Introduction
Hearing impairment is one of the most common human sensory defects, affecting approximately one in 1000 children at birth. In more than half these cases, the defect can be attributed to a genetic cause.Reference Cohen, Gorlin, Gorlin, Toriello and Cohen1, Reference Morton2 Eighty per cent of hereditary hearing impairment is categorised as non-syndromic, based on the absence of other symptoms.Reference Cohen, Gorlin, Gorlin, Toriello and Cohen1 Forms of isolated deafness which are transmitted in the recessive mode deafness, autosomal dominant nonsyndromic-sensorineural (DFNB) are the most frequent (85 per cent) and most severe.Reference Morton2 Autosomal dominant hearing impairment accounts for approximately 22 per cent of cases of profound, non-syndromic hearing impairment at birth.Reference Cohen, Gorlin, Gorlin, Toriello and Cohen1
To date, 54 loci for autosomal dominant, non-syndromic, sensorineural hearing loss have been mapped, and 20 genes coding for a wide variety of proteins have been identified (see hereditary hearing loss homepage: http://webhost.ua.ac.be/hhh/).3 The genes responsible for autosomal dominant, non-syndromic, sensorineural hearing loss are diverse, and encode proteins including members of the gap junction family, cytoskeletal proteins, transcription proteins, ion channels and molecular motors; genes of unknown function have also been implicated.
Despite such advances, the underlying mechanisms of such hearing loss are still unclear. Hearing loss associated with recessive, non-syndromic, sensorineural hearing loss is generally congenital and profound; on the other hand, dominant, non-syndromic, sensorineural hearing loss is characterised by delayed-onset, high-frequency hearing impairment that progresses to involve all frequencies.
Previously, we have mapped a new, novel DFNA locus, DFNA52 (OMIM 607683), to a region of chromosome 5q31.1-q32, using a genome-wide scan.Reference Xia, Deng, Feng, Zhang, Pan and Dai4 The phenotype was characterised by bilateral, postlingual, progressive, non-syndromic, sensorineural hearing impairment involving all frequencies. Fine mapping indicated that the disease gene was located within an 8.8-cM region between the markers D5S2056 and D5S638, with a maximum two-point logarithm of differences score of 6.89 (theta = 0) at D5S2017. No mutations could be detected in either the POU4F3 or DIAPH1 genes, both of which map to 5q31. Furthermore, the genetic interval of the DFNA52 locus overlaps with the DFNA54 and DFNB60 regions;Reference Gurtler, Kim, Mhatre, Schlegel, Mathis and Lalwani5 therefore, it is likely that there is another gene in 5q31 which is associated with nonsyndromic, sensorineural hearing impairment.
Materials and methods
Family data
The clinical characteristics of the DFNA52 family have been described by Xia et al. Reference Xia, Deng, Feng, Zhang, Pan and Dai4 Thirty-eight subjects, representing four generations, were available for testing, 17 of whom were clinically affected (Figure 1).Reference Xia, Deng, Feng, Zhang, Pan and Dai4 Peripheral blood samples were collected from the 38 subjects and deoxyribonucleic acid (DNA) extraction was performed, following the standard phenol-chloroform method. Informed consent was obtained from all subjects, and from the parents of subjects younger than 18 years. Clinical history interviews and physical examinations of subjects ruled out the presence of causative environmental factors and syndromes. Pure tone audiometry was used to test air conduction and bone conduction. Tympanometry and caloric tests were also performed.
Fig. 1 Haplotype analysis of the non-syndromic, sensorineural hearing loss pedigree, illustrating the recombination events between the disease locus and chromosome 5q markers. 17 markers from chromosome 5 are shown in the upper left corner. The haplotype linked to deafness is boxed. Inferred alleles are shown in parentheses. ? = individual possibly affected (i.e. IV:10 and V:6); * = individual with a phenocopy (i.e. III:6)
Candidate gene selection
The NCBI map, view 36.1 (see http://www.ncbi.nlm.nih.gov/mapview/), was used to identify candidate genes in the DFNA52 interval defined by the markers D5S2056 (139.33 cM) and D5S638 (148.63 cM). The corresponding region of DFNA52 is very gene-rich, containing 152 named genes and 26 uncharacterised segments of Complementary DNA. The genes responsible for autosomal dominant, non-syndromic, sensorineural hearing loss are diverse, encoding for many proteins, including: members of the gap junction family, cytoskeletal proteins, transcription proteins, ion channels and molecular motors; genes of unknown function have also been implicated. It is difficult to select likely candidates from such a large number of genes. The selection of candidate genes was based on gene expression information, function, the disease phenotype, genetic studies in a mouse model (regarding phenotypes and expression), and other findings reported in the literature.
