A multiplex PCR amplicon sequencing assay to screen genetic hearing loss variants in newborns

Background: Congenital hearing loss is one of the most common birth defects. Early identification and management play a crucial role in improving patients' communication and language acquisition. Previous studies demonstrated that genetic screening complements newborn hearing screening in clinical settings.

Methods: We developed a multiplex PCR amplicon sequencing assay to sequence the full coding region of the GJB2 gene, the most pathogenic variants of the SLC26A4 gene, and hotspot variants in the MT-RNR1 gene. The sensitivity, specificity, and reliability were validated via samples with known genotypes. Finally, a pilot study was performed on 300 anonymous dried blood samples.

Results: Of 103 samples with known genotypes, the multiplex PCR amplicon sequencing assay accurately identified all the variants, demonstrating a 100% sensitivity and specificity. The consistency is high in the analysis of the test-retest reliability and internal consistency reliability. In the pilot study, 12.3% (37/300) of the newborns were found to carry at least one pathogenic variant, including 24, 10, and 3 from the GJB2, SLC26A4, and MT-RNR1 gene, respectively. With an allele frequency of 2.2%, the NM_004004.6(GJB2):c.109G>A was the most prevalent variant in the study population.

Conclusion: The multiplex PCR amplicon sequencing assay is an accurate and reliable test to detect hearing loss variants in the GJB2, SLC26A4, and MT-RNR1 genes. It can be used to screen genetic hearing loss in newborns.