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. E-mail: firstname.lastname@example.org Published online Sep 19, 2005, in Wiley InterScience (www.interscience.wiley.com). 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 767 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. Results 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 768 Annals of Neurology Vol 58 No 5 November 2005 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 dilatation. 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 micrognathia. 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 weeks. 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- CARRIER FEMALE SUBJECTS. 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 769 Table. Muscle Fiber Measurements Type 1 Fibers Patient No. III:7 IV:1:A IV:1:B IV:2 IV:8 II:3 Sex M M M M F Type 2A Fibers Type 2B Fibers Age at Biopsy Biopsy Site %a Diam (SD) % Diam (SD) % Diam (SD) 2 mo 1 yr 2.8 yr 3 mo 5 weeks 46 yr Uncertain Deltoid Quad Quad Quad Uncertain 48 59 58 55 42 63 9 (1.5) 19.7 (4.4) 23.8 (6.2) 12.7 (1.8) 13.4 (3.0) 41.8 (7.9) 21 25 (42c) 43 (58c) 37 13.5 (2) 27.3 (4.3) 29.2 (5.8) 13.1 (1.6) 19.4 (3.4) 47.0 (8.6) 19 16 13.4 (2.4) 29.1 (3.0) 1 12.6 (0.9) c c 0 Type 2C Fibers (%) 12 0 c 1.4 c 0 %FSDb 33 30 18.5 3 31 11 a 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. b c 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- 770 Annals of Neurology Vol 58 No 5 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. Discussion 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. References 1. Brooke MH. Congenital fiber type disproportion. In: Kakulas BA, ed. Clinical studies in myology. Proceedings of the 2nd International Congress on Muscle Diseases, Perth, Australia, Nov. 22-29, 1971. 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