Establishment of a Flexible Real-Time Polymerase Chain Reaction-Based Platform for Detecting Prevalent Deafness Mutations Associated with Variable Degree of Sensorineural Hearing Loss in Koreans

Abstract

Many cutting-edge technologies based on next-generation sequencing (NGS) have been employed to identify candidate variants responsible for sensorineural hearing loss (SNHL). However, these methods have limitations preventing their wide clinical use for primary screening, in that they remain costly and it is not always suitable to analyze massive amounts of data. Several different DNA chips have been developed for screening prevalent mutations at a lower cost. However, most of these platforms do not offer the flexibility to add or remove target mutations, thereby limiting their wider use in a field that requires frequent updates. Therefore, we aimed to establish a simpler and more flexible molecular diagnostic platform based on ethnicity-specific mutation spectrums of SNHL, which would enable bypassing unnecessary filtering steps in a substantial portion of cases. In addition, we expanded the screening platform to cover varying degrees of SNHL. With this aim, we selected 11 variants of 5 genes (GJB2, SLC26A4, MTRNR1, TMPRSS3, and CDH23) showing high prevalence with varying degrees in Koreans and developed the U-TOP™ HL Genotyping Kit, a real-time PCR-based method using the MeltingArray technique and peptide nucleic acid probes. The results of 271 DNA samples with wild type sequences or mutations in homo- or heterozygote form were compared between the U-TOP™ HL Genotyping Kit and Sanger sequencing. The positive and negative predictive values were 100%, and this method showed perfect agreement with Sanger sequencing, with a Kappa value of 1.00. The U-TOP™ HL Genotyping Kit showed excellent performance in detecting varying degrees and phenotypes of SNHL mutations in both homozygote and heterozygote forms, which are highly prevalent in the Korean population. This platform will serve as a useful and cost-effective first-line screening tool for varying degrees of genetic SNHL and facilitate genome-based personalized hearing rehabilitation for the Korean population.

Mutational and phenotypic spectrum of OTOF-related auditory neuropathy in Koreans: eliciting reciprocal interaction between bench and clinics

“…Molecular genetic diagnoses of 5 families with 6 probands was made through a screening kit for detection of prevalent 11 deafness-causing variants [ 20 ] and Sanger sequencing of previously reported OTOF variants among Koreans including p.Arg1939Gln followed by either targeted exome sequencing (TES), or whole exome sequencing (WES), as previously described [ 12 , 21 – 24 ]. In detail, genomic DNA was extracted from the peripheral blood samples or buccal cells using standard protocols (Gentra Puregene Blood Kit, Qiagen, cat.…”

One-step noninvasive prenatal testing (NIPT) for autosomal recessive homozygous point mutations using digital PCR

“…In the SH197 family, allele-specific PCR-based universal array from the second baby confirmed a wildtype of GJB2 c.235delC 16 . In the SB275 and SH162 families, Sanger sequencing from the second baby confirmed a homozygous mutant of GJB2 c.235delC and SLC26A4 IVS7-2A > G, respectively ( Fig.…”

Flexible Real-Time Polymerase Chain Reaction-Based Platforms for Detecting Deafness Mutations in Koreans: A Proposed Guideline for the Etiologic Diagnosis of Auditory Neuropathy Spectrum Disorder

“…We previously developed a genetic diagnostic kit that enabled screening of the prevalent variants of five major prelingual deafness genes in the Korean population [13]. The kit was highly flexible, time efficient, and cost effective, ideal for the everyday clinical setting [13].…”

“…We previously developed a genetic diagnostic kit that enabled screening of the prevalent variants of five major prelingual deafness genes in the Korean population [13]. The kit was highly flexible, time efficient, and cost effective, ideal for the everyday clinical setting [13]. Nevertheless, given an extreme etiologic heterogeneity, there is still demand for the development of additional convenient screening kits that ensure coverage of other important deafness genes.…”