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. Author manuscript; available in PMC: 2016 May 18.
Published in final edited form as: Genet Med. 2015 Jul 30;18(4):364–371. doi: 10.1038/gim.2015.89

Comprehensive Analysis via Exome Sequencing Uncovers Genetic Etiology in Autosomal Recessive Non-Syndromic Deafness in a Large Multiethnic Cohort

Guney Bademci 1, Joseph Foster II 1, Nejat Mahdieh 2, Mortaza Bonyadi 3, Duygu Duman 4, FBasak Cengiz 4, Ibis Menendez 1, Oscar Diaz Horta 1, Atefeh Shirkavand 5, Sirous Zeinali 5,6, Asli Subasioglu 7, Suna Tokgoz-Yilmaz 8, Fabiola Huesca Hernandez 9, Maria de la Luz Arenas Sordo 9, Juan Dominguez-Aburto 9, Edgar Hernandez-Zamora 9, Paola Montenegro 10, Rosario Paredes 10, Germania Moreta 10, Rodrigo Vinueza 10, Franklin Villegas 10, Santiago Mendoza Benitez 11, Shengru Guo 1, Nazim Bozan 12, Tulay Tos 13, Armagan Incesulu 14, Gonca Sennaroglu 8, Susan H Blanton 1, Hatice Ozturkmen Akay 15, Muzeyyen Yildirim-Baylan 16, Mustafa Tekin 1
PMCID: PMC4733433  NIHMSID: NIHMS693478  PMID: 26226137

Abstract

Purpose

Autosomal recessive non-syndromic deafness (ARNSD) is characterized by a high degree of genetic heterogeneity with reported mutations in 58 different genes. This study was designed to detect deafness causing variants in a multiethnic cohort with ARNSD by using whole-exome sequencing (WES).

Methods

After excluding mutations in the most common gene, GJB2, we performed WES in 160 multiplex families with ARNSD from Turkey, Iran, Mexico, Ecuador and Puerto Rico to screen for mutations in all known ARNSD genes.

Results

We detected ARNSD-causing variants in 90 (56%) families, 54% of which had not been previously reported. Identified mutations were located in 31 known ARNSD genes. The most common genes with mutations were MYO15A (13%), MYO7A (11%), SLC26A4 (10%), TMPRSS3 (9%), TMC1 (8%), ILDR1 (6%) and CDH23 (4%). Nine mutations were detected in multiple families with shared haplotypes suggesting founder effects.

Conclusion

We report on a large multiethnic cohort with ARNSD in which comprehensive analysis of all known ARNSD genes identifies causative DNA variants in 56% of the families. In the remaining families, WES allows us to search for causative variants in novel genes, thus improving our ability to explain the underlying etiology in more families.

Keywords: Autosomal Recessive, Deafness, Exome, Next-Generation Sequencing

Introduction

Deafness is a global public health concern which affects 1 to 3 per 1,000 newborns.1 In more than half of the cases with congenital or prelingual deafness, the cause is genetic and most demonstrate an autosomal recessive inheritance pattern.1 Mutations in 58 different genes have been reported to cause autosomal recessive non-syndromic deafness (ARNSD) (http://hereditaryhearingloss.org/).

Except for one relatively common gene, GJB2 (MIM 121011), most reported mutations are present in only a single or a few families.2 Whole-exome sequencing (WES) allows resequencing of nearly all exons of the protein-coding genes in the genome.3 A growing number of research and clinical diagnostic laboratories are successfully using WES for gene/variant identification, owing to its comprehensive analysis advantages.4,5 In this study, we present the results of WES in a large multiethnic cohort consisting of 160 families with ARNSD that were negative for GJB2 mutations.

Material and Methods

Statement of Ethics

This study was approved by the University of Miami Institutional Review Board (USA), Ankara University Medical School Ethics Committee (Turkey), Growth and Development Research Ethics Committee (Iran), Bioethics Committee of FFAA (HE-1) in Quito (Ecuador) and the Ethics Committee of National Institute of Rehabilitation (Mexico). A signed informed consent form was obtained from each participant or, in the case of a minor, from parents.

