Non-syndromic hearing loss gene identification: A brief history and glimpse into the future

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Non-syndromic hearing loss gene identification: A brief history and glimpse into the future Barbara Vona a, *, Indrajit Nanda a, Michaela A.H. Hofrichter a, Wafaa Shehata-Dieler b, Thomas Haaf a a

Institute of Human Genetics, Julius Maximilians University, Würzburg, Germany Comprehensive Hearing Center, Department of Otorhinolaryngology, Plastic, Aesthetic and Reconstructive Surgery, University Hospital, Würzburg, Germany

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 February 2015 Accepted 23 March 2015 Available online xxx

From the first identified non-syndromic hearing loss gene in 1995, to those discovered in present day, the field of human genetics has witnessed an unparalleled revolution that includes the completion of the Human Genome Project in 2003 to the $1000 genome in 2014. This review highlights the classical and cutting-edge strategies for non-syndromic hearing loss gene identification that have been used throughout the twenty year history with a special emphasis on how the innovative breakthroughs in next generation sequencing technology have forever changed candidate gene approaches. The simplified approach afforded by next generation sequencing technology provides a second chance for the many linked loci in large and well characterized families that have been identified by linkage analysis but have presently failed to identify a causative gene. It also discusses some complexities that may restrict eventual candidate gene discovery and calls for novel approaches to answer some of the questions that make this simple Mendelian disorder so intriguing. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Keywords: Copy number variation (CNV) Deafness GJB2 Homozygosity mapping Linkage analysis Missing heritability Next generation sequencing (NGS) Non-syndromic hearing loss (NSHL) Positional cloning

1. Introduction to non-syndromic hearing loss Hearing loss (HL) is the most prevalent sensory deficit primarily originating from genetic etiologies and clinically occurring without any additional phenotypes [1]. Non-syndromic hearing loss (NSHL) generally follows simple Mendelian inheritance and is predominantly transmitted as an autosomal recessive trait (75e80%), although autosomal dominant (20%), X-linked (2e5%) and mitochondrial mutations (1%) can also cause HL [1]. With certain exceptions, the onset and severity of these inherited types of HL follow a similar clinical pattern. For example, autosomal recessive NSHL is generally prelingual, non-progressive (stable) and severeto-profound [2], whereas autosomal dominant NSHL is primarily characterized as post-lingual (with an onset often spanning the second through fifth decades of life) and progressive [3]. The prevalence of NSHL, beginning in prelingual children, doubles from approximately 1.33 per every 1000 newborns to 2.7 per every 1000

* Corresponding author. Institute of Human Genetics, Julius-Maximilians-Uni€t Würzburg, Biozentrum, Am Hubland, 97074 Würzburg, Germany. Tel.: þ49 versita 931 3184244; fax: þ49 931 3187398. E-mail address: [email protected] (B. Vona).

children at five years of age [4]. With the steadily aging global population, the number of individuals with various types of HL is expected to reach approximately 1 billion by the year 2020 [5], with age-related hearing loss (ARHL) accounting for a large proportion of this estimate. ARHL is thought to be multifactorial and the result of unclarified genetic susceptibility and environmental factors such as excessive noise exposure, exposure to ototoxic chemicals, and medical conditions that exacerbate HL in advanced age, specifically diabetes and cardiovascular disease [5]. Furthermore, ARHL shows a high degree of heritability within nuclear families and twin studies [6,7], and males are generally more affected than females [5]. The apparently complex pathological processes of ARHL can often mimic those of late-onset autosomal dominant NSHL, clouding their strict distinctions [8]. Genotype-phenotype correlations have intrigued physicians and scientists for as long as these principles have known to exist. The wealth of information that has emerged from connecting genetic mutations to impact on health has dramatically expanded the general understanding of inherited diseases. However, in the case of NSHL, full comprehension of these correlations is a great challenge due to extreme clinical and genetic heterogeneity [9]. Mutations in broad subsets of genes confer HL and the initial development and progression due to diverse genetic underlying

http://dx.doi.org/10.1016/j.mcp.2015.03.008 0890-8508/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article in press as: Vona B, et al., Non-syndromic hearing loss gene identification: A brief history and glimpse into the future, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.03.008

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causes are often indistinguishable. To resolve the molecular basis of a process as complex and delicate as hearing necessitates a fundamental understanding of participating genes. The systematic elucidation of NSHL genes has translational consequence to improved diagnostic, prognostic, and therapeutic options. The identification of new NSHL genes has been met with astonishing success, uncovering a total of 85 genes (Fig. 1). Many of the recently identified genes have been established using massively parallel sequencing (MPS) or next generation sequencing (NGS) (Table 1); a process in which targeted enrichment and MPS with the so-called “next generation” of sequencing technologies posteSanger sequencing era, permit the scaling up of sequencing data by orders of magnitude [10]. Before expanding on how this relatively new method has forever transformed the face of molecular genetics by alleviating a significant data generation bottleneck, it is necessary to review the classical methods of NSHL gene discovery. 2. A brief history of the identification of non-syndromic hearing loss genes Whole-genome linkage analysis within a large, clinically wellcharacterized family provides a powerful approach for mapping critical chromosomal intervals. This process integrates informative marker co-segregation in a large family for localizing a genetic region encompassing a disease-causing mutation to a defined chromosomal position [11] for a variety of subsequent positional cloning methods. Similarly, homozygosity mapping is an alternative approach for delineating autosomal recessive loci by mapping

long stretches of homozygous genotypes that has proven successful with consanguineous families [12] and homogeneous populations sharing similar clinical features [13]. Due to limiting meiotic crossing-over events, these locus mapping approaches typically identify large chromosomal regions spanning several mega bases that include hundreds of genes for positional cloning strategies, namely bacterial artificial chromosome (BAC)-mediated cloning [14,15], cDNA library preparation from tissues of interest for expression analysis [16,17], as well as selection and sequencing of candidate genes based on a priori hypotheses linking a candidate gene to a certain phenotype [18,19] or candidate gene selection with the aid of pre-existing deafness mouse models for selection of human orthologous genes to sequence [20,21]. Candidate genes in the locus are ranked and sequentially sequenced to pinpoint mutations. The year 1988 marked the first reported NSHL locus mapping to chromosome Xq using a linkage approach [22] that served as the first step in a seven year journey toward identifying the gene POU3F4 (MIM: *300039) [23]. Similarly, the first autosomal locus was linked to chromosome 5q31 in 1992 [24] leading to the identification of DIAPH1 (MIM: *602121) five years later [25]. To grasp the full appreciation of this work, these loci were mapped at a time when human molecular genetics was still in its infancy and newly developed physical maps still unknowingly harboured serious errors [26], increasing the risk of incorrect map distances and reduced linkage analysis power [27]. This emerging work served as an example from which many classical NSHL candidate gene studies materialized.

Fig. 1. Timeline of NSHL genes identified according to method of discovery and major achievements in the development of NGS technologies per year [97,98]. Abbreviations: D, Q5 dominant form of HL; R, recessive form of HL; WES, whole exome sequencing; WGS, whole genome sequencing.

Please cite this article in press as: Vona B, et al., Non-syndromic hearing loss gene identification: A brief history and glimpse into the future, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.03.008

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Table 1 Bilateral non-syndromic hearing loss genes discovered through the use of next generation sequencing methods. Gene symbol

DFN locus

Gene name

MIM

Discovery method

Clinical features and audiogram profiles

Ref

Autosomal dominant inheritance CEACAM16 DFNA4B Carcinoembryonic antigen-related cell adhesion molecule 16

*614591

WGL, candidate gene sequencing, WES

[114,115]

P2RX2

DFNA41

Purinergic receptor P2X, ligand gated ion channel 2

*600844

WGL, TE and MPS

OSBPL2

DFNA67

Oxysterol binding protein-like 2

*606731

WGL and WES

TBC1D24

DFNA65

TBC1 domain family, member 24

*613577

TNC

DFNA56

Tenascin C

*187380

IBD mapping and WES; WGL and WES WGL and WES

Postlingual, progressive, moderate SNHL, flat or gently down-sloping audiogram Postlingual, progressive, moderateto-severe SNHL, accelerated presbycusis, _:down-sloping, \: ascending audiogram; over time, sex differences converge Postlingual, progressive, downsloping audiogram Postlingual, progressive, downsloping audiogram Postlingual, progressive, severe low-frequency SNHL, ascending audiogram

Autosomal recessive inheritance ADCY1 DFNB44 Adenylate cyclase 1

*103072

WGL and WES

[123,124]

BDP1

DFNB49

CABP2

Prelingual (probably congenital), profound, mild-to-moderate mixed and SNHL, highly variable audiogram Prelingual, progressive, moderateto-severe SNHL, down-sloping audiogram Prelingual, moderate-to-severe SNHL, flat to shallow U-shaped audiogram Prelingual, severe-to-profound, mixed HL, variable audiogram Congenital, profound, flat audiogram Prelingual, severe-to-profound SNHL, gently down-sloping audiogram Early onset, moderate-to-severe SNHL, progressive, down-sloping audiogram Prelingual, moderate-to-profound, stable, variable audiogram Prelingual, congenital, moderate SNHL, stable, down-sloping audiogram Prelingual, profound SNHL, flat audiogram Congenital, severe-to-profound SNHL Prelingual, severe-to-profound SNHL, down-sloping or flat audiogram Congenital, profound SNHL, flat audiogram Prelingual, severe-to-profound, down-sloping audiogram, vestibular areflexia

*607012

WGL and WES

DFNB93

B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB Calcium binding protein 2

*607314

WGL, TE and MPS

ELMOD3

DFNB88

ELMO/CED-12 domain containing 3

*615427

WGL, HM and WES

EPS8

DFNB102

*600206

WES

GPSM2

DFNB32/82

Epidermal growth factor receptor pathway substrate 8 G-protein signaling modulator 2

*609245

WGL, HM and WES

GRXCR2

DFNB101

Glutaredoxin, cysteine rich 2

*615762

WGL, HM and WES

KARS

DFNB89

Lysyl-tRNA synthetase

*601421

OTOGL

DFNB84B

Otogelin-like

*614925

TBC1D24

DFNB86

TBC1 domain family, member 24

*613577

WGL, candidate gene sequencing, and WES Autozygosity mapping, WGL, candidate gene sequencing and WES WGL, HM and WES

