# Mutation Analysis of the Common Deafness Genes in Patients with Nonsyndromic Hearing Loss in Linyi by SNPscan Assay

Authors: Fengguo Zhang; Yun Xiao; Lei Xu; Xue Zhang; Guodong Zhang; Jianfeng Li; Huaiqing Lv; Xiaohui Bai; Haibo Wang
Journal: BioMed Research International
 (2016)

---

## Abstract

Hearing loss is a common sensory disorder, and at least 50% of cases are due to a genetic etiology. Although hundreds of genes have been reported to be associated with nonsyndromic hearing loss,GJB2,SLC26A4, andmtDNA12SrRNA are the major contributors. However, the mutation spectrum of these common deafness genes varies among different ethnic groups. The present work summarized mutations in these three genes and their prevalence in 339 patients with nonsyndromic hearing loss at three different special education schools and one children’s hospital in Linyi, China. A new multiplex genetic screening system “SNPscan assay” was employed to detect a total of 115 mutations of the above three genes. Finally, 48.67% of the patients were identified with hereditary hearing loss caused by mutations inGJB2,SLC26A4, andmtDNA12SrRNA. The carrying rate of mutations in the three genes was 37.76%, 19.75%, and 4.72%, respectively. This mutation profile in our study is distinct from other parts of China, with high mutation rate ofGJB2 suggesting a unique mutation spectrum in this area.


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## Body

## 1. Introduction

Hearing loss or deafness is one of the most common neurosensory disorders in humans affecting about 1 to 3 in 1000 children worldwide [1]. It has been estimated that 30,000 newborns are detected with congenital hearing loss per 20 million live births every year in China [2], and approximately 50% of hearing loss is caused by a genetic etiology. At least 70% of all cases are classified as nonsyndromic hearing loss (NSHL) manifesting with isolated hearing loss without other associated clinical features. Of all NSHL, 75%–80% are autosomal recessive hereditary hearing loss, 15%–20% are autosomal dominant, 5% are X-linked, and 1% are inherited by mitochondrial genes [3].Many previous genetic screening studies have shown that a large proportion of NSHL is caused by the mutations of a few genes, such as gap junction beta-2 protein (GJB2), solute carrier family 26 member 4 (SLC26A4), andmitochondrial DNA (mtDNA)12SrRNA [4, 5]. Therefore, mutation screening of these common deafness-causing genes is of vital importance during genetic testing and counseling of deafness.China harboring the highest population in the world is consisting of 56 nationalities. The mutation spectrum of the common deafness genes may vary among different ethnic nationalities due to geographical and language separation. The city Linyi with the biggest population and the largest area in Shandong Province locates in the east part of China. Almost all the populations in this area are Han nationality. People in Linyi are always impressed by introversion and rusticity. Because of its desolate geographical condition, Linyi city is a relatively conservative area with a less migration of population and an obstructive communication with other regions. Also, it has a less-developed economics for a long time. But to our knowledge, no systematic mutation analysis of deaf patients in Linyi has been reported previously. Therefore, there is a reason to believe that mutation spectrum of common deafness genes in patients with NSHL in this area must have a unique and special characteristic.Up to now, many screening methods are used to detect the mutations of hearing loss, for example, direct sequencing, microarray, and PCR-restriction fragment length polymorphism (PCR-RFLP). These methods are either too expensive or time-consuming. Du et al. established a universal genetic screening system of hearing loss called the SNPscan assay which could detect 115 mutations ofGJB2,SLC26A4, andmtDNA12SrRNA genes [6]. These selected mutations accounted for up to 90% of hearing loss caused by the most common deafness-causing genes in Chinese population.In this study, we performed a comprehensive analysis of 3 common deafness-causing genes,GJB2,SLC26A4, andmtDNA12SrRNA, by SNPscan assay in 339 patients with NSHL from Linyi in eastern China. The aim was to investigate the molecular etiology in order to provide effective genetic counseling for hearing loss patients in this area.


## 2. Materials and Methods

### 2.1. Recruitment of the Subjects

A total of 339 subjects with NSHL from unrelated families were included in this study. They came from three different special education schools and one children’s hospital in Linyi. Clinical questionnaire and informed consent were obtained from all subjects or their parents on their behalf. Clinical questionnaire showed basic information, including name, age, family history, health condition of the mother during pregnancy, and a clinical record of the patient, such as infections, possible head or brain injury, and the use of aminoglycoside antibiotics [7]. Physical and neurological examination was performed with special attention to renal, electrocardiac, and ophthalmologic differences to exclude those with syndromic hearing loss. An audiologic evaluation was performed including otoscope examination, tympanometry, and pure-tone audiometry (PTA). Auditory brainstem response (ABR) was employed in subjects when they were too young to accomplish the PTA test, and temporal bone computed tomography (CT) scan was also performed on participants for diagnosis of enlarged vestibular aqueduct (EVA) or inner ear malformation.The onset of hearing impairment was categorized as prelingual/early (≤6 years) and late (>6 years). The degree of hearing impairment was calculated on the average of PTA in the speech frequencies 0.25, 0.5, 1, 2, and 4 kHz. Normal hearing was classified as PTA ≤ 25 dB nHL, mild hearing loss as PTA > 25 dB nHL and ≤40 dB nHL, moderate hearing loss as PTA > 40 dB nHL and ≤60 dB nHL, severe hearing loss as PTA > 60 dB nHL and ≤80 dB nHL, and profound hearing loss as PTA > 80 dB nHL. To enroll in this study, all subjects should have bilateral, permanent sensorineural hearing impairment. This study was approved by the Ethics Committee of Shandong Provincial Hospital.


