Sequence variants in genes causing nonsyndromic hearing loss in a Pakistani cohort.

BACKGROUND: Hearing loss or hearing impairment is a clinically and genetically heterogeneous disorder. More than 117 genes were discovered to date in hereditary, nonsyndromic hearing loss (NSHL). Identifying novel gene variants and their frequency in specific populations is valuable for public health and potentially for genetic screening of NSHL. AIMS: To identify the gene variants underlying NSHL in a Pakistani cohort. METHODS AND
RESULTS: A cohort of 40 school-aged children with NSHL was initially screened for variants in GJB2, the gene with the highest incidence of variants in other populations with NSHL. We found known homozygous as well as compound heterozygous GJB variants in 15 individuals. Next, we used targeted next generation sequencing (TNGS) for the remaining 25 individuals and identified 20 different variants in 14 genes (SLC26A4, KCNQ4, MYO7A, MYO15A, TMPRSS3, ESPN, TMC1, GIPC3, LHFPL5, WFS1, DFNB59, GRXCR1, ESRRB, and LRTOMT).
CONCLUSIONS: We described common and novel variants in 15 genes in a Pakistani cohort of NSHL.

To the Editor:

Hearing loss is a clinically and genetically heterogeneous disorder and is the most common human disability (Shrivastava, Shrivastava, & Ramasamy, 2016). Both environmental and genetic factors were shown in the pathogenesis of hearing loss such as iodine deficient diet, infections, ototoxic drug treatment, or gene variants (http://hereditaryhearingloss.org/) (Morton & Nance, 2016; Shrivastava et al., 2016; Wonkam et al., 2013). Nonsyndromic hearing loss (NSHL) can be inherited as an autosomal recessive (AR), autosomal dominant (AD), X‐linked (dominant or recessive), or mitochondrial trait (Liu et al., 2005; Snoeckx et al., 2005; Wang, Han, Khan, & Zhang, 2017). Identifying novel sequence variants in populations provide valuable information for public health, particularly for genetic screening of the NSHL.

In our study, we recruited a total of 40 unrelated individuals affected by NSHL from Bannu and Kohat districts in Khyber Pakhtunkhwa (KPK) province, Pakistan. A clinical questionnaire was used to collect medical history and rule out the history of other diseases potentially affecting hearing and environmental factors such as antibiotic use, excessive noise exposure, and infection that could cause hearing loss. Detailed physical examination did not reveal other abnormal findings than hearing loss. Palpable goiters and other dysmorphic features were not observed in any of the participants with hearing loss; their hearing was ranging from severely impaired to deaf, listed in Figure 1 and Table 1. All of the probands had bilateral, prelingual hearing loss consistent with a congenital origin. There was no previous family history of hearing loss in the participants. The study design and protocol were approved by the Institutional Review Board of (IRB) Quaid‐i‐Azam University Islamabad Pakistan, the Ethical Review Committee (ERC) of Peking Union Medical College (Beijing, China), and China Medical University (Shenyang, China). Written informed consents to conduct and publish the study were obtained from all participating individuals and their parents. All 40 individuals were screened by polymerase chain reaction (PCR) and Sanger sequencing for variants in GJB2 (gap junction protein beta 2; NM_004004.5) gene which is the most commonly mutated gene in recessive NSHL (Liu et al., 2005; Snoeckx et al., 2005). We found known homozygous as well as compound heterozygous variants in 15 individuals. Four homozygous patients had GJB2; c. 370C > T; p.Gln124*, six had GJB2; c.35delG; p.Gly12Valfs*2, three compound heterozygotes had GJB2; c.[71G > A];c.[231G > A]; p.[p.Trp24*];p.[Trp77*], and two had GJB2; c.[−23 + 1G>A]; c.[231G > A]; p.[N/A]; p.[Trp77*] variants respectively (Table 1). The 25 individuals, who were negative for GJB2 gene variants, were screened by targeted next generation sequencing (TNGS) technology using the Ion Torrent platform for a panel of 63 genes implicated in hearing loss as described previously (Wang et al., 2017). We identified 20 variants in 14 genes (SLC26A4, KCNQ4, MYO15A, TMPRSS3, ESPN, TMC1, GIPC3, LHFPL5, WFS1, DFNB59, GRXCR1, ESRRB, and LRTOMT). Ten were novel such as two homozygous missense variant in MYO15A c.[9518‐2A > G];p.[N/A], c.3944G > A;p.Gly1315Glu, one novel variant c.199T > G; p.Tyr67Asp in LHFPL5 in two unrelated individuals, one novel variant c.2519G > A; p.Trp840* in ESPN, one in KCNQ4 c.1288G > A; p.Glu430Lys, one in ESRRB c521G > A; p.Arg174His, one in DFNB59 c.147T > A; p.Tyr49*, one in GIPC3 c.759C > G; p.Ser253Arg, one in TMC1 c.662A > G; p.Tyr221Cys, and one novel variant in LRTOMT c.154C > T; p.Arg52Trp (Figure 1, Table 1). The functional effect of the variants was predicted using the in silico tools including Polyphen2 (http://genetics.bwh.harvard.edu/pph2/), Sorting Intolerant from Tolerant (SIFT) (http://sift.jcvi.org/), PROVEAN (http://www.provean.jcvi.org) Mutation Taster (http://www.mutationtaster.org/), Human Splicing Finder (HSF) (http://www.umd.be/HSF) and CADD (https://cadd.gs.washington.edu) (Table 1).

