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A Truncating Mutation in SERPINB6 Is Associated with Autosomal-Recessive Nonsyndromic Sensorineural Hearing Loss

A Truncating Mutation in SERPINB6 Is Associated with Autosomal-Recessive Nonsyndromic Sensorineural Hearing Loss
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  REPORT  A Truncating Mutation in  SERPINB6   Is Associatedwith Autosomal-Recessive NonsyndromicSensorineural Hearing Loss Asl ı  S ı rmac ı , 1,2 Seyra Erbek, 3  Justin Price, 1,2 Mingqian Huang, 4 Duygu Duman, 5 F. Bas x ak Cengiz, 5 Gu¨ney Bademci, 1,2 Suna Tokgo¨z-Y ı lmaz, 5 Burcu His x mi, 5 Hilal O¨zda g ˘ , 6 Banu O¨ztu¨rk, 7 Sevsen Kulaks ı zo g ˘ lu, 8 Erkan Y ı ld ı r ı m, 9 Haris Kokotas, 10 Maria Grigoriadou, 10 Michael B. Petersen, 10 Hashem Shahin, 11 Moien Kanaan, 11 Mary-Claire King, 12 Zheng-Yi Chen, 4 Susan H. Blanton, 1,2 Xue Z. Liu, 2,13 Stephan Zuchner, 1,2 Nejat Akar, 5 and Mustafa Tekin 1,2, * Morethan270millionpeopleworldwidehavehearinglossthataffectsnormalcommunication.Althoughastonishingprogresshasbeenmadeintheidentificationofmorethan50genesfordeafnessduringthepastdecade,themajorityofdeafnessgenesareyettobeidentified.Inthisstudy,wemappedapreviouslyunknownautosomal-recessivenonsyndromicsensorineuralhearinglosslocus(DFNB91)tochromo-some6p25inaconsanguineousTurkishfamily.Thedegreeofhearinglosswasmoderatetosevereinaffectedindividuals.Wesubsequentlyidentifiedanonsensemutation(p.E245X)in SERPINB6 ,whichislocatedwithinthelinkageintervalforDFNB91andencodesforanintra-cellularproteaseinhibitor.Thep.E245Xmutationcosegregatedinthefamilyasacompletelypenetrantautosomal-recessivetraitandwasabsent in 300 Turkish controls. The mRNA expression of   SERPINB6  was reduced and production of protein was absent in the peripheralleukocytesofhomozygotes,suggestingthatthehearinglossisduetolossoffunctionofSERPINB6.WealsodemonstratedthatSERPINB6was expressed primarily in the inner ear hair cells. We propose that SERPINB6 plays an important role in the inner ear in the protectionagainst leakage of lysosomal content during stress and that loss of this protection results in cell death and sensorineural hearing loss. Genetic causes of hearing loss are estimated to account for68% of cases in newborns and 55% of cases by the age of four. 1 Autosomal-recessive,dominant,andX-linkedinheri-tance accounts for 77%, 22%, and 1% of the genetic deaf-ness, respectively. 2 Most cases of genetic deafness fall intothe category of sensorineural hearing loss and are causedby pathologies of the inner ear or auditory nerves; thesecan be identified with audiological investigations. Hear-ing loss can be classified into syndromic (20%–30%) andnonsyndromic (70%–80%) forms based on the presence orabsence of distinctive clinical or laboratory features. Morethan50dominantand/orrecessivegenesfornonsyndromicsensorineuralhearinglosshavebeenidentifiedandmostof the35genesfortheautosomal-recessiveformwereinitiallymappedinconsanguineousfamiliesorpopulationisolates.During our studies on hereditary deafness, whichwere approved by Ankara University Ethics Committee(Turkey), by the IRB at the University of Miami (USA),and by the Ethics Committee of the Institute of ChildHealth, Athens (Greece), we ascertained a Turkish family,family 728, in which four children with sensorineuralhearing loss were born to consanguineous parents. Thefather, whose parents were also first cousins, had sensori-neural hearing loss as well (Figure 1A). Diagnosis of senso-rineural hearing loss was established via standard audiom-etry. Audiograms of family 728 showed that the hearingloss was moderate to severe with some residual hearinginall affectedindividuals (Figure 1B). Allaffected membersof the family had oral communication with partial help of lip reading. Individual 201, at 54 years old the eldestaffected in the family, had the most severe hearing losswith