### 2.2. DNA Samples

Genomic DNA for all 339 patients was prepared from 2 mL of peripheral blood with AxyPrep Genomic Blood DNA Extraction Kit (AXYGEN, USA). For the evaluation of the quantity and quality of extracted DNA, spectrophotometry (UNICO 2100, USA) and 1% agarose gel electrophoresis were carried out according to routine methods [8].


### 2.3. SNPscan for Mutation Detection

Two customized multiplex SNPscan assays from Genesky Biotechnologies Inc. (Shanghai, China) were designed to capture a total of 115 mutations of the three common deafness-causing genes as previously described (Table S1 in Supplementary Material available online athttp://dx.doi.org/10.1155/2016/1302914): one is 53-plex and the other is 62-plex [6]. As a high-throughput and cost-saving SNP genotyping method, the SNPscan assay is based on double ligation and multiplex fluorescence PCR as described [9–11]. The SNPscan assays were done according to the detailed protocol described by Du et al. [6].


### 2.4. Statistical Analysis

The statistical analysis was performed using SAS 9.1.3 software (SAS, USA).


## 2.1. Recruitment of the Subjects

A total of 339 subjects with NSHL from unrelated families were included in this study. They came from three different special education schools and one children’s hospital in Linyi. Clinical questionnaire and informed consent were obtained from all subjects or their parents on their behalf. Clinical questionnaire showed basic information, including name, age, family history, health condition of the mother during pregnancy, and a clinical record of the patient, such as infections, possible head or brain injury, and the use of aminoglycoside antibiotics [7]. Physical and neurological examination was performed with special attention to renal, electrocardiac, and ophthalmologic differences to exclude those with syndromic hearing loss. An audiologic evaluation was performed including otoscope examination, tympanometry, and pure-tone audiometry (PTA). Auditory brainstem response (ABR) was employed in subjects when they were too young to accomplish the PTA test, and temporal bone computed tomography (CT) scan was also performed on participants for diagnosis of enlarged vestibular aqueduct (EVA) or inner ear malformation.The onset of hearing impairment was categorized as prelingual/early (≤6 years) and late (>6 years). The degree of hearing impairment was calculated on the average of PTA in the speech frequencies 0.25, 0.5, 1, 2, and 4 kHz. Normal hearing was classified as PTA ≤ 25 dB nHL, mild hearing loss as PTA > 25 dB nHL and ≤40 dB nHL, moderate hearing loss as PTA > 40 dB nHL and ≤60 dB nHL, severe hearing loss as PTA > 60 dB nHL and ≤80 dB nHL, and profound hearing loss as PTA > 80 dB nHL. To enroll in this study, all subjects should have bilateral, permanent sensorineural hearing impairment. This study was approved by the Ethics Committee of Shandong Provincial Hospital.


## 2.2. DNA Samples

Genomic DNA for all 339 patients was prepared from 2 mL of peripheral blood with AxyPrep Genomic Blood DNA Extraction Kit (AXYGEN, USA). For the evaluation of the quantity and quality of extracted DNA, spectrophotometry (UNICO 2100, USA) and 1% agarose gel electrophoresis were carried out according to routine methods [8].


## 2.3. SNPscan for Mutation Detection

Two customized multiplex SNPscan assays from Genesky Biotechnologies Inc. (Shanghai, China) were designed to capture a total of 115 mutations of the three common deafness-causing genes as previously described (Table S1 in Supplementary Material available online athttp://dx.doi.org/10.1155/2016/1302914): one is 53-plex and the other is 62-plex [6]. As a high-throughput and cost-saving SNP genotyping method, the SNPscan assay is based on double ligation and multiplex fluorescence PCR as described [9–11]. The SNPscan assays were done according to the detailed protocol described by Du et al. [6].


## 2.4. Statistical Analysis

The statistical analysis was performed using SAS 9.1.3 software (SAS, USA).


## 3. Result

### 3.1. Characterization of the Deaf Probands

A total of 339 probands with NSHL were recruited in this sequencing study, including 304 simplex and 35 multiplex probands. Simplex probands refer to the sporadic patients whose families have no one else suffering from hearing loss. Multiplex probands mean that there is at least one first- or second-degree deaf relative in the family. Clinical characterization of the deaf probands was summarized in Table1. The patient cohort consisted of 196 males and 143 females, and they were all Han Chinese. The ages of the probands varied between a few months and 24 years (mean 8.9 years, 95% CI 8.3–9.5 years). Only one proband had late-onset, progressive, and moderate sensorineural hearing loss. All other probands had prelingual or early-onset sensorineural hearing loss. Hearing tests demonstrated that the level of hearing loss was severe to profound in 328 patients. The remaining 11 patients showed moderate hearing loss.Table 1
Deaf patients (n
=
339) categorized by their clinical characteristics.
Simplex (n
=
304)
Multiplex (n
=
35)
Sex
Male
179
17
Female
125
18
Age at the test
0–6 years
153
13
6–18 years
122
21
18–35 years
29
1
Age of onset
Early onset (≤6 years)
303
35
Late onset (>6 years)
1
0
Severity of hearing impairment
Mild
0
0
Moderate
11
0
Severe
56
5
Profound
237
30Among all these patients, 48.67% were identified with hereditary hearing loss by means of the SNPscan assay (Figure1). There were 104 patients (30.68%) affected byGJB2 mutations: 49 homozygotes and 55 compound heterozygotes. 45 (13.27%) patients carried twoSLC26A4 pathogenic mutations: fourteen homozygotes and 31 compound heterozygotes. 16 (4.72%) patients harboredmtDNA12SrRNA mutations consisting of 15 homoplasmic mutations and 1 heteroplasmic mutation.Figure 1
Distribution of the common deafness-causing genes in 339 NSHL patients.