Figure 1

(a) Novel sequence variants identified in ESPN, KCNQ4, ESRRB, MYO15A, LHFPL5, DFNB59, GIPC3, TMC1, and LRTOMT in our cohort from Pakistan. (b) Audiometry of individuals in our cohort with hearing loss

Table 1

Clinical features and genotyping in hearing loss individuals

Subjects	Sex	Age	Gene	Transcript ID	Variant	Allele	Effect on protein	Mutation type	Known/Novel	Variant interpretation prediction scores					ACMG classification
										Polyphen2	PROVEAN	SIFT	Mutation taster	CADD
1	M	7	GJB2	NM_004004.5	c.[71G > A]; [71G > A]	Hom	p.[Trp24*];[Trp24*]	Nonsense	Known	–	–	–	1	36	PVS1
2	M	7	GJB2	NM_004004.5	c.[71G > A];[71G > A]	Hom	p.[Trp24*];[Trp24*]	Nonsense	Known	–	–	–	1	36	PVS1
3	M	9	GJB2	NM_004004.5	c.[71G > A];c.[231G > A]	Comp Het	p.[Trp24*];p.[Trp77*]	Nonsense	Known	–	–	–	1	36/38	PVS1
4	F	6	GJB2	NM_004004.5	c.[370C > T];[370C > T]	Hom	p.[Gln124*];[Gln124*]	Nonsense	Known	–	–		1	34	PVS1
5	F	5	GJB2	NM_004004.5	c.[370C > T];[370C > T]	Hom	p.[Gln124*];[Gln124*]	Nonsense	Known	–	–	–	1	34	PVS1
6	M	11	GJB2	NM_004004.5	c.[370C > T];[370C > T]	Hom	p.[Gln124*];[Gln124*]	Nonsense	Known	–	–	–	1	34	PVS1
7	F	13	GJB2	NM_004004.5	c.[370C > T];[370C > T]	Hom	p.[Gln124*];[Gln124*]	Nonsense	Known	–	–	–	1	34	PVS1
8	M	9	GJB2	NM_004004.5	c.[35delG];[35delG]	Hom	p.[(Gly12Valfs*2)];[(Gly12Valfs*2)]	Frameshift	Known	–	–	–	–	–	PVS1
9	F	8	GJB2	NM_004004.5	c.[35delG];[35delG]	Hom	p.[(Gly12Valfs*2)];[(Gly12Valfs*2)]	Frameshift	Known	–	–	–	–	–	PVS1
10	F	10	GJB2	NM_004004.5	c.[35delG];[35delG]	Hom	p.[(Gly12Valfs*2)];[(Gly12Valfs*2)]	Frameshift	Known	–	–	–	–	–	PVS1
11	F	7	GJB2	NM_004004.5	c.[35delG];[35delG]	Hom	p.[(Gly12Valfs*2)];[(Gly12Valfs*2)]	Frameshift	Known	–	–	–	–	–	PVS1
12	M	5	GJB2	NM_004004.5	c.[35delG];[35delG]	Hom	p.[(Gly12Valfs*2)];[(Gly12Valfs*2)]	Frameshift	Known	–	–	–	–	–	PVS1
13	M	8	GJB2	NM_004004.5	c.[35delG];[35delG]	Hom	p.[(Gly12Valfs*2)];[(Gly12Valfs*2)]	Frameshift	Known	–	–	–	15	–	PVS1
14	M	9	GJB2	NM_004004.5	c.[−23 + 1G>A];c.[231G > A]	Comp Het	p.[N/A];p.[Trp77*]	Splice site; Frameshift	Known	–	–	–	–	−/38	PVS1
15	M	10	GJB2	NM_004004.5	c.[−23 + 1G>A];[−23 + 1G>A]	Hom	p.[N/A];[N/A]	Splice site	Known	–	–	–	–	–	PVS1
16	M	8	SLC26A4	NM_000441.1	c.[679G > C];[679G > C]	Hom	p.[Ala227Pro];[Ala227Pro]	Missense	Known	1	−4.94	0.001	1	30	PM2
17	F	7	SLC26A4	NM_000441.1	c.[679G > C];[679G > C]	Hom	p.[Ala227Pro];[Ala227Pro]	Missense	Known	1	−4.94	0.001	1	30	PM2
18	F	7	SLC26A4	NM_000441.1	c.[679G > C];[679G > C]	Hom	p.[Ala227Pro];[Ala227Pro]	Missense	Known	1	−4.94	0.001	1	30	PM2
