Limb girdle muscular dystrophy type 2A in India: A study based on semi-quantitative protein analysis, with clinical and histopathological correlation
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.68675
Source of Support: None, Conflict of Interest: None
Background : Limb girdle muscular dystrophy (LGMD) type 2A is caused by mutation in the gene encoding for calpain-3 resulting in total or partial loss of protein. Diagnosis of LGMD2A, the most prevalent form of LGMD, is established by analyzing calpain-3 protein deficiency or CAPN3 gene mutation. Since there is no data from India regarding the incidence of LGMD2A, this study was undertaken. Aims : To study the frequency of LGMD2A in Indian population on the basis of protein analysis by immunoblotting and to correlate pathological and clinical features with protein analysis. Settings and Design : One hundred and seventy-one muscle biopsies of clinically suspected LGMD, unclassified muscular dystrophy or myopathy were analyzed in a tertiary national referral centre for neurosciences. Materials and Methods : Histopathological, immunohistochemical and enzyme histochemical analysis of muscle biopsies was performed followed by protein analysis for calpain-3 and dysferlin by immunoblotting. Results : Immunoblot identified 75 patients (43.8%) with calpain-3 deficiency, of which 36 (45%) had complete loss and 39 (55%) had partial loss of calpain-3 protein. In patients with LGMD phenotype alone, the incidence of LGMD2A was 47%. The biopsies of these patients displayed variety of morphological changes ranging from dystrophic pattern with presence of active fibre necrosis, regeneration and lobulated fibres to end stage muscle disease. The mean age of presentation and disease onset was 24 and 18 years respectively. Conclusions : This series of 75 patients is probably the first confirmed cases of LGMD2A (calpainopathy) from India. Our study suggests that LGMD2A is the most frequent form of LGMD in India, similar to the Western data, thus, highlighting the importance of immunoblotting for an accurate diagnosis.
Keywords: LGMD2A, calpain-3, calpainopathy, immunoblotting, immunohistochemistry, lobulated fibers, India
Muscular dystrophies comprise a genetically heterogeneous group of muscle disorders characterized by progressive muscle wasting and weakness. Limb girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive form of LGMD characterized by symmetrical and selective weakness of pelvic, scapular, and trunk muscles, and moderate to gross elevation of serum creatine kinase (CK). The course of the disease is highly variable. , The gluteus maximus, thigh adductors, and posterior compartment of the limbs are the most commonly affected muscles. ,,, Facial and neck muscles are usually spared, while calf hypertrophy is very uncommon. In the advanced stage of the disease, climbing stairs, rising up from the chair, or getting up from the floor are the difficulties. Joint contractures are also common features.  Rarely, there can be only distal muscle weakness.  The disease is invariably progressive, and loss of ambulation occurs approximately 10-30 years after the onset of symptoms. ,, The electromyogram (EMG) pattern is typically myopathic, although a normal EMG can also be observed in presymptomatic individuals. However, establishing a diagnosis of LGMD2A on firm grounds on the basis of clinical features alone is difficult at times. Clinically, involvement of the posterior compartment of the thigh is the best clue, but it may not be present at the later stages.
Some studies on small groups of sarcoglycanopathies and dyferlinopathies are available from India but, in the absence of calpain-3 and dysferlin immunoblotting, it is impossible to assess the prevalence of LGMD2A and different forms of LGMD. ,,,, The present study was conducted to ascertain the frequency of LGMD2A among patients with a clinical phenotype of LGMD, unclassified muscular dystrophy, and myopathy.
Patient inclusion criteria
Patients fulfilling the following criteria were included: (i) patients with a clinical phenotype consistent with LGMD but with biopsies showing normal dystrophin, sarcoglycans, dysferlin, emerin, lamin A/C, caveolin, and merosin; (ii) muscle histopathology consistent with a dystrophic process; (iii) moderate to highly increase serum CK level; (iv) EMG findings consistent with myopathic pattern; (v) patients with unclassified muscular dystrophy or myopathy; and (vi) patients having distal myopathy with dystrophic features (excluding the known causes of distal myopathies such as Miyoshi and Nonaka type distal myopathies).
