Somatic Mosaicism for Duchenne Dystrophy:
Evidence for Genetic Normalization Mitigating
Akanchha Kesari,1Robert Neel,2Lynne Wagoner,3Brennan Harmon,1
Christopher Spurney,4and Eric P. Hoffman1*
1Research Center for Genetic Medicine, Children’s National Medical Center, Washington, District of Columbia
2Department of Neurology, University of Cincinnati Medical Center, Cincinnati, Ohio
3Department of Cardiology, University of Cincinnati Medical Center, Cincinnati, Ohio
4Department of Cardiology, Children’s National Medical Center, Washington, District of Columbia
Received 12 February 2009; Accepted 18 March 2009
We describe a young adult male presenting with cardiac failure
necessitating cardiac transplantation 7 months after presenta-
tion. Skeletal muscle biopsy showed mosaic immunostaining
for dystrophin. DNA studies showed somatic mosaicism for a
nonsense mutation in the dystrophin gene (Arg2905X). The
frequency of normal versus mutant genes were determined in
blood/DNA (50:50), muscle/DNA (80:20) and muscle/mRNA
(90:10). These data are consistent with genetic normalization
processes that may biochemically rescue skeletal muscle in male
somatic mosaic patients mitigating muscle symptoms (gradual
loss of dystrophin-negative skeletal muscle tissue replaced by
dystrophin-positive stem cells). To our knowledge, this is only
the second reported case of a clinically ascertained patient
showing somatic mosaicism for Duchenne muscular dystrophy
(DMD). We hypothesize that many somatic mosaic males
for DMD exist, yet they are not detected clinically due to
genetic normalization. Somatic mosaicism for DMD should be
genetic normalization in heart is unlikely to occur.
? 2009 Wiley-Liss, Inc.
Key words: genetic normalization; somatic mosaic; dystrophin
Duchenne muscular dystrophy (DMD) is an X linked recessive
disorder that occurs in 1 in 3,500 males in all world populations
[Hoffman et al., 1988]. DMD is a progressive proximal muscular
dystrophy with early calf hypertrophy, elevations of serum
creatine kinase, and dystrophic muscle pathology [Ishpekova
et al., 1999]. Dilated cardiomyopathy develops relatively late in
the disease process. Patients with less severe Becker muscular
dystrophy (BMD) variant can show cardiomyopathy as the pre-
senting symptom. Female carriers also show heart abnormalities
upon electrocardiographic analysis; however it is rarely life
threatening [Politano et al., 1996; Cox and Kunkel, 1997;
Hoogerwaard et al., 1999].
rate of the large DMD gene(2.4Mb), with 1/10,000 of all germ line
Haldane, 2004]. The high germ-line (meiotic) mutation rate
leads to a high proportion of boys with DMD with no previous
family history, where the mother is not a carrier of her son’s
mutation. Typically, high germ line mutation rates predict high
somatic (mitotic) mutation rates, leading to cases of somatic
mosaicism. For example, in DMD, male embryos may sustain a
mutation of the DMD gene early in embryonic development,
leading to a patient with populations of both abnormal
(dystrophin-negative) and normal (dystrophin-positive) in tissues
expressing the gene (muscle, heart, smooth muscle). Despite the
Grant sponsor: NIH; Grant number: 3R01 NS29525; Grant number:
5R24HD050846; Grant number: 1P30HD40677.
Eric P.Hoffman, Ph.D.,Research Centerfor Genetic Medicine, Children’s
NationalMedicalCenter, 111Michigan Ave NW,Washington, DC20010.
Published online 15 June 2009 in Wiley InterScience
How to Cite this Article:
Kesari A, Neel R, Wagoner L, Harmon B,
Spurney C, Hoffman EP. 2009. Somatic
mosaicism for Duchenne dystrophy:
Evidence for genetic normalization
mitigating muscle symptoms.
Am J Med Genet Part A 149A:1499–1503.
? 2009 Wiley-Liss, Inc.
DMD, two cases ascertained at autopsy [Saito et al., 1995; Uchino
somatic mosaics could reflect a lower mutation rate in somatic cells
relative to germ line cells, or alternatively, somatic mosaics may be
poorly ascertained due to mild or unexpected clinical symptoms.
In this report we present a case of somatic mosaicism in
DMD gene presenting with acute cardiac failure. This broadens
the clinical phenotypes associated with dystrophinopathy. We
also show data consistent with genetic normalization processes
previously reported in female carriers, where the muscle becomes
increasingly dystrophin-positive (normal) with advancing age
[Pegoraro et al., 1995]. We hypothesize that genetic normalization
somatic mosaics for DMD.