Subjects

We included 160 families with at least two members with nonsyndromic sensorineural hearing loss with a pedigree structure suggestive of autosomal recessive inheritance (affected siblings born to unaffected parents with or without parental consanguinity) and GJB2 mutations were negative. Hearing loss was congenital or prelingual onset with a severity ranging from mild to profound. One hundred and one families from Turkey, fifty-four from Iran, two from Mexico, two from Ecuador and one from Puerto Rico were included. Sensorineural hearing loss was diagnosed via standard audiometry in a sound-proof room according to standard clinical practice. Clinical evaluation of all affected individuals by a geneticist and an otolaryngologist included a thorough physical examination, otoscopy, and ophthalmoscopy. Tandem walking and the Romberg test were used for initial vestibular evaluation with more detailed tests if needed based on symptoms and findings. Laboratory investigation included but was not limited to an EKG, urinalysis, and, when available, a high resolution CT scan of the temporal bone or an MRI to identify inner ear anomalies. DNA was extracted from peripheral leukocytes of each member of the family by standard protocols.

Whole-Exome Sequencing

Agilent SureSelect Human All Exon 50 Mb versions 3, 4, and 5 (Agilent Technologies Santa Clara, CA) were used for in-solution enrichment of coding exons and flanking intronic sequences following the manufacturer's standard protocol. The enriched DNA samples were subjected to standard sample preparation for the HiSeq 2000 instrument (Illumina San Diego, CA). The Illumina CASAVA v1.8 pipeline was used to produce 99 bp sequence reads. BWA6 was used to align sequence reads to the human reference genome (hg19) and variants were called using the GATK (https://www.broadinstitute.org/gatk/) software package.7 All single nucleotide variants (SNVs) and insertion/deletions (INDELs) were submitted to SeattleSeq137 for further characterization and annotation. Sanger sequencing was used for confirmation and segregation of the variants in each family.

Bioinformatics Analysis

We analyzed WES data using our in house tool (https://genomics.med.miami.edu). Our workflow is seen in Figure 1. The analysis started with QC checks including the coverage and average read depth of targeted regions, numbers of variants in different categories, and quality scores. All variants were annotated and categorized into known and novel variants. As previously recommended, we filtered variants based on minor allele frequency of <0.005 in dbSNP141.8 We also filtered out variants that are present in >10 samples in our internal database of >3,000 exomes from European, Asian, and American ancestries that includes Turkish, Iranian, Mexican, Ecuadorian, and Puerto Rican samples (Figure 1). Autosomal recessive inheritance with both homozygous and compound heterozygous inheritance models, and a genotype quality (GQ) score >35 for the variant quality were chosen. Missense, nonsense, splice site, in-frame INDEL and frame-shift INDELs in the known ARNSD genes (supplementary data) were selected. Missense variants that remained after these filters were later analyzed for presence in the Human Gene Mutation Database (HGMD) (www.hgmd.cf.ac.uk) and having a pathogenic prediction score at least in two of the following tools: PolyPhen29, SIFT10, MutationAssessor11, and MutationTaster12. Finally, we used CoNIFER13 (Copy Number Inference From Exome Reads) and XHMM14 (eXome-Hidden Markov Model) to detect CNVs.15 After this filtering, only those variants co-segregated with the phenotype in the entire family was considered pathogenic.

Fig.1. Overall workflow of our WES pipeline.

Fig.1

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Results

On average, each exome had 99%, 95% and 88% of mappable bases of the Gencode defined exome represented by coverage of 1X, 5X and 10X reads, respectively. Average coverage of the mappable bases for the 58 known ARNSD genes (exons and the first and last 20 bps of introns) were 99%, 95%, 87% for the 1X, 5X, 10X reads, respectively.

We detected pathogenic or likely pathogenic variants that can explain ARNSD in 90 (56%) families. All identified variants co-segregated with deafness as an autosomal recesive trait. 54% of the mutations were not previously reported in HGMD. Mutations were identified in 31 ARNSD genes. The genes with mutations identified in at least three families are MYO15A (MIM 602666) (13%), MYO7A (MIM 276903) (11%), SLC26A4 (MIM 605646) (10%), TMPRSS3 (MIM 605551) (9%), TMC1 (MIM 606706) (8%), ILDR1 (MIM 609739) (6%), CDH23 (MIM 605516) (4%), OTOF (MIM 603681) (4%), PCDH15 (MIM 605514) (3%), and TMIE (MIM 607723) (3%). During the course of this study we reported mutations in OTOGL (MIM 614925) and FAM65B (MIM 611410) as novel causes of ARNSD16,17 (Figure 1)(Table 1).

Table 1. Mutations identified in known ARNSD genes*.