Patient exclusion criteria
Patients of all other types of muscular dystrophies: (i) Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), fascioscapulohumeral muscular dystrophy(FSHMD), and Emery-Dreifuss muscular dystrophy More Details and other dystrophies; (ii) patients showing features of neurogenic atrophy on muscle biopsy; (iii) patients with inflammatory myopathies; and (iv) patients with congenital and metabolic myopathies.
One hundred and seventy-one patients meeting the inclusion and exclusion criteria were included for the analysis. Clinical phenotypes were: LGMD (124), unclassified proximal myopathy (26), unclassified muscular dystrophy with elevated serum CK (12), and distal myopathy (9). As controls for immunoblot, 29 muscle biopsies with clinically/histopathologically and/or molecularly diagnosed diseases were used. In these cases, calpain-3 and dysferlin were normal on immunoblot. These cases included: DMD (5), unclassified muscular dystrophies showing normal calpain-3 and dysferlin equivalent to other control subjects (8), inflammatory myopathy (7), sarcoglycanopathy (2), FSHMD (4), and metabolic myopathy (3).
The biopsy from the left vastus lateralis was obtained under local anaesthesia after taking written informed consent from patients or their parents. Each muscle biopsy specimen was received in a fresh state without any fixative or additive. One small piece was fixed in 10% neutral buffered formalin, routinely processed, and paraffin embedded. Five-micron thick sections were cut and stained with hematoxylin and eosin. A second, bigger, piece was immediately snap frozen in isopentane precooled in liquid nitrogen at −80°C. Serial sections of 5-μm thickness were cut for routine immunohistochemistry and enzyme histochemistry.
Immunohistochemistry (IHC) and enzyme histochemistry (EHC)
Various histochemical stains were done to exclude other muscle diseases. These included: modified Gomori trichrome (MGT); oil red O; ATPase at pH 9.6, 4.6, and 4.3; nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR); succinic dehydrogenase (SDH), alone as well as combined with cytochrome oxidase (cox with SDH); myophosphorylase amylopectinase, phosphofructokinase (PFK); and adenylate deaminase. Immunohistochemical analysis was performed by streptavidin-biotin immunoperoxidase complex method using various monoclonal antibodies (obtained from M/s Novocastra, UK). These antibodies included dystrophin 1, 2, 3; sarcoglycans α, β, γ, δ; emerin; lamin A/C; dysferlin (Hamlet 1); caveolin; and merosin.
Muscle biopsy sections were homogenized in treatment buffer (0.125 M Tris, 10% glycerol, 4% SDS, and 0.001% bromophenol blue; pH 8.0), kept on a heating block at 100°C for 3 minutes, and centrifuged for 15 minutes at 4°C and 13000 rpm. The supernatant was taken out and protein was estimated using standard Bradford assay (Bio-Rad). A volume corresponding to 30-40 μg of protein was loaded in each lane of 1.5-mm-thick 8% polyacrylamide gel. Proteins were separated by SDS electrophoresis (80-100 Volt current for 3 hours) and electroblotted (25 V constant) to nitrocellulose membrane for 13 minutes in a dry blotting system (Invitrogen). Post transfer gels were stained for 1 hour with Coomassie blue (Bio-Rad). Nitrocellulose membrane blots were blocked overnight with 5% non-fat milk dissolved in phosphate-buffered saline with Tween-20, and then cut into two pieces according to the reference prestained molecular mass bands, so that calpain-3 (molecular weight: 94 kD) remained in one half and dysferlin (molecular weight: 237 kD) in other half. The membranes were processed separately. Anti-calpain-3 (diluted 1:100) (Calp3c/12A2, Novocastra, UK) and anti-dysferlin (diluted 1:50) (NCL-Hamlet-1, Novocastra, UK) antibodies were incubated at room temperature for 2 hours with shaking. Immunoreactive bands were detected using appropriate alkaline phosphatase-conjugated secondary antibody from Dako ( diluted 1:300 PBST prepared in 5% non-fat milk for 1 hour). Subsequently membrane was developed by incubating with BCIP and NBT (Promega, CA, USA) for 5-10 minutes. The amount of protein in patients was determined by densitometry (Alpha Innotech software v 126.96.36.199) and expressed as a percentage of control.
