild Prinz
49
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Rappaport Faculty of Medicine and Research Institute, Technion and Rambam Medical Center, Haifa,
Israel (Jewish populations) Doron Behar
Royal Institute of Technology, Stockholm, Sweden (canine genetics), Peter Savolainen
SANE POWIC, Oxford (sex chromosomes biology), Tim Crow
Smurfit Institute, Trinity College, Dublin (genetics of domesticates), Dan Bradley
UniGene, IBMC Porto (genetic diseases), Jorge Sequeiros
Univ Pompeu Fabra (population genetics modelling), Francesc Callafel
Univ. Oxford (gene function & evolution), Rosalind Harding
Univ. Tunis (population genetics), Houssein Khodjet, Lotfi Cherni
Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, USA
(mtDNA), David C. Samuels
Jackson Laboratory, Bar Harbor, USA (lab mouse genetics), Molly Bogue
PRICAI-Fundacion Favaloro, Buenos Aires, Argentina (population genetics), Eduardo Raimondi
Lab. Genes, Medellin, Colombia (population genetics), Mª Luisa Bravo
Lab. Imunogenetica, Univ. Cartagena, Colombia (population genetics), Luiz Caraballo
Lab. Diagnóstico DNA, Univ. Estadual Rio de Janeiro (population genetics), Eliseu Carvalho
Inst. Higiene e Medicina Tropical, Lisboa (malaria and PKLR polymorphisms), Ana Paula Arez
Institute of Integrative and Comparative Biology, Univ. Leeds (mtDNA), Martin Richards
Visiting researchers at IPATIMUP
Houssein Khodjet, Tunis University, 4-8 Dec
Karina Maxzud, CEPROCOR (Centro de Excelencia en Productos y Procesos) do Complejo Hospitalario
Santa María de Punilla, Córdoba, Argentina, March
Patrícia Machado, Inst. Higiene e Medicina Tropical, Jul/Aug
Dayse Aparecida da Silva, University of Rio de Janeiro, Brazil, Nov
Elizeu Fagundes de Carvalho, University of Rio de Janeiro, Brazil, May
Angélica Bertagnolli, Universidade Federal de Minas Gerais, Brazil, October 2006 to March 2007
Slah Ouerhani, Tunis University, September
David Samuels, Virginia Bioinformatics Institute, 25-30 Jan
Visits / Courses Abroad
A Goios: Dep. Statistics, Univ. Glasgow (host: Vincent Macaulay), 3-30 May
I Gomes: Office of Chief Medical Examiner, Dep. Forensic Biology, New York (host: Mechthild Prinz),
July-August
S Martins: Center for the Study of Brain Diseases, CHUM Research Center, Notre Dame Hospital,
Montreal, Canada (host: Guy A. Rouleau), Feb 6 – Apr 24
A Lopes: Cheung Lab, University of Pennsylvania, Philadelphia (host: Vivian Cheung), June.
L Gusmão: Curso de Genética de populações. Curso de Maestría en Ciencias Básicas Biomédicas.
Facultad de Salud. Universidade Industrial de Santander, Bucaramanga, Colombia, 27Feb-3Mar and
6-17Nov
L Gusmão: Laboratório de Diagnóstico por DNA. UERJ. Rio de Janeiro, Brasil (Programa de cooperação
científica na área da genética forense), 22 Apr - 8 May
Organization of Scientific Meetings
• Portugaliae Genetica 9th. edition (GENES AND POPULATIONS IN HEALTH AND DISEASE), 16-18
March
• ABZ da Genética Humana – Identificação Genética (31 Mar – 1 Apr, SPGH/ IPATIMUP)
50
Coordination of QC/QA cooperative exercises
• Colaborative exercise on canine mtDNA, Non-human Forensic Genetics Workgroup, António
Amorim and José Pestano Brito, GEP- ISFG
• Colaborative exercise on Y-chromosome STR markers mutation rates, Sex chromosomes
Genetics Workgroup, Leonor Gusmão and António Amorim, GEP- ISFG
• Colaborative exercise on X-chromosome STRs, Sex chromosomes Genetics Workgroup, Leonor
Gusmão and António Amorim, GEP- ISFG
National Projects
Lead by IPATIMUP
POCTI/MGI/45076/2002 “Tratamento e susceptibilidade na leucemia linfoblástica aguda infantil:
influência de factores genéticos em enzimas destoxificadoras”
01-05-2003 - 01-05-2006
PI: MJ Prata
total budget: €56200
POCI/ANT/57037/2004 – “Estudo evolutivo e epidemiológico de focos de anemias hereditárias em
Portugal”.
01-10-2005 - 01-10-2008
PI: MJ Prata
total budget: €30000
POCI/AFR/62242/2004 – “Etnias e genética na fronteira da rota de migração Bantu: o caso dos
Karamojong”. Ethnicity and genetics in the fringe of Bantu migration route: The Karamojong case
01-01-2005 - 01-01-2008
PI: L Gusmão total budget:
€30000
1931/2006 Programa Saúde XXI/Medida 1.1 “Registo da deficiência genética da metabolização de
tiopurinas na população portuguesa e prevenção da toxicidade em terapêuticas citostáticas”
01/05/ 2006 – 31/12/2007 PI: MJ Prata
total budget: €150000
IPATIMUP as participant
POCI/SAU-ESP/55110/2004 “Avaliação da epidemiologia da malária na República de Cabo Verde”
01-07-2005 - 01-07-2008
PI: Ana Paula Martins dos Reis Arez (IHMT)
POCI/DES/62499/2004 - Aspectos genéticos da actividade física, aptidão física associada à saúde,
sobrepeso e obesidade. Um estudo em gémeos dos 6 aos 20 anos de idade.
PI: José António Ribeiro Maia (FCDEF, UP)
International Projects
Factores de riesgo en asma: genes relacionados con la remodelación bronquial. Instituto de
Investigaciones Inmunológicas, Universidad de Cartagena, Colombia.
Editorial Boards
Forensic Science International (A Amorim)
Forensic Science International: Genetics (L Gusmão)
Patents
Pereira F, van Asch B, Gusmão L.
“Processo de identificação de espécies animais em amostras contendo material genético, baseado na
determinação do tamanho de sequências do ADN mitocondrial”
Número de Pedido: 103 599; Data de Depósito: 6 de Novembro de 2006
Invited talks
L Gusmão - Marcadores genéticos de importancia forense: STRs del cromosoma Y. IV Congresso
Internacional y VII Colombiano de Genética. Simposio precongresso: Interpretación de la prueba de
ADN en la administración de justicia. Bucaramanga, Colombia, 22/02.
L Gusmão - Marcadores genéticos en estudios de genética poblacional: cuatro modos de transmisión
distinta. IV Congresso Internacional y VII Colombiano de Genética. Simposio genética de poblaciones y
evolución. Bucaramanga, Colombia, 23-25/02.
L Gusmão - Linhagens masculinas em estudos de genética populacional e forense. Curso de
Doutoramento da Faculdade de Medicina da Universidade de Santiago de Compostela. Santiago de
Compostela, Espanha, 24/03.
L Gusmão - Variabilidade genética humana: aplicação a estudos de genética populacional e investigação
de parentescos. Faculdade de Medicina da UERJ. Rio de Janeiro, Brasil, 28/04.
51
International Juries
L Gusmão: MSc thesis Patrícia Mariana Gonçalves da Rocha Porto Domingues, “Contribuição ao estudo
da constituição genética da população brasileira: Análise de regiões STR do cromossomo Y em uma
amostra populacional de indivíduos afro-descendentes do Rio de Janeiro”. Instituto de Biologia
Roberto Alcântara Gomes da UERJ-Universidade do Estado do Rio de Janeiro, 4 May.
52
Tumor Evolution and Development
Group Leader
Luís Teixeira da Costa, PhD, Researcher at IPATIMUP
Staff members
Ângela Costa, BSc – BD
Catarina Osório, BSc – BTI (through October 2006)
Elisabete Figueiredo, BSc – BTI, MSc student
Inês Barbosa – Undergraduate student (Graduated October 2006)
Isabel Castro, BSc – MSc Student
Joana Macedo, MD – MSc Student (part-time)
Nuno Camboa – Undergraduate student (Graduated October 2006)
Objectives/Goals of the research activity
Our main long term goal is to understand the molecular mechanisms underlying tumor
evolution. Rather than focusing on an organ or tissue-specific type of tumor, we intend
to pursue that goal by using different models to try to answer specific fundamental
questions about tumor evolution and development. At the initial stage of “group
establishment”, this was translated into focusing on problems with which we had
previous experience and felt we have the technical and financial means to tackle: a)
the regulation of TCF4, which is a key player in intestinal tumorigenesis; b) the role
played by p53 mutations in chromosomal instability in tumors.
A second goal, derived from IPATIMUP’s profile and objectives, is to directly put our
knowledge of and technical expertise in cancer molecular genetics to the service of
patients.
Background and major achievements during 2006
2006 was a consolidation year for the lab. Setting up and expansion of our “technical
base” were still significant, but lab composition was much more stable than previously.
It was the year in which our research started to come to fruition, with the completion of
our first project, and the acceptance of our first paper.
Project 1: “Identification of TCF4-interacting proteins” (FCT)
Our interest in TCF4 stems from the PI’s previous experience in colorectal cancer, a
tumor type initiated, in an overwhelming majority of cases, by inactivating mutations of
the tumor suppressor gene APC or “activating” mutations of β-catenin. Both result in an
abnormal increase in the cellular levels of a dimeric transcription factor including by βcatenin and members of the TCF-LEF transcription factor family, in particular TCF4.
TCF4 has also been shown to be a key player in the differentiation of mouse gut
epithelial cells, a role consistent with the fact that β-catenin is an effector of the "WntWingless" signaling pathway, whose importance in a number of developmental
processes in multiple animal species is well established.
53
Our approach to identify new TCF4 partners involved the use of a yeast two-hybrid
system. Yeast work (primary and secondary screens, identification and individual retesting of candidates) and testing of the candidates in mammalian cells (by coimmunoprecipitation/western blot and a mammalian two-hybrid system) were carried
out in 2004-2005 and led us to concentrate on two candidates: Grg5 and “clone 149”, a
previously uncharacterized gene. Subsequent work, initiated in 2005, was mostly done
during 2006.
The identification of the Grg5-TCF4 interaction was actually surprising, as it was
previously thought that the two proteins did not interact, but we confirmed it in
mammalian cells by co-immunoprecipitation. We went on to map the “core” Grg5binding region to a 69aa fragment of TCF4 by deletion analysis, identify 4 residues
critical for the interaction by site-directed/alanin-scan and random mutagenesis
affecting conserved amino-acids, and finally demonstrated that Grg5 can repress
TCF4-mediated transcription (from two different promoters) in mammalian cells. These
results complement previous data on Grg-TCF interactions, raise important questions
concerning the mechanisms of transcription repression by TCF-Grg complexes, and
open the way for a more detaile analysis of the role of Grg family members in normal
and pathological intestinal epithelial development. They will be submitted for
publication in the form of an article entitled “Identification and characterization of the
interaction between TCF4 and Grg5”.
As for the C149-TCF4 interaction, we have confirmed it in mammalian cells by both
two-hybrid and one-hybrid systems. We have demonstrated it is phylogenetically
conserved, as TCF1 also binds C149, and C149’s zebrafish homolog binds human
TCF4. Deletion analysis has shown that TCF4’s Grg5 and C149 binding domains are
similar, but not identical. Interestingly, we have shown that human C149 can also bind
Grg5, both in yeast and in mammalian cells, suggesting that the three proteins might
form a multimeric complex. Immunolocalization of C149 in human cell lines showed a
nuclear, punctate, distribution, similar to that of other TCF4 interactors, suggesting of a
role for C149 as a transcription factor. This was corroborated by co-transfection assays
demonstrating that C149 has the ability to repress TCF4-mediated transcription.
Expression analysis in a panel of human cell lines has shown that C149 in expressed
in a variety of tumor types. Particularly interesting, in view of recent studies suggesting
a transcription repression role for TCF4 in breast cancer, was the fact that C149’s
expression was most prominent in breast cell lines. These results will be submitted for
publication in the form of an article entitled “Cloning and characterization of a novel
human gene that interacts with TCF4”.
In the course of our studies of TCF4-interacting proteins, we detected a variety of
undescribed Grg4 transcripts, due to alternative splicing in the 3’ portion of the RNA.
This was intriguing, because the sequences involved code for the protein’s domains
responsible for both multimerization and TCF-binding. A more detailed analysis
indicated that, in the cell line used for cDNA synthesis, only about one third of the
cDNA molecules correspond to the sequence used in studies showing repression of
beta-catenin/TCF by Grg4, raising the possibility that the majority of the protein in
human cells functions differently from what has been described. These (preliminary)
results have been presented as an oral communication at the Annual SPGH
(Portuguese Society of Human Genetics) Meeting and are now being extended, both to
other cell lines and by cloning the various splice forms of Grg4 and evaluating them for
TCF- and “canonical” Grg4-binding by the two-hybrid system.
54
As in previous years, we maintained a commitment to technical improvements. (Mostly)
Within the framework of this project, this translated in 2005-2006 in the development of
a family of user-friendly, low-cost, high-efficiency vectors for PCR-product cloning.
Project 2: “Analysis of the role of p53 in cancer's chromosomal instability” (FCT)
As stated in the previous report, the execution of this project was significantly delayed
by continuing problems with tissue culture facilities. With the improvements in those
facilities in 2006, it was possible to start working on the most time-consuming part of
the project: the generation of knock-out and knock-in human tumor cell lines. However,
(given the legth of time involved) we should only be able to start performing the main
experiments for this project during 2007.