TMEM132E

DFNB99

Transmembrane protein 132E

*616178

HM and WES

TPRN

DFNB79

Taperin

*613354

WGL, candidate gene sequencing, TE and MPS; WGL, HM

TSPEAR

DFNB98

*612920

WGL, HM and WES

CLIC5

DFNB103

Thrombospondin-type laminin G domain and EAR repeats Chloride intracellular channel 5

*607293

HM, WES

X-linked COL4A6

DFNX6

Collagen, type IV, alpha 6

*303631

Linkage and WES of the X chromosome

SMPX

DFNX4

Small muscle protein, X-linked

*300226

Linkage and TE and MPS of linked locus

_: Congenital, severe SNHL, flat audiogram; \: (carriers) 3rd and 4th decade onset, mild-to-moderate mixed or conductive HL, flat audiogram _: Postlingual, severe-to-profound SNHL, progressive, down-sloping or flat audiogram; \: (carriers) 2nd to 4th decade onset, moderate-tosevere high frequency SNHL, downsloping audiogram

[116,117]

[118,119] [120,121] [122]

[125]

[126,127]

[128] [129] [37,130]

[131]

[132,133] [134]

[135,136] [137] [36,138,139]

[140] [141]

[142]

[68,143,144]

Abbreviations: HM, homozygosity mapping; IBD, identity-by-descent; MPS, massively parallel sequencing; SNHL, sensorineural hearing loss; TE, targeted enrichment; WES, whole exome sequencing; WGL, whole-genome linkage.

Please cite this article in press as: Vona B, et al., Non-syndromic hearing loss gene identification: A brief history and glimpse into the future, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.03.008

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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Table 2 Linked NSHL loci remaining candidate gene clarification. All cases were described as causing bilateral sensorineural hearing loss unless otherwise described. Locus

Severity

Audiogram profile

Additional features

Ref

Autosomal dominant (DFNA) DFNA7a 1q21q23 2nd decade DFNA16a 2q23q24.3 Between 9 and 10 years of age

Chr. position

Onset

Mild-to-moderately severe Fluctuating; mild-to-profound

Sloping in high frequencies Sloping in high frequencies

[57] [145]

DFNA18a

3q22

Severe

Progressive Progressive, steroid therapy treatment, occasional tinnitus/ vertigo, possible correlation between physical stress/HL Progressive

DFNA21

6p24.1p22.3

Progressive

[147,148]

Progressive

[58,149]

Progressive

[150]

Progressive, reduced penetrance

[71]

Progressive Progressive, can include tinnitus

[26] [151]

Progressive, can include tinnitus

[152]

Progressive

[153]

Progressive

[154]

Progressive Progressive, substantial phenotypic expressivity

[59] [67]

Progressive, vestibular symptoms occasionally reported Progressive, one report of tinnitus (7th decade) and vestibular symptoms (8th decade) Progressive, one affected individual reported tinnitus

[61]

Non-progressive Progressive

[156] [157]

1st decade, postlingual

DFNA24

4q35qter

DFNA27

4q12q13.1

DFNA30a

15q25q26

DFNA31 DFNA33

6p21.3 13q34qter

DFNA43

2p12

DFNA47a

9p21p22

DFNA49

1q21q23

DFNA52a DFNA53

5q31.1q32 14q11.2q12

DFNA54

5q31

Onset: high frequencies, progresses to mid- and low frequencies, gently downsloping 1st to 5th decades ND Non-specific mid- to midehigh frequencies Congenital Birth: moderate, 8th decade: Mid- to high frequencies; profound steeply down-sloping 1st to 3rd decades Moderate-to-profound < 40 years, Onset: high frequencies, then severe-to-profound > 40 years shifts to include all frequencies; flat 2nd to 5th decades Variable Onset: high frequencies, then progresses to mid-frequencies; down-sloping 1st to 2nd decades ND Mid- to high frequencies 2nd to 3rd decades Mild-to-profound Onset: high frequencies, then includes all frequencies; downsloping, flat in 7th decade 2nd decade Moderate-to-profound Onset: high frequencies, progresses to include all frequencies; down-sloping 3rd to 4th decades Moderate-to-severe Onset: high frequencies, progresses to include all frequencies; down-sloping 1st decade, postlingual Moderate-to-severe Onset: low to mid-frequencies, then progresses in middle frequencies; U-shaped audiogram by 4th decade 2nd to 3rd decade Progresses to profound High frequency; down-sloping 2nd decade Variable High frequencies primarily affected; audiograms can be down-sloping or flat 1st and 5th decades Moderate-to-severe Low frequencies; ascending

DFNA57

19p13.2

1st decade, postlingual Mild-to-moderate

DFNA58

2p12p21

2nd to 5th decades

Mild-to-profound

DFNA59 DFNA60

11p14.2q12.3 2q23.1q23.3

Congenital 2nd to 5th decades

Severe-to-profound Mild-to-severe

Autosomal DFNB5a DFNB13 DFNB14a DFNB17a DFNB20a DFNB26a,c DFNB27a DFNB33 DFNB38 DFNB40 DFNB45 DFNB46 DFNB47 DFNB51 DFNB55 DFNB62 DFNB65 DFNB68 DFNB71 DFNB80

recessive (DFNB) 14q12 7q34q36 7q31 7q31 11q25qter 4q31 2q23q31 10p11.23q21.1 6q26q27 22q11.21q12.1 1q43q44 18p11.32p11.31 2p25.1p24.3 11p13p12 4q12q13.2 12p13.2p11.23 20q13.2q13.32 19p13.2 8p22p21.3 2p16.1p21

Congenital Congenital Congenital Congenital Prelingual Congenital Prelingual Prelingual Prelingual Prelingual Prelingual Prelingual Prelingual Prelingual Prelingual Congenital Prelingual Prelingual Prelingual Prelingual

Severe-to-profound Severe-to-profound Profound Profound Moderate-to-profound Profound ND Severe Profound Profound Profound Profound Profound Profound Profound Profound Profound Profound Profound Profound

a

Low and mid-frequencies, progresses to include high frequencies; flat or gently ascending High frequencies at onset, progresses to include all frequencies; flat Sloping to 1 kHz, then flat Low and mid-frequencies; flat, U-shaped, down-sloping possible ND ND ND ND ND Down-sloping ND ND Flat Flat or down-sloping ND ND Flat ND Flat Flat Flat ND Flat Down-sloping

Progressive

[146]

[155]

[60]

[158,159] [160,161] [162] [51] [163] [72] [164] [165,166] [167] [168] [169] [170] [52] [53] [171] [172] [173] [54] [174] [175]

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Table 2 (continued ) Locus

Chr. position

Onset

Severity

Audiogram profile

Additional features

Ref

DFNB81

19p

Prelingual

Severe-to-profound

Down-sloping

Mixed and sensorineural HL possible

[55,56]

DFNB83 9p23p21.2 DFNB85 17p12q11.2 DFNB90 7p22.1p15.3 DFNB96 1p36.31p36.13 X-linked (DFNX) a DFNX3 Xp21.2

Prelingual Prelingual Prelingual Prelingual

ND ND Severe-to-profound Severe-to-profound

ND ND Flat Flat

_: congenital; \: 2nd to 3rd decades _: 2nd decade \: ND

_: profound, \: mild-to-moderate _: mild-to-severe, \: ND

_: flat; \: variable Variable

1st to 3rd decades, postlingual

Moderate-to-severe

Flat or U-shaped

Modifier (DFNM) DFNM1a,c 1q24

Congenital

Profound

Down-sloping

DFNM2a

Congenital

Severe

ND

DFNX5

Xq23q27.3

Y-linked (DFNY) DFNY1b Y

8p23

[176] [176] [177] [178] Incomplete penetrance and [69] variable expressivity in females [179] Auditory neuropathy and progressive peripheral neuropathy [180,181]

Contains a modifier gene suppressing the development of DFNB26 deafness Contains a modifier gene for m.1555A > G and HL with and without aminoglycoside exposure

[72]

[73,182]

Abbreviation: ND, not described. a Loci identified before 2003. b DFNY1 was determined to be a complex chromosomal rearrangement with a ~160 kb insertion from chromosome 1 that maps to a DFNA49 locus [181]. c DFNM1 is a dominant modifier of DFNB26 and suppresses deafness in those with homozygous DFNB26 haplotypes [72].

As new NSHL loci were mapped, they were sequentially numbered by order of discovery and grouped according to inheritance; a process still continued today. Loci with dominant transmission are represented by the abbreviation DFNA, followed by a HUGO Gene Nomenclature Committee accession. Likewise, loci with recessive inheritance are abbreviated DFNB, X-linked inheritance is represented by DFNX, modifier loci altering expression of other HL genes are represented by DFNM, and even one Y-linked locus is called DFNY1 [9]. To date, 141 loci have been identified and published in peer-reviewed journals. Occasionally, loci merge when multiple independently linked loci overlap and it is discovered that the same underlying gene causes HL. Furthermore, it has been established that the same gene can be responsible for both dominant and recessive forms of HL depending on the mutation. Studies have been particularly successful when investigating the fundamental cause of NSHL in families from regions of the world with high consanguineous marriage rates and have uncovered a significant number of recessive deafness genes [28]. As the search for NSHL genes is quite young, the population distribution for many of the newly identified HL genes is unclear. Nonetheless, it is well known that the molecular epidemiology of deafness can vary considerably among populations. A common example is the gene GJB2 (MIM: *121011), the most prominent deafness gene, and for many years, one of the few genes routinely screened in diagnostic testing. In hearing impaired individuals with European ethnicity, GJB2 mutations account for between 28 and 63% of HL [29]. However, in the Saudi Arabian, African American, Caribbean Hispanic, Pakistani and Moroccan populations, mutations in GJB2 contribute very little to the underlying genetic cause of NSHL [30e33]. It is thought that up to 1% of human genes are necessary for hearing [34], making the high frequency of GJB2 mutations disproportionately high in the European population. These findings highlight that it should not be expected that necessarily all populations carry the same mutational load in NSHL genes. While there has been much success using these classical strategies, a profound metamorphosis in the field of genetics has allowed for a breakthrough in candidate gene research and has increased the pace of gene discovery from a low-throughput brisk