Appropriate statistical analysis was done wherever required. Statistical significance was established at P<.05.
The mean age at presentation was 24 years (range: 3-59 years) and the mean age at onset of symptoms was 18 years (range: 2-49 years). On the basis of age at onset, patients were categorized as early (<12 years), adult (aged 12-30 years), and late (>30 years).  Thirty-two patients (43%) had early onset, 33 (44%) had adult onset, and ten (13%) had late onset. Among the 75 calpain-3-deficient patients, there was a male preponderance (M:F ratio of 1.8:1). The mean age at presentation and age at onset of symptoms was slightly higher in males (24 and 20 years, respectively, in males vs 19 and 13.5 years in females). In 90% of patients, the age at presentation ranged from 3 to 40 years, with a mean age of 21 years and the mean age at onset was 14 years. Among the 75 calpain-3-deficient patients the clinical presentation was: LGMD phenotype in 64 (85%) patients, distal myopathy in three (4%), and pseudometabolic myopathy with myalgia in one (1.3 %); the remaining seven (9%) were unclassified muscular dystrophies/myopathies. Advanced stage symptoms, such as difficulty in climbing stairs, rising up from a chair, or getting up from the floor, were observed in 17 (23%) patients. Laxity of the abdominal muscles was observed in 2 (3%) patients, calf hypertrophy in 9 (12%), scapular winging in 12 (16%), and joint contracture in 7 (9%) patients. Involvement of the posterior compartment of the thigh was observed in 40 (53%) patients. Six patients (8%) were wheelchair-bound and nonambulatory at the time of presentation and had attained this state 10-31 years after onset of symptoms. All the patients with calpainopathy were sporadic cases, except for three patients from North India who had a family history of the condition. The mean CK was 2026 IU/L (range 121 to 30000 IU/L). EMG was myopathic pattern in 70 (93%) patients.
Patients with total protein loss displayed a severe form of muscular dystrophy with typical features of an active dystrophic process viz. increased fiber size variability, internalization of nuclei, split fibers, increased fibrosis, necrotic fibers, regenerating and degenerating fibers, and end-stage muscle disease. Patients with partial deficiency of protein displayed a moderate or relatively milder dystrophic process viz. increased central nuclei, fibers splitting, and degenerative fibers [Figure 1]. Most lobulated fibers and a less active necrotic process were seen in patients with long-standing disease. The disease duration in this group ranged from 2-41 years, with mean of 18.4 years.
Calpain-3 immunoblot analysis
Of the 200 muscle biopsies subjected to combined calpain-3 and dysferlin protein analysis by immunoblot, 171 were suspected cases of calpainopathy with normal or partial dysferlin immunostaining and 29 were control subjects with normal calpain-3 and dysferlin on immunoblot. Of the 171 patients, 124 patients were clinically LGMDs (excluding 33 cases of dysferlinopathy and 9 cases of sacroglycanopathies). Of the 171 muscle biopsies, 75 (43.8%) muscle biopsies showed calpain-3 deficiency, which comprised the major subgroup of 47% (58/124) of clinically defined unclassified muscular dystrophy alone, and 45.1% (75/166) of all LGMDs (including 33 cases of dysferlinopathy and 9 cases of sarcoglycanopathies).
Complete calpain-3 deficiency
Quantitative analysis of calpain-3 using immunoblot showed complete loss of calpain-3 protein in 36 (48%) of 75 muscle biopsies and additional partial deficiency of dysferlin in 16 (44.4%) [Figure 2] a-h. In the patients with total calpain-3 protein loss, the mean age at presentation was 23.5 years and mean age at onset of symptoms was 17.5 years.
