A Common Variant in CLDN14 Causes Precipitous, Prelingual Sensorineural Hearing Loss in Multiple Families Due to Founder Effect

Hum Genet DOI 10.1007/s00439-016-1746-7

ORIGINAL INVESTIGATION

A common variant in CLDN14 causes precipitous, prelingual sensorineural hearing loss in multiple families due to founder effect

Justin A. Pater1 · Tammy Benteau1 · Anne Griffin1 · Cindy Penney1 · Susan G. Stanton2 · Sarah Predham1 · Bernadine Kielley3 · Jessica Squires1 · Jiayi Zhou1 · Quan Li1 · Nelly Abdelfatah1 · Darren D. O’Rielly1,4 · Terry‑Lynn Young1,2,4

Received: 9 August 2016 / Accepted: 7 November 2016 © The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract Genetic isolates provide unprecedented opportu- Targeted sequencing of 169 deafness probands identified nities to identify pathogenic mutations and explore the full one homozygote and one heterozygous carrier. Genea- natural history of clinically heterogeneous phenotypes such logical studies, cascade sequencing and haplotype analysis as hearing loss. We noticed a unique audioprofile, char- across four unrelated families showed all subjects with the acterized by prelingual and rapid deterioration of hearing unique audioprofile n( 12) were also homozygous for = thresholds at frequencies >0.5 kHz in several adults from p.(Ala163Val) and shared a 1.4 Mb DFNB29-associated unrelated families from the island population of New- haplotype on chromosome 21. Most significantly, sequenc- foundland. Targeted serial Sanger sequencing of probands ing 175 population controls revealed 1% of the population for deafness alleles (n 23) that we previously identified are heterozygous for CLDN14 p.(Ala163Val), consistent = in this founder population was negative. Whole exome with a major founder effect in Newfoundland. The young- sequencing in four members of the largest family (R2010) est CLDN14 [c.488C>T; p.(Ala163Val)] homozygote identified a CLDN14 (DFNB29) variant [c.488C>T; p. passed newborn screening and had normal hearing thresh- (Ala163Val)], likely pathogenic, sensorineural hearing loss, olds up to 3 years of age, which then deteriorated to a pre- autosomal recessive. Although not associated with deaf- cipitous loss >1 kHz during the first decade. Our study ness or disease, CLDN14 p.(Ala163Val) has been previ- suggests that genetic testing may be necessary to identify ously reported as a variant of uncertain significance (VUS). at-risk children in time to prevent speech, language and developmental delay.

Electronic supplementary material The online version of this article (doi:10.1007/s00439-016-1746-7) contains supplementary material, which is available to authorized users. Introduction

* Terry‑Lynn Young Hearing loss is one of the most common and genetic of all [email protected] human phenotypes. Permanent bilateral sensorineural hear- 1 Craig L. Dobbin Genetics Research Centre, Discipline ing loss affects 1/500 newborns, and almost twice as many of Genetics, Faculty of Medicine, Memorial University, 300 adolescents (Smith et al. 1999; Morton and Nance 2006). Prince Phillip Drive, St. John’s, NL A1B 3V6, Canada Although approximately two-thirds of prelingual severe 2 Communication Sciences and Disorders, Western University, hearing loss cases are recessive, and 94 deafness loci have Elborn College, 1201 Western Road, London, ON N6G 1H1, been reported, only a minority of hearing loss cases with a Canada presumed recessive inheritance pattern can be conclusively 3 Department of Education and Early Childhood Development, diagnosed with a clear genetic etiology (Sloan-Heggen Government of Newfoundland and Labrador, St. John’s, NL et al. 2016). Therefore, many recessive cases may be due A1B 4J6, Canada to genetic defects in genes yet to be identified. However, 4 Molecular Diagnostic Laboratory, Eastern Health, Craig recent studies using new high-throughput technologies L. Dobbin Genetics Research Centre, Faculty of Medicine, Memorial University, 300 Prince Phillip Drive, St. John’s, and broader application in multi-ethnic populations report NL A1B 3V6, Canada GJB2 yields of less than 25%, suggesting a larger role for