walk to a high-throughput full sprint, with the core of this change fueled by NGS. 3. A paradigm shift: next generation sequencing Fifty years after the double-helix structure of DNA was elucidated, the completion of the Human Genome Project marked the beginning of the 21st century and delivered a human reference genome that would play a fundamental importance in the shift from single amplicon Sanger sequencing to high-throughput sequencing of millions or even billions of base pairs en masse. A few years later in 2005, the 454 GS FLX (Roche) was launched, followed by a continuous trickle of alternative new sequencing platforms shortly thereafter [35]. While there were differences in the working chemistries, platform designs, and data output capabilities, the research and development behind these NGS platforms all shared one prevailing goal: to develop an accurate and powerful technology to reduce cost and manpower for a high-throughput sequencing system [35]. The quick development of these technologies has had a clear consequence on the speed and proficiency of detecting variants for all Mendelian disorders, including NSHL (Table 1) and come in a variety of different formats ranging from small targeted gene panels to sequencing all coding exons as in whole exome sequencing (WES) or the complete genomic DNA sequence as with whole genome sequencing (WGS). With the realization of these goals came the revolutionized approaches for new gene identification. Linked loci, occasionally containing hundreds of genes for screening, could now undergo parallel sequencing of all linked genes through either custom targeted capture and MPS of the single locus or WES to reduce the need for designing customized panels. Additionally, small families where theoretical linkage significance was not attainable were often excluded from conventional approaches and consequently never analyzed. Many of these challenges, namely single gene screening in small families without opportunity for linkage analysis, have been alleviated through the use of NGS powered methods such as WES. Utilizing these approaches have proven successful in the identification of 21 NSHL genes (between 2010 and early 2015)

Please cite this article in press as: Vona B, et al., Non-syndromic hearing loss gene identification: A brief history and glimpse into the future, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.03.008

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(Table 1) since the first publications began describing targeted enrichment and MPS of a linked locus leading to the description of TPRN (MIM: *613354) [36] and WES resulting in the recognition of GPSM2 (MIM: *609245) [37] in 2010. While this technology was initially intended for research purposes, it quickly branched out as an indispensable tool for targeted enrichment panel diagnostics. As is especially the case with NSHL, there are hundreds of genes implicated in phenotypes and it is very difficult correlating pre-diagnostic hypotheses through clinical history and audiogram data [2]. For example, the age of onset and audiogram profiles appear indistinguishable in many forms of autosomal dominant and recessive NSHL despite different underlying genetic etiologies [2,3]. Screening hundreds of clinically relevant genes in a single experiment has been a cost and time effective strategy to approach diagnoses in genetically heterogeneous NSHL. There have already been a number of studies reporting success from employing small gene panels [38e45] and WES [46e48]. Furthermore, these tools enable diagnostic competence to deviate away from typical exclusionary testing of only a limited number of genes to comprehensive diagnostic testing including syndromic genes with significant clinical overlap during onset that mimic NSHL but later progresses to multiple organ system involvement [49]. These distinctions are of critical importance to discriminate, since the genetic counselling session for NSHL is very different from that of a syndrome [50]. A recent and comprehensive review on managing diagnostic testing is presented in a recent publication [49]. 4. Hiding in plain sight: linked loci's second chance Linkage and mapping are highly effective strategies for delimiting loci for positional cloning gene prioritization; however, as it currently stands, these approaches do not have a perfect track record of identifying new NSHL genes. Throughout the relatively short history of NSHL gene identification, there are currently 49 loci (19 DFNA, 25 DFNB, 2 DFNX, 1 DFNY, and 2 DFNM loci) published in peer-reviewed journals without a causative gene assigned (Table 2). This currently comprises 35% of total linked loci [9], which means it is quite likely that there are, at the very least, 49 new NSHL genes awaiting discovery. In many cases, these loci have been linked by analyzing more than one multigenerational pedigree showing significance to one chromosomal region [51e56] and included pedigrees with extensive numbers of family members having undergone analysis, occasionally exceeding 30 to 40 [57e61]. Owing to the large size of many of these intervals, one-by-one screening of genes with known expression in the ear is a costly and time consuming endeavour, and the risk for missing a causative gene or mutation is high [62]. Furthermore, nearly one-third of these reported loci (Table 2) have been mapped before the completion of the Human Genome Project in 2003. Given that the early versions of linkage maps were known to contain occasional errors since a complete understanding of the reference genome was still beyond full comprehension [26], it would be worth re-mapping many of these families to confirm or re-establish linkage, or to even refine the linkage interval for subsequent follow-up including either targeted enrichment MPS or WES, as these families hold great promise for novel gene identification. The advent of targeted capture and MPS technologies present a unique opportunity for re-visiting these loci. 5. The question of missing heritability Albert Einstein's comment on Occam's razor, “Everything should be made as simple as possible, but not simpler,” presents an

interesting paradox applicable to “simple” Mendelian disorders [63]. Genotype-phenotype correlation inconsistencies have long been described in single-gene disorders, complicating management and therapy [64]. Similarly, intra-familial variability in NSHL is common not only from parent to child in dominant cases, but also between sibs. Some of these inconsistencies are thought to be due to environmental or secondary genetic factors [65]. As early as 1999, Grifa and colleagues [66] described the differing expressivity accounting for variability of HL among autosomal dominant hearing impaired individuals as “almost the rule for dominant deafness.” However, these hypothetical differences in expressivity [67,68], incomplete or reduced penetrance [68e71] have yet to be accounted for on the molecular level. Two modifier loci have been identified on chromosomes 1 (DFNM1) and 8 (DFNM2) [72,73]. DFNM1 was identified through linkage analysis of a 141 member highly consanguineous Pakistani family that simultaneously localized DFNB26 [72]. DFNM1 is described as a dominant modifier and suppressor of DFNB26, accounting for the high degree of reported intra-familial HL variability, as well as protecting the seven normal hearing individuals with homozygous DFNB26 haplotypes [72]. Furthermore, linkage analysis of 11 nuclear families of ArabeIsraeli, Spanish, and Italian descent with co-segregating matrilineal NSHL disclosed the DFNM2 locus that is described as modifying the mitochondrial gene MTRNR1 (MIM: *561000) m.1555G > A mutation [73]. The variable penetrance of this mitochondrial mutation was concluded to require either environmental exposures such as aminoglycoside antibiotics or other additive effects of several nuclear genes that are presently unclear in order to cause a greater severity of HL [73]. The hearing impaired individuals in these families confirmed no previous aminoglycoside exposure. The precise genetic determinant and mechanism for these two modifier loci remains to be elucidated; however, these data suggest a “complex” dimension to a normally regarded “simple” disorder. Mutational effect in more than one gene is an understudied aspect of many Mendelian disorders. Digenic inheritance has already been associated with mutations in GJB2 and GJB3 (MIM: þ603324) for NSHL [74], and KCNJ10 (MIM: *602208) and SLC26A4 (MIM: *605646) with HL and enlarged vestibular aqueduct [75]. Moreover, GJB6 (MIM: *604418) is a cis-regulatory element of GJB2 expression that can cause NSHL when a GJB2 mutation exists in trans [76]. These digenic configurations explain only a small proportion of cases. Different layers of genetic complexity have long been thought to account for a large part of missing heritability. Considering the more than 3 million variants present in the average human genome [77,78], only a small fraction of common variants are expected to be functional [79] and it is unclear as to how they contribute to the great variability in human phenotypes [80]. Likewise, it is thought that a significant proportion of missing heritability is neither due to common, nor rare variants, but rather rare combinations of common variants [81]. Several studies have supported that rare mildly deleterious missense mutations are likely to be damaging and may have an impact on health [80,82]. Additionally, there is strong evidence that the impact of rare variants and structural variation such as deletions, duplications and inversions, although individually rare, are collectively common in the human population [83,84]. In fact, it has been determined that approximately five to ten percent of the healthy population carries a large deletion or duplication >500 kb with a frequency of <0.05% [84]. Although the presence and frequency of such changes create extremely complicated interpretation scenarios in a clinical setting, the conclusion that such events have insignificant impact on genotypeephenotype correlations would be incomplete [81]. Furthermore, polymorphic duplications in regions recognized as undergoing rapid

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evolutionary and/or adaptive bursts have been identified [85]; however, as these actively evolving regions are characterized by a repetitive and multicopy nature, they are generally inaccessible by sequencing technologies [81]. Whether mechanisms for genetic variation reside in these largely unexplored regions remains unknown, but it seems quite logical. For example, despite significant evidence that epigenetic mechanisms are heavily implicated in human disease [86], hearing-related epigenetic research has been neglected to a great extent. Limited reports on epigenetic modifications have been presented in the context of age-related and syndromic HL [87,88]. Additionally, only one NSHL gene, MIR96 (MIM: 611606) (DFNA50), encoding a microRNA, has been reported to date and described as important for maintaining posttranscriptional regulation of gene expression in the hair cells and adult cochlea [89]. Through advances in sequencing technology, it is becoming increasingly routine to identify mutations in cis-regulatory elements; however, assessing their impact on gene regulation remains challenging [90]. Two additional NSHL genes highlight the importance of regulation. The gene POU3F4 contains a hot spot for proximal deletions ranging from 8 kb to 1.75 Mb that removes a transcriptional regulation element [91,92]. Secondly, the gene HGF (MIM: *142409) (DFNB39) has shown two mutations in the 30 untranslated region of a short isoform (c.482 þ 1986_1988delTGA and c.482 þ 1991_2000delGATGATGAA) that causes gene dysregulation [93]. Coordinated gene expression mechanisms are central to normal functioning biological processes and are influenced by a variety of environmental and genetic factors. As epigenetic control is central to the appropriately coordinated gene expression and gene silencing in normal biological processes, why would it be expected that hearing be markedly simpler? It is, therefore, premature to conclude that variation in sequence content and structural arrangement in these “sequestered” regions have either minimal or no impact on human health until they are fully analyzed [94]. These areas demonstrate that in spite of the remarkable progress identifying NSHL genes in recent years, there are still understudied avenues for improving the mechanistic understanding of normal hearing and the structural, mutational, and epigenetic changes underscoring NSHL. If causative mutations are not detected in many of the 49 NSHL loci with unassigned cause of deafness through conventional methods that now include MPS, it would be of high interest to explore these alternative explanations for unaccounted “phantom heritability,” as target enrichment MPS technology alone may not be able to resolve all cases [81]. 6. Look how far we have come. Look how far we have to go The past twenty years after the first identified NSHL gene have produced a fruitful understanding and greater appreciation of the diverse array of genes and specialized proteins required for normal hearing (Fig. 1). The gradual discovery of novel NSHL genes meant that for the first time, genetic diagnostic testing was possible. However, the diverse array of unknown NSHL genes marginalized these initial diagnostic successes. The compounded cost and labour intensive low-throughput capabilities together with genetic and clinical heterogeneity meant that clinicians had to rely on audiological, vestibular, and radiological cues that were usually inconclusive, but were the best way to guide genetic testing [2]. As initial reports using NGS technology for NSHL research and diagnostic testing yielded exceptional precision and output, the quick transition from “bench to bedside” revolutionized clinical genetic approaches from low-throughput single gene strategies to the testing of all validated NSHL genes. The genetic testing of HL has transitioned to a high priority test for patient management and is now recommended immediately following physical examination, audiometry,

