Targeted sequencing of the DMD locus: A comprehensive diagnostic tool for all mutations

Abstract

Background & objectives:Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder and is caused mainly by deletion, duplication and point mutations in the DMD gene. Diagnosis of DMD has been a challenge as the mutations in the DMD gene are heterogeneous and require more than one diagnostic strategy for the validation of the mutation. This study was planned to evaluate the targeted next-generation sequencing (NGS) as a single platform to detect all types of mutations in the DMD gene, thereby reducing the time and costs compared to conventional sequential testing and also provide precise genetic information for emerging gene therapies.Methods:The study included 20 unrelated families and 22 patients from an Indian population who were screened for DMD based on phenotypes such as scoliosis, toe walking and loss of ambulation. Peripheral blood DNA was isolated and subjected to multiplex ligation-dependent probe amplification (MLPA) and targeted NGS of the DMD gene to identify the nature of the mutation.Results:In the study patients, 77 per cent of large deletion mutations and 23 per cent single-nucleotide variations (SNVs) were identified. Novel mutations were also identified along with reported deletions, point mutations and partial deletions within the exon of the DMD gene.Interpretation & conclusions:Our findings showed the importance of NGS in the routine diagnostic practice in the identification of DMD mutations over sequential testing. It may be used as a single-point diagnostic strategy irrespective of the mutation type, thereby reducing the turnaround time and cost for multiple diagnostic tests such as MLPA and Sanger sequencing. Though MLPA is a sensitive technique and is the first line of a diagnostic test, the targeted NGS of the DMD gene may have an advantage of having a single diagnostic test. A study on a larger number of patients is needed to highlight NGS as a single, comprehensive platform for the diagnosis of DMD.

Molecular Diagnosis of Muscular Dystrophy Patients in Western Indian Population: A Comprehensive Mutation Analysis Using Amplicon Sequencing

“…Previously, many studies have been published for NGS-based approaches for MD ( Lim et al, 2011 ; Wang et al, 2014 ; Wei et al, 2014 ; Alame et al, 2016 ). However, there are very few such studies reported for the Indian population ( Aravind et al, 2019 ; Ganapathy et al, 2019 ; Polavarapu et al, 2019 ).…”

“…Further, the NGS platforms also identified the novel variants as well as confirmation of hard-to-detect variants ( Sheikh and Yokota, 2020 ). Recent studies suggest that the utilization of a high-throughput method using NGS platform is more suitable for clinical diagnosis ( Okubo et al, 2016 ; Aravind et al, 2019 ). Moreover, the detection of large duplications is a major challenge for single-point diagnostic strategy ( Okubo et al, 2016 ).…”

A Review on the Challenges in Indian Genomics Research for Variant Identification and Interpretation

“…Diagnostic genetic tests, on the other hand, help in identification of the molecular cause of the disease. These tests are used to confirm known or suspected diagnosis, carrier status determination, identification of at-risk genetic relatives, optimize treatments, and clinical decisions (Gupta et al, 2017;Aravind et al, 2019;Uttarilli et al, 2019). There are different types of diagnostic genetic tests currently available in India such as single-gene and multigene testing, exome, and genome sequencing, carrier and newborn screening (Puri et al, 2017;Singh et al, 2018).…”

Current Pharmacological Strategies for Duchenne Muscular Dystrophy

“…The severity of DMD depends on the mutation type. ‘Out of frame’ mutations disrupt the reading frame and further produce the dysfunctional dystrophin, which result in the severe DMD phenotype ( Aravind et al, 2019 ). On the other hand, ‘In-frame’ mutations perverse the frame of DMD gene and generate partial functional dystrophin protein, which lead to less severe Becker muscular dystrophy (BMD) phenotype ( Haidet et al, 2008 ).…”