1 3 Hum Genet other recessive genes in prelingual severe cases (Yan et al. alleles are born with normal hearing thresholds and expe- 2016; Sloan-Heggen et al. 2016). rience a rapid and progressive loss by 3–4 years of age. Sensorineural hearing loss is characterized by both Extensive clinical recruitment and targeted screening sug- degree (mild, moderate, severe or profound) and configu- gest that CLDN14 p.(Ala163Val) represents a major founder ration (low, mid and/or high frequency) using the standard variant in the Newfoundland population. behavioral audiogram. Although clinically heterogeneous, rare pathognomonic audiograms may present with surpris- ing regularity in clinics within genetically isolated popu- Materials and methods lations and where patients often share a common ancestor due to founder effects. For example, the Finnish and Study participants and audiometric evaluations Pakistani populations have been invaluable for discovery of deafness genes as population bottlenecks (genetic drift) This project is part of a large study of hereditary hearing loss and/or inbreeding increase the likelihood of inheriting in the Canadian province of Newfoundland and Labrador. recessive alleles that are identical by descent. These popu- Informed consent, family history and permission to access lations are often characterized by large sibships, deep gene- medical records and audiograms were obtained from all par- alogies and higher consanguineous rates. The population of ticipants as per approved institutional review board protocol Newfoundland and Labrador (NL) was founded by ~20,000 #01.186 (Human Research Ethics Board, St. John’s, NL, Protestant English and Roman Catholic Irish settlers. Reli- Canada). Sensorineural hearing loss was determined when gious and geographic isolation within small coastal fish- hearing thresholds were abnormal, and the air and bone con- ing (outport) communities (Manion 1977) has resulted in duction results within 10 decibels (dB) of each other (i.e., a higher inbreeding coefficient in the NL population (Bear air–bone gaps of 10 dB or less). Both retrospective and pro- et al. 1987, 1988; Zhai et al. 2015). We have previously spective audiograms and health records were obtained. identified several founder deafness mutations in the NL In the course of ongoing clinical recruitment, we noted populations (Abdelfatah et al. 2013a, b; Ahmed et al. 2004; a rare but consistent clinical audioprofile characterized as Doucette et al. 2009; Young et al. 2001). steeply sloping, sensorineural hearing loss above 0.5 kHz A unique clinical audioprofile of steeply sloping sensori- with mid- and high-frequency thresholds in the severe to neural hearing loss was noted in several unrelated families. profound range (Fig. 1a–d). On the premise that subjects Herein, we report a founder missense variant in CLDN14 with this hearing loss pattern also shared a recent common causing precipitous prelingual sensorineural hearing loss in ancestor, we used the distinct audioprofile to guide clini- children born with normal hearing thresholds. The essential cal recruitment, and a research team visited several small role of CLDN14, a component of tight junctions, was first fishing villages (outports) to measure hearing thresholds, discovered through studies in consanguineous families from extend pedigrees, provide genetic counseling and collect the genetically isolated population of Pakistan. Tight junc- blood samples (Fig. 2). tions have been shown to play a significant role in maintain- ing the structural integrity of cells within the inner ear. Other DNA preparation, targeted sequencing genes encoding tight junction proteins, such as MARVELD2 and audioprofiling (DFNB49) (Riazuddin et al. 2006; Nayak et al. 2016), have also been implicated in recessive hearing loss. Clau- Genomic DNA was extracted from peripheral blood using din-14 is essential for the formation of tight junctions and a simple salting out protocol (Miller et al. 1988). All is expressed in both hair cells and supporting cells of the probands recruited to the study were screened for popu- organ of Corti; however, CLDN14 exhibits preferential gene lation-specific deafness alleles (Supplementary Table 1; expression in sensory hair cells over supporting cells (Wil- Abdelfatah et al. 2013a, b; Ahmed et al. 2004; Doucette cox et al. 2001; Ben-Yosef et al. 2003; Scheffer et al. 2015). et al. 2009; Young et al. 2001). To identify other candidate Initially, CLDN14 was considered the cause of congenital genes to screen, audiograms were submitted to Audiogene recessive and profound deafness (Wilcox et al. 2001), and (Hildebrand et al. 2009) for computerized comparison with more recently of milder forms of hearing loss (Bashir et al. known average audiograms of 16 autosomal dominant loci 2013). The CLDN14 c. 488C>T p.(Ala163Val) allele has (under the assumption that hearing loss was segregating as previously been reported in multiple studies as a variant of an autosomal dominant trait in these NL families). Bidi- uncertain significance (VUS; Thorleifsson et al. 2009; Toka rectional Sanger sequencing (ABI PRISM 3500XL DNA et al. 2013; Purcell et al. 2014) and recently identified by Analyzer; Applied Biosystems, Foster City, CA, USA) with Sloan-Heggen et al. (2016) as one of two VUS in a patient standard PCR assay using Primer3 (Untergasser et al. 2012) with congenital hearing loss. Our study shows children was used to screen candidate mutations and genes (Merner inheriting two copies of CLDN14 c. 488C>T p.(Ala163Val) et al. 2008). We used Mutation Surveyor Software (version

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Fig. 1 Rare, precipitous audiologic phenotype caused by CLDN14 (c.488C>T; p.(Ala163Val)) in an Irish clan. a Pure tone audiogram of Family 2010 proband (PID V-9) and sister (PID V-10), b pure tone audiogram series for PID VI-2 (Family 2075) showing normal hearing at age 2 years and a progressive hearing loss apparent by 4 years of age, c first and d second decade pure tone audiogram of affected subjects. Yellow shaded area indicates range of normal hearing. Hearing thresholds are measured in decibels hearing level (dB HL), X left ear (air conduction), O = right ear (air conduction), > = left ear (bone = conduction), ↘ no response at the limits of the= audiometer. * 8 kHz was not measured =

4.07, SoftGenetics LLC State College, PA 16803) to select pseudodominant inheritance pattern; therefore, we con- quality reads and analyze DNA sequences. ducted both autosomal dominant and recessive analyses.

Whole exome sequencing and variant filtration Cascade sequencing and haplotype analysis

We prepared whole exome libraries for four members of Potential pathogenic mutations were subjected to cascade Family 2010 using the Ion Torrent AmpliSeq RDY Exome screening in all available relatives across three families Kit (Life Technologies, Cat. #A27193) (Fig. 2). Exome observed to have the same rare audioprofile (Families 2010, library purification, adapter ligation and barcoding were 2033 and 2075) and also in 175 ethnically matched con- done using the Ion PI Hi-Q OT2 200 kit (Life Technolo- trols. Microsatellites flanking candidate genes were geno- gies, Cat. #A26434). Purified libraries were quantified typed according to standard procedures (Abdelfatah et al. using the Ion Library Quantification Kit (Life Technolo- 2013a) and alleles called using GeneMapper software v4.0. gies, Cat. #4468802) and then loaded onto a PI v3 chip Haplotypes were reconstructed manually and compared and sequenced with the Ion Torrent Proton Sequencer. across families. Variants of interest were also screened in Single-nucleotide variants (SNVs) and insertion/dele- 169 deafness probands with Newfoundland ancestry. tions (INDELs) were called (GATK, v3.5) and annotated using SnpEff (v4.1; http://snpeff.sourceforge.net/) and SNVs were filtered against publically available SNP data- Results bases (ExAC Browser, http://exac.broadinstitute.org/; dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/; 1000 Clinical evaluation genomes, http://www.1000genomes.org). We assessed the impact of SNVs at the protein level with SIFT, Poly- Our research audiologist (AG) noted that probands (from Phen-2, and MutationTaster. Filtered SNVs had a mini- Families 2010, 2075 and 2033) all shared a unique hear- mum of 20 coverage, a predicted moderate/high impact ing loss pattern. The proband of Family 2010 (V-9; Fig. 2) × (nonsense, frameshift, missense, splice sites) and a minor presented at 36 years of age with the characteristic pat- allele frequency (MAF) of <1%. The apparent vertical tern of normal low-frequency thresholds, steeply sloping transmission of hearing loss in several branches of the to severe bilateral, symmetrical, sensorineural hearing loss clan pedigree (Fig. 2) could be due to either a dominant throughout mid and high frequencies (Fig. 1a). Age appro- gene with reduced penetrance, or a recessive gene with a priate audiologic tests of the proband’s son (VI-4; Fig. 2) at

