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Anovel X-linked form of congenital fiber-type disproportion.

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A Novel X-Linked Form of Congenital
Fiber-Type Disproportion
Nigel F. Clarke, FRACP,1 Robert L. L. Smith, FRACP,2 Melanie Bahlo, PhD,3 and Kathryn N. North, MD1
We describe a four-generation family with a previously unreported form of congenital fiber-type disproportion that
follows an X-linked inheritance pattern. Affected male family members have a striking pattern of weakness. From birth
there is marked ptosis, facial weakness, poor sucking, hypotonia, respiratory weakness, and relatively preserved limb
strength. Most affected male individuals die of respiratory failure within the first months of life. A mild dilated cardiomyopathy developed in infancy in the sole surviving affected male member of this family. Some carrier female individuals
manifest milder signs. We have demonstrated linkage to two regions of the X chromosome, Xp22.13 to Xp11.4 and
Xq13.1 to Xq22.1, with a maximum logarithm of odds score of 3.25 in the latter region. We propose that clinical clues
can differentiate this disorder from other forms of congenital fiber-type disproportion so that affected families can receive
appropriate genetic counseling.
Ann Neurol 2005;58:767–772
Congenital fiber-type disproportion (CFTD) is a rare
form of congenital myopathy in which the defining
pathological feature is that type 1 (slow twitch) muscle
fibers are at least 12% smaller than type 2 (fast twitch)
muscle fibers as the main histological abnormality.1,2
CFTD is likely to be genetically heterogeneous with
reported sporadic cases and families suggestive of either
autosomal dominant or autosomal recessive inheritance.3–7 Only one genetic cause has been identified to
date. Heterozygous mutations in the ACTA1 gene, encoding ␣-skeletal actin, were found in three sporadic
cases with severe congenital onset weakness.8 Until
now there have been no reports of families that follow
X-linked inheritance.2 Here, we report a family with
CFTD associated with a distinctive pattern of weakness
and a high risk for death from respiratory failure during the neonatal period in affected male family members.
Subjects and Methods
This study was conducted as part of a research project into
CFTD that was approved by the ethics committees of the
Children’s Hospital at Westmead (ID: 2000/068), the
Hunter Area Health Service (ID: 02/09/11/3.09), and the
University of Sydney (ID: 01/11/50). We identified a large
family in whom severe congenital muscle weakness followed
an X-linked inheritance pattern (Fig 1). Seventeen of the 22
From the 1Institute for Neuromuscular Research, Children’s Hospital at Westmead, Discipline of Paediatrics and Child Health, University of Sydney, Sydney; 2John Hunter Children’s Hospital and
University Discipline of Paediatrics and Child Health, Newcastle;
New South Wales; and 3The Walter and Eliza Hall Institute of
Medical Research, Melbourne, Victoria, Australia.
living blood relatives who provided DNA samples were examined by at least one of the authors; written consent was
obtained before blood collection. Genomic DNA was extracted from circulating blood lymphocytes using standard
protocols in all but one case; DNA for this subject was obtained from an archived frozen muscle biopsy. Muscle biopsies had standard histochemical stains, and fiber measurements were performed from frozen muscle biopsies stained
for ATPase after preincubation at pH 4.3 and 4.6.
Linkage Analysis
Forty-eight dinucleotide microsatellite markers on the X
chromosome were selected from the HD-5 ABI PRISM
Linkage Mapping Set v2.5 (Applied Biosystems, Foster City,
CA). Genotyping was performed using the ABI 377 DNA
sequencers, and alleles were called with the ABI Genotyper
V2.1 software using standard procedures. Genotyping data
were examined with PEDCHECK, which detects genotyping
errors through violation of Mendelian inheritance rules.9 In
the absence of reliable estimates for the allele frequencies,
each microsatellite marker was assumed to have 10 alleles
with equal frequency of 0.1. The marker map was estimated
using the DeCode genetic map and the University of California Santa Cruz physical map.10,11
We performed both parametric and nonparametric linkage
analysis with the software ALLEGRO.12 Nonparametric
linkage analysis was performed with the sharing statistic,
Srobdom, suitable for dominant disease models. Parametric
Address correspondence to Dr North, Children’s Hospital at Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia.
Published online Sep 19, 2005, in Wiley InterScience
( DOI: 10.1002/ana.20644
Received Feb 22, 2005, and in revised form Jul 23. Accepted for
publication Jul 29, 2005.
