Sarcoglycanopathy: Clinical and histochemical characteristics in 66 patients
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.72164
Source of Support: None, Conflict of Interest: None
Background: Sarcoglycanopathies are a group or autosomal recessive muscular dystrophies designated as α, β, γ, or δ sarcogycanopathy. Materials and Methods: It is a retrospective analysis of case series. Results: Sixty six patients immunohistochemically confirmed to have sarcoglycan deficiency were included in the analysis. The study period extended from 1997-2008. The male to female ratio was 1.5:1. Mean age at the onset of muscle complaints was 6.2±3.7 years (range 1-18). Mean age at evaluation was 10.0±4.8 years (range 3-31). Mean duration of illness was 47.02±44.80 months (range 3-325). Onset in the first decade was seen in 59 (89.4%) and 25 (42.4%) of these had onset before five years of age. The remaining seven (10.6%) had onset in second decade and none after 20 years of age. Consanguinity was present in 54 (81.8%). In 34 of 66 cases only a-SG was carried out and this had shown total absence of staining in all fibers. In the remaining 32 cases where the entire panel was performed, absence of all sarcoglycans was noted in 10 (15.1%), isolated a-SG deficiency in 7 (10.6%), isolated b-SG deficiency in 6 (9.1%), and isolated g-SG deficiency in 3 (4.5%). Combination deficiency was also observed: absence of α and β (n=4), β and γ (n=2), and α and γ (n=1). Conclusions: Our series was a large series and with predominantly pediatric age group. Sarcoglycanopathy should be particularly suspected in a child born to consanguineous parents and who presents with proximal muscle weakness and calf hypertrophy, elevated CK level, and myopathic pattern on EMG.
Keywords: Immunohistochemistry, sarcoglycan, sarcoglycanopathy, southern India
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders and they affect more than two-thirds of the total number of patients suffering from muscular dystrophy,  and alterations in the components of the sarcoglycan (SG) complex cause different types of muscular dystrophies. ,,, Matsumura et al. in 1992 reported adhalin deficiency or α-sarcoglycanopathy (SGP) in muscle biopsy specimens from Algerian and Lebanese patients presenting with severe childhood autosomal recessive muscular dystrophies (SCARMD)  The clinical phenotype is close to DMD and BMD as reported by Ben Hamida et al. in 1983.  Several authors have subsequently reported on adhalin deficiency in European, ,, Brazilian, , and Asian ,, patients with SCARMD or milder dystrophies.
The gene for α-SG (SG) is located at 17q21,  for β-SG at 4q12, , for γ-SG at 13q12,  and for δ-SG at 5q33  Alpha and gamma SGs are specific to skeletal muscle and heart; β-SG is expressed in multiple tissues, although expression is prominent in skeletal and cardiac muscle; ,,, and δ-SG gene is expressed mainly in striated and smooth muscle.  Diagnosis of SGP starts with accurate clinical assessment of the patients, including family history, signs and symptoms, histopathology, and genetic studies. Routine immunohistochemical analysis in typical SGPs demonstrates absence or severe reduction of single or multiple SGs.
The age at onset of SGP is usually between 3 and 15 years and the severity is heterogeneous.  Patients present with proximal weakness most often associated with hypertrophy of calves. The serum creatine kinase (CK) levels are usually very high. We identified 66 patients with SG deficiency by immunohistochemistry and report the clinical characteristics and histopathological findings in these cases.
All patients who were immunohistochemically confirmed to have SG deficiency were included in the analysis. The medical records maintained from 1997-2008 were reviewed by a resident in neurology training and neurologist (A.N). The 66 patients included in this analysis fulfilled the following selection criteria: all had (i) clinical examination by a neurologist, (ii) progressive proximal muscle weakness, and (iii) undergone a muscle biopsy, which demonstrated normal dystrophin and isolated or multiple SG deficiency by immunohistochemistry. For all subjects following parameters: age, sex, consanguinity, family history, age of onset, topography of muscle involvement, presence of calf hypertrophy, age at loss of ambulation, and manual muscle testing (Medical research Council, 1943), were recorded. Electromyography, motor and sensory conduction studies, ECG, 2D echocardiography, and serum CK determinations were done.