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Fig. 2 Combined pedigrees of three families (2033, 2075 and 2010) symbols precipitous sensorineural hearing loss. Half-shaded symbols with rare, precipitous audiologic phenotype connect to a founding unspecified hearing loss couple and share an ancestral DFNB29-associated haplotype. Shaded

1 month and 1 year of age were normal. Serial audiograms Targeted sequencing and audioprofiling on PID VI-2 (Fig. 1b; Family 2075) show normal hearing thresholds across frequencies up to 3 years of age, and sub- Targeted sequencing was carried out on probands for known sequent rapid progression of hearing loss affecting high deafness alleles (previously identified in this population; Sup- frequencies first. Significant hearing loss of variable sever- plementary Table 1) but none were found. Several gene can- ity is already present in children aged 5–7 years (Fig. 1c), didates, as suggested by Audiogene (Hildebrand et al. 2009), which include probands of families 2033 and 2075. By the were also Sanger sequenced, including COCH, KCNQ4 and middle of the second decade of life, these children uni- TMC1. We identified a rare variant in TMC1 (c.421C>T; formly exhibit the distinctive steeply sloping audiogram MAF of 0.01%) predicted to cause the substitution of argi- (Fig. 1d). The hearing loss progresses slowly during subse- nine to a tryptophan residue at position 141 and to be delete- quent decades, primarily in the mid–high frequencies, with rious by SIFT (and probably damaging by PolyPhen-2 and relatively well-preserved low-frequency thresholds. For Panther). Although identified in both the probands (V-11) of adults, some variation in thresholds at 0.5 kHz is observed Family 2010 and transmitted to her affected son (V-14), the (PID V-10; Fig. 1a) but otherwise the adult presentation is c.421C>T variant did not co-segregate with mid–high-fre- relatively uniform. quency loss in this family (data not shown).

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Fig. 3 a Pedigree of family R2072, identified in screening of the (XP_015155717.1), Gekko japonicas (XP_015277878.1), Pelodis- NL deafness cohort, with the rare, precipitous audiologic phenotype cus sinensis (XP_006126056.1), Xenopus laevis (NP_001086045.1), who also share the CLDN14 [c.488C>T; p.(Ala163Val)] variant and Danio rerio (NP_001004559.2). Red font and arrow indicate a highly ancestral DFNB29-associated haplotype, b Conservation of the Clau- conserved alanine residue at position 163, c sequence electrophero- din-14 protein using Clustal Omega and WebLogo display. Homo grams of CLDN14 [c.488C>T; p.(Ala163Val)]. Box highlights vari- sapiens (NP_001139551.1), Pan paniscus (XP_008975916.1), Mus ant, d wild-type/normal CLDN14 c.488C. Box highlights normal musculus (NP_001159398.1), Rattus norvegicus (NP_001013447.1), sequence Canis lupus familiaris (XP_013965166.1), Gallus gallus

Whole exome sequencing isoforms and encodes a protein containing four transmembrane domains. The CLDN14 p.(Ala163Val) point vari- Whole exome sequencing on Family 2010 using three ant (Fig. 3c) identified in Family 2010 predicts substitu- affecteds (V-9, VI-4 and V-17) and one unaffected par- tion of an alanine to a valine at the beginning of the fourth ent (V-8) (Fig. 2) yielded >35,000 total variants. Under a transmembrane domain (Fig. 4a) and is highly conserved dominant model, 34 heterozygous variants were filtered (Fig. 3b). The CLDN14 c.488C>T allele was first identi- (data not shown). However, none of these variants resided fied in the Icelandic population (Thorleifsson et al. 2009). within known deafness genes/loci (http://hereditaryhear- Globally, the CLDN14 c.488C>T variant has an MAF ingloss.org/). Under a recessive model, we filtered four of 0.02564% (ExAC Browser, http://exac.broadinstitute. homozygous variants (in PRKDC, ZNF404, CUL7 and org/) and has been reported in both European and African CLDN14). One of these, CLDN14 (DFNB29) is a known populations. The heterozygous CLDN14 (human GRCh37/ deafness gene expressed in the sensory epithelium of the hg19: g. 37833506 G>A, NM_012130.3: c.488 C>T) organ of Corti of the inner ear (Wilcox et al. 2001; Schef- allele is reported as a variant of uncertain significance in fer et al. 2015). CLDN14 consists of three exons and two dbSNP (rs143797113), ExAC browser (MAF: 0.02564%),

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Fig. 4 a Location of pathogenic mutations in Claudin-14. tional diagram illustrating the anatomical location of the cochlear Colored amino acid residues indicate previously reported clau- canals and their respective ionic composition. *CLDN14 expression, din-14 mutations. Arrow indicates position of CLDN14 c.488C>T c Schematic diagram demonstrating the molecular structure of tight [p.(Ala163Val)]. Adapted from: Bashir et al. (2013), b Cross-sec- junctions

1000 genomes (MAF: 0.04%), and the Grand Opportunity (Fig. 4a; Bashir et al. 2013; Charif et al. 2013; Lee et al. Exome Sequencing Project (MAF: 0.05%). This allele has 2012; Wattenhofer et al. 2005; Wilcox et al. 2001). Func- also been reported in several control samples from other tional studies of CLDN14 mutations have demonstrated study cohorts within the USA (Toka et al. 2013), Swe- the importance of transmembrane domains with respect den (Purcell et al. 2014), and Africa (ExAC browser). The to protein topology and folding, as well as proper spatial majority of known pathogenic CLDN14 mutations reside localization within cells. For example, previous localiza- within one of the transmembrane domains in Claudin-14 tion experiments showed that the p.V85D and p. G101R