Gene expression
Genes expressed specifically in auditory tissues are likely to be good candidates to screen for genetic alterations in patients with deafness. To identify genes within the DFNA52 loci which are expressed in the cochlea, we searched each gene expression profile supplied in the UniGene website (see http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene). We also used three ear gene expression databases, which provide insight into cochlear gene expression and identify candidate genes for deafness: the Table of Gene Expression in the Developing Ear (see http://www.ihr.mrc.ac.uk/hereditary/genetable/), the Human Cochlear Complementary DNA Library, and the Expressed Sequence Tag Database (see http://hearing.bwh.harvard.edu/estinfo.htm), and the ear libraries on the Neibank website (see http://neibank.nei.nih.gov/cgi-bin/libList.cgi?tissue=ear).6–Reference Pompeia, Hurle, Belyantseva, Noben-Trauth, Beisel and Gao8 Tables of gene expression in the developing ear comprise collections of the many genes that are expressed at different times during inner-ear development, for different animal species. The Human Cochlear cDNA Library and the Expressed Sequence Tag Database (2006 update) were obtained from the Fetal Cochlear cDNA library of the Morton Hearing Research Group. The Neibank website contains an expressed sequence tag database for the mouse organ of Corti.
These resources provide an overview of the cochlear gene expression profile, and constitute tools with which to identify candidate genes for deafness. Of the genes located in the candidate chromosome interval which are expressed in the inner ear, 34 were considered as potential candidate genes for the DFNA52 locus (Table I). These results indicated that screening genes that are highly expressed in the inner ear might be an efficient approach to identifying aberrant proteins involved in deafness.
Sequence of mouse cochlear cDNA analysis
We analysed sequences of mouse organ of Corti cDNA, pBluescript (The Single Nucleotide Polymorphism Database (dbEST) library identity number 10920) (see http://www.ncbi.nlm.nih.gov/UniGene/library.cgi?ORG=Mm&LID=10920), and subtracted the cochlear cDNA library (dbEST library identity number 12768) found at the UniGene. DNA sequence similarity (the basic local alignment search tool) analysis was performed, in order to compare nucleotide sequences of interest with the mouse genome database, and also to determine the chromosomal location of the relevant human orthologues. Sixteen genes in the target interval were considered to be potential functional candidate genes for DFNA52.
Human–mouse homology relationships and mouse deafness model
Interspecies homologies and mouse–human comparative mapping in the vicinity of the human disease loci can also provide a powerful approach to accelerate positional cloning of disease genes. In hearing impairment research, this approach has been proven effective in the cases of MYO7A (USH1B, DFNB2 and DFNA11),Reference Weil, Blanchard, Kaplan, Guilford, Gibson and Walsh9–Reference Liu, Walsh, Tamagawa, Kitamura, Nishizawa and Steel12 MYO15 (DFNB3) and POU4F3 (DFNA15).Reference Wang, Liang, Fridell, Probst, Wilcox and Touchman13, Reference Vahava, Morell, Lynch, Weiss, Kagan and Ahituv14 Therefore, it is recommended to investigate the mouse homologue region and the availability of a relevant mouse mutant which maps to the homologue region. There are more than 200 mouse mutations associated with inner-ear dysfunction (see Hereditary Hearing Impairment in Mice; http://www.jax.org/hmr/). Some human hearing disorders have been identified that have parallel mouse disorders due to mutations in orhologous genes. The human chromosome 5q31 DFNA52 locus seems to be homologous to a region on mouse chromosome 13,18. It has been reported that the mouse NEUROG1 and SMAD5 gene mutations are related to deafness and inner-ear defects.Reference Ma, Anderson and Fritzsch15, Reference Guo, Yang, Liu, Sun, Yang and Han16
Literature review
Through the analysis of expression profiles for human cochlear and vestibular tissues on a cDNA microarray, Abe et al. identified a set of 52 genes that were specifically or preferentially expressed in the inner ear, and investigated their possible roles in patients with non-syndromic deafness.Reference Abe, Katagiri, Saito-Hisaminato, Usami, Inoue and Tsunoda17 Of these 52 genes, the PCDHGC3 gene (encoding protocadherin gamma subfamily C 3) was located in the same chromosomal region as the DFNA52 loci.