© 2005 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Fig 1. Family tree of reported family.
analysis of X chromosome linkage data requires sex-specific
disease penetrance models. We assumed 100% penetrance
for male subjects. Two models were tried for the penetrance
in female subjects. The first model had 80% penetrance for
both heterozygotes and homozygotes for the disease allele
and a 5% phenocopy rate (Model 1). The second model had
90% penetrance for the heterozygotes and homozygotes and
a reduced phenocopy rate of 1% (Model 2). The disease allele frequency was assumed to be 0.0001, reflecting a rare
X-linked dominant disorder.
Case Reports
AFFECTED MALE SUBJECTS. There have been seven
male members with a clinical myopathy in this family
(see Fig 1). One further male infant (Subject II:5) died
of uncertain causes during the neonatal period and was
likely to have had the same condition, but clinical information is unavailable.
Subject IV:1 is the only affected male member to
have lived beyond 4 months old; he is now 5.5 years
old (Fig 2). Pregnancy and delivery were uneventful,
and birth weight, length, and head circumference were
in the 3rd to 10th centile. At birth he had bilateral
ptosis, facial weakness, generalized hypotonia, and mild
generalized weakness. He required oxygen supplementation for several weeks but was never intubated. He
was tube-fed for 3 weeks and had difficulty sucking
from the bottle thereafter. A chest radiograph showed
hypoplasia of both the lung fields and second ribs bilaterally. A gastrostomy tube was sited at age 1 year for
food refusal (with no clear dysphagia). As an infant he
underwent surgeries for bilateral ptosis and undescended testes. He rolled at 9 months old and walked
independently at 17 months old. There was a mild delay in the acquisition of fine motor skills and both expressive and receptive language. He still receives bolus
high-energy food supplementation via a gastrostomy
Annals of Neurology
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tube and eats small amounts. He is in the third centile
for height and head circumference and is significantly
below the first centile for weight. Mild dilated cardiomyopathy was first identified at age 3.5 years and has
remained stable. At age 5.5 years he has micrognathia,
bilateral ptosis, full extraocular movements, frontalis
weakness, moderate lower facial weakness, a horizontal
smile, and an immobile upper lip. He can manage a
fast walk, is just able to jump, and does not have a
Gowers’ sign on rising from supine to standing. He has
mild generalized limb weakness and relatively rigidity
of the lower spine but no scoliosis or other joint contractures.
Repeated measures of serum creatine kinase and lactate have been normal. A modified barium swallow at
age 5 years showed a mild delay and increased fatigability swallowing thin fluids but no aspiration. Red
blood cell Kell antigens were normal. Results of nerve
conduction studies (including repetitive nerve stimulation) and electromyography were normal. Echocardiography at age 4.5 years showed a mild dilated cardiomyopathy with left ventricle fractional shortening of
29% (lower limit of normal) and mild left ventricular
Subjects III:1, III:7, IV:2, IV:3, IV:4, and IV:5 had
similar clinical presentations in the neonatal period. All
were born between 30 and 37 weeks gestation by normal vaginal delivery, and most pregnancies were complicated by polyhydramnios in the final weeks. All male
infants required either intubation or continuous positive airway pressure within an hour of birth. Typically
they were hypotonic and had marked bilateral ptosis,
moderate facial weakness, poor suck, micrognathia, a
weak gag reflex, and a weak cry. None had ophthalmoplegia, and all had relatively preserved limb strength
with antigravity movements. All required tube feeding
and drooled prominently. One infant had bilateral flex-
Fig 2. Subject III:5 (left) has absent frontalis movement and mild bilateral ptosis (with previous ptosis surgery on the right). Subject IV:1 (right) has bilateral ptosis (after bilateral ptosis surgery), moderate generalized facial weakness, a horizontal smile, and
ion contractures of the index fingers and mild contractures of other fingers. Most affected male infants had
small chests and hypoplastic lung fields on chest radiograph. Echocardiograms on four affected male members were normal in the neonatal period. Subject III:1
died of respiratory failure 12 hours after birth. Subject
IV:3 was born at 30 weeks gestation and died of respiratory failure at 3 hours old. Subjects III:7, IV:2, IV:4,
and IV:5 were initially stabilized on continuous positive airway pressure and at times required only oxygen
by nasal cannula. The respiratory function gradually
worsened in each infant, and despite intubation, they
died of respiratory failure between ages 6 and 14
Two women in the family
(Subjects II:3 and III:5) are obligate carriers because
they have had affected male children and both have
mild weakness. Subject I:2 is also likely to be a carrier
because she had a male child who died of unknown
causes in the neonatal period. When examined at age
81 years, she was frail with an unsteady, broad-based
gait, but she had no significant facial or limb weakness.
Subject II:3 was born at term by breech delivery, but
she was well at birth. She has had lifelong facial asymmetry and required a right ptosis operation in childhood. Her motor development was otherwise normal.
