Attitudes toward genetic testing

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1 DOI /jbcpp J Basic Clin Physiol Pharmacol 2013; 24(3): Mini Review Natali Idan, Zippora Brownstein, Shaked Shivatzki and Karen B. Avraham* Advances in genetic diagnostics for hereditary hearing loss Abstract: Hereditary hearing loss affects a significant proportion of the hearing impaired, with genetic mutations estimated to be responsible for its etiology in over 50% of this population. The methods for molecular diagnostics are changing as a result of the transition from linkage analysis to next generation sequencing to identify the genes responsible for hearing loss in affected families. In this review, we summarize the attitudes of the hearing impaired towards genetic testing, the latest techniques for identifying mutations, and provide a comprehensive list of the mutations found in the Israeli Jewish hearingimpaired population. Keywords: deafness; exome sequencing; massively parallel sequencing; mutations; targeted capture. *Corresponding author: Prof. Karen B. Avraham, Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel, Phone: , Fax: , karena@post.tau.ac.il Natali Idan, Zippora Brownstein and Shaked Shivatzki: Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine; Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel Genetics of hearing loss Genetic testing and counseling are the cornerstones of medical genetics, providing crucial answers for patients with hereditary diseases. The considerations and details differ, depending on the disease, but the questions remain the same: What are the recurrence rates, risks, benefits, and possible consequences of each available option? For hereditary hearing loss (HL), a sensorineural disorder that is compatible with life, many of the questions remain the same, but the approach differs, depending on whether the parents are deaf or hearing, the age of onset, and whether the HL is isolated or part of a syndrome. Sensorineural HL is the most common sensory disorder, affecting 4% of the population before the age of 45 [1]. This form of HL is caused by various defects in the inner ear and associated nerve fibers, and the onset ranges from birth through childhood to late adulthood. HL can be due to genetic mutations, a result of exposure to environmental factors, or a combination of both. Most early-onset HL disorders are genetic [2], and a major portion of late-onset HL is as well or at least has genetic factors contributing to a multifactorial etiology [3]. Hereditary HL is a heterogeneous condition, with more than 700 forms of deafness associated with other clinical abnormalities (syndromic HL, SHL) (Online Mendelian Inheritance in Man; Over 100 loci have been linked to dominant, recessive, and X-linked forms of nonsyndromic HL (NSHL), and over 60 genes have been identified, some of which are associated with both recessive and dominant forms of deafness, as well as SHL [4]. Attitudes toward genetic testing A large effort has been made in the past two decades to discover the genes involved in hereditary HL. Although the principles defining the function of the cochlea, the mammalian auditory organ, have been recognized for some time, knowledge about the molecular mechanisms of the auditory system has only recently come to light [5]. Revealing the genetic basis of hereditary HL is crucial for patients eager to know the cause of their HL. Known etiology enables preimplantation decisions or prenatal diagnosis in order to prevent recurrence. In addition, it is important in promoting the understanding of the molecular basis of HL and the auditory system, which may facilitate new methods of treatment. In recent years, deaf individuals attitudes and perceptions of genetic testing for deafness have shifted toward greater personal interest [6, 7]. Deaf individuals and parents of deaf children, in general, are interested in pursuing genetic testing [8, 9]. However, the reasons and motivations behind this interest vary. It is multifaceted, with cultural considerations playing a major role. The most important reasons for pursuing genetic testing