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and family history [50]. The availability of NGS based tests has allowed comprehensive genetic testing to become an indispensable part of the clinical work up. Although there are still many NSHL genes remaining to be discovered, as reflected by diagnostic yields well under 100%, these strategies are now being implemented into the clinical setting at a never before seen pace [95]. It has been thought that approximately 85% of Mendelian disease-causing mutations occur in coding, or exoneintron canonical splice junction regions, making a targeted enrichment and MPS approach highly efficient [96]. Furthermore, since 2008, the estimated costs of NGS-based sequencing have been outpacing Moore's law [97], and the $1000 genome has become a reality [98]. Its increased affordability supports widespread application usage in clinical and research settings. Along with the seemingly unlimited possibilities that are now at our finger-tips arise the complexities and uncertainties that emerge when having gigabytes of data to sift through. The limitations of WES approaches are well described, and despite many clear benefits, some consistent drawbacks remain [99]. Firstly, the definition of an exome is constantly evolving as our knowledge of all the truly protein-coding exons is defined [99]. The target capture design only targets exons that are annotated, and not all genes or gene isoforms have been determined. This means that despite analyzing the maximum number of informative family members in a wellcharacterized pedigree, there is always a chance that region of interest harbouring mutation(s), is not included in the whole exome capture design. Secondly, exon-dropout of GC rich exons is a wellknown occurrence and, therefore, despite proper capture design, certain regions are simply inaccessible using target capture technologies. In spite of some of the described limitations, diagnostic success rates ranging between 23 and 50% have been reported for WES study cohorts of Mendelian disorders [100e102]. Looking to the not so distant future, WGS will become more economical than WES because the target capture step is entirely omitted [10] producing a genuine exome output, as well as including conserved non-coding regions. However, data storage problems that are already encountered with WES datasets will become exacerbated, as the WGS output is around 100 times greater than the already staggering amount of data produced by WES [10]. Regardless of these challenges, it is of critical importance to study the non-coding regions of the genome to increase understanding of these largely ignored areas [99]. The elegantly penned phrase, “The $1000 genome, the $100,000 analysis,” accentuates the reality much of the scientific community is currently facing: when it comes to genomics, sequencing is by far the cheapest and simplest part [103]. The full interpretation of data constitutes a major expense and demands expertise from a variety of different backgrounds including geneticists, statisticians, bioinformaticians, biologists, and physicians to translate the impact of a variant into a clinically relevant context [97]. It is clear that the biggest area for improvement resides in the computational infrastructure domain and development of efficient algorithms for alignment and querying complicated statistical questions. While there is a long road ahead in these areas, major progress has already paved the way to alleviating these burdens. For example, one recently developed bioinformatics pipeline enabling computationally efficient WGS analysis that includes alignment, postalignment processing and genotyping, produces a variant list ready for clinical interpretation in less than 2 h [104]. Another area undergoing rapid development is the CNV calling from NGS data. CNV calling in WES is heavily complicated by experimental noise, as well as biases and artifacts introduced during target capture and amplification and thus WES depth and coverage may not always accurately reflect true copy number [105]. There are a growing collection of recently developed algorithms that aim to resolve CNV

Please cite this article in press as: Vona B, et al., Non-syndromic hearing loss gene identification: A brief history and glimpse into the future, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.03.008

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issues to reduce false positive and false negative results [105e107]. CNVs are an especially important contributor to NSHL and it has been determined from one study that approximately 15% of NSHL patients carry at least one CNV within a known deafness gene [108]. A chronic problem plaguing all NGS-based datasets is the clear need for improved interpretation of pathogenic variants. Increased availability of MPS is resulting in an increased number of variants with unknown clinical significance or unclassified variants (UVs) that require interpretation. There is great difficulty in assigning a strong heterozygous, in silico predicted pathogenic heterozygous mutation, as in the case of autosomal dominant NSHL, since it is well-known that even phenotypically normal individuals carry disease causing mutations. Consider the well-studied tumor suppressor genes BRCA1 (MIM: *113705) and BRCA2 (MIM: *600185), for breast and ovarian cancer risk. While frameshift indels and nonsense mutations result in protein truncation and are thus pathogenic, it is less clear the result of missense variants. These missense UVs constitute a large majority of coding variants and pose the greatest challenge in calculating hereditary cancer risks, especially if there are insufficient epidemiological data for pathogenic or benign classification [109]. With regard to NSHL, one such epidemiological study recategorized pathological variants using a 66 NSHL gene targeted enrichment and MPS strategy [110]. Shearer and colleagues sequenced nearly 8600 subjectively normal hearing individuals from twelve populations to calculate MAF thresholds for the included dominant and recessive NSHL genes. These data were compared against pathogenic variants from Human Gene Mutation Database (HGMD), ClinVar and dbSNP to evaluate the variants reported in these control individuals against those classified as pathogenic. The resulting conclusion was able to reassign 93 (4.2%) previously reported pathogenic variants as benign [110]. Previous work specifically attempting to understand the variability in the human genome has only begun shedding light on this problem. In a general study unspecific to HL, an extensive variant cross-comparison of WGS data between presumably healthy control individuals in the 1000 Genomes Project (TGP) database and pathogenic variants reported in symptomatic individuals in HGMD, uncovered that many frequently reported pathogenic variants were occurring with minor allele frequencies (MAF)  0.05 and 0.01, for a total of 3.5% and 4.6% of HGMD variants, respectively [111]. Furthermore, this study concluded that only 10.6% of variants genome-wide met the strictest criteria and scientific credibility for reporting in a clinical setting [112]. The primary benefit of universal screening of all known genes is that they are now put to the ultimate test of co-segregation in independently identified families. This recently was the case with the disqualification of MYO1A (MIM: *601478) (DFNA48), wherein three families with different predicted pathogenic MYO1A mutations demonstrated co-segregation incompatible with pathogenicity [113]. Furthermore, two additional genes, namely GJB3 and SLC26A5 (MIM: *604943) are recently raised as questionable for causing fully penetrant NSHL, but require further validation [110]. These distinctions are important to identify, since the gene content of focused NSHL panels heavily relies on qualified genes for content. Such studies evaluating variant pathogenicity are of critical importance to support or discredit reported damaging mutations. It is clear that the data analysis bottleneck carries many uncertainties and that much work is required until a more comprehensive understanding of these variants become apparent. It is safe to conclude that the wide availability and decreasing cost of NGS has changed the way research is conducted. Improved habilitation, calculation of recurrence, and prognostic information are all critical components that are the direct result of determining a genetic diagnosis of NSHL [38]. Establishing genetic causes for NSHL

are especially important in prelingual children to establish the best medical course of action. Now, more than ever, there is great sense of urgency to continue research in this field with tools and resources our scientific ancestors would have never dreamed possible.

