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Limb-girdle weakness in a marfanoid man: distinguishing calpainopathy from Becker's muscular dystrophy
  1. Gasnat Shaboodien1,2,
  2. David A Watkins1,2,
  3. Komala Pillay3,
  4. Peter Beighton4,
  5. Jeannine M Heckmann5,
  6. Bongani M Mayosi1,2
  1. 1Cardiovascular Genetics Laboratory, Hatter Institute of Cardiovascular Research in Africa, Cape Town, South Africa
  2. 2Department of Medicine, Groote Schuur Hospital and University of Cape Town, Cape Town, South Africa
  3. 3Division of Anatomical Pathology, Department of Clinical Laboratory Sciences, National Health Laboratory Service and University of Cape Town, Cape Town, South Africa
  4. 4Division of Human Genetics, University of Cape Town, Cape Town, South Africa
  5. 5Division of Neurology, Department of Medicine, Groote Schuur Hospital and University of Cape Town, Cape Town, South Africa
  1. Correspondence to Professor Jeannine M Heckmann, Division of Neurology, University of Cape Town, E8-74, New Groote Schuur Hospital, Observatory, Cape Town 7925, South Africa; jeanine.heckmann{at}uct.ac.za

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Case report

A 24-year-old man had experienced hip-girdle weakness since the age of 15 years and exercise-induced muscle cramps with ‘toe-walking’ since aged 12 years. He was thought to have Marfan's syndrome but had no known family history of muscle disease. His marfanoid features prompted a transthoracic echocardiogram at 18 years, showing a normal aortic root and cardiac size/function without mitral valve prolapse.

On examination at the age of 24, he was tall (6 feet, 7 inches; 201 cm) with an asthenic habitus, disproportionate arm length to height (1.06), high-arched palate, arachnodactyly, marked pectus excavatum and pes planus, but no other features of Marfan's syndrome. Neuromuscular examination showed hypertrophy of calf muscles and shortening of the Achilles’ tendons as well as selective atrophy of the sternal heads of sternocleidomastoid muscles, proximal arms and quadriceps muscles. There was no scapular winging. The limb-girdle pattern of weakness was of moderate severity (Medical Research Council (MRC) grade 3) in the hip extensors and adductors, and knee flexors and mild weakness (MRC grade 4) in neck flexors, hip flexors and abductors, as well as knee extensors. The strength in other muscle groups and the remaining neurological examination, including tendon reflexes, was normal.

He remained fully ambulant and functional at the age of 31 years, albeit with a moderately waddling gait.

His 12-lead ECG at age 24 was abnormal, with a right-bundle branch pattern and a prominent R wave in chest lead V2. Echocardiography showed borderline dilatation and reduced function of both ventricles. The ECG and echocardiogram were unchanged when re-examined 7 years later.

Laboratory tests showed a serum creatine kinase of 1640 IU/L (>10 times the upper limit of normal). Biopsy of the quadriceps femoris showed an increase in endomysial connective tissue, infiltration of adipose tissue and focal necrotic fibres. Immunohistochemistry showed normal spectrin and caveolin staining but decreased intensity (∼50%) in the staining of the following proteins: dysferlin, N-, R- and C-dystrophin as well as α-, β- and γ-sacroglycan. We performed Western blotting to detect expression levels of dysferlin (using the NCL-Hamlet (230 kDa)) and calpain 3 (using NCL-Calp-2C4 (94 and 30 kDa)) antibodies. Despite normalisation of protein loading by using myosin detection levels, and normal dysferlin levels compared with control muscle, there was reduced intensity of the two calpain 3 bands: the 94 kDa band in the patient's muscle was <50% of the control muscle, and the 30 kDa bands in both patient and control were similarly faint.

Our provisional diagnosis of calpainopathy did not explain his marfanoid features or his cardiomyopathy. Calpainopathy results from recessive mutations in CAPN3 and deletions in FBN1 are known to cause Marfan's syndrome. Due to the presumed serendipitous finding of two phenotypes (Marfan's and calpain 3), both mapping to chromosome 15q15–21.1, we postulated a common genetic defect, such as a large chromosomal deletion. Using a multiplex ligation-dependent probe amplification (MLPA) kit, we screened for possible insertions, deletions or small copy number variations in the genes fibrillin 1 (FBN1), calpain 3 (CAPN3) and transforming growth factor β-receptor II (TGFBR2), but found no abnormalities. MLPA can also detect point mutations very close to the probe ligation site. The ratios approximating 1 in the Coffalyser output indicated normal function and distribution of all probes.

We proceeded with a genome-wide high-resolution single nucleotide polymorphism (SNP) array, CytoScan HD (Affymetrix), with >99% sensitivity and specificity for copy number changes >400 kilobases (kb), and the highest probe density (>2.6 million SNPs) to detect gene-level, copy-neutral loss of heterozygosity, uniparental isodisomy and regions identical-by-descent, in addition to low-level mosaicism and sample heterogeneity. We detected a 60 kb inframe deletion in the dystrophin (DMD) gene, spanning exons 48 and 49.

Discussion

We describe a young man with marfanoid features and an initial erroneous diagnosis of limb-girdle muscle dystrophy 2a (or calpainopathy), based upon misleading results from the muscle biopsy's immunohistochemistry and Western blot analysis, and because both calpain and Marfan genes are located on chromosome 15q15–21.1. Calpainopathy can show wide phenotypic variation and muscle biopsy interpretation can be problematic due to technical factors (such as rapid degradation of calpain 3) or secondary calpain 3 deficiency associated with other types of muscular dystrophies.1 After a negative mutation screen of the CAPN3 gene, a genome-wide high-resolution SNP array identified an inframe deletion of exons 48–49 in the DMD gene, which secured a diagnosis of Becker's muscular dystrophy. This case highlights several important practical points that warrant brief discussion.

