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Deficiency of the 50 kDa dystrophin-associated-glycoprotein (adhalin) in an Indian autosomal recessive limb girdle muscular dystrophy patient : immunochemical analysis and clinical aspects.
Correspondence Address:
Abnormalities of dystrophin are a common cause of muscular dystrophy and testing for dystrophin gene or protein has become a part of routine diagnostic evaluation of patients who present with progressive proximal muscle weakness, high serum creatine kinase concentrations, and histopathological evidence of a dystrophic process. Patients who have no dystrophin abnormalities are assumed to have autosomal recessive muscular dystrophy. In a family consisting of 5 sibs, 2 mentally normal brothers presented with abnormal gait and protrusion of chest and hips. Muscle biopsy from one of them showed dystrophic changes and reduced patchy binding of dystrophin. No detectable deletion was observed in the patient's DNA and his brother with cDMD probes. Dystrophin associated proteins, beta-dystroglycan showed discontinuous immunostaining in the sarcolemma and alpha-sarcoglycan (adhalin) was totally absent, while beta-, gamma-, and delta-sarcoglycans were highly reduced. Immunoblot analysis showed dystrophin of normal molecular weight but of decreased quantity, beta-dystroglycan was reduced by about 37% while alpha-sarcoglycan was completely absent. This study is a first attempt for a systematic clinical, genetic and molecular investigation of the autosomal recessive LGMD in India.
Autosomal recessive limb girdle muscular dystrophies (AR-LGMD) are a heterogenous group of genetic disorders in which there is a progressive weakness of the pelvic and shoulder girdle musculature. Eight genes have already been mapped for AR-LGMDs which are : LGMD2A at 15q, LGMD2B at 2p, LGMD2C at 13q, LGMD2D at 17q, LGMD2E at 4q, LGMD2F at 5q, LGMD2G at 17q and LGMD2H at 9q.[1],[2],[3],[4],[5],[6],[7],[8],[9] Among these AR-LGMDs : LGMD2D, LGMD2E, LGMD2C and LGMD2F arise from mutations in genes encoding four different dystrophin-associated glycoproteins [DAG / dystrophin-associated proteins (DAP)], -(adhalin), -, - and -sarcoglycans respectively.[3],[4],[5],[6],[7],[10],[11],[12] In 1992, Matsumura et al[13] reported adhalin deficiency in patients presenting with severe childhood autosomal recessive muscular dystrophy (SCARMD) from Algeria and Lebanan. The clinical phenotype of this disease was close to Duchenne (DMD) and Becker (BMD) muscular dystrophies as reported earlier by Ben-Hamida et al.[14] Presence of dystrophin in muscle biopsy specimens led to a search for deficiency in one of the DAPs (adhalin).[13],[14],[15] Several authors have subsequently reported adhalin deficiency in European, Brazilian and Asian patients with SCARMD or milder muscular dystrophies.[16],[17],[18],[19],[20],[21],[22],[23] Genetic studies indicated a heterogeneity of adhalin deficiencies. Ben-Othmane et al[24] mapped the defective gene, responsible for the form of the SCARMD prevalent in north Africa, to chromosome 13q12 and this was confirmed by Azibi et al.[25] Roberds et al26 mapped the adhalin gene to chromosome 17q12-q21.[33] and identified the first missense mutations in a previously reported French family.[4],[26] Different groups subsequently reported other cases of 'primary adhalinopathy'.[20],[22],[27] Piccolo et al[27] described 12 biochemical deficient - sarcoglycan families and showed -sarcoglycan gene mutations in 9 of them while in a molecular screen of 30 north American muscular dystrophy patients with normal dystrophin, a single African-American girl with -sarcoglycan gene mutation and a phenotype consistent with childhood-onset muscular dystrophy was identified.28 The frequency of -sarcoglycan deficiency in Japanese patients has been studied using immunohistochemical data. However, the primary gene defect was not known and these patients may include secondary adhalin deficiency.[23] Secondary adhalin deficiencies are due to mutations affecting -, - and -sarcoglycan genes.[3],[5],[6],[12] We report here one Indian patient whose intellectual development was normal and there was no heart dysfunction. A complete absence of 50 DAG, partial deficiency of dystrophin and -dystroglycan, and variable reactivity with merosin was observed. This case illustrates the potential risk of misdiagnosis in patients exhibiting a patchy pattern of dystrophin.
