Detailed four-way comparative mapping and gene order analysis of the canine ctvm locus reveals evolutionary chromosome rearrangements

Canine tricuspid valve malformation (CTVM) maps to canine chromosome 9 (CFA9), in a region syntenic with gene-dense human chromosome 17q. To define synteny blocks, we analyzed 148 markers on CFA9 using radiation hybrid mapping and established a four-way comparative map for human, mouse, rat, and dog. We identified a large number of rearrangements, allowing us to reconstruct the evolutionary history of individual synteny blocks and large chromosomal segments. A most parsimonious rearrangement scenario for all four species reveals that human chromosome 17q differs from CFA9 and the syntenic rodent chromosomes through two macroreversals of 9.2 and 23 Mb. Compared to a recovered ancestral gene order, CFA9 has undergone 11 reversals of <3 Mb and 2 reversals of >3 Mb. Interspecies reuse of breakpoints for micro- and macrorearrangements was observed. Gene order and content of the ctvm interval are best extrapolated from murine data, showing that multispecies genome rearrangement scenarios contribute to identifying gene content in canine mapping studies.     

Physical and radiation hybrid mapping of canine chromosome 12, in a region corresponding to human chromosome 6p12-q12

The positional cloning of the hypocretin receptor 2, the gene for autosomal recessive canine narcolepsy, has led to the development of a physical map spanning a large portion of canine chromosome 12 (CFA12), in a region corresponding to human chromosome 6p12-q13. More than 40 expressed sequence tags (ESTs) were used in homology search experiments, together with chromosome walking, to build both physical and radiation hybrid maps of the CFA12 13-21 region. The resulting map of bacterial artificial chromosome ends, ESTs, and microsatellite markers represents the longest continuous high-density map of the dog genome reported to date. These data further establish the dog as a system for studying disease genes of interest to human populations and highlight feasible approaches for positional cloning of disease genes in organisms where genomic resources are limited.     

Comparative sequence analysis and radiation hybrid mapping of the canine keratin 10 gene.

The type I keratin, K10, is expressed in epidermal keratinocytes undergoing terminal differentiation to form the stratum corneum, a barrier essential for life. In order to facilitate the study of keratinization disorders in the dog, the sequence and mapping of KRT10 is reported. The coding region of KRT10 is 1707 bp and is comprised of eight exons. Although the length of KRT10 has been reported to be polymorphic in humans, this was not observed in the eight domestic dog breeds studied, although one wild canid displayed a size difference. The structure and sequence of this gene is highly conserved across mammalian species. Canine K10 had an 86% amino acid identity with the human gene. KRT10 was localized to the on-going canine radiation hybrid map to chromosome 9 in the type I keratin gene cluster.     

Structure,chromosomal location,and analysis of the canine Cu/Zn superoxide dismutase (SOD1) gene

Mutations in Cu/Zn superoxide dismutase (SOD1), a major cytosolic antioxidant enzyme in eukaryotic cells, have been reported in approximately 20% of familial amyotrophic lateral sclerosis (FALS) patients. Hereditary canine spinal muscular atrophy (HCSMA), a fatal inherited motor neuron disease in Brittany spaniels, shares many clinical and pathological features with human motor neuron disease, including FALS. The SOD1 coding region has been sequenced and cloned from several animal species, but not from the dog. We have mapped the chromosomal location, sequenced, and characterized the canine SOD1 gene. Extending this analysis, we have evaluated SOD1 as a candidate for HCSMA. The 462 bp SOD1 coding region in the dog encodes 153 amino acid residues and exhibits more than 83% and 79% sequence identity to other mammalian homologues at both the nucleotide and amino acid levels, respectively. The canine SOD1 gene maps to CFA31 close to syntenic group 13 on the radiation hybrid (RH) map in the vicinity of sodium myo/inositol transporter (SMIT) gene. The human orthologous SOD1 and SMIT genes have been localized on HSA 21q22.1 and HSA 21q21, respectively, confirming the conservation of synteny between dog syntenic group 13 and HSA 21. Direct sequencing of SOD1 cDNA from six dogs with HCSMA revealed no mutations. Northern analysis indicated no differences in steady-state levels of SOD1 mRNA.     

ATP6H, a subunit of vacuolar ATPase involved in metal transport: evaluation in canine copper toxicosis

Copper toxicosis (CT), resulting in liver disease, occurs commonly in Bedlington terriers. Canine CT is of particular interest because identification of the causative gene may lead to the discovery of another important gene in the copper transport pathway possibly related to human copper diseases not yet identified. Homologs of the copper transporting ATPase ATP7B, defective in Wilson disease, and the copper chaperone ATOX1 were potential candidates, but both have been excluded. The CT locus in Bedlington terriers has been mapped to canine chromosome region CFA10q26, which has a syntenic human chromosome region, HAS2p13-21. The gene ATP6H, for human vacuolar proton-ATPase subunit M9.2, is associated with copper and iron transport in yeast and has been mapped to HAS2p21 and suggested as a candidate gene for CT. We cloned canine ATP6H, which encodes a predicted protein with 99% amino acid sequence identity to the orthologous human protein. Canine ATP6H shows a conserved potential metal binding site, CSVCC, and a glycosylation site, NET. The canine ATP6H is organized into four exons, with a 246-bp open reading frame. Sequence analysis of the coding regions showed no mutations in ATP6H from genomic DNA of an affected dog. We have also identified two, apparently non-transcribed canine ATP6H pseudogenes. Mapping of the true ATP6H gene and a marker closely linked to the CT locus on a canine radiation hybrid panel indicted lack of close physical association. We have therefore excluded canine ATP6H as a candidate gene for canine copper toxicosis, indicating that some other unidentified gene is responsible for this copper storage disease. Received: 8 February 2001 / Accepted: 12 April 2001     

Comparative sequence analysis and radiation hybrid mapping of two epidermal type II keratin genes in the dog: keratin 1 and keratin 2e

In order to extend knowledge of the process of cornification across species and to be better able to recognize inborn errors in keratin synthesis in the dog, we describe the organization and chromosome mapping of canine KRT1 and KRT2E and compare these results to human and murine sequence data. The coding regions of KRT1 and KRT2E are 1,860 bp and 1,902 bp respectively, distributed over nine exons. Both genes are localized on the canine radiation hybrid map to chromosome 27 in the type II keratin gene cluster close to polymorphic markers. These genes are highly conserved across species and based on both genomic and amino acid sequences, canine KRT1 and KRT2E share greater homology with humans than with mice.     

RXRA and HSPA5 map to the telomeric end of dog chromosome 9

Previous results showed that loci from human chromosome 17q (HSA17q) map to the centromeric two-thirds of dog chromosome 9 (CFA9). In these studies fluorescence in situ hybridization (FISH) using a human total chromosome 17 painting probe, indicated that the telomeric one-third of CFA9 must have homology to one or more human chromosomes other than HSA17. Here we report that this distal part of CFA9 contains a segment syntenic to the telomeric end of HSA9q and mouse chromosome 2 (MMU2). The gene loci encoding retinoid X receptor, alpha (RXRA) and heat shock protein 5 (HSPA5 or GRP78), which are found on HSA9q34 and MMU2, occupy a region on CFA9 distal to NF1 and CRYBA1. FISH of a canine specific genomic cosmid clone for RXRA demonstrated the more telomeