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Genetic Basis of Y-Linked Hearing Impairment

Qiuju Wang,1,2 Yali Xue,2 Yujun Zhang,2,5 Quan Long,2,6 Asan,2,3 Fengtang Yang,2 Daniel J. Turner,2,7 Tomas Fitzgerald,2 Bee Ling Ng,2 Yali Zhao,1 Yuan Chen,2 Qingjie Liu,4 Weiyan Yang,1 Dongyi Han,1 Michael A. Quail,2 Harold Swerdlow,2 John Burton,2 Ciara Fahey,2 Zemin Ning,2 Matthew E. Hurles,2 Nigel P. Carter,2 Huanming Yang,3 and Chris Tyler-Smith2,*

A single Mendelian trait has been mapped to the human Y : Y-linked hearing impairment. The molecular basis of this disorder is unknown. Here, we report the detailed characterization of the DFNY1 Y chromosome and its comparison with a closely related Y chromosome from an unaffected branch of the family. The DFNY1 chromosome carries a complex rearrangement, including duplication of several noncontiguous segments of the Y chromosome and insertion of ~160 kb of DNA from , in the peri- centric region of Yp. This segment of chromosome 1 is derived entirely from within a known hearing impairment locus, DFNA49. We suggest that a third copy of one or more from the shared segment of chromosome 1 might be responsible for the hearing-loss phenotype.

The human Y chromosome is transmitted directly from Y chromosome of a closely related but unaffected branch father to son for most of its length, generating a male- of the family. specific pattern of inheritance that is distinct from the In the seven-generation DFNY1 pedigree reported in rest of the genome. Y-chromosomal phenotypic traits 2004, all adult males were affected.6 Subsequently, the should thus be readily recognizable from their male-line pedigree was traced back two more generations, and inheritance, and early studies claimed to identify several additional male-line descendants of an earlier ancestor such traits, including notable examples such as "hairy were identified.7 Strikingly, their hearing was unaffected ears." However, as long ago as 1957, critical examination (Figure 1). We had previously demonstrated the identity of these claims failed to find support for any of the 17 of the Y from the two branches of the family traits under consideration,1 a finding reinforced by subse- at 67 Y-STR loci, and we therefore reasoned that the pheno- quent molecular-genetic analysis of "hairy ears" itself.2 typic difference between the branches must be associated This perhaps surprising lack of Y-chromosomal traits can with a genetic variant carried specifically by the Y chromo- be understood at least in part as the consequence of two some of the affected branch. We sorted a representative factors. First, when a reference sequence for the male- Y chromosome from each branch by flow cytometry and specific region of the human Y chromosome was gener- then sequenced it. Only four base-substitutional differ- ated in 2003,3 it revealed that the chromosome carries ences were identified between the chromosomes.8 Three few male-specific genes and codes for only 23 distinct of these had arisen on the unaffected branch. The fourth . Subsequent work has found that three of these had arisen on the affected branch and segregated with are absent from about 2% of South Asian men, whose Y the phenotype, but it provided a poor candidate for the chromosomes thus code for just 20 male-specific causal mutation because it lay outside any annotated proteins.4 Second, the Y chromosome has specialized . Although this analysis could not evaluate the functions in male sex determination and fertility, in repeated regions of the chromosome, the known repeated which Mendelian variation would be unlikely to be genes are involved mainly in spermatogenesis,3,9 so in the heritable. Thus, in early 2004 it was possible to write current study we have explored the possibility that the ‘‘pedigree analysis has yet to reveal a single Y-linked causal mutation might not be a point mutation. This study gene.’’5 Later that year, however, Y-linked hearing impair- was approved by the sample donors, who provided written ment (DFNY1, MIM 400043) was reported in a Chinese informed consent, and by the Committee of Medical family6 and remains the sole documented Mendelian Ethics of the Chinese PLA General Hospital, Beijing, disorder showing Y-linkage in humans. Its basis thus China. seems likely to be unusual and is of considerable interest. We examined the relative read depth along the chromo- Here, we investigate this basis by examining the sequence some by aligning high-quality duplicate-filtered reads to of the DFNY1 Y chromosome and comparing it with the the chrY reference sequence by using Sequence Search

1Department of Otolaryngology, Head and Neck Surgery, Chinese PLA Institute of Otolaryngology, Chinese PLA General Hospital, Beijing 100853, China; 2The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, UK; 3Beijing Genomics Institute at Shenzhen, Shenzhen 518000, China; 4National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Deshengmenwai, Beijing 100088, China 5Present address: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100094, China 6Present address: Department of Genetics and Genomic Sciences, Institute of Genomics and Multiscale Biology, Mount Sinai School of Medicine, New York, NY 10029-6574, USA 7Present address: Oxford Nanopore Technologies, Oxford Science Park, Oxford OX4 4GA, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ajhg.2012.12.015. Ó2013 by The American Society of Human Genetics. All rights reserved.