1 3 Hum Genet deafness mutations within domain II (Fig. 4a) fail to form which was incorporated into our DFNB29-associated deaf- tight junctions due to the mislocalization of Claudin-14 ness haplotype. protein to the cytoplasm, in vitro (Wattenhofer et al. 2005). Since p. (Ala163Val) is predicted to change a highly con- Genealogical analysis served amino acid within the fourth transmembrane domain (Fig. 3b), we suspect a similar impact regarding the spatial Extension of the pedigrees and review of all clinical audio- localization of claudin-14 to the plasma membrane, leading grams identified 16 subjects with hearing loss; 10/16 sub- to the cells’ inability to form tight junctions. While previ- jects showed the distinct precipitous mid–high-frequency ous research have demonstrated the importance of amino hearing loss (Fig. 2). Subjects with hearing impairment not acid conservation within in claudin-14 transmembrane consistent with the distinct precipitous mid–high-frequency domains, experimental functional studies are warranted to pattern include PID IV-3 (whom we have not connected prove CLDN14 c.488C>T, p.(Ala163Val) pathogenicity. to the founding couple) and all descendants of PID IV-4 (Fig. 2). In these cases, the audiogram can be characterized Cascade sequencing and haplotype analysis as a flat loss across all frequencies: PID IV-4 had a profound flat loss and his descendants (V-5, V-7, VI-3) show Cascade sequencing in all available subjects from Families a mild flat loss (Fig. 5). Family interviews determined the 2033 and 2075 show that affecteds with the distinct precipi- surnames suggestive of Irish descent (Seary 1977) and con- tous mid–high-frequency hearing loss (Fig. 2, filled sym- nected Families 2010, 2033 and 2075 to a single founding bols) were also homozygous for CLDN14 p.(Ala163Val) couple six generations ago. We noted that the inheritance (Fig. 3c). Subjects with a flat loss, such as PID IV-4 and pattern in the combined pedigree suggested either auto- his descendants (V-5, V-7 and VI-4) lacked the recessive somal dominant (with reduced penetrance) or autosomal CLDN14 variant (Fig. 3d). This pattern is consistent with recessive (pseudodominant) inheritance (Fig. 2). In sum- our hypothesis that CLDN14 p.(Ala163Val) is a likely mary, this population-based study using a targeted and pathogenic, recessive allele where homozygosity results in whole exome sequencing approach identified a CLDN14 a distinct precipitous mid–high-frequency hearing loss and (DFNB29) variant (c.488C>T, p. (Ala163Val), likely patho- relatives inheriting a single copy (carriers) or wild type do genic, sensorineural hearing loss, autosomal recessive. not have this pattern. According to the American College of Medical Genetics standards and guideline (Richards et al. 2015), CLDN14 p.(Ala163Val) is a strong PS4 PM2 likely Discussion pathogenic variant. The cause of hearing loss in subjects with flat audioprofiles is not known, but is clearly not due We have determined that a known VUS (CLDN14, to homozygosity for CLDN14 c.488C>T. Future studies c.488C>T, p. (Ala163Val)) is likely pathogenic, and causes will explore the genetic etiology of their hearing loss. Fur- a precipitous, bilateral and rapid deterioration of hearing thermore, screening our cohort of 169 deafness probands thresholds at frequencies >0.75 kHz in children, progress- identified an additional homozygous subject (Family ing gradually in adults. We have also determined that this 2072) and two heterozygous carriers. In Family 2072, the likely pathogenic variant is amplified in the founder popu- proband’s mother (with the distinct audioprofile) was also lation of the island of Newfoundland and is present in ~1% found to be homozygous for CLDN14 p.(Ala163Val) and of the population. The role of CLDN14 in nonsyndromic her father a carrier (Fig. 3a). Screening population controls hearing loss was first described in two large consanguin- identified four carriers out of 175 subjects, estimating an eous families from Pakistan with recessive profound con- MAF of 1.15% in the Newfoundland population and sug- genital deafness (Wilcox et al. 2001). Recessive CLDN14 gesting that this likely pathogenic variant is not rare. alleles manifest as nonsyndromic sensorineural hearing Extensive genotyping in the vicinity of DFNB29 loss with considerable phenotypic variability and may revealed that p.(Ala163Val) resides on a 1.4 Mb ances- present as congenital or prelingual, and mild, moderate– tral haplotype shared across all four families (Figs. 2, 3a). severe or profound (Bashir et al. 2010, 2013). In this study, Haplotype analysis shows affected individuals in the four homozygous children had normal hearing thresholds up to families inherit an ancestral DFNB29-associated haplo- 3 years of age and overall, a remarkably conserved hearing type on chromosome 21q22.1, signifying clan member- phenotype. A combination of pedigree extension and geno- ship, although biological connection for Family 2072 was typing linked four families of Irish ancestry to a founding not found (Fig. 3a). Additionally, we sequenced all coding couple six generations back. sequences of the CLDN14 gene, including the exon/intron The claudin family of proteins consists of 24 mem- boundaries and 5′ and 3′ UTRs. We identified a common bers with tissue-specific expression. Claudin-14 plays a synonymous variant (c.243C>T; rs219799) within the clan, critical role in the formation of tight junction barriers that

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Fig. 5 Clan members lacking the recessive CLDN14 [c.488C>T; ease, d at age 39, PID VI-3 presents with mild hearing loss, and e a p.(Ala163Val)] variant do not present with the characteristic steeply heterozygous CLDN14 [c.488C>T; p.(Ala163Val)] carrier (PID V-4) sloping hearing phenotype, exhibiting a different age of onset and exhibits mild hearing loss at age 60. Yellow shaded area indicates hearing threshold progression a Profound, flat sensorineural hearing range of normal hearing. Hearing thresholds are measured in decibels loss with an unknown etiology at age 63 (PID IV-4), b PID V-5 (age: hearing level (dB HL), X left ear (air conduction), O right ear 53) presents with borderline hearing thresholds, c PID V-7 (age: 58) (air conduction) = = presents with mild hearing loss with a diagnosis of Meniere’s dis- regulate paracellular ion transport (Mineta et al. 2011) increasing the potassium concentration around the hair cell and is highly expressed in the kidney, liver and the inner body, compromising mechanotransduction and causing hair ear (Ben-Yosef et al. 2003; Wilcox et al. 2001). Moreover, cell toxicity and eventual cell death. preferential gene expression has been observed in the inner The CLDN14 p.(Ala163Val) variant reported in this ear, as CLDN14 expression is lower in supporting cells, study has been identified in previous studies but not in asso- relative to sensory hair cells (Wilcox et al. 2001; Scheffer ciation with disease. It was first reported as a VUS by Thor- et al. 2015). Normal hearing function and hair cell depo- leifsson et al. (2009) in a large Iceland/Netherlands GWAS larization are dependent on tight junctions in the reticular cohort study examining SNPs associated with kidney lamina. In the organ of Corti, hair cell stereocilia are bathed stones and bone mineral density and more recently by Toka in potassium-rich endolymph, while the basolateral surface et al. (2013), who detected the CLDN14 p.(Ala163Val) of the hair cell body is surrounded by an intercellular (or allele in 3 of 1230 study participants for another kidney extracellular) fluid continuous with the perilymph (Fig. 4b, function study. The heterozygous p.(Ala163Val) allele c). The reticular lamina, formed in part by tight junctions was also found in a Swedish GWAS study examining the between the apical surfaces of hair cells and supporting polygenic nature of schizophrenia (Purcell et al. 2014). cells of the sensory epithelium, creates a barrier isolating The heterozygous p.(Ala163Val) allele was submitted 31 the endolymphatic fluid from other cochlear compartments, times to ExAC browser, 29 alleles from European descent which contain perilymph. Maintenance of this ionic gradi- and 2 from the African population. In a recent American ent is essential for mechanotransduction, which depends study including 1119 deafness probands, a cohort made on the modulation of potassium current flowing from the up of 62.3% autosomal recessive cases (Sloan-Heggen endolymph into the hair cells through the stereocilia as et al. 2016) used a targeted sequencing approach and the they are displaced by sound-induced vibrations. Disrup- most commonly implicated genes were GJB2, MYH9, tion of this tight junction barrier alters the ionic gradient, OTOA, PCDH15, SLC26A4, STRC, TMC1, TMPRSS3 and

