Analysis of mutation spectrum of common deafness-causing genes in Hakka newborns in southern China by semiconductor sequencing

Abstract Hearing loss is a common neurosensory disorder, approximately half of the cases are caused by genetic factors, and approximately 70% of hereditary hearing impairments are nonsyndromic hearing loss (NSHL). The mutations of GJB2 (gap junction beta-2 protein), GJB3 (gap junction beta-3 protein), SLC26A4 (solute carrier family 26 member 4), and MT-RNR1 (mitochondrially encoded 12S RNA) are the most common inherited causes of NSHL. Because of different genetic backgrounds, the mutation spectrum of these common deafness-causing genes varies among different regions in China. Because no data are known on these mutations among the Hakka population of Southern China, we aim to investigate the mutation spectrum to add these to neonatal screening and genetic counseling. A total of 1252 blood samples from newborns have been detected by semiconductor sequencing for 100 mutations loci of 18 deafness-causing genes. Of the participants, 95 subjects carried deafness-causing genes mutations with the carrier rate of 7.59%. The mutation frequencies of GJB2, SLC26A4, GJB3, and mitochondrial genes were 3.04%, 3.51%, 0.16%, and 0.88%, respectively. We followed up subjects with single-gene homozygous or compound heterozygous mutations. Our study firstly analyzed deafness-causing genes mutation spectrum in Hakka population, providing evidence for future neonatal screening and genetic counseling in this area.


Introduction
Hearing loss is one of the most common neurosensory disorders, affecting approximately 1 to 3 newborns in every 1000 live births. [1][2][3] In China, there are approximately 800,000 children younger than 7 years who are hearing impaired, and this number continues to grow, with an increase of more than 30,000 deaf children every year. [3,4] According to whether other organ systems are abnormal, sensorineural hearing loss can be classified into nonsyndromic hearing loss (NSHL) and syndrome-induced hearing loss. If infants with profound hearing loss are not detected and treated within the first year of life, they may experience permanent hearing impairment with major and irreversible defects in linguistic and cognitive development. However, this situation can be improved if it is detected and intervention started before 6 months of age (cochlear implants can help patients with severe hearing loss to recover their hearing ability, implanting an electronic medical device that sends sound signals to the brain to replace the damaged inner ear.).
Previous studies have confirmed that hearing loss can be congenital or caused by environmental factors, such as infection, trauma, or ototoxic drugs. [5,6] However, hearing loss is etiologically uneven. Some studies have shown that at least two-thirds of the cases of childhood-onset hearing loss have a genetic cause, and approximately 70% of hereditary hearing impairments are NSHL. [7][8][9] NSHL can be inherited by autosomal recessive, autosomal dominant, X-linked trait, or mitochondrial deafness. [10][11][12] At present, previous genetic screening studies have shown that a few genes mutations are known to cause hereditary hearing loss or deafness, such as GJB2 (gap junction beta-2 protein; OMIM: 121011), GJB3 (gap junction beta-2 protein; OMIM: 603324), SLC26A4 (solute carrier family 26 member 4; OMIM: 605646), and the mitochondrial gene MT-RNR1 (mitochondrially encoded 12S RNA; OMIM: 561000). [9,[13][14][15][16][17][18][19][20][21][22][23][24] Knowledge of the gene mutation can help to identify hearing impairment at birth, and the educational programs for auditory stimulation and sufficient language exposure in early childhood can begin immediately. Furthermore, it can also provide warning to avoid taking certain types of aminoglycosides antibiotics, such as streptomycin, gentamicin, and tobramycin, which are known to cause deafness in children carrying certain mitochondrial gene mutations.
China is the most populous country in the world, consisting of 56 nationalities. Due to geographical separation, Chinese people from different regions may have different genetic backgrounds. [25,26] The Hakka population is a Han Chinese that mainly living in southern China with unique culture. [26] The city Meizhou with the most Hakka population in Guangdong Province locates in the south part of China. Because of its remote location, Meizhou city is a relatively conservative area with a less migration of population. Hearing loss in infancy is a common sensory disorder, of which about is hereditary, caused by known mutations such as GJB2, SLC26A4, GJB3, and mitochondrial genes. Although some genetic studies have been performed on Chinese patients with deafness, [8,9,27,28] the large-scale deafness population and racial differences require regional and individual genetic analysis; these data cannot simply be inferred from the conclusions of other groups. For example, 1 study with a comprehensive investigation of the molecular etiology of nonsyndromic deafness in 2 typical areas from northern and southern China (Chifeng City in Inner Mongolia and Nantong City in Jiangsu Province), GJB2 gene mutations account for approximately 18.31% of patients with hearing impairment, SLC26A4 gene mutations account for approximately 13.73%, and the mitochondrial m.1555A > G mutation accounts for 1.76%. [8] However, common molecular etiologies are rare in the Tibetan Chinese deaf population. [27] The prevalence of mutations varies among different regions in China. We aim to explore the prevalence of these mutations among the Hakka population in southern China, which might be helpful to neonatal screening and genetic counseling.