more severe involvement of high frequencies. Theyoungest affected family member, 106, was 23 yearsold with hearing loss involving all frequencies. He self-reportedthathestarted havingmoredifficulties inhearingafter age 20. A progressive nature of hearing loss wasreported by affected individuals but has not been verifiedwithaudiograms.Theageattheonsetofhearinglosscouldnot be precisely determined because previous audiogramswere not available. The remainder of the examination wascompletely normal including normal anterior chamberand fundus of the eyes. Affected individuals did not havedelays in gross motor development. Neither did theyhave balance problems, vertigo, dizziness, or spontaneousand positional nystagmus. Tandem walking was normaland Romberg test was negative. CT scans of the temporal 1 Dr. John T. Macdonald Department of Human Genetics, University of Miami, Miller School of Medicine, Miami, FL 33136, USA;  2  John P. Hussman Insti-tute for Human Genomics, University of Miami, Miller School of Medicine, Miami, FL 33136, USA;  3 Department of Otorhinolaryngology, Baskent Univer-sity School of Medicine, Ankara 06490, Turkey;  4 Eaton-Peabody Laboratory, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, HarvardMedical School, Boston 02114, USA;  5 Division of Genetics, Department of Pediatrics, Ankara University School of Medicine, Ankara 06100, Turkey; 6 Biotechnology Institute, Ankara University, Ankara 06100, Turkey;  7 Department of Ophthalmology, Selcuk University Meram School of Medicine, Konya42080, Turkey;  8 Department of Biochemistry, Baskent University School of Medicine, Konya 42080, Turkey;  9 Department of Radiodiagnostics, BaskentUniversity School of Medicine, Konya 42080, Turkey;  10 Department of Genetics, Institute of Child Health, ‘Aghia Sophia’ Children’s Hospital, Athens11527, Greece;  11 Department of Life Sciences, Bethlehem University, Bethlehem, Palestinian Authority;  12 Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA;  13 Department of Otorhinolaryngology, University of Miami, Miller School of Medicine,Miami, FL 33136, USA*Correspondence: 10.1016/j.ajhg.2010.04.004. ª 2010 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics  86  , 1–8, May 14, 2010  1 Please cite this article in press as: S ı rmac ı  et al., A Truncating Mutation in  SERPINB6  Is Associated with Autosomal-Recessive NonsyndromicSensorineural Hearing Loss, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2010.04.004  bone in two affected family members were normal as well.EKGs, liver and kidney function tests, serum electrolytes,urinalysis, CBC, and leukocyte subgroups were all withinnormal limits in affected subjects.DNA was extracted from peripheral leukocytes of eachmember of family 728 via phenol chloroform method.Two affected individuals (728-101 and 728-201) were pre-screened and found to be negative for mutations in  GJB2 (MIM 121011) via direct sequencing of both exons andfor the m.1555A > G mutation in  MTRNR1  (MIM 561000)via an RFLP method via previously reported protocols. 3,4 Genome-wideSNPgenotypingwasdoneineightmembers Figure 1. The DFNB91 Locus, Audiograms, and Molecular Studies in Family 728 (A) Haplotypes created with SNP markers show complete cosegregation of a locus at 6p25 (DFNB91) in family 728.(B) Audiograms of seven members of family 728. 301, 102, and 103 have heterozygous, 201, 101, 105, and 106 have homozygousp.E245X mutation in  SERPINB6 .(C) Electropherograms showing the p.E245X (c.733G > T) mutation in  SERPINB6 .(D) Relative quantity values of   SERPINB6  cDNA obtained from peripheral blood leukocytes of homozygotes and heterozygotes in family728. Difference between homozygotes and heterozygotes is statistically significant (p  ¼  0.034).