PATIENT AND METHODS
A 20-year-old African American male sought evaluation after a
2-week history of increasing fatigue, shortness of breath and a
asthma. At initial evaluation, an echocardiogram showed severely
depressed left ventricular systolic function. The patient was placed
on inotropic support and underwent cardiac catheterization
where an intra-aortic balloon pump was placed. Possible muscular
a neurological consult that found mild weakness (4/5 MRC) of the
deltoids and hip flexors, but was otherwise normal. An EMG
was obtained that was myopathic and biopsies of skeletal and
cardiac muscle were obtained. The patient was discharged home
on a continuous inotrope infusion. His cardiac function did not
improve and he underwent cardiac transplantation approximately
7 months later.
Biochemical and Molecular Analysis
The patient’s muscle biopsy was studied by immunostaining for
dystrophin, alpha-sarcoglycan and merosin, and immunoblotting
for dystrophin and dysferlin (CNMC IRB protocol #2405).
muscle biopsy using Genomic DNA Purification Kit by Qiagen
(Valencia, CA). Peripheral blood samples were obtained from the
patient, and his parents, and genomic DNA was isolated. Muscle
biopsy RNA was purified, and converted to cDNA. One hundred
nanograms of genomic DNA and biopsy cDNA were used for
MLPA reactions and cDNA–MLPA as previously described
[Kesari et al., 2008]. Mutation numbering is based on the DMD
coding DNA reference sequence GenBank ID NM_004006.1. The
Allele Specific Expression Assays
Applied Biosystem’s Taqman?Assays-on-Demand was used for
quantitation of normal versus mutant alleles in each sample. The
template (both cDNA and the corresponding genomic DNA) was
mixed with 900nM forward and reverse PCR primers, 200nM
fluorescent allele discrimination probes and TaqMan?Universal
Foster City, CA). Five replicates were run for each reaction.
The amplification and probe release was done in the ABI 7900
and rare alleles are taken after each of the 44 amplification cycles.
Data analysis for allele expression is done using Ct values, normal-
that same allele.
Right ventricle endomyocardial biopsies showed nonspecific
changes of focal myocyte hypertrophy with patchy subendocardial
fibrosis, and no significant inflammation. The skeletal muscle
showed mild chronic myopathic changes including fiber size
variation and increased central myonuclei. Dystrophin immnuo-
staining showed clear subpopulations of dystrophin-negative and
positive myofibers with positive fibers predominating (Fig. 1A;
left panel). Serial sections also showed secondary deficiency of
a-sarcoglycan in dystrophic negative fibers (Fig. 1A; right panel).
Both heart and skeletal muscle tissue tested negative for enter-
oviruses and influenza B.
Pathological analysis of the explanted heart showed no gross
abnormalities of the coronary arteries. There was four chamber
dilation and subendocardial fibrosis. H&E stained tissue sections
displayed myocyte hypertrophy, subendocardial fibroelastosis and
tissue of the diseased heart was available for molecular studies.
Genomic DNA and cDNA from muscle biopsy tested negative
for deletions and duplications of the 79 exons of the DMD
gene using MLPA. Complete cDNA sequencing was done, and
apparently heterozygosity for a C>T (U) change identified at
position 8713 (r.8713c>u), predicted to cause a nonsense muta-
tion (Arg2905X; Fig. 1, Panel B). This mutation was not seen in
either parent by MLPA or sequence analyses. A series of known
DMD gene polymorphisms were identified in the patient that were
shared with the mother, and were hemizygous in the patient
(Table I), proving somatic mosaicism for the mutation.
To determine if genetic normalization had occurred in the male
somatic mosaic here, we tested the ratio of normal versus mutant
genes in peripheral blood, muscle genomic DNA, and muscle
RNA (cDNA; Fig. 2). Muscle showed a much lower proportion of
mutant genes, in both DNA (20%) and cDNA (10%). This result
is consistent with genetic normalization of muscle, as previously
shown for female carriers, and may explain the mild muscle
pathology and biochemical findings.
We describe a young male adult presenting with cardiac failure
disease (dystrophy, myositis), resulted in the discovery of
mosaic immunostaining for dystrophin. Subsequent molecular
1500AMERICAN JOURNAL OF MEDICAL GENETICS PART A
dystrophin gene (Arg2905X). To our knowledge, this is the first
report of DMD somatic mosaicism in a living patient.
The young man’s presentation was most consistent with acutely
decompensated heart failure secondary to muscular dystrophy
associated dilated cardiomyopathy. A similar presentation could
be expected with viral myocarditis, but the patient had no signi-
and influenza B. The cardiac histological findings, approximately
2 weeks after presentation, do not meet the Dallas criteria for
TABLE I. Biochemical and Molecular Feature of Patient
Very mild dystrophy
and frequent variants
(p.Thr279Thr) E9; r.7096c>a
(p.Gln2366Lys) E48 FV
The reference sequence NM_004006.1 has been used to name the alterations in the sequence, FV—frequent variable as reported in the Leiden database.