Participants
This retrospective clinical study included 1252 newborns who born in Meizhou People's Hospital (Huangtang Hospital), Meizhou Hospital Affiliated to Sun Yat-sen University between May 2016 and January 2018. The inclusion and exclusion criteria are shown in Figure 1. All the blood samples from participants have been detected by semiconductor sequencing. Before blood sampling, informed consent was obtained from all participants' parents. The study was approved by the Committee of Ethics and Research of the Meizhou People's Hospital, Meizhou Hospital Affiliated to Sun Yat-sen University for experiments involving humans. Before participants recruited for the study, their guardians signed a written informed consent form according to the ethical guidelines of the Helsinki Declaration.

DNA extraction and detection of deafness-causing genes mutations
Peripheral blood samples were collected from the study participants and stored in 2-mL evacuated vacuum tubes containing ethylenediaminetetraacetic, or heel blood was collected to the blood spot card. These blood samples are stored at 4°C for not more than 3 days before testing. QIAamp DNA Blood Mini Kit (Qiagen, Germany) was used to extract genomic DNA from each blood sample following the manufacturer's instructions, and NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA) was used to evaluate the quantity and quality of extracted DNA. DNA from peripheral blood sample was used for library construction according to the Ion Plus Fragment Library Kit (Life Technologies, Carlsbad, CA), and semiconductor sequencing was performed using an Ion Proton instrument according to the manufacturer's instructions (Life Technologies). High-throughput sequencing technology and bioinformatics analysis methods were used to detect the presence of deafness-causing gene mutation in subjects. The detection contents include 18 deafness-causing genes, including

Results
Among the 1252 participants enrolled in this study (669 men and 583 women), 95 subjects carried common deafness-causing genes mutations, total carrier rate was 7.59% (Table 1). A total of 38 participating subjects carried mutations in GJB2, and the carrier rate was 3.04% in the population. SLC26A4 mutations were detected in 44 participants; the carrier rate was 3.51%. Two participants carried mutations in GJB3. Mitochondrial gene mutations carrier rates were MT-RNR1 (0.64%), MT-TL1

Mutations in SLC26A4 gene
Forty-two newborns carried monoallelic variants in the heterozygous form. One newborns carried homozygous mutation for c.IVS7-2A > G and 1 with c.2168A > G heterozygote compound c.1229C > T heterozygote. Totally, 44 subjects had molecular defects in SLC26A4 gene (Table 5).

Mutations in GJB3 and mitochondrial genes
Two neonates carried mutations in GJB3, one was heterozygous mutation for c.538C > T and another one with c.547G > A heterozygote. In addition, 11 subjects were detected to be mitochondrial gene mutation carriers, accounting for 0.88% (11/ 1252) of the group. Eight newborns carried mutations in MT-RNR1, containing 1 heteroplasmic mutation for m.827A > G, 2 homoplasmic mutation for m.827A > G, 4 homoplasmic mutation for m.1555A > G, and 1 homoplasmic mutation for m.1494C > T. One newborns carried a heteroplasmic mutation for m.3243A > G in MT-TL1. Another 2 newborns carried a homoplasmic mutation for m.7444G > A in MT-CO1. Since the mutation of mitochondrial gene MT-RNR1 is important mechanism of genetic susceptibility to aminoglycoside ototoxicity, screened carriers were provided with detailed drug using guide.

