Background & objectives: Duchenne (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders, caused by mutations in the dystrophin gene. Genetic diagnosis of the proband becomes crucial, and forms the base for carrier analysis, genetic counselling, prediction of natural history and prognosis, and eligibility for therapeutic strategies. Traditional multiplex PCR assay is the common method used in India to detect DMD gene deletions, mainly in the hot-spot region. Deletions of exons outside the usual 18 or 21 exons in the hot-spot, duplications and carrier analysis are often left without precise genetic diagnosis and require efficient dosage/quantitative analysis. In this study we evaluated the efficacy of using multiplex PCR (mPCR) of 30 exons followed by multiplex ligation-dependent probe amplification (MLPA), to study deletions and duplications in the DMD gene in patients clinically diagnosed as BMD/ DMD. Methods: Using an algorithm of mPCR and MLPA which was less invasive and cost-effective, we performed retrospective and prospective analysis on 150 male patients. Results: Multiplex PCR could pick up deletions in 103 of the 150 cases. MLPA was able to detect deletions and duplications including nine additional mutations. Further, the borders of the deletions and duplications were more accurately defined by this recent methodology, which enables one to determine the effect of the mutation on the reading frame. In all, including the single exon deletions, MLPA was efficient in accurately confirming mutations in 35 per cent of all cases. Ten novel mutations were identified in this study. Overall, this approach confirmed mutations in 75 per cent of the patients in our study. Interpretations & conclusions: The systematic approach/algorithm used in this study offers the best possible economical mutation analysis in the Indian scenario.

Use of multiplex ligation-dependent probe amplification (MLPA) for Duchenne muscular dystrophy (DMD) gene mutation analysis.

Background : Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy that affects young boys and the dystrophin gene on the X chromosome has been found to be associated with the disorder. Materials and Methods : In this prospective study, 112 clinically diagnosed DMD patients had muscle biopsy and were tested for exon deletions. Genotyping was also carried out at STR44, STR45, STR49 and STR 50 markers in 15 families. Results : Of the 112 clinically suspected DMD patients, the diagnosis of DMD was confirmed by histopathology and/or genetics in 101 patients. The mean age of onset was 3.1±1.44 years (1-6 years) and the mean age at presentation was 8.0±3.1 years (1.1-18.0 years). Delayed motor milestones were present in 63 (62.3%) patients. The mean creatine kinase value was 11822.64±8206.90 U/L (1240-57,700). Eighty-four patients had muscle biopsy and immunohistochemistry was done in 60 muscle samples, all of which demonstrated absence of dystrophin staining. Of the 60 dystrophin-negative cases, 73% showed deletion of at least one exon. Single exon deletion was found in 20.4%. Distal hotspot Exons 45, 47, 49 and 50 were the commonly deleted xenons and the deletion rates were 36%, 35%, 33.7% and 38.5% respectively. Conclusions : In this study population in south India the deletion rate was 73% and were more frequent in the distal end exon. With the availability of genetic analysis, the first investigation of choice in DMD should be genetic studies and muscle biopsy should be considered only if the genetic tests are negative or not available.

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Duchenne muscular dystrophy: A clinical, histopathological and genetic study at a neurology tertiary care center in southern India

Families of children with Duchenne muscular dystrophy (DMD) go through significant challenges in dealing with the condition. Few studies have looked into the situation, especially in the sociocultural scenario that is unique to India. The authors' aim was to identify the psychosocial challenges for caregivers of children with DMD. A cross-sectional descriptive study was perfomed among the caregivers of 60 children with DMD who were attending the neuromuscular disorders clinic of a national tertiary referral center for neurological disorders. The knowledge and attitude, psychosocial needs, burden, and coping patterns were assessed in an interview. The findings showed that parents of children with DMD tended to have inadequate understanding of the disease but had a positive attitude, had a moderate family burden, and tended to rely more on religion, focus on and venting of emotions, and instrumental and emotional social support for coping. Caregivers of children with DMD would benefit from psychosocial intervention to address their understanding of and attitude toward the disease, as well as burden of dealing with it, and to help them develop their coping skills and meet their children's and their own needs.

Psychosocial Challenges in Family Caregiving with Children Suffering from Duchenne Muscular Dystrophy

We report a dystrophinopathy patient with an in-frame deletion of DMD exons 45-47, and therefore a genetic diagnosis of Becker muscular dystrophy, who presented with a more severe than expected phenotype. Analysis of the patient DMD mRNA revealed an 82 bp pseudoexon, derived from intron 44, that disrupts the reading frame and is expected to yield a nonfunctional dystrophin. Since the sequence of the pseudoexon and canonical splice sites does not differ from the reference sequence, we concluded that the genomic rearrangement promoted recognition of the pseudoexon, causing a severe dystrophic phenotype. We characterized the deletion breakpoints and identified motifs that might influence selection of the pseudoexon. We concluded that the donor splice site was strengthened by juxtaposition of intron 47, and loss of intron 44 silencer elements, normally located downstream of the pseudoexon donor splice site, further enhanced pseudoexon selection and inclusion in the DMD transcript in this patient.

https://researchrepository.murdoch.edu.au/27730/1/Greer_et_al%2D2015%2DMolecular_Genetics_%26_Genomic_Medicine.pdf