In adulthood, bilateral ptosis gradually worsened, and
she noticed difficulty climbing stairs and right facial
numbness. At age 48 years she had bilateral ptosis,
minimal movement of frontalis, resting facial asymme-
try, reduced movement of the right lower face, mild
thoracic kyphosis, moderate obesity, and difficulty
squatting but no definite limb weakness or reduced reflexes in her upper limbs and knees. Results of nerve
conduction studies were normal. Small polyphasic
units and rapid recruitment were present in the deltoid
and triceps, consistent with a myopathy. Brain magnetic resonance imaging was normal.
Subject III:5 was described as a “small, sickly child”
with a poor appetite. She underwent the first of several
operations to correct a right ptosis at age 10 months.
As a child, she was poor at sports, but motor developmental milestones were normal. As an adult, she has
reduced exercise tolerance and uses handrails for stairs.
At age 24 years she had a flat forehead with absent
frontalis movement, bilateral ptosis, normal lower facial
movements, mild obesity, mild proximal limb weakness, and reduced knee and ankle reflexes (see Fig 2). A
karyotype from blood lymphocytes was normal.
All other close adult female relatives (except for one)
were examined, and none had definite myopathic signs.
Muscle Histology
A total of six muscle biopsies were taken from five affected male members; the fiber size parameters of five
biopsies are reported in the Table. Four of six biopsies
were consistent with CFTD in that type 1 fibers were at
least 12% smaller than type 2 fibers as the primary histological abnormality (Fig 3). In three of these four biopsies, hypertrophy of type 2 fibers accounted for the
difference in size between fiber types. In one biopsy, hy-
Clarke et al: A Novel X-Linked Form of CFTD
Table. Muscle Fiber Measurements
Type 1 Fibers
Type 2A Fibers
Type 2B Fibers
Age at
Diam (SD)
Diam (SD)
Diam (SD)
2 mo
1 yr
2.8 yr
3 mo
5 weeks
46 yr
9 (1.5)
19.7 (4.4)
23.8 (6.2)
12.7 (1.8)
13.4 (3.0)
41.8 (7.9)
13.5 (2)
27.3 (4.3)
29.2 (5.8)
13.1 (1.6)
19.4 (3.4)
47.0 (8.6)
13.4 (2.4)
29.1 (3.0)
12.6 (0.9)
Type 2C
Percentage of total fibers.
%FSD ⫽ percentage fiber-size disproportion ⫽ 100 ⫻ (mean type 2 diameter ⫺ mean type 1 diameter)/mean type 2 diameter.
Type 2 fiber subtyping was not possible. Total type 2 fibers are recorded under type 2A fibers.
Diam ⫽ mean diameter; Quad ⫽ quadriceps; FSD ⫽ fiber-size disproportion; SD ⫽ standard deviation.
potrophy of type 1 fibers accounted for the difference.
Of the two remaining biopsies, the biopsy from Subject
IV:2 showed only a 3% difference in size between type 1
and 2 fibers. Type 1 and 2 fibers could not be distinguished reliably in the biopsy from Subject IV:3 (taken
at 30 weeks gestation), probably because of immaturity.
In general, darker fibers were much smaller than paler
fibers on ATPase stains (with preincubation at pH 4.3),
which is consistent with the pattern seen in CFTD. In
none of the biopsies were increased central nuclei, degenerating or regenerating fibers, increased connective
tissue, nemaline bodies, or cores noted. In Subject IV:1,
electron microscopy, mitochondrial enzyme analysis, and
immunohistochemistry for dystrophin, sarcoglycans (␣,
␤, ␥, ␦), laminin-␣2, ␤-dystroglycan, and caveolin-3
were normal.
Two carrier women (Subjects II:3 and III:5) had a
total of three muscle biopsies that showed increased
variation in fiber size or type 1 fiber predominance but
no other abnormalities.
Linkage Analysis
Nonparametric linkage analysis defined two regions of
maximum non-parametric linkage score (Zmean ⫽
8.27) on the X chromosome. All affected male members and obligate carrier female members shared a large
portion of the X chromosome, except for one male
member (Subject III:7) who had two crossovers within
this region, thereby excluding the central portion. This
accounts for the presence of two candidate regions in
the nonparametric analysis. These two regions are
Xp22.13 to Xp11.4 (between markers DXS8019 and
DXS993, spanning a region of 34cM) and Xq13.1 to
Xq22.1 (between markers DXS1216 and DXS8020,
spanning 18cM). These regions encompass at least 320
known genes. Parametric analysis took into account the
presence of four asymptomatic female members who
shared portions of the candidate regions and identified
the second region as being the one most likely to har-
Annals of Neurology
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November 2005
bor the disease gene. Maximum logarithm of odds
scores were 3.25 (adjusted p ⫽ 0.01) and 3.17 (adjusted p ⫽ 0.01) for parametric Models 1 and 2, respectively. Haplotype analysis confirmed these results.