(E) The western blotting results of SERPINB6 in peripheral blood leukocytes in homozygous and heterozygous members of family 728.The42 kDaproteinis clearlyvisibleinNIH/3T3,HeLacells,andahealthypersonwithwild-type SERPINB6. AreducedSERPINB6bandisvisible in a heterozygote. The SERPINB6 protein band is completely absent in homozygotes. 2  The American Journal of Human Genetics  86  , 1–8, May 14, 2010 Please cite this article in press as: S ı rmac ı  et al., A Truncating Mutation in  SERPINB6  Is Associated with Autosomal-Recessive NonsyndromicSensorineural Hearing Loss, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2010.04.004  of family 728 (201, 301, 101, 102, 103, 104, 105, and 106)via Illumina 1M duo beadchips and assays (Illumina, CA).The DNA samples were processed according to Illuminaprocedures for processing of the Infinium II assay, theBeadChips were scanned on the Illumina BeadArrayReader,anddatawereextractedbytheIlluminaBeadstudiosoftware (Illumina). Before analysis, overall sample callrate,genderconsistencychecks,relationshipinference(viathe Graphical Representation of Relationships program), 5 and the Mendelian inconsistency rates were used forquality assessment. Genotypes were transferred into Excelfiles and sorted according to genomic positions along withall 35 previously identified autosomal-recessive nonsyn-dromic deafness genes. The cosegregation of the flankinggenotypes for each gene was visually evaluated. None of the known deafness loci cosegregated with the phenotype,thereby excluding these as possible causes and suggestingthat a mutation in a previously unknown deafness genewas responsible in this family. Copy number variants(CNVs) were assessed by determining the relative loss orgain of fluorescent signal intensity from SNP or CNVprobes on the array via PennCNV program 6 and no segre-gating CNVs were detected.Regions of autozygosity from the SNP screen were firstsought with PLINK. 7 All five affected members of family728 were concordant for homozygous SNP marker blocksthat were larger than one megabase in five chromosomallocations (Table S1 available online). Haplotypes were con-structed for homozygous genomic segments and theircosegregationwithdeafnesswasassessedvisually.Onlythehomozygous block between 289,878 bp (rs7762811) and3,908,468 bp (rs13205752) (total length was 3,618,590bp) at 6p25 cosegregated with hearing loss in the family(Figure 1A) (NCBI Build 36.1; hg18). The other four largeshared homozygous streches in affected family membersdid not cosegregate with the phenotype and were notfurther analyzed. A genome-wide two-point linkage anal-ysis was performed with SuperLink. 8 Sixteen SNP markerson chromosomes 6, 8, 16, and 21 were detected witha LOD score of more than 3 (Table S2). Visual evaluationof haplotypes in these regions confirmed that the largestautozygous segment cosegregating with the phenotypewas the same 3,618,590 bp region at 6p25. There werefive other cosegregating segments on chromosomes 8,16, and 21 with much smaller sizes ranging from 8,791bp to 273,430 bp (Table S2). Before concentrating on thelargest autozygous region on chromosome 6, the otherfive cosegregating regions were evaluated for their genecontent with the UCSC genome browser. There were noknown genes within the regions at chromosomes 16 and21. The cosegregating segment on chromosome 8 was14,852 bp long and partially included  MSRA  (MIM601250). Sequencing of all six exons as well as intron-exon boundaries of this gene in two affected members of family 728 did not detect a mutation.The only remaining autozygous segment that couldcontain a previously unrecognized deafness gene in family728 was at 6p25. Multipoint linkage analysis of this seg-ment was conducted with Fastlink  9 assuming a fully pene-trant autosomal-recessive disease, with a disease allele fre-quency of 0.0001. Both inbreeding loops were retainedin the analysis. SNPs spanning the homozygous regionwere chosen for linkage analysis based on tagging andheterozygosity in the