### 3.2. Mutations inGJB2 Gene

Eleven variants were identified in this cohort. They were three frameshift deletions (c.176_191del16, c.235delC, and c.299_300delAT), two frameshift insertions (c.34_35insG and c.511_512insAACG), four missense mutations [c.109G>A (p.V37I), c.257C>G (p.T86R), c.427C>T (p.R143W), and c.571T>C (p.F191L)], one nonsense mutation c.9G>A (p.W3X), and one splicing site mutation IVS1+1G>A (shown in Table2). Nine of them were pathological mutations which have been determined previously. The category of two nucleotide changes was unknown (c.9G>A, c.571T>C). The mutant alleles ofGJB2 accounted for 34.37% (233/678) of the total alleles in all patients (Table 3). The most common mutation allele ofGJB2 in this area was c.235delC with a mutant frequency of 20.65% (140/678). The second common was c.299_300delAT of 5.60% (38/678), followed by c.109G>A of 2.51% (17/678), c.34_35insG of 1.77% (12/678), c.176_191del16 of 1.33% (9/678), c.511_512insAACG of 1.18% (8/678), c.257C>G of 0.29% (2/678), c.427C>T of 0.29% (2/678), and IVS1+1G>A of 0.15% (1/678) (1/678) (Table 3).Table 2
GJB2 genotypes of deaf patients from Linyi.
Allele 1
Allele 2
Number of patients
Nucleotide change
Consequence or amino acid change
Category
Nucleotide change
Consequence or amino acid change
Category
IVS1+1G>A
Splicing site
Pathogenic
c.9G>A
p.W3X
Unknown
1
34_35insG
Frameshift
Pathogenic
109G>A
p.V37I
Pathogenic
1
34_35insG
Frameshift
Pathogenic
176_191del16
Frameshift
Pathogenic
1
34_35insG
Frameshift
Pathogenic
235delC
Frameshift
Pathogenic
8
34_35insG
Frameshift
Pathogenic
299_300delAT
Frameshift
Pathogenic
2
109G>A
p.V37I
Pathogenic
109G>A
p.V37I
Pathogenic
3
109G>A
p.V37I
Pathogenic
235delC
Frameshift
Pathogenic
1
109G>A
p.V37I
Pathogenic
299_300delAT
Frameshift
Pathogenic
1
109G>A
p.V37I
Pathogenic
—
8
176_191del16
Frameshift
Pathogenic
235delC
Frameshift
Pathogenic
8
235delC
Frameshift
Pathogenic
235delC
Frameshift
Pathogenic
42
235delC
Frameshift
Pathogenic
257C>G
p.T86R
Pathogenic
2
235delC
Frameshift
Pathogenic
299_300delAT
Frameshift
Pathogenic
22
235delC
Frameshift
Pathogenic
427C>T
p.R143W
Pathogenic
2
235delC
Frameshift
Pathogenic
511_512insAACG
Frameshift
Pathogenic
3
235delC
Frameshift
Pathogenic
—
10
299_300delAT
Frameshift
Pathogenic
299_300delAT
Frameshift
Pathogenic
4
299_300delAT
Frameshift
Pathogenic
511_512insAACG
Frameshift
Pathogenic
4
299_300delAT
Frameshift
Pathogenic
—
1
511_512insAACG
Frameshift
Pathogenic
—
1
571T>C
p.F191L
Unknown
—
3Table 3
Allele frequencies ofGJB2 mutations in 339 deaf patients from Linyi.
Mutations
Consequence
Number of alleles
Allele frequency (%)
IVS1+1G>A
Splice site
1
0.15
c.34_35insG
Frameshift
12
1.77
c.109G>A
p.V37I
17
2.51
c.176_191del16
Frameshift
9
1.33
c.235delC
Frameshift
140
20.65
c.257C>G
p.T86R
2
0.29
c.299_300delAT
Frameshift
38
5.60
c.427C>T
p.R143W
2
0.29
c.511_512insAACG
Frameshift
8
1.18A total of 104 patients (30.68%) were confirmed to be associated with hereditary hearing loss caused byGJB2 mutation: 49 homozygotes (42 with the c.235delC allele, 4 with c.299_300delAT, and 3 with the c.109G>A allele) and 55 compound heterozygotes. Twenty patients (5.90%) had monoallelic variants in the heterozygous form: ten with c.235delC, eight with c.109G>A, one with c.299_300delAT, and one with c.511_512insAACG. Totally, 128 (37.76%, 128/339) patients had molecular defects inGJB2 gene including three patients with c.571T>C mutation (Table 2).The most common mutation allele ofGJB2 was c.235delC with a mutant frequency of 20.65% (140/678) affecting 98 patients. Among these patients carrying at least one c.235delC allele, there were 42 homozygotes, 46 compound heterozygotes, and 10 single heterozygotes.


