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|Year : 2013 | Volume
| Issue : 5 | Page : 481-487
A large series of immunohistochemically confirmed cases of congenital muscular dystrophy seen over a period of one decade
Madhu Nagappa1, Nalini Atchayaram1, Gayathri Narayanappa2
1 Department of Neurology, National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, India
2 Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, Karnataka, India
|Date of Submission||10-Apr-2013|
|Date of Decision||06-Jun-2013|
|Date of Acceptance||11-Oct-2013|
|Date of Web Publication||22-Nov-2013|
Department of Neurology, National Institute of Mental Health and Neurosciences, Bengaluru - 560 029, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Although congenital muscular dystrophies (CMD) is a common condition among primary muscle disorders, there are only a few small series reported from India. Aims, Settings, and Design: Retrospective analysis to characterize histopathologically and/or immunohistochemically confirmed cases of CMD. Materials and Methods: Patients were identified retrospectively from the archived muscle biopsy reports between 1997 and 2007 at the Department of Neuropathology of the institute. Medical records were scrutinized for all details. Results: There were 102 cases which were characterized by clinical phenotype and histopathology. Among these 56 had immunohistochemical staining and were included in the final analysis. Merosin staining performed in 53 samples identified nine patients with merosin negative CMD. The male to female ratio (M:F) was 2:1 and the mean age at presentation was 69.7 ± 62.2 months. All had grossly delayed motor milestones. There were 13 cases of Ullrich CMD confirmed by absent staining for collagen 6A1 in muscle. Mean age at diagnosis was 63.7 ± 27.9 months. Onset of symptoms was in infancy in 12 patients. All had significant delay in motor milestones and had classical features of proximal contractures, distal hyperextensibility, prominent calcaneum, velvety palms and soles with absent palmar creases. Mean creatine kinase (CK) value was 259.1 ± 109.4 IU/l. Alpha-dystroglycan (α-DG) deficiency was identified in three cases. Illness onset was in infancy. Classical magnetic resonance imaging (MRI) features were seen in all. Large group of 31 cases of merosin positive CMD had clinical findings of early onset limb weakness, hypotonia, and contractures; with histopathological evidence of dystrophy, and normal staining pattern of merosin, collagen 6A1 and α-DG. Mean age at evaluation was 58.61 ± 48.4 months. Majority (87.1%) had onset of symptom in infancy with delay in motor milestones. Conclusions: This study provides a significant data on one of the largest cohort of patients with CMDs from India. Immunohistochemistry (IHC) has definitely helped us to categorize 56 patients into specific subtypes of CMDs. This is essential for directing genetic analysis which is imperative for definitive diagnosis and also prenatal diagnosis.
Keywords: Alpha-dystroglycan, COL 6A1, congenital muscular dystrophy, merosin, Ullrich muscular dystrophy
|How to cite this article:|
Nagappa M, Atchayaram N, Narayanappa G. A large series of immunohistochemically confirmed cases of congenital muscular dystrophy seen over a period of one decade. Neurol India 2013;61:481-7
|How to cite this URL:|
Nagappa M, Atchayaram N, Narayanappa G. A large series of immunohistochemically confirmed cases of congenital muscular dystrophy seen over a period of one decade. Neurol India [serial online] 2013 [cited 2019 Feb 20];61:481-7. Available from: http://www.neurologyindia.com/text.asp?2013/61/5/481/121913
| » Introduction|| |
Congenital muscular dystrophies (CMDs) are clinically and genetically heterogeneous and one of the commonest autosomal recessive neuromuscular disorders. It was first described by Frederick Eustace Batten in 1903.  The illness manifests in children with weakness, hypotonia, and contractures from birth.  In Japan Fukuyama-type (FCMD), represents the most common form followed by collagen VI (COL6)-deficient CMD. , From India short series have been reported on various forms of CMD's. ,,,,, Specific diagnosis and subclassification of these disorders is challenging as histopathology is not distinctive. Immunohistochemistry (IHC) using a battery of antibodies can aid in the specific diagnosis and goal directed genetic testing which is confirmatory. Although CMDs are reported to be common, large studies are very few. Currently, the establishment of a molecular diagnosis is of great importance not only for phenotype-genotype correlation, genetic and prenatal counseling, prognosis, and aspects of management, but also for the imminent availability of clinical trials and treatments. There is a paucity of clinical data from India and hence, this study.
