Microarray-captured next-generation sequencing as a diagnostic platform for detecting small mutations in Duchenne muscular dystrophy

Abstract

Objective To set up a new diagnostic platform based on microarray exon-capture and next-generation sequencing for detecting small mutations in dystrophin gene.The sensitivity and specificity of the method were assessed in clinical settings and the distribution of small mutations in Chinese Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD) patients were also analyzed. Methods Forty-one DMD/BMD patients diagnosed by the clinical criteria without large deletion or duplication (≥1 exon) were recruited from Peking Union Medical College Hospital consecutively.Genomic DNA was extracted from blood samples.The libraries were prepared.Then exon and intron-exon flanking sequences of DMD gene were captured by custom microarray.Targeted next-generation sequencing and Sanger Sequencing were conducted.The patients who were not detected any disease-causing mutation were performed muscle biopsy. Results Thirty-eight subjects were detected small mutations in DMD gene.All single nucleotide variants (SNVs) and insertion & deletions (INDELs) were validated by Sanger sequencing.Twenty-one novel mutations were reported.The distribution of SNVs and INDELs was similar to other international DMD databases.Upon immunohistochemistry staining of dystrophin protein，1 of 3 mutation-undetected patients was diagnosed as DMD，2 of them were excluded.The specificity of the method was 100%，while the sensitivity was 97.4%. Conclusions Our microarray-captured next-generation sequencing assay could detect SNVs and INDELs with high sensitivity and specificity.Its advantages are economic，timesaving and stable.The platform is suitable for clinical gene diagnosis. Key words: Muscular dystrophy，duchenne; Oligonucleotide array sequence analysis; Mutation; Exons; High-throughput nucleotide sequencing