19	M	6	SLC26A4	NM_000441.1	c.[716T > A];[716T > A]	Hom	p.[Val239Asp];[Val239Asp]	Missense	Known	0.845	−5.67	0.001	1	29.5	PP2
20	M	7	SLC26A4	NM_000441.1	c.[716T > A];[716T > A]	Hom	p.[Val239Asp];[Val239Asp]	Missense	Known	0.845	−5.67.	0.001	1	29.5	PP2
21	M	8	MYO7A	NM_000260.3	c.[1258A > T];[1258A > T]	Hom	p.[Lys420*];[Lys420*]	Nonsense	Known	–	–	–	1	41	PVS1
22	M	9	MYO7A	NM_000260.3	c.[1258A > T];[1258A > T]	Hom	p.[Lys420*];[Lys420*]	Nonsense	Known	–	–	–	1	41	PVS1
23	F	14	MYO7A	NM_000260.3	c.[4838delA];[4838delA]	Hom	p.[Asp1613Valfs*32];[Asp1613Valfs*32]	Frameshift	Known	–	–	–	–	–	PVS1
24	M	13	MYO15A	NM_016239.3	c.[9518−2A > G]; [9518−2A > G]	Hom	p.[N/A];[N/A]	Splice site	Novel	–	–	–	1	19	PVS1
25	F	15	MYO15A	NM_016239.3	c.[3944G > A]; [3944G > A]	Hom	p.[Gly1315Glu]; [Gly1315Glu]	Missense	Known	1	−7.7	0.0	1	24.6	PM2
26	M	7	GRXCR1	NM_001080479.2	c.[784C > T];[784C > T]	Hom	p.[Arg262*];[Arg262*];	Nonsense	Known	–	–	–	1	50	PVS1
27	M	12	GRXCR1	NM_001080479.2	c.[784C > T];[784C > T]	Hom	p.[Arg262*];[Arg262*];	Nonsense	Known	–	–	–	1	50	PVS1
28	M	10	LHFPL5	NM_182548.3	c.[199T > G];[199T > G]	Hom	p.[Tyr67Asp];[Tyr67Asp]	Missense	Novel	1	−6.12	0.032	1	27.8	PM2
29	M	8	LHFPL5	NM_182548.3	c.[199T > G];[199T > G]	Hom	p.[Tyr67Asp];[Tyr67Asp]	Missense	Novel	1	−6.12	0.032	1	27.8	PM2
30	M	7	TMPRSS3	NM_024022.2	c.[727G > A];[727G > A]	Hom	p.[Gly243Arg];[Gly243Arg]	Missense	Known	1	−7.53	0.01	1	35	PM2
31	F	9	TMPRSS3	NM_024022.2	c.[1219T > C];[1219T > C]	Hom	p.[Cys407Arg];[Cys407Arg]	Missense	Known	0.997	−3.98	0.122	1	23.3	PM2
32	M	6	WFS1	NM_006005.3	c.[2338G > A];[1219T > C]	Hom	p.[Gly780Ser];[Gly780Ser]	Missense	Known	0.896	−1.34	0.060	1	23.7	PM2
33	M	16	WFS1	NM_006005.3	c.[2590G > A];[2590G > A]	Hom	p.[Glu864Lys];[Glu864Lys]	Missense	Known	1	−1.68	0.045	1	28.6	PM2
34	M	22	ESPN	NM_031475.2	c.[2519G > A];[2519G > A]	Hom	p.[Trp840*];[Trp840*]	Nonsense	Known	–	–	–	1	42	PVS1
35	M	13	KCNQ4	NM_004700.3	c.[1288G > A];[1288G > A]	Hom	p.[Glu430Lys];[Glu430Lys]	Missense	Novel	0.956	−0.72	0.376	1	18.19	PP2
36	M	16	ESRRB	NM_004452.3	c.[521G > A];[521G > A]	Hom	p.[Arg174His];[Arg174His]	Missense	Novel	0.860	−4.27	0.046	1	27.5	PM2
37	M	10	DFNB59	NM_001042702.4	c.[147T > A];[521G > A]	Hom	p.[Tyr49*];[Tyr49*]	Nonsense	Novel	–	–	–	1	35	PVS1
38	M	8	GIPC3	NM_133261.2	c.[759C > G];[759C > G]	Hom	p.[Ser253Arg];[Ser253Arg]	Missense	Novel	0.995	−3.87	0.043	0.9994	23.2	PM2
39	F	9	TMC1	NM_138691.2	c.[662A > G];[662A > G]	Hom	p.[Tyr221Cys];[Tyr221Cys]	Missense	Novel	1	−6.34	0.045	1	27.3	PM2
40	F	11	LRTOMT	NM_001145308.4	c.[154C > T];[154C > T]	Hom	p.[Arg52Trp];[Arg52Trp]	Missense	Known	0.988	−1.79	0.001	1	26.5	PVS1

