Hearing loss, which severely affects patients’ daily quality of life, is one of the most common sensory impairments, affecting approximately one to three newborns per every 1,000 live births[1, 2]. In China, there are approximately 0.8 million children (less than 7 years of age) with hearing impairments, and this proportion exhibits an annual increase of 30,000 children[3]. Hearing loss is etiologically heterogeneous, and it has been estimated that at least two thirds of the cases of childhood-onset hearing loss have genetic causes[4,5]. To date, more than 80 genes and more than a hundred genetic loci have been mapped and associated with hereditary hearing loss (http://hereditaryhearingloss.org/).

However, previous epidemiological studies have shown that nearly half of NSHL cases are related to the following genes with recurrent mutations in the Chinese population: GJB2 (OMIM: 121011), GJB3 (OMIM: 603324), SLC26A4 (OMIM: 605646) and the mitochondrial gene MT-RNR1 (OMIM: 561000) [6–8]. With respect to genetic factors of NSHL in China, GJB2 variants appear to be the most common (23.37%), followed by variants in the SLC26A4 (14.74%), MT-RNR1 (2.56%) and GJB3(1.97%) genes[9,10]. Mutations in the GJB2 gene are the most frequent causes of nonsyndromic autosomal recessive sensorineural hearing loss in most Asian populations, including Han Chinese [6]. Four common mutations (c.235delC, c.299_ 300delAT, c.176_ 191del16, and c.35delG) account for 88.0% of GJB2 mutant alleles in the Chinese population [11]. A SLC26A4 mutation is the second most common cause of deafness in China [12]. Mutations in the SLC26A4 gene are responsible for Pendred syndrome (PDS) and enlarged vestibular aqueduct syndrome (EVA), with hearing loss detected at birth or in early childhood [13,14]. In China, the most common mutation of SLC26A4 is IVS7-2 A>G, with an allele frequency ranging from 5.13% to 11.06% [10, 15]. The GJB3 gene, which is related to hereditary NSHL, was first cloned in the Chinese population, and mutations in GJB3 are associated with progressive hearing loss [7]. The most common mutations in GJB3 are c.538C>T and c.547G>A [16]. Although nuclear gene defects constitute the majority of cases of hereditary hearing loss, it has become clear that mutations in mtDNA can also cause NSHL. The best-studied mutations related to aminoglycoside susceptibility are m.1555A>G and m.1494T>C in the MT-RNR1 gene [17–19]. In a previous study, we showed that the carrier frequency of common mutations of the four above-mentioned genes among 9317 newborns was 3.84% [20], and another study conducted by Zhang showed a higher carrier frequency of 5.52% in northern China [21]. Congenital deafness accounts for the overwhelming majority of the population with prelingual deafness. Because it can affect language capacity, the timing of the detection of hearing impairment is very important. Therefore, early detection, diagnosis, and intervention are necessary for newborns who are susceptible to deafness. Deafness gene screening might identify the cause of deafness at the molecular level and is thus a reliable and effective method for identifying NSHL-associated gene mutation carriers.

At present, different methods are used for the detection of NSHL-associated gene mutations, including classic polymerase chain reaction/restriction enzyme analysis (PCR-RFLP)[22], denaturing high-performance liquid chromatography (DHPLC) [23], matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) [24] and direct sequencing. Such methods present variable challenges; for example, the PCR-RFLP method can detect a limited number of mutations in a single run, making it insufficient for multi-allele detection. Other methods, however, require expensive equipment and personnel with specialized abilities, making them unsuitable for first-line screening in a clinic setting.

In this study, we aimed to develop a more efficient genetic diagnostic assay based on the PCR-RDB technique that could be used as a first-line screening tool for prevalent mutations in the Chinese population. We selected 20 variants of four genes (GJB2, GJB3, SLC26A4 and MT-RNR1) that are frequently found in the Chinese population and cause NSHL to varying degrees and with differing phenotypes. This platform will serve as a useful and cost-effective first-line screening tool for genetic NSHL in the Chinese population.

We developed a platform that can be easily performed with common equipment, has a low cost of less than US$3 and can be completed within 6 h. It is an ideal method for smaller clinics and laboratories with limited resources that could allow the front-line detection of NSHL genes without having to resort to direct DNA sequencing. Our results for 255 unrelated subjects showed 100% concordance with the results of independent direct sequencing.

At present, the MassARRAY system combined with an iPLEX assay, which is a high-throughput genotyping technology based on MALDI-TOF mass spectrometry, is the most common deafness gene-detecting method used in clinical settings[26–29]. This MassARRAY system can detect a large amount of variants in multiple samples simultaneously. Nevertheless, the MassARRAY system requires a very expensive platform for the analysis, which limits its use.

Genetic testing can potentially allow an accurate diagnosis of NSHL, and a diagnosis of the genetic etiology will aid the clinical management of patients, including the selection of the most appropriate treatment. Combined newborn hearing screeningand genetic screening of common deafness-related genes will benefit the early detection of infants at high risk of developing delayed hereditary sensorineural deafness or sensitivity to deafness-related drugsas well as the diagnosis of hearing loss in children. The screening of deafness-related genes might effectively prevent the occurrence and development of deafness. The molecular etiology, diagnosis, and effective genetic counseling of the probandare important for the prevention and treatment of deafness.

Because GJB2, GJB3, SLC26A4, and MT-RNR1 are the four most common causative genes of prelingual NSHL in the Chinese population and hotspot mutations of these four genes are included, our assay is expected to cover a substantial portion of prelingual severe-to-profound genetic NSHL in the Chinese population. The restricted targeting reduces the possibility of incidental findings but allows higher coverage at a lower cost compared with genome-wide approaches. This study shows that this simple and inexpensive method can be used for routine molecular diagnosis and potentially for large-scale genetic screening. However, it is possible that the RDB will be affected by the SNP near the mutation. Although this did not occur in the 225 positive samples, we will take test samples in the future study.

We propose that the PCR-RDB assay is a highly accurate, rapid and economical clinical assay that can be potentially used for neonatal hearing screening and personalized medicine. Children with the MT-RNR1mutation are recommended stay away from amino glycoside antibiotics to avoid the development of drug-induced hearing loss. Infants with homozygous or compound heterozygous forms of the same autosomal recessive gene may show the symptoms of deafness at birth, and clinical and audiology intervention should be performed as soon as possible. In infants with homozygous or heterozygous mutations in autosomal dominant genes, the mutations can cause deafness, However, some individuals mayexhibit normal hearing at birth and develop delayed deafness, and they should be guided regarding optimizing their listening environment to refrain from developing hearing impairment during their hearing development, e.g., avoidance of head trauma, and timely administration of treatment if their auditory threshold changes.

In conclusion, we developed a new method for the genetic screening of hearing loss and successfully detected hearing loss-related genetic mutations prevalent in the Chinese population in homozygousand heterozygous forms, as validated by Sanger sequencing. This assay is expected to serve as a primary screening tool for genetic hearing loss and could be helpful in gene-based and personalized hearing rehabilitation.