| » Materials and Methods|| |
The study was approved by the Institutional Ethics Committee. Patients were identified retrospectively from the archived muscle biopsy records from 1997 to 2007 at the Department of Neuropathology of our institute and all data were collected from the medical records. Patients with clinical evidence of very early onset generalized weakness and wasting, contractures, ophthalmologic abnormalities, elevated creatine kinase (CK) level, and abnormal brain imaging consistent with CMD were prospectively recruited from 2008 to 2010, after obtaining written informed consent for all procedures. Muscle biopsy slides were analyzed for routine histology and IHC findings.
Cryosections were utilized for enzyme histochemical staining. Paraffin sections were stained with hematoxylin and eosin (H and E), Massons trichrome (MAT), and modified Gomori trichrome (MGT). IHC was carried out using monoclonal antibodies to dystrophin (1, 2, and 3), sarcoglycans (α, β, γ, and δ), merosin, collagen 6, and alpha-dystroglycan (α-DG) as primary antibodies and peroxidase labeled LSAB+ (labeled streptavidin biotin) as secondary antibody. Final diagnosis was based on phenotypic characteristics, muscle morphologic features and IHC findings.
| » Results|| |
The entire group consisted of 102 patients with clinical and histopathological features compatible with a diagnosis of CMD. As 46 did not have IHC studies, they were excluded from the final analysis. Thus, based on IHC, 56 patients could be subclassified into four groups: (i) Merosin negative CMD; (ii) Ullrich CMD; (iii) alpha-dystroglycanopathy; and (iv) merosin positive CMD. The various features are summarized in [Table 1].
|Table 1: Clinical features of 102 cases of congenital muscular dystrophy|
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Merosin negative congenital muscular dystrophies
Of the 53 patients in whom merosin IHC was done, nine patients were negative for merosin (complete deficiency-8, partial deficiency-1). The male to female ratio (M:F) was 2:1 and the mean age at presentation was 69.7 ± 62.2 months (15 days ± 16 years). Presenting symptom was delayed acquisition of motor milestones in all, except for one male infant who presented on the 15 th day of life with stridor. Seven patients were aged 2 years or above. One patient had undescended testes. Details are represented in [Table 1]. Classical features, multiple contractures, wasting, severe weakness, and preserved mental functions were noted in all the patients [Figure 1]. Mean CK level was 860.5 ± 567.7 IU/l (207-1801). All demonstrated dystrophic features in muscle biopsy with increase in connective tissue [Figure 2]a-f.
|Figure 1: (a) Two siblings with merosin negative congenital muscular dystrophy show generalized wasting and multiple joint contractures, (b) cranial computed tomography (CT), and (c and d) T2-weighted magnetic resonance imaging (T2W MRI) show diffuse white matter abnormalities|
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|Figure 2: (a) Hematoxylin and eosin stain (H and E) showing rounding, variation in fiber size, and increased endomysial collagen. (a1) Showing normal muscle with uniform membrane labeling to merosin. (b) Patchy discontinuous labeling to merosin in all fibers indicating partial deficiency. (b1) Complete absence of merosin labeling on immunohistochemistry|
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Thirteen patients (M:F 10:3) were diagnosed to have Ullrich CMD with deficiency of collagen 6A1 in muscle biopsy. Mean age at diagnosis was 63.7 ± 27.9 months (36.0-108). Onset of symptoms was in infancy in 12 patients. Significant delay in acquiring motor milestones was present in all the patients. Salient findings are given in [Table 1]. Subluxation of joints were reported in three patients. Bifacial weakness, global areflexia, prominent calcaneum, and velvety palms and soles with absent palmar creases were noted in all the patients. Weakness of limbs was more pronounced proximally than distally and distal hyperextensibility was seen in the upper limbs in 11 patients and in lower limbs in nine patients [Figure 3]. Mean CK value was 259.1 ± 109.4 IU/l (117-518). In addition to classical dystrophic features, moth eaten fibers and lobulated fibers were found in four and three patients, respectively. IHC studies using monoclonal antibodies to collagen 6A1 (COL6A1) revealed complete absence of staining in eight patients, and sarcolemmal specific deficiency in the remaining [Figure 4]. There was no difference in clinical or pathological findings between these two groups.
