Rapid Direct Sequence Analysis of the Dystrophin Gene

Abstract

Mutations in the dystrophin gene result in both Duchenne and Becker muscular dystrophy (DMD and BMD), as well as X-linked dilated cardiomyopathy. Mutational analysis is complicated by the large size of the gene, which consists of 79 exons and 8 promoters spread over 2.2 million base pairs of genomic DNA. Deletions of one or more exons account for 55%-65% of cases of DMD and BMD, and a multiplex polymerase chain reaction method-currently the most widely available method of mutational analysis-detects approximately 98% of deletions. Detection of point mutations and small subexonic rearrangements has remained challenging. We report the development of a method that allows direct sequence analysis of the dystrophin gene in a rapid, accurate, and economical fashion. This same method, termed "SCAIP" (single condition amplification/internal primer) sequencing, is applicable to other genes and should allow the development of widely available assays for any number of large, multiexon genes.

Experience and Strategy for the Molecular Testing of Duchenne Muscular Dystrophy

“…Recently a single condition amplification/internal primer sequencing technique was described for point mutation detection in the dystrophin gene. 46 The method relied on amplification of dystrophin gene exons at a single set of PCR conditions followed by sequencing using a second set of internal primers. The analysis was both automated and high throughput, with all of the dystrophin exons being sequenced within 3 working days at a reasonable cost.…”

Clinical Utility Gene Card for: Becker muscular dystrophy

“…Approximately 5-10% of BMD cases are a result of point variants. The majority of these are small indels and splice site variants, and less commonly missense variants; however, they can also affect dystrophin function [11,12]. Intronic variants can also negatively influence splicing and lead to BMD.…”

Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans, mice, and zebrafish

“…There have been some recent improvements though, as methods have been developed to scan the dystrophin locus for mutations by SSCP (Hayashi and Yandell 1993) and Wave technology (Bennett et al 2001). In addition, a group has recently developed an alternative sequencing approach for the dystrophin locus (Flanigan et al 2003), which is robust and economical, but has not been refined for other loci and has some limitations on its use.…”