### 3.3. Mutations inSLC26A4 Gene

Nineteen variants were identified in this cohort, including 12 missense mutations [c.147C>G (p.S49R), c.269C>T (p.S90L), c.563T>C (p.I188T), c.589G>A (p.G197R), c.1173C>A (p.S391R), c.1174A>T (p.N392Y), c.1225C>T (p.R409C), c.1226G>A (p.R409H), c.1975G>C (p.V659L), c.1985G>A (p.C662Y), c.2027T>A (p.L676Q), and c.2168A>G (p.H723R)], four nonsense mutations [c.235C>T (p.R79X), c.946G>T (p.G316X), c.1318A>T (p.K440X), and c.1540C>T (p.Q514X)], two splicing site mutations (c.919-2A>G, c.1707+5 G>A), and one frameshift insertion mutation (c.1547_1548insC) (Table5). Combining the prediction of SIFT and Polyphen-2 with the results of previous studies, all variants analyzed in our cohort were considered pathogenic mutations except c.147C>G (p.S49R) for an unknown significant pathogenicity (Table 4).Table 4
SLC26A4 genotypes of deaf patients from Linyi.
Allele 1
Allele 2
Number of patients
Nucleotide change
Consequence or amino acid change
Category
Nucleotide change
Consequence or amino acid change
Category
c.147C>G
p.S49R
Unknown
—
1
c.235C>T
p.R79X
Pathogenic
c.919-2A>G
Splice site
Pathogenic
1
c.235C>T
p.R79X
Pathogenic
—
1
c.269C>T
p.S90L
Pathogenic
—
1
c.563T>C
p.I188T
Pathogenic
c.919-2A>G
Splice site
Pathogenic
1
c.589G>A
p.G197R
Pathogenic
c.919-2A>G
Splice site
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.919-2A>G
Splice site
Pathogenic
13
c.919-2A>G
Splice site
Pathogenic
c.946G>T
p.G316X
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.1174A>T
p.N392Y
Pathogenic
2
c.919-2A>G
Splice site
Pathogenic
c.1226G>A
p.R409H
Pathogenic
3
c.919-2A>G
Splice site
Pathogenic
c.1318A>T
p.K440X
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.1540C>T
p.Q514X
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.1547_1548InsC
Frameshift
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.1707+5 G>A
Splice site
Pathogenic
3
c.919-2A>G
Splice site
Pathogenic
c.1975G>C
p.V659L
Pathogenic
3
c.919-2A>G
Splice site
Pathogenic
c.1985G>A
p.C662Y
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.2027T>A
p.L676Q
Pathogenic
2
c.919-2A>G
Splice site
Pathogenic
c.2168A>G
p.H723R
Pathogenic
6
c.919-2A>G
Splice site
Pathogenic
—
12
c.946G>T
p.G316X
Pathogenic
c.2168A>G
p.H723R
Pathogenic
1
c.1173C>A
p.S391R
Pathogenic
c.2168A>G
p.H723R
Pathogenic
1
c.1225C>T
p.R409C
Pathogenic
—
1
c.1226G>A
p.R409H
Pathogenic
c.1975G>C
p.V659L
Pathogenic
1
c.1226G>A
p.R409H
Pathogenic
c.2168A>G
p.H723R
Pathogenic
1
c.1226G>A
p.R409H
Pathogenic
—
1
c.1975G>C
p.V659L
Pathogenic
—
1
c.2168A>G
p.H723R
Pathogenic
c.2168A>G
p.H723R
Pathogenic
1
c.2168A>G
p.H723R
Pathogenic
—
4Table 5
Allele frequencies ofSLC26A4 mutations in 339 deaf patients from Linyi.
Mutations
Consequence
Number of alleles
Allele frequency (%)
c.235C>T
p.R79X
2
0.29
c.269C>T
p.S90L
1
0.15
c.563T>C
p.I188T
1
0.15
c.589G>A
p.G197R
1
0.15
c.919-2A>G
Splice site
65
9.59
c.946G>T
p.G316X
2
0.29
c.1173C>A
p.S391R
1
0.15
c.1174A>T
p.N392Y
2
0.29
c.1225C>T
p.R409C
1
0.15
c.1226G>A
p.R409H
6
0.88
c.1318A>T
p.K440X
1
0.15
c.1540C>T
p.Q514X
1
0.15
c.1547_1548InsC
Frameshift
1
0.15
c.1707+5 G>A
Splicing site
3
0.44
c.1975G>C
p.V659L
5
0.74
c.1985G>A
p.C662Y
1
0.15
c.2027T>A
p.L676Q
2
0.29
c.2168A>G
p.H723R
15
2.21There were 45 (13.27%) patients who were confirmed to have inherited hearing loss because they carried twoSLC26A4 pathogenic mutations: fourteen homozygotes (13 c. IVS 7-2A>G and 1 c.2168A>G) and 31 compound heterozygotes. And 22 (6.49%) patients who were identified carried oneSLC26A4 pathogenic mutation. Thus, the detection rate ofSLC26A4 mutations was 19.76% (67/339) in this patient cohort. All patients carrying two mutations were diagnosed with EVA syndrome by CT scan of the temporal bone. And 17 out of 22 patients carrying one pathogenic mutation were diagnosed with EVA syndrome.The most common mutation allele ofSLC26A4 was c.919-2A>G with a mutant frequency of 9.59% (65/678) affecting 52 patients. Among these patients carrying at least one c.919-2A>G mutation allele, there were 13 homozygotes, 27 compound heterozygotes, and 12 single heterozygotes. The second common mutation allele ofSLC26A4 was c.2168A>G with a mutant frequency of 2.21% (15/678).