Abbreviations: Comp. Het, Compound Heterozygous; F, Female; Hom, Homozygous; M, Male.

Furthermore, these novel variants was neither present in the dbSNP (http://www.ncbi.nlm.nih.gov/SNP/),Exome Variant Server (http://evs.gs.washington.edu/EVS/), GnomAD (https://gnomad.broadinstitute.org), Human gene mutation database (HGMD, http://www.hgmd.cf.ac.uk/ac/index.php), 1,000 Genomes (http://www.1000genomes.org/), and Exome aggregation consortium (ExAC) (http://www.exac.broadinstitute.org). Finally, for the interpretation of variants, the American College of Medical Genetics and Genomics (ACMG) 2015 guidelines were used (Table 1) (Richards et al., 2015).

None of the novel variants were present in 200 ethnically matched control individuals confirmed by PCR and Sanger sequencing. The '15 of 40 patients had known variants in GJB2 (described above), 5  of 40 patients had known variants in SLC26A4 c.679G > C; p.Ala227Pro, 3 of 40 had known variants in MYO7A c.1258A > T; p.Lys420*, c.4838delA; p.Asp1613Valfs*32, 2 of 40 had known variants in GRXCR1 c.784C > T; p.Arg262*, 2 of 40 had known variants in TMPRSS3 c.727G > A; p.Gly243Arg, c.1219T > C; p.Cys407Arg, 2 of 40 had known variants in WFS1 c.2338G > A; p.Gly780Ser, c.2590G > A; p.Glu864Lys, 1 of 40 had known variant in ESPN c.2519G > A; p.Trp840*,1  of 40 had known variant in LRTMOT c.154C > T; p.Arg52Trp and were already known to be associated with syndromic or NSHL (Salman et al., 2015; Wang et al., 2017). The GJB2 variants were the most common variants in our patient cohort. Our study showed that GJB2 is the most common gene found mutated in our Pakistani cohort which is similar to other cohorts around the world (Salman et al., 2015; Snoeckx et al., 2005; Wang et al., 2017). We found the GJB2 variants c.35delG; p.Gly12Valfs*2, c.71G > A; p.Trp24* and c. [71G > A]; c. [231G > A]; p. [p.Trp24*]; p. [Trp77*] as the most common, 15 of 40 patients in our cohort however their frequency is variable worldwide (Salman et al., 2015).

In our patient cohort we found 15/40 patients had GJB2 variants, (5/40) had SLC26A4, and (3/40) had MYO7A variants responsible for NSHL. We found the incidence of GJB2 variants is higher compared to other populations. While this would argue for screening for GJB2 variants in the first place, we found a comparably high incidence for SLC26A4 and MYO7A variants together. Future research could specifically take advantage of using TNGS in the Pakistani cohort to capture a larger proportion of NSHL cases of genetic origin. We expect our study will enhance public awareness toward this hearing loss trouble and genetic counseling significance for the affected families and their members. This study expanded a spectrum of disease causing variants in genes involved in causing hearing loss.

WEB RESOURCES

The URLs for data presented herein are as follows:

1,000 Genomes: http://www.1000genomes.org/

Exome Variant Server: http://evs.gs.washington.edu/EVS/

ExAC: http://exac.broadinstitue.org/

dbSNP: http://www.ncbi.nlm.nih.gov/SNP/

OMIM: http://www.omim.org/

HGMD: http://www.biobase-international.com/products/hgmd

SIFT: http://sift.jcvi.org/

Polyphen2: http://genetics.bwh.harvard.edu/pph2/

IGV: http://www.broadinstitute.org/igv/

ANNOVAR: http://annovar.openbioinformatics.org/en/latest/

Mutation Taster: http://www.mutationtaster.org/

OMIM: http://www.omim.org/

UCSC Genome Browser: http://genome.ucsc.edu

CONFLICT OF INTEREST

The authors declare no conflicts of interest.