2 166 Idan et al.: Genetic diagnostics for hereditary hearing loss are to understand the cause of the HL to determine if it is genetic in origin and to evaluate what might be the consequences for other family members. In some cases, patients also want to help advance research, due to an interest in long-term solutions such as prevention in future family members, and advanced rehabilitation and therapeutics based on research outcome [7, 10, 11]. However, genetic testing for deafness is complex for both scientific and social reasons, which introduces complexity into genetic counseling as well. The scientific complexity is derived from the underlying etiological and genetic heterogeneity of deafness and the limitations in the past to conduct a comprehensive genetic test of all deafness genes for each deaf individual as a routine in the clinic. However, targeted capture and next-generation sequencing (NGS), also known as massively parallel sequencing and deep sequencing, of all known deafness genes [12, 13], and whole exome sequencing [14] is now being performed in research as well as in private laboratories, yielding impressive results. These new technologies may change the face of genetic diagnosis for HL, although they bring new challenges. Interpreting and analyzing the NGS test results are one of the obstacles that have to be faced in order to increase the portion of solved cases. The social complexity stems from diverse perceptions and beliefs of the deaf in different societies and cultures. Deafness is not a life-threatening illness. Moreover, many individuals do not consider it a medical condition or a disorder that needs to be treated, but as a nonmedical trait that does not require medical intervention [15]. A complicated picture of deaf individuals reasons for genetic testing has been drawn, where cultural affiliation appears to play an important role in constructing their motivations for testing [10, 16]. Individuals who associate with the Deaf Community, a community that has developed its own social beliefs, behavior, art, literature, history, values, and institutions, are less likely to endorse genetic testing at birth, compared to individuals with equal involvement in both the deaf and hearing communities [17]. Members of the Deaf Community tend to believe that genetic testing for HL would have a negative effect on their community; they have no preference regarding the hearing status of their children and therefore are not interested in prenatal diagnosis for HL. They are also more likely to feel that termination of pregnancy based on hearing status should be illegal [18]. A small percentage polled would consider termination of pregnancy if the fetus was found to be hearing [19]. Others, who are culturally associated with the hearing community, would see HL as a medical condition, would support prenatal diagnosis, and would consider a termination of pregnancy [18, 20]. For some of them, as well as for the growing elderly population, finding ways to cure or improve hearing is essential. Molecular diagnostics for deafness-causing genes Worldwide, the most frequent causative genes for HL are GJB2, SLC26A4, MYO15A, OTOF, CDH23, and TMC1, most of which are large genes with many deafness-causing mutations in each. Because of the size of the genes and the cost of Sanger sequencing, until recently, only the known mutations were screened in clinics, based on relevance according to ethnic origin and HL phenotype. Searching for new genes or mutations was performed mostly in research laboratories, using linkage analysis followed by Sanger sequencing of the coding regions of each candidate gene in a critical interval. The genes were sequenced one by one after ranking them on the basis of hypotheses about gene function or expression patterns. More than 60 genes were found to be associated with human deafness in this way, but this technique, both time- and cost-consuming, was not optimal as it did not allow for examination of a large number of genes simultaneously. It has been difficult, if not impossible, to offer clinically useful screening for each of the 64 genes currently implicated in NSHL. Recent advances in technology, using NGS, have made it possible to rapidly and cost-effectively sequence all the genes of interest, including the noncoding regions. NGS is changing the face of gene identification and is making it possible to screen for mutations in hundreds of genes, allowing one to provide precise information about the genetic cause and the recurrence risk and offer precise treatment [21]. For example, identification of individuals with mutations in CACNA1D, who are usually initially misdiagnosed with NSHL, should undergo regular cardiological care to avoid cardiac complications that are part of SANDD, a syndrome associated with sinoatrial node dysfunction and deafness [22]. In the Middle East, as in most other regions in the world, the most common gene involved in HL is GJB2, responsible for 27% of congenital HL among Israeli Jews [23] and 14% among Palestinian Arabs [24]. Therefore, in most clinics, the routine test for each deaf individual includes restriction enzyme analysis of the most common mutations in this gene and, occasionally, sequencing of