References [1] Smith RJ, Bale Jr JF, White KR. Sensorineural hearing loss in children. Lancet 2005;365:879e90. [2] Bitner-Glindzicz M. Hereditary deafness and phenotyping in humans. Br Med Bull 2002;63:73e94. [3] Petersen MB. Non-syndromic autosomal-dominant deafness. Clin Genet 2002;62:1e13. [4] Morton CC, Nance WE. Newborn hearing screeningea silent revolution. N Engl J Med 2006;354:2151e64. [5] Van Eyken E, Van Camp G, Van Laer L. The complexity of age-related hearing impairment: contributing environmental and genetic factors. Audiol Neurootol 2007;12:345e58. [6] Gates GA, Couropmitree NN, Myers RH. Genetic associations in age-related hearing thresholds. Arch Otolaryngol Head Neck Surg 1999;125:654e9. [7] Karlsson KK, Harris JR, Svartengren M. Description and primary results from an audiometric study of male twins. Ear Hear 1997;18:114e20. [8] Bowl MR, Dawson SJ. The mouse as a model for age-related hearing loss - a mini-review. Gerontology 2014. Q1 [9] Van Camp G, Smith RHJ. Hearing loss homepage. [10] Majewski J, Schwartzentruber J, Lalonde E, Montpetit A, Jabado N. What can exome sequencing do for you? J Med Genet 2011;48:580e9. [11] Botstein D, Risch N. Discovering genotypes underlying human phenotypes: past successes for mendelian disease, future approaches for complex disease. Nat Genet 2003;(33 Suppl):228e37. [12] Friedman TB, Liang Y, Weber JL, Hinnant JT, Barber TD, Winata S, et al. A gene for congenital, recessive deafness DFNB3 maps to the pericentromeric region of chromosome 17. Nat Genet 1995;9:86e91. [13] Schraders M, Ruiz-Palmero L, Kalay E, Oostrik J, del Castillo FJ, Sezgin O, et al. Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment. Am J Hum Genet 2012;91: 883e9. [14] Wang A, Liang Y, Fridell RA, Probst FJ, Wilcox ER, Touchman JW, et al. Association of unconventional myosin MYO15 mutations with human nonsyndromic deafness DFNB3. Science 1998;280:1447e51. [15] Mburu P, Mustapha M, Varela A, Weil D, El-Amraoui A, Holme RH, et al. Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31. Nat Genet 2003;34:421e8. [16] Zwaenepoel I, Mustapha M, Leibovici M, Verpy E, Goodyear R, Liu XZ, et al. Otoancorin, an inner ear protein restricted to the interface between the apical surface of sensory epithelia and their overlying acellular gels, is defective in autosomal recessive deafness DFNB22. Proc Natl Acad Sci U. S. A 2002;99:6240e5. [17] Robertson NG, Lu L, Heller S, Merchant SN, Eavey RD, McKenna M, et al. Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction. Nat Genet 1998;20:299e303. [18] von Ameln S, Wang G, Boulouiz R, Rutherford MA, Smith GM, Li Y, et al. A mutation in PNPT1, encoding mitochondrial-RNA-import protein PNPase, causes hereditary hearing loss. Am J Hum Genet 2012;91:919e27. [19] Liu X, Han D, Li J, Han B, Ouyang X, Cheng J, et al. Loss-of-function mutations in the PRPS1 gene cause a type of nonsyndromic X-linked sensorineural deafness, DFN2. Am J Hum Genet 2010;86:65e71. [20] Naz S, Griffith AJ, Riazuddin S, Hampton LL, Battey Jr JF, Khan SN, et al. Mutations of ESPN cause autosomal recessive deafness and vestibular dysfunction. J Med Genet 2004;41:591e5. [21] Kurima K, Peters LM, Yang Y, Riazuddin S, Ahmed ZM, Naz S, et al. Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function. Nat Genet 2002;30:277e84. [22] Wallis C, Ballo R, Wallis G, Beighton P, Goldblatt J. X-linked mixed deafness with stapes fixation in a mauritian kindred: linkage to Xq probe pDP34. Genomics 1988;3:299e301. [23] de Kok YJ, van der Maarel SM, Bitner-Glindzicz M, Huber I, Monaco AP, Malcolm S, et al. Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4. Science 1995;267:685e8. [24] Leon PE, Raventos H, Lynch E, Morrow J, King MC. The gene for an inherited form of deafness maps to chromosome 5q31. Proc Natl Acad Sci U S A 1992;89:5181e4. [25] Lynch ED, Lee MK, Morrow JE, Welcsh PL, Leon PE, King MC. Nonsyndromic deafness DFNA1 associated with mutation of a human homolog of the Drosophila gene diaphanous. Science 1997;278:1315e8. [26] Snoeckx RL, Kremer H, Ensink RJ, Flothmann K, de Brouwer A, Smith RJ, et al. A novel locus for autosomal dominant non-syndromic hearing loss, DFNA31, maps to chromosome 6p21.3. J Med Genet 2004;41:11e3. [27] He C, Weeks DE, Buyske S, Abecasis GR, Stewart WC, Matise TC. Enhanced genetic maps from family-based disease studies: population-specific comparisons. BMC Med Genet 2011;12:15.

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B. Vona et al. / Molecular and Cellular Probes xxx (2015) 1e11 [28] Avraham KB, Kanaan M. Genomic advances for gene discovery in hereditary hearing loss. J Basic Clin Physiol Pharmacol 2012;23:93e7. [29] Mahdieh N, Rabbani B. Statistical study of 35delG mutation of GJB2 gene: a meta-analysis of carrier frequency. Int J Audiol 2009;48:363e70. [30] Imtiaz F, Taibah K, Ramzan K, Bin-Khamis G, Kennedy S, Al-Mubarak B, et al. A comprehensive introduction to the genetic basis of non-syndromic hearing loss in the Saudi Arabian population. BMC Med Genet 2011;12:91. [31] Samanich J, Lowes C, Burk R, Shanske S, Lu J, Shanske A, et al. Mutations in GJB2, GJB6, and mitochondrial DNA are rare in African American and Caribbean Hispanic individuals with hearing impairment. Am J Med Genet A 2007;143A:830e8. [32] Santos RL, Wajid M, Pham TL, Hussan J, Ali G, Ahmad W, et al. Low prevalence of Connexin 26 (GJB2) variants in Pakistani families with autosomal recessive non-syndromic hearing impairment. Clin Genet 2005;67:61e8. [33] Nahili H, Ridal M, Boulouiz R, Abidi O, Imken L, Rouba H, et al. Absence of GJB3 and GJB6 mutations in moroccan familial and sporadic patients with autosomal recessive non-syndromic deafness. Int J Pediatr Otorhinolaryngol 2008;72:1633e6. [34] Friedman TB, Griffith AJ. Human nonsyndromic sensorineural deafness. Annu Rev Genomics Hum Genet 2003;4:341e402. [35] Liu L, Li Y, Li S, Hu N, He Y, Pong R, et al. Comparison of next-generation sequencing systems. J Biomed Biotechnol 2012;2012:251364. [36] Rehman AU, Morell RJ, Belyantseva IA, Khan SY, Boger ET, Shahzad M, et al. Targeted capture and next-generation sequencing identifies C9orf75, encoding taperin, as the mutated gene in nonsyndromic deafness DFNB79. Am J Hum Genet 2010;86:378e88. [37] Walsh T, Shahin H, Elkan-Miller T, Lee MK, Thornton AM, Roeb W, et al. Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet 2010;87:90e4. [38] Shearer AE, DeLuca AP, Hildebrand MS, Taylor KR, Gurrola 2nd J, Scherer S, et al. Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing. Proc Natl Acad Sci U S A 2010;107:21104e9. [39] Baek JI, Oh SK, Kim DB, Choi SY, Kim UK, Lee KY, et al. Targeted massive parallel sequencing: the effective detection of novel causative mutations associated with hearing loss in small families. Orphanet J Rare Dis 2012;7:60. [40] Krawitz PM, Schiska D, Kruger U, Appelt S, Heinrich V, Parkhomchuk D, et al. Screening for single nucleotide variants, small indels and exon deletions with a next-generation sequencing based gene panel approach for Usher syndrome. Mol Genet Genomic Med 2014;2:393e401. [41] Miyagawa M, Naito T, Nishio SY, Kamatani N, Usami S. Targeted exon sequencing successfully discovers rare causative genes and clarifies the molecular epidemiology of Japanese deafness patients. PLoS One 2013;8: e71381. €der J, et al. Tar[42] Vona B, Müller T, Nanda I, Neuner C, Hofrichter MA, Schro geted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations. Genet Med 2014;16:945e53. [43] Brownstein Z, Abu-Rayyan A, Karfunkel-Doron D, Sirigu S, Davidov B, Shohat M, et al. Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing. Eur J Hum Genet 2014;22:768e75. [44] Parzefall T, Shivatzki S, Lenz DR, Rathkolb B, Ushakov K, Karfunkel D, et al. Cytoplasmic mislocalization of POU3F4 due to novel mutations leads to deafness in humans and mice. Hum Mutat 2013;34:1102e10. [45] Brownstein Z, Friedman LM, Shahin H, Oron-Karni V, Kol N, Abu Rayyan A, et al. Targeted genomic capture and massively parallel sequencing to identify genes for hereditary hearing loss in middle Eastern families. Genome Biol 2011;12:R89. [46] Woo HM, Park HJ, Park MH, Kim BY, Shin JW, Yoo WG, et al. Identification of CDH23 mutations in Korean families with hearing loss by whole-exome sequencing. BMC Med Genet 2014;15:46. [47] Gao X, Zhu QY, Song YS, Wang GJ, Yuan YY, Xin F, et al. Novel compound heterozygous mutations in the MYO15A gene in autosomal recessive hearing loss identified by whole-exome sequencing. J Transl Med 2013;11:284. [48] Sirmaci A, Edwards YJ, Akay H, Tekin M. Challenges in whole exome sequencing: an example from hereditary deafness. PLoS One 2012;7:e32000. [49] Hoefsloot LH, Feenstra I, Kunst HP, Kremer H. Genotype phenotype correlations for hearing impairment: approaches to management. Clin Genet 2014;85:514e23. [50] Shearer AE, Smith RJ. Genetics: advances in genetic testing for deafness. Curr Opin Pediatr 2012;24:679e86. [51] Greinwald Jr JH, Wayne S, Chen AH, Scott DA, Zbar RI, Kraft ML, et al. Localization of a novel gene for nonsyndromic hearing loss (DFNB17) to chromosome region 7q31. Am J Med Genet 1998;78:107e13. [52] Hassan MJ, Santos RL, Rafiq MA, Chahrour MH, Pham TL, Wajid M, et al. A novel autosomal recessive non-syndromic hearing impairment locus (DFNB47) maps to chromosome 2p25.1-p24.3. Hum Genet 2006;118: 605e10. [53] Shaikh RS, Ramzan K, Nazli S, Sattar S, Khan SN, Riazuddin S, et al. A new locus for nonsyndromic deafness DFNB51 maps to chromosome 11p13-p12. Am J Med Genet A 2005;138:392e5. [54] Santos RL, Hassan MJ, Sikandar S, Lee K, Ali G, Martin Jr PE, et al. DFNB68, a novel autosomal recessive non-syndromic hearing impairment locus at chromosomal region 19p13.2. Hum Genet 2006;120:85e92.