parents. Allele frequencies were ob-tained from the CEU HapMap. Linkage analysis detectedamaximumlod scoreof5.0 betweenrecombinantmarkersrs7762811 and rs13205752 at 6p25 (Figure 1A). Examina-tion of haplotypes between these two SNPs not includedin the multipoint analysis did not identify any recombina-tions. This locus was designated as DFNB91 by the HUGONomenclature Committee.The linkage interval at 6p25 included 24 known proteincoding genes listed on the UCSC database (Figure S1).None of these genes have been previously reported tocause any forms of hearing loss in humans or in animals.The coding exons and intron-exon boundaries of all24 genes were sequenced in family 728 (Figure S1 andTable S3). A homozygous nonsense mutation, p.E245X(c.733G > T) (according to GenBank accession numberNM_004568.4), was identified in  SERPINB6  (MIM 173321)inallaffected members offamily 728(Figure1C).No otherDNA sequence change in this gene was detected. Thismutationremoves131aminoacidsinthecarboxyterminalof the protein including the reactive center loop. It com-pletelycosegregatedwithdeafnessinfamily728.Theiden-tified mutation was not found in 300 Turkish controlsvia an amplification refractory mutation detection system(ARMS)protocolthatwasshowntobesensitiveandspecificwith DNA sequence analysis-proven homozygous, hetero-zygous, and wild-type samples (Table S3). One previouslyunreported intronic change and 21 previously reportedSNPs were detected in other sequenced genes. None of thesechanges werepredicted to havean effect onfunction(Table S4).A premature stop codon was likely to activate nonsense-mediated mRNA decay response, thus leading to adecreased  SERPINB6  mRNA expression.  SERPINB6  wasknown to be expressed in white blood cells. Total RNAwas isolated from peripheral blood samples of sevenmembers of family 728 and cDNA was synthesized. Thesequencing of   SERPINB6  in cDNA containing the pointmutation with exonic primers detected the p.E245X(c.733G > T) mutation in both homozygotes and heterozy-gotes, showing that the identified mutation was present atthe mRNA level. The quantitative expression analysis of  SERPINB6  was performed with TaqMan PCR assays inquadriplicate for each individual on cDNA samples viaABIPrism7900HTSequenceDetectionsystem2.3(AppliedBiosystems Inc., CA) (for methods, see Table S3). ThemRNA expression was significantly reduced in homozy-gotes compared to those in heterozygotes (Figure 1D)(p  ¼  0.034; Mann-Whitney U test), indicating that thepremature stop codon destabilizes mRNA, leading tomRNA decay. The American Journal of Human Genetics  86  , 1–8, May 14, 2010  3 Please cite this article in press as: S ı rmac ı  et al., A Truncating Mutation in  SERPINB6  Is Associated with Autosomal-Recessive NonsyndromicSensorineural Hearing Loss, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2010.04.004  The protein expression of the mutant  SERPINB6  wasevaluated in peripheral leukocytes of homozygotes andheterozygotes (for methods, see Table S3). The expectedSERPINB6bandwasnotdetectedinhomozygotes,whereasthe 42 kDa protein was visible in a normal control anda heterozygote (Figure 1E). The trunctated protein productof   SERPINB6  with the p.E245X mutation was estimated tobe 27 kDa via Current Protocols database dna-rna-proteinmolecular weight calculator software. A truncated proteinwas not observed in heterozygotes or homozygotes, sug-gesting that the truncated protein is either not producedor rapidly degraded.For identification of other families with  SERPINB6  muta-tions, the probands of a total of 256 unrelated multiplexfamilies with autosomal-recessive nonsyndromic sensori-neural hearing loss were screened. These families werefrom Turkey (202), Greece (26), and the USA (28). Thevast majority of affected members of these families hadsevere to profound congenital or prelingual onset sen-sorineural hearing