Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs

The objective of the present study was to localize the gene or genomic region that is involved in the development of canine congenital sensorineural deafness (CCSD) in Dalmatian dogs. In man as in different dog breeds deafness is an often diagnosed disorder with the Dalmatian dog showing the highest incidence. Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate a high level of clinical and molecular similarity. Altogether 39 genes have already been found causative for sensorineural non-syndromic hearing impairment in humans. Out of this 39 deafness causing genes a total of 32 candidate genes were selected for canine congenital deafness, which showed an appropriate clinical and histological disease pattern in comparison to deafness in Dalmatians dogs. An existing set of 43 microsatllite markers for in total 24 candidate genes were used for a non-parametric linkage analysis, among them the claudin-14 (CLDN14) gene on canine chromosome (CFA) 31 and the myosin, heavy polypeptide 9 (MYH9) gene on CFA10. The gap junction protein, alpha 1 (GJA1) gene on CFA1 was also considered as a candidate gene for CCSD and thus GJA1-associated microsatellites were part of the existing set. Recently it turned out, that GJA1 is not responsible for human sensorineural non-syndromic deafness, but for a human syndromic disorder that can be related with conductive deafness. In the last few years more human deafness genes have been identified, among them eight genes that were considered as appropriate candidates for CCSD. For these eight genes a total of 21 SNPs were newly developed and used for non-parametric linkage and association analyses. The used microsatellite and SNP markers derived either from a partial sequence analysis of BAC clones each containing a different candidate gene, or from sequences deposited in the current dog genome assembly (Boxer genome assembly 2.1) of the NCBI GenBank. We found significant linkage to markers associated to CLDN14 on CFA31, MYH9 on CFA10 and GJA1 on CFA1. To substantiate the linkage we searched for sequence variations within these three genes. SNPs found in intronic sequences of either gene were included in the linkage analyses. Sequence analysis neither revealed a causative mutation nor significant linkage disequilibrium of SNP markers with CCSD. Subsequently, CFA1, 10 and 31 were scanned completely with microsatellite markers derived from the NCBI database with the purpose to see if other regions on this three chromosomes harbour a gene that is involved in the development of CCSD. The analyses of SNPs and more microsatellite markers on CFA1 revealed no significant linkage to CCSD. Thus, it is unlikely that the canine chromosome and the GJA1 gene are responsible for the CCSD phenoptype. As deafness in dogs, especially in Dalmatians, is almost exclusively caused by sensorineural non-syndromic forms, the GJA1 gene should not be considered as a candidate gene for CCSD anymore. On CFA10 we could exclude MYH9 for being causal for deafness, but by adding more microsatellites covering CFA10 completely we found significant linkage to the CCSD phenotype in the region distal of MYH9. Hence, new SNP-markers for fine mapping the region spanning 36 to 48 Mb were developed by sequence analyses of different Dalmatian dogs. The search for SNPs was carried out on genomic sequences of genes located in the significantly linked region. The sequences of these genomic sequences were derived from the NCBI GenBank. The SNPs confirmed the linkage and narrowed the region harbouring a causative CCSD gene down to 5 Mb spanning from 39 to 44 Mb. After scanning CFA31 we could not exclude CLDN14 for being responsible for the CCSD phenotype as microsatellite markers associated to CLDN14 indicated linkage. However, to clarify the importance of CLDN14 in the CCSD phenotype, more SNPs have to be developed within the CLDN14 gene as well as in its flanking regions with the aim to find linkage disequilibrium for SNP markers.