This study presents the retrospective analysis of genetic testing for 415 clinically suspected DMD/BMD patients in our centre located in southern India using MLPA. MLPA is a rapid and highly sensitive technique used to detect deletions and duplications in the DMD gene [
17‐
21]. In this cohort, the overall detection rate by MLPA was 75.42%. Our findings are comparable to the study Wang et al., [
22], who reported a mutation rate of 72.5% in the Chinese population. The present study showed deletions in 91.6% cases and duplications in 8.30% cases in the dystrophin gene. The frequency of deletion was more common than duplications, similar to frequency reported from other parts of India [
23‐
27]. The reported deletion rates in Pakistanis is 40.7%, Chinese 66.25%, Korean 45.5% and in Taiwanese patients 36%, thus showing possible variations among different populations [
22,
28‐
30]. The duplication rate in our cases mainly involved larger fragments and the pattern of duplication was more towards the distal part of the gene unlike other populations [
22]. Random age distribution was observed in this cohort, i.e. there was no correlation between extent of deletion/duplication or position of mutation, and the age of onset of clinical symptoms. This finding was similar to the Dubowitz study where no correlations could be drawn between age of onset or severity to the extent of mutation [
31].
Muscle biopsy was undertaken for patients who tested negative by MLPA. Immunohistochemically, the diagnosis of DMD was established for the patients with complete absence of staining along the sarcolemma. However, BMD patients showed heterogenous dystrophin expression ranging from reduced patchy staining to normal staining on IHC [
32‐
34]. The dystrophin – glycoprotein complex is responsible for stabilizing the muscle fiber, a perturbation in any of its components may result in overlapping clinical presentation. Six patients with suspected DMD showed normal dystrophin labelling, but absence of sarcoglycans expression. Immunohistochemistry thus still remains the gold standard method for diagnosing muscular dystrophies [
24]. IHC should be considered to detect dysfunctional dystrophin expression when genetic testing results are negative.
Genotype- Phenotype correlation
Age of onset, CK values, age at wheel chair bound and IQ score was evaluated in this study to define genotype and clinical phenotype correlation. Patients who lost ambulation at an average age of 9.5 years were seen to have deletions in the exon 45–55 region of the DMD gene (
n = 30). A lower IQ score was noted largely in patients who had distal gene deletions. This was keeping with expectation as the full length isoform Dp427 is minimally expressed in the brain [
35]. The dystrophin isoforms Dp140 & Dp71 which are highly expressed in the brain lack the proximal exons. The role of dystrophin in the brain remains unclear, however mutations at the 3’ end of the gene have been associated with compromised brain function. Ricotti et al [
36] observed that mutations disrupting the isoform Dp140 & Dp70 are more frequently associated with lower IQ scores. There was no correlation noted in CK values with gene mutation as this was a cross sectional study [
37].
The PROVEAN analysis predicts effect of mutation based on the changed aminoacid sequence of mutated dystrophin protein. Mutations in exon 45–54 (Out of frame) and exon 46–54 (In-frame) region in the central rod domain of dystrophin showed more negative scores compared to other domains in the present study. Previous reports demonstrated that the phosphorylation sites of dystrophin present within the central rod domain including T2621 which is encoded by exon 53 might affect the structure of this N terminal domain. Dystrophin upon phosphorylation is believed to undergo a conformational change in the N-terminal actin binding domain, thereby enhancing its affinity for myofibrillar actin [
38,
39]. Actin also binds the central rod domain encoded by exon 31–45 which is located between spectrin type repeats 11–17 [
40]. This reconfirms the role of rod domain in dystrophin function [
41].
Dystrophin protein interacts with integral membrane proteins to form the dystrophin- glycoprotein complex (DGC). The role of DGC is to stabilize the sarcolemma and protect the muscle fibers from long term damage. The hydrophobic region of dystrophin plays an important role in maintaining the stability and interaction with other proteins. There are four hydrophobic regions in dystrophin coded by exons 3–6 (region I), 42 (region II), 51 (region III), and 65–68 (region IV) which are found on the calponin homology CH2 domain on the actin-binding domain (ABD), spectrin-type repeat 16, hinge III and the EF Hand domain respectively. Liang et al [
16] observed that mutational disruption in the hydrophobic region I, II, IV directly impairs the DGC function which leads to severe DMD phenotype, whereas, region III disruption leads to a less severe BMD phenotype. Carsana et al [
42] demonstrated that an in-frame deletion of the hinge region in the distal rod domain shows a milder phenotype compared with deletions that do not include hinge III region. Further analysis by PROVEAN programme showed the deletion of hinge III region has more negative score compared to deletions which do not include the hinge III region. This suggests that clinical severity of the BMD maybe determined by the presence or absence of hinge III region in the dystrophin protein. However, all patients (
n = 12) with exon 51 deletion /duplication corresponding to region III with age of onset ranging from 1–8 years had a severe DMD phenotype as predicted by reading frame rule.
Dystrophin is a large cytoskeletal protein comprised of four domains. The larger central rod domain has 24 repeating units similar to spectrin-like repeats. The repeat is a triple coiled coil structure made up of three helices with heptad pattern of amino acids [
43,
44]. This filamentous protein acts as a scaffold for several interacting partners and also provides resistance to the stress of muscle contraction. Any mutation altering this structure of dystrophin might be expected to affect its function along with that of its binding partners. The
eDystrophin programme provides a computational model for each in-frame mutation and shows whether an approximate 3D filamentous structure is reconstituted (hybrid repeat) or a more deleterious structure (fractional) repeat is formed. Nicholas et.al [
44] reported the differences in the structure of mutant dystrophin protein may be responsible for clinical heterogeneity in BMD patients. They observed earlier wheel chair dependency and early development of cardiomyopathy in patients with exon 45–47 (Fractional repeat) deletion compared to exon 45–48 (Hybrid repeat) deletion. Fractional repeat has slower refolding dynamics and higher molecular surface hydrophobicity compared to hybrid repeat. In this study, the most prevalent in-frame deletion observed was exon 45–47 deletion which was associated with age of onset 4–20 years and exon 45–48 deletion which was associated with age of onset 5–20 years. Analysis of hinge III deletion in
e-dystrophin programme also results in retention of typical filamentous structure of dystrophin (hybrid repeat). The hybrid repeat reconstitution depends on exon phasing and though the presence of hybrid repeat does not restore the dystrophin function completely, it results in a more functional protein compared to fractional repeat [
15]. Exon phasing if considered along with restoration of reading frame for exon-skipping therapy might result in improved clinical outcome.
To assess the effect of mutation on clinical severity, we did correlations between pathogenicity score and the age of onset of the clinical symptoms primarily, observed muscle weakness. Both DMD & BMD patients showed no definite correlation between sequence variation as assessed by PROVEAN score and clinical symptoms. In this cohort, we observed ‘neutral effect’ both in patients having exon 51 deletion/duplication which would produce truncated protein and duplications in exon 2–11 region, where the entire amino acid sequence is disturbed. We hypothesize that this mild phenotype seen as milder disease progression despite a large predicted ‘out of frame’ mutation in the proximal part of the protein could be due to compensatory changes in the downstream region. Further, the possibility of false positive deletion calls due to variations at the site of primer binding cannot be ruled out. These mutations which cannot be detected by MLPA should be further evaluated by sequencing.