### 3.4. Mutations inmtDNA12SrRNA

Sixteen patients (4.72%) carried anmtDNA12SrRNA mutation, where all of which were the m.1555A>G mutation containing 15 homoplasmic mutations and 1 heteroplasmic mutations. Ten of these patients had a clear history of aminoglycoside antibiotic use.


## 3.1. Characterization of the Deaf Probands

A total of 339 probands with NSHL were recruited in this sequencing study, including 304 simplex and 35 multiplex probands. Simplex probands refer to the sporadic patients whose families have no one else suffering from hearing loss. Multiplex probands mean that there is at least one first- or second-degree deaf relative in the family. Clinical characterization of the deaf probands was summarized in Table1. The patient cohort consisted of 196 males and 143 females, and they were all Han Chinese. The ages of the probands varied between a few months and 24 years (mean 8.9 years, 95% CI 8.3–9.5 years). Only one proband had late-onset, progressive, and moderate sensorineural hearing loss. All other probands had prelingual or early-onset sensorineural hearing loss. Hearing tests demonstrated that the level of hearing loss was severe to profound in 328 patients. The remaining 11 patients showed moderate hearing loss.Table 1
Deaf patients (n
=
339) categorized by their clinical characteristics.
Simplex (n
=
304)
Multiplex (n
=
35)
Sex
Male
179
17
Female
125
18
Age at the test
0–6 years
153
13
6–18 years
122
21
18–35 years
29
1
Age of onset
Early onset (≤6 years)
303
35
Late onset (>6 years)
1
0
Severity of hearing impairment
Mild
0
0
Moderate
11
0
Severe
56
5
Profound
237
30Among all these patients, 48.67% were identified with hereditary hearing loss by means of the SNPscan assay (Figure1). There were 104 patients (30.68%) affected byGJB2 mutations: 49 homozygotes and 55 compound heterozygotes. 45 (13.27%) patients carried twoSLC26A4 pathogenic mutations: fourteen homozygotes and 31 compound heterozygotes. 16 (4.72%) patients harboredmtDNA12SrRNA mutations consisting of 15 homoplasmic mutations and 1 heteroplasmic mutation.Figure 1
Distribution of the common deafness-causing genes in 339 NSHL patients.


## 3.2. Mutations inGJB2 Gene

Eleven variants were identified in this cohort. They were three frameshift deletions (c.176_191del16, c.235delC, and c.299_300delAT), two frameshift insertions (c.34_35insG and c.511_512insAACG), four missense mutations [c.109G>A (p.V37I), c.257C>G (p.T86R), c.427C>T (p.R143W), and c.571T>C (p.F191L)], one nonsense mutation c.9G>A (p.W3X), and one splicing site mutation IVS1+1G>A (shown in Table2). Nine of them were pathological mutations which have been determined previously. The category of two nucleotide changes was unknown (c.9G>A, c.571T>C). The mutant alleles ofGJB2 accounted for 34.37% (233/678) of the total alleles in all patients (Table 3). The most common mutation allele ofGJB2 in this area was c.235delC with a mutant frequency of 20.65% (140/678). The second common was c.299_300delAT of 5.60% (38/678), followed by c.109G>A of 2.51% (17/678), c.34_35insG of 1.77% (12/678), c.176_191del16 of 1.33% (9/678), c.511_512insAACG of 1.18% (8/678), c.257C>G of 0.29% (2/678), c.427C>T of 0.29% (2/678), and IVS1+1G>A of 0.15% (1/678) (1/678) (Table 3).Table 2
GJB2 genotypes of deaf patients from Linyi.
Allele 1
Allele 2
Number of patients
Nucleotide change
Consequence or amino acid change
Category
Nucleotide change
Consequence or amino acid change
Category
IVS1+1G>A
Splicing site
Pathogenic
c.9G>A
p.W3X
Unknown
1
34_35insG
Frameshift
Pathogenic
109G>A
p.V37I
Pathogenic
1
34_35insG
Frameshift
Pathogenic
176_191del16
Frameshift
Pathogenic
1
34_35insG
Frameshift
Pathogenic
235delC
Frameshift
Pathogenic
8
34_35insG
Frameshift
Pathogenic
299_300delAT
Frameshift
Pathogenic
2
109G>A
p.V37I
Pathogenic
109G>A
p.V37I
Pathogenic
3
109G>A
p.V37I
Pathogenic
235delC
Frameshift
Pathogenic
1
109G>A
p.V37I
Pathogenic
299_300delAT
Frameshift
Pathogenic
1
109G>A
p.V37I
Pathogenic
—
8
176_191del16
Frameshift
Pathogenic
235delC
Frameshift
Pathogenic
8
235delC
Frameshift
Pathogenic
235delC
Frameshift
Pathogenic
42
235delC
Frameshift
Pathogenic
257C>G
p.T86R
Pathogenic
2
235delC
Frameshift
Pathogenic
299_300delAT
Frameshift
Pathogenic
22
235delC
Frameshift
Pathogenic
427C>T
p.R143W
Pathogenic
2
235delC
Frameshift
Pathogenic
511_512insAACG
Frameshift
Pathogenic
3
235delC
Frameshift
Pathogenic
—
10
299_300delAT
Frameshift
Pathogenic
299_300delAT
Frameshift
Pathogenic
4
299_300delAT
Frameshift
Pathogenic
511_512insAACG
Frameshift
Pathogenic
4
299_300delAT
Frameshift
Pathogenic
—
1
511_512insAACG
Frameshift
Pathogenic
—
1
571T>C
p.F191L
Unknown
—
3Table 3
Allele frequencies ofGJB2 mutations in 339 deaf patients from Linyi.
Mutations
Consequence
Number of alleles
Allele frequency (%)
IVS1+1G>A
Splice site
1
0.15
c.34_35insG
Frameshift
12
1.77
c.109G>A
p.V37I
17
2.51
c.176_191del16
Frameshift
9
1.33
c.235delC
Frameshift
140
20.65
c.257C>G
p.T86R
2
0.29
c.299_300delAT
Frameshift
38
5.60
c.427C>T
p.R143W
2
0.29
c.511_512insAACG
Frameshift
8
1.18A total of 104 patients (30.68%) were confirmed to be associated with hereditary hearing loss caused byGJB2 mutation: 49 homozygotes (42 with the c.235delC allele, 4 with c.299_300delAT, and 3 with the c.109G>A allele) and 55 compound heterozygotes. Twenty patients (5.90%) had monoallelic variants in the heterozygous form: ten with c.235delC, eight with c.109G>A, one with c.299_300delAT, and one with c.511_512insAACG. Totally, 128 (37.76%, 128/339) patients had molecular defects inGJB2 gene including three patients with c.571T>C mutation (Table 2).The most common mutation allele ofGJB2 was c.235delC with a mutant frequency of 20.65% (140/678) affecting 98 patients. Among these patients carrying at least one c.235delC allele, there were 42 homozygotes, 46 compound heterozygotes, and 10 single heterozygotes.