Partial calpain-3 deficiency
In 39 (52%) of 75 muscle biopsies, calpain-3 loss was partial as compared to normal control [Figure 2] a-h. Arbitrarily, biopsies with less than 70% reduction in intensity of 94 kDa protein band of calpain-3 compared to normal control was considered as partial loss. In the patients with partial calpain-3 protein loss, the mean age at presentation and onset was 22.4 years and 18 years, respectively.
This difference in the mean ages at onset and presentation of symptoms between the two groups (complete loss and partial loss of calpain-3) was not statistically significant. There was no difference between the patients with early, adult, or late onset of disease in the incidence of total or partial loss of calpain-3.
LGMD2A is caused by a defect in a protein with an enzymatic rather than structural function. The pathophysiological role of calpain-3 is not exactly known. Immunohistochemical (IHC) analysis of calpainopathy is still not possible as there is no anti-calpain-3 antibody available that is compatible for IHC on muscle biopsy cryosections. Calpain-3 immunoblot analysis of muscle biopsy is the most useful diagnostic tool in calpainopathy and the validity of this protein testing has been fully recognized as most patients with protein defect do indeed have CAPN3 gene mutation. Approximately 80% of individuals with a CAPN3 mutation show variable levels of calpain-3 protein deficiency and in 10%-20% of individuals, muscle biopsies show a normal amount of protein. ,,,, In the latter group of patients, the amount of protein is normal but calpain-3 has lost its autocatalytic activity and is functionally inactive. ,, Protein estimation by immunoblotting is reliable for diagnosis as, in individuals with immunoblot-confirmed calpain-3 deficiency, the detection rate for one or two pathogenic CAPN3 mutations is approximately 80%-84%. ,
The present study was conducted with the objective of establishing the diagnosis and frequency of LGMD2A and to correlate the clinical and histopathological findings with immunoblot. The muscle biopsy histopathology results in our calpain-3-deficient cases are quite similar to that previously reported. , We observed that patients with long-standing disease show a greater number of lobulated fibers in muscle biopsies; this observation is similar to some of the earlier studies.  In these cases, the presence of lobulated fibers could be an important clue to the differential diagnosis and indicate the need to perform immunoblot in these patients. Hence, the presence of lobulated fibers may be suggestive of LGMD2A; it is a marker of chronic disease stage and severe clinical manifestations but it is not pathognomic of the disease.
Immunoblot analysis revealed the deficiency of calpain-3 protein in 75 cases, which could therefore be characterized as primary calpainopathy. However, in 16 cases, in addition to complete deficiency of calpain-3, there was also secondary dysferlin deficiency on immunoblot. Relatively weak dysferlin pattern in most of the cases on muscle biopsy section were also evident by IHC. Secondary dysferlin loss may be due to a possible signalling pathway in which these two molecules along with AHNAK, a protein involved in subsarcolemmal cytoarchitecture and membrane repair in the dysferlin protein complex, are involved. AHNAK can act as substrate for CAPN3 and this could lead to secondary dysferlin protein loss.  As not much is known about the incidence of secondary calpain-3 deficiency in the Asian population, our results need to be complemented by studies at DNA level. We have performed dysferlin immunoblot in each of these cases to rule out secondary calpain-3 deficiency, and only 3 of 171 cases showed secondary calpain-3 deficiency. All three cases had complete deficiency of dysferlin and were categorized as dyferlinopathy and, hence, we suspect that all of our cases showing calpain-3 deficiency are primary defects.
