We selected male children with following features: proximal muscle weakness with positive Gower sign and valley sign, supported by an elevated serum CPK level and a positive EMG finding of myopathic pattern. The patients clinically suspected but having normal CPK level and with no historical evidence of X-linked inheritance; or having unrelated co-morbidity which may influence the course and progression of the disease were excluded from the study.

After selection, the patients were subjected to genetic analysis after pretest counseling of parents and obtaining necessary informed consent from them. Muscle biopsy was performed in patients where the genetic analysis was negative.

Muscle biopsy: This was carried out after taking written informed consent from parents. Muscle biopsies were obtained by the open method from quadriceps or biceps muscles. The specimen was subjected to microscopic examination after hematoxylin and eosin staining and also for Immunostaining with monoclonal antibodies to dystrophin (1-3).

Statistical analysis: All statistical analysis was done by using SPSS software version 19. Comparisons of variables were done using independent t–test. Correlation of variables was done using Spearman and Pearson’s test wherever applicable with P value <0.05 considered statitstically significant.

A total 100 of clinically suspected DMD patients were included in this study. Of them 73 patients (73%) were confirmed by genetic analysis. Nine (33.33%) out of 27 genetically undiagnosed cases underwent muscle biopsy. Eight of them proved to be cases of DMD by absence of dystrophin staining, while one patient proved to have congenital dystrophy. Rest of the cases (n=18) could not be confirmed as they refused to undergo muscle biopsy. We included only genetically or biopsy proven DMD cases (n=81) for further study of genetic pattern, clinical profile and their correlation. The demographic and clinical profile is given in Table I.

TABLE  I	Demographic and Clinical Features of Study Patients (N=81)

In majority of the cases (80%), the onset of the disease symptoms was 5 yrs or below. Seventy three (90.12%) patients showed positive Valley sign while 76 (93.82%) patients showed calf hypertrophy and 46 (56.79%) showed hypertrophy of extensor digitorum brevis (EDB). Gower’s sign was observed in 57(70.37%) cases. Non-ambulatory patients had historical suggestion of Gower’s sign but could not be confirmed.

The patients with inability to get up without support, inability to walk and or wheel-chair bound and with severe contracture, and marked wasting were considered in advanced stage of the disease. Twenty patients (24.69%) were non-ambulatory at the time of presentation.

Among non-ambulatory patients, the youngest patient presented to us was 8.5-year-old. The mean (SD) age of onset of the symptoms in non-ambulatory patients was 4.71 (0.72) y, while the mean age of presentation to our clinic was 10.23 (1.15) y. The mean (SD) age of loss of ambulation in our patients was 8.81 (0.81) y. We also observed mild facial weakness in 41(51.3%) cases. Although there was an increase incidence of facial weakness in older children, there was no correlation of facial weakness with age (P=0.057). Similarly the facial weakness was equally common in ambulatory and non-ambulatory patients.

The highest and lowest serum CPK values among these 100 patients are recorded as 38160 IU/L and 388 IU/L, respectively.

Detailed genetic pattern is shown in Table II. Exon 48 was the most commonly deleted exon in our study. Although no specific pattern of deletion was found, most of our patients with severe clinical features had deletion of distal exons. We also did not find any difference of size and pattern of exon deletion with loss of early ambulation (P=0.089) in our patients.

TABLE  II	Exon Deletion Pattern in Duchenne Muscular Dystrophy (N=73) 

This study presents the clinical and genetic pattern of confirmed (genetically or biopsy proven) DMD cases from a tertiary referral centre of eastern India. The age of onset of DMD was distributed randomly among this small group of patients similar to earlier studies [11]. The distribution of weakness, inter-individual variability of weakness pattern [12], and presence of calf hypertrophy and Valley sign were similar to other studies [13]. Similar to our study, mental retardation has been reported in about one-third of DMD patients [14].

The frequency of dystrophin gene deletions is reported to vary from 22% to 86% [6-9, 15-19], which is in agreement with our study. Possibilities in the remaining cases were either point mutation and duplication or other rare variants of congenital dystrophy which mimic DMD in clinical presentation.

Our observation of dystrophin gene deletion mutations is relatively higher than that reported in most of the other studies including geographically contiguous neighbouring areas like Pakistan (40.7%), Sri Lanka (62.5%) and China (66%) [15-17]. Low rate of deletion in frequency in some studies may be due to screening of fewer exons than recommended and small group of patients studied. However, studies from various regions of India like South [5], North [8] and West [9] showed similar deletional rates of 73.1%, 72% and 72% respectively, while previous study from eastern India by Basak, et al. [4] reported lower rate of deletion (65.7%). The varying rate of deletion in different parts of country may be due to highly selective group, different population group and ethnic differences in these populations.

In our study, 72.6% cases showed distal exon involvement, which is in agreement with literature [4-7]. The patients with advanced clinical features showed variable genetic pattern with distal exon deletion being the most common pattern. It is difficult to correlate clinical severity with deletion type within DMD as patients are being seen at different points in the natural history of their disease making assessment difficult, especially in the young isolated case. Any particular deletion pattern is rarely seen in DMD as DMD deletions are varied in position and extent. Our data clearly shows that DMD deletions are inhomogeneously distributed among our patients. Our observation was similar to other studies like Banerjee, et al. [20] and Swaminathan, et al. [5] where they could not establish any phenotypic and genotype correlation considering the size of exon deletion and pattern of deletion. Moreover, Baumbach, et al. [21] and Lindlof, et al. [22] found no significant correlation between clinical phenotype and number of exon deletion.

The diagnosis of DMD is based on clinical, biochemical and histopathological studies and further confirmed by molecular analysis. Other than multiplex PCR, detection of duplication can be done by Southern blot analysis [23], dosimetric PCR based methods [24] or techniques such as multiplex ligation dependent probe amplification [25] (MLPA), but all these methods can not identify point mutation. We could not analyze point mutation or duplication in our laboratory as they require sophisticated facilities and are being performed in very few places and for research purpose only. The identification of female carrier or prenatal diagnosis is important for preventing the birth of children affected by DMD, when the index case is alive.

The availability of genetic testing makes it possible to confirm the diagnosis early without going for any invasive procedure. Hence, the genetic studies should be the investigation of choice in suspected cases of DMD and muscle biopsy should be reserved for cases where genetic study couldn’t confirm the disease.

Contributors: SD: data collection, statistical analysis and manuscript writing; AKS, AP, AB and GG: study design, data collection and manuscript writing; DSG: data collection and manuscript writing; AJ and SR: genetic testing.

Funding: None; Competing interest: None stated.

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