Skript - Institut für Humangenetik

Transcrição

Master Study Molecular Medicine
Functional Tumor and
Molecular Human Genetics
Uniparental Disomies, Rapid
Prenatal Testing, Contamination and
Paternity
Ferdinand von Eggeling
von Eggeling, Tumor + Mol
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How does this fit together?
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PCR
Polymerase Chain Reaction
Folie: 3
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What are Microsatellites or Short Tandem Repeats (STR)?
Microsatellites, also known as simple sequence
repeats (SSRs) or short tandem repeats (STRs),
are repeating sequences of 2-5 base pairs of
DNA. It is a type of Variable Number Tandem
Repeat (VNTR). Microsatellites are typically codominant. They are used as molecular markers
in STR analysis, for kinship, population and other
studies. They can also be used for studies of
gene duplication or deletion, marker assisted
selection, and fingerprinting.
4 repeats CGT (Trinucleotidrepeat)
CTGGATCGTCGTCGTCGTAATAGCAT
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How do we perform an STR analysis?
Sketch of a Tetranukleotidrepeat
..NNNNN GATC GATC NNNNNNN..
Allele mother I
..NNNNN GATC GATC GATC NNNNNNN..
Allele mother II
..NNNNN GATC NNNNNNN..
Allele father I
..NNNNN GATC GATC GATC GATC NNNNNNN..
Allele father II
Gel view after PCR
M
V
K
M
M
V
K
Gelgild nach PCR
V
K
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What we can analyse with Mikrosatellites (short tandem
repeats, STR)?
•
Test for Paternity
•
Identification of Deliquents (e.g. murder, raper) in
forensic medicine
•
Analysis of Uniparental Disomies
•
…. And rapid Prenatal Testing
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Paternity test
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….and of course Amazon
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Paternity Test
At least 10% of children are not from the father who
believe that he is the father……
…..but this is done at the Institute of Forensic
Medicine, where also other delinquents can be
identified
But the same test set (10 STR, in USA 13) is used
for…..
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Identification of Deliquents (e.g. murder, raper) in
forensic medicine
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Coton swabs
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……..Analysis of Contamination / Exclusion of a
Contamination
For what this is needed?
• Abortion
• Chorionic villus sampling (CVS)
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Chorionic villus sampling (CVS)
peritoneal
trans-vaginal
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Placenta
Mother
Child
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How do we perform an STR analysis?
Sketch of a Tetranukleotidrepeat
..NNNNN GATC GATC NNNNNNN………………….
Allel Mutter I
..NNNNN GATC GATC GATC NNNNNNN………………….….
Allel Mutter II
..NNNNN GATC NNNNNNN…………………..
Allel Vater I
Allel Vater II
..NNNNN GATC GATC GATC GATC NNNNNNN………………………...
Gelgild nach PCR
M
V
K
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No maternal contamination
M K M K M K M K M K M K M K M K M
K
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Maternal contamination
M K M K
M K M K M K
M K M K M K
M K Amelogenin
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The rapid prenatal screening
(STR based)
„Aneuploidy Test, PCR based“
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Case:
An amniocentesis was performed by a 38 year old pregnant
women. The gynaecologist send the amniotic fluid to Human
Genetics Institute
To make a “rapid prenatal screening”
To analyse chromosomes (Karyogramm)
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Why rapid prenatal screening?
Normal karyotyping take about 8 -14 days!
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Rapid prenatal screening
Marker for #21
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Rapid prenatal screening
From mother
Three peaks: in Meiosis II
From mother
Two peaks: in Meiosis I
Double height
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Advantages / Disadvantages
Advantage
• fast
• Cost effective
Disadvantage
• Mosaics are difficult to see
• Numerical Aberrations of the gonosomes can´t be seen
(z. B. X0, Turner)
• If it is positive, women are earlier worried
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Uniparental Disomy
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Uniparental Disomies (UPD):
What’s that?
Uni: (from one) single
Parental: from parent
Disomy: 2x the same Chromosomes
= both Chromosomes are from one parent
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Uniparental Disomy
Maternal Chromosome
Paternal Chromosome
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Uniparental Disomy
Definition:
Uniparental disomy (UPD) occurs when a person receives two
copies of a chromosome, or part of a chromosome, from one parent
and no copies from the other parent
UPD can occur as a random event during the formation of egg or
sperm cells or may happen in early fetal development. It can also
occur during transonic rescue.
When the child receives the (two) homologous chromosomes
(inherited from both grandparents) from one parent, this is called an
heterodisomic UPD. Heterodisomy (Heterozygous) indicates a
meiosis I error.
When the child receives, two (identical) replica copies of a single
homolog of a chromosome, this is called an isodisomic UPD.
Isodisomy (homozygous) indicates either a meiosis II error or
postzygotic duplication.
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Mikrosatellitenanalyse
PCR
PCR
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The Normal Case
Ovum
Sperm
Zygote
Biparental
(normal)
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Development of a UPD: Trisomy Rescue
Ovum
Sperm
Zygote
biparental
(normal)
biparental
(normal)
Uniparental,
heterodisom
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Development of a UPD: Monosomy Rescue
Ovum
Sperm
Zygote
Uniparental, isodismom
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Development of a UPD: Trisomy Rescue with a Robertson Translation
Ovum
Sperm
Zygote
biparental
(normal)
Uniparental,
heterodisome
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Problems with UPD
With a uniparental pair of chromosomes the following
problems can be associated:

Mosaic Trisomy

Homozygotisation of autosomal recessive inherited
mutations

Unphysiological genomic Imprinting
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Mosaic Trisomy
Ovum
Sperm
Klon 1
Zygote
Klon 2
biparental
(normal)
biparental
(normal)
Uniparental,
heterodisom
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Homozygotisation of an autosomal rezessive inherited mutation
Ovum
Sperm
Recessive Disorder
Zygote
Mutation is now present on both alleles
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What is a Iso-Disomy, what a Hetero-Disomy?
Ovum Sperm
Zygote
Uni-parental, iso-disom
Ovum Sperm
Zygote
biparental biparentalUniparental,
(normal) (normal) heterodisom
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Uniparental Disomy
Definition:
Uniparental disomy (UPD) occurs when a person receives two
copies of a chromosome, or part of a chromosome, from one parent
and no copies from the other parent
UPD can occur as a random event during the formation of egg or
sperm cells or may happen in early fetal development. It can also
occur during transonic rescue.
When the child receives the (two) homologous chromosomes
(inherited from both grandparents) from one parent, this is called an
heterodisomic UPD. Heterodisomy (Heterozygous) indicates a
meiosis I error.
When the child receives, two (identical) replica copies of a single
homolog of a chromosome, this is called an isodisomic UPD.
Isodisomy (homozygous) indicates either a meiosis II error or
postzygotic duplication.
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Until now known clinically relevant UPDs

upd(6)pat: Transient neonatal Diabetes mellitus

upd(7)mat: Russell-Silver Syndrom

upd(11)pat: Beckwith-Wiedemann Syndrom (BWS)*

upd(14)mat: Dwarfism, Hypotonia, extensible joints, Scoliose,
facial Dysmorphisms, mild retardation of developement, early puberty

a
upd(14)pat: stronger characteristics of upd(14)mat features as well
additional mental retardation and skeletal malformations which cause
dwarfism

upd(15)mat: Prader-Willi Syndrom (PWS)*

upd(15)pat: Angelman Syndrom (AS)*
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upd(7)mat: Russell-Silver Syndrom
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upd(11)pat: Beckwith-Wiedemann Syndrom (BWS)*
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UPD 15
Prader-Willi-Syndrom
Angelman-Syndrom
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Unphysiological Genomic Imprinting
Normal case
PWS
AS
mat
pa
t
Prader-Willi-Syndrom
PWS
AS
mat
mat
Angelman-Syndrom
PWS
AS
pat
pat
active Gene
inaktive Gene
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When a UPD Diagnostic is performed?
•
Fetus with Robertson Translocation or an Isochromosome for
chromosomes 14 und 15
•
Children with Robertson Translocation or an Isochromosome for
chromosomes 14 und 15 and growth retardation and/or mental
retardation
•
Fetus with chromosomal markers, which were identified as
chromosome 6, 7, 11, 14 or 15
•
Abnormalities in ultra sound, which ar consistent with die UPD
syndrome features
•
Fetus with Mosaic trisomy
•
New born with a neonatal Diabetes mellitus
•
New born with features of a Silver-Russell Syndrome
•
New born with suspicion of BWS with normal karyotyp and no
FISH proven duplication von 11p15.5
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How a UPD Diagnostic is performed?
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Vereinfachte Darstellung eines Tetranukleotidrepeats
..NNNNN GATC GATC NNNNNNN..
Allel Mutter I
..NNNNN GATC GATC GATC NNNNNNN..
Allel Mutter II
..NNNNN GATC NNNNNNN..
Allel Vater I
Allel Vater II
..NNNNN GATC GATC GATC GATC NNNNNNN..
Gelgild nach PCR
M
V
K
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M
ab
F
cc
P
ab
a
b
M
bc
F
ac
P
bb
a
b
c
c
Uniparental Hetero-disomy 12
Uniparental Iso-disomy 12
D12S2078
D12S1042
M
aa
F
bb
a
b
M
bc
P
cc
F
ab
P
bc
Uniparental disomy 12
(not informative for iso
or hetero)
a
b
c
Not informative at all
D12S100
D12S372
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M
ab
F
aa
P
bb
M
ab
F
bc
P
aa
b
c
F
ab
D20S1084
P
cd
M
ac
F
bb
D20S470
P
ac
a
a
b
c
d
M
aa
F
bc
P
aa
a
b
b
c
c
D20S601
P
dd
c
d
D20S486
M
cd
F
ac
a
b
a
a
b
M
bd
D20S481
D20S485
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Results of a UPD testing
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Please see also connections to the lecture:
• Epigenetic
• Imprinting
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The END
Questions?
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Skript - Institut für Humangenetik

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