Alsaber et al. established a list of strong candidate genes within regions linked to various non-syndromic hereditary hearing loss phenotypes, by using a novel bioinformatics approach.Reference Alsaber, Tabone and Kandpal18 Eleven genes (FGF1, GFRA3, IK, PCDH1, DIAPH1, POU4F3, TTID, NRG2, PCDHAC1, PCDHAC2 and NDFIP1) were identified as possible candidates located within the DFNA52 locus. The candidates presented here provide a starting point for mutational analysis of the DFNA52 locus.
Gene prediction according to phenotype of known deafness genes
The phenotype of the DFNA52 locus is characterised by bilateral, postlingual, progressive, non-syndromic, sensorineural hearing impairment involving all frequencies. These clinical features are similar to the hearing loss caused by mutations in genes encoding the myosin superfamily and in KCNQ4 in autosomal dominant, non-syndromic, sensorineural hearing loss. There were no genes encoding the myosin superfamily within the critical region of the DFNA52 locus. The KCTD16 gene (encoding a potassium channel tetramerisation domain containing 16), which maps to this region, was considered a candidate because mutations of the KCNQ4 gene, a member of the potassium channel genes, are known to cause the DFNA2.Reference Coucke, Van Hauwe, Kelley, Kunst, Schatteman and Van Velzen19, Reference Kubisch, Schroeder, Friedrich, Lutjohann, El-Amraoui and Marlin20
We also selected SLC23A1, PCDHB3, TRPC7, LRRTM2, TAF7, ARHGAP26, ETF1 and PACAP as candidate genes. These genes are known to be likely to share similarities with known deafness genes, or else their gene function is possibly associated with deafness.
Sequencing and analysing
For mutation analysis, all exons and intron–exon boundaries of selected genes were amplified by polymerase chain reaction, using genomic DNA of the proband as a template and primers designed according to the genomic sequences obtained from Genbank (NIH (National Institutes of Health) genetic sequence database). The polymerase chain reaction products were sequenced using an ABI Prism BigDye terminator cycle sequencing reaction kit (Applied Biosystems, Foster City, California, USA). An automated sequencer (ABI 3100, Applied Biosystems) was used for direct sequencing. Sequence assembly was conducted using DNAStar (DNASTAR Inc., Madison, WI). When a variation was detected, single nucleotide polymorphism BLAST software (see http://www.ncbi.nlm.nih.gov/blast/) was used to exclude polymorphism. Then, all subjects' DNA samples were screened to test whether the variation co-segregated with the disease.
Results and analysis
Screening of candidate genes
We located 52 genes which we considered to be potential functional candidate genes for the DFNA52 locus, from either the above mentioned expression databases, our own analysis or other study results (Table I).
Table I Candidate genes in the DFNA52 interval
cDNA = copy deoxyribonucleic acid ; EST = expressed sequence tag
Mutational analysis
Mutation detection for the 52 candidate genes located in the critical region of the DFNA52 locus was performed in two subjects. We found no disease-causing mutations in the coding and splice site regions of these genes, which segregated with the disease. However, 108 single nucleotide polymorphisms were identified, of which 15 were novel. Eleven of the 108 single nucleotide polymorphisms changed the encoded amino acid (Table II).
