A reverse dot blot assay for the screening of twenty mutations in four genes associated with NSHL in a Chinese population

• Published in: PloS one 2017-05, Vol.12 (5), p.e0177196-e0177196
• Main Authors: Li, Siping, Peng, Qi, Liao, Shengyun, Li, Wenrui, Ma, Qiang, Lu, Xiaomei
• Format: Article
• Language: English
• Subjects: Asian Continental Ancestry Group; Assaying; Biology and Life Sciences; Children & youth; China; Chromatography; Deafness; Defects; Deoxyribonucleic acid; Diagnosis; Disorders; DNA; DNA Mutational Analysis; DNA probes; Fragments; Gene mutation; Genes; Genetic aspects; Genetic Association Studies; Genetic disorders; Genetic Predisposition to Disease; Genetic screening; Genetic testing; Genetic Testing - methods; Genetics; Genomics; Health aspects; Hearing loss; Hearing Loss - diagnosis; Hearing Loss - epidemiology; Hearing Loss - genetics; Humans; Hybridization; Identification and classification; Incidence; Laboratories; Mass spectrometry; Medical screening; Medicine and Health Sciences; Methods; Multiplexing; Mutation; Mutation hot spots; Neonates; Newborn babies; Nucleotide sequence; Oligonucleotides; Pediatrics; Polymerase Chain Reaction; Population; Population Surveillance; Probes; Reproducibility of Results; Research and Analysis Methods; Scientific imaging; Screening
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0177196

Congenital deafness is one of the most distressing disorders affecting humanity and exhibits a high incidence worldwide. Most cases of congenital deafness in the Chinese population are caused by defects in a limited number of genes. A convenient and reliable method for detecting common deafness-related gene mutations in the Chinese population is required. We developed a PCR-reverse dot blot (RDB) assay for screening 20 hotspot mutations of GJB2, GJB3, SLC26A4, and MT-RNR1, which are common non-syndromic hearing loss (NSHL)-associated genes in the Chinese population. The PCR-RDB assay consists of multiplex PCR amplifications of 10 fragments in the target sequence of the four above-mentioned genes in wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. We applied our method to a set of 225 neonates with deafness gene mutations and 30 normal neonates. The test was validated through direct sequencing in a blinded study with 100% concordance. The results demonstrated that our reverse dot blot assay is a reliable and effective genetic screening method for identifying carriers and individuals with NSHL among the Chinese population.