Log-PCR: A New Tool for Immediate and Cost-Effective Diagnosis of up to 85% of Dystrophin Gene Mutations

Abstract

Background: Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the dystrophin gene. Despite the progress in the technologies of mutation detection, the disease of one third of patients escapes molecular definition because the labor and expense involved has precluded analyzing the entire gene. Novel techniques with higher detection rates, such as multiplex ligation-dependent probe amplification and multiplex amplifiable probe hybridization, have been introduced. Methods: We approached the challenge of multiplexing by modifying the PCR chemistry. We set up a rapid protocol that analyzes all dystrophin exons and flanking introns (57.5 kb). We grouped exons according to their effect on the reading frame and ran 2 PCR reactions for DMD mutations and 2 reactions for BMD mutations under the same conditions. The PCR products are evenly spaced logarithmically on the gel (Log-PCR) in an order that reproduces their chromosomal locations. This strategy enables both simultaneous mapping of all the mutation borders and distinguishing between DMD and BMD. As a proof of principle, we reexamined samples from 506 patients who had received a DMD or BMD diagnosis. Results: We observed gross rearrangements in 428 of the patients (84.6%; 74.5% deletions and 10.1% duplications). We also recognized a much broader spectrum of mutations and identified 14.6% additional cases. Conclusions: This study is the first exhaustive investigation of this subject and has made possible the development of a cost-effective test for diagnosing a larger proportion of cases. The benefit of this approach may allow more focused efforts for discovering small or deep-intronic mutations among the few remaining undiagnosed cases. The same protocol can be extended to set up Log-PCRs for other high-throughput applications.

Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center

“…The largest institutional database reported was from Italy; that database contained 506 cases, but some cases remained for which no dystrophin mutation had been identified. 35 The KUCG database is the second-largest institutional database, but the first completed mutation database. Even in smallscale studies, the mutation detection rate has previously only reached up to 96% of examined cases.…”

Motor Chip: A Comparative Genomic Hybridization Microarray for Copy-Number Mutations in 245 Neuromuscular Disorders

“…In NMD molecular diagnosis (16 -18 ), aCGH applications initially focused on Duchenne and Becker muscular dystrophies, which are caused predominantly by DMD 10 (dystrophin) deletions (75%) or duplications (10%) on the X chromosome (19 ). Other aCGH designs have focused on sarcoglycanopathies (20 ), collagen VI-related myopathies (21 ), and amyotrophic lateral sclerosis (22 ).…”

A resolved discrepancy between multiplex PCR and multiplex ligation‐dependent probe amplification by targeted next‐generation sequencing discloses a novel partial exonic deletion in the Duchenne muscular dystrophy gene

“…In these methods, loss of the amplified product of a corresponding exon is commonly interpreted as a deletion of that entire exon. Contradictory amplification results between these two methods may occur if there are nucleotide changes within PCR primer or MLPA probe binding sites, especially when the loss of amplification only involves a single exon. Here, we propose an alternative possibility that the discrepancy reflects partial exonic deletion that escapes MLPA detection.…”