Newborn genetic screening for high risk deafness-associated mutations with a new Tetra-primer ARMS PCR kit

Previous epidemiological studies indicate that GJB2, SLC26A4 or mtDNA 12S rRNA mutations were chiefly responsible for the hearing loss in children. A cost-effective method for screening deafness-associated mutations at early age is needed. This study aimed to develop a simple kit for screening of high risk deafness-associated mutations in newborns using tetra-primer amplification refractory mutation system PCR.

The screening kit was designed to detect high risk deafness-associated mutations (GJB2 c.235delC, SLC26A4 c.919-2A>G, mtDNA 12S rRNA mt.1555A>G and mt.1494C>T). The kit was able to amplify both wild-type and mutant alleles with a control fragment. The proposed method was conducted to genotype the above four deafness gene mutations in four PCR reactions. Each mutation was genotyped by a set of four primers, two allele-specific inner primers, and two common outer primers. A mismatch at the penultimate or antepenult nucleotide of the 3' terminus was introduced in order to maximize specificity. The 16 primers were used for the amplification of genomic DNA as a template. Amplified fragments were separated by electrophoresis. We designed and validated the kit with wild and mutant type DNA samples that had been previously been confirmed by Sanger sequencing. Then 1181 newborns were enrolled, and those samples with mutations were further validated with sequencing too.

Among 1181 newborns, 29 individuals had one or two mutant alleles, with the carrier rate being 2.46% (29/1181). For GJB2 c.235delC mutation, one case was homozygote and 12 cases were heterozygote carriers. For SLC26A4 c.919-2A>G mutation, 12 cases were heterozygotes carriers, and no homozygotes were found; for mtDNA 12S rRNA mt.1555A>G mutation, one case was identified; three cases of mtDNA 12S rRNA mt.1494C>T mutation were detected. All mutations were detected with high specificity. Mutation samples were confirmed via Sanger sequencing. No false positive was found.

A user-friendly screening kit for deafness-associated mutations was successfully developed. It provided rapid, reproducible, and cost-effective detection of deafness gene mutation without special equipment. The kit allowed the detection of the four high risk deafness-associated mutations with only 4 single tube PCR reactions. In the future, the kit could be applied to large population-based epidemiological studies for newborn hearing defects screening.