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USH2A. Interestingly, Sloan-Heggen et al. 2016 reported not find them beneficial. For example, PIDs V-9 (age: 50), the p.(Ala163Val) allele in a patient with congenital hear- V-10 (age: 51) and V-17 (age: 57; Fig. 2) reported some ing loss; however, in a compound heterozygous state with a additional sound with hearing aids but no improvement of second CLDN14 allele (p.P28L). In summary, these studies speech comprehension, consistent with the extreme erosion suggest that the likely pathogenic CLDN14 p.(Ala163Val) of mid and high frequencies. Older affected adults with allele is both rare and widely distributed around the globe. well-preserved low-frequency sensitivity have limited com- Many reports claim that approximately 50% of autosomal munication by phone. PIDs V-10 and V-17 (Fig. 2) whose recessive deafness is caused by either homozygous or com- threshold at 0.5 kHz is deteriorated can no longer compound heterozygous mutations in the DFNB1 locus (GJB2), municate by phone. Adult members of this clan are highly which is often the only gene that is routinely screened in skilled speech readers who can detect speech initiation and the clinical setting when there is a family history of hear- turn quickly to maximize the use of visual clues. Unfortu- ing loss. This represents a massive ascertainment bias, as nately, these skills can be mistaken for hearing and subjects children who are DFNB1 negative are not followed up, due have voiced concerns regarding safety in the workplace. to the expenses associated with genetic testing. Recently, The development of the organ of Corti is unidirectional, a large, ethnically diverse, cohort study demonstrated the and follows a base-to-apex hair cell degeneration in the importance of investigating DFNB1-negative deaf probands Cldn14-null mouse cochlea. This may explain why we (Yan et al. 2016). This study took a targeted panel approach observe a sensorineural threshold loss progressing from in 342 probands (185 simplex and 157 multiplex families), the high to low frequencies in affected clan members. The sequenced 180 known hearing loss genes, and identified cochlea discerns high- from low-frequency sound, based 151 variants in 119 families. Fifty-three families had patho- on a stiffness gradient along the basilar membrane (Ehret genic or likely pathogenic mutations within 27 genes, while 1978; Teudt and Richter 2014). In Cldn14-null mice, the the remaining were variants of uncertain significance. This organ of Corti undergoes a base-to-apex deterioration study solved 25 and 7% of multiplex and simplex families, beginning around postnatal day 10, with a more severe and respectively, emphasizing the importance of large families rapid degeneration of outer hair cells compared to inner and strong histories of disease in genetic studies. hair cells. By day 13, the three rows of outer hair cells are Pediatric hearing programs strive to identify and treat almost completely absent in the cochlear base, with partial early to prevent delay in language, learning and social loss and stereociliar disorganization in the middle and api- development. However, the detection of delayed onset cal turns (Ben-Yosef et al. 2003). The cochlear lesion then and progressive forms of hearing loss remain a signifi- proceeds towards the cochlear apex, with a rapid deterio- cant challenge. Children who are homozygous CLDN14 ration of the outer hair cells accompanied by the onset of p.(Ala163Val) pass newborn and early hearing tests. The inner hair cell damage. By day 18, outer hair cell deterio- proband’s son (R2010, PID VI-4) was discharged after his ration is severe with only a few remaining outer hair cells test at 1 year of age indicated normal hearing. Preschool exhibiting damaged stereocilia in the most apical region; testing 4 years later showed significant deterioration of in contrast, only partial inner hair cell loss is reported both mid and high frequencies (Fig. 1c). Delayed identifi- throughout the cochlea by this age. Auditory brainstem cation could result from limited testing of high frequencies responses measured in 4-week-old Cldn14-null mice indi- in the preschool years, often complicated by limited testing cate a significant hearing loss in comparison to their wild- tolerance in children. In this study, PID VI-2 had normal type and heterozygous littermates (Ben-Yosef et al. 2003). hearing at 8 kHz at 2 years of age. At 3 years, hearing was reported normal although thresholds at 8 kHz were not performed. By 4 years, a 55 dBHL threshold at 8 kHz and mild Summary to moderate loss at all high frequencies required immedi- ate hearing aid fitting. Retrospectively, if 8 kHz thresholds A population-based study of hearing loss in the NL popu- had been performed at 3 years, diagnosis and therapy could lation has clarified the role of CLDN14 p.(Ala163Val), a have been offered a year earlier (Fig. 1b). Conversely, VUS previously identified in the USA, Iceland and Swe- genetic testing or prenatal/preconception parental carrier den. CLDN14 p.(Ala163Val) appears to be of Irish origin screening could provide appropriate hearing surveillance and causes a precipitous, prelingual recessive sensorineu- and minimize the risk of delays in language development ral hearing loss. This likely pathogenic variant is frequent and learning from rapidly progressing hearing loss. in this island population of Northern European decent, and Adults who are homozygous for CLDN14 p.(Ala163Val) CLDN14 p.(Ala163Val) homozygotes have normal hear- also have a consistent phenotype but challenges in manage- ing thresholds at birth, then experience rapid, progressive ment remain. Hearing aids benefit affected children and nonsyndromic hearing loss in early childhood. Although young adults (up to the third decade), but most adults do missed by newborn hearing screening, genetic testing