We sequenced the coding regions of five candidate
genes within the candidate regions (SMPX, ITGB1BP2,
PDHA1, PDK3, and AFX1) but found no mutations.
The gene associated with X-linked myotubular myopathy (MTM1) is located at Xq28, which is well outside
the candidate regions.
Affected male members in this family fulfill the two
central diagnostic features of CFTD: Type 1 fibers are,
on average, at least 12% smaller than type 2 fibers as
the primary histological abnormality, and there are
clinical features of a congenital myopathy without evidence of other neuromuscular pathology. The oldest
surviving affected male member (Subject IV:1) has
been investigated extensively for alternative causes of
small type 1 fibers.2 There is no evidence of a peripheral neuropathy, neuromuscular junction abnormality,
muscular dystrophy, or central nervous system dysfunction in this boy (or in his deceased brothers). The cardiac involvement in Subject IV:1 also supports a primary myopathic process.
CFTD is a heterogenous condition regarding both
clinical course and genetic basis, and this report provides
further evidence of that. Most cases of CFTD follow a
relatively benign course with generalized or proximal
limb weakness that improves with age.2 Severe weakness
with respiratory muscle involvement in the neonatal period has been reported, but usually the weakness is generalized. Recently, Laing and colleagues8 identified mutations in the ACTA1 gene in three such patients. The
family reported here differs from previously reported
CFTD patients with severe disease by having a consistent, distinctive pattern of weakness. We believe the pattern of marked ptosis, facial weakness (particularly of
Fig 3. ATPase stains with preincubation at pH 4.3. Type 1 fibers are dark, type 2 (A⫹B) fibers are pale, and type 2C fibers are
intermediate. (A) Muscle from Subject IV:1 taken at age 1 year. (B) Muscle from Subject III:7.
frontalis), and respiratory muscle weakness, together
with relatively strong limbs in the neonatal period, is an
important clinical clue to this disorder. Cardiac involvement also sets this family apart from most other CFTD
patients, although the dilated cardiomyopathy was only
seen in the surviving affected male member from age 3.5
years and was not apparent in the neonatal period. A
dilated cardiomyopathy has been reported in only one
other case of CFTD.13
X-linked CFTD is not fully penetrant for female individuals because only two of the three obligate carrier
women in our family had myopathic signs. The degree
of penetrance in carrier women cannot be accurately
determined because of small numbers. This information is important for neurologists and geneticists who
must advise CFTD families on the recurrence risk in
further pregnancies, because women who carry
X-linked CFTD have a one in four chance of having
another affected male child each time they conceive.
The lack of unaffected male members in the affected
branch of this family is likely to be a chance occurrence.
We believe this family has a previously unreported
X-linked disorder. Affected male members share several
clinical and histological characteristics in common with
X-linked myotubular myopathy, but linkage analysis
conclusively excludes the MTM1 gene as causing disease
in this family. The genes for two other X-linked myopathies lie within the candidate regions. We consider the
DMD and XK genes to be unlikely candidate genes for
X-linked CFTD because of the marked differences in
clinical course between our family and either Duchenne
muscular dystrophy or McLeod neuroacanthocytosis, together with the normal dystrophin immunohistochemis-
try and Kell antigen test result.14 Similarly, we consider
it unlikely that X-linked CFTD is allelic to X-linked episodic weakness due to significant differences in the phenotypes.15 The genes for Danon’s disease (LAMP2),
Kennedy’s disease (SMAX1), and X-linked dominant
congenital isolated ptosis16 are outside the candidate regions for X-linked CFTD.
The linkage analysis results suggest that X-linked
CFTD is likely to be caused by a novel disease gene.
Because many factors can alter fiber size, it is difficult
to predict the causative gene from our knowledge of
muscle biology. The best hope for identifying the gene
responsible for X-linked CFTD is likely to be through
refining the candidate regions using linkage analysis in
other families.
Genotyping was performed at the Australian Genome Research Facility (AGRF), Melbourne, Australia, which receives support from
the Australian Commonwealth Government. This research was supported by the National Health and Medical Research Council of
Australia (206529) and the Muscular Dystrophy Association of New
South Wales, Australia (N.F.C).
We thank Dr M. Ryan for performing the repetitive stimulation
nerve conduction tests, Dr G. K. Herkes for contributing clinical
information, Dr N. Yang for his assistance in sequencing candidate
genes, and J. Silver for his contributions to the linkage analysis.
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