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

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A Truncating Mutation in SERPINB6 Is Associated with Autosomal-Recessive Nonsyndromic Sensorineural Hearing Loss View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector REPORT A Truncating Mutation in SERPINB6 Is Associated with Autosomal-Recessive Nonsyndromic Sensorineural Hearing Loss Aslı Sırmacı,1,2 Seyra Erbek,3 Justin Price,1,2 Mingqian Huang,4 Duygu Duman,5 F. Basxak Cengiz,5 Gu¨ney Bademci,1,2 Suna Tokgo¨z-Yılmaz,5 Burcu Hisxmi,5 Hilal O¨ zdag˘,6 Banu O¨ ztu¨rk,7 Sevsen Kulaksızog˘lu,8 Erkan Yıldırım,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 Tekin1,2,* More than 270 million people worldwide have hearing loss that affects normal communication. Although astonishing progress has been made in the identification of more than 50 genes for deafness during the past decade, the majority of deafness genes are yet to be identified. In this study, we mapped a previously unknown autosomal-recessive nonsyndromic sensorineural hearing loss locus (DFNB91) to chromo- some 6p25 in a consanguineous Turkish family. The degree of hearing loss was moderate to severe in affected individuals. We subsequently identified a nonsense mutation (p.E245X) in SERPINB6, which is located within the linkage interval for DFNB91 and encodes for an intra- cellular protease inhibitor. The p.E245X mutation cosegregated in the family as a completely penetrant autosomal-recessive trait and was absent in 300 Turkish controls. The mRNA expression of SERPINB6 was reduced and production of protein was absent in the peripheral leukocytes of homozygotes, suggesting that the hearing loss is due to loss of function of SERPINB6. We also demonstrated that SERPINB6 was expressed primarily in the inner ear hair cells. We propose that SERPINB6 plays an important role in the inner ear in the protection against 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 for neural hearing loss as well (Figure 1A). Diagnosis of senso- 68% of cases in newborns and 55% of cases by the age of rineural hearing loss was established via standard audiom- four.1 Autosomal-recessive, dominant, and X-linked inheri- etry. Audiograms of family 728 showed that the hearing tance accounts for 77%, 22%, and 1% of the genetic deaf- loss was moderate to severe with some residual hearing ness, respectively.2 Most cases of genetic deafness fall into in all affected individuals (Figure 1B). All affected members the category of sensorineural hearing loss and are caused of the family had oral communication with partial help of by pathologies of the inner ear or auditory nerves; these lip reading. Individual 201, at 54 years old the eldest can be identified with audiological investigations. Hear- affected in the family, had the most severe hearing loss ing loss can be classified into syndromic (20%–30%) and with more severe involvement of high frequencies. The nonsyndromic (70%–80%) forms based on the presence or youngest affected family member, 106, was 23 years absence of distinctive clinical or laboratory features. More old with hearing loss involving all frequencies. He self- than 50 dominant and/or recessive genes for nonsyndromic reported that he started having more difficulties in hearing sensorineural hearing loss have been identified and most of after age 20. A progressive nature of hearing loss was the 35 genes for the autosomal-recessive form were initially reported by affected individuals but has not been verified mapped in consanguineous families or population isolates. with audiograms. The age at the onset of hearing loss could During our studies on hereditary deafness, which not be precisely determined because previous audiograms were approved by Ankara University Ethics Committee were not available. The remainder of the examination was (Turkey), by the IRB at the University of Miami (USA), completely normal including normal anterior chamber and by the Ethics Committee of the Institute of Child and fundus of the eyes. Affected individuals did not have Health, Athens (Greece), we ascertained a Turkish family, delays in gross motor development. Neither did they family 728, in which four children with sensorineural have balance problems, vertigo, dizziness, or spontaneous hearing loss were born to consanguineous parents. The and positional nystagmus. Tandem walking was