## 3.3. Mutations inSLC26A4 Gene

Nineteen variants were identified in this cohort, including 12 missense mutations [c.147C>G (p.S49R), c.269C>T (p.S90L), c.563T>C (p.I188T), c.589G>A (p.G197R), c.1173C>A (p.S391R), c.1174A>T (p.N392Y), c.1225C>T (p.R409C), c.1226G>A (p.R409H), c.1975G>C (p.V659L), c.1985G>A (p.C662Y), c.2027T>A (p.L676Q), and c.2168A>G (p.H723R)], four nonsense mutations [c.235C>T (p.R79X), c.946G>T (p.G316X), c.1318A>T (p.K440X), and c.1540C>T (p.Q514X)], two splicing site mutations (c.919-2A>G, c.1707+5 G>A), and one frameshift insertion mutation (c.1547_1548insC) (Table5). Combining the prediction of SIFT and Polyphen-2 with the results of previous studies, all variants analyzed in our cohort were considered pathogenic mutations except c.147C>G (p.S49R) for an unknown significant pathogenicity (Table 4).Table 4
SLC26A4 genotypes of deaf patients from Linyi.
Allele 1
Allele 2
Number of patients
Nucleotide change
Consequence or amino acid change
Category
Nucleotide change
Consequence or amino acid change
Category
c.147C>G
p.S49R
Unknown
—
1
c.235C>T
p.R79X
Pathogenic
c.919-2A>G
Splice site
Pathogenic
1
c.235C>T
p.R79X
Pathogenic
—
1
c.269C>T
p.S90L
Pathogenic
—
1
c.563T>C
p.I188T
Pathogenic
c.919-2A>G
Splice site
Pathogenic
1
c.589G>A
p.G197R
Pathogenic
c.919-2A>G
Splice site
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.919-2A>G
Splice site
Pathogenic
13
c.919-2A>G
Splice site
Pathogenic
c.946G>T
p.G316X
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.1174A>T
p.N392Y
Pathogenic
2
c.919-2A>G
Splice site
Pathogenic
c.1226G>A
p.R409H
Pathogenic
3
c.919-2A>G
Splice site
Pathogenic
c.1318A>T
p.K440X
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.1540C>T
p.Q514X
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.1547_1548InsC
Frameshift
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.1707+5 G>A
Splice site
Pathogenic
3
c.919-2A>G
Splice site
Pathogenic
c.1975G>C
p.V659L
Pathogenic
3
c.919-2A>G
Splice site
Pathogenic
c.1985G>A
p.C662Y
Pathogenic
1
c.919-2A>G
Splice site
Pathogenic
c.2027T>A
p.L676Q
Pathogenic
2
c.919-2A>G
Splice site
Pathogenic
c.2168A>G
p.H723R
Pathogenic
6
c.919-2A>G
Splice site
Pathogenic
—
12
c.946G>T
p.G316X
Pathogenic
c.2168A>G
p.H723R
Pathogenic
1
c.1173C>A
p.S391R
Pathogenic
c.2168A>G
p.H723R
Pathogenic
1
c.1225C>T
p.R409C
Pathogenic
—
1
c.1226G>A
p.R409H
Pathogenic
c.1975G>C
p.V659L
Pathogenic
1
c.1226G>A
p.R409H
Pathogenic
c.2168A>G
p.H723R
Pathogenic
1
c.1226G>A
p.R409H
Pathogenic
—
1
c.1975G>C
p.V659L
Pathogenic
—
1
c.2168A>G
p.H723R
Pathogenic
c.2168A>G
p.H723R
Pathogenic
1
c.2168A>G
p.H723R
Pathogenic
—
4Table 5
Allele frequencies ofSLC26A4 mutations in 339 deaf patients from Linyi.
Mutations
Consequence
Number of alleles
Allele frequency (%)
c.235C>T
p.R79X
2
0.29
c.269C>T
p.S90L
1
0.15
c.563T>C
p.I188T
1
0.15
c.589G>A
p.G197R
1
0.15
c.919-2A>G
Splice site
65
9.59
c.946G>T
p.G316X
2
0.29
c.1173C>A
p.S391R
1
0.15
c.1174A>T
p.N392Y
2
0.29
c.1225C>T
p.R409C
1
0.15
c.1226G>A
p.R409H
6
0.88
c.1318A>T
p.K440X
1
0.15
c.1540C>T
p.Q514X
1
0.15
c.1547_1548InsC
Frameshift
1
0.15
c.1707+5 G>A
Splicing site
3
0.44
c.1975G>C
p.V659L
5
0.74
c.1985G>A
p.C662Y
1
0.15
c.2027T>A
p.L676Q
2
0.29
c.2168A>G
p.H723R
15
2.21There were 45 (13.27%) patients who were confirmed to have inherited hearing loss because they carried twoSLC26A4 pathogenic mutations: fourteen homozygotes (13 c. IVS 7-2A>G and 1 c.2168A>G) and 31 compound heterozygotes. And 22 (6.49%) patients who were identified carried oneSLC26A4 pathogenic mutation. Thus, the detection rate ofSLC26A4 mutations was 19.76% (67/339) in this patient cohort. All patients carrying two mutations were diagnosed with EVA syndrome by CT scan of the temporal bone. And 17 out of 22 patients carrying one pathogenic mutation were diagnosed with EVA syndrome.The most common mutation allele ofSLC26A4 was c.919-2A>G with a mutant frequency of 9.59% (65/678) affecting 52 patients. Among these patients carrying at least one c.919-2A>G mutation allele, there were 13 homozygotes, 27 compound heterozygotes, and 12 single heterozygotes. The second common mutation allele ofSLC26A4 was c.2168A>G with a mutant frequency of 2.21% (15/678).