In the present study, in 90% of cases, the age ranged from 3 to 40 years and the mean age at presentation and onset of symptoms were 21 and 14 years, respectively. This is in concordance with other studies. , In large series of 300 patients reported by Zatz et al. and 238 patients of LGMD by Saenz et al., the mean age at onset was 13.7 years and 14 years, respectively. , In our series, the mean ages at presentation of males and females were 19.5 and 18 years respectively, showing homogeneity in both genders. The mean age at onset in males and females was 14.5 and 13 years, respectively; this is unlike the observations by Zatz et al. and Groen et al. who found marked differences in the ages at onset in males and females. , The early motor milestones were typically normal. The incidence of involvement of the posterior compartment of the thigh and joint contractures in the hips, knees, elbows, and fingers were similar to other reports. Calf hypertrophy was observed in 12% of cases. Only 6 of the 75 patients were wheelchair-bound or had loss of ambulation and this indicates a slower progression of disease than in European populations.  One patient died at the age of 61 years. All the patients included in the study were from the northern and central parts of India and presented with sporadic disease (except three cases in which there was a family history).
From the present study it appears that LGMD2A could be the most prevalent form of LGMD in India, accounting for 43.8% of all patients with unclassified LGMD, unclassified muscular dystrophies, or myopathy, and 45.1% of all LGMD. Our data support the hypothesis that, in any population in the world, LGMD occurs most frequently in the sporadic form. ,,,,,,,, The possible reason for the high frequency of calpainopathy could be the allelic frequency differences between groups in India, a country that is larger than Europe, reflecting strong founder effects whose signatures have been maintained for thousands of years owing to endogamy. Thus, it is predicted that there will be an excess of recessive diseases in India. , In most of the published series, calpainopathy is considered the most common form of LGMD, representing approximately 40% of all LGMD cases depending on the geographic region. , Estimates based on molecular data indicate that the frequency ranges from 10% of LGMD cases in a Caucasian population from the US to 21% in the Netherlands, 26% in Japan, 50% in Turkey, and 80% in the Basque country and Russia. ,,,,,,, In individuals with immunoblot-confirmed calpain-3 deficiency, the detection rate for pathogenic CAPN3 mutations is approximately 80%-84%. , Our data suggest that prevalence of LGMD2A is similar to that in the Italian population, where approximately 40% with the LGMD phenotype were LGMD2A.  Our clinical characterization and the distribution of partial and complete calpain-3-deficient cases in our series is also similar to that seen in an Italian series in which, among 66 calpain-3-deficient patients, the LGMD phenotype was seen in 80%, proximal myopathy in 11%, and distal myopathy in 3%; complete loss of calpain-3 in 47%; and partial deficiency of calpain-3 in 53%.  In immunoblot, the 94 kDa band intensity of calpain-3 compared to control muscle was the mainstay for determination of partial or complete deficiency. Patients with complete and partial loss of calpain-3 essentially showed lower densitometric values of 60, 58, and 55 kDa band of calpain-3 as compared to control. Stringent procedural precautions were taken while performing immunoblot and that greatly improved the interpretation of results on the basis of 94 kDa calpain-3 band intensity; our results are in concordance with existing literature. ,,
In all the 75 calpain-3-deficient patients, the mean CK was 2026 IU/L (range: 121-30000 IU/L). An elevated CK value is one of the biochemical features observed in LGMD2A cases. Our observations were similar to that in a UK-based study, which reported a CK range of 193-38620 IU/L.  Elevated CK level is seen particularly in the active stage or at the onset of disease; it decreases with progression of disease though, at times, it may remain high at all the stages.
In conclusion, these 75 cases are the first documented cases of calpainopathy in India, and this pioneering study suggests that calpainopathy is one of the most frequent forms of autosomal recessive LGMDs in Indian. We have established the value of this technique for the diagnosis of LGMD2A; however, it needs to be supplemented with mutational analysis of the CAPN3 gene. More studies from different parts of the country are needed to confirm the actual frequency of this disease in India. Accurate diagnosis of LGMD2A is important for genetic counselling, prediction of progression outcome, and for therapeutic purposes.
This original work by the authors is supported by ICMR, New-Delhi, Project 3/1/2/1/Neuro/2008-NCD-I. The authors thank Mr. Rajeshwar Khadia and Anil Bisht for technical assistance with IHC and EHC.
[Figure 1], [Figure 2]