Becker's and Duchenne's muscular dystrophy are allelic disorders caused by mutations in the DMD gene. Duchenne's muscular dystrophy is a severe childhood-onset disease that is fatal in early adulthood. In Becker's muscular dystrophy, the onset is later, the phenotype milder and affected men often remain ambulant until at least the third decade. Some men with Becker's muscular dystrophy remain asymptomatic into late adulthood (reviewed in2).

The DMD gene is located on the X chromosome and comprises 79 exons. The encoded protein, dystrophin, localises to the inner muscle fibre sarcolemma, where it binds to several proteins, forming the dystrophin-associated protein complex.2 DMD gene mutations affect ∼1 in 3500 live male births and new mutations in the DMD gene are not infrequent. Mutations in the DMD gene most commonly comprise inframe deletions (∼65%)2 and 70% of deletion mutations are clustered in the distal region between exons 45 and 51.3 Importantly, mutations in this region are three times more likely to be new mutations arising later in embryonic development. This situation results in somatic mosaicism and therefore may occur as sporadic cases with a low risk of recurrence (∼4%).3

In Becker's muscular dystrophy, the deletions are usually inframe and predominantly between exons 45 and 55.2 Most people with exon 45–51 deletions manifest mild phenotypes or even remain without symptoms into late adulthood.2 Reports of cases harbouring the same mutation and manifesting varied phenotypes suggest additional influences, such as epigenetic factors.2 For instance, a person with the same exon deletion and similar age at presentation as our patient had cardiomyopathy as the only manifestation of dystrophinopathy.4 Although not all persons with Becker's dystrophy develop cardiac involvement, cardiac complications frequently occur and can vary from mild electrocardiographic abnormalities to dilated cardiomyopathy developing, on average, in mid-adulthood.2

Cramps and myalgia, often induced by exercise or cold, occur in a third of people with Becker's muscular dystrophy.2 As in our patient, teenagers with Becker's dystrophy frequently develop exercise-induced muscle cramps (often involving calves), myalgia or fatigue without weakness as their first symptoms.2 Exercise-induced muscle cramps may be due to abnormal neuronal nitric oxide (nNOS)-mediated regulation of muscle blood flow during exercise.2 Although exons 42–45 of DMD encode spectrin-like repeats that are necessary for securing nNOS to the sarcolemma—and thereby facilitating the regulation of vasodilation during muscular activity—persons with deletions distal to this domain may also experience myalgia or cramps, with or without myoglobinuria (reviewed in2).

Muscle biopsies in Becker's dystrophy can show dystrophin staining patterns that vary between 50% and 100% compared with controls.2 In addition, altered dystrophin expression (in dystrophinopathies) can cause substantial secondary reductions of many of the dystrophin-associated complex components including α-, β- and γ-sarcoglycan expression.2 To complicate matters further, muscle biopsies from cases with molecularly confirmed sarcoglycanopathies, either α-, β-, γ- or δ-sarcoglycanopathy, can show a secondary reduction in the expression levels of the remaining normal sarcoglycan proteins as well as dystrophin and β-dystroglycan.5 Muscle biopsy results therefore may not accurately predict genotype. Indeed, even in specialised laboratories, the quantified analysis of calpain 3 protein expression levels lack precision; for example, of 57 cases with CAPN3 mutations, Western blotting of muscle calpain 3 was accurate in 23%.1 Furthermore, altered/reduced expression of more than one protein suggests involvement of secondary mechanisms.5

Clinical pointers towards molecularly-confirmed calpainopathy—but absent in our patient—are scapular winging and weakness of elbow flexors.1 Typically in calpainopathy, the posterior leg muscles (hip extensors and knee flexors) as well as hip adductors are the most severely affected muscle groups.1 The person adopts a characteristic broad-based stance with locked knees (relatively preserved knee extensors) and a pronounced lumbar lordosis,1 which was absent in our patient. Joint contractures (ankle dorsiflexion, finger-, wrist- and elbow flexors) occur in 68% with calpainopathy and may be severe.1 In contrast to Becker's muscle dystrophy, calf pseudohypertrophy is uncommon and cardiac function, including ECG and echocardiography, is essentially normal in calpainopathy.1

Conclusions

The important messages of this report are that unexplained symptoms of muscle fatigue, limb-girdle weakness, quadriceps myopathy, exercise-induced muscle cramps (± calf pseudohypertrophy), asymptomatic hyperCKemia, recurrent myoglobinuria or cardiomyopathy in men warrant screening for mutations in the DMD gene.2 Muscle biopsy analysis may be misleading. Molecular investigation and mutation detection facilitate accurate genetic counselling and appropriate prognostication and management.

Acknowledgments

We thank Dr Franclo Henning for performing the muscle biopsy at Tygerberg Hospital.

References

Footnotes

  • Contributors All the authors had access to the data and a role in the writing of the manuscript. GS and JMH wrote the draft manuscript. KP provided the muscle biopsy text. DAW, BMM and PB contributed to the drafting of the manuscript.

  • Funding This work was funded in part by the International Centre for Genetic Engineering and Biotechnology (ICGEB) and the Lily and Ernst Hausmann Research Trust.

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval University of Cape Town Health Sciences Research Ethics Committee.

  • Provenance and peer review Not commissioned; externally peer reviewed. This paper was reviewed by Jon Walters, Swansea, UK.

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