Pseudoexon activation increases phenotype severity in a Becker muscular dystrophy patient

Studies on monogenic diseases are considered valuable because they give insights and expand our knowledge on gene function and regulation. Despite all the current advancement in science and technology , a deep understanding and knowledge as to why only those particular genes are affected in a disease is still vague. We also lack profound illumination as to why only certain mutations are seen in a disease. Though useful from a research perspective, a majority of these diseases are lethal resulting in death of the affected individual. Unfortunately, in the fast – growing land of India, the incidence of monogenic diseases is very high with few counter-measures in place. This article encompasses a list of all monogenic diseases ever to be reported in India with special focus on five diseases which has been stated to have the highest incidence in India. Here, we discuss about the limited research carried out in India on these high incidence monogenic diseases, the other diseases related to those genes, the range of treatments available for these diseases in India in contrast to its availability around the world and the need to develop treatment strategies to reduce the mortality and morbidity due to these rare but daunting diseases.

Monogenic diseases in India.

Background: Becker muscular dystrophy (BMD) is caused by mutations in the dystrophin gene with variable phenotypes. Becker muscular dystrophy patients have low levels of nearly full-length dystrophin and carry in-frame mutations, which allow partial functioning of the protein. Aim: To study the deletion patterns of BMD and to correlate the same with reading frame rule and different phenotypes. Setting: A tertiary care teaching hospital. Design: This is a prospective hospital-based study. Materials and Methods: Thirty-two exons spanning different hot spot regions using Multiplex PCR techniques were studied in 347 patients. Two hundred and twenty-two showed deletions in one or more of the 32 exons. Out of these, 46 diagnosed as BMD patients were analyzed. Results: Forty-six BMD patients showed deletions in both regions of the dystrophin gene. Out of these 89.1% (41/46) were in-frame deletions. Deletions starting with Exon 45 were found in 76.1% (35/46) of the cases. Mutations in the majority of cases i.e. 39/46 (84.8%) were seen in 3' downstream region (Exon 45-55, distal rod domain). Few, i.e. 5/46 (10.8%) showed deletions in 5' upstream region (Exons 3-20, N-terminus and proximal rod domain) of the gene, while in 2/46 (4.4%) large mutations (>40 bp) spanning both regions (Exons 3-55) were detected. Conclusion: This significant gene deletion analysis has been carried out for BMD patients particularly from Western India using 32 exons.

/pdf/becker-muscular-dystrophy-in-indian-patients-analysis-of-5er2k4gpii.pdf

Becker muscular dystrophy in Indian patients: analysis of dystrophin gene deletion patterns.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder involving degeneration of anterior horn cells of spinal cord resulting in progressive muscle weakness and atrophy. Aims: The molecular analysis of two marker genes for spinal muscular atrophy (SMA) i.e., the survival motor neuron gene (SMN) and the neuronal apoptosis inhibitory protein gene (NAIP) was conducted in 39 Indian patients with clinical symptoms of SMA. Out of these, 28 showed homozygous deletions and the phenotypic features of these SMA patients were compared with the corresponding genotypes. Settings: A tertiary care teaching Hospital. Design: This is a prospective hospital based study. Materials and Methods: Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was used to detect the deletion of exon 7 and exon 8 of SMN1 gene, as well as multiplex PCR for exon 5 and 13 of NAIP gene. Results: Exons 7 and 8 of SMN and NAIP (exon 5) were homozygously deleted in 73% of SMA I and 27% of SMA II patients. SMN exon 7 and 8 deletions without NAIP deletions were seen in 27% of type I SMA and 46% of SMA type II patients. Two patients of type III SMA showed single deletion of SMN exon 7 along with 27% of SMA type II patients. Conclusion: With the advent of molecular biology techniques, SMN gene deletion studies have become the first line of investigation for confirmation of a clinical diagnosis of SMA. The findings of homozygous deletions of exons 7 and/or 8 of SMN1 gene confirms the diagnosis of SMA, even in patients with atypical clinical features. Deletions of NAIP gene were mainly seen in severely affected patients, hence is useful for predicting the prognosis.

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Correlation between deletion patterns of SMN and NAIP genes and the clinical features of spinal muscular atrophy in Indian patients.

The application of molecular diagnostic techniques has greatly improved the diagnosis, carrier detection, prenatal testing and genetic counseling for families with Duchenne and Becker muscular dystrophy (D/BMD) in India. The prediction of Duchenne muscular dystrophy (DMD) patients to have out-framed deletions and Becker's muscular dystrophy (BMD) patients to have in-frame deletions of dystrophin gene holds well in the vast majority of cases. Mutation detection is obviously critical for diagnosis but it may also be important for future therapeutic purposes. These factors underscore the need for earlier referral, genetic counseling and provision of support and rehabilitation services which are the main priorities for psychosocial assessment and intervention at medical and social levels.

/pdf/duchenne-and-becker-muscular-dystrophies-an-indian-update-on-260z6xy413.pdf

Jayshree J Nadkarni

Papers

Becker muscular dystrophy in Indian patients: analysis of dystrophin gene deletion patterns.

Correlation between deletion patterns of SMN and NAIP genes and the clinical features of spinal muscular atrophy in Indian patients.

Duchenne and Becker muscular dystrophies: an Indian update on genetics and rehabilitation