Family ID Country of origin Genotype cDNA Protein NM Transcript Gene Reference
543 Turkey Homozygous c.4441T>C p.S1481P NM_016239.3 MYO15A (Cengiz 2010)23
724 Turkey Homozygous c.4652C>A p.A1551D NM_016239.3 MYO15A (Diaz-Horta 2012)4
765 Turkey Homozygous c.4273C>T p.Q1425X NM_016239.3 MYO15A (Diaz-Horta 2012)4
723 Turkey Homozygous c.8307_8309delGGA p.E2770del NM_016239.3 MYO15A Novel
795 Turkey Homozygous c.5808_5814delCCGTGGC p.R1937TfsX10 NM_016239.3 MYO15A (Cengiz 2010)23
793 Turkey Homozygous c.5808_5814delCCGTGGC p.R1937TfsX10 NM_016239.3 MYO15A (Cengiz 2010)23
1209 Puerto Rico Heterozygous c.7226delC p.P2409QfsX8 NM_016239.3 MYO15A Novel
Heterozygous c.9620G>A p.R3207H NM_016239.3 MYO15A Novel
1083 Turkey Homozygous c.5183T>C p.L1728P NM_016239.3 MYO15A Novel
1332 Turkey Homozygous c.10361delT p.V3454GfsX5 NM_016239.3 MYO15A Novel
489 Turkey Homozygous c.5286_5287delTC p.R1763AfsX45 NM_016239.3 MYO15A Novel
1023 Iran Homozygous c.8638_8641delCCTG p.P2880RfsX19 NM_016239.3 MYO15A Novel
862 Turkey Heterozygous c.7894G>T p.V2632L NM_016239.3 MYO15A Novel
Heterozygous c.5133+1G>A splice NM_016239.3 MYO15A Novel
974 Iran Homozygous c.6487G>A p.G2163S NM_000260.3 MYO7A (Janecke 1999)24
1391 Turkey Homozygous c.6487G>A p.G2163S NM_000260.3 MYO7A (Janecke 1999)24
435 Turkey Homozygous c.3935T>C p.L1312P NM_000260.3 MYO7A Novel
472 Turkey Homozygous c.1556G>T p.G519V NM_000260.3 MYO7A Novel
1370 Turkey Homozygous c.722G>A p.R241H NM_000260.3 MYO7A (Cremers 2007)25
432 Turkey Homozygous c.5362_5363delAG p.R1788DfsX13 NM_000260.3 MYO7A Novel
637 Turkey Heterozygous c.5838delT p.F1946LfsX24 NM_000260.3 MYO7A Novel
Heterozygous c.5573T>C p.L1858P NM_000260.3 MYO7A (Bharadwaj 2000)26
996 Iran Homozygous c.5785C>T p.Q1929X NM_000260.3 MYO7A Novel
1019 Iran Homozygous c.1708C>T p.R570X NM_000260.3 MYO7A (Yoshimura 2014)27
1404 Turkey Heterozygous c.1708C>T p.R570X NM_000260.3 MYO7A (Yoshimura 2014)27
Heterozygous c.6025G>A p.A2009T NM_000260.3 MYO7A Novel
786 Turkey Homozygous c.1001G>T p.G334V NM_000441.1 SLC26A4 (Landa 2013)28
634 Turkey Homozygous c.1001G>T p.G334V NM_000441.1 SLC26A4 (Landa 2013)28
1418 Turkey Homozygous c.1061T>C p.F354S NM_000441.1 SLC26A4 (Blons 2004)29
238 Turkey Homozygous c.1226G>A p.R409H NM_000441.1 SLC26A4 (Van hauwe 1998)30
973 Iran Homozygous c.1334T>G p.L445W NM_000441.1 SLC26A4 (Van hauwe 1998)30
905 Turkey Homozygous c.2168A>G p.H723R NM_000441.1 SLC26A4 (Van hauwe 1998)30
1417 Turkey Heterozygous c.665G>A p.G222D NM_000441.1 SLC26A4 Novel
Heterozygous c.1198delT p.C400VfsX32 NM_000441.1 SLC26A4 Novel
1346 Turkey Homozygous c.919-2A>G splice NM_000441.1 SLC26A4 (Coucke 1999)31
1321 Turkey Homozygous c.1198delT p.C400VfsX32 NM_000441.1 SLC26A4 Novel
395 Turkey Homozygous c.36dupC p.F13LfsX10 NM_024022.2 TMPRSS3 (Diaz-Horta 2012)4
777 Turkey Homozygous c.913A>T p.I305F NM_024022.2 TMPRSS3 Novel
674 Turkey Homozygous c.271C>T p.R91X NM_024022.2 TMPRSS3 Novel
629 Turkey Homozygous c.399G>C p.W133C NM_024022.2 TMPRSS3 Novel
1368 Turkey Homozygous c.1126G>A p.G376S NM_024022.2 TMPRSS3 Novel
1410 Turkey Homozygous c.436G>A p.G146S NM_024022.2 TMPRSS3 Novel
910 Turkey Homozygous c.616G>T p.A206S NM_024022.2 TMPRSS3 Novel
633 Turkey Homozygous c.616G>T p.A206S NM_024022.2 TMPRSS3 Novel
52 Turkey Homozygous c.1589_1590CT p.S530X NM_138691.2 TMC1 (Hildebrand 2010)32
123 Turkey Homozygous c.1080_1084delGATCA p.R362PfsX6 NM_138691.2 TMC1 Novel
662 Turkey Homozygous c.2050G>A p.D684N NM_138691.2 TMC1 Novel
1268 Ecuador Heterozygous c.1718T>A p.I573N NM_138691.2 TMC1 Novel