We studied one family whose two affected siblings (BJ and BJB) born of nonconsanguineous marriage were non ambulant at the time of presentation. They had similar phenotypes with normal developmental milestones. They had two younger brothers and one sister in good health. The sibs BJ and BJB, 16 and 14 year old males; mentally normal, presented with progressive difficulty in walking and frequent falls for the last 5 years. The trunk, arms and legs of both the probands were globally thin. On examination, there was proximal muscle weakness of the upper limbs with limited range of movements in all joints. The hip, knee, and the ankle joints were fixed due to contractures. Deep tendon reflexes were ellicitable in the upper limbs but not in lower limbs due to contractures. ECG was normal. EMG examination showed low voltage, short amplitude, polyphasic action potentials. Serum creatine kinase (CK) was elevated to 1680 U/L (BJ) and 4881 U/L (BJB).
DNA Analysis : Peripheral blood samples were obtained from BJ and BJB and unrelated healthy control subject. Genomic DNA was digested with restriction endonuclease Hind III and separated by electrophoresis through 1.2% agarose gel, 20-24cm in length and transferred to hybond N+ membrane (Amersham,UK) by Southern blotting technique with some modifications.29 cDMD probes 1-2a, 2b-3, 4-5a, 5b-7, 8 and 9-14 were obtained from ATCC, USA. Probe 9-14 was further digested with BamHI to separate probes 9, 10 and 11-14. DNA analysis was also carried out by mPCR using three different multiplex primer sets comprising 9 primer pairs each (IMMCO Corporation, USA). The PCR products were separated on 2.5% agarose gels. Multiplex I (exons ; 45, 48, 19, 17, 51, 8, 12, 44, 4) and Multiplex II (exons; Pm, 3, 43, 50, 13, 6, 47, 60, 52) were designed by Chamberlain et al30 and Beggs et al respectively.31 Multiplex III (exons: 49, 16, 41, 32, 42, 2,79, 25, 66) was designed in our laboratory based on the sequences by Beggs (personal communication). No other family member agreed to undergo a blood test for determination of CK and DNA analysis. Immunocytochemistry : Skeletal muscle biopsy was obtained from the gastrocnemius muscle of the patient BJ. Muscle biopsy was snap frozen in liquid nitrogen. The cryostat sections were processed for immunocytochemistry with indirect immunoperoxidase technique.32 Frozen muscle sections, 6 m thick were incubated with monoclonal antibodies against dystrophin (N-terminal, rod domain and Cterminal), -dystroglycan, -, -, - and - sarcoglycans and laminin- 2 (Novacastra, Newcastle, UK). These antibodies were used at dilution shown in [Table I]. Incubation with primary antibodies was performed for 1 hr, followed by an incubation with 1:50 diluted peroxidase labelled goat anti mouse Ig 20 (Novacastra, Newcastle, UK), with TBS; (0.05 M TrisHCI pH 7.6) for 1 hour at room temperature. Each incubation step was followed by extensive washing with TBS. Visualisation was achieved by exposure to freshly prepared 0.05% (w/v) diaminobenzidiene in TBS containing 0.1% hydrogen peroxide. Muscle sections were examined under Leitz Orthoplan, microscope. Negative controls were always included. Western Blot : Western blotting was carried out using thick SDS-polyacrylamide gel with discontinuous system modified by the use of 0.2% (w/v) of SDS throughout.33,34 The separated proteins were blotted onto PVDF membrane (Amresco, USA) using transfer buffer of Otter et al at 400 mA overnight.35 Immunoreaction was performed using monoclonal antibodies against dystrophin (rod-domain, Cterminal), -dystroglycan and -sarcoglycan. These antibodies were used at dilution shown in [Table I]. Visualisation was achieved using chemiluminescence working solution (Pierce, USA). Quantification of dystrophin and its associated proteins : The quantity (relative cellular abundance) of dystrophin, -dystroglycan and -sarcoglycan was also determined as a percentage of the adjacent normal control samples. This approximation was completely dependent on the amount of muscle tissue loaded in each lane, determined by post-transfer myosin stain.[36]
DNA Analysis The restriction pattern observed in patient's DNA sample and his brother with dystrophin cDNA probes completely matched that of a control DNA sample, indicating that no structural alteration such as deletion or duplication were present in the dystrophin gene. Immunocytochemistry (Dystrophin and Spectrin) Immuno-cytochemistry of dystrophin : Patchy labelling with reduced intensity was seen with the antibody against N-terminal domain, but the pattern was more severe against the rod-domain which also revealed more negative fibres. Staining with Cterminal domain antibody also showed patchy labelling but with reduced intensity as compared to Nterminal. Spectrin immunostaining in the muscle fibres was almost identical to that of dystrophin. The staining was patchy and the intensity was slightly less than that seen with N-terminal domain of dystrophin. Very few negative fibres were observed. Dystrophin associated proteins and Laminin- 2 : Immunostainability of -dystroglycan antibody in the muscle was moderately reduced as compared to normal controls. In contrast, staining for - sarcoglycan was completely absent, while -, - and -sarcoglycans were highly reduced in the sarcolemma of the muscle fibres [Figure - 1]. A reduction in the intensity of immunostaining for laminin- 2 around each muscle fibre was seen [Figure - 2]. Immunoblot analysis Dystrophin and Dystrophin associated proteins [Figure - 3]: Immunoblot analysis of muscle proteins showed dystrophin of normal molecular weight (427 kDa), but of decreased quantities by densitometric analysis. On being compared to normal control, presence of 90% and 89% of dystrophin was observed with antibodies against mid-rod and carboxyl terminal, respectively. Presence of 63% of - dystroglycan was observed, while no immunoreactivity was seen with - sarcoglycan.
