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

ABSTRACT: Muscular Dystrophies (MDs) are a group of inherited diseases and heterogeneous in nature. To date, 40 different genes have been reported for the occurrence and/or progression of MDs. This study was conducted to demonstrate the application of next-generation sequencing (NGS) in developing a time-saving and cost-effective diagnostic method to detect single nucleotide variants (SNVs) and copy number variants (CNVs) in a single test. A total of 123 cases clinically suspected of MD were enrolled in this study. Amplicon panel-based diagnosis was carried out for 102 (DMD/BMD) cases and the results were further screened using multiplex ligation-dependent probe amplification (MLPA). Whilst in the case of LGMD (N = 19) and UMD (N = 2), only NGS panel-based analysis was carried out. We identified the large deletions in 74.50% (76/102) of the cases screened with query DMD or BMD. Further, the large deletion in CAPN3 gene (N = 3) and known SNV mutations (N = 4) were identified in LGMD patients. Together, the total diagnosis rate for this amplicon panel was 70.73% (87/123) which demonstrated the utility of panel-based diagnosis for high throughput, affordable, and time-saving diagnostic strategy. Collectively, present study demonstrates that the panel based NGS sequencing could be superior over to MLPA.

SUBMITTER: Patel KM

PROVIDER: S-EPMC8679082 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput.An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper.This automated process allows laboratories to discover DNA variations in a short time and at low cost.

Project description:Recently DNA sequencing analysis has played a vital role in the unambiguous diagnosis of clinically suspected patients with Duchenne Muscular Dystrophy (DMD). DMD is a monogenic, X-linked, recessive, degenerative pediatric neuromuscular disorder affecting males, invariably leading to fatal cardiopulmonary failure. Early and precise diagnosis of the disease is an essential part of an effective disease management strategy as care guidelines and prevention through counseling need to be initiated at the earliest particularly since therapies are now available for a subset of patients. In this manuscript we report the DMD gene mutational profiles of 961 clinically suspected male DMD patients, 99% of whom were unrelated. We utilized a molecular diagnostic approach which is cost-effective for most patients and follows a systematic process that sequentially involves identification of hotspot deletions using mPCR, large deletions and duplications using MLPA and small insertions/ deletions and point mutations using an NGS muscular dystrophy gene panel. Pathogenic DMD gene mutations were identified in 84% of patients. Our data compared well with the frequencies and distribution of deletions and duplications reported in the DMD gene in other published studies. We also describe a number of rare in-frame mutations, which appeared to be enriched in the 5' proximal hotspot region of the DMD gene. Furthermore, we identified a family with a rare non-contiguous deletion mutation in the DMD gene where three males were affected and two females were deemed carriers. A subset of patients with mutations in the DMD gene who are likely to benefit therapeutically from new FDA and EMA approved drugs were found in our cohort. Given that the burden of care for DMD patients invariably falls on the mothers, particularly in rural India, effective genetic counseling followed by carrier screening is crucial for prevention of this disorder. We analyzed the carrier status of consented female relatives of 463 probands to gauge the percentage of patients with familial disease. Our analysis revealed 43.7% of mothers with DMD gene mutations. Our comprehensive efforts, involving complete genetic testing coupled with compassionate genetic counseling provided to DMD patients and their families, are intended to improve the quality of life of DMD patients and to empower carrier females to make informed reproductive choices to impede the propagation of this deadly disease.

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

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