The data were analyzed using descriptive statistics such as mean standard deviation for continuous variables, and frequency percentage for categorical variables. Comparison between the different phenotypes was carried out by independent sample 't' test for continuous variables and Fisher's exact test for categorical variables (P≤0.05).
Histopathological and immunohistochemical studies
Open muscle biopsy was performed in all 66 patients after obtaining written informed consent and morphologic and immunohistochemical studies were done. Samples were obtained from biceps in 34 cases, quadriceps in 31, gastrocnemius in one. Muscle samples were frozen in isopentane precooled liquid nitrogen and serial cryostat sections were stained for enzyme and nonenzyme stains (Hematoxylin-Eosin, modified Gomori trichrome, periodic acid Schiff, NADH-TR, SDH, ATpase at pH 9.5 and 4.6). Immunohistochemical staining was carried out using monoclonal antibodies to dystrophin (1, 2 , 3), SGs (α, β, γ), dysferlin, merosin, and β-dystroglycan as primary and HRP-tagged secondary antibody (Novolink). In 34 of 66 cases, dystrophin (1, 2, 3), merosin, and α-SG were utilized, while the aforementioned entire panel was used for the remaining 32 samples.
During the period from 1997-2008, 3232 patients underwent muscle biopsies, of which dystrophy was diagnosed in 1322. The breakup for dystrophinopathies and nondystrophinopathies was not possible as all samples were not subjected to immunohistochemistry. Of these patients with possible limb-girdle muscular dystrophies (LGMDs), 66 specimens showed SG deficiency and this group formed the basis of the present study. Majority of patients hailed from Southern India. There were 40 boys and 26 girls. The mean age at onset of muscle complaints was 6.2±3.7 years (range 1-18). Mean age at onset was 5.9±3.8 years in boys and 6.9±3.5 years in girls. Mean age at time of evaluation was 10.0±4.8 years (range 3-31). Mean duration of illness at presentation was 47.02±44.80 months (range 3-325). Onset in the first decade was seen in 59 (89.4%) and 25 (42.4%) of these had onset before five years of age. The remaining seven (10.6%) had onset in second decade and none after 20 years of age. Consanguinity was present in 54 (81.8%) while in 12 (18.1%) it appeared to be sporadic. More than one member was affected in 24 families but no second sibling underwent muscle biopsy. The initial symptom at onset was progressive lower limb proximal muscle weakness in 63 (95.4%), calf muscle hypertrophy in 2 (3.0%), and cramps in 1 (1.5%). The salient clinical characteristics of the 66 SGP patients are summarized in [Table 1]. Wasting of the thigh muscles was noted in 18 (27.3%), and of the shoulder girdle muscles in 7 (10.6%). All patients had weakness of pelvic girdle muscles and in addition pectoral girdle weakness was noted in 57 (86.3%). Time elapsed between lower and upper girdle weakness was difficult to assess as the functional motor disability in upper limb was mild and frequently not noticed by the child or parents. Lower limb distal muscle weakness was present in 21 (31.8%) and in the upper limbs in 18 (27.3%). Scapular winging was noted in 20 (30.3%) patients, calf hypertrophy was present in 58 (87.9%) patients. Tendo Achilles contracture was seen in 36 (54.5%), hamstrings in 6 (9.1%), and biceps in 2 (3.0%). Electroneuromyography was performed in 43 (65.1%) cases which showed myopathic process. Motor and sensory parameters were normal. ECG and 2D Echo done in 32 (48.4%) showed evidence of mild cardiomyopathy in only one patient. ECG was reported to be normal in all.
Majority of patients were seen in the early phase of the illness, 59 (89.3%) were ambulant at time of evaluation, 6 (9.0%) required assistance to walk, and only one (1.5%) patient was bed bound. In these patients, time period between onset of disease and loss of independent ambulation ranged from two to five years. No relationship was found between age at onset and age at loss of ambulation. Serum CK was elevated in all patients except one case and the value ranged from 428-27186 IU/L (mean±SD, 7853±5270). All had CK values more than five times elevated above the upper limit of normal. The only patient with low CK value had β-SGP and had the milder form.
