[Application of PCR-MALDI-TOF MS in genetic detection of non-syndromic hearing loss and genotype-audiometric phenotype analysis].

Objective

This study aimed to evaluate the diagnostic efficacy, clinical feasibility, and performance of PCR-coupled Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry（PCR-MALDI-TOF MS） in screening 20 common mutations across four deafness-associated genes in patients with non-syndromic hearing loss（NSHL）. Furthermore, it investigated the correlation between specific gene mutation types and the severity of hearing loss（genotype-phenotype correlation） was investigated.

Methods

A total of 298 patients with NSHL（141 males and 157 females; age range: 3 months to 72 years） presenting to the Department of Otolaryngology Head and Neck Surgery at Xiangya Hospital, Central South University, between July, 2022, and July, 2024, were enrolled. The PCR-MALDI-TOF MS assay was employed to detect 20 hotspot mutations in four major deafness genes（GJB2 , SLC26A4 , GJB3 , and mtDNA 12S rRNA）. Sanger sequencing was used as the "gold standard" for bidirectional validation of all samples to assess performance metrics, including sensitivity, specificity, overall concordance rate, and the Kappa coefficient. Additionally, the audiometric phenotypes of patients carrying different pathogenic mutations were statistically analyzed.

Results

Among the 298 subjects, pathogenic mutations were identified in 84 cases by PCR-MALDI-TOF MS, yielding a total detection rate of 28.19%. The mutation spectrum included 13 cases with homozygous variants, 42 with heterozygous variants, 16 with compound heterozygous variants, and 13 with homoplasmic mitochondrial DNA variants. The assay showed complete concordance with Sanger sequencing, with overall sensitivity, specificity, and concordance all reaching 100%. Among the 84 positive carriers, the distribution of hearing loss severity was as follows: mild（n=11）, moderate（n=20）, moderately severe（n=15）, severe（n=13）, and profound（n=25）. Notably, patients with profound hearing loss predominantly carried SLC26A4 mutations（13/25） and GJB2 mutations（8/25）, while those with severe hearing loss were primarily associated with SLC26A4 mutations（7/13）.

Conclusion

The PCR-MALDI-TOF MS technology demonstrates high accuracy, sensitivity, and specificity, making it an ideal tool for rapid first-line screening of common deafness gene hotspots in the Chinese population. This study reveals a significant association between GJB2 truncating mutations and SLC26A4 splice site mutations with severe-to-profound hearing loss phenotypes. These findings suggest that early genetic diagnosis is crucial for predicting auditory prognosis and guiding timely clinical intervention for patients with these specific genotypes.