Discussion
Previous studies have reported that GJB2, SLC26A4, GJB3, and mitochondrial genes are the most common causes NSHL in Chinese people. [14,[29][30][31] Here we studied retrospectively 100 loci of 18 genes known to cause hearing impairment. The actual hearing tests of the subjects were not complete in our study. This is a limitation due to our study design.

GJB2 mutation analysis
In the present study, GJB2 mutations were detected in 3.04% (38/1252) of all subjects. Like most areas of China, [8,32,33] the c.235delC was the most prevalent mutation in Hakka population  [34] The second frequent mutation was c.109G > A, with a carrier rate of 1.20% (15/1252). We followed up 2 newborns with homozygous mutation in c.109G > A, the result was that they have normal hearing at the time of 9 months after birth. Another infant who with c.235delC heterozygote compound c.109G > A heterozygote also has normal hearing ( Table 6). The mutation in c.109G > A is common in East Asians. The mutation frequency of c.109G > A in deaf population had been reported to be 4.2% in China, [9] 4.3% in Thailand, [35] 1.0% in Japan, [36] and 0.6% in Korea. [37] At present, the pathogenicity of this mutation site is controversial, because some individuals with normal hearing also carry the homozygous mutation. [38,39]

SLC26A4 mutation analysis
In this study, SLC26A4 with higher mutation rate 3.51% (44/ 1252) compared to GJB2 3.04% (38/1252). The mutation hotspots of SLC26A4 differed among different nations and areas. In our group, the hotspot mutation of SLC26A4 was c.919-18T > G, with a carrier rate of 1.12% (14/1252), but this variant has been proposed to as benign variation according to the reported data (http://deafnessvariationdatabase.org/). The carrier rate of c.IVS7-2A > G was 0.96% (12/1252), was similar with the study that c.IVS7-2A > G mutation carrier rate in the Chinese hearing population is 1.24% (90/7263). [34] We followed up 1 subject with homozygous mutation in c.IVS7-2A > G; the result was that the infant has normal hearing at the time of 7 months after birth. Another infant with c.2168A > G heterozygote compound c.1229C > T heterozygote also has normal hearing ( Table 6). Pendred syndrome (PDS) is classically described as bilateral sensorineural hearing loss and thyroid enlargement, and PDS is caused by mutations of SLC26A4 gene, disease may occurs at any age from birth to adolescence, inducement including colds, fever, mild craniocerebral trauma, barotrauma, or other causes of increased intracranial pressure. [40] We have informed the parents of the subject with homozygous mutation in c.IVS7-2A > G, to closely observe the child's behavior and its response to sound. If in doubt the parents should make a new hospital appointment, and we will follow-up this case next time.

GJB3 and mitochondrial genes mutation analysis
In our study, 2 subjects were found carrying mutation in GJB3, one was heterozygous mutation for c.538C > T and another one was heterozygous mutation for c.547G > A. This result indicates that deafness-associated variation in GJB3 was considered not common in Hakka population. The mutations of m.1555A > G and m.1494C > T for the MT-RNR1 gene are considered as the most common mutations of mitochondrial genes. The mutation frequency of m.1555A > G was observed in 2.9% in China, Japan 3%, [41] and Indonesia 5.3%. [42] In our study, the mutation carrier rate of m.1555A > G and m.1494C > T accounted for 0.32% (4/1252) and 0.08% (1/1252), respectively, was similar with the study that m.1555A > G and m.1494C > T mutation carrier rate in the Chinese hearing population are 0.25% (18/7263), 0.04% (3/7263), respectively. [34] Since the mutation of mitochondrial gene MT-RNR1 is important mechanism of genetic susceptibility to aminoglycoside ototoxicity, screened carriers should be provided with detailed drug using guide. In our study, we have given out a warning to these carriers' parents that these children may be prone to ototoxic effects of aminoglycoside, and informed the physician.

Conclusions
Considering that 1 to 3 newborns in every 1000 could be hearing impaired, screening for these mutations causing genetic hearing loss is relevant and may be applied if the gene mutation spectrum of the Hakka Chinese population is known. With the results of our study, the basis for neonatal screening is laid: of the 1252 participants, 95 subjects carried deafness-causing genes mutations with the carrier rate 7.59%. The mutation frequencies of GJB2, SLC26A4, GJB3, and mitochondrial genes were 3.04%, 3.51%, 0.16%, and 0.88%, respectively.