Project 3: “Clinical Use of Tobacco’s Genetic Targets” (FCG)
The goal of this project, accepted for funding in 2006 by Fundação Calouste
Gulbenkian, is to use molecular genetic data obtained from clinical samples to help in
diagnosis, prognosis and clinical management of lung cancer patients. Following up on
our 2005 preliminary studies, in 2006 we focused on: 1) trying to identify the major
limitations to the routine clinical use of molecular genetic screening in lung cancer in
Portugal; 2) Establishing reliable and cost-effective methods for the detection of rare
genetic variants in clinical samples. The results obtained so far have been the subject
of a poster presentation at Annual SPGH Meeting and an article (Rev Port Pneumol.
2007;13:9-34.).
Collaborations
Several collaborations were established in 2006, both “in-house” and with researchers
abroad, including: 1) “Carcinogenesis” group (auto-regulation of CDX2 expression); 2)
“Tumour Biology” group (targeted genetic manipulation of RET/PTC); 2) Victor
Velculescu, The Johns Hopkins University (lung cancer mutation detection); 3) Phil
Buckhaults, University of South Carolina (C149 status in breast cancer; lung cancer
mutation detection).
Financing
As of January 1, 2007, available funding for current lab expenses was approximately
50,000€, which is close to the total spent in the three year period 2004-2006.
55
Genetic Diversity and Bioinformatics
Group Leader: Luísa Pereira
Staff members: Sandra Oliveira (Master degree student); Carla Afonso (Master degree
student); Verónica Fernandes (Undergraduate Student in Biochemistry – University of Beira
Interior); Joana Pereira (Undergraduate Student in Biology – University of Aveiro)
Establishment: July 2006
Objectives/Goals of the research activity
The basic aim of the group is to establish a bridge between population genetics and clinical
genetics. This symbiosis is of major importance when analysing non recombining genetic
markers, such as mitochondrial DNA and Y-chromosome. For these markers it is extremely
difficult to disentangle between neutral and pathologic diversities because of its transmission in
block and its haplotypic distribution in human populations (many rare haplotypes). We will
pursuit in a detailed characterisation of worldwide genetic diversity, for the uniparental markers,
and in the design of studies of complex phenotypes, namely fertility and longevity. New
developments in biostatistics and bioinformatics will be essential for an efficient evaluation of
the genetic diversity.
Background and major achievements during 2006:
The group was established in July 2006. During this half year, efforts consisted mainly in (a)
forming new staff, (b) applying for funding, and (c) renovating and establishing international
collaborations. Presently, the staff consists in undergraduate and master students, but we intend
by the middle of 2007 to apply for PhD grants in order to begin a more solid team. For funding,
we submitted two projects to the Portuguese Science Foundation (FCT) in the areas of
“Anthropology” and “African studies”, and the PI is collaborator in a project submitted to an
English Foundation (Natural Environment Research Council - NERC). The active international
collaborations are listed bellow; some are established for some years now; the PI was invited to
visit a group in Geneve, and we are now conducting a theoretical analysis of Y-chromosome
STRs in a broad geographical scale.
With respect to our line of research of mtDNA influence in male fertility, we achieved to
publish two papers (listed bellow). The field is bursting with claims of false associations with
mtDNA haplogroups, mainly due to disregarding the phylogenetic information and keeping on
performing an “allelic” approach for a genetic marker that does not recombine. In one paper we
performed the complete sequencing of mtDNA (16,569bp) in 20 asthenozoospermic (and in 23
control individuals), demystifying a previous reported positive association between this
phenotype (lower sperm motility) and the European haplogroup T. This was the first study of
complete mtDNA sequencing in the field of fertility. In the other paper, we showed that
mutation ‘C11994T’ in the mitochondrial ND4 gene is not a cause of low sperm motility in
Portugal, as opposed to what was reported in India.
Work plan for 2007:
We are conducting the following lines of research:
. Applications to population genetics:
a) Refining the Out-of-Africa model with high-resolution genetic techniques. This is a wide
project, in which we will perform the complete mtDNA sequencing in some haplogroups
56
matching the Out-of-Africa migration (especially L3) in East African, Arabian and
Levantine populations. We have already 21 L3 complete sequences screened in Sudan.
b) Evaluation of the Neolithic genetic influence in the Great Mediterranean. The same
strategy described above will be applied to haplogroups matching the dispersion of
agriculture (J and T1) throughout the Mediterranean.
c) Evaluation of genetic exchanges between Morocco and Portugal. The last place in North
Africa to be left by the Portuguese, in the 17th century, was Mazagão, now called El
Jadida (meaning “the new one”). We have a small project with El Jadida University in
order to evaluate if the extant population bears more genetic resemblance to the
Portuguese population than the rest of Morocco. The analysis of mtDNA is finished (and
data show that El Jadida is a characteristic Moroccan population) and we are now
beginning the Y-chromosome diversity analysis.
d) Characterisation of the genetic diversity in el-Hayez oasis population (Egypt) in
collaboration with a group from University of Prague, Czech Republic.
. Applications to clinical genetics:
a) Evaluation of the mitochondrial influence in male fertility. Some in vivo evidence has
shown that glycolysis is more important than oxidative phosphorylation for the
production of energy for sperm motility. We will evaluate the genetic diversity of the
sperm-specific glycolytic enzyme (GAPD2) in infertile males.
b) Evaluation of longevity in humans of African origin. Aging is a multifactorial
phenomenon, but the mitochondrial theory of aging has been the centre of investigation
especially after evidence that (1) accumulation of mtDNA mutations in mice did cause
early aging and (2) European and Asian mtDNA haplogroups are positively associated
with longevity. We will perform the first study in African populations, namely in
Dominica, a Caribbean Island inhabited by descendents of sub-Saharan slaves and
presenting the highest frequency of very old people (more than 100 years old) in the
world. We will sequence the complete mtDNA molecule and survey the genetic
diversity in Polymerase gamma enzyme gene in centenarians versus controls.
. Bioinformatics
a) Evaluation of the relationship between mtDNA binding energy and mutability. The
research of Samuels (2005) found a relationship in mammals between the average
binding energy of the two strands of mtDNA and maximum lifespan of each species.
Samuels proposed the hypothesis that the mechanism responsible for this relationship is
the temporary and local separation of the two mtDNA strands due to thermal
fluctuations. Recent experimental work has confirmed that reducing the body
temperature in mice does extend their maximum lifespan in agreement with this
hypothesis. The aim of this short project is to develop a computational prediction of the
locations in the human mitochondrial genome which have low binding energy, and
therefore would be more often vulnerable to mutation processes, and to compare this to
the distribution of variable sites in the human mtDNA sequence. A new staff member
(Fernando Freitas, undergraduate student in University of Porto) will spend 3 months
(from March to May 2007) in Virginia Bioinformatics Institute, under the supervision of
Dr. David C. Samuels.
b) Testing a new algorithm for sequence alignment. Thesis Project of Sandra Oliveira, in
collaboration with the Faculty of Sciences, University of Porto.
c) Analysing Y-STR diversity in a broad geographic scale, centred in Arabian Peninsula. In
collaboration with Mathias Currat and Estella Poloni from Départment d’anthropology et
57
d’ecologie, Université de Geneve, Switzerland and Farida Alshamali from Dubai Police
General Headquarters.
Financing/projects:
1- 1/4/2006-31/03/2007 - “Y-chromosome phylogeography in Southeast Asia and the Pacific”;
The British Academy; PI: Martin Richards; Colaborators: Luísa Pereira, Jim Wilson; 43,783 £
2- 2006-2007 - “Genetic exchanges between Morocco and Portugal”, GRICES /CNRST; PIs:
Luísa Pereira and Nourdin Harich; 1,800 € plus 14,000 dirham for the Portuguese team
3- 01/05/2003-30/04/2006 - “Reassessment of European mitochondrial DNA diversity: present
and past distributions of female lineages”; FCT; PI: Luísa Pereira; 70,000 €
Theses – ongoing:
- co-supervision of PhD - Ana Goios: “Dynamics of the mitochondrial genome – the moving
boundary between normal and pathogenic diversity”; Faculty of Sciences, University of Porto,
IPATIMUP and Department of Statistics, University of Glasgow. Fundação para a Ciência e a
Tecnologia (SFRH/BD/16518/2004). Since November 2004.
- supervision of Master thesis in Forensic Medicine – Carla Afonso: “Characterisation of
mitochondrial DNA and Y-chromosome diversities in Sudan (East Africa): forensic and
population approaches”; ICBAS, IPATIMUP. Since July 2006.
- co-supervision of Master thesis in Mathematical Engineering – Sandra Oliveira:
“Development of computational tools for mitochondrial DNA alignment: application to the
analysis of mammal diversity”. Faculty of Sciences, University of Porto, IPATIMUP. Since July
2006.
Main international collaborations
Vincent Macaulay, Department of Statistics, University of Glasgow, UK
Martin Richards, Institute of Integrative & Comparative Biology, Faculty of Biological
Sciences, University of Leeds, UK
Doron Behar, Rappaport Faculty of Medicine and Research Institute, Technion and Rambam
Medical Center, Haifa, Israel
Farida Alshamali, Dubai Police General Headquarters
David C. Samuels, Virginia Bioinformatics Institute, Virginia, USA
Mathias Currat and Estella Poloni, Départment d’anthropology et d’ecologie, Université de
Geneve, Switzerland
Main national collaborations
João Gonçalves, Centro de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge,
Lisboa
Visiting researchers at IPATIMUP
Professor Nourdin Harich, University of El Jadida, Morocco
Professor Mostafa Kandil, University of El Jadida, Morocco
58
Publications
1- Yacoubi B, Cherni L, Enaafaa H, Khodjet el-Khil H, Pereira L, Amorim A, Ben Ayed F, El
Gaaied A (2006) Islands inside an island: reproductive isolates in Jerba Island. Am. J. Hum.
Biol. 18: 149-153. IF: 1.489
2- Behar DM, Metspalu E, Kivisild T, Achilli A, Hadid Y, Tzur S, Pereira L, Amorim A,
Quintana-Murci L, Majamaa K, Herrnstadt C, Howell N, Balanovsky O, Kutuev I,
Pshenichnov A, Gurwitz D, Bonne-Tamir B, Torroni A, Villems R, Skorecki K (2006) The
matrilineal ancestry of Ashkenazi Jewry: portrait of a recent founding event. Am. J. Hum.
Genet. 78: 487-497. IF: 12.649
3- Pereira L, Richards M, Goios A, Alonso A, Albarrán C, Garcia O, Behar DM, Gölge M,
Hatina J, Al-Gazali L, Bradley DG, Macaulay V, Amorim A (2006) Evaluating the forensic
informativeness of mtDNA haplogroup H sub-typing on a Eurasian scale. Forensic Sci. Int.
159: 43-50. IF: 1.577
4- Pereira F, Davis SJ, Pereira L, McEvoy B, Bradley DG, Amorim A (2006) Genetic
Signatures of a Mediterranean Influence in Iberian Peninsula Sheep Husbandry. Mol Biol
Evol. 23: 1420-1426. IF: 6.233
5- Frigi S, Pereira F, Pereira L, Yacoubi B, Gusmão L, Alves C, Khodjet el Khil H, Cherni L,
Amorim A, El Gaaied A (2006) Data for Y-chromosome haplotypes defined by 17 STRs
(AmpFLSTR® Yfiler™) in two Tunisian Berber communities. Forensic Sci. Int. 160: 8083. IF: 1.577
6- Goios A, Meirinhos J, Rocha R, Lopes R, Amorim A, Pereira L (2006) RepeatAround: a
software tool for finding and visualizing repeats in circular genomes and its application to a
human mtDNA database. Mitochondrion 6: 218-224. IF: 0.821
7- Alonso A, Albarran C, Martín P, García P, Capilla J, García O, de la Rua C, Izaguirre N,
Pereira F, Pereira L, Amorim A, Sancho M (2006) Usefulness of microchip electrophoresis
for the analysis of mitochondrial DNA in forensic and ancient DNA studies. Electrophoresis
27: 5101-5109. IF: 3.850
Accepted:
8- Goios A, Pereira L, Bogue M, Macaulay V, Amorim A (2007) MtDNA phylogeny and
evolution of laboratory mouse strains. Genome Res. 17: 293-298. IF: 10.139
9- Pereira L, Gonçalves J, Franco-Duarte R, Silva J, Rocha T, Arnold C, Richards M, Macaulay
V (2007) No evidence for an mtDNA role in sperm motility: data from complete sequencing
of asthenozoospermic males. Mol Biol Evol.. 24: 868-874. IF: 6.233
10- Pereira L, Gonçalves J, Bandelt H-J (2007) Mutation ‘C11994T’ in the mitochondrial ND4
gene is not a cause of low sperm motility in Portugal. Fertil Steril. (in press). IF: 3.114
Invited communications:
1- Pereira, L. (2006) mtDNA diversity in Portugal and in the Mediterranean basin. Division of
Environmental and Evolutionary Biology, University of Glasgow, UK. 27/04.
2- Pereira, L. (2006) mtDNA complete sequencing: refining inferences on past human
migrations and on clinical genetics. Laboratory of Genetics and Biometry, Department of
Anthropology and Ecology, University of Geneva, Switzerland. 18/10.
59
Public Health and Cancer
(Department of Hygiene and Epidemiology, Porto University Medical School – IPATIMUP)
Activity report of 2006
The Public Health group is a result of the cooperation between the Department of Hygiene
and Epidemiology from the Porto University Medical School (SHE-FMUP) and the IPATIMUP. It
aims the transfer of knowledge across different levels of scientific production – from bench side to
populations, and back – promoting the interaction between researchers with expertise on
molecular pathology, molecular and population genetics, and epidemiology.
