Milder course in Duchenne patients with nonsense mutations

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Neuromuscular Disorders xxx (2014) xxx–xxx
www.elsevier.com/locate/nmd
Clinical Casebook
Milder course in Duchenne patients with nonsense mutations and no
muscle dystrophin
M. Zatz ⇑, R.C.M. Pavanello, M. Lazar, G.L. Yamamoto, N.C.V. Lourencßo, A. Cerqueira,
L. Nogueira, M. Vainzof
Human Genome Center, Biosciences Institute, University of São Paulo, São Paulo, Brazil
Received 21 May 2014; accepted 1 June 2014
Abstract
Duchenne muscular dystrophy (DMD), a severe and lethal condition, is caused by the absence of muscle dystrophin. Therapeutic
trials aiming at the amelioration of muscle function have been targeting the production of muscle dystrophin in affected Duchenne
patients. However, how much dystrophin is required to rescue the DMD phenotype remains an open question. We have previously
identified two exceptional golden retriever muscular dystrophy (GRMD) dogs with a milder course despite the total absence of
muscle dystrophin. Here we report two unusual patients carrying nonsense mutations in the DMD gene and dystrophin deficiency
but with an unexpectedly mild phenotype. Three reported polymorphisms, respectively in genes LTBP4, SPP1 and ACTN3 were
excluded as possible DMD genetic modifiers in our patients. Finding the mechanisms that protect some rare patients and dogs from
the deleterious effect of absent muscle dystrophin is of utmost importance and may lead to new avenues for treatment. Importantly,
these observations indicate that it is possible to have a functional large muscle even without dystrophin.
Ó 2014 Elsevier B.V. All rights reserved.
Keywords: Duchenne dystrophy; Milder progression; Nonsense mutation; No dystrophin
1. Introduction
Duchenne muscular dystrophy (DMD) is a lethal
X-linked condition caused by mutations in the dystrophin
gene which results in the absence of muscle dystrophin
protein. The course is usually severe and very similar in
affected patients. Onset occurs around 2 to 3 years of age
and without careful management, loss of ambulation
between 9 and 12. On the other hand, in Becker muscular
dystrophy (BMD), there is a wide variability (intra and
inter familial) in the severity of the phenotype which has
been mainly associated with the site of the deletion and
⇑ Corresponding author. Address: Human Genome and Stem Cell
Research Center, Institute of Biosciences, University of São Paulo, Rua do
Matão, 106, Cidade Universitária, São Paulo, SP 05508-090, Brazil. Tel.:
+55 11 30917563.
E-mail address: [email protected] (M. Zatz).
to the amount of muscle dystrophin. Therefore, the
quantity/quality of muscle dystrophin has been strongly
associated with appropriate muscle function.
Some exceptional patients [1] and dystrophic animal
models, however, deserve special attention. For instance,
understanding why the mdx mice, the murine model for
DMD is almost asymptomatic, despite total absence of
dystrophin has been a great challenge. Possible
explanations are their smaller size and shorter lifespan.
However, we have identified two exceptional golden
retriever muscular dystrophy (GRMD) dogs with no
muscle dystrophin and a very mild phenotype [2]. Here we
report two very rare examples of patients carrying
nonsense mutations and a milder phenotype despite the
absence of muscle dystrophin. These observations indicate
that it is possible to have a functional large muscle even
without dystrophin.
http://dx.doi.org/10.1016/j.nmd.2014.06.003
0960-8966/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Zatz M et al., Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin, Neuromuscul
Disord (2014), http://dx.doi.org/10.1016/j.nmd.2014.06.003
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2. Clinical and laboratory exams
2.1. Case 1
Two half-brothers called our attention due to their
strikingly discordant phenotype (Fig. 1A). Onset of
symptoms in the younger brother (II) was at age 3, a
DMD diagnosis was established at age 7, and he was
wheelchair-bound at age 9. His serum CK is 3260 U/l
(normal up to 189 U/l). His older half-brother (I) was
noticed to have some weakness at age 13, when his
younger brother was diagnosed. Currently, at age 15, he
Fig. 1. (A) The two brothers, illustrating their calf hypertrophy. (B) The 15 year-old isolated DMD patient; C-Muscle histological and
immunohistochemical analysis for dystrophin, using DYS 2. (C) Terminal antibody. (D) Western blot analysis showing the reaction for dystrophin Nterminal antibody (DYS3, Vector), rod domain antibody (DYS1, Vector) and C-terminal antibody (DYS2, vector). A reaction with antibody for alpha
actinin 3 (ACTN3, generous gift from Dr. Alan Beggs, Boston, USA), showed the presence of the protein in the 3 analyzed patients. (C) normal control
muscle, Myosin-band observed in the Ponceau S pre-stained blot.