|Figure 3: A 4-year-old girl with Ullrich congenital muscular dystrophy. (a) Proximal contractures at elbows, hips, and knees. (b-d) Hyperextensibility at toes, ankles, and fingers. (e) Prominent calcaneum. (f) Soft velvety palmar skin. (g and h) Faint major creases and fine mesh like creases in palm and sole|
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|Figure 4: (a) H and E showing normal polygonal fibers and (b) normal labeling to COL6A1. (c and e) H and E stain showing rounding, variation in diameter, and increased endomysial collagen. (d) Sarcolemmal specific COL6A1 deficiency. (f) Total absence of COL6A1|
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Of the 13 patients in whom IHC for α-DG was done, three (M:F = 2:1) patients had absent staining [Figure 5]. All the three patients had normal staining for merosin and COL6A1 and the symptoms onset was in infancy with severe delay in motor, language, and social milestones. 
|Figure 5: (a) H and E showing rounding and variation in fiber diameter. (b) Total absence of ƒ¿-DG in all fibers|
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Merosin positive congenital muscular dystrophies
Patients diagnosed to have CMD on the basis of clinical findings of early onset limb weakness, hypotonia, and contractures; histopathological evidence of dystrophy and normal staining pattern of merosin, COL6A1, and α-DG were included in this group. There were 31 patients with 21 males. Mean age at evaluation was 58.61 ± 48.4 months (9 months-16 years). Majority (87.1%) had onset of symptom in infancy with delayed acquisition of motor milestones. Delay in language and social milestones was noted in five of 28 (17.9%) and in three of 28 (10.7%) patients, respectively. Six patients had feeding difficulty and five had recurrent lower respiratory tract infection. One patient had primary generalized seizures. Mean CK level was 1,689.5 ± 3,302.5 IU/l (53-16,500). There were 10 patients whose CK was above 1,000 IU/l. IHC for merosin, COL 6, and α-DG showed positive staining.
| » Discussion|| |
Congenital muscular dystrophies are autosomal recessive (AR) disorders, with considerable heterogeneity in clinical presentation, severity, and evolution.  The classification of CMD's has undergone several revisions; the most recent one is based on the defects in various genetic and biochemical pathways. Besides it is well-recognized that some forms of CMD's have characteristic features permitting a clinical diagnosis, and which can be further supported by IHC on muscle biopsy and/or genetic studies. , Although CMD's are one of the commonest AR inherited neuromuscular disorders, large studies are very few.