The SHE-FMUP researchers are involved in this common project according to their
expertise and research interests. From March to December 2006, Nuno Lunet (Degree in
Pharmaceutical Sciences, Master in Public Health, PhD in Public Health), Bárbara Peleteiro
(Degree in Biochemistry, Master in Epidemiology), Joana Bastos (Degree in Mathematics), Marta
Pereira (Degree in Biochemistry), Cristina Pereira (Degree in Pharmaceutical Sciences), Raquel
Lucas (Degree in Pharmaceutical Sciences, Master in Epidemiology) and Milton Severo (Degree
in Mathematics, Master in Data Analysis and Decision Support Systems) were involved in this
cooperation. The publications and communications in the first ten months largely reflect the
research that has been developed by these elements at the SHE-FMUP.
• Ongoing common research projects:
- Characterization of gastric lesions associated with Helicobacter pylori infection in Viana do
Castelo shipyard workers (Fundação Calouste Gulbenkian).
- Gastric lesions in Moçambique and Portugal: the African Enigma (Fundação Calouste
Gulbenkian).
- Environmental exposures and CDX2 expression in Helicobacter pylori-positive gastric
cancer (POCTI/SAU-ESP/61685/2004).
- Risk of gastric cancer and its precursor lesions associated with salt consumption and
Helicobacter pylori infection (Agência Portuguesa de Segurança Alimentar).
• Research projects submitted to the FCT for funding:
- Genetic and non-genetic factors contributing for phenylketonuria phenotype diversity: a
study based on the Portuguese Neonatal Screening Program.
• Publications
Chapters in books:
1.
Lunet N, Barros H. Helicobacter pylori infection and gastric cancer in developing
countries: revisiting the “enigmas”. In: Ly A, Khayat D, editors. About cancer in Africa: from
epidemiology to biomedical applications and perspectives. Paris: INCa, 2006.
Papers in national periodicals with referees
60
1.
Severo M, Santos AC, Lopes C, Barros H. Fiabilidade e Validade dos
Conceitos Teóricos das Dimensões de Saúde Física e Mental da Versão Portuguesa do MOS
SF-36. Acta Med Port 2006; 19:281-8.
2.
Canhão H, Ferreira R, Costa L, Romeu JC, Fonseca JE, Branco J, Barros H.
[Normative data for quantitative ultrasound measurement of the calcaneus in a Portuguese
population]. Acta Reumatol Port 2006; 31(1):65-73.
3.
Vale MJ, Borges T, Alexandrino A, Gesta C, Casanova C, Lunet N, Paz Dias
C. Criança em risco: estudo multicêntrico. Nascer e Crescer 2006;XV(4):S255-61.
4.
Lunet N, Severo M, Barros H. Desvio padrão ou erro padrão?. Arq Med
2006 (in press).
5.
Barros H, Lunet N. Cancro: uma aproximação de Saúde Pública. Arq Med
2006 (in press).
6.
Pina F, Lunet N, Macedo Dias A. Carcinoma da próstata e envelhecimento:
aspectos preocupantes. Arq Med 2006 (in press).
Papers in international scientific periodicals with referees
1. Lunet N, Williams L, Govind M, Silinto A, Zucua I, Damasceno A, Barros H. Tobacco
advertising in Maputo, Mozambique: how will they keep pressing?. Gac Sanit 2006; 20(3):2512.
2. Xavier P, Beires J, Belo L, Rebelo I, Martinez-de-Oliveira J, Lunet N, Barros H. Serum
levels of VEGF and TNF-α and their association with C-reactive protein in patients with
endometriosis. Arch Gynecol Obstet 2006; 273(4):227-31.
3. Botelho F, Lunet N, Barros H. Coffee and gastric cancer: systematic review and metaanalysis. Cad Saude Publica 2006; 22(5):889-900.
4. Mesquita P, Almeida R, Lunet N, Reis CA, Santos Silva LF, Serpa J, van Seuningen I,
Barros H, David L. Metaplasia - a transdifferentiation process that facilitates cancer
development. The model of gastric intestinal metaplasia. Crit Rev Oncog 2006; 12(1-2):3-26.
5. Lunet N, Valbuena C, Carneiro F, Lopes C, Barros H. Antioxidant vitamins and risk of
gastric cancer: a case-control study in Portugal. Nutr Cancer 2006; 55(1):71-7.
6. Rocha O, Lunet N, Costa L, Barros H. [Osteoporosis treatment in Portugal: trends and
geographical variation]. Acta Med Port 2006; 19:373-80.
7. Mansilha A, Araújo F, Severo M, Sampaio SM, Toledo T, Albuquerque R. Combined Factor
V Leiden (R506Q) and prothrombin G20210A genotyping in young patients presenting with
deep venous thrombosis. Phlebology 2006; 21(1).
8. Severo M, Gama J. Change detection with Kalman filter and CUSUM. Lecture Notes In
Artificial Intelligence 2006; 4265:243-254.
Abstracts published in national periodicals
1. Lucas R, Azevedo A, Barros H. Factores associados à fadiga grave em adultos
portugueses. Acta Reum Port 2006; 31:61(sup).
2. Barcelos A, Santos RA, Costa L, Lucas R, Barros H. Prevalência de alterações
radiológicas e diferença na sintomatologia musculo-esquelética de acordo com o sexo em
adultos portugueses. Acta Reum Port 2006; 31:52(sup).
3. Santos RA, Costa L, Barcelos A, Lucas R, Barros H. Concordância entre os diagnósticos
clínico e radiológico de osteoartrose. Acta Reum Port 2006; 31:66(sup).
4. Costa L, Rocha O, Lunet N, Barros H. Tratamento da Osteoporose em Portugal: tendência
e variação geográfica. Acta Reum Port 2006; 31(Supl):131-2.
5. Severo M, Lucas R, Costa L, André R, Barros H. Algoritmo para o Rastreio da
Osteoartrose do Joelho, Coluna, Mão e Anca. Acta Reum Port 2006; nº3 Especial do XIII
Congresso Português de Reumatologia; 2006.
61
6. Severo M, Costa L, André R, Barros H; Determinantes de Osteopenia e Osteoporose em
Mulheres Pós-Menopaúsicas; Acta Reum Port 2006; 31:66(sup).
Abstracts published in international periodicals
Santos AC, Gaio R, Severo M, Lopes C, Barros H. No dietary pattern is specific for
1.
metabolic syndrome: A cross-sectional study within the EPIPorto cohort. Eur J Epidemiol 2006;
21(suppl):147.
2.
Lucas R, Barros H. Life prevalence and determinants of hormone replacement therapy
among Portuguese women. Pharmacoepidemiol Drug Saf 2006; 15:S66.
3.
Bastos J, Lucas R, Azevedo A, Barros H. Use of antidepressants in an urban sample of
Portuguese adults. Pharmacoepidemiol Drug Saf 2006; 15:S238.
4.
Azevedo A, Lucas R, Barros H. Prevalence, awareness, treatment and control of
hypertension in the Portuguese general population. Pharmacoepidemiol Drug Saf 2006;
15:S255.
5.
Pina F, Figueiredo G, Lunet N, Tomada N, Silva A, Cruz F, Barros H. Soluble receptor
of Human cytokine IL-6 (SIL-6R) on 123 patients with untreated prostate cancer (PCA). Eur
Urol Suppl 2006; 5(2):275.
6.
Pina F, Figueiredo G, Lunet N, Silva A, Silva P, Cruz F. Transforming growth factor
alpha (TGF-a) level is associated with both to serum androgen and primary androgen
insensibility prostate cancer (PCA) status. Eur Urol Suppl 2006; 5(2):278.
7.
Canedo P, Castanheira-Vale AJ, Lunet N, Pereira F, Figueiredo C, Barros H, Suriano G,
Carneiro F, Machado JC. Interleukin 8-251T/A polymorphism is associated with risk for gastric
carcinoma development in populations of Asian origin but not of Caucasian origin. Helicobacter
2006; 11:365.
8.
Peleteiro B, Lunet N, Figueiredo C, David L, Barros H. High-virulence H. pylori strains
increase risk of all intestinal metaplasias but smoking increases only complete type. Eur J
Epidemiol 2006; 21(s13):148.
9.
Pereira J, Lunet N, Carvalho D, Pinto M, Medina J; Are high-density lipoproteins
associated with a lower risk of periodontitis?. J Clin Periodontol 2006; 33(s7):152.
10.
Azevedo A, Lucas R, Barros H. Prevalence, awareness, treatment and control of
hypertension in the Portuguese general population. Eur J Epidemiol 2006; 21
(Supplement):131.
11.
Azevedo A, Lucas R, Barros H. Determinants of antihypertensive treatment in the
general population. Eur J Epidemiol 2006; 21 (Supplement):137.
12.
Lucas R, Azevedo A, Barros H. Gender and education explain a large fraction of the
variability in fatigue in adults. Eur J Epidemiol 2006; 21 (Supplement):146.
13.
Pereira F, Magalhães B, Canedo P, Peleteiro B, Lunet N, Barros H, Machado JC. Proinflammatory genetic polymorphisms and the risk of developing colorectal câncer. Gut 2006;
55(Suppl V):A185.
• Communications
International
1. Santos AC, Gaio R, Severo M, Lopes C, Barros H. No dietary pattern is specific for
metabolic syndrome: A cross-sectional study within the EPIPorto cohort. European Congress of
Epidemiology. Utrecht 2006.
2. Lucas R, Barros H. Life prevalence and determinants of hormone replacement therapy
among Portuguese women. 22nd International Conference on Pharmacoepidemiology &
Therapeutic Risk Management. Lisboa 2006.
62
3. Pina F, Figueiredo G, Lunet N, Silva A, Silva P, Cruz F. Transforming growth factor alpha
(TGF-a) level is associated with both to serum androgen and primary androgen insensibility
prostate cancer (PCA) status. 21st Annual EAU Congress. Paris 2006.
4. Pereira J, Lunet N, Carvalho D, Pinto M, Medina J. Are high-density lipoproteins associated
with a lower risk of periodontitis?. Europerio 5. Madrid 2006.
5. Peleteiro B, Lunet N, Figueiredo C, David L, Barros H. High-virulence H. pylori strains
increase risk of all intestinal metaplasias but smoking increases only complete type. European
Congress of Epidemiology. Utrecht 2006.
6. Matos C, Damasceno A, Lunet N, Diogo D, Prista A, Kahozi Sangwa P. Prevalence,
Awareness, Treatment and Control of Hypertension in Mozambique. 21st Scientific Meeting of
International Society of Hypertension. Fukuoka 2006.
7. Damasceno A, Matos C, Carrilho C, Duarte A, Lobo V, Lopes H, Madede T, Mihajlovic J,
Pravinrai P, Ismail M, Lunet N. The Epidemiology of Stroke in Maputo: Report of a Survey of
Hospitalized Events During 5 Months. 21st Scientific Meeting of International Society of
Hypertension. Fukuoka 2006.
8. Pereira F, Magalhães B, Canedo P, Peleteiro B, Lunet N, Barros H, Machado JC. Proinflammatory genetic polymorphisms and the risk of developing colorectal cancer. 14th United
European Gastroenterology Week "UEGW 2006". Berlin 2006.
9. Bastos J, Lucas R, Azevedo A, Barros H. Use of Antidepressants in an Urban Sample of
Portuguese Adults. 22nd International Conference on Pharmacoepidemiology and Therapeutic
Risk Management. Lisboa 2006.
10. Azevedo A, Lucas R, Barros H. Prevalence, awareness, treatment and control of
hypertension in the Portuguese general population. The IEA-EEF European Congress of
Epidemiology. Utrecht 2006.
11. Azevedo A, Lucas R, Barros H. Determinants of antihypertensive treatment in the general
population. The IEA-EEF European Congress of Epidemiology. Utrecht 2006.
12. Lucas R, Azevedo A, Barros H. Gender and education explain a large fraction of the
variability in fatigue in adults. The IEA-EEF European Congress of Epidemiology. Utrecht 2006.
National
1. Oliveira A, Severo M, Santos AC, Ramos E, Lopes C. Contributo dos Alimentos para a
Ingestão Nutricional da população adulta do Porto. V Congresso de Nutrição e Alimentação.
Porto 2006.
2. Lucas R, Azevedo A, Barros H. Factores associados à fadiga grave em adultos portugueses.
XIII Congresso Português de Reumatologia. Ponta Delgada 2006.
3. Lucas R. Epidemiologia da gonartrose. Congresso Porto século XXI: 2º Congresso de
artroscopia, cirurgia do joelho e traumatologia desportiva. Porto 2006.
4. Lucas R, Azevedo A, Severo M, Barros H. Propriedades psicométricas da escala de
gravidade da fadiga em adultos portugueses. XIII Congresso Português de Reumatologia. Ponta
Delgada 2006.
5. Lucas R, Cordoeiro N, Santos RA, Ramos I, Barros H. Concordância entre osteoartrose autodeclarada e diagnosticada. IV Congresso Português de Epidemiologia. Cascais 2006.
6. Lucas R, Pereira D, Santos RA, Ramos I, Barros H. Sintomatologia depressiva em adultos
com osteoartrose radiográfica e queixas musculo-esqueléticas. IV Congresso Português de
Epidemiologia. Cascais 2006.
7. Lucas R, Cardoso P, Santos RA, Ramos I, Barros H. Osteoartrose radiográfica e parâmetros
ultrassonográficos do calcâneo em adultos portugueses. IV Congresso Português de
Epidemiologia. Cascais 2006.
8. Costa L, Rocha O, Lunet N, Barros H. Tratamento da Osteoporose em Portugal: tendência e
variação geográfica. Congresso Português de Reumatologia. Ponta Delgada 2006.
9. Bastos J, Barros H, Lunet N. Evolução da mortalidade por cancro da mama em Portugal
(1955-2002). IV Congresso de Epidemiologia. Cascais 2006.
63
10. Pereira M, Lunet N, Azevedo A, Barros H. Revisão sistemática de estudos que avaliaram a
prevalência, conhecimento, tratamento e controlo da Hipertensão Arterial em diferentes regiões
do mundo. IV Congresso de Epidemiologia. Cascais 2006.