Routine histology revealed dystrophic features with rounding, hyalinization, polyfocal necrosis, myophagocytosis, splitting, and moderate to marked fibrosis. Adipose tissue infiltration was also noted. In addition, clusters of regenerating fibers were present in majority (n=25). The other findings included atrophic fibers (n=13), and perivascular inflammation (n=13). Type I predominance (n=3) and type II fiber predominance (n=6) was noted on enzyme stains. Presence of ragged red fibers was seen in one case. Immunostaining to dystrophin and merosin showed normal labeling along the membrane in all the cases. In 34 of the 66 cases only α-SG was carried out and this had shown total absence of staining in all the fibers. In the remaining 32 cases where the entire panel was performed absence of all SGs was noted in 10 (15.1%), isolated α-SG deficiency in 7 (10.6%) [Figure 1], isolated β-SG deficiency in 6 (9.1%) [Figure 2], and isolated γ-SG deficiency in 3 (4.5%) [Figure 3]. Combination deficiencies were also noted: absence of α and β (n=3), β and γ (n=2), and α and γ (n=1).
Among the patients who had very rapid progression requiring assistance within few years after onset, muscle immunostaining showed that two had γ-SGP, one β-SGP, two had combined absence of γ and β-SG. Two had absent α-SG staining but neither γ or β-SG was done in these two specimens. Age at onset was less than five years in 25 patients but its correlation with multiple or isolated deficiencies could not be done as 34 samples had only α-SG staining. However, even among the 32 patients who had all three immunostainings done there was no correlation between age at onset, disease severity, and the pattern of SG deficiency.
In the present study on SGPs, α-SG was found to be the commonest SG among our patients. This has been reported earlier in population-based studies.  Majority of our patients had onset in the first decade of life with mean age at onset of 6.2 years. In the report from Northern India the mean age was 7.2 years.  In studies from France and Italy also majority had onset in the first decade of life. , In contrast, in a report from Mumbai in West India and another from Hyderabad in Southern India the mean age at onset was reported to be in the third decade. ,
Autosomal recessive (AR) pattern of inheritance was the commonest with consanguinity seen in 81.8%. We presume the remaining sporadic cases and those with multiple siblings affected also have an AR inheritance. The presence of equal number of males and females also confirms the AR mode of inheritance. This high incidence of consanguinity is also reported in other series. ,,, Calf muscle hypertrophy was common in majority, and this is also reported earlier. ,,,, The clinical characteristics in our series fit well with those described previously by Fardeau et al. and Van der Kooi et al. ,
Ten of our patients showed all three SG deficiencies and 70% of these had onset of illness before five years of age and also had severe weakness. All those with isolated deficiency of γ-SG or in combination with others had onset at or before 10 years of age. In a report by Rita et al. all patients lacking all three SGs had an early onset of symptoms, and those with disease onset before 10 years of age as well as absence of γ-SG or an absence of all three SGs had a severe phenotype. 
When all three SG stainings were performed, our muscle samples showed multiple SG deficiencies. In earlier reports immunohistochemical analysis of α-, β-, and γ-SG confirmed that loss of one SG leads to reduction or absence of others. This is reported by several authors. ,,, It is reported that when gamma-SG is absent, α and β can be partially expressed  and this is also reported by Jung et al.  while they mention that absence of α- or β-SG alone is never found. Mizuno et al.  reported that deficiency of one of the SG components leads to a complete deficiency of the SG complex, but this was found to be untrue by others.  We did find isolated α- or β-SG deficiency when all three immunostains were performed. This was also seen in other studies. ,,,,
Muscle weakness usually began in the pelvic girdle and these muscles were the most severely affected. The classic feature of SG which are preferential and early involvement of pelvic girdle muscles followed later by shoulder girdle and associated scapular winging were all noted in our patients. The distal groups were mildly affected in a small number, but rather involved early in the course of illness. The hip adductors, and flexors of hip, knee and arm were earliest and most affected muscles. The hip abductor sign, previously reported in childhood onset gamma-SG , and in adults, , was indeed observed in certain patients in our series, but it was not consistently documented in the medical records. A larger group has been reported to have prominent distal muscle involvement in a report from India but these were predominantly older patients.  Classically, the patients with α-SG had onset of illness in the first decade of life and progression was rapid. This is typically described by earlier authors. , Interestingly, the three patients who were aged between 20 and 31 years at presentation had β-SG deficiency and had a slow progression with mild disability. Such benign form is also described by Vainzoff et al . 