Heterozygous c.2130-1delG splice NM_138691.2 TMC1 Novel
911 Turkey Homozygous c.1534C>T p.R512X NM_138691.2 TMC1 (Kurima 2002)33
490 Turkey Homozygous c.1959C>G p.Y653X NM_138691.2 TMC1 Novel
393 Turkey Heterozygous c.63+2T>A splice NM_138691.2 TMC1 (Duman 2011)18
Heterozygous c.236+1G>A splice NM_138691.2 TMC1 (Duman 2011)18
988 Iran Homozygous c.3215C>A p.A1072D NM_022124.5 CDH23 (Duman 2011)18
1165 Mexico Heterozygous c.2959G>A p.D987N NM_022124.5 CDH23 Novel
Heterozygous c.3628C>T p.Q1210X NM_022124.5 CDH23 Novel
1015 Iran Homozygous c.5851G>A p.D1951N NM_022124.5 CDH23 Novel
1032 Iran Heterozygous c.7822C>T p.R2608C NM_022124.5 CDH23 Novel
Heterozygous c.8120C>T p.P2707L NM_022124.5 CDH23 Novel
968 Iran Homozygous c.820C>T p.Q274X NM_001199799.1 ILDR1 (Diaz-Horta 2012)4
800 Turkey Homozygous c.942C>A p.C314X NM_001199799.1 ILDR1 Novel
799 Turkey Homozygous c.942C>A p.C314X NM_001199799.1 ILDR1 Novel
782 Turkey Homozygous c.942C>A p.C314X NM_001199799.1 ILDR1 Novel
969 Iran Homozygous c.82delG p.V28SfsX31 NM_001199799.1 ILDR1 Novel
1297 Turkey Homozygous c.5431A>T p.K1811X NM_194248.2 OTOF (Romanos 2009)34
98 Turkey Homozygous c.5431A>T p.K1811X NM_194248.2 OTOF (Romanos 2009)34
1398 Turkey Homozygous c.3679C>T p.R1227X NM_194248.2 OTOF Novel
909 Turkey Homozygous c.765G>C p.Q255H NM_194248.2 OTOF (Rodriguez 2008)35
725 Turkey Homozygous c.3918T>G p.C1306W NM_033056.3 PCDH15 Novel
1238 Turkey Homozygous CNV CNV NM_033056.3 PCDH15 Novel
1044 Iran Homozygous c.3101G>A p.R1034H NM_033056.3 PCDH15 Novel
1369 Turkey Homozygous c.250C>T p.R84W NM_147196.2 TMIE (Naz 2002)36
1354 Turkey Homozygous c.250C>T p.R84W NM_147196.2 TMIE (Naz 2002)36
1402 Turkey Homozygous c.250C>T p.R84W NM_147196.2 TMIE (Naz 2002)36
1239 Turkey Homozygous c.490-1G>T splice NM_016366.2 CABP2 Novel
1366 Turkey Homozygous c.1018G>T p.E340X NM_004452.3 ESRRB Novel
1372 Turkey Homozygous c.1018G>T p.E340X NM_004452.3 ESRRB Novel
794 Turkey Homozygous c.508C>A p.H170N NM_133261.2 GIPC3 Novel
1356 Turkey Homozygous c.508C>A p.H170N NM_133261.2 GIPC3 Novel
182 Turkey Homozygous c.4480C>T p.R1494X NM_144612.6 LOXHD1 (Diaz-Horta 2012)4
779 Turkey Homozygous c.2863G>T p.E955X NM_144612.6 LOXHD1 (Diaz-Horta 2012)4
303 Turkey Homozygous c.628A>T p.K210X NM_005709.3 USH1C Novel
994 Iran Homozygous c.876+2delTA splice NM_005709.3 USH1C Novel
661 Turkey Homozygous c.330T>A p.Y110X NM_006383.3 CIB2 Novel
262 Turkey Homozygous c.2662C>A p.P888T NM_080680.2 COL11A2 (Chakchouk 2015)37
448 Turkey Homozygous c.499C>T p.R167X NM_001042702.3 DFNB59 (Collin 2007)38
908 Turkey Homozygous c.102-1G>A splice NM_014722.2 FAM65B (Diaz-Horta 2014)17
1289 Turkey Homozygous c.2956A>T p.K986X NM_032119.3 GPR98 Novel
820 Turkey Homozygous c.79C>T p.R27X NM_001080476.2 GRXCR1 Novel
67 Turkey Homozygous c.1498C>T p.R500X NM_001038603.2 MARVELD2 (Riazuddin 2006)39
1364 Turkey Homozygous c.1015C>T p.R339W NM_004999.3 MYO6 (Yang 2013)40
63 Turkey Homozygous CNV CNV NM_144672.3 OTOA (Bademci 2014)15
338 Turkey Homozygous c.1430delT p.V477EfsX25 NM_173591.3 OTOGL (Yariz 2012)16
1294 Turkey Homozygous c.1108C>T p.R370X NM_002906.3 RDX Novel
850 Turkey Homozygous CNV CNV NM_153700.2 STRC (Bademci 2014)15
1035 Iran Homozygous c.5210A>G p.Y1737C NM_005422.2 TECTA (Diaz-Horta 2012)4
7 Turkey Homozygous c.705_709dupCCTGC p.R237PfsX215 NM_001128228.2 TPRN Novel
23 Turkey Homozygous c.2335_2336delAG p.R785SfsX50 NM_001039141.2 TRIOBP (Diaz-Horta 2012)4
5 Turkey Homozygous c.387_388insC p.K130QfsX5 NM_173477.2 USH1G Novel
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*