loss. Sequencing of all seven exonsrevealed seven additional previously unreported single-nucleotide changes (Table S5). However, these changesare unlikely to be the cause of phenotype because (1) theydid not cosegregate with deafness in the families and (2)in silico analyses with PolyPhen and SIFT predicted thatthey do not affect protein function and are therefore likelyto be benign. An additional 286 Palestinian probandsfrom multiplex autosomal-recessive nonsyndromic deaf-ness families were screened for the p.E245X mutationwith a restriction fragment length polymorphism (RFLP)method because the mutation removes the recognitionsite of the  BslI   restriction enzyme (Table S3). None of the samples were found to be positive for the screenedmutation.Given the observed hearing loss in humans, immunos-taining and in situ hybridization were used to identifythesiteforSerpinb6expressionindevelopingmouseinnerear. Immunohistochemistry was performed with devel-oping mouse inner ear tissues according to the protocolas described before. 10 In brief, frozen sections of the innerear tissues were dried for 15 min at 37  C and rehydrated in1 3 PBS for 5 min. Then, the sections were subjected to anantigen unmasking treatment via the Antigen UnmaskingSolution (Vector Laboratories, UK) according to the manu-facturer’s protocol. The blocking, primary antibody andsecond primary antibody incubation followed the stan-dard protocol. Antibodies used in the study are goat anti-SERPINB6 (human) (sc-21143, Santa Cruz Biotechnology,CA); rabbit anti-Myo7a (Proteus BioSciences Inc., CA),and rabbit anti-Ptprq (a gift from Dr. Guy Richardson,University of Sussex, UK). The secondary antibodies wereanti-goat Alexa 594 and anti-rabbit Alexa 488 (InvitrogenCorporation, CA) for fluorescent labeling. Data visualiza-tion and acquisition were performed with a Zeiss Axio-scope 2 fluorescent microscope. A weak Serpinb6 signalwas detected in E13.5 utricle sensory epithelium but notin hair cells (Figures 2A–2D). At E16.5, more prominentSerpinb6 was detected in crista hair cells (Figures 2E–2H).Hair cell expression of Serpinb6 was sustained in postnatalmice (data not shown). In cochlea, Serpinb6 was detectedin cochlear hair cells in embryo (Figures 2I–2L) and inhair cells and the greater epithelial ridge (GER) region inearly postnatal age (Figures 2M–2P). In both cochlear andutricular hair cells, Serpinb6 was found in cytoplasm(Figures 2E and 2M). There are five mouse Serpinb6 ortho-logs including Serpinb6a, b, c, d, and e, and the humanSERPINB6 antibody recognizes mouse Serpinb6a andSerpinb6c. Mouse Serpinb6a has the highest homologytohumanSERPINB6with75%identity,whereasSerpinb6cis68%identicaltoSERPINB6.Tofurtherconfirmimmuno-histochemistry results from the human SERPINB6 anti-body study, we performed in situ hybridization with themouse Serpinb6a antisense probe. The mouse Serpinb6acDNAwas purchased from Thermo Scientific Open Biosys-tems (clone ID: 5342439), and the preparation of ribop-robes and in situ hybridization followed the protocoldescribed. 11 The Serpinb6a in situ results matched thatfrom immunostaining with weak expression in E18.5 andsignificantly increased expression in P6 GER and cochlearhair cells (Figures 2Q and 2S).Proteases are important components of a number of physiological processes, including coagulation, cell migra-tion, phagocytosis, fibrinolysis, cell-mediated cytotoxicity,complement fixation, and apoptosis. To avoid nonspecifictissue damage, protease activity is regulated by inhibitors,such as members of the serpin superfamily. Inhibitory ser-pinsbindtheirtargetproteasesirreversibly,viaaconforma-tional change that deforms the protease and stabilizesthe serpin-protease complex. 12 Serpin dysfunction or defi-ciency is the underlying factor in a variety of human dis-eases, including emphysema, angioedema, thrombosis,and dementia 13,14 because of uncontrolled tissue damageby different proteases.In humans, the largest serpin clade (clade B) contains 13intracellular proteins. 