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[55] Ain Q, Nazli S, Riazuddin S, Jaleel AU, Riazuddin SA, Zafar AU, et al. The autosomal recessive nonsyndromic deafness locus DFNB72 is located on chromosome 19p13.3. Hum Genet 2007;122:445e50. [56] Rehman AU, Gul K, Morell RJ, Lee K, Ahmed ZM, Riazuddin S, et al. Mutations of GIPC3 cause nonsyndromic hearing loss DFNB72 but not DFNB81 that also maps to chromosome 19p. Hum Genet 2011;130:759e65. [57] Fagerheim T, Nilssen O, Raeymaekers P, Brox V, Moum T, Elverland HH, et al. Identification of a new locus for autosomal dominant non-syndromic hearing impairment (DFNA7) in a large Norwegian family. Hum Mol Genet 1996;5: 1187e91. [58] H€ afner FM, Salam AA, Linder TE, Balmer D, Baumer A, Schinzel AA, et al. A novel locus (DFNA24) for prelingual nonprogressive autosomal dominant nonsyndromic hearing loss maps to 4q35-qter in a large Swiss German kindred. Am J Hum Genet 2000;66:1437e42. [59] Xia J, Deng H, Feng Y, Zhang H, Pan Q, Dai H, et al. A novel locus for autosomal dominant nonsyndromic hearing loss identified at 5q31.1-32 in a Chinese pedigree. J Hum Genet 2002;47:635e40. [60] Lezirovitz K, Braga MC, Thiele-Aguiar RS, Auricchio MT, Pearson PL, Otto PA, et al. A novel autosomal dominant deafness locus (DFNA58) maps to 2p12p21. Clin Genet 2009;75:490e3. [61] Gürtler N, Kim Y, Mhatre A, Schlegel C, Mathis A, Lalwani AK. DFNA54, a third locus for low-frequency hearing loss. J Mol Med Berl 2004;82:775e80. [62] Elkan-Miller T, Avraham KB. Deafness gene discovery in the genomic era. 1 ed. Nova Scientific Publishers, Inc; 2013. [63] Sobie EA, Guatimosim S, Song LS, Lederer WJ. The challenge of molecular medicine: complexity versus Occam's razor. J Clin Invest 2003;111:801e3. [64] Dipple KM, McCabe ER. Modifier genes convert “simple” mendelian disorders to complex traits. Mol Genet Metab 2000;71:43e50. [65] Kim TB, Isaacson B, Sivakumaran TA, Starr A, Keats BJ, Lesperance MM. A gene responsible for autosomal dominant auditory neuropathy (AUNA1) maps to 13q14-21. J Med Genet 2004;41:872e6. [66] Grifa A, Wagner CA, D'Ambrosio L, Melchionda S, Bernardi F, Lopez-Bigas N, et al. Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus. Nat Genet 1999;23:16e8. [67] Yan D, Ke X, Blanton SH, Ouyang XM, Pandya A, Du LL, et al. A novel locus for autosomal dominant non-syndromic deafness, DFNA53, maps to chromosome 14q11.2-q12. J Med Genet 2006;43:170e4. [68] del Castillo I, Villamar M, Sarduy M, Romero L, Herraiz C, Hernandez FJ, et al. A novel locus for non-syndromic sensorineural deafness (DFN6) maps to chromosome Xp22. Hum Mol Genet 1996;5:1383e7. [69] Lalwani AK, Brister JR, Fex J, Grundfast KM, Pikus AT, Ploplis B, et al. A new nonsyndromic X-linked sensorineural hearing impairment linked to Xp21.2. Am J Hum Genet 1994;55:685e94. [70] Greene CC, McMillan PM, Barker SE, Kurnool P, Lomax MI, Burmeister M, et al. DFNA25, a novel locus for dominant nonsyndromic hereditary hearing impairment, maps to 12q21-24. Am J Hum Genet 2001;68:254e60. [71] Mangino M, Flex E, Capon F, Sangiuolo F, Carraro E, Gualandi F, et al. Mapping of a new autosomal dominant nonsyndromic hearing loss locus (DFNA30) to chromosome 15q25-26. Eur J Hum Genet 2001;9:667e71. [72] Riazuddin S, Castelein CM, Ahmed ZM, Lalwani AK, Mastroianni MA, Naz S, et al. Dominant modifier DFNM1 suppresses recessive deafness DFNB26. Nat Genet 2000;26:431e4. [73] Bykhovskaya Y, Estivill X, Taylor K, Hang T, Hamon M, Casano RA, et al. Candidate locus for a nuclear modifier gene for maternally inherited deafness. Am J Hum Genet 2000;66:1905e10. [74] Liu XZ, Yuan Y, Yan D, Ding EH, Ouyang XM, Fei Y, et al. Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31. Hum Genet 2009;125:53e62. [75] Yang T, Gurrola 2nd JG, Wu H, Chiu SM, Wangemann P, Snyder PM, et al. Mutations of KCNJ10 together with mutations of SLC26A4 cause digenic nonsyndromic hearing loss associated with enlarged vestibular aqueduct syndrome. Am J Hum Genet 2009;84:651e7. [76] Rodriguez-Paris J, Schrijver I. The digenic hypothesis unraveled: the GJB6 del(GJB6-D13S1830) mutation causes allele-specific loss of GJB2 expression in cis. Biochem Biophys Res Commun 2009;389:354e9. [77] Kim JI, Ju YS, Park H, Kim S, Lee S, Yi JH, et al. A highly annotated wholegenome sequence of a Korean individual. Nature 2009;460:1011e5. [78] Levy S, Sutton G, Ng PC, Feuk L, Halpern AL, Walenz BP, et al. The diploid genome sequence of an individual human. PLoS Biol 2007;5:e254. [79] Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 2009;361:1058e66. [80] Boyko AR, Williamson SH, Indap AR, Degenhardt JD, Hernandez RD, Lohmueller KE, et al. Assessing the evolutionary impact of amino acid mutations in the human genome. PLoS Genet 2008;4:e1000083. [81] Eichler EE, Flint J, Gibson G, Kong A, Leal SM, Moore JH, et al. Missing heritability and strategies for finding the underlying causes of complex disease. Nat Rev Genet 2010;11:446e50. [82] Kryukov GV, Pennacchio LA, Sunyaev SR. Most rare missense alleles are deleterious in humans: implications for complex disease and association studies. Am J Hum Genet 2007;80:727e39. [83] Sebat J, Lakshmi B, Troge J, Alexander J, Young J, Lundin P, et al. Large-scale copy number polymorphism in the human genome. Science 2004;305: 525e8.

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[84] Itsara A, Cooper GM, Baker C, Girirajan S, Li J, Absher D, et al. Population analysis of large copy number variants and hotspots of human genetic disease. Am J Hum Genet 2009;84:148e61. [85] Alkan C, Kidd JM, Marques-Bonet T, Aksay G, Antonacci F, Hormozdiari F, et al. Personalized copy number and segmental duplication maps using nextgeneration sequencing. Nat Genet 2009;41:1061e7. [86] Brookes E, Shi Y. Diverse epigenetic mechanisms of human disease. Annu Rev Genet 2014;48:237e68. [87] Wolber LE, Steves CJ, Tsai PC, Deloukas P, Spector TD, Bell JT, et al. Epigenome-wide DNA methylation in hearing ability: new mechanisms for an old problem. PLoS One 2014;9:e105729. [88] Provenzano MJ, Domann FE. A role for epigenetics in hearing: establishment and maintenance of auditory specific gene expression patterns. Hear Res 2007;233:1e13.  Modamio-Høybjør S, Redshaw N, Morín M, Mayo-Merino F, [89] Mencía A, Olavarrieta L, et al. Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss. Nat Genet 2009;41: 609e13. [90] Mathelier A, Shi W, Wasserman WW. Identification of altered cis-regulatory elements in human disease. Trends Genet 2015;31:67e76. [91] Choi BY, An YH, Park JH, Jang JH, Chung HC, Kim AR, et al. Audiological and surgical evidence for the presence of a third window effect for the conductive hearing loss in DFNX2 deafness irrespective of types of mutations. Eur Arch Otorhinolaryngol 2013;270:3057e62. [92] de Kok YJ, Vossenaar ER, Cremers CW, Dahl N, Laporte J, Hu LJ, et al. Identification of a hot spot for microdeletions in patients with X-linked deafness type 3 (DFN3) 900 kb proximal to the DFN3 gene POU3F4. Hum Mol Genet 1996;5:1229e35. [93] Schultz JM, Khan SN, Ahmed ZM, Riazuddin S, Waryah AM, Chhatre D, et al. Noncoding mutations of HGF are associated with nonsyndromic hearing loss, DFNB39. Am J Hum Genet 2009;85:25e39. [94] Craddock N, Hurles ME, Cardin N, Pearson RD, Plagnol V, Robson S, et al. Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls. Nature 2010;464:713e20. [95] Hayden EC. Sequencing set to alter clinical landscape. Nature 2012;482:288. [96] Johansen Taber KA, Dickinson BD, Wilson M. The promise and challenges of next-generation genome sequencing for clinical care. JAMA Intern Med 2014;174:275e80. [97] Sboner A, Mu XJ, Greenbaum D, Auerbach RK, Gerstein MB. The real cost of sequencing: higher than you think! Genome Biol 2011;12:125. [98] Sheridan C. Illumina claims $1,000 genome win. Nat Biotechnol 2014;32: 115. [99] Bamshad MJ, Ng SB, Bigham AW, Tabor HK, Emond MJ, Nickerson DA, et al. Exome sequencing as a tool for mendelian disease gene discovery. Nat Rev Genet 2011;12:745e55. [100] Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, et al. Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med 2013;369:1502e11. [101] Atwal PS, Brennan ML, Cox R, Niaki M, Platt J, Homeyer M, et al. Clinical whole-exome sequencing: are we there yet? Genet Med 2014;16:717e9. [102] Need AC, Shashi V, Hitomi Y, Schoch K, Shianna KV, McDonald MT, et al. Clinical application of exome sequencing in undiagnosed genetic conditions. J Med Genet 2012;49:353e61. [103] Mardis ER. The $1,000 genome, the $100,000 analysis? Genome Med 2010;2:84. [104] Kelly BJ, Fitch JR, Hu Y, Corsmeier DJ, Zhong H, Wetzel AN, et al. Churchill: an ultra-fast, deterministic, highly scalable and balanced parallelization strategy for the discovery of human genetic variation in clinical and population-scale genomics. Genome Biol 2015;16:6. [105] Jiang Y, Oldridge DA, Diskin SJ, Zhang NR. CODEX: a normalization and copy number variation detection method for whole exome sequencing. Nucleic Acids Res 2015. [106] Miyatake S, Koshimizu E, Fujita A, Fukai R, Imagawa E, Ohba C, et al. Detecting copy-number variations in whole-exome sequencing data using the eXome Hidden markov model: an 'exome-first' approach. J Hum Genet 2015. [107] Yang JF, Ding XF, Chen L, Mat WK, Xu MZ, Chen JF, et al. Copy number variation analysis based on AluScan sequences. J Clin Bioinforma 2014;4: 15. [108] Shearer AE, Kolbe DL, Azaiez H, Sloan CM, Frees KL, Weaver AE, et al. Copy number variants are a common cause of non-syndromic hearing loss. Genome Med 2014;6:37. [109] Akbari MR, Zhang S, Fan I, Royer R, Li S, Risch H, et al. Clinical impact of unclassified variants of the BRCA1 and BRCA2 genes. J Med Genet 2011;48: 783e6. [110] Shearer AE, Eppsteiner RW, Booth KT, Ephraim SS, Gurrola 2nd J, Simpson A, et al. Utilizing ethnic-specific differences in minor allele frequency to recategorize reported pathogenic deafness variants. Am J Hum Genet 2014;95: 445e53. [111] Cassa CA, Tong MY, Jordan DM. Large numbers of genetic variants considered to be pathogenic are common in asymptomatic individuals. Hum Mutat 2013;34:1216e20. [112] Cassa CA, Savage SK, Taylor PL, Green RC, McGuire AL, Mandl KD. Disclosing pathogenic genetic variants to research participants: quantifying an emerging ethical responsibility. Genome Res 2012;22:421e8.