Families with compound heterozygous mutations are italicized.

Discussion

Identifying causative variants in ARNSD is challenging because of (1) the extreme genetic heterogeneity of ARNSD; (2) the presence of different categories of genetic variants such as SNVs, INDELs and CNVs; (3) the presence of a high proportion of non-recurrent mutations and (4) the variability in mutation frequencies in individual ARNSD genes across ethnicities.18 Consequently, we performed a comprehensive analysis to detect pathogenic SNVs, INDELs and CNVs in the ARNSD genes.

Targeted resequencing allows identification of mutations in the interested gene sets. Recent studies pioneered by Shearer et al. have shown the effectiveness of the targeted resequencing of deafness genes.8,19 An advantage of the targeted resequencing over WES is having better coverage with higher depth and significantly lowered costs, which is suitable for clinical diagnostic labs. However, a main limitation of the targeted sequencing is the need for revalidation of the panel after adding each new gene. In contrast, many laboratories around the world offer WES as a diagnostic tool requiring validation only when a new WES version is introduced. Our analysis using three different versions of an exome capture kit during the four year period shows that the depth of coverage of WES has improved to reliably identify most mutations in known ARNSD genes (Figure 2) (Table S1 and Table S4). Recently developed WES approaches provide more coverage for genes that are known to cause Mendelian disease. They are expected to cover deafness genes more efficiently. In addition, adding in baits to improve coverage over poorly covered regions may be considered if a better coverage is desired. It was recently shown via targeted sequencing that CNVs are a common cause of deafness.20 While CNV analysis of the WES data is being still optimized for clinical usage, we integrated two currently available tools, XHMM and CoNIFER into our WES analysis pipeline and identified large OTOA (MIM 607038), STRC (MIM 606440) and PCDH15 (exon 27-28) homozygous deletions in our cohort, supporting a significant role of CNVs to in deafness etiology.

Fig.2.

Fig.2

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Overview of coverage of 58 known ARNSD genes according to 3 different versions (Version 3=V3, Version 4=V4 and Version 5=V5) of the exome enrichment kit (A,B,C). Numbers of samples studied with different capture kits (D).

