PCR‐based strategies for delimiting large mutations in the DMD gene

Abstract

Large mutations such as deletions, inversions and duplications are causative factors in many genetic diseases, in particular Duchenne muscular dystrophy (DMD). Due to the complexity of pre‐mRNA splicing, the genomic span of such mutations is not readily predictable from the sequence of their mature mRNAs, nor vice versa. The difficulty of delimiting these mutations is compounded further in large genes with substantial intronic complements, such as the human dystrophin gene DMD, and the cost of whole genome sequencing, although continuing to fall, remains prohibitive for routine diagnosis and researchers attempting to define multiple large mutations. We outline several polymerase chain reaction‐based strategies for defining the precise junctions of large gene rearrangements. These techniques represent a practical alternative to whole genome sequencing for defining such mutations to a single gene. We report a pilot study designed to verify the feasibility of these techniques on a subset of patients with large mutations to the DMD gene. This study also analyses the mRNAs of these patients and attempts to draw inferences about the effect these mutations have on pre‐mRNA processing. The data gleaned from this project and similar future studies will contribute towards our understanding of pre‐mRNA splicing, the spliceosome and motifs that impact on gene expression. In a proportion of DMD cases, the phenotype and genotype do not correlate, and elucidating the mechanisms responsible will allow more accurate diagnosis and inform personalized treatment strategies