Table II Candidate Gene Screening and Variant Detection
refSNP ID = In the first column, numbers prefixed with “rs” are public reference SNP numbers from the dbSNP database. Item not found in this database are listed as “Novel”; Ex = exon; IVS = intron; UTR = untranslated region; A = adenine; C = cytosine; G = guanine; T = thymine; Gly =glycine; Ser = serine; Pro = proline; Gln = glutamine; Lys = lysine; Leu = leucine; Asn = asparagine; Ala = alanine; Phe = phenylalanine; Arg = arginine; Ile = isoleucine; Thr = threonine; Val = valine; Tyr = tyrosine; SNP = single nucleotide polymorphism
Discussion
Our previous genome-wide scan had shown linkage to the DFNA52 locus within an interval defined by D5S2056 and D5S638 on chromosome 5q31.1-q32.Reference Xia, Deng, Feng, Zhang, Pan and Dai4 No mutations could be detected in either the POU4F3 or DIAPH1 genes, both of which map to the DFNA52 interval. Moreover, the genetic interval of the DFNA52 locus overlaps with the DFNA55 and DFNB60 regions, and it is likely that there is another gene (or genes) in 5q31 associated with non-syndromic hearing impairment. This DFNA52 interval spans 12 Mb and comprises 178 genes, including known genes and several predicted or poorly characterised genes, according to the National Center for Biotechnology Information database.
We selected 52 genes for mutation screening, within the DFNA52 interval. Analysis of coding and splice site regions of these genes did not reveal any potentially pathogenic mutations segregating with the disease. This in return implies that these genes are unlikely candidates for DFNA52.
Because of the similarities between the physiology and morphology of their auditory systems, mouse models have been used as tools in the discovery and characterisation of genes for non-syndromic deafness in humans. The NEUROG1 and SMAD5 genes are plausible candidates within the DFNA52 locus.
The proneuronal gene neurogenin 1 (official symbol: NEUROGI; previous name: NGN1) is essential for the development of inner-ear sensory neurons, and these are completely absent in NGN1 null mutants. NGN1 null mutant ears develop smaller sensory epithelial cells, with hair cells that are morphologically normal but disorganised and reduced in number.Reference Ma, Anderson and Fritzsch15 We sequenced all the coding regions and the untranslated region (UTR) and conserved sequences in upstream regions of the gene, in order to search for any pathogenic variants.Reference Blader, Lam, Rastegar, Scardigli, Nicod and Simplicio21 The results of mutation analysis showed that there was only one sequence alteration, an rs8192558 single nucleotide polymorphism in the 5′ UTR region, which did not segregate with the disease.
In the mouse, knockout of the SMAD5 gene can cause a moderate or severe auditory threshold decline.Reference Guo, Yang, Liu, Sun, Yang and Han16 Morphological assessment indicates that hair cells (mainly outer hair cells) in the mouse cochlea basal membrane become deficient.Reference Sun, Yang, Sun, Yang, Han and Yang22 The SMAD5 gene expresses in the cochlea at a high level. SMAD5 gene-encoded proteins occur in the mouse cochlea and may be involved in cochlear formation and hair cell differentiation. The SMAD5 gene may be essential to the development of a normal cochlea. SMAD5 gene knockout causes deafness and hair cell deficiency, although the mechanism requires further study. We sequenced and analysed three transcripts of the SMAD5 gene. We found five resulting single nucleotide polymorphisms, but none of these segregated with the disease. Wang et al. analysed SMAD5 mutation in 143 Chinese patients with different phenotypes of hearing loss, and identified five single nucleotide polymorphisms in the introns of the SMAD5 gene.Reference Wang, Li, Zong, Guo, Lan, Yuan and Zhao23 The results implied that the SMAD5 gene might not be the direct causative gene for deafness in these patients.Reference Wang, Li, Zong, Guo, Lan, Yuan and Zhao23
Another interesting candidate gene within the DFNA52 interval is SLC23A1, a member of the solute carrier family. Mutations in another solute carrier family member, SLC26A4, are responsible for DFNB4,Reference Li, Everett, Lalwani, Desmukh, Friedman and Green24 Pendred syndromeReference Everett, Glaser, Beck, Idol, Buchs and Heyman25 and enlarged vestibular aqueduct syndrome.Reference Usami, Abe, Weston, Shinkawa, Van Camp and Kimberling26 SLC26A4 mutations account for approximately 10 per cent of hereditary deafness in diverse populations, including eastern and southern Asians.Reference Park, Shaukat, Liu, Hahn, Naz and Ghosh27 However, despite the fact that all 15 protein-coding exons of SLC23A1 have been sequenced (using DNA samples from two affected family members), no mutation has been found.
