Analysis of deafness susceptibility gene of neonates in northern Guangdong, China

This study aimed to explore the molecular epidemiology characteristics of deafness susceptibility genes in neonates in northern Guangdong and provide a scientific basis for deafness prevention and control. A total of 10,183 neonates were recruited between January 2018 and December 2022 at Yuebei People's Hospital. Among these, a PCR hybridization screening group of 8276 neonates was tested for four deafness genes: GJB2, SLC26A4, mtDNA, and GJB3 by PCR hybridization. Another group used next-generation sequencing (NGS) to detect genetic susceptibility genes in 1907 neonates. In PCR hybridization screening group, 346 (4.18%) of 8276 neonates were found to be carriers of the deafness gene. Among these, 182 (2.2%) had GJB2 variants, 114 (1.38%) had SLC26A4 variants, 35 (0.42%) had mtDNA variants, and 15 (0.18%) had GJB3 variants. In NGS Screening Group, 195 out of 1907 neonates were found to be carriers of the deafness gene, with a positive rate of 10.22%. Among these, 137 (7.18%) had GJB2 variants, 41 (2.15%) had SLC26A4 variants, 11 (0.58%) had mtDNA variants, and 6 (0.31%) had GJB3 variants. The prevalence of deafness gene variants was high in Northern Guangdong Province. The most common gene for deafness was GJB2, followed by SLC26A4 and mtDNA. GJB3 variants are rare. Compared with PCR hybridization method, NGS technology can expand the screening scope and greatly improve the detection rate of deafness genes. The c.109G>A of GJB2 was found to occur at a high frequency, which should be considered. Therefore, it is important to conduct neonatal deafness gene screening to prevent and control hereditary deafness.


Subjects
A total of 10,183 neonates were enrolled between January 2018 and December 2022 at the Yuebei People's Hospital.Screening of four deafness genes (GJB2, SLC26A4, mtDNA and GJB3) without knowing the result of hearing test.Among them, 8276 cases were detected by PCR hybridization, and 1907 cases were detected by NGS.This study was approved by the Ethics Committee of Yuebei People's Hospital (KY-2021-219).Informed consent was obtained from the neonatal guardian, and an informed consent form was signed.All methods followed relevant guidelines and standard operating procedures (SOPs).

NGS screening
The target exons of four deafness susceptibility genes were sequenced using the hereditary deafness genetic test kit (BGI Genomics, China).The test kit has been approved by the National Medical Products Administration (NMPA, no.20203400432).Within ten bases both ends of the exons were analyzed.The basis for estimating the pathogenicity of gene variants is the joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) 7 .

Statistical analysis
Data analysis was performed using the SPSS22.0software (IBM, USA).Data are presented as case (n) or percentage (%).

Ethics statement
This research was approved by the Ethics Committees of Yuebei People's Hospital.

Discussion
The detection rate of deafness genes by PCR hybridization screening was only 4.18%, which was lower than the 4.78% of genetic deafness testing results in Chinese newborns 8 .However, that of NGS screening increased to 10.22%, which improved detection efficiency.Both methods found GJB2 to be the most common deafness gene, followed by SLC26A4 and mtDNA, GJB3 variants were rare, with positive rates of 2.2%, 1.38%, 0.42%, and 0.18%, respectively, consistent with other reports 9,10 .GJB2 is the most common variant associated with hereditary deafness 11 , which encodes the gap junction protein connexin-26 (CX26) on chromosome 13q12.11,which variants and causes congenital sensorineural deafness 12 .The c.235delC was the most common variant in PCR hybridization screening.Significantly, we found that the c.109G>A variant was the most common in NGS screening.It is possible that the c.109G>A variant was not included in PCR hybridization screening.The pathogenicity of the c.109G>A variant is controversial because of the heterogeneity of the hearing phenotype resulting from the variant at this locus.The c.109G>A homozygous variant, which is considered a polymorphism, can also be detected in individuals with normal hearing.Others may even display severe-to-extreme hearing loss 13 .The ClinGen hearing loss expert panel determined that c.109G>A is a causative factor for autosomal recessive non-syndromic hearing loss with variable expression and incomplete penetrance 14 .According to ACMG, the c.109G>A variant is predicted to be a pathogenic variant 15 .In this study, five patients with the c.109G>A homozygous variant and two with heterozygous variants did not  pass hearing screening.Further follow-up and investigation are needed to determine whether their hearing can be restored.SLC26A4 is the second most mutated gene in hereditary deafness, and c.919-2A>G is a hotspot variant.SLC26A4 is located on 7q22.3 and encodes the pendrin protein, which causes non-syndromic deafness of the vestibular aqueduct to dilate 16 .Such patients should avoid hard blows, sneezing, blowing nose, head trauma, and other inducements that can effectively prevent deafness 17 .
MtDNA variants are one of the causes of drug-induced deafness, leading to cochlear and vestibular cell dysfunction 18 .Using aminoglycosides can lead to deafness.Patients with delayed deafness and their maternal members should be banned from aminoglycoside antibiotics for life 19 .Therefore, screening for deafness genes before aminoglycoside antibiotics can effectively prevent the tragedy of one-needle deafness.
GJB3 variant is rare, is located on 1p34.3 and encodes gap junction protein 31.Variants cause acquired delayed-onset sensor-neural deafness 20 .The carrier rate of GJB3 variant is low in China 4,9 .In this study, the variant rate of GJB3 was also low, at only 0.19%.
Studies have revealed that the results of deafness gene screening can also guide the evaluation of the effectiveness of hearing aids and cochlear implants.For example, patients with deafness associated with GJB2 or SLC26A4 have a good prognosis after cochlear implantation 21 .The limitation of this study is insufficient information on the subjects, such as sex, twins, hearing loss, and other clinical features.Making it difficult to directly compare the results with previous data.

Conclusions
In brief, the key finding of our investigation pertains to the use of NGS, which surpasses the constraints of conventional screening methods in identifying previously overlooked diagnoses.NGS technology has expanded the scope of deafness gene screening and greatly improved the detection rate.Therefore, it is necessary to include deafness gene testing in hearing screening.High-risk groups can be identified early for intervention or diagnostic guidance.This is significant for individualized and accurate prevention and treatment of deafness.

Table 2 .
Results of deafness gene in NGS screening group (n = 1907).Significant values are in bold.