11. Martins E, Lunet N, Oliveira A, Azevedo A, Barros H. Prevalência, conhecimento e tratamento
da diabetes: estudo EpiPorto. IV Congresso de Epidemiologia. Cascais 2006.
12. Fonseca T, Lunet N, Damasceno A, Matos C, Barros H. Consumo de tabaco, álcool, frutas e
vegetais no Porto e em Maputo. IV Congresso de Epidemiologia. Cascais 2006.
13. Bastos J, Barros H, Lunet N. Meta-análise de estudos observacionais que quantificam a
associação entre a ocorrência de cancro gástrico e o consumo de frutas e vegetais: comparação
de três métodos. IV Congresso de Epidemiologia. Cascais 2006.
14. Lunet N, Cumaio F, Silva P, Dias E, Barros H. Avaliação do uso de medicamentos antimaláricos: impacto do desenho do questionário. IV Congresso de Epidemiologia. Cascais 2006.
15. Vale M, Soares S, Ramos E, Severo M, Borges T, Machado C. Obesidade em idade préescolar: uma realidade previsível?. 17º Congresso da Sociedade Europeia de Pediatria
Ambulatória e 9ª Reunião da Secção de Pediatria Ambulatória da SPP. Coimbra 2006.
16. Fraga S, Severo M, Sousa S, Ramos E, Barros E. Álcool e tabaco: representações sociais
dos adolescentes. IV Congresso de Epidemiologia. Cascais 2006.
17. Severo M, Lucas R, Costa L, André R, Barros H. Algoritmo para o Rastreio da Osteoartrose
do Joelho, Coluna, Mão e Anca. XIII Congresso Português de Reumatologia. Ponta Delgada
2006.
18. Severo M, Costa L, André R, Barros H. Determinantes de Osteopenia e Osteoporose em
Mulheres Pós-Menopaúsicas. XIII Congresso Português de Reumatologia. Ponta Delgada 2006.
19. Severo M, Santos AC, Lopes C, Barros H. Fiabilidade e Validade dos Conceitos Teóricos das
Dimensões Saúde Física e Mental da Versão Portuguesa do MOS SF-36. IV Congresso
Português de Epidemiologia. Cascais 2006.
20. Severo M, Lucas R, Costa L, André R, Branco J, Barros H. Avaliação dos conhecimentos
sobre doenças reumáticas numa amostra da população adulta Portuguesa. IV Congresso
Português de Epidemiologia. Cascais 2006.
21. Bastos J, Barros H. Evolução da mortalidade infantil em Portugal (1950-2004). IV Congresso
Português de Epidemiologia. Cascais 2006.
22. Bastos J, Rocha C. Análise estatística da sobrevivência de pacientes com melanoma maligno
da pele caso estudo de utilização do software R. XIV Congresso da Sociedade Portuguesa de
Estatística. Covilhã 2006.
23. Peleteiro B, Barros H, Lunet N. Prevalência de metaplasia intestinal em diferentes países:
revisão sistemática e análise ecológica. IV Congresso de Epidemiologia. Cascais 2006.
24. Pereira M, Azevedo A, Severo M, Barros H. Long-term stability of endogenous BNP during
storage at -20º C. Congresso Nacional de Bioquímica. Aveiro 2006.
64
Activity plans of 2007
• Maintenance and improvement of the cooperation with other IPATIMUP groups through the
ongoing research projects and establishment of new projects.
• Organization of the Porto Cancer Meeting 2007 (Cancer Etiology: bridging worlds).
• Organization of statistics courses open to the IPATIMUP researchers.
65
Relatório de actividades da Unidade de Educação Contínua e Difusão
Científica
A UNIDADE DE EDUCAÇÃO CONTÍNUA E DIFUSÃO CIENTÍFICA do IPATIMUP (UECDC-IPATIMUP)
desenvolveu durante o ano de 2005 um conjunto de iniciativas em diversos domínios da promoção do
pensamento e cultura científica:
A- Projectos
1- Projecto “Autolaboratório”
A UECDC do IPATIMUP iniciou a execução do projecto “AUTOLABORÁTORIO” (ref# CV / 138 / 2005)
financiado pela Ciência Viva. Mantendo os princípios da iniciativa o projecto sofreu uma profunda
reestruturação organizativa e funcional relativamente ao passado recente. Foi ainda efectuada uma total
remodelação das vertentes de imagem e dos conteúdos de forma a existir uma maior aproximação ao
público alvo e um real enquadramento das actividades com as orientações curriculares do ministério da
educação.
Apesar do início conturbado do ano lectivo nas escolas foram já efectuadas sessões para diferentes
níveis de ensino envolvendo 520 alunos em estabelecimentos de ensino da região norte.
Monitora: Carolina Ferraz.
2- Projecto “A Magia da Ciência”
A UECDC-IPATIMUP deu continuidade ao projecto “A magia da Ciência – POCTI/DIV/2005/00061”
financiado pela FCT – Programa POCTI do III Quadro comunitário de apoio - Medida 3.1. - “Atribuição
de Financiamento para projectos de divulgação da cultura científica e tecnológica”. Foi produzido um
conjunto de DVDs com sessões dos cursos de “Medicina Preventiva” e “Introdução à Medicina
Molecular” que será um elemento fundamental para a aprendizagem dos conceitos e dos métodos
experimentais da biologia molecular a nível do ensino secundário. Em simultâneo encontra-se em
desenvolvimento um software – “MicE – Microscópio Educativo” que possibilitará às escolas uma
abordagem virtual à biologia celular.
3- Projecto “Despertar para a Ciência”
Foi aprovada pela Agência Ciência Viva, a candidatura do projecto “Despertar para a Ciência” que a
UECDC-IPATIMUP em articulação com a Câmara Municipal da Trofa, preparou e submeteu em
Dezembro de 2005. O projecto “Despertar para a Ciência” iniciar-se-á durante o ano de 2007 e tem
como objectivo implantar nos diversos níveis de ensino da comunidade educativa da Trofa, um modelo
pedagógico baseado na prática sustentada e consequente de uma abordagem experimental da ciência
que contribuirá decisivamente para uma qualificação do capital humano e progresso do concelho da
Trofa.
4- Projecto “EEA – Grants”
A UECDC-IPATIMUP participou na preparação da proposta de divulgação científica, da candidatura do
projecto “EEA - Grants” ao concurso público para apresentação de propostas de projectos individuais no
âmbito do Mecanismo Financeiro do Espaço Económico Europeu em Portugal.
4- Projecto “Laboratório Aberto”
Projecto entre a Câmara Municipal do Porto, Ciência Viva e o IPATIMUP com o objectivo de criar um
centro de Ciência Viva. Este projecto foi aprovado em Outubro de 2006 e terá início em Junho de 2007;
o Laboratório situar-se-á nas proximidades do IPATIMUP.
B- Programas
1- Programa “Ciência Viva em Férias”
A UECDC-IPATIMUP promoveu durante as seis quinzenas das Férias de Verão o programa “Ciência
Viva em Férias” para 22 alunos do Ensino Secundário, que participaram em estágios de iniciação
científica, acompanhando activamente e com tarefas distribuídas, o trabalho dos investigadores do
IPATIMUP.
66
2- Programa “Porto de Crianças”
No ano lectivo de 2005/2006, o IPATIMUP renovou o protocolo com a Câmara Municipal do Porto –
Pelouro da Educação, de modo a levar, em sessões mensais, o ensino experimental aos alunos do
primeiro ciclo. Foram contemplados cerca de 90 alunos.
Neste âmbito, a UECDC-IPATIMUP, com o apoio do Pelouro da Educação da Câmara Municipal do
Porto, estabeleceu um protocolo de cooperação com o Centro de Formação de Professores da Escola
Secundária António Nobre. A formação Decorreu entre Janeiro a Maio de 2006.
3- Escola das Ciências da Vida e da Saúde – Universidade Júnior
A UECDC-IPATIMUP participou na iniciativa “Escola das Ciências da Vida e da Saúde”, integrada no
programa “Universidade Júnior” promovido pela Universidade do Porto de 4 a 8 de Setembro. Os 5
alunos
participantes,
desenvolveram
2
projectos,
um
no
âmbito
da
genética
("Genómica comparativa de diversas espécies de mamíferos através do estudo de
sequencias codificantes e não codificantes do DNA") e outro no âmbito da biologia celular ("Efeitos
celulares de mutações inactivantes em genes supressores tumorais").
C- Conferências, Colóquios e Palestras
1. Curso livre sobre - Medicina Molecular e Cancro
A UECDC-IPATIMUP em colaboração com a Associação de Estudantes da Faculdade de Medicina da
Universidade do Porto, organizou o “Curso Livre sobre Medicina Molecular e Cancro” constituído por 5
sessões (20h) que decorreram durante o mês de Outubro de 2005 nos auditórios do IPATIMUP, Museu
de Serralves e Faculdade de Medicina do Porto.
2. Colóquios sobre - A Medicina Preventiva Do Cancro - 2006
A UECDC-IPATIMUP em colaboração com a Fundação Calouste Gulbenkian e a Fundação de
Serralves organizou, a 18 e 31 de Outubro na Fundação Calouste Gulbenkian e a 29 de Novembro e 6
de Dezembro na Fundação de Serralves, o terceiro Ciclo de Colóquios (2006) sobre Medicina e Cancro,
este ano subordinado ao tema “Multifactoriedade genética e ambiental no cancro familiar: implicações
na prevenção e no diagnóstico precoce”.
3- X Conferencia do Equinócio
A UECDC-IPATIMUP organizou a X conferência do Equinócio intitulada “Público e privado”, realizada a
9 de Outubro de 2006, com a coordenação da Dr. Jorge Sampaio e os conferencistas José Pacheco
Pereira e Artur Santos Silva.
4. Palestras
A UECDC-IPATIMUP promoveu durante o ano de 2006, a realização de palestras sobre temas como a
Biologia, a Genética, o Cancro, em várias escolas secundárias (Esc. Sec. Lourinhã – Maio 2006; Esc.
Sec. Gondomar – Novembro de 2006; Escola Sec. Cinfães – Novembro de 2006).
D- Actividades de Divulgação Científica
1. 4ª Mostra de Ciência, Ensino e Inovação da Universidade do Porto
A UECDC-IPATIMUP participou na “4ª Mostra de Ciência, Ensino e Inovação da Universidade do Porto”,
promovida pela Universidade do Porto, que decorreu de 16 a 19 de Março de 2006, no Pavilhão Rosa
Mota – Porto. O stand fez a divulgação das diversas vertentes de actividade do IPATIMUP
(Investigação, Formação, Diagnóstico e Divulgação), proporcionando ainda um conjunto de
experiências sobre os temas a célula e o ADN.
2. “Semana da Ciência e da Tecnologia”
A UECDC-IPATIMUP na “Semana da Ciência e da Tecnologia”, de 20 a 24 de Novembro, o IPATIMUP
aderiu ao programa “Portas Abertas” do Ciência Viva, na qual os alunos que frequentaram o “Ciência
Viva em Férias – edição - 2006” puderam trazer amigos para visitar as instalações, bem como
esclarecer dúvidas com os investigadores.
67
3. “Dia do IPATIMUP”
No Dia do IPATIMUP (16 de Fevereiro) foram convidadas 3 escolas: Escola Secundária de Rio Tinto,
Escola Secundária de Aveiro e Escola Secundária de Aguiar da Beira, com 30 alunos de cada escola.
E- Participação em iniciativas de Divulgação Científica
A UECDC-IPATIMUP participou a 16 de Dezembro de 2006, no simpósio " Tecnologias de Informação e
Comunicação
(TIC)
e
o
ensino
de
ciências:
diálogos
entre
as
literacias
digital e científica”, do “International Postgraduation Program – Life and Health Sciences” organizado,
pela Escola de Ciências da Saúde da Universidade do Minho.
ÁREA: PROJECTOS/MULTIMÉDIA
A área de projectos/multimédia da UNIDADE DE EDUCAÇÃO CONTÍNUA E DIFUSÃO CIENTÍFICA do
IPATIMUP (UECDC-IPATIMUP) desenvolveu durante o ano de 2006 um conjunto de actividades em
diversos domínios da divulgação da cultura científica e tecnológica.
A UECDC-IPATIMUP foi responsável pela implementação de diversos projectos e pela divulgação
multimédia (web / podcasts) de iniciativas promovidas pelo IPATIMUP. Esta estratégia de divulgação
teve muito sucesso como demonstram os dados relativos à página da Conferência do Equinócio
(www.ipatimup-uecdc.com/equinocio2006) com 1.125 visitas e a presença dos podcasts nos destaques
e tops de downloads do iTunes.
Responsável: Filipe Santos Silva
Equipa:
Jorge Oliveira (Tecnologias da Comunicação)
Rui Oliveira (Design gráfico)
Nuno Ribeiro (Programação multimédia e produção de conteúdos)
José Rui Fernandes (coordenador/web design)
68
Unidade de Prestação de Serviços (UPS)
Relatório de Actividades 2006
Introdução
O principal objectivo da Unidade de Prestação de Serviços (UPS) em 2006 foi o de manter o Sistema de Gestão de
Qualidade definido desde 2003, de acordo com as normas propostas pelo CAP e realizar a nossa primeira auditoria
interna antes da segunda inspecção pelos inspectores da CAP prevista para 2007. O número de exames foi de
13414 (menos 257 exames do que 2005). Esta diminuição numérica deve-se ao facto de não registarmos todos os
testes de proficiência com números de rotina em 2006 e cessação do protocolo de citologias cérvico-vaginais com o
Dr. Paulo Figueiredo em Novembro de 2006. Como vem sucedendo há vários anos, continuámos a actuar como um
centro de formação profissional pós-graduado, tendo recebido em 2006, 5 patologistas e 7 técnicos. Cinco destes
técnicos estagiaram por um período de 10 semanas cada dentro de protocolo estabelecido com a CESPU e ESTESP.