Table also shows the point mutation (heterozygous) and polymorphisms and frequent variants (hemizygous) detected in the patient by cDNA sequencing.
FIG. 1. Molecular analysis of a somatic mosaic of DMD. Panel A: Shown is immunoflurorescence staining of serial sections of skeletal muscle of the
patient for dystrophin (left panel), and alpha-sarcoglycan (right panel). Arrows show dystrophin-negative fibers, and these same fibers show
secondary deficiency of alpha-sarcoglycan. Panel B: Shown is automated sequence analysis of dystrophin gene exon 59 in cDNA from the patient.
patient’s genes. Quantitative TaqMan assay data proved that the patient was a somatic mosaic for this mutation.
KESARI ET AL.
less consistent with ischemic heart disease since there were no
noregional wallmotionabnormalities on echocardiogram and the
coronaries were normal on evaluation of the explanted heart.
The patient had a mixture of normal and mutant genes in
tion was in heart. In the patient’s blood, we found the ratio of
normal/mutant to be 50:50, functionally the same as most female
carriers (due to random X inactivation) [Pegoraro et al., 1995].
Cardiac muscle shows only two or so per cardiocyte, and thus
the somatic mosaic male patient, like female carriers, would be
expected to have populations of both dystrophin-positive and
dystrophin-negative cardiocytes. Cardiocytes are less affected
by dystrophin-deficiency than myofibers, however cardiocytes
are generally incapable of regeneration leading to a late-stage
cardiomyopathy. We suspect that the acute cardiac failure seen in
the somatic mosaic patient presented here is a combination of the
failure of cardiac muscle to regenerate, and a disproportionate
number of dystrophin-negative cells comprising his heart. This
interpretation is consistent with a reported high incidence of heart
manifesting carriers, 5 of 7 ECGs were reported as abnormal, and
1990; Mirabella et al., 1993; Kinoshita et al., 1995; Politano et al.,
1996]. We cannot rule out that male sex and exercise did not
exacerbate the cardiac symptoms as well.
Of note, we found that the patient’s muscle tissue showed a
much lower relative amount of mutant genes (20%) compared to
peripheral blood (50%). There are two possible explanations for
the region of muscle biopsies. An alternative explanation is the
somatic loss (necrosis) of dystrophin-negative muscle fibers
with subsequent regeneration by dystrophin-positive stem cells.
This process has been demonstrated in the majority of manifesting
female carriers (functionally somatic mosaics, but due to
X inactivation rather than somatic mutation) [Pegoraro et al.,
1995]. This previous study found that 11/14 of clinically manifest-
ing carriers (80%) showed an average of threefold increase in
muscle dystrophin-positive nuclei compared to blood nuclei,
with dystrophin positive skeletal muscle over time (genetic
normalization). This process explains the reduction in serum
creatine kinase in female carriers with advancing age, and the
age-related improvement in clinical symptoms that can be seen
withsomemanifesting carriers[Pegoraroetal.,1995]. Hearttissue
is unable to regenerate, and thus heart is incapable of showing
genetic normalization (other than loss of dystrophin-negative
regions tofibrosis). Nonsense-mediateddecayis acommon mech-
anism by which mRNA levels that contain premature stop muta-
tions are reduced in tissue. This seems a likely explanation for the
the proportion in genomic DNA (20%).
Our data suggesting genetic normalization in skeletal muscle in
mosaics for DMD, despite the expected high incidence of such
cases based on mutation rates. The genetic normalization
process, where dystrophin-negative muscle is gradually replaced
FIG. 2. Quantitative allele discrimination assays suggests genetic normalization in muscle. Shown is the relative percentage of normal versus
mutant (Arg2905X) genes in the patient’s parents, and the patient (peripheral blood DNA, muscle DNA, muscle RNA [cDNA]). Peripheral blood DNA
shows equal proportions of normal versus mutant genes (50:50), whereas muscle genomic DNA shows a reduction in mutant genes to 20%, and
muscle RNA to 10%. These data are consistent with genetic normalization causing the muscle to become increasingly dystrophin-positive with
1502 AMERICAN JOURNAL OF MEDICAL GENETICS PART A
by dystrophin-positive muscle as a function of age, would be
expected to preclude the onset of skeletal muscular symptoms
(weakness). This is in contrast to the heart, where the lack of
regeneration in the heart prevents genetic normalization from
occurring, and then leading to preferential expression of cardiac
symptoms. This case suggests that male somatic mosaics for DMD
may present with cardiomyopathy, not skeletal muscle symptoms,
and that this diagnosis should be considered in idiopathic cardio-
myopathy. Possible screening tests for identifying male somatic
mosaics for DMD could include a persistence of the muscle (MM)
isoforms of creatine kinase (reflective of a subclinical muscular
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