The American Journal of Human Genetics 92, 301–306, February 7, 2013 301 Figure 1. The DFNY1 Pedigree Males, squares; females, circles; diagonal line, deceased. Filled symbols indicate hearing impairment (including from family records); question marks indicate two individuals whose hearing phenotype is unknown; and asterisks indicate individuals who are on the affected branch but who were below the age of onset of symptoms at the time of examination. Arrows indicate the two individuals whose Y chromosomes were sequenced. The structural rearrangement occurred during one of the four meioses marked by red stars. Spouses are omitted in generations VII-IX and in generation VI on the unaffected branch. and Alignment by Hashing Algorithm 2 (SSAHA2)10 sequence: RP11-334D2 (including TSPY1), RP11-182H20, and comparing the numbers of reads per 10 kb bin RP11-155J5, RP11-160K17, and RP11-108I14 (including between Y chromosomes from the affected individual centromeric sequences). The results (Figure 2C) showed VIII-2 (Figure 1) and the unaffected individual VII-24 that the unaffected chromosome was organized in the (Figure 1). This revealed three discontinuous segments same way as the reference sequence, which can be duplicated in the affected chromosome, and all were summarized as TSPY1-182H20-centromere. In contrast, derived from the limited region between the TSPY1 (MIM the affected chromosome (Figure 2D) was interrupted 480100) gene cluster ending at ~9.3 Mb and the centro- within the 182H20 sequence to produce the organization meric gap at ~10.1 Mb (Figure 2A). These duplications TSPY1-partial 182H20-gap-centromeric duplication- were confirmed by conventional oligonucleotide array TSPY1 duplication-182H20-centromere. The fiber-FISH comparative genomic hybridization (CGH) with an results thus demonstrated that the duplicated Y Agilent 1 million array covering the whole genome and sequences were reinserted locally in a rearranged form, a custom NimbleGen 385K array spanning positions chrY: but they also revealed the presence of a segment that 2,000,000–10,715,000 (1,990,000–10,105,000 hg19) at did not hybridize to any probe from the TSPY1-centro- high (~20 bp) resolution (Figure 2B). Because they included mere region. part of the TSPY1 gene cluster, we verified an increase in In order to identify these unknown sequences, we per- TSPY1 gene number by qPCR (Table S1 in the Supplemental formed thermal asymmetric interlaced PCR (TAIL-PCR)12 Data available online) and pulsed-field gel electrophoresis on the segment extending from the 182H20 breakpoint (PFGE; Figure S1). These analyses showed that the duplica- into the gap by using the primers shown in Table S2.In tion was shared by all affected family members tested and consecutive amplifications, this procedure pairs nested was absent from all unaffected members and also that the primers specific to the known sequence with degenerate duplication lay in a separate restriction fragment from the primers expected to prime in the unknown flanking original TSPY1 cluster and was thus noncontiguous. sequence. Candidate junction products are recognized Although the combined evidence confirmed a complex because their size differences in consecutive reactions duplication of the 9.3–10.1 Mb region, it provided no infor- reflect the known primer positions. The adjacent mation about where the duplicated sequences were in- sequences, confirmed with breakpoint-specific primers serted. Metaphase fluorescence in situ hybridization (Table S3 and Figures S2 and S3), were derived from chro- (FISH) with a TSPY1 probe showed a single signal (not mosome 1 (160.16 Mb), and examination of the read shown), so we used fiber-FISH as described previously11 depth, CGH profiles (Figures 2E and 2F), and sequence sug- for higher resolution. Y-chromosomal probes were BAC gested the involvement of a contiguous region of ~160 kb; clones spanning most of ~9.3–10.1 Mb of the reference such involvement was supported by the identification of

302 The American Journal of Human Genetics 92, 301–306, February 7, 2013 Figure 2. Characterization of the Structural Rearrangement Carried by the DFNY1 Y Chromosome (A) Relative read depth (affected/unaffected) in 10 kb bins mapped to the Y chromosome reference sequence between 9.3 and 10.1 Mb. Note the assembly gaps at each end of this region. (B) CGH log2 ratio (affected/unaffected) for the same region. (C) Fiber-FISH of the three BAC clones indicated to the unaffected Y chromosome, showing that it carries the reference structure in this region. Both 334D2 and 108I14 detect tandem arrays larger than the size of the BAC clone. (D) Fiber-FISH of the same BAC clones to the affected Y chromosome. This chromosome carries an insertion that interrupts the 182H20- hybridizing region and contains partial duplications of both 334D2- and 108I14-hybridizing sequences. (E) Absolute read depth of unaffected and affected Y chromosome reads to the 160.15–160.35 Mb region of the chromosome 1 reference sequence. (legend continued on next page)