1.
Recrutamento de pessoal:
•
Não houve alterações no quadro de pessoal.
2. Aquisição de Equipamento e Testes de Proficiência::
Com a finalidade de equipar o laboratório de rotina diagnostica, no âmbito do Programa de Acreditação da Unidade
via CAP, foram realizados os seguintes gastos com equipamentos e testes de proficiência do CAP
Aparelho/Empresa – Modelo
Armário Labor Security System A-100
Microtomo rotatório HM325 e acessórios
Processador Automático de Tecidos STP120-3
Banho Maria de Extensão
Autostainer 360-2D Imuno-histoquímica
Testes de Proficiência do Colégio Americano de
Patologistas
Preço c/ IVA
1.452,00 Euros
8.224,37 Euros
17.720,45 Euros
1.150,00 Euros
38.115,00 Euros
3.984,00 Euros
Adquiridos integralmente com recursos da Unidade.
3. Estágios de internos:
Estágios
Nome
Alesso Sartoreli, Interno de Anatomia
Patológica, Faculdade de Medicina de
Botucatu, UNESP, São Paulo, Brasil
Lidiane Martins, Interna de Anatomia
Patológica, Faculdade de Medicina de
Botucatu, UNESP, São Paulo, Brasil
Marina
Debrot, Interna de Anatomia
Patológica da Faculdade de Medicina da
UFMG, Belo Horizonte, MG, Brasil
Melissa Vita, Interna de Anatomia
Patológica, Instituto Nacional do Câncer do
Rio de Janeiro, Brasil.
Período
04/09/2006 a
27/10/2006
01/06/2006 a
21/07/2006
15/02/2006 a
7/04/2006
03/04/2006 a
31/05/2006
Tipo de Estágio
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Patologia Cirúrgica e
Citopatologia
Punção Aspirativa
Situação Actual
Concluído
Concluído
Concluído
Concluído
69
Rita de Cássia Alencar, Hospital Araújo
Jorge, Goiânia, GO, Brasil
01/10/2006 a
31/10/2006
Imunocitoquímica
Concluído
4. Publicações com material da U.P.S.
Reis-Filho JS, Steele D, Di Palma S, Jones RL, Savage K, James M, Milanezi F, Schmitt FC, Ashworth A.
Distribution and significance of nerve growth factor receptor (NGFR/p75) in normal, benign and malignant breast
tissue. Modern Pathol 19: 307-319, 2006.
Gouvea AP, Milanezi F, Olsen SJ, Leitão D, Schmitt FC, Gobbi H. Selecting antibodies to detect HER2
overexpression by immunohistochemistry in invasive mammary carcinomas. Appl Immunohistochem Mol
Morphol 14: 103-108, 2006.
Moreira MAR, Longatto-Filho A, Taromaru E, Queiroz G, Jube LF, Pinto SA, Schmitt FC. Investigation of human
papillomavirus by hybrid capture II in cervical carcinomas including 113 adenocarcinomas and related lesions. Int
J Gynecol Cancer 16: 586-590, 2006.
Reis-Filho JS, Milanezi F, Steele D, Savage K, Simpson PT, Nesland JM, Pereira EM, Lakhani SR, Schmitt FC.
Metaplastic breast carcinomas are basal-like tumours. Histopathology 49: 10-21, 2006.
Utagawa ML, Loreto C, Freitas C, Milanezi F, Longatto-Filho A, Pereira SM, Maeda MY, Schmitt FC. Pêro Vaz
de Caminha. An interchange program for quality control between Brazil and Portugal. Acta Cytol 50: 303-308,
2006.
Schmitt FC. Thyroid cytology: FNA is still the best diagnostic approach. Cytopathology 17: 210-216, 2006.
Reis-Filho JS, Pinheiro C, Lambros MBK, Milanezi F, Carvalho S, Savage K, Simpson PT, Jones C, Swift S,
Mackay A, Reis RM, Hormick JL, Pereira EM, Baltazar F, Fletcher CDM, Ashworth A, Lakhani SR, Schmitt FC.
EGFR amplification and lack of activating mutations in metaplastic breast carcinomas. J Pathol 209: 445-453,
2006.
Kocjan G, Feichter G, Hagmar B, Kapila K, Kardum-Skelin I, Kloboves V, Kobayashi TK, Koutselini H, Majak B,
Schenck U, Schmitt F, Tani E, Totch M, Onal B, Vass L, Vielh P, Weynand B, Herbert A. Fine needle aspiration
cytology: a survey of current European practice. Cytopathology 17: 219-226, 2006.
Paredes J, Lopes N, Milanezi F, Schmitt FC. P-cadherin and cytokeratin 5: useful adjunct markers to distinguish
basal-like ductal carcinomas in situ. Virchows Arch 2006: DOI 10.1007/s00428-006-0334-y
Ricardo S, Milanezi F, Carvalho S, Leitão D, Schmitt F. HER2 evaluation through the novel rabbit monoclonal
antibody SP3 and CISH in tissue microarrays of invasive breast carcinoma. J Clin Pathol 2006: DOI 10.1136
5.
Exames realizados na U.P.S.
Nº total de exames: 13.413
Captura Híbrida: 65
Citologias ginecológicas: 8.780
Citologias não ginecológicas: 60
Citologias Aspirativas: 1.987
Histológicos: 1.859 (620 Autópsias)
Histoquímicos: 80
Imuno-histoquímicos: 226 (inclui hepáticas com imuno)
Hibridização in situ: (Projecto ROCHE): 120
Imunofluorescência Directa: 14
Relatório Complementar: 29
Exames de proficiência da CAP
70
CAP-MK: 4
CAP-NGC: 10
CAP-PAPM: 5
CAP-PIP: 10
Casos em consulta*: 164
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Os casos em consulta foram oriundos das seguintes instituições:
A. Verhest - Institut Jules Bordet – Bruxelas – Bélgica
Afaf M. Elhag - The Laboratory Mafrag Hospital - Abu Dhabi - U.A.E.
Agostinho Sanches - Hospital Senhora de Oliveira Guimarães - Portugal
Ailton Cabral Fraga Júnior – Goiana – Brasil
Albino Oliveira - Lab. Anatomia Patológica Dr. Albino Oliveira, Lda. - Portugal
Albino Verçosa Magalhães – Brasília – Brasil
Ana Carvalho – ANPAT – Lisboa - Portugal
Ana Filipa Capelinha - Centro Hospitalar do Funchal – Madeira – Portugal
Ana Maria Duarte - Hospital de Matosinhos - Portugal
Ana Paula F. Pesinato – IDAP - Florinoples – Brasil
Ana Paula Farina – UFSC – Florianópolis - Brasil
Andrea Schultheis - Kaiserin Elisabeth Spital – Viena - Austria
Anne Couvelard - Hopital Beaujon – Clichy – França
Anne Hoorens - Academis Hospital - Free University of Brussels – Bélgica
B. Iglesias - Centro Médico POVIS - Vigo (Pontevedra) - Espanha
Beatriz Elizaguirre - Hospital de Galdakao - Espanha
Carla Carrilho – Hospital Central de Maputo - Moçambique
Célia Fazzio - Laboratório Patologia - Hospital Base - São Paulo - Brasil
Celso Ruben - Hospital Aliança – Salvador – Bahia – Brasil
Christophe Girardet - Institut Central des Hôpitaux Valaisans – Sion - Suiça
Daniela Aldovi - Ospedale S. Chiara – Trento – Itália
Décio Fausto Gorini - Histolab -Lab. Anatomia Patológica e Citopatologia – Brasília - Brasil
Dores Pombinho - Hospital Santo António – Porto - Portugal
Dott Daniela Aldovini - Ospedale S. Chiara –Trento - Itália
Élbio C. de Paula – GOIAS - Brasil
Espregueira Mendes – Hospital São Sebastião – Santa Maria da Feira – Portugal
Eva Sigstad - Norwegian Radium Hospital – Oslo - Noruega
Fernando Pardal – Hospital S. Marcos – Braga – Portugal
Gustavo Sales Barbosa - Caruaru – PE - Brasil
Hanifa Bouzourene - Centre Hospitalier Universitaire Vaudois – Lausanne - Suiça
Hélcio Miziara – CIAP – Brasília - Brasil
Hélène Trouette - CHU - Hôpitaux de Bordeaux - França
Hospital Santa Maria – Lisboa - Portugal
Isabel Amendoeira – Hospital S. João - Portugal
Jean Louis Dargent - Institute Jules Bordet - Bélgica
Jean Pialat - Cabinet d'Anatomie et Cytologie Pathologiques – Limonest - França
José Luiz Henriquez - Hospital Distrital de Santarém - Portugal
José Maria Rivera Pomar - Hospital de Cruces - Espanha
Juliano Carvalho Freitas - Hospital Portugês – Salvador – Bahia - Brasil
Leonora Vianna - Hospital Universitário - Brasília - Brasil
Linda Giannikaki - University Hospital of Crete - Crete - Grécia
Luiz Carlos Takita - Campo Grande – MS - Brasil
M. Akif Demir - Celal Bayar University - School of Medicine – Manisa - Turquia
M. Manichal - Academisch Ziekenhuis V. U.B. – Bruxelas - Bélgica
M. Pruszczynski - Radboud University Nijmegen Medical Center - Nijmegen – Holanda
Maísa Quintal - Instituto de Anatomia Patológica - São Paulo - Brasil
Maja Jerse - Faculdade de Medicina - Eslovénia
Marcello Franco - UNIFESP/EPM - São Paulo - Brasil
Maria Jeis Arias - Hospital Garcia da Orta, S.A. Almada – Portugal
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Maria José Brito - Hospital Garcia da Orta, S.A. Almada - Portugal
Maria Teresa Dias Carvalho - Hospital São Teotónio – Viseu - Portugal
Mary Toner - University Teaching Hospit. Trinity College Dublin - Irlanda
Melissa Vita - INCA - Instituto Nacional de Câncer – Rio de Janeiro – Brasil
Odete Real – IPO – Coimbra - Portugal
P. Anani - Laboratoire Argot Lab – Lausanne - Suiça
Palmira Lima – Porto - Portugal
Paola Souza - Souza Anatomia Patológica - Brasil
Patrícia Sabino de Matos - FCM/ Unicamp – Campinas - Brasil
Paula Guerra - Hospital SAMS - Portugal
Pinar Firat – Turquia
Ricardo Fonseca – IPO – Lisboa – Portugal
Roderik Simpson - Royal Devon and Exeter Healthcare - NHS Trust - UK
Rogério de Almeida Ribeiro – Brasília - Brasil
Ronald de Krijger - Erasmus MC - Holanda
Ronchin Georges - Universidade de Bruxelas - Bélgica
Rosemary Nascimento - Rio de Janeiro - Brasil
Salete Silva - Hospital Fernando Fonseca – Amadora - Portugal
San Miguel - Centro Médico POVI - Vigo - Espanha
Santiago Ramón Cajal - Hospital Vall d’Hebron – Barcelona - Portugal
Sigurd Lax - Landeskrankenhaus Graz West – Graz - Austria
Silene Barreto Roters – Salvador – Bahia - Brasil
Simona Stolnico – Roménia
Sofia Loureiro Santos - Hospital Garcia da Orta, S.A. Almada - Portugal
Suna Erkilic - Gaziantep University (School of Medicine) - Turkey
Th. Rüdiger - Pathologisches Institut der Universität Würzburg – Alemanha
Thais Mavad - Faculdade de Medicina USP - São Paulo - Brasil
Tiemo Katzenberger - Pathologisches Institut der Universität Würzburg – Alemanha
Umbelina Ramos - Maternidade Júlio Dinis - Porto - Portugal
Umit Ince - Oruc Patoloji ve Sitoloji Lab. - Turquia
Walter Seelentag – Lausanne - Suiça
Yesim Ertan - University of Ege - Faculty of Medicine - Turquia
6.
Controle de Qualidade
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Data de Instituição 19-01-1998
Membros
Prof. Fernando Schmitt
Drª Fernanda Milanezi
D. Susana Silva
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Nº de Casos Revistos em 2005 – 311 casos de patologia cirúrgica e citopatologia (não ginecológica)
Nº de Casos Revistos em 2005 de citologias ginecológicas: 2.123
•
Principais Conclusões no Final do 9º Ano:
Este foi o primeiro ano em que todo o Sistema de Controle de Qualidade foi realizado através do sistema
informático Sislab, cujos resultados estão publicados no Manual da Qualidade da UPS-2005. Os principais achados
comparativamente a 2004 foram:
Total nº de casos
Casos Revistos
2004
12.755
302
2005
13.573
311
2006
13.413
573
Tipo de Exames
Citologia aspirativa por agulha fina
Citologia não ginecológica
2004
121
6
2005
164
6
2006
375
18
72
Histológico de biópsias
Histológico de Peças cirúrgicas
Biópsia hepatica sem imuno-histoquímica
Biópsia hepatica com imuno-histoquímica
Biópsia gástrica com pesquisa de H. Pylori
Consultas
Citologia ginecológica a)
Imunofluorescência
Relatório Complementar/de Revisão
27
68
4
11
3
3
54a)
24
91
2
11
9
3
N/A
1
33
97
7
25
11
2
N/A
1
4
a)
Desde Abril de 2004 os casos de citologia ginecológica foram avaliados separadamente de acordo com o
procedimento PR.MED.01
Valores de discordância:
Critério
Identificação do espécime, arquivo e macroscopia
Diagnóstico
Codificação
2004
2.1%
0.4%b)
0.9%
2005
2.6%
0.6%c)
0.6%
2006
1.7%
0.0%
0.2%
“Turn-around-time”
2004
2.8 dias
4.5 dias
4.9 dias
9.5 dias
3.2 dias
7.2 dias
6.9 dias
0.3 dias
14.7 dias
Citologia aspirativa por agulha fina
Citologia não ginecológica
Histológico de biópsias
Histológico de Peças cirúrgicas
Biópsia hepatica sem imuno-histoquímica
Biópsia hepatica com imuno-histoquímica
Biópsia gástrica com pesquisa de H. Pylori
Consultas
Citologia ginecológica a)
2005
1.8 dias
4.7 dias
2.4 dias
3.2 dias
5.0 dias
5.2 dias
1.9 dias
N/A
2.4 dias
2006
0.8 dias
1.7 dias
1.3 dias
1.7 dias
3.8 dias
4.6 dias
1.5 dias
N/A
N/A
a)
(Janeiro a Março)
Na análise do controle de qualidade de citologia ginecológica, os principais resultados foram os seguintes:
Total de casos
Casos revistos
Concordância entre
patologistas
e
citotécnico
Discordância entre
patologista
e
citotécnico
2004
5920
1404
2005
8952
2123
2006
8.780
1970
2004
1301
(92.6%)
2005
1962
(92.4%)
2006
1758
(89.2%)
103
(7.4%)
161
(7.5%)
214
(10.8%)
O número total de citologias consideradas não satisfatórias para a análise foi de 1.4% (3.5% em 2005), o que
representa uma diminuição conforme um dos objectivos propostos no último ano.