In this study after excluding GJB2 mutations we detected pathogenic variants in the known ARNSD genes in 56% of the studied families. The advantage of this study is to have large multiplex autosomal recessive families (including affected and unaffected children) that can be tested for co-segregation of all variants. While we identified more novel variants than those reported in Table 1 through WES, only those variants co-segregated in the family with deafness were considered pathogenic. Similarly heterozygous variants didn't explain the phenotype since they did not co-segregate with deafness and were not included. WES facilitates the cataloguing of mutations in different populations. Population characteristics such as the rate of consanguineous marriages may affect the distribution of deafness mutations in different populations. As expected, the vast majority of Turkish and Iranian probands from consanguineous marriages are homozygous for the pathogenic variants (Table 2). However, there is a marked difference between the rates of solved families in Turkey (73%) vs. Iran (24%) (Figure 3). As seen in figure 3, the distribution of genes is also different between the two countries. In our study, the top five genes explain 39 out of 101 families (39%) in Turkey, while only 10 out of 54 families (19%) in Iran. Moreover, our analysis of the WES data in the unsolved Iranian families shows that there are no common mutations in genes that are not known to be deafness genes (data not shown). Unless there are common mutations in regions that are not well covered by WES, our data suggest that many rare genes are responsible for the majority of hereditary deafness in the Iranian cohort. It is likely that there are undetected rare variants specific to certain ethnicities in Iran.21 Another advantage of WES is to allow surveying of mutations for founder effects. We detected TMIE c.250C>T (p.R84W) in three unrelated Turkish families, which all shared a flanking haplotype as noted previously.22 Furthermore MYO15A, MYO7A, SLC26A4, TMPRSS3, ILDR1, OTOF, ESRRB (MIM 602167) and GIPC3 (MIM 608792) genes had recurrent mutations with shared haplotypes indicating founder effects (Table S2).

Table 2. Overview of mutation detection and parental consanguinity.

Countries Number of Families Reported Parental Consanguinity Number of Homozygous Probands (consanguineous) Number of Compound Heterozygous Probands (consanguineous)
Turkey 101 82 67 (59) 5 (2)
Iran 54 31 12 (10) 1 (1)
Ecuador 2 0 0 1 (0)
Mexico 2 0 0 1 (0)
Puerto Rico 1 0 0 1 (0)
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Fig.3.

Fig.3

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Distribution of causative DNA variants in known ARNSD genes according to the family origin (A) and variant categories (B).

There is no correlation between the size of transcript and number of mutant alleles (Table S3). There may be some deafness genes that are more prone to have mutations. Founder effects appear to play a role because some small genes such as TMIE, ESRRB, and GIPC3 ranked high in mutation frequency because of founder mutations. Some discrepancy between the size of a gene and number of mutations can be explained by the fact that only certain mutations cause nonsyndromic deafness for some genes. For instance, CDH23, PCDH15, MYO7A are big genes but many mutations in those genes cause Usher syndrome (MIM 276900) instead of ARNSD. An interesting example is TMC1 that ranks the 20th based on size but the 5th for mutation frequency. Nonsyndromic deafness is the only phenotype caused by TMC1 mutations and none of the TMC1 mutations are recurrent in our cohort. These may suggest that TMC1 is relatively more prone to have de novo mutations or it is a highly conserved gene and its variants are rarely tolerated.

In conclusion, WES is a an effective tool for identifying pathogenic SNVs, INDELs and CNVs simultaneously in ARNSD genes and provides further analysis of the unsolved families for novel gene discovery. Identification of two novel ARNSD genes16,17 during the course of this study testifies its power.

Supplementary Material

Supplementary 1 _Appendix_ online only material_ etc._
Supplementary 2 _Appendix_ online only material_ etc._

Acknowledgments

We are grateful to the participating families.

This work was supported by National Institutes of Health grant R01DC009645 to M.T.

Footnotes

Author Disclosure Statement: Authors declare that there is no conflict of interest to report.

Supplementary Material:Supplementary information is available at the Genetics in Medicine website