In our study, the genes PCDHB5, PCDHAC1, PCDHAC2, PCDHB3 and CTNNA1 were selected as good functional candidates for the DFNA52 locus. The cadherin 23 gene (CDH23) has been confirmed to be the cause of type 1D Usher syndrome and DFNB12.Reference Bolz, von Brederlow, Ramirez, Bryda, Kutsche and Nothwang28, Reference Bork, Peters, Riazuddin, Bernstein, Ahmed and Ness29 Mutations of PCDH15, encoding protocadherin 15, are known to cause Usher syndrome type 1F and DFNB23.Reference Ahmed, Riazuddin, Ahmad, Bernstein, Guo and Sabar30, Reference Ahmed, Riazuddin, Bernstein, Ahmed, Khan and Griffith31 Moreover, two gene mutations cause deafness in the deaf mouse mutants Waltzer (CDH23 gene) and Ames–Waltzer (PCDH15 gene).Reference Di Palma, Holme, Bryda, Belyantseva, Pellegrino and Kachar32, Reference Alagramam, Murcia, Kwon, Pawlowski, Wright and Woychik33 However, the results of mutation screening for five genes within the DFNA52 locus were negative. This apparently excluded them as candidates. However, there are three closely linked tandem protocadherin gene clusters (alpha, beta and gamma) on human chromosome 5q31, including 57 genes and six pseudogenes. It is difficult to screen all protocadherin genes, and other protocadherin genes mapping to the region have demonstrated themselves as suitable candidates for the DFNA52 locus.
In conclusion, we screened for mutations 52 genes linked to the DFNA52 locus, within a Chinese family with congenital sensorineural hearing loss. Although these genes were plausible candidates due to their expression profiles and functions, no potentially pathogenic mutations segregating with the disease were found. These findings suggest that these 52 genes are unlikely to be involved in DFNA52 locuas. However, these genes cannot be completely ruled out as pathogenic; as we did not screen the regulatory regions (i.e. promoter, 5′ and 3′ UTRs) of these genes, the possibility of a functional variant in the intronic regions cannot be discounted. It may also be the case that a new, novel gene within this interval is responsible for the pathology; if so, mutation detection of this novel candidate gene needs to be confirmed. Expression profiling of the remaining known and predicted genes should lead to new candidate genes.
• Hearing impairment is one of the most common human sensory defects, affecting approximately one in 1000 children at birth. In more than half of these cases, the defect can be attributed to a genetic cause
• To date, 54 loci for autosomal dominant, non-syndromic, sensorineural hearing loss have been mapped, and 20 genes, coding for a wide variety of proteins, have been identified
• This study attempted to locate specific gene mutations responsible for deafness in a large, consanguineous Chinese family with congenital sensorineural hearing loss
• No disease-causing mutation in the coding and splice site regions of these genes, which segregated with the disease, was found
Future work required includes the screening of other candidate genes, recruiting future generations within the same family pedigree, and the detection of new families with hearing impairment linked to the DFNA52 locus, in order to refine the genetic interval involved, which will assist in the task of isolating the DFNA52 gene responsible.
Conclusion
Bioinformatics analysis was used to identify 52 candidate disease genes, within the DFNA52 locus, based on gene expression data, deafness phenotype, mouse model findings and relevant literature. Although we identified a number of nucleotide changes in affected patients, by analysis of the coding and splice site regions of these genes, none of these changes are likely to be pathogenic mutations segregating with the disease. The results imply that these genes are unlikely to be the cause of DFNA52-related sensorineural hearing loss; this includes two identified mouse deafness genes (NEUROG1 and SMAD5).
Acknowledgements
We would like to thank our subjects for their participation. We also thank Fengxiao Bu, Lingqian Wu, Desheng Liang and Qian Li at the National Laboratory of Medical Genetics of China, and Chufeng He in the Department of Otorhinology, Xiangya Hospital, Central South University, for their contribution. This study was supported by the National Natural Science Foundation of China (grants 30630062, 30600334 and 30470954) and the ‘973’ programme of China (grant 2004CB518601).