Ascus foi diagnosticado em 2.67% dos casos com uma redução da relação ASCUS/Lesão para 1.4 (4.1 em 2005).
Para além da participação com aprovação em todos os testes de proficiência da CAP, continuamos a participar de
forma positiva nos programas de controlo de qualidade externo das técnicas de imuno-histoquímica do UKNEQAS e do Programa de Qualidade da Sociedade Brasileira de Patologia (PIQ). Todas estas actividades estão
registadas de acordo com o Procedimento Regulamentador PR MED-05 – Programas Externos de Educação e
Avaliação Contínua.
73
Unidade de Prestação de Serviços (UPSI)
Relatório de Actividades 2006
1. Exames realizados
1.1
131
0
20
Nº de exames requeridos
Pedidos cancelados
Em curso (ainda por efectuar ou por falta de 1 ou mais colheitas)
1.2
Tipos de exame:
Caracterizações/Identificações genéticas
3
Paternidades
1.3
Com 1 pretenso pai, sem análise da mãe
Com 2 filhos
Com 3 filhos
Com 1 pretenso pai e com análise da mãe
Com 2 filhos
Com 2 pretensos pais e com análise da mãe
Com 3 pretensos pais e com análise da mãe
Maternidades
Com 2 pretensas mães, sem análise do pai
14
1
1
97
1
5
1
Outros parentescos
Comparação de perfis genéticos (amostra/indivíduo)
6
1
1
Locais de requisição:
Local
Angra do Heroísmo
Arouca
Aveiro
Braga
Bragança
Faro
Gondomar
Lisboa
Macedo de Cavaleiros
Matosinhos
Oliveira de Azeméis
Penafiel
Ponta Delgada
Porto
Póvoa de Varzim
Santa Maria da Feira
Santarém
Santo Tirso
São João da Madeira
Viana do Castelo
Vila do Conde
Vila Nova de Famalicão
Vila Nova de Gaia
Vila Real
Total
Tribunais
0
3
0
0
3
0
1
0
3
1
0
4
0
35
1
1
0
2
1
0
5
0
27
0
87 (66,4%)
Particulares
1
0
1
3
1
1
0
16
0
0
1
0
2
9
1
2
1
0
0
1
0
2
1
1
44 (33,6%)
Total
1
3
1
3
4
1
1
16
3
1
1
4
2
44
2
3
1
2
1
1
5
2
28
1
131
74
1.4
Tipos de requerentes
Tribunais
Averiguações Oficiosas de Paternidade/Maternidade
Outras
Particulares
1.5
80
7
44
Nº exames por requerente
Clientes
Tribunais:
Trib. Família e Menores do Porto
Varas Cíveis do Porto
Trib. Família e Menores de Vila Nova de Gaia
Trib. Santo Tirso
Trib. Penafiel
Trib. Bragança
Trib. Vila do Conde
Trib. Arouca
Trib. Macedo de Cavaleiros
Trib. Póvoa de Varzim
Trib. Matosinhos
Trib. Gondomar
Trib. São João da Madeira
Trib. Santa Maria da Feira
Particulares
Clínica Dr. Joaquim Chaves - Lisboa
Internos (IPATIMUP)
Outros
Total
Total
34
1
27
2
4
3
5
3
3
1
1
1
1
1
16
2
26
131
2. Publicações
2.1
Artigos em Proceedings
Amorim A, Alves C, Gusmão L, Pereira L (2006): Extended Northern Portuguese database on 21
autosomal STRs used in genetic identification. Progress in Forensic Genetics 11: 364-366.
Alves C, Gusmão L, Meirinhos J, Amorim A (2006): Making the most of Y-STR haplotypes. The HapYDive.
Progress in Forensic Genetics 11: 201-203.
Alves C, Coelho M, Rocha J, Amorim A (2006): The Amelogenin locus displays a high frequency of X
homologue failures in São Tomé island (West Africa). Progress in Forensic Genetics 11: 271-273.
Soares PA, Pereira F, Brion M, Alves C, Carracedo A, Amorim A, Gusmão L (2006): Relative Y-STR
mutation rates from the variance inside SNP defined lineages. Progress in Forensic Genetics 11: 82-84.
Gusmão L, Sánchez-Diz P, Gomes I, Alves C, Carracedo A, Prata MJ, Amorim A (2006): Genetic analysis
of autosomal and Y-specific STRs in the Karimojong population from Uganda. Progress in Forensic Genetics 11:
213-215.
2.2
Revistas Internacionais
Frigi S, Pereira F, Pereira L, Yacoubi B, Gusmão L, Alves C, Khodjet el Khil H, Cherni L, Amorim A, El
Gaaied A (2006): Data for Y-chromosome haplotypes defined by 17 STRs (AmpFLSTR® Yfiler™) in two Tunisian
Berber communities. Forensic Sci Int 160: 80-83.
Beleza S, Gusmão L, Lopes A, Alves C, Gomes I, Giouzeli M, Calafell F, Carracedo A, Amorim A (2006):
Micro-phylogeographic and demographic history of Portuguese male lineages. Ann Hum Genet. 70(Pt 2): 181-94.
Trovoada MJ, Tavares L, Gusmão L, Alves C, Abade A, Amorim A, Prata MJ (2007): Dissecting the Genetic
History of Sao Tome e Principe: A New Window from Y-Chromosome Biallelic Markers. Ann Hum Genet. 71: 77-85.
Alves C, Gomes V, Prata MJ, Amorim A, Gusmão L: Population data for Y-chromosome haplotypes defined
by 17 STRs (AmpFlSTR YFiler) in Portugal. Forensic Sci Int (2007), doi:10.1016/j.forsciint.2006.10.022.
75
3. Participação em Congressos/Comunicações
3.1
Gusmão L, Alves C, Maxzud K, Gomes I, Amorim A: Análise populacional de 10 X-STRs em 3 populações
africanas. XI Jornadas de Genética Forense, 1 e 2 de Junho de 2006, Madrid, Espanha.
3.2
Gomes I, Alves C, Maxzud K, Pereira R, Prata MJ, Sánchez-Diz P, Carracedo A, Amorim A Gusmão L:
Analysis of 10 X-STRs in three African Populations. DNA in Forensics 2006, 28 a 30 de Setembro de 2006,
Innsbruck, Austria.
3.3
8th STR Promega WG Meeting, Porto, Portugal, 24 a 26 de Outubro de 2006. Participação de Cíntia Alves
e Leonor Gusmão.
3.4
Seminário sobre Bases de Dados de Perfis de ADN com Fins Forenses, Coimbra, Portugal, 27 de Outubro
de 2006. Participação da Unidade a convite da Comissão nomeada para elaborar proposta de base de dados
genéticos com fins forenses em Portugal.
4. Actividades/Outros
4.1
Participação no Exercício de Controlo de Qualidade de 2006 (Paternidade e Forense) do Grupo Espanhol e
Português da International Society for Forensic Genetics, GEP-ISFG (Certificados em anexo).
4.2
Formação de Cíntia Alves em Acreditação de Laboratórios e a NP EN ISO/IEC 17025 no ITQB em Oeiras,
de 31 de Janeiro a 3 de Fevereiro 2006, com duração de 28 horas, organizado pela Specanalítica e pela UBiA.
4.3
Organização e realização de seminários no ABZ da Genética Humana (SPGH) – Identificação Genética, no
IPATIMUP a 31 de Março e 1 de Abril de 2006.
4.4
Aula “A genética em identificação humana” (Cíntia Alves) ao Curso de Mestrado em Ciências do Desporto
no FCDEF, Porto, a 10 de Julho de 2006.
76
Unidade de Prestação de Serviços de Susceptibilidade Genética (UPSS)
Relatório de Actividades 2006
Resumo
O objectivo fundamental da UPSS para o ano 2006 continuou a ser a consolidação da sua
actividade como prestador de serviços. Este objectivo foi conseguido através:
1. Do aumento do número de testes de diagnóstico genético realizados em áreas que
consideramos chave para a unidade, como a gastrenterologia, oncologia e
cardiovascular;
2. Do estabelecimento e implementação de novos testes.
Para 2007 a UPSS estabeleceu como principais objectivos:
3. Aumentar o número de testes de diagnóstico genético realizados em áreas que
consideramos chave para a unidade, como a gastrenterologia, oncologia e
cardiovascular;
4. Dar continuidade ao programa de estabelecimento e implementação de novos testes.
Em 2007 iniciaremos a nossa actividade na área da hipertensão;
5. Implementar um programa de acreditação e certificação da unidade.
Publicações com material da UPSS
1. Oliveira C, Velho S, Moutinho C, Ferreira A, Preto A, Domingo E, Capelinha AF, Duval
A, Hamelin R, Machado JC, Schwartz S Jr, Carneiro F, Seruca R. KRAS and BRAF
oncogenic mutations in MSS colorectal carcinoma progression. Oncogene 26: 158-63,
2007.
2. Roviello F, Corso G, Pedrazzani C, Marrelli D, De Falco G, Berardi A, Garosi L, Suriano
G, Vindigni C, De Stefano A, Leoncini L, Seruca R, Pinto E. Hereditary diffuse gastric
cancer and E-cadherin: Description of the first germline mutation in an Italian family.
Eur J Surg Oncol. 2006 Nov 23; [Epub ahead of print]
Exames realizados na UPSS
Exame
alfa1-Antitripsina
BRCA
caderina - E
CARD15/NOD2
H. Pylori
hMLH1 e hMSH2
HPV
MSI
N-myc/1p
OTC
P53
polimorfismos pró-inflamatórios
Prader-Willi/Angelman
RET
Nº de testes realizados
2005
2006
95
132
15
13
5
12
3
1
2
7
1
1
6
3
19
22
3
3
5
15
1
1
1
0
64
100
4
12
77
Translocações
UBE3A
X-Frágil
Braf
C-KIT/PDGFRA
EGFR
Estenose Supravalvular
Marfan
Miocardiopatia Dilatada
Miocardiopatia hipertrófica
Síndrome QT-Longo
SMAD4
Tipagem de HLA
Síndrome de Brugada
Total
8
10
3
0
0
0
0
0
0
0
0
0
0
0
245
2
9
4
8
5
20
1
4
4
14
2
1
3
13
412
As figuras 1 e 2 representam a distribuição e o número total, respectivamente, de exames
realizados na UPSS por área médica. O facto de maior relevo é o claro aumento da actividade
da UPSS, independentemente da área considerada. Ainda assim, a área cardiovascular
2006
25%
9%
66%
Oncologia
Cardiovasculares
Def. Enzimáticas / D. metabólicas /
Outras
Fig. 1: Distribuição dos exames realizados pela UPSS por área médica.
78
UPSS 2005 vs. 2006
450
400
350
Nº de casos
300
250
2005
2006
200
150
100
50
0
Oncologia
Cardiovasculares
Def. Enzimáticas / D. metabólicas
/ Outras
Total
Exame
Fig. 2: Número de exames realizados pela UPSS por área médica.
Novos exames implementados na UPSS
Área Cardiovascular
•
Hipercolesterolemia familiar - pesquisa de mutações nos genes LDLR, APOB e
PCSK9
•
Cardiomiopatia hipertrófica - pesquisa de mutações nos genes MYH7, MYBPC3 e
TNNT2
•
Cardiomiopatia dilatada - pesquisa de mutações nos genes MYH7, LMNA/C e TNNT2
•
Síndrome de Marfan - pesquisa de mutações nos genes FBN1, TGFBR1 e TGFBR2
•
Trombose, factor genético predisponente - inibidor do activador do plasminogénio 1
(PAI 1) - pesquisa de variante PAI1 4G
•
Trombose, factor genético predisponente - metilenotatrahidrofolato reductase pesquisa das variantes MTHFR 677T e MTHFR 1298C
•
Trombose, factor genético predisponente - pesquisa de Factor V de Leiden
•
Trombose, factor genético predisponente - Protrombina - pesquisa de variante
PT20210A
•
Síndrome de Brugada - pesquisa de mutações no gene SCN5A
•
Síndrome QT-curto - pesquisa de mutações nos genes SCN5A, KCNQ1 e KCNH2
•
Síndrome QT-longo - pesquisa de mutações nos genes SCN5A, KCNQ1 e KCNH2
79
Área Oncologia
•
Cancro do Pulmão - Mutações EGFR
•
GIST - Pesquisa de mutações do gene KIT
•
GIST - Pesquisa de mutações do gene PDGFRA
Outros
•
Doença Celíaca - Genotipagem HLA
80
Recém-doutorados
Maria José Costa
Estudou nas células epiteliais tireoideas humanas a contribuição de microdomínios da membrana plasmática ("rafts" lipídicos e cavéolas) para a
activação da via de transdução de sinal tirotropina (TSH) -AMP cíclico e para
a organização funcional das enzimas intervenientes na produção da
hormona da tireóide (ThOx e TPO).