3 Idan et al.: Genetic diagnostics for hereditary hearing loss 167 Table 1 Mutations for HL found in the Jewish population. Gene RefSeq ID Inheritance Name of mutation a cdna location Origin Phenotype References CDH23 NM_ Recessive V2635F c.7903g > T Algeria NSHL [12] GJB2 NM_ Recessive IVS1+1 G to A c.-3172g > A Iraq NSHL [23] Recessive 35delG c.35del All NSHL [34] Recessive 51del12insA c.51del12insa Buchara NSHL [34] Recessive W24X c.72g > A Buchara NSHL [23] Recessive V27I+E114G c.79g > A+341A > G Ashkenazi NSHL Personal communication Recessive R32C c.94c > T Ashkenazi NSHL [23] Recessive M34T c.101t > G Ashkenazi NSHL Personal communication Recessive V37I c.109g > A Ashkenazi NSHL [23] Recessive 167delT c.167del Ashkenazi, Syria NSHL [35] Recessive L90P c.269t > C Iraq NSHL [23] Recessive R127H c.380g > A India NSHL Personal communication Recessive W134R c.400t > A Iraq NSHL Personal communication Recessive V153I c.457g > A Tunisia NSHL Personal communication Dominant R75Q c.224g > A Ashkenazi NSHL Personal communication GJB6 NM_ Recessive 342kb del(gjb6-d13s1830) 342kb del(gjb6-d13s1830) Ashkenazi NSHL [25] MYO3A NM_ Recessive 1777(-12)G > A c.1777(-12)g > A Iraq NSHL [27] Recessive 732(-2)A > G c.732(-2)a > G Iraq NSHL [27] Recessive Y1042X c.3126t > G Iraq NSHL [27] MYO15A NM_ Recessive 373delCG c.373_374del Ashkenazi NSHL [12] Recessive R2728H c.8183g > A Ashkenazi NSHL [12] PCDH15 NM_ Recessive R245X c.733c > T Ashkenazi USH1F [26] POU3F4 NM_ X-linked Q79X c.235c > T Ashkenazi NSHL, Mondini [33] X-linked 853delAT c.853_854del Bulgaria NSHL, Mondini [33] POU4F3 NM_ Dominant 884del8 c.884_891del Libya NSHL [30] SLC26A4 NM_ Recessive InsT c.1458_1459inst Iran NSHL, EVA b [28] SYNE4 NM_ Recessive 228delAT c.228_229del Iraq NSHL [32] TECTA NM_ Dominant T1866M c.5497c > T Turkey NSHL [12] TJP2 NM_ Dominant Inverted duplication Whole gene Tunisia NSHL [31] TMC1 NM_ Recessive R389X c.1165c > T Morocco NSHL [12] Recessive W404R c.1210t > C Morocco NSHL [12] Recessive R604X c.1810c > T Morocco NSHL [12] Recessive S647P c.1939t > C Morocco NSHL [12] USH1C NM_ Recessive InsC c.238_239insc Ashkenazi USH1C [23] WFS1 NM_ Dominant E864K c.2590g > A Ashkenazi NSHL [12] a The review aims to bring information to clinicians to use routinely in the clinic; therefore, we have referred to the mutations by their common names. b EVA, enlarged vestibular aqueduct.

4 168 Idan et al.: Genetic diagnostics for hereditary hearing loss Figure 1 Representative family with hereditary HL evaluated for genetic diagnosis and counseling. A c.7903g > T mutation was identified in the CDH23 gene encoding the cadherin 23 protein, predicted to lead to a p.v2635f missense mutation, by deep sequencing [12]. (A) The congenital HL is inherited in a recessive mode. (B) Representative pure-tone audiogram from an affected individual with severe to profound HL. Rt, right; Lt, left; AC, air conduction; BC, bone conduction; NR, no response. (C) The mutation was validated by Sanger sequencing, demonstrated in a chromatogram. (D) Residue V2635 in ectodomain 25 of cadherin 23 is buried in the protein core, and the evolutionary conservation analysis shows that it is conserved (ConSurf grade of 7, consurf.tau.ac.il/). The mutation V2635F appears to cause a significant change from a hydrophobic residue of medium size to phenylalanine, which is aromatic, rigid, and much larger. (E) Cadherin 23, along with protocadherin 15, is expressed in the tip links of the stereocilia [36]. the single coding exon of GJB2. The other deafness genes that were known in the Middle East prior to the deep sequencing era included mutations in eight genes for HL in the Israeli Jewish population, which were identified over a period of more than 15 years: GJB6 [25], PCDH15 [26], USH1C (harmonin) [23], MYO3A [27], SLC26A4 [28], LOXHD1 [29], POU4F3 [30], and the inverted duplication of TJP2 [31]. Because most of these genes were found only in one extended family, screening for them was not practical. Only the known mutations in these genes are routinely tested in the clinic, and then only if relevant to a specific ethnic group, mode of inheritance, and type of HL. These routine clinical tests miss other mutations in the known genes and potential new deafness genes, which are most likely the cause of deafness in two-thirds of patients with hereditary HL that remain unsolved with respect to etiology in the Middle East [12] and worldwide. Advances in gene discovery due to NGS Recent experiments, using NGS, added six genes to the list in a short period of time, including CDH23, MYO15A, WFS1, and TECTA [12], SYNE4 [32], and POU3F4 [33]. The genes and mutations involved in HL in the Israeli Jewish population known to date are listed in Table 1. NGS has