Please cite this article in press as: Zatz M et al., Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin, Neuromuscul
Disord (2014), http://dx.doi.org/10.1016/j.nmd.2014.06.003
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is only mildly affected, with discrete calveś hypertrophy,
some difficulties for running and climbing stairs but with
normal ability for walking. His serum CK is 3620 U/l.
MLPA
(multiplex
ligation
dependent
probe
amplification) DNA analysis revealed that both brothers
carry an “out of frame” duplication in exon 2 of the
dystrophin gene which was not present in the mother
blood lymphocytes, indicating a gonadal mosaicism.
Despite the clinical differences, muscle histology
analyzed in blind test showed a similar pattern in both
brothers with variation in fiber size, degeneration,
splitting fibers, centrally located nuclei and connective
tissue replacement (Fig. 1C).
Dystrophin immunostaining with at least 3 antibodies
against the N-terminal, rod domain and C-terminal,
showed a negative pattern, with a cluster of 6–8 positive
fibers (reverting fibers) per section, in both of them
(Fig. 1C). Merosin was positive, and sarcoglycans were
faints, as observed in most DMD dystrophin deficient
patients.
When normalized for the myosin content and compared
to normal controls in the same blot, dystrophin amount
through WB revealed a very faint band, of less than 5%
of normal and very similar in both of them, using an
antibody against the rod domain. No dystrophin bands
were identified with the N-terminal and C-terminal
antibodies (Fig. 1D).
Therefore, although the two brothers exhibited equally
low amounts of dystrophin they presented different
phenotypes.
2.2. Case 2
This isolated DMD patient who, at age 16, is able to
walk without difficulties and climb stairs with the aid of
the banister was first seen in our center at age 7. At that
time, he had a typical DMD phenotype with difficulties
for climbing stairs, running or raising up from the floor
(Fig. 1B). His serum CK was grossly elevated (30,756 U/
l). DNA analysis through MLPA revealed the presence of
an out-of-frame deletion, encompassing exons 51–54, in
the dystrophin gene. Currently, although he has a visible
lordosis he has discrete calf hyperthophy and he does not
walk on his toes.
Muscle biopsy showed a histological pattern typical of a
dystrophic process, with variation in fiber size, centrally
located nuclei, degeneration and significant connective
tissue replacement (Fig. 1C).
Dystrophin immunostaining with three antibodies
against the N-terminal, rod domain and C-terminal,
showed a negative pattern (Fig. 1C), also observed
through western blot analysis, using the same antibodies
(Fig. 1D).
In the 3 patients, alpha-actinin 3 protein was present,
and DNA analysis confirmed the absence of the R577X
variant.
3
Analysis of the LTBP4 haplotype in the three patients
showed the following genotypes: IV-AT-AT-MT, for case
I mildly affected brother, II-AT-AT-TT for case II more
severely affected brother, and IV-AT-AT-MT for case III.
Therefore, all 3 cases were heterozygous for this
haplotypes.
Analysis of the SNP-66T > G (rs28357094) in the
osteopontin gene (SPP1), where the G allele (GG or TG)
has been apparently associated with a more severe
phenotype, showed the TG, TT and TT genotypes in
patients I, II and III, respectively.
3. Discussion
Patients with a discordant Duchenne and Becker
phenotype belonging to the same family have been
previously reported by us and others. In one situation,
the milder progression was associated with growth
hormone deficiency [3,4]. More recently, a DMD patient
with significant growth delay due to corticotherapy, who
was also still ambulant at age 19, was reported [5].
However, the two 15 and 16 year old patients with a
milder course here reported have normal growth.