## 3.4. Mutations inmtDNA12SrRNA

Sixteen patients (4.72%) carried anmtDNA12SrRNA mutation, where all of which were the m.1555A>G mutation containing 15 homoplasmic mutations and 1 heteroplasmic mutations. Ten of these patients had a clear history of aminoglycoside antibiotic use.


## 4. Discussion

Currently, many studies have reported thatGJB2,SLC26A4, andmtDNA12SrRNA genes are the most common causes in Chinese NSHL population [12–14]. In the present study we have screened for the common deafness gene mutations of 339 NSHL children from Linyi, east part of China. SNPscan assay was used to perform genotyping detection of these three common deafness-causing genes. Compared with other methods, this technique is accurate, rapid, and economically effective. As the novel mutations have been reported successively, more mutation alleles should be added in the SNPscan assay.


### 4.1.GJB2 Mutation Analysis

In this present study,GJB2 mutations were detected in 37.76% (128/339) of all patients, including 30.68% (104/339) with two pathogenic mutations and 7.08% (24/339) with only one mutant allele. The c.235delC and c.299_300delAT were the most frequent mutations in the NSHL patients in Linyi, whereas the hotspot mutation in Xiamen, China, was c.109G>A [7].The frameshift mutation c.235delC has been reported as the most common mutation causing premature protein termination in hearing impaired patients in East and Southeast Asia, while lower frequencies were reported in Europe and Oceania [15–24]. The c.235delC mutation allele frequency was 20.65% (140/678) in this work. In an earlier nationwide study, Dai et al. analyzed theGJB2 mutation of 2063 unrelated NSHL students from 23 different regions of China, and the c.235delC mutation allele frequency was 12.34% (509/4,126) [25]. Compared with 12.34% in his study, the difference in the c.235delC mutation allele frequency was significant (P
=
0.001), reflecting a certain distinction of the c.235delC mutation frequency in Linyi and other areas. There was a higher carrying rate of c.235delC in the Linyi deaf population.


### 4.2.SLC26A4 Mutation Analysis

According to previous study, more than 200 mutations have been described in theSLC26A4 gene with Pendred syndrome (PS) and enlarged vestibular aqueduct (EVA) syndrome (http://www.healthcare.uiowa.edu/labs/pendredandbor/) showing specific distinctions among racial backgrounds. Most of them are missense mutations, in addition to frameshift mutations, splice site mutations, insertions, or deletions [26].The mutation hotspots ofSLC26A4 differed among different nations and areas. In the present study, the most common mutation in our patient cohort was c.IVS7-2A>G and the mutation allele frequency was 9.59% (65/678), whereas the mutation hot spots are p.T416P and c.IVS8+1G>A [27] in Northern Europe, p.H723R and c. IVS7-2A>G in South Korea [15], and p.H723R in Japan [28]. It was reported in a mutation study of a large cohort in Chinese deaf population that the most commonSLC26A4 mutation was also IVS7-2A>G (8.65%, 566/6,542) [12]. Although the mutation allele frequency was a little higher (9.59%) in our study, these frequencies of c.IVS7-2A>G are not significantly different (P = 0.41).