Merosin negative CMD, first described by Tomé, et al.,  is often cited to be the single most common form of CMD, [15a],[15b] with a relatively homogenous presentation characterized by severe neonatal hypotonia, joint contractures, marked delay in motor milestones, elevated serum CK, and abnormal white matter signal changes on brain imaging.  Regional variation in the frequency of merosin-negative CMD has been recognized. , The clinical findings in our patients were consistent with the described features in other studies. , However, at the time of evaluation the age range was wide, (15 days to more than 10 years). One of the older patients had partial merosin deficiency possibly accounting for the milder course. Clinically, milder phenotypes resulting from partial merosin expression have been described earlier. , None of the patients in our cohort achieved the ability to stand independently or walk, although there are rare reports of child walking with support transiently.  Epilepsy was noted in one patient, however other rare features such as ophthalmoplegia,  reduced cardiac function,  and severe mental subnormality  described previously were not observed in our cohort. Abnormalities of white matter in brain imaging is said to be invariably found in patients with merosin negative CMD after the age of 6 months,  and this may be a clue in patients with partial deficiency and mild phenotypes.  In the current study, magnetic resonance imaging (MRI) done in four patients including the patient with partial deficiency, between the ages of 9 months and 10 years, showed extensive periventricular and subcortical white matter abnormalities. Rare imaging findings include cortical anomaly and polymicrogyria. 
Thirteen patients had the classical clinical features of Ullrich CMD and were confirmed on IHC. Ullrich CMD is the second most common CMD described in literature. In our study, this was the commonest type. Our cohort had a uniform phenotype with the presence of proximal contractures, striking distal laxity, generalized thin habitus, global areflexia, velvety palms, and soles with mesh-like creases replacing the major creases of palms and soles and prominent calcaneum. There was no difference noted between patients with complete vs sarcolemmal specific deficiency of Col 6A1. The specific and notable finding on routine histopathology was the presence of clumped and discontinuous collagen. The clinical feature of nine of these patients has been described in an earlier report.  Functionally these patients fared better and seven of them attained ability to stand and walk short distances.
Alpha-dystroglycanopathies are characterized by the presence of characteristic central nervous system abnormalities coupled with ocular and muscle abnormalities, caused by genes involved in the glycosylation of α-DG.  In the current study, five patients presented with clinical features of α-DG and in three, IHC confirmed the absence of α-DG. These three cases have been reported earlier. 
Merosin positive CMD is a clinically heterogenous group, with milder manifestations and more slow progression compared to merosin negative group. There are no specific characteristics or salient dysmorphic features described in this group.  In the current 31 of 56 cases with normal expression of merosin, collagen 6A1 and α-DG were subclassified as merosin positive CMD. Sensitive techniques like immunoblot to detect deficiency of α-DG and genetic studies do help in better categorization of such patients is necessary.
Minimal reduction of COL 6A1 may be difficult to identify. In one study, [15b] IHC reduction of COL 6 was observed in only six patients and it was noted that age and presentation at onset, distribution, and severity of weakness and contractures, and frequency of non-ambulant patients were similar in patients with and without COL 6 involvement. Hence, COL 6 deficiency may not be a uniform feature of all patients with Ullrich CMD. In the current study, antibodies to collagen 6A1 only were used. The involvement of other collagen peptides viz 6A2 and A3, cannot be ruled out.
Until the last decade, the diagnosis of CMD was based on a combination of clinical signs coupled with the presence of dystrophic changes in muscle biopsy. The discovery of patients with deficient merosin staining and the fact that such patients formed a homogenous group allowed the first classification of CMD's. Thereafter, 13 different types of CMD's based on specific protein deficit and gene defect have been identified. Despite these advances, there are still a large number of patients who fulfill the clinical criteria for CMD's, but do not have defects in the genes described so far.
The main drawback of the study is the retrospective nature. Also IHC could not be performed in a proportion of patients due to nonavailability of stored muscle tissue. Genetic testing, which is the confirmatory test could not be done in this cohort. Follow-up was limited which could be attributed to lack of motivation among parents as these disorders are untreatable and also have limited survival. Despite these shortcomings, this study provides a significant data on one of the largest cohort of patients with CMD's from India. IHC has definitely helped us to categorize 56 patients in this study into specific subtypes of CMD. This is essential for directing genetic analysis which is essential for a definitive diagnosis. Further studies are necessary to characterize the natural outcome including survival patterns and correlation with pathogenic mutations in this group of disorders.
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