A family with clinically discordant relatives have been
previously reported by us in a maternal uncle and his
nephew. But, differently from the present cases proteins
and molecular analyses revealed that the uncle with a
milder course had some muscle dystrophin as a result of
an alternative splicing in the dystrophin gene [6,7]. Kesari
et al. [8] reported a high rate of exceptions in the
reading-frame rule in a study of 75 patients, mainly in
BMD patients involving the N-terminal region of the
gene. This is explained by the high incidence of 50 gene
deletions/duplications in a region known as a hotspot for
exceptions due to complex splicing patterns [8]. However,
it is important to point out that all these exceptional
BMD patients with apparently nonsense mutations had
some amount of dystrophin in muscle biopsy in
accordance with their milder phenotype. More recently,
one more patient with a nonsense mutation in exon 2
and a milder course was reported [9] but he presented a
visible muscle dystrophin band through western blot,
differently from our two here reported cases.
In the two milder affected patients, DNA mutations
were compatible with dystrophin deficiency observed in
their muscles, which showed a very similar pattern in the
three of them, as well as in more than 300 DMD patients
with nonsense mutations we have analyzed in our center
so far. Therefore, the milder phenotype could not be
related to differences in the amount of expressed protein.
Consistently with the muscle dystrophin deficiency, all
complementary
exams,
in
particular
muscle
histopathology, and other studied proteins showed a very
similar pattern in the two half-brothers as well as in the
other unrelated 16-years old patient, despite their clinical
difference. In short, in both families there is a
Please cite this article in press as: Zatz M et al., Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin, Neuromuscul
Disord (2014), http://dx.doi.org/10.1016/j.nmd.2014.06.003
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controversy between molecular findings and the clinical
course.
This raises the question whether other mechanisms
could maintain muscle functioning without dystrophin in
mdx mice and exceptional GRMD dogs [2].
The great challenge is to identify modifier genes or
molecular mechanisms which could explain these
findings, that is, protect skeletal muscles with dystrophin
deficiency against degeneration and weakness. Three
possible genetic modifiers of the DMD phenotype,
respectively in the SPP1, LTBP4 and ACTN3 genes were
studied in our patients. Alpha actinin 3 was present in
normal quantity in muscle and the a-actinin R577X
variant, the possible modifier polymorphism associated to
muscle function, was also excluded. The GG/GT
genotypes in the SPP1 gene, apparently associated with a
more severe progression of the DMD phenotype was also
excluded, since the more severely affected brother
presented the TT genotype, and the mildly affected one,
the T/G genotype in the SPP1 gene [10]. More recently,
Flanigan et al. [11] reported that individuals homozygous
for the IAAM LTBP4 haplotype remained ambulatory
significantly longer than those heterozygous or
homozygous for the VTTT haplotype. Since the three
patients here reported were heterozygous for the LTBP4
haplotype, this would not explain their clinical
differences. Interestingly, whole exome analysis performed
recently in 604 healthy Brazilian individuals revealed that
the frequency of the LTBP4 IAAM haplotype
polymorphism is 8% (50 of 604) in our population.
Therefore we would expect that about 8% of our DMD
patients should have this haplotype, and in this case, also
would have a milder course. However, in a cohort of
more than 2600 DMD patients (diagnosed through DNA
or muscle biopsies) that have been followed in our center
in the last 30 years, we ascertained less than 1% of
patients with a mild course at age 14 among those with
nonsense mutations or no muscle dystrophin in muscle
biopsies.
The severity of the clinical course in dystrophinopathies
has been correlated with the amount of muscle dystrophin
[12]. Therefore, therapeutic trials aiming the amelioration
of muscle function have been targeting the production of
muscle dystrophin in affected Duchenne patients.
However, how much dystrophin is required to rescue the
DMD phenotype remains an open question. Finding the
mechanisms that protect some rare patients and dogs
from the deleterious effect of absent muscle dystrohin is
of utmost importance and may lead to new avenues for
treatment. Importantly, these observations indicate that it
is possible to have a functional large muscle even without
dystrophin.
Acknowledgements
We would like to thank Dr. Kevin Flanigan for helpful
discussion on the LTBP4 variants and to Dr. João Paulo
Kitajima for the analysis of this variant in our normal
population. We are very grateful to FAPESP/CEPID,
CNPq, Duchenne research fund and ABDIM for
financial support.
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Please cite this article in press as: Zatz M et al., Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin, Neuromuscul
Disord (2014), http://dx.doi.org/10.1016/j.nmd.2014.06.003