Sarcoglycanopathies are muscular dystrophies due to disruption of the dystrophin-associated sarcoglycan complex caused by mutations in any of the four already identified sarcoglycan genes ( -, -, - and - SG).[3],[4],[5],[6],[7] Mutations in any one of these sarcoglycans lead to more or less pronounced secondary deficiencies of the other components of the complex, indicating the importance of the integrity of the entire complex for the prevention of muscle cell degeneration.37 The clinical severity of primary - sarcoglycanopathy varies strikingly. The most severe course has been observed in whom -SG was completely absent. A pronounced but variable decrease in -SG, were usually observed in milder forms of variable severity.[27],[28],[29],[30],[31],[32],[33],[34],[35],[36],[37],[38] The characteristic of the affected members of this family are: (i) the peculiar clinical presentation in being able to slide on their buttocks; (ii) with late onset but rapid progression resulting in contractures of hip, knee and ankle joints. In agreement with Beckmann and Bushby, to discriminate between different LGMD2 entities, both immunocytochemistry and western blot are helpful for the initial screening.[39] -SG immunocytochemistry can be taken as the first step to screen all the cases for sarcoglycanopathy and to separate these cases from others. Mutations of any component of the sarcoglycan complex result in the secondary loss of the other components of the complex. In many cases, the defective sarcoglycan protein was not detected by immunocytochemical analysis, whereas other sarcoglycans were weakly or strongly stained.40 In patients with -sarcoglycan deficiency on immunostaining, a complete deficiency (absence of staining) was found to be more specific for mutations in the -sarcoglycan gene than for mutations in - or -sarcoglycan gene. On the other hand, a partial deficiency of -sarcoglycan on immunostaining was not specific for mutations in any of the three sarcoglycan gene. It is most likely that patient BJ suffered from primary adhalinopathy, since immunoreactivity for -sarcoglycan was completely absent while -, - and -sarcoglycans were highly reduced. Immunocytochemistry revealed a patchy pattern with antibodies against dystrophin in BJ while slightly reduced amount of dystrophin was observed (90% of normal with rod-domain and 89% of the normal with C-terminal domain) in western blot. Normal amount of dystrophin has been observed in SCARMD.[15],[16],[27] In contrast, studies by Duggan et al[41] in LGMD2D patients showed reduced to variable staining with antibodies directed against dystrophin while muscle protein studies by Passos-Bueno et al[42] in Brazilian population for seven autosomal recessive LGMDs revealed that dystrophin was reduced in quantity in all LGMD2C, 2E, and 2F patients. However, among the seven LGMD2D only one patient who was severely affected showed a reduction in dystrophin quantity by western blot. Staining for -dystroglycan in BJ was found to be patchy while by immunoblot a moderate reduction of -dystroglycan (63% of the normal) was observed. Duggan et al[41] reported one patient with partial deficiency of -sarcoglycan who also showed variable staining with -dystroglycan out of 30 cases of childhood muscular dystrophy. -dystroglycan was found to be preserved in SCARMD patients from north-Africa, Europe and Japan.[13],[16],[23] In conclusions, our results suggest that the severity of the clinical course in patient BJ correlated with the total absence of adhalin and that the stability of dystrophin in the myofibre plasma membrane is somewhat dependent upon -sarcoglycan.
A Fellowship to VH from University Grants Commission, and financial help from Department of Biotechnology and Indian Council of Medical Research, New Delhi is gratefully acknowledged.
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