5 Idan et al.: Genetic diagnostics for hereditary hearing loss 169 proven to be the optimal genetic diagnostic approach to identify deafness mutations (Figure 1). In addition to the immediate consequences of genetic counseling and guiding clinical decisions, identification of the causative genes is the basis for understanding auditory mechanisms, which, in turn, paves the way not only for successful rehabilitation, but in the future, perhaps for therapeutics as well. Initial steps toward therapy have already been taken and show promising results. In vitro induction of new hair cells and partial recovery of hearing in ears damaged by noise trauma when Notch signaling was inhibited suggests a method to replace lost hair cells by pharmacological inhibition of Notch [37]. Hearing and vestibular function were rescued in a mouse model for Usher syndrome with an antisense oligonucleotide that corrects defective pre-mrna splicing of transcripts from the USH1C gene with the c.216g > A mutation [38]. The optimal approaches to alleviating HL may depend on identifying the genes underlying deafness. In conclusion, the discovery of all deafness genes is now more feasible than ever. A change of concept is about to occur regarding the routine molecular testing for HL, with comprehensive multigene testing likely to become more commonplace. Moreover, identifying all genes involved in deafness might facilitate a greater understanding of auditory complexity. Acknowledgments: We are indebted to the families and physicians who have contributed to our research over the years, allowing us to determine the genetic basis for hearing impairment in Israel. Research in the Karen Avraham laboratory is supported by the National Institutes of Health (NIDCD) R01DC011835, I-CORE Gene Regulation in Complex Human Disease Center No. 41/11, Israel Science Foundation 1320/11, Human Frontier Science Program RGP0012/2012, and the Hedrich Charitable Trust. We thank Maya Shushan and Nir Ben-Tal for the Consurf analysis. Conflict of interest statement Authors conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received May 10, 2013; accepted August 8, 2013 References 1. Morton CC, Nance WE. Newborn hearing screening a silent revolution. N Engl J Med 2006;354: Nance WE. The genetics of deafness. Ment Retard Dev Disabil Res Rev 2003;9: Bovo R, Ciorba A, Martini A. Environmental and genetic factors in age-related hearing impairment. Aging Clin Exp Res 2011;23: Van Camp G, Smith RJ. Hereditary Hearing Loss Homepage. Available at: Accessed: July Richardson GP, de Monvel JB, Petit C. How the genetics of deafness illuminates auditory physiology. Annu Rev Physiol 2011;73: Dagan O, Hochner H, Levi H, Raas-Rothschild A, Sagi M. Genetic testing for hearing loss: different motivations for the same outcome. Am J Med Genet 2002;113: Withrow KA, Tracy KA, Burton SK, Norris VW, Maes HH, Arnos KS, et al. Impact of genetic advances and testing for hearing loss: results from a national consumer survey. Am J Med Genet A 2009;149A: Brunger JW, Murray GS, O Riordan M, Matthews AL, Smith RJ, Robin NH. Parental attitudes toward genetic testing for pediatric deafness. Am J Hum Genet 2000;67: Parker MJ, Fortnum HM, Young ID, Davis AC. Genetics and deafness: what do families want? J Med Genet 2000;37:E Boudreault P, Baldwin EE, Fox M, Dutton L, Tullis L, Linden J, et al. Deaf adults reasons for genetic testing depend on cultural affiliation: results from a prospective, longitudinal genetic counseling and testing study. J Deaf Stud Deaf Educ 2010;15: Burton SK, Withrow K, Arnos KS, Kalfoglou AL, Pandya A. A focus group study of consumer attitudes toward genetic testing and newborn screening for deafness. Genet Med 2006;8: 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:R Shearer AE, DeLuca AP, Hildebrand MS, Taylor KR, Gurrola J, 2nd, Scherer S, et al. Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing. Proc Natl Acad Sci USA 2010;107: 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: Robertson JA. Extending preimplantation genetic diagnosis: the ethical debate. Ethical issues in new uses of preimplantation genetic diagnosis. Hum Reprod 2003;18:

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