1 3 Hum Genet would ensure identification of at-risk children, allowing for Abdelfatah N, Merner N, Houston J, Benteau T, Griffin A, Doucette appropriate monitoring and timely intervention, aural reha- L, Stockley T, Lauzon JL, Young TL (2013b) A novel deletion in SMPX causes a rare form of X-linked progressive hearing loss bilitation and counseling for families. Although GJB2 is in two families due to a founder effect. Hum Mutat 34:66–69. routinely screened in the regional hospital diagnostic clinic, doi:10.1002/humu.22205 we recommend targeted screening of CLDN14, as well. Ahmed ZM, Li XC, Powell SD, Riazuddin S, Young TL, Ramzan K, Ahmad Z, Luscombe S, Dhillon K, MacLaren L, Ploplis B, Shotland LI, Ives E, Riazuddin S, Friedman TB, Morell RJ, Wil- Limitations cox ER (2004) Characterization of a new full length TMPRSS3 isoform and identification of mutant alleles responsible for non- While exome sequencing is a powerful tool to elucidate dis- syndromic recessive deafness in Newfoundland and Pakistan. ease causing, coding variant, it does not explore non-coding BMC Med Genet 5:24. doi:10.1186/1471-2350-5-24 Bashir R, Fatima A, Naz S (2010) Mutations in CLDN14 are associ- regions. Additionally, there is no experimental proof of the ated with different hearing thresholds. J Hum Genet 55:767–770. predicted amino acid substitution, and without functional doi:10.1038/jhg.2010.104 data, we cannot be certain that the point mutation impacts Bashir ZE, Latief N, Belyantseva IA, Iqbal F, Riazuddin SA, Khan protein location within tight junctions. For example, this SN, Friedman TB, Riazuddin S, Riazuddin S (2013) Pheno- typic variability of CLDN14 mutations causing DFNB29 hear- variant could cause alternative splicing or alter gene expres- ing loss in the Pakistani population. J Hum Genet 58:102–108. sion. Although less likely, it is plausible that a causal, non- doi:10.1038/jhg.2012.143 coding variant at the DFNB29 locus is in linkage disequi- Bear JC, Nemec TF, Kennedy JC, Marshall WH, Power AA, Kolo- librium with p.(Ala163Val). Even though our study presents nel VM, Burke GB (1987) Persistent genetic isolation in outport Newfoundland. Am J Med Genet 27:807–830. doi:10.1002/ several lines of evidence to suggest pathogenicity, experi- ajmg.1320270410 mental functional analysis of p.(Ala163Val) is required. Bear JC, Nemec TF, Kennedy JC, Marshall WH, Power AA, Kolonel VM, Burke GB (1988) Inbreeding in outport Newfoundland. Am Acknowledgements We are grateful to the individuals who partici- J Med Genet 29:649–660. doi:10.1002/ajmg.1320290324 pated in this study and to Elspeth Drinkell, Carol Negrijn, Jim Hou- Ben-Yosef T, Belyantseva IA, Saunders TL, Hughes ED, Kawamoto ston and Dante Galutira for technical assistance. K, Van Itallie CM, Beyer LA, Halsey K, Gardner DJ, Wilcox ER, Rasmussen J, Anderson JM, Dolan DF, Forge A, Raphael Compliance with ethical standards Y, Camper SA, Friedman TB (2003) Claudin 14 knockout mice, a model for autosomal recessive deafness DFNB29, are deaf due to cochlear hair cell degeneration. Hum Mol Genet Funding This work was supported by a grant from the Canadian 12:2049–2061 Institutes for Health Research-Regional Partnership Program with Bespalova IN, Van Camp G, Bom SJ, Brown DJ, Cryns K, DeWan the Research and Development Corporation of Newfoundland and AT, Erson AE, Flothmann K, Kunst HP, Kurnool P, Sivakumaran Labrador, the Canadian Foundation for Innovation (New Investiga- TA, Cremers CW, Leal SM, Burmeister M, Lesperance MM tor Award no. 9384 and Leaders Opportunity Fund no. 13120) and (2001) Mutations in the Wolfram syndrome 1 gene (WFS1) are Genome Canada (Atlantic Medical Genetics and Genomics Initiative). a common cause of low frequency sensorineural hearing loss. The authors also gratefully acknowledge financial support from the Hum Mol Genet 10:2501–2508 Janeway Children’s Hospital Foundation, Memorial University and the Charif M, Bakhchane A, Abidi O, Boulouiz R, Eloualid A, Roky Government of Newfoundland and Labrador. N.A. is a CIHR-RPP Fel- R, Rouba H, Kandil M, Lenaers G, Barakat A (2013) Analy- lowship awardee. sis of CLDN14 gene in deaf Moroccan patients with nonsyndromic hearing loss. Gene 523:103–105. doi:10.1016/j. Conflict of interest The authors declare that they have no conflict of gene.2013.03.123 interest. Chaussenot A, Rouzier C, Quere M, Plutino M, Ait-El-Mkadem S, Bannwarth S, Barth M, Dollfus H, Charles P, Nicolino M, Chabrol B, Vialettes B, Paquis-Flucklinger V (2015) Mutation Open Access This article is distributed under the terms of the Crea- update and uncommon phenotypes in a French cohort of 96 tive Commons Attribution 4.0 International License (http://crea- patients with WFS1-related disorders. Clin Genet 87:430–439. tivecommons.org/licenses/by/4.0/), which permits unrestricted use, doi:10.1111/cge.12437 distribution, and reproduction in any medium, provided you give Doucette L, Merner ND, Cooke S, Ives E, Galutira D, Walsh V, Walsh appropriate credit to the original author(s) and the source, provide a T, MacLaren L, Cater T, Fernandez B, Green JS, Wilcox ER, link to the Creative Commons license, and indicate if changes were Shotland LI, Li XC, Lee M, King MC, Young TL (2009) Pro- made. found, prelingual nonsyndromic deafness maps to chromosome 10q21 and is caused by a novel missense mutation in the Usher syndrome type IF gene PCDH15. Eur J Hum Genet 17:554–564. doi:10.1038/ejhg.2008.231 References Ehret G (1978) Stiffness gradient along the basilar membrane as a basis for spatial frequency analysis within the cochlea. J Acoust Abdelfatah N, McComiskey DA, Doucette L, Griffin A, Moore SJ, Soc Am 64:1723–1726 Negrijn C, Hodgkinson KA, King JJ, Larijani M, Houston J, Goncalves AC, Matos TD, Simoes-Teixeira HR, Pimenta Machado Stanton SG, Young TL (2013a) Identification of a novel in-frame M, Simao M, Dias OP, Andrea M, Fialho G, Caria H (2014) deletion in KCNQ4 (DFNA2A) and evidence of multiple pheno- WFS1 and non-syndromic low-frequency sensorineural hearing copies of unknown origin in a family with ADSNHL. Eur J Hum loss: a novel mutation in a Portuguese case. Gene 538:288–291. Genet 21:1112–1119. doi:10.1038/ejhg.2013.5 doi:10.1016/j.gene.2014.01.040