The American Journal of Human Genetics 92, 301–306, February 7, 2013 303 Figure 3. Structure and Gene Content of the Unaffected and Affected Y Chromosomes (A) The structure of the unaffected (VII-24 in Figure 1) Y chromosome. Grey and black: errors and gaps, respectively, in the GRCH37 assembly. Blue: region matching the reference sequence at the level of resolution used. Shown below are part of the TSPY1 array (blue arrowheads) and the location of BAC clone signals identified in Figure 2. (B) Structure of the affected (VIII-2 in Figure 1) Y chromosome. This chromosome contains a segment that is not in the unaffected chromosome; this segment is derived from duplications of sequences from both chromosome 1 (yellow) and the Y chromosome (blue). Again, the gene content and BAC signals are shown below. The arrowheads above indicate the orientation of the duplicated segments relative to the reference sequence. (C) Detailed view of the two chromosome 1-Y junctions studied at the sequence level (Figures S2 and S3), showing the difference between the simple structure of junction 1 and the complex structure of junction 2. a second more complex chromosome 1-Y junction at some 1 sequences carry five RefSeq genes (CASQ1 [MIM 160.32 Mb. The translocation of chromosome 1 sequences 114250], PEA15 [MIM 603434], DCAF8 [MIM NA], PEX19 to the affected Y was confirmed by metaphase FISH [MIM 600279], and COPA [MIM 601924]) and the 50 end (Figures 2G and 2H), and their location within the gap in of another gene, NCSTN (MIM 605254) (exons 1–3; the Y duplication was confirmed by fiber-FISH (Figures 2I Figure 3). Mechanisms by which the duplication might and 2J) with BACs RP11-574F21, RP11-179G5, and W12- lead to the deafness phenotype include an increase in 1365F20 from chromosome 1 as probes. The combined gene copy number, inappropriate expression resulting data thus revealed a complex duplication structure consist- from novel flanking DNA, or formation of an altered ing of both a chromosome 1 fragment and multiple product through truncation, fusion, or point mutation. segments of Y DNA (Table S4 and Figures S2 and S3). No expression data are available from the DFNY1 family, None of the sequenced breakpoints exhibited extensive and no nonsynonymous mutations were found in the lengths of homology, but all revealed microhomology, chromosome 1 RefSeq genes (Table S5). TSPY1 copy a pattern indicative of FoSTeS (fork stalling and template number is polymorphic within the population,14 and switching), which is a mechanism that combines disparate larger numbers of TSPY1 copies have been found without genomic fragments during replication.13 reported deafness, including in 47,XYY individuals; one The duplicated structure observed here has not been re- study associated high TSPY1 copy number with a different ported elsewhere and must have arisen during one of just phenotype, infertility.15 Increased TSPY1 copy number four meioses, which encompass the meiosis in which the thus provides a poor candidate for deafness. In contrast, DFNY1 mutation itself occurred (Figure 1). It is therefore the chromosome 1 region lies entirely within an approxi- likely to be causal. The duplicated Y-chromosomal mately 900 kb interval, DFNA49 (MIM %608372), previ- sequences code for only one known , TSPY1, from ously associated with hearing loss; the causal mutation in a tandem array of ~10 TSPY1 genes, whereas the chromo- this interval remains unknown.16 We therefore propose

(F) CGH log2 ratio (affected/unaffected) for the same chromosome 1 region. (G) Metaphase FISH of the chromosome 1 BAC clone 179G5 on the unaffected Y chromosome (yellow). Hybridization is seen only at the reference location on chromosome 1. (H) Metaphase FISH of the same chromosome 1 probe on the affected Y chromosome (yellow). Hybridization is seen at an additional locus on the Y chromosome. (I) Fiber-FISH of the two Y BAC clones indicated plus the chromosome 1 clones 574F21, 179G5 and 1365F20 to the unaffected Y chro- mosome. No chromosome 1 signal is detected on the Y. (J) Fiber-FISH of the same clones to the unaffected Y chromosome. The chromosome 1 clones detect a signal on the Y between the partial 182H20 signal and the 108I14 signal. Genome coordinates refer to GRCh37/hg19.

304 The American Journal of Human Genetics 92, 301–306, February 7, 2013 that the same gene or genes might underlie both the Received: October 16, 2012 DFNA49 and DFNY1 phenotypes. The most parsimonious Revised: November 19, 2012 mechanism would be dosage-sensitivity of one or more of Accepted: December 21, 2012 these genes, such that increased expression resulting from Published: January 24, 2013 three copies leads to hearing loss, although other mecha- nisms are not excluded. Web Resources We found the cause of the human Y-linked Mendelian The URLs for data presented herein are as follows: disorder was associated with insertion of chromosome 1 sequences rather than mutation of a Y-chromosomal Online Mendelian Inheritance in Man (OMIM), http://www. gene. This is consistent with the observations that neither omim.org duplication (47,XYY karyotype) nor deletion (45,X karyo- NCBI RefSeqGene, http://www.ncbi.nlm.nih.gov/refseq/rsg/ type) of the entire Y chromosome and thus of all its genes is associated with hearing impairment, implying that loss Accession Numbers of function or duplication of a Y-chromosomal gene is unlikely to underlie the DFNY1 phenotype. The com- The GenBank accession numbers for the two junction sequences reported in this paper are KC489797–KC489798. plexity of the rearrangement is also consistent with its rarity: Although male-line deafness should be an easily recognizable phenotype, to our knowledge only a single References additional family with a similar phenotype has been re- 17 1. Stern, C. (1957). The problem of complete Y-linkage in man. ported. The relationship between the two families is Am. J. Hum. Genet. 9, 147–166. unknown; the second family is also Chinese, but from 2. 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