Mostrou, in vitro, que a estimulação prolongada das células tireoideas
humanas pela TSH-AMP cíclico conduz a uma diminuição da expressão das
caveolinas 1 e 2. Identificou ainda que a regulação da expressão da
caveolina-1 nestas condições é controlada ao nível da degradação do
respectivo RNA mensageiro, num processo mediado por um RNA (ainda
desconhecido). Mostrou ainda que ratinhos knockout para a caveolina-1
apresentam um aumento da proliferação das células epiteliais tireoideas.
Estes resultados permitiram explicar a diminuição da expressão das
caveolinas em casos de adenomas autonomos da tireoide (patologia na qual
as células epiteliais tireoideas são cronicamente estimuladas pelo AMP
cíclico) e sugerem que a caveolina-1 actua como um gene onco-supressor
nessas células.
Bioquímica, doutorou-se em 20 de Janeiro de 2006 com a tese "Papel da caveolina-1 e das cavéolas na biologia celular da
tireóide". Actualmente, está a fazer pós-doutoramento na Universidade da California.
81
Alexandra Lopes
Estudou a dinâmica evolutiva e os padrões de diversidade no bloco de
homologia Xq21.3/Yp11.2 entre os cromossomas sexuais humanos,
mostrando que marcadores do cromossoma X nesta região do genoma são
mais diversos do que os seus homólogos no cromossoma Y, tanto em
populações africanas como europeias.
Demonstrou que o gene que codifica a Protocaderina11X,
localizado neste bloco de homologia especificamente humano,
escapa a inactivação do cromossoma X, sendo expresso no
cérebro a um nível significativamente superior em indivíduos do
sexo feminino. Este facto poderá estar relacionado com o
dimorfismo sexual do cérebro humano. Poderá também contribuir
para o fenótipo de indivíduos com aneuploidias dos cromossomas
sexuais, nos quais também demonstrou a ausência de metilação da
citosina na região promotora deste gene.
Bióloga, doutorou-se em 14 de Julho de 2006 com a tese “Gene evolution and regulation within the Xq21.3/Yp11.2 hominidspecific homology block”. Actualmente está a fazer um pós-doutotamento na Universidade de Cambridge.
82
Marina Leite
Analisou a expressão de um conjunto de receptores designados por “Natural
Killer Receptors” (NKR) em células NK e linfócitos T CD8+ de doentes com
Leucemia Linfocítica Crónica de células B (LLC-B) e em dadores saudáveis.
Este estudo revelou, pela primeira vez, que os doentes com LLC-B, quando
comparados com os dadores, apresentavam múltiplas alterações na expressão
dos receptores NKR, principalmente no compartimento das células NK, e que
essas alterações se manifestavam em diferenças na proporção de células
positivas para diversos receptores, mas não afectava a densidade de expressão
desses receptores à superfície.
Bióloga, doutorou-se em 14 de Julho de 2006 com a tese “O Papel das Células Natural Killer e dos Linfócitos T Citotóxicos na
Vigilância Imunológica da Leucemia Linfocítica Crónica – Estudo da Expressão dos Receptores NKR em Indivíduos Normais e
Doentes”.
83
Jorge Lima
Estudou mutações nos genes SDHB e SDHD, os quais codificam as
subunidades homónimas da proteína mitocondrial desidrogenase do
succinato, em tumores de origem neuroendócrina, nomeadamente
paragangliomas, feocromocitomas e carcinomas medulares da tireóide.
Demonstrou que a vasta maioria dos casos de paragangliomas familiares
eram causados por mutações patogénicas germinativas nos genes SDHB e
SDHD. Além disso, uma percentagem significativa de paragangliomas
aparentemente esporádicos apresentavam também mutações patogénicas
germinativas naqueles genes, pelo que estes casos eram, de facto, casos
familiares ocultos. Demonstrou que os casos com mutações germinativas
nos genes SDHB e SDHD apresentavam características clínicas diferentes
dos casos que não tinham aquelas mutações.
Demonstrou ainda que determinadas alterações no gene SDHD (variantes
polimórficas) podem conferir susceptibilidade genética para o
desenvolvimento de carcinoma medular da tireóide.
Biólogo, doutorou-se em 19 de Dezembro de 2006 com a tese "A DESIDROGENASE DO SUCCINATO EM TUMORES
HUMANOS: ESTUDO DE MUTAÇÕES DOS GENES SDHB, SDHC E SDHD EM PARAGANGLIOMAS,
FEOCROMOCITOMAS E CARCINOMAS MEDULARES DA TIREÓIDE".
Actualmente está a fazer um pós-doutoramento no IPATIMUP e no "Roswell Park Cancer Institute" em Buffalo, E.U.A.
84
Resumo dos projectos e seu financiamento
PROJECTOS DE INVESTIGAÇÃO EM CURSO EM 2006
Fundação para a Ciência e Tecnologia
“Identificação e caracterização de mecanismos envolvidos na aquisição de motilidade e invasibilidade das células
do carcinoma papilar da tireóide”– 2003-2006 – 36 meses - IR: Manuel Sobrinho Simões
“Mitochondrial DNA deletions and mautations in tumours and in the respective normal thyroid prenchyma.” – 20042006 – 36 meses - IR: Manuel Sobrinho Simões
“Papel da segregação membranar em rafts na modulação/alteração das vias de transdução de sinal em tireóide
normal e em carcinoma papilar da tireóide”– 2003-2006 – 36 meses - IR: Paula Soares
“Papel da activação oncogénica do BRAF na carcinogénese da tireoide” – 2005-2008 – 36 meses - IR: Paula
Soares
“Identificação dos mecanismos de escape à acção da P53 na carcinogénese humana usando as neoplasias da
tiréoide como modelo de estudo” – 2003-2006 – 36 meses - IR: Paula Soares
“Caracterização dos mecanismos moleculares responsáveis pelas alterações da glicosilação das mucinas na
metaplasia intestinal” - 2003-2006 – 36 meses - IR: Celso Reis
“Identificação de genes associados a glicosilação induzidos em células gástricas pela Helicobacter pylori:
“Glicómica” – 2005-2008 – 36 meses - IR: Celso Reis
“Helicobacter pylori virulence and interleukin-1 polymorphisms: interplay in gastric carcinogenesis” – 2003-2006 –
36 meses - IR: José Carlos Machado
“Antropogenética do arquipélago de São Tomé e Príncipe: um caso exemplar de microevolução humana” –
Fundação para a Ciência e Tecnologia - 2003-2006 – 36 meses - IR: Jorge Rocha
“Adaptação bio-cultural: respostas evolutivas humanas a alterações relevantes na economia de subsistência –
2005-2008 – 36 meses - IR: Jorge Rocha
“Estudo evolutivo e epidemiológico de focos de anemias hereditárias em Portugal” – 2005-2008 – 36 meses - IR:
Maria João Prata
“Etnias e genética na fronteira da rota de migração Bantu: o caso dos Karamojong” – 2005-2008 – 36 meses - IR:
Leonor Gusmão
“ Reavaliação da diversidade mitocondrial europeia: distribuições das linhagens femininas no presente e no
passado” – 2003-2006 – 36 meses - IR: Luísa Pereira
“Genes associados à metaplasia intestinal da mucosa gástrica (mucina MUC2 e fucosiltransferase FUT3):
regulação da transcrição e relavância para a adesão do Helicobacter pylori”.”– 2005-2008 – 36 meses - IR: Leonor
David
“Clarificação do papel biológico da variabilidade da mucina MUC1 na carcinogénese gástrica” – 2004-2007 – 36
meses - IR: Luís Filipe Santos Silva
“Clarificação da importância do polimorfismo da mucina MUC1 na infecção por helicobacter pylori” – 2005-2008 –
36 meses - IR: Luís Filipe Santos Silva
85
“Identificação de vias de sinalização envolvidas na regulação do Cdx2 em dois modelos humanos de diferenciação
intestinal e polipose juvenil”– 36 meses – 2005-200 – IR: Raquel Almeida
“Tumores mamários de gata – Análise Patológica, Molecular e Citigenética”– 36 meses – 2005-2008 – IR: Fátima
Gartner
“Caracterização biológica de tumores mamários mistos caninos: histogénese, progressão tumoral e alterações
genéticas” – 2005-2008 – 36 meses – IR: Fátima Gartner
“Caracterização genética de carcinomas ductais in situ (DCIS)” – 2004-2007 – 36 meses – IR: Luís Teixeira da
Costa
“No cancro colorectal com instabilidade de microssatélites são os genes BRAF e KRAS novos marcadores de
prognóstico e novas ferramentas terapêutica? (KRAFT)” – 2005-2008 – 36 meses – IR: Raquel Seruca
“Análise funcional dos repressores da caderina-E (Slug, ZEB1 e E12/E47) em carcinomas do estômago”.)” – 20052008 – 36 meses – IR: Fátima Carneiro
“P-caderina no Cancro da Mama: o que regula a sua expressão e qual o seu papel na invasão de células
neoplásicas?”– 2005-2008 – 36 meses – IR: Fernando Schmitt.
“Diferenciação mioepitelial em carcinomas da mama: caracterização e implicações clínicas” - 2003-2006 – 36
meses – IR: Fernando Schmitt.
“Efeitos da infecção por Helicobacter pylori em células epiteliais gástricas”– 2005-2008 – 36 meses – IR: Maria do
Céu Figueiredo
“Identificação de mecanismos moleculares subjacentes ao desenvolvimento de cancro gástrico em famílias
portadoras e não-portadoras de mutações germinativas da caderina-E” – 2005-2008 – 36 meses – IR: Carla
Oliveira
“Estudo da heterogeneidade fenotípica patológica usando a proteína ornitina transcarbamilase (OTC) como
modelo” - POCI/CVT/58082/2004 – 2005-2007 – 24 meses – IR: Gianpaolo Suriano
“E-cadherin germline missense mutations and hereditary diffuse gastric câncer: a model for the identificationh of
the E-cadherin-dependent sigmaling pathways pivotal for cell invasion”– 2005-2008 – 36 meses IR: Gianpaolo
Suriano
“Inactivação de genes supressores tumorais do complexo II mitocondrial em paragangliomas e feocromocitomas
esporádicos e familiares”– 12 meses – 2005-2006 – IR: Ginesa Róstan
“Caracterização molecular genómica e pós-genómica das vias de sinalização RAS/RAF/ERK e P13K/AKT em
tumores agressivos da Tireóide”– 36 meses – 2005-2008 – IR: Ginesa Róstan
2. AGÊNCIA DE INOVAÇÃO
“Mecanismos que interferem na invasão celular e sua aplicação a estudos de oncologia e diagnóstico pré-natal”,
ADI, Programa Ideia – Promotor GDPN - Genética Médica e Diagnóstico Pré-Natal Professor Doutor Sérgio
Castedo, Lda.; IR: Raquel Seruca- 36 meses. 2005-2007
“Teste de susceptibilidade genética para determinação de risco de desenvolvimento de Doença de Crohn na
população portuguesa”, ADI, Programa Ideia – Promotor GENETEST - Prestação de Serviços de Testes de
Diagnóstico Genético S.A.”; IR:José Carlos Machado – 36 meses – 2005-2008
86
3. MINISTÉRIO DA SAÚDE
“Prevenção do carcinoma gástrico na população portuguesa através da identificação de marcadores genéticos” –
Programa Saúde XXI – 2004-2006 – 36 meses - IR: José Carlos Machado
“Registo de deficiência genética da metabolização de tiopurinas na população portuguesa e prevenção da
toxicidade em terapêuticas citostáticas”, - Programa Saúde XXI – 2005-2006 - IR: Maria João Prata
4. AGÊNCIA PORTUGUESA DE SEGURANÇA ALIMENTAR
"Avaliação do risco do consumo de sal e da infecção por Helicobacter pylori no desenvolvimento de carcinoma
gástrico e das suas lesões precursoras em Portugal” – 24 meses – 2006-2007 IR: Manuel Sobrinho Simões
5. FUNDAÇÃO CALOUSTE GULBENKIAN
“Caracterização de factores biológicos e estilos de vida com impacto de ocorrência e na forma de apresentação do
cancro – “Lesões da mucosa gástrica em Moçambique e Portugal: “o enigma Africano” – 36 meses – 2005-2008
IR: Leonor David
“Helicobacter Pylori: da Biologia à clínica” – 24 meses – 2006-2008 IR: Maria do Céu Figueiredo
“Utilização clínica dos alvos genéticos do tabaco”- 2006-2009 – 36 meses – IR: Luís Costa
“Irradiação por tinea capitis e risco de cancro” - 2006-2009 – 36 meses – IR: José Teixeira Gomes
6. PROJECTOS EM COLABORAÇÃO COM A INDÚSTRIA FARMACÊUTICA
“Diminuição da resistência ao imatinib (mesilato) em modelos de linhas celulares de leucemia através da
diminuição específica de MDR1 com siRNAs” – Novartis Farma, S.A. - IR: Helena Vasconcelos
“Papel biológico da activação do BRAF na carcinogénese do cólon ” – Novartis Farma, S.A. - 2004 – 12 meses IR: Paula Soares
“Determinação do estado do HER2 em carcinomas da mama por Hibridização in situ” - Roche Farmacêutica, S.A.