1 3 Hum Genet

Haywood AF, Merner ND, Hodgkinson KA, Houston J, Syrris P, mutations in schizophrenia. Nature 506:185–190. doi:10.1038/ Booth V, Connors S, Pantazis A, Quarta G, Elliott P, McKenna nature12975 W, Young TL (2013) Recurrent missense mutations in TMEM43 Riazuddin S, Ahmed ZM, Fanning AS, Lagziel A, Kitajiri S, Ramzan (ARVD5) due to founder effects cause arrhythmogenic cardio- K, Khan SN, Chattaraj P, Friedman PL, Anderson JM, Belyant- myopathies in the UK and Canada. Eur Heart J 34:1002–1011. seva IA, Forge A, Riazuddin S, Friedman TB (2006) Tricellu- doi:10.1093/eurheartj/ehs383 lin is a tight-junction protein necessary for hearing. Am J Hum Hildebrand MS, DeLuca AP, Taylor KR, Hoskinson DP, Hur IA, Tack Genet 79: 1040–1051. doi:10.1086/510022 D, McMordie SJ, Huygen PL, Casavant TL, Smith RJ (2009) A Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody contemporary review of AudioGene audioprofiling: a machine- WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL, based candidate gene prediction tool for autosomal dominant Committee ALQA (2015) Standards and guidelines for the inter- nonsyndromic hearing loss. Laryngoscope 119:2211–2215. pretation of sequence variants: a joint consensus recommenda- doi:10.1002/lary.20664 tion of the American College of Medical Genetics and Genom- Hodgkinson KA, Howes AJ, Boland P, Shen XS, Stuckless S, Young ics and the Association for Molecular Pathology. Genet Med TL, Curtis F, Collier A, Parfrey PS, Connors SP (2016) Long- 17:405–424. doi:10.1038/gim.2015.30 Term Clinical Outcome of Arrhythmogenic Right Ventricu- Scheffer DI, Shen J, Corey DP, Chen ZY (2015) Gene Expression lar Cardiomyopathy in Individuals With a p.S358L Mutation by Mouse Inner Ear Hair Cells during Development J Neurosci in TMEM43 Following Implantable Cardioverter Defibrilla- 35:6366–6380. doi:10.1523/JNEUROSCI.5126-14.2015 tor Therapy. Circ Arrhythm Electrophysiol 9. doi:10.1161/ Seary ER (1977) Family Names of the Island of Newfoundland. CIRCEP.115.003589 McGill-Queen’s University Press, St. John’s Lee K, Ansar M, Andrade PB, Khan B, Santos-Cortez RL, Ahmad W, Sloan-Heggen CM, Babanejad M, Beheshtian M, Simpson AC, Booth Leal SM (2012) Novel CLDN14 mutations in Pakistani families KT, Ardalani F, Frees KL, Mohseni M, Mozafari R, Mehrjoo Z, with autosomal recessive non-syndromic hearing loss. Am J Med Jamali L, Vaziri S, Akhtarkhavari T, Bazazzadegan N, Nikzat Genet A 158A:315–321. doi:10.1002/ajmg.a.34407 N, Arzhangi S, Sabbagh F, Otukesh H, Seifati SM, Khodaei H, Manion JJ (1977) The Peopleing of Newfoundland: Essays in His- Taghdiri M, Meyer NC, Daneshi A, Farhadi M, Kahrizi K, Smith torical Geography. Institute of Scoial and Economic Research RJ, Azaiez H, Najmabadi H (2015) Characterising the spectrum Memorial University of Newfoundland, St. John’s of autosomal recessive hereditary hearing loss in Iran. J Med Merner ND, Hodgkinson KA, Haywood AF, Connors S, French VM, Genet 52:823–829. doi:10.1136/jmedgenet-2015-103389 Drenckhahn JD, Kupprion C, Ramadanova K, Thierfelder L, Sloan-Heggen CM, Bierer AO, Shearer AE, Kolbe DL, Nishimura CJ, McKenna W, Gallagher B, Morris-Larkin L, Bassett AS, Parfrey Frees KL, Ephraim SS, Shibata SB, Booth KT, Campbell CA, PS, Young TL (2008) Arrhythmogenic right ventricular cardio- Ranum PT, Weaver AE, Black-Ziegelbein EA, Wang D, Azaiez myopathy type 5 is a fully penetrant, lethal arrhythmic disorder H, Smith RJ (2016) Comprehensive genetic testing in the clinical caused by a missense mutation in the TMEM43 gene. Am J Hum evaluation of 1119 patients with hearing loss. Hum Genet 135: Genet 82: 809–821. doi:10.1016/j.ajhg.2008.01.010 441-50. doi:10.1007/s00439-016-1648-8 Miller SA, Dykes DD, Polesky HF (1988) A simple salting out pro- Smith RJH, Shearer AE, Hildebrand MS, et al. Deafness and Heredi- cedure for extracting DNA from human nucleated cells. Nucleic tary Hearing Loss Overview. 1999 Feb 14 [Updated 2014 Jan 9]. Acids Res 16:1215 In: Pagon RA, Adam MP, Ardinger HH, et al., editors. GeneRe- Milting H, Klauke B, Christensen AH, Musebeck J, Walhorn V, views® [Internet]. Seattle (WA): University of Washington, Seat- Grannemann S, Munnich T, Saric T, Rasmussen TB, Jensen HK, tle; 1993-2016. Available from: http://www.ncbi.nlm.nih.gov/ Mogensen J, Baecker C, Romaker E, Laser KT, zu Knyphausen books/NBK1434/. Accessed May 21 2016 E, Kassner A, Gummert J, Judge DP, Connors S, Hodgkinson Sun Y, Cheng J, Lu Y, Li J, Lu Y, Jin Z, Dai P, Wang R, Yuan H K, Young TL, van der Zwaag PA, van Tintelen JP, Anselmetti D (2011) Identification of two novel missense WFS1 mutations, (2015) The TMEM43 Newfoundland mutation p. S358L caus- H696Y and R703H, in patients with non-syndromic low-fre- ing ARVC-5 was imported from Europe and increases the stiff- quency sensorineural hearing loss. J Genet Genomics 38:71–76. ness of the cell nucleus. Eur Heart J 36:872–881. doi:10.1093/ doi:10.1016/j.jcg.2011.01.001 eurheartj/ehu077 Teudt IU, Richter CP (2014) Basilar membrane and tectorial mem- Mineta K, Yamamoto Y, Yamazaki Y, Tanaka H, Tada Y, Saito K, brane stiffness in the CBA/CaJ mouse. J Assoc Res Otolaryngol Tamura A, Igarashi M, Endo T, Takeuchi K, Tsukita S (2011) 15:675–694. doi:10.1007/s10162-014-0463-y Predicted expansion of the claudin multigene family. FEBS Lett Thorleifsson G, Holm H, Edvardsson V, Walters GB, Styrkarsdottir U, 585:606–612. doi:10.1016/j.febslet.2011.01.028 Gudbjartsson DF, Sulem P, Halldorsson BV, de Vegt F, d’Ancona Morton CC, Nance WE (2006) Newborn hearing screening - FC, den Heijer M, Franzson L, Christiansen C, Alexandersen P, silent revolution. N Engl J Med 354:2151в164. doi:10.1056/ Rafnar T, Kristjansson K, Sigurdsson G, Kiemeney LA, Bod- NEJMra050700 varsson M, Indridason OS, Palsson R, Kong A, Thorsteinsdot- Nayak G, Varga L, Trincot C, Shahzad M, Friedman PL, Klimes I, tir U, Stefansson K (2009) Sequence variants in the CLDN14 Greinwald JH, Riazuddin SA, Masindova I, Profant M, Khan gene associate with kidney stones and bone mineral density. Nat SN, Friedman TB, Ahmed ZM, Gasperikova D, Riazuddin S, Genet 41:926–930. doi:10.1038/ng.404 Riazuddin S (2015) Molecular genetics of MARVELD2 and Toka HR, Genovese G, Mount DB, Pollak MR, Curhan GC (2013) clinical phenotype in Pakistani and Slovak families segregating Frequency of rare allelic variation in candidate genes among DFNB49 hearing loss. Hum Genet 134(4):423–437. doi:10.1007/ individuals with low and high urinary calcium excretion. PLoS s00439-015-1532-y One 8:e71885. doi:10.1371/journal.pone.0071885 Purcell SM, Moran JL, Fromer M, Ruderfer D, Solovieff N, Roussos Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm P, O’Dushlaine C, Chambert K, Bergen SE, Kahler A, Duncan M and Rozen SG (2012) Primer3–new capabilities and inter- L, Stahl E, Genovese G, Fernandez E, Collins MO, Komiyama faces. Nucleic Acids Res 40(15):e115 NH, Choudhary JS, Magnusson PK, Banks E, Shakir K, Gari- Van Camp G, Smith RJH (2015) Hereditary Hearing Loss Homepage. mella K, Fennell T, DePristo M, Grant SG, Haggarty SJ, Gabriel http://hereditaryhearingloss.org/. Accessed May 21, 2016 S, Scolnick EM, Lander ES, Hultman CM, Sullivan PF, McCa- Wattenhofer M, Reymond A, Falciola V, Charollais A, Caille D, Borel rroll SA, Sklar P (2014) A polygenic burden of rare disruptive C, Lyle R, Estivill X, Petersen MB, Meda P, Antonarakis SE