[113] Eisenberger T, Di Donato N, Baig SM, Neuhaus C, Beyer A, Decker E, et al. Targeted and genomewide NGS data disqualify mutations in MYO1A, the “DFNA48 gene”, as a cause of deafness. Hum Mutat 2014;35:565e70. [114] Chen AH, Ni L, Fukushima K, Marietta J, O'Neill M, Coucke P, et al. Linkage of a gene for dominant non-syndromic deafness to chromosome 19. Hum Mol Genet 1995;4:1073e6. [115] Zheng J, Miller KK, Yang T, Hildebrand MS, Shearer AE, DeLuca AP, et al. Carcinoembryonic antigen-related cell adhesion molecule 16 interacts with alpha-tectorin and is mutated in autosomal dominant hearing loss (DFNA4). Proc Natl Acad Sci U S A 2011;108:4218e23. [116] Blanton SH, Liang CY, Cai MW, Pandya A, Du LL, Landa B, et al. A novel locus for autosomal dominant non-syndromic deafness (DFNA41) maps to chromosome 12q24-qter. J Med Genet 2002;39:567e70. [117] Yan D, Zhu Y, Walsh T, Xie D, Yuan H, Sirmaci A, et al. Mutation of the ATPgated P2X(2) receptor leads to progressive hearing loss and increased susceptibility to noise. Proc Natl Acad Sci U S A 2013;110:2228e33. [118] Xing G, Yao J, Wu B, Liu T, Wei Q, Liu C, et al. Identification of OSBPL2 as a novel candidate gene for progressive nonsyndromic hearing loss by wholeexome sequencing. Genet Med 2015;17:210e8. [119] Thoenes M, Zimmermann U, Ebermann I, Ptok M, Lewis MA, Thiele H, et al. OSBPL2 encodes a protein of inner and outer hair cell stereocilia and is mutated in autosomal dominant hearing loss (DFNA67). Orphanet J Rare Dis 2015;10:15. [120] Azaiez H, Booth KT, Bu F, Huygen P, Shibata SB, Shearer AE, et al. TBC1D24 mutation causes autosomal-dominant nonsyndromic hearing loss. Hum Mutat 2014;35:819e23. [121] Zhang L, Hu L, Chai Y, Pang X, Yang T, Wu H. A dominant mutation in the stereocilia-expressing gene TBC1D24 is a probable cause for nonsyndromic hearing impairment. Hum Mutat 2014;35:814e8. [122] Zhao Y, Zhao F, Zong L, Zhang P, Guan L, Zhang J, et al. Exome sequencing and linkage analysis identified tenascin-C (TNC) as a novel causative gene in nonsyndromic hearing loss. PLoS One 2013;8:e69549. [123] Ansar M, Chahrour MH, Amin Ud Din M, Arshad M, Haque S, Pham TL, et al. DFNB44, a novel autosomal recessive non-syndromic hearing impairment locus, maps to chromosome 7p14.1-q11.22. Hum Hered 2004;57:195e9. [124] Santos-Cortez RL, Lee K, Giese AP, Ansar M, Amin-Ud-Din M, Rehn K, et al. Adenylate cyclase 1 (ADCY1) mutations cause recessive hearing impairment in humans and defects in hair cell function and hearing in zebrafish. Hum Mol Genet 2014;23:3289e98. [125] Girotto G, Abdulhadi K, Buniello A, Vozzi D, Licastro D, d'Eustacchio A, et al. Linkage study and exome sequencing identify a BDP1 mutation associated with hereditary hearing loss. PLoS One 2013;8:e80323. [126] Tabatabaiefar MA, Alasti F, Shariati L, Farrokhi E, Fransen E, Nooridaloii MR, et al. DFNB93, a novel locus for autosomal recessive moderate-to-severe hearing impairment. Clin Genet 2011;79:594e8. [127] Schrauwen I, Helfmann S, Inagaki A, Predoehl F, Tabatabaiefar MA, Picher MM, et al. A mutation in CABP2, expressed in cochlear hair cells, causes autosomal-recessive hearing impairment. Am J Hum Genet 2012;91:636e45. [128] Jaworek TJ, Richard EM, Ivanova AA, Giese AP, Choo DI, Khan SN, et al. An alteration in ELMOD3, an Arl2 GTPase-activating protein, is associated with hearing impairment in humans. PLoS Genet 2013;9:e1003774. [129] Behlouli A, Bonnet C, Abdi S, Bouaita A, Lelli A, Hardelin JP, et al. EPS8, encoding an actin-binding protein of cochlear hair cell stereocilia, is a new causal gene for autosomal recessive profound deafness. Orphanet J Rare Dis 2014;9:55. [130] Masmoudi S, Tlili A, Majava M, Ghorbel AM, Chardenoux S, Lemainque A, et al. Mapping of a new autosomal recessive nonsyndromic hearing loss locus (DFNB32) to chromosome 1p13.3-22.1. Eur J Hum Genet 2003;11:185e8. [131] Imtiaz A, Kohrman DC, Naz S. A frameshift mutation in GRXCR2 causes recessively inherited hearing loss. Hum Mutat 2014;35:618e24. [132] Basit S, Lee K, Habib R, Chen L, Umm e K, Santos-Cortez RL, et al. DFNB89, a novel autosomal recessive nonsyndromic hearing impairment locus on chromosome 16q21-q23.2. Hum Genet 2011;129:379e85. [133] Santos-Cortez RL, Lee K, Azeem Z, Antonellis PJ, Pollock LM, Khan S, et al. Mutations in KARS, encoding lysyl-tRNA synthetase, cause autosomalrecessive nonsyndromic hearing impairment DFNB89. Am J Hum Genet 2013;93:132e40. [134] Yariz KO, Duman D, Seco CZ, Dallman J, Huang M, Peters TA, et al. Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss. Am J Hum Genet 2012;91:872e82. [135] Ali RA, Rehman AU, Khan SN, Husnain T, Riazuddin S, Friedman TB, et al. DFNB86, a novel autosomal recessive non-syndromic deafness locus on chromosome 16p13.3. Clin Genet 2012;81:498e500. [136] Rehman AU, Santos-Cortez RL, Morell RJ, Drummond MC, Ito T, Lee K, et al. Mutations in TBC1D24, a gene associated with epilepsy, also cause nonsyndromic deafness DFNB86. Am J Hum Genet 2014;94:144e52. [137] Li J, Zhao X, Xin Q, Shan S, Jiang B, Jin Y, et al. Whole-exome sequencing identifies a variant in TMEM132E causing autosomal-recessive nonsyndromic hearing loss DFNB99. Hum Mutat 2015;36:98e105. [138] Khan SY, Riazuddin S, Shahzad M, Ahmed N, Zafar AU, Rehman AU, et al. DFNB79: reincarnation of a nonsyndromic deafness locus on chromosome 9q34.3. Eur J Hum Genet 2010;18:125e9. [139] Li Y, Pohl E, Boulouiz R, Schraders M, Nurnberg G, Charif M, et al. Mutations in TPRN cause a progressive form of autosomal-recessive nonsyndromic hearing loss. Am J Hum Genet 2010;86:479e84.