References

  • 1.Morton CC, Nance WE. Newborn hearing screening--a silent revolution. N Engl J Med. 2006;354:2151–2164. doi: 10.1056/NEJMra050700. [DOI] [PubMed] [Google Scholar]
  • 2.Duman D, Tekin M. Autosomal recessive nonsyndromic deafness genes: a review. Front Biosci (Landmark Ed) 2012;17:2213–2236. doi: 10.2741/4046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ng SB, Turner EH, Robertson PD, et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature. 2009;461:272–276. doi: 10.1038/nature08250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Diaz-Horta O, Duman D, Foster J, 2nd, et al. Whole-exome sequencing efficiently detects rare mutations in autosomal recessive nonsyndromic hearing loss. PLoS One. 2012;7:e50628. doi: 10.1371/journal.pone.0050628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Atik T, Bademci G, Diaz-Horta O, Blanton S, Tekin M. Whole-exome sequencing and its impact in hereditary hearing loss. Genet Res (Camb) 2015;97:e4. doi: 10.1017/S001667231500004X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26:589–595. doi: 10.1093/bioinformatics/btp698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.McKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–1303. doi: 10.1101/gr.107524.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Shearer AE, Eppsteiner RW, Booth KT, et al. Utilizing ethnic-specific differences in minor allele frequency to recategorize reported pathogenic deafness variants. Am J Hum Genet. 2014;95:445–453. doi: 10.1016/j.ajhg.2014.09.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–249. doi: 10.1038/nmeth0410-248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009;4:1073–1081. doi: 10.1038/nprot.2009.86. [DOI] [PubMed] [Google Scholar]
  • 11.Reva B, Antipin Y, Sander C. Predicting the functional impact of protein mutations: application to cancer genomics. Nucleic Acids Res. 2011;39:e118. doi: 10.1093/nar/gkr407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods. 2014;11:361–362. doi: 10.1038/nmeth.2890. [DOI] [PubMed] [Google Scholar]
  • 13.Krumm N, Sudmant PH, Ko A, et al. Copy number variation detection and genotyping from exome sequence data. Genome Res. 2012;22:1525–1532. doi: 10.1101/gr.138115.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Fromer M, Moran JL, Chambert K, et al. Discovery and statistical genotyping of copy-number variation from whole-exome sequencing depth. Am J Hum Genet. 2012;91:597–607. doi: 10.1016/j.ajhg.2012.08.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Bademci G, Diaz-Horta O, Guo S, et al. Identification of copy number variants through whole-exome sequencing in autosomal recessive nonsyndromic hearing loss. Genet Test Mol Biomarkers. 2014;18:658–661. doi: 10.1089/gtmb.2014.0121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Yariz KO, Duman D, Seco CZ, et al. Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss. Am J Hum Genet. 2012;91:872–882. doi: 10.1016/j.ajhg.2012.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Diaz-Horta O, Subasioglu-Uzak A, Grati M, et al. FAM65B is a membrane-associated protein of hair cell stereocilia required for hearing. Proc Natl Acad Sci U S A. 2014;111:9864–9868. doi: 10.1073/pnas.1401950111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Duman D, Sirmaci A, Cengiz FB, Ozdag H, Tekin M. Screening of 38 genes identifies mutations in 62% of families with nonsyndromic deafness in Turkey. Genet Test Mol Biomarkers. 2011;15:29–33. doi: 10.1089/gtmb.2010.0120. [DOI] [PubMed] [Google Scholar]
  • 19.Shearer AE, DeLuca AP, Hildebrand MS, et al. Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing. Proc Natl Acad Sci U S A. 2010;107:21104–21109. doi: 10.1073/pnas.1012989107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Shearer AE, Kolbe DL, Azaiez H, et al. Copy number variants are a common cause of non-syndromic hearing loss. Genome Med. 2014;6:37. doi: 10.1186/gm554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Mahdieh N, Rabbani B, Wiley S, Akbari MT, Zeinali S. Genetic causes of nonsyndromic hearing loss in Iran in comparison with other populations. J Hum Genet. 2010;55:639–648. doi: 10.1038/jhg.2010.96. [DOI] [PubMed] [Google Scholar]
  • 22.Sirmaci A, Ozturkmen-Akay H, Erbek S, et al. A founder TMIE mutation is a frequent cause of hearing loss in southeastern Anatolia. Clin Genet. 2009;75:562–567. doi: 10.1111/j.1399-0004.2009.01183.x. [DOI] [PubMed] [Google Scholar]