1 3 Hum Genet

(2005) Different mechanisms preclude mutant CLDN14 proteins AS, Blanton SH, Liu XZ, Tekin M (2016) Spectrum of DNA from forming tight junctions in vitro. Hum Mutat 25:543–549. variants for non-syndromic deafness in a large cohort from doi:10.1002/humu.20172 multiple continents. Hum Genet 135(8):953–961. doi:10.1007/ Wilcox ER, Burton QL, Naz S, Riazuddin S, Smith TN, Ploplis B, s00439-016-1697-z Belyantseva I, Ben-Yosef T, Liburd NA, Morell RJ, Kachar B, Young TL, Ives E, Lynch E, Person R, Snook S, MacLaren L, Cater T, Wu DK, Griffith AJ, Riazuddin S, Friedman TB (2001) Muta- Griffin A, Fernandez B, Lee MK, King MC (2001) Non-syndro- tions in the gene encoding tight junction claudin-14 cause auto- mic progressive hearing loss DFNA38 is caused by heterozygous somal recessive deafness DFNB29. Cell 104:165–172 missense mutation in the Wolfram syndrome gene WFS1. Hum Yan D, Tekin D, Bademci G, Foster J, Cengiz FB, Kannan-Sundhari Mol Genet 10:2509–2514 A, Guo S, Mittal R, Zou B, Grati M, Kabahuma RI, Kameswaran Zhai G, Zhou J, Woods MO, Green JS, Parfrey P, Rahman P, Green M, Lasisi TJ, Adedeji WA, Lasisi AO, Menendez I, Herrera M, RC (2015) Genetic structure of the Newfoundland and Labra- Carranza C, Maroofian R, Crosby AH, Bensaid M, Masmoudi S, dor population: founder effects modulate variability. Eur J Hum Behnam M, Mojarrad M, Feng Y, Duman D, Mawla AM, Nord Genet. doi:10.1038/ejhg.2015.256

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