title: Hybrid minigene splicing assay verified the pathogenicity of a novel splice site variant in the dystrophin gene of a Chinese patient with typical Duchenne muscular dystrophy phenotype.
abstract: BACKGROUND
  Duchenne muscular dystrophy (DMD) is typically caused by disrupting the reading frame of the dystrophin gene: approximately 70%-80% of mutational events are represented by deletions or duplications of one or more exons in the dystrophin gene, and the remaining cases by subtle mutations, including point mutations, small indels, small inversions, and complex small rearrangements. The dystrophin gene is the largest known gene with one of the highest known rates of new mutations.
  
  
  METHODS
  Deletions and duplications were detected in the DMD gene of the proband by using multiple ligation-dependent probe amplification (MLPA). Targeted next-generation sequencing (NGS) was used in the subtle mutation detection, followed by Sanger sequencing confirmation. The effect of the mutation on the splicing of the DMD gene was assessed by bioinformatics prediction and hybrid minigene splicing assay (HMSA).
  
  
  RESULTS
  Neither duplication nor deletion was found in the DMD gene of the proband. While a novel splice site mutation c.6762+1G>C was identified in the proband by NGS and Sanger sequencing, and his mother was heterozygous at the same site. Bioinformatics predicted that the 5' donor splice site of intron 46 disappeared because of the mutation, which would lead to aberrant splicing and introduce premature stop codon. The HMSA results were in agreement with the prediction.
  
  
  CONCLUSIONS
  The novel splice site mutation caused DMD in the proband by aberrant splicing. We suggested that combined applications of MLPA, NGS, HMSA and bioinformatics are comprehensive and effective methods for diagnosis and aberrant splicing study of DMD.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Wang
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Lin
authors.forename: Liping
authors.initials: L
authors.lastname: Qiu
authors.forename: Dezhu
authors.initials: D
authors.lastname: Zheng
authors.forename: Aizhen
authors.initials: A
authors.lastname: Yan
authors.forename: Jian
authors.initials: J
authors.lastname: Zeng
authors.forename: Fenghua
authors.initials: F
authors.lastname: Lan
mesh_terms: D044466:Asian People / Q000235:genetics*; D002648:Child; D002681:China; D019295:Computational Biology; D004252:DNA Mutational Analysis; D016189:Dystrophin / Q000235:genetics*; D017353:Gene Deletion; D014644:Genetic Variation / Q000235:genetics*; D006801:Humans; D008297:Male; D020388:Muscular Dystrophy, Duchenne / Q000175:diagnosis / Q000235:genetics*; D010641:Phenotype; D012326:RNA Splicing / Q000235:genetics*
chemical_list: D016189:Dystrophin