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B. Vona et al. / Molecular and Cellular Probes xxx (2015) 1e11 [140] Delmaghani S, Aghaie A, Michalski N, Bonnet C, Weil D, Petit C. Defect in the gene encoding the EAR/EPTP domain-containing protein TSPEAR causes DFNB98 profound deafness. Hum Mol Genet 2012;21:3835e44. [141] Seco CZ, Oonk AM, Dominguez-Ruiz M, Draaisma JM, Gandia M, Oostrik J, et al. Progressive hearing loss and vestibular dysfunction caused by a homozygous nonsense mutation in CLIC5. Eur J Hum Genet 2015;23:189e94. [142] Rost S, Bach E, Neuner C, Nanda I, Dysek S, Bittner RE, et al. Novel form of Xlinked nonsyndromic hearing loss with cochlear malformation caused by a mutation in the type IV collagen gene COL4A6. Eur J Hum Genet 2014;22: 208e15. [143] Schraders M, Haas SA, Weegerink NJ, Oostrik J, Hu H, Hoefsloot LH, et al. Next-generation sequencing identifies mutations of SMPX, which encodes the small muscle protein, X-linked, as a cause of progressive hearing impairment. Am J Hum Genet 2011;88:628e34. [144] Huebner AK, Gandia M, Frommolt P, Maak A, Wicklein EM, Thiele H, et al. Nonsense mutations in SMPX, encoding a protein responsive to physical force, result in X-chromosomal hearing loss. Am J Hum Genet 2011;88: 621e7. [145] Fukushima K, Kasai N, Ueki Y, Nishizaki K, Sugata K, Hirakawa S, et al. A gene for fluctuating, progressive autosomal dominant nonsyndromic hearing loss, DFNA16, maps to chromosome 2q23-24.3. Am J Hum Genet 1999;65: 141e50. €nsch D, Scheer P, Neumann C, Lang-Roth R, Seifert E, Storch P, et al. [146] Bo A novel locus for autosomal dominant, non-syndromic hearing impairment (DFNA18) maps to chromosome 3q22 immediately adjacent to the DM2 locus. Eur J Hum Genet 2001;9:165e70. [147] Kunst H, Marres H, Huygen P, van Duijnhoven G, Krebsova A, van der Velde S, et al. Non-syndromic autosomal dominant progressive non-specific mid-frequency sensorineural hearing impairment with childhood to late adolescence onset (DFNA21). Clin Otolaryngol Allied Sci 2000;25:45e54. [148] de Brouwer AP, Kunst HP, Krebsova A, van Asseldonk K, Reis A, Snoeckx RL, et al. Fine mapping of autosomal dominant nonsyndromic hearing impairment DFNA21 to chromosome 6p24.1-22.3. Am J Med Genet A 2005;137: 41e6. [149] Santos RL, H€ afner FM, Huygen PL, Linder TE, Schinzel AA, Spillmann T, et al. Phenotypic characterization of DFNA24: prelingual progressive sensorineural hearing impairment. Audiol Neurootol 2006;11:269e75. [150] Peters LM, Fridell RA, Boger ET, San Agustin TB, Madeo AC, Griffith AJ, et al. A locus for autosomal dominant progressive non-syndromic hearing loss, DFNA27, is on chromosome 4q12-13.1. Clin Genet 2008;73:367e72. €nsch D, Schmidt CM, Scheer P, Bohlender J, Neumann C, Am Zehnhoff[151] Bo Dinnesen A, et al. A new gene locus for an autosomal-dominant nonsyndromic hearing impairment (DFNA 33) is situated on chromosome 13q34-qter. HNO 2009;57:371e6. [152] Flex E, Mangino M, Mazzoli M, Martini A, Migliosi V, Colosimo A, et al. Mapping of a new autosomal dominant non-syndromic hearing loss locus (DFNA43) to chromosome 2p12. J Med Genet 2003;40:278e81. [153] D'Adamo P, Donaudy F, D'Eustacchio A, Di Iorio E, Melchionda S, Gasparini P. A new locus (DFNA47) for autosomal dominant non-syndromic inherited hearing loss maps to 9p21-22 in a large Italian family. Eur J Hum Genet 2003;11:121e4. [154] Moreno-Pelayo MA, Modamio-Høybjør S, Mencía A, del Castillo I, ndez-Burriel M, et al. DFNA49, a novel locus for autoChardenoux S, Ferna somal dominant non-syndromic hearing loss, maps proximal to DFNA7/ DFNM1 region on chromosome 1q21-q23. J Med Genet 2003;40:832e6. €nsch D, Schmidt CM, Scheer P, Bohlender J, Neumann C, am Zehnhoff[155] Bo Dinnesen A, et al. A new locus for an autosomal dominant, non-syndromic hearing impairment (DFNA57) located on chromosome 19p13.2 and overlapping with DFNB15. HNO 2008;56:177e82. [156] Chatterjee A, Jalvi R, Pandey N, Rangasayee R, Anand A. A novel locus DFNA59 for autosomal dominant nonsyndromic hearing loss maps at chromosome 11p14.2-q12.3. Hum Genet 2009;124:669e75. [157] van Beelen E, Schraders M, Huygen PL, Oostrik J, Plantinga RF, van Drunen W, et al. Clinical aspects of an autosomal dominantly inherited hearing impairment linked to the DFNA60 locus on chromosome 2q23.1-2q23.3. Hear Res 2013;300:10e7. [158] Fukushima K, Ramesh A, Srisailapathy CR, Ni L, Chen A, O'Neill M, et al. Consanguineous nuclear families used to identify a new locus for recessive non-syndromic hearing loss on 14q. Hum Mol Genet 1995;4:1643e8. [159] Petit C. Genes responsible for human hereditary deafness: symphony of a thousand. Nat Genet 1996;14:385e91. [160] Mustapha M, Chardenoux S, Nieder A, Salem N, Weissenbach J, el-Zir E, et al. A sensorineural progressive autosomal recessive form of isolated deafness, DFNB13, maps to chromosome 7q34-q36. Eur J Hum Genet 1998;6:245e50.

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[161] Masmoudi S, Charfedine I, Rebeh IB, Rebai A, Tlili A, Ghorbel AM, et al. Refined mapping of the autosomal recessive non-syndromic deafness locus DFNB13 using eight novel microsatellite markers. Clin Genet 2004;66: 358e64. [162] Mustapha M, Salem N, Weil D, el-Zir E, Loiselet J, Petit C. Identification of a locus on chromosome 7q31, DFNB14, responsible for prelingual sensorineural non-syndromic deafness. Eur J Hum Genet 1998;6:548e51. [163] Moynihan L, Houseman M, Newton V, Mueller R, Lench N. DFNB20: a novel locus for autosomal recessive, non-syndromal sensorineural hearing loss maps to chromosome 11q25-qter. Eur J Hum Genet 1999;7:243e6. [164] Pulleyn LJ, Jackson AP, Roberts E, Carridice A, Muxworthy C, Houseman M, et al. A new locus for autosomal recessive non-syndromal sensorineural hearing impairment (DFNB27) on chromosome 2q23-q31. Eur J Hum Genet 2000;8:991e3. [165] Medlej-Hashim M, Mustapha M, Chouery E, Weil D, Parronaud J, Salem N, et al. Non-syndromic recessive deafness in Jordan: mapping of a new locus to chromosome 9q34.3 and prevalence of DFNB1 mutations. Eur J Hum Genet 2002;10:391e4. [166] Belguith H, Masmoudi S, Medlej-Hashim M, Chouery E, Weil D, Ayadi H, et al. Re-assigning the DFNB33 locus to chromosome 10p11.23-q21.1. Eur J Hum Genet 2009;17:122e4. [167] Ansar M, Ramzan M, Pham TL, Yan K, Jamal SM, Haque S, et al. Localization of a novel autosomal recessive non-syndromic hearing impairment locus (DFNB38) to 6q26-q27 in a consanguineous kindred from Pakistan. Hum Hered 2003;55:71e4. [168] Delmaghani S, Aghaie A, Compain-Nouaille S, Ataie A, Lemainque A, Zeinali S, et al. DFNB40, a recessive form of sensorineural hearing loss, maps to chromosome 22q11.21-12.1. Eur J Hum Genet 2003;11:816e8. [169] Bhatti A, Lee K, McDonald ML, Hassan MJ, Gutala R, Ansar M, et al. Mapping of a new autosomal recessive non-syndromic hearing impairment locus (DFNB45) to chromosome 1q43-q44. Clin Genet 2008;73:395e8. [170] Mir A, Ansar M, Chahrour MH, Pham TL, Wajid M, Haque S, et al. Mapping of a novel autosomal recessive nonsyndromic deafness locus (DFNB46) to chromosome 18p11.32-p11.31. Am J Med Genet A 2005;133A:23e6. [171] Irshad S, Santos RL, Muhammad D, Lee K, McArthur N, Haque S, et al. Localization of a novel autosomal recessive non-syndromic hearing impairment locus DFNB55 to chromosome 4q12-q13.2. Clin Genet 2005;68: 262e7. [172] Ali G, Santos RL, John P, Wambangco MA, Lee K, Ahmad W, et al. The mapping of DFNB62, a new locus for autosomal recessive non-syndromic hearing impairment, to chromosome 12p13.2-p11.23. Clin Genet 2006;69: 429e33. [173] Tariq A, Santos RL, Khan MN, Lee K, Hassan MJ, Ahmad W, et al. Localization of a novel autosomal recessive nonsyndromic hearing impairment locus DFNB65 to chromosome 20q13.2-q13.32. J Mol Med Berl 2006;84: 484e90. [174] Chishti MS, Lee K, McDonald ML, Hassan MJ, Ansar M, Ahmad W, et al. Novel autosomal recessive non-syndromic hearing impairment locus (DFNB71) maps to chromosome 8p22-21.3. J Hum Genet 2009;54:141e4. [175] Ali Mosrati M, Schrauwen I, Ben Saiid M, Aifa-Hmani M, Fransen E, Mneja M, et al. Genome-wide analysis reveals a novel autosomal-recessive hearing loss locus DFNB80 on chromosome 2p16.1-p21. J Hum Genet 2013;58: 98e101. [176] Shahin H, Walsh T, Rayyan AA, Lee MK, Higgins J, Dickel D, et al. Five novel loci for inherited hearing loss mapped by SNP-based homozygosity profiles in Palestinian families. Eur J Hum Genet 2010;18:407e13. [177] Ali G, Lee K, Andrade PB, Basit S, Santos-Cortez RL, Chen L, et al. Novel autosomal recessive nonsyndromic hearing impairment locus DFNB90 maps to 7p22.1-p15.3. Hum Hered 2011;71:106e12. [178] Ansar M, Lee K, Naqvi SK, Andrade PB, Basit S, Santos-Cortez RL, et al. A new autosomal recessive nonsyndromic hearing impairment locus DFNB96 on chromosome 1p36.31-p36.13. J Hum Genet 2011;56:866e8. [179] Wang QJ, Li QZ, Rao SQ, Lee K, Huang XS, Yang WY, et al. AUNX1, a novel locus responsible for X linked recessive auditory and peripheral neuropathy, maps to Xq23-27.3. J Med Genet 2006;43:e33. [180] Wang QJ, Lu CY, Li N, Rao SQ, Shi YB, Han DY, et al. Y-linked inheritance of non-syndromic hearing impairment in a large Chinese family. J Med Genet 2004;41:e80. [181] Wang Q, Xue Y, Zhang Y, Long Q, Asan Yang F, et al. Genetic basis of Y-linked hearing impairment. Am J Hum Genet 2013;92:301e6. Q2 [182] Guan MX, Fischel-Ghodsian N, Attardi G. Biochemical evidence for nuclear gene involvement in phenotype of non-syndromic deafness associated with mitochondrial 12S rRNA mutation. Hum Mol Genet 1996;5: 963e71.

Please cite this article in press as: Vona B, et al., Non-syndromic hearing loss gene identification: A brief history and glimpse into the future, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.03.008

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