13 Although their physiological rolesremain largely unknown, there is evidence that theyare involved in the regulation of cell growth, differentia-tion, and cytoprotection. A distinct subset of clade B ser-pins comprises  SERPINB1  (MIM 130135),  SERPINB6 , and SERPINB9  (MIM 601799), which are encoded by a geneclusteronchromosome6. 15 SERPINB6(alsonamedplacen-tal thrombin inhibitor, PTI, cytoplasmic antiproteinase,andproteaseinhibitor-6)wasdiscoveredin1993inhumanplacental tissue. 16 Its reactive center is located at Arg-341and Cys-342, and it lacks a classical N-terminal signalsequence, showing that it is not an extracellular protein. 17 Intracellular serpins, Serpins b6, b9, and b1 in particular,have been implicated in cytoprotective roles. For exam-ple, Serpinb9 inhibits the cytotoxic lymphocyte proteasegranzyme B. 18,19 Serpinb6 is a potent inhibitor of themonocyte/granulocyte protease cathepsin G, which isstoredinazurophilicgranulesandthenreleasedintophag-olysosomes or secreted during inflammation, 20 and kalli-kreins, 21 which are stored in cytoplasmic storage vesicles 4  The American Journal of Human Genetics  86  , 1–8, May 14, 2010 Please cite this article in press as: S ı rmac ı  et al., A Truncating Mutation in  SERPINB6  Is Associated with Autosomal-Recessive NonsyndromicSensorineural Hearing Loss, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2010.04.004  analogous to leukocyte granules. Any proteases releasedinto the interior of the cell during cellular stress wouldbe rapidly inactivated by intracellular serpins. Althoughcells can tolerate and repair lysosomal ruptures to a certaindegree, as the amount of damage increases, cells mayundergo apoptosis or necrosis. A dramatic support forthis model has recently been reported where a  C. elegans intracellular serpin, srp-6, exhibited a prosurvival functionby blocking necrosis. Minutes after hypotonic shock, srp-6null animals underwent a catastrophic series of events cul-minatinginlysosomaldisruption,cytoplasmicproteolysis,and death. This ‘‘hypo-osmotic stress lethal’’ phenotypewas dependent upon calpains and lysosomal cysteinepeptidases, two in vitro targets of srp-6. By protectingagainst both the induction of and the lethal effects fromlysosomal injury, srp-6 also blocked death induced by Figure 2. Detection of Serpinb6 in the Developing Mouse Inner Ear by Immunohistochemistry and In Situ Hybridization (A–D) Weak Serpinb6 was detected in E13.5 utricular sensory epithelium. However, Serpinb6 was not in utricular hair cells, which werelabeled with myo7a (B). Lines show examples of hair cells that are devoid of Serpinb6 (A).(E–H) Distinct Serpinb6 was detected in E16.5 crista hair cells (lines).(I–L) Weak Serpinb6 was detected in E16.5 cochlear hair cells. Multiple inner and outer hair cells were shown due to a section angle (J).(M–P) Serpinb6 was upregulated in P6 inner and outer hair cells (IHC, OHC), with mainly cytoplasm distribution. Ptprq labeled hairbundles (N) 40 . In addition, Serpinb6 was detected in the GER at this stage.(Q–T)InsituhybridizationshowedveryweakSerpinb6aexpressioninE18.5cochlearhaircellsandGER(Q),whereastheexpressionwassignificantly increased in P6 cochlear hair cells and GER (S). Control sense probe did not produce any signal (R, T).Abbreviations: Coch, cochlea; Cr, crista; Ut, utricle; anti-sen, antisense probe; sen, sense probe. Magnification: 40 3 . The American Journal of Human Genetics  86  , 1–8, May 14, 2010  5 Please cite this article in press as: S ı rmac ı  et al., A Truncating Mutation in  SERPINB6  Is Associated with Autosomal-Recessive NonsyndromicSensorineural Hearing Loss, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2010.04.004
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