normal father, whose parents were also first cousins, had sensori- and Romberg test was negative. CT scans of the temporal 1Dr. John T. Macdonald Department of Human Genetics, University of Miami, Miller School of Medicine, Miami, FL 33136, USA; 2John P. Hussman Insti- tute for Human Genomics, University of Miami, Miller School of Medicine, Miami, FL 33136, USA; 3Department of Otorhinolaryngology, Baskent Univer- sity School of Medicine, Ankara 06490, Turkey; 4Eaton-Peabody Laboratory, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston 02114, USA; 5Division of Genetics, Department of Pediatrics, Ankara University School of Medicine, Ankara 06100, Turkey; 6Biotechnology Institute, Ankara University, Ankara 06100, Turkey; 7Department of Ophthalmology, Selcuk University Meram School of Medicine, Konya 42080, Turkey; 8Department of Biochemistry, Baskent University School of Medicine, Konya 42080, Turkey; 9Department of Radiodiagnostics, Baskent University School of Medicine, Konya 42080, Turkey; 10Department of Genetics, Institute of Child Health, ‘Aghia Sophia’ Children’s Hospital, Athens 11527, Greece; 11Department of Life Sciences, Bethlehem University, Bethlehem, Palestinian Authority; 12Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195, USA; 13Department of Otorhinolaryngology, University of Miami, Miller School of Medicine, Miami, FL 33136, USA *Correspondence: [email protected] DOI 10.1016/j.ajhg.2010.04.004. ª2010 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 86, 797–804, May 14, 2010 797 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 homozygous p.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 family 728. 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. The 42 kDa protein is clearly visible in NIH/3T3, HeLa cells, and a healthy person with wild-type SERPINB6. A reduced SERPINB6 band is visible in a heterozygote. The SERPINB6 protein band is completely absent in homozygotes. bone in two affected family members were normal as well. Two affected individuals (728-101 and 728-201) were pre- EKGs, liver and kidney function tests, serum electrolytes, screened and found to be negative for mutations in GJB2 urinalysis, CBC, and leukocyte subgroups were all within (MIM 121011) via direct sequencing of both exons and normal limits in affected subjects. for the m.1555A>G mutation in MTRNR1 (MIM 561000) DNA was extracted from peripheral leukocytes of each via an RFLP method via previously reported protocols.3,4 member of family 728 via phenol chloroform method. Genome-wide SNP genotyping was done in eight members 798 The American Journal of Human Genetics 86, 797–804, May 14, 2010 of family 728 (201, 301, 101, 102, 103, 104, 105, and 106) 728 was at 6p25. Multipoint linkage analysis of this seg- via Illumina 1M duo beadchips and assays (Illumina, CA). ment was conducted with Fastlink9 assuming a fully pene- The DNA samples were processed according to Illumina trant autosomal-recessive disease, with a disease allele fre- procedures for processing of the Infinium II assay, the quency of 0.0001. Both inbreeding loops were retained BeadChips were scanned on the Illumina BeadArray in the analysis. SNPs spanning the homozygous region Reader, and data were extracted by the Illumina Beadstudio were chosen for linkage analysis based on tagging and software (Illumina). Before analysis, overall sample call heterozygosity in the parents. Allele frequencies were ob- rate, gender consistency checks, relationship inference (via tained from the CEU HapMap. Linkage analysis detected the Graphical Representation of Relationships program),5 a maximum lod score of 5.0 between recombinant markers and the Mendelian inconsistency rates were used for rs7762811 and rs13205752 at 6p25 (Figure 1A). Examina- quality assessment. Genotypes were transferred into Excel tion of haplotypes between these two SNPs not included files and sorted according to genomic positions along with in the multipoint analysis did not identify any recombina- all 35 previously identified autosomal-recessive nonsyn- tions. This locus was designated as DFNB91 by the HUGO dromic deafness genes. The cosegregation of the flanking Nomenclature Committee. genotypes for each gene was visually evaluated. None of The linkage interval at 6p25 included 24 known protein the known deafness loci cosegregated with
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