The prevalence of LGMD in the world is estimated to be 20-40 per million.  Later in a study from Netherlands, a much lower prevalence rate of 8.1 per million was reported.  The prevalence of ARLGMD is estimated to be about 1 in 15,000.  In a study from Northern India, over a period of six years only 13 cases of immunohistochemically confirmed SGPs (2.4% of muscular dystrophies) and 11.8% of all LGMD were seen in the pathology department.  This low prevalence was attributed to a low rate of consanguineous marriages in this region. In a study from Japan, 8.8% of all LGMDs had SGP.  While a study from Netherlands reports 25% and much higher incidence of 55% in Brazil. , A high incidence of 46.2%  and 53.8% are reported in two other studies from India. 
The first reported case of β-SG in a three-year-old girl was by Bonnemann et al.  There were six cases of isolated β-SGP in our series. In a series of 25 cases of SG reported from India β-SG formed the predominant group.  A single case of β-SG had been reported in a four-year-old boy by Kapoor et al. from India.  Later Meena et al. reported β-SG in four cases.  Interestingly, the group from Northern India reported γ-SG to be the commonest SGP. 
Lim et al. have shown that rapidly progressive α-SG cases become wheelchair bound at a mean age of 11.7 years, while for the slowly progressive one there is no uniform time to attain wheelchair bound state. For β-SG it is about 14.3 years and for γ-SG it is 17.5 years.  Two of the 40 patients in our series with α-SG had become wheelchair bound in the early second decade. The remaining patients were still ambulant, but they were in their early phase of the disease course. Nevertheless, majority of patients with α-SG had Duchenne like phenotype with similar progression of muscle weakness and disability suggesting that most of them resembled SCARMD. However, α-SG is reported to generally show a milder course as compared to other SGPs.  Three of our six patients with isolated β-SG had a long duration of illness and minimal motor disability while the remaining two were very early in the course of illness. The three patients with isolated γ-SG had duration of illness between two and three years and were ambulant. There was no clear phenotypic differentiation among the various SGPs. The pattern of muscle involvement and course of illness varied across the various SGPs.
Among the 32 patients who underwent ECG and 2D echo, only one patient had evidence of dilated cardiomyopathy on 2D echo. None showed abnormality on ECG. None of our patients at time of diagnosis had clinical evidence of cardiac involvement. Moreover, our patients were evaluated in the early phase of their illnesses. In the report by Khadilkar et al., cardiomyopathy was noted in only one case.  Melacini et al. reported cardiac involvement in 30% of their patients  while subclinical cardiac involvement was reported in 50% of patients by Meena et al. 
Dincer et al. in a series of 20 Turkish families, found SGP as the second common type of ARLGMD.  In the series reported by VanderKooi et al. , SG constituted 25% of the ARLGMD. In our series of clinical and histopathologically confirmed ARLGMD, SGP was the commonest followed by dysferlinopathy.  The total number of biopsies performed during the study period was 3232 and dystrophy was diagnosed in 1322. However, it is not possible to give the exact distribution of the various ARLGMD as immunohistochemical studies were not performed on all muscle samples and we also do not do immunoblot for calpain deficiency. In United States the most common LGMD was dysferlinopathy (18%), followed by SG (15%), dystroglycanopathy (15%), and calpainopathy (12%). 
Sarcoglycanopathy should be particularly suspected in a child born to consanguineous parents and who presents with proximal muscle weakness and calf hypertrophy, raised CK level, and myopathic pattern on EMG. SGP is unique in that in principle the four muscular dystrophies belonging to this group are indistinguishable not only in terms of clinical features but also in terms of immunohistochemical examination of the muscles. , Genetic studies are necessary for discriminating individual SGPs as the same phenotype results from the mutation of various genes. Unfortunately, we were unable to perform genetic analysis on our patients. Immunohistochemical analysis of SG should be included in routine screening of muscle dystrophies. Molecular analysis of the genes coding for the individual components of the SG complex is necessary to identify the primary defect. Immunostaining is a prerequisite prior to genetic analysis, and will be useful in indicating which gene to investigate. In the absence of molecular testing for LGMD in India and not so easily available Western blot studies, a relatively good degree of diagnosis is possible with immunohistochemical methods but it is imperative to start molecular diagnosis and genetic counseling should be considered due to its possibility of prenatal genetic diagnosis.
[Figure 1], [Figure 2], [Figure 3]