Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort

Abstract

Mutations in the DMD gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Readily available clinical tests detect only deletions of one exon or greater, which are found in approximately 60% of cases. Mutational analysis of other types of DMD mutations, such as premature stop codons and small frameshifting insertions or deletions, has historically been hampered by the large size of the gene. We have recently reported a method that allows the rapid and economical sequencing of the entire coding region of the DMD gene, and that is more sensitive than methods based on single-strand conformational polymorphism (SSCP) screening or other preliminary screening steps. Here we use single condition amplification/internal primer (SCAIP) sequencing analysis, in combination with multiplex amplifiable probe hybridization (MAPH) analysis of duplications, to report the frequency of mutations in a large cohort of unselected dystrophinopathy patients from a single clinic. Our results indicate that 7% of dystrophinopathy patients do not have coding region mutations, suggesting that intronic mutations are not uncommon. The availability of rapid and thorough mutation analysis from peripheral blood samples, along with an improved estimate of the percentage of non-coding region mutations, will be of benefit for improved genetic counseling and in identification of cohorts for clinical trials.

DMD pseudoexon mutations: splicing efficiency, phenotype, and potential therapy

“…The exact prevalence of these mutations is unknown, but they may account for many of the 4 to 7% of mutations not detected by genomic mutation analysis methods. 8,9 All three patients we describe carry mutations that result in aberrant mRNA that would be predicted by the reading-frame rule 1 alone to cause DMD. Patient DC0160 has an in-frame pseudoexon in which a premature stop signal is encoded.…”

“…6,7 Combining these modern methods of molecular analysis results in detection of about 93 to 96% of mutations via genomic DNA derived from blood samples. 8,9 The availability of detailed genomic mutation anal-ysis has resulted in the increasingly frequent identification of individuals with DMD but no coding region mutations in the DMD gene, and thus to the increasing recognition of a previously difficult to identify category of DMD mutations: intronic point mutations that create novel splice sites, resulting in the inclusion of intronic sequence as a "pseudoexon" within the mRNA. 10 -12 The large size of the DMD introns still precludes direct intronic sequencing as a practical assay.…”

Female Carriers of Duchenne Muscular Dystrophy

“…46,47) Furthermore, dystrophin gene sequencing is used to analyze single-condition amplification/internal primers and multiplex amplifiable probe hybridization for detecting point mutations or small deletions/insertions if deletion/ duplication testing is negative. 48,49) Full characterisation of the mutation (deletion endpoints or exact position of any point mutation) is helpful to investigate the clinical variability of dystrophinopathy 50,51) and recent trials about mutation-specific treatment.…”

Targeting post-transcriptional control for drug discovery

“…[20][21][22][23] in muscle myofiber membranes, 57 with consequent loss of muscle integrity and generalized muscle weakness. PTC124 treatment of mdx mice and cultured mdx myotubes promotes production of full-length dystrophin (Fig.…”