– IR: Fernando Schmitt (prestação de serviços)
“Approaching basal-like breast carcinomas to target theraphy. A project combining the reinforcement of logistics
facilities with translational research” – GlaxoSmithKline – 36 meses (jan2006 a Dez2008) – IR: Manuel Sobrinho
Simões
PROTOCOLOS DE COLABORAÇÃO COM A INDÚSTRIA E SERVIÇOS
UNICER - Bebidas de Portugal, SGPS, S.A.
“Desenvolvimento de estudos que identifiquem compostos com propriedades quimioprotectoras da carcinogênese
ou com propriedades inibidoras da invasão de células tumorais” 2006 – 2009 IR: Paula Soares
Millennium BCP
“Criação de um Observatório/Consultório de Risco de cancro familiar e Ambiental” – 24 meses – 2006-2007 IR:
Raquel Seruca
87
PROTOCOLOS DE COLABORAÇÃO COM ASSOCIAÇÔES
APCL – Associação Portuguesa contra a Leucemia
“Registo de deficiência genética da metabolização de tiopurinas na população portuguesa e prevenção de
toxicidade em terapêuticas citostáticas” – IR: Maria João Prata – 36 meses
ADRIMINHO – Associação de Desenvolvimeto Rural Integrado do Vale do Minho
“Estudo genético de preservação da raça do Cão de Castro Laboreiro mediante a identificação autossómica e
mitocondrial” – IR: Prof. António Amorim - 18 meses – 1-7-2006 a 31-12-2007
CONCURSOS E/OU ENQUADRAMENTOS INTERNACIONAIS*
Association for International Cancer Research (Inglaterra) Projecto “Molecular mechanisms of biosynthesis of
sialylated cancer-associated mucin carbohydrate antigens in gastric carcinoma” – 36 meses – 2005-2008 PI Celso
Reis.
Comissão Europeia – “Prevention, diagnosis and molecular characterisation of mismatch repair defect-related
hereditary cancers of digestive systems” – 2006-2008 – 36 M - Raquel Seruca.
Comissão Europeia – “The role of chronic infections in the development of cancer” – 2006-2010 – 48 M – IR: José
Carlos Machado
Comissão Europeia – Projecto “Evaluation and Control of Neglected Mucosal Enteric Infections in Childhood
(CONTENT)” – 2006 – 2009 - 48 meses – IR José Carlos Machado Donativo do “The late David and Esther Bernstein Halpern fund”, Israel - Projecto “Mapping of genes predisposing
to familial thyroid tumours” – 36 meses - 2006 – 2009 - IR Valdemar Máximo
PROJECTOS ESTRATÉGICOS
FUNDAÇÃO ORIENTE
“O cancro e as lesões precancerosas do estômago na China " – 36 MESES – 2004-2006
“Acções de colaboração com instituições Norte-Americanas (renovação anual) – Fundação Luso Americana para o
Desenvolvimento
PROJECTOS DE REEQUIPAMENTO
1. Fundação para a Ciência e a Tecnologia
“Espectometria de massa (MALDI TOF) para caracterização de proteínas e análise proteómica no norte de
Portugal” – 24 meses- 2006-2007 IR: Celso Reis
“Interacções genético- ambientais na carcinog´renese humana com ênfase no cancro gástrico”, 24 meses – 20062007 IR: Maria do Céu Figueiredo
88
Trabalhos publicados
1.
Agudo A, Sala N, Pera G, Capella G, Berenguer A, Garcia N, Palli D, Boeing H, Del Giudice G, Saieva C,
Carneiro F, Berrino F, Sacerdote C, Tumino R, Panico S, Berglund G, Siman H, Stenling R, Hallmans G,
Martinez C, Amiano P, Barricarte A, Navarro C, Quiros JR, Allen N, Key T, Bingham S, Khaw KT, Linseisen J,
Nagel G, Overvad K, Tjonneland A, Olsen A, Bueno-de-Mesquita HB, Boshuizen HC, Peeters PH, Numans ME,
Clavel-Chapelon F, Boutron-Ruault MC, Trichopoulou A, Lund E, Blaker H, Jenab M, Ferrari P, Norat T, Riboli E,
Gonzalez CA. No association between polymorphisms in CYP2E1, GSTM1, NAT1, NAT2 and the risk of gastric
adenocarcinoma in the European prospective investigation into cancer and nutrition. Cancer Epidemiol
Biomarkers Prev 15: 1043-5, 2006. IF: 4.5
2.
Agudo A, Sala N, Pera G, Capella G, Berenguer A, Garcia N, Palli D, Boeing H, Del Giudice G, Saieva C,
Carneiro F, Berrino F, Sacerdote C, Tumino R, Panico S, Berglund G, Siman H, Stenling R, Hallmans G,
Martinez C, Bilbao R, Barricarte A, Navarro C, Quiros JR, Allen N, Key T, Bingham S, Khaw KT, Linseisen J,
Nagel G, Overvad K, Tjonneland A, Olsen A, Bueno-de-Mesquita HB, Boshuizen HC, Peeters PH, Numans ME,
Clavel-Chapelon F, Boutron-Ruault MC, Trichopoulou A, Lund E, Offerhaus J, Jenab M, Ferrari P, Norat T, Riboli
E, Gonzalez CA. Polymorphisms in metabolic genes related to tobacco smoke and the risk of gastric cancer in
the European prospective investigation into cancer and nutrition. Cancer Epidemiology & Biomarkers Prevention
15:2427-2434, 2006. IF: 4.5
3.
Alonso A, Albarran C, Martin P, Garcia P, Capilla J, Garcia O, De La Rua C, Izaguirre N, Pereira F, Pereira L,
Amorim A, Sancho M. Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic
and ancient DNA studies. Electrophoresis. 27(24): 5101-9, 2006. IF: 3.9
4.
Alves S, Mangas M, Prata MJ, Ribeiro G, Lopes L, Ribeiro H, Pinto-Basto J, Lima Mr, Lacerda L. Molecular
characterization of Portuguese patients with mucopolysaccharidosis type II shows evidence that the IDS gene is
prone to splicing mutations. J Inherit Metab Dis 29:743-754, 2006. IF: 1.7
5.
Azevedo L, Soares PA, Quental R, Vilarinho L, Teles EL, Martins E, Diogo L, Garcia P, Cenni B, Wermuth B,
Amorim A. Mutational Spectrum and Linkage Disequilibrium Patterns at the Ornithine Transcarbamylase Gene
(OTC) Ann Hum Genet 70(Pt 6):797-801, 2006. IF: 3.2
6.
Azevedo L, Suriano G, van Asch B, Harding RM, Amorim A. Epistatic interactions: how strong in disease and
evolution? Trends Genet 22: 581-5, 2006. IF: 12.0
7.
Babu SD, Jayanthi V, Devaraj N, Reis CA, Devaraj H: Expression profile of mucins (MUC2, MUC5AC and
MUC6) in Helicobacter pylori infected pre-neoplastic and neoplastic human gastric epithelium. Molecular Cancer
5: 10-17, 2006.
8.
Behar DM, Metspalu E, Kivisild T, Achilli A, Hadid Y, Tzur S, Pereira L, Amorim A, Quintana-Murci L, Majamaa K,
Herrnstadt C, Howell N, Balanovsky O, Kutuev I, Pshenichnov A, Gurwitz D, Bonne-Tamir B, Torroni A, Villems
R, Skorecki K. The Matrilineal Ancestry of Ashkenazi Jewry: Portrait of a Recent Founder Event. Am J Hum
Genet 78: 487-497, 2006. IF: 12.6
9.
Beleza S, Gusmao L, Lopes A, Alves C, Gomes I, Giouzeli M, Calafell F, Carracedo A, Amorim A. Microphylogeographic and demographic history of Portuguese male lineages. Ann Hum Genet 70: 181-94, 2006. IF:
3.2
10.
Botelho CH, Magalhaes AV, Mello PA, Schmitt FC, Casulari LA. Expression of p53, Ki-67 and c-erb B2 in growth
hormone-and/or prolactin-secreting pituitary adenomas. Arq Neuropsiquiatr 64: 60-6, 2006. IF: 4.0
11.
Bruggemann M, White H, Gaulard P, Garcia-Sanz R, Gameiro P, Oeschger S, Jasani B, Ott M, Delsol G, Orfao
A, Tiemann M, Herbst H, Langerak AW, Spaargaren M, Moreau E, Groenen PJ, Sambade C, Foroni L, Carter GI,
Hummel M, Bastard C, Davi F, Delfau-Larue MH, Kneba M, van Dongen JJ, Beldjord K, Molina TJ. Powerful
strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED2 Concerted Action BHM4 CT98-3936. Leukemia. 2006 Dec 14; [Epub ahead of print] IF: 6.6
12.
Builes JJ, Bravo ML, Gomez C, Espinal C, Aguirre D, Gomez A, Rodriguez J, Castaneda P, Montoya A, Moreno
M, Amorim A, Gusmao L. Y-chromosome STRs in an Antioquian (Colombia) population sample. Forensic Sci Int.
164: 79-86, 2006 IF: 1.6
89
13.
Builes JJ, Martinez B, Gomez A, Caraballo L, Espinal C, Aguirre D, Montoya A, Moreno M, Amorim A, Gusmao
L, Bravo ML Y chromosome STR haplotypes in the Caribbean city of Cartagena (Colombia). Forensic Sci Int.
2006 Jan 30; [Epub ahead of print] IF: 1.6
14.
Cameselle-Teijeiro J, Abdulkader I, Barreiro-Morandeira F, Ruiz-Ponte C, Reyes-Santias R, Chavez E, SobrinhoSimoes M. Breast tumor resembling the tall cell variant of papillary thyroid carcinoma: a case report. Int J Surg
Pathol14:79-84, 2006. IF: 0.9
15.
Cameselle-Teijeiro JF, Cortizo-Torres ME, Schmitt FC. Medical chronobiology: inheritance and cancer. Rev Clin
Esp 206: 60-1; author reply 61, 2006. IF: 0.3
16.
Cardoso H, Nunes AC, Carneiro F, Tavarela Veloso F. Successful infliximab therapy for oral Crohn's disease.
Inflamm Bowel Dis 12: 337-8, 2006. IF: 3.0
17.
Carneiro F, Chaves P. Pathologic risk factors for therapy for adenocarcinoma of the gastric cardia and
gastroesophageal junction. Surg Oncol Clin N Am 15: 697-714, 2006
18.
Carvalho B, Buffart TE, Reis RM, Mons T, Moutinho C, Silva P, van Grieken NC, Grabsch H, van de Velde CJ,
Ylstra B, Meijer GA, Carneiro F. Mixed gastric carcinomas show similar chromosomal aberrations in both their d
fuse and glandular components. Cell Oncol 28: 283-94, 2006. IF: 4.2
19.
Castro P, Soares P, Gusmão L, Seruca R, Sobrinho-Simões M. H-RAS 81 polymorphism is sign icantly
associated with aneuploidy in follicular tumors of the thyroid. Oncogene 25:4620-4627, 2006. IF: 6.9
20.
Chen C-Y, Chi K-H, George RW, Cox DL, Srivastava A, Silva MR, Carneiro F, Lauwers GY, Ballard RC.
Diagnosis of gastric syphilis by direct immunofluorescence staining and real-time PCR testing. J Clinical
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21.
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REVISTAS CIENTÍFICAS
INTERNACIONAIS COM
MEMBROS DO
IPATIMUP
NOS SEUS EDITORIAL
BOARDS
Acta Cytologica (SCI PRINTERS & PUBL INC)
Advances in Anatomic Pathology (LIPPINCOTT WILLIAMS & WILKINS)
Archives of Pathology and Laboratory Medicine (COLLEGE OF AMERICAN
PATHOLOGISTS)
Breast Cancer Research (BIOMED CENTRAL LTD)
Cancer Genetics and Cytogenetics (ELSEVIER SCI IRELAND LTD)
Critical Review in Oncogenesis (BEGELL HOUSE, USA)
Current Diagnostic Pathology (CHURCHILL LIVINGSTONE)
Cytojournal (BIOMED CENTRAL)
Cytopathology (BLACKWELL PUBLISHING LTD)
Diagnostic Cytopathology (WILEY-LISS)
Endocrine Pathology (BLACKWELL PUBLISHING LTD)
European Journal of Cancer Prevention (LIPPINCOTT WILLIAMS & WILKINS)
Forensic Science International (ELSEVIER SCI IRELAND LTD)
Hereditary Cancer in Clinical Practice (UICC GLOBAL CANCER CONTROL)
Histopathology (BLACKWELL PUBLISHING LTD)
Human Biology (WAYNE STATE UNIVERSITY PRESS)
International Journal of Surgical Pathology (WESTMINSTER PUBL INC)
Journal of Cellular and Molecular Medicine (CAROL DAVILA UNIV PRESS)
Journal of Pathology (JON WILEY & SONS LTD)
Pathology Research & Practice (URBAN & FISCHER VERLAG)
Seminars in Diagnostic Pathology (W B SAUNDERS CO)
Ultrastructural Pathology (TAYLOR & FRANCIS INC)
Virchows Archiv (SPRINGER)
96
NÚCLEO DE AMIGOS
DO
IPATIMUP
Allianz Portugal, SA
Amorim Inv. e Participações
Astrazeneca Produtos Farmacêuticos Lda
Banco BPI, SA
Bayer Portugal, SA
BES
Bial - Portela & C.ª, SA
Fundação Millennium BCP
GlaxoSmithKline Prod. Farmacêuticos, Lda
Ipsen Portugal - Produtos farmacêuticos, SA
Merck Sharp & Dohme, Lda.
Mota Engil, SGPS, SA
Novartis Farma - Prod. Farmacêuticos, SA
Olinveste SGPS
RAR - Sociedade de Controle (Holding), SA
Roche Farmacêutica Química, Lda
SAG GEST - Soluções Automóvel Globais, SA
Sonae SGPS, SA
Têxtil Manuel Gonçalves SA
Unicer
97