### 4.3.mtDNA12SrRNA Mutation Analysis

Patients with mutations inmtDNA12SrRNA can be affected with aminoglycoside antibiotic-induced deafness showing a hereditary model of maternal inheritance. And m.1494C>T and m.1555A>G are the most common mutations of this mitochondrial gene. Mutate rate of this gene varies among racial and geographic origins in populations with NSHL, with a frequency of 0.6–2.5% in Caucasians [29–32], 1.8% in Turks [30], 0.7% in Germans, 1.8% in Hungarians, and 2.4% in Poles [29]. In our study, we found that 4.72% of the patients were identified to have NSHL caused by the m.1555A>G mutation ofmtDNA12SrRNA gene which was close to that observed by Dai et al. (3.87%) (P
=
0.53).In summary, we performed the first genetic analysis of hearing loss patients from Linyi, eastern part of China. According to genetic detections ofGJB2,SLC26A4, andmtDNA12SrRNA via the SNPscan assay, the fact that almost half of the patients with nonsyndromic hearing loss appeared to have a genetic etiology shows that the SNPscan assay is an available diagnosis tool for studies on genetic hearing loss. The results of the detection suggest thatGJB2 mutations appear to be a major cause of congenital hearing loss in Linyi. The prevalence ofSLC26A4 mutations andmtDNA12SrRNA mutations was very close to other previous studies in Chinese deaf population. However, about 50% of NSHL patients are still not identified with a molecular etiology. For this reason, copy number variations (CNVs) of the three common deafness-causing genes need to be detected. Also, next generation sequencing (NGS) will be employed in screening other deafness-causing genes in the future.


## 4.1.GJB2 Mutation Analysis

In this present study,GJB2 mutations were detected in 37.76% (128/339) of all patients, including 30.68% (104/339) with two pathogenic mutations and 7.08% (24/339) with only one mutant allele. The c.235delC and c.299_300delAT were the most frequent mutations in the NSHL patients in Linyi, whereas the hotspot mutation in Xiamen, China, was c.109G>A [7].The frameshift mutation c.235delC has been reported as the most common mutation causing premature protein termination in hearing impaired patients in East and Southeast Asia, while lower frequencies were reported in Europe and Oceania [15–24]. The c.235delC mutation allele frequency was 20.65% (140/678) in this work. In an earlier nationwide study, Dai et al. analyzed theGJB2 mutation of 2063 unrelated NSHL students from 23 different regions of China, and the c.235delC mutation allele frequency was 12.34% (509/4,126) [25]. Compared with 12.34% in his study, the difference in the c.235delC mutation allele frequency was significant (P
=
0.001), reflecting a certain distinction of the c.235delC mutation frequency in Linyi and other areas. There was a higher carrying rate of c.235delC in the Linyi deaf population.


## 4.2.SLC26A4 Mutation Analysis

According to previous study, more than 200 mutations have been described in theSLC26A4 gene with Pendred syndrome (PS) and enlarged vestibular aqueduct (EVA) syndrome (http://www.healthcare.uiowa.edu/labs/pendredandbor/) showing specific distinctions among racial backgrounds. Most of them are missense mutations, in addition to frameshift mutations, splice site mutations, insertions, or deletions [26].The mutation hotspots ofSLC26A4 differed among different nations and areas. In the present study, the most common mutation in our patient cohort was c.IVS7-2A>G and the mutation allele frequency was 9.59% (65/678), whereas the mutation hot spots are p.T416P and c.IVS8+1G>A [27] in Northern Europe, p.H723R and c. IVS7-2A>G in South Korea [15], and p.H723R in Japan [28]. It was reported in a mutation study of a large cohort in Chinese deaf population that the most commonSLC26A4 mutation was also IVS7-2A>G (8.65%, 566/6,542) [12]. Although the mutation allele frequency was a little higher (9.59%) in our study, these frequencies of c.IVS7-2A>G are not significantly different (P = 0.41).


## 4.3.mtDNA12SrRNA Mutation Analysis

Patients with mutations inmtDNA12SrRNA can be affected with aminoglycoside antibiotic-induced deafness showing a hereditary model of maternal inheritance. And m.1494C>T and m.1555A>G are the most common mutations of this mitochondrial gene. Mutate rate of this gene varies among racial and geographic origins in populations with NSHL, with a frequency of 0.6–2.5% in Caucasians [29–32], 1.8% in Turks [30], 0.7% in Germans, 1.8% in Hungarians, and 2.4% in Poles [29]. In our study, we found that 4.72% of the patients were identified to have NSHL caused by the m.1555A>G mutation ofmtDNA12SrRNA gene which was close to that observed by Dai et al. (3.87%) (P
=
0.53).In summary, we performed the first genetic analysis of hearing loss patients from Linyi, eastern part of China. According to genetic detections ofGJB2,SLC26A4, andmtDNA12SrRNA via the SNPscan assay, the fact that almost half of the patients with nonsyndromic hearing loss appeared to have a genetic etiology shows that the SNPscan assay is an available diagnosis tool for studies on genetic hearing loss. The results of the detection suggest thatGJB2 mutations appear to be a major cause of congenital hearing loss in Linyi. The prevalence ofSLC26A4 mutations andmtDNA12SrRNA mutations was very close to other previous studies in Chinese deaf population. However, about 50% of NSHL patients are still not identified with a molecular etiology. For this reason, copy number variations (CNVs) of the three common deafness-causing genes need to be detected. Also, next generation sequencing (NGS) will be employed in screening other deafness-causing genes in the future.


## 5. Conclusions

In this study, we used the SNPscan assay technique to detect the 115 mutations of the three common deafness-causing genes in 339 nonsyndromic hearing loss patients from Linyi, China. And we found that the mutation profile was distinct from other parts of China suggesting a unique mutation spectrum in this area. Also, the SNPscan assay was an available diagnosis tool for studies on genetic hearing loss.



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*Source: 1302914-2016-05-09.xml*