  • 23.Cengiz FB, Duman D, Sirmaci A, et al. Recurrent and private MYO15A mutations are associated with deafness in the Turkish population. Genet Test Mol Biomarkers. 2010;14:543–550. doi: 10.1089/gtmb.2010.0039. [DOI] [PubMed] [Google Scholar]
  • 24.Janecke AR, Meins M, Sadeghi M, et al. Twelve novel myosin VIIA mutations in 34 patients with Usher syndrome type I: confirmation of genetic heterogeneity. Hum Mutat. 1999;13:133–140. doi: 10.1002/(SICI)1098-1004(1999)13:2<133::AID-HUMU5>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  • 25.Cremers FP, Kimberling WJ, Kulm M, et al. Development of a genotyping microarray for Usher syndrome. J Med Genet. 2007;44:153–160. doi: 10.1136/jmg.2006.044784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Bharadwaj AK, Kasztejna JP, Huq S, Berson EL, Dryja TP. Evaluation of the myosin VIIA gene and visual function in patients with Usher syndrome type I. Exp Eye Res. 2000;71:173–181. doi: 10.1006/exer.2000.0863. [DOI] [PubMed] [Google Scholar]
  • 27.Yoshimura H, Iwasaki S, Nishio SY, et al. Massively parallel DNA sequencing facilitates diagnosis of patients with Usher syndrome type 1. PLoS One. 2014;9:e90688. doi: 10.1371/journal.pone.0090688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Landa P, Differ AM, Rajput K, Jenkins L, Bitner-Glindzicz M. Lack of significant association between mutations of KCNJ10 or FOXI1 and SLC26A4 mutations in Pendred syndrome/enlarged vestibular aqueducts. BMC Med Genet. 2013;14:85. doi: 10.1186/1471-2350-14-85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Blons H, Feldmann D, Duval V, et al. Screening of SLC26A4 (PDS) gene in Pendred's syndrome: a large spectrum of mutations in France and phenotypic heterogeneity. Clin Genet. 2004;66:333–340. doi: 10.1111/j.1399-0004.2004.00296.x. [DOI] [PubMed] [Google Scholar]
  • 30.Van Hauwe P, Everett LA, Coucke P, et al. Two frequent missense mutations in Pendred syndrome. Hum Mol Genet. 1998;7:1099–1104. doi: 10.1093/hmg/7.7.1099. [DOI] [PubMed] [Google Scholar]
  • 31.Coucke PJ, Van Hauwe P, Everett LA, et al. Identification of two different mutations in the PDS gene in an inbred family with Pendred syndrome. J Med Genet. 1999;36:475–477. [PMC free article] [PubMed] [Google Scholar]
  • 32.Hildebrand MS, Kahrizi K, Bromhead CJ, et al. Mutations in TMC1 are a common cause of DFNB7/11 hearing loss in the Iranian population. Ann Otol Rhinol Laryngol. 2010;119:830–835. doi: 10.1177/000348941011901207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Kurima K, Peters LM, Yang Y, et al. Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function. Nat Genet. 2002;30:277–284. doi: 10.1038/ng842. [DOI] [PubMed] [Google Scholar]
  • 34.Romanos J, Kimura L, Favero ML, et al. Novel OTOF mutations in Brazilian patients with auditory neuropathy. J Hum Genet. 2009;54:382–385. doi: 10.1038/jhg.2009.45. [DOI] [PubMed] [Google Scholar]
  • 35.Rodriguez-Ballesteros M, Reynoso R, Olarte M, et al. A multicenter study on the prevalence and spectrum of mutations in the otoferlin gene (OTOF) in subjects with nonsyndromic hearing impairment and auditory neuropathy. Hum Mutat. 2008;29:823–831. doi: 10.1002/humu.20708. [DOI] [PubMed] [Google Scholar]
  • 36.Naz S, Giguere CM, Kohrman DC, et al. Mutations in a novel gene, TMIE, are associated with hearing loss linked to the DFNB6 locus. Am J Hum Genet. 2002;71:632–636. doi: 10.1086/342193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Chakchouk I, Grati M, Bademci G, et al. Novel mutations confirm that COL11A2 is responsible for autosomal recessive non-syndromic hearing loss DFNB53. Mol Genet Genomics. 2015 doi: 10.1007/s00438-015-0995-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Collin RW, Kalay E, Oostrik J, et al. Involvement of DFNB59 mutations in autosomal recessive nonsyndromic hearing impairment. Hum Mutat. 2007;28:718–723. doi: 10.1002/humu.20510. [DOI] [PubMed] [Google Scholar]
  • 39.Riazuddin S, Ahmed ZM, Fanning AS, et al. Tricellulin is a tight-junction protein necessary for hearing. Am J Hum Genet. 2006;79:1040–1051. doi: 10.1086/510022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Yang T, Wei X, Chai Y, Li L, Wu H. Genetic etiology study of the non-syndromic deafness in Chinese Hans by targeted next-generation sequencing. Orphanet J Rare Dis. 2013;8:85. doi: 10.1186/1750-1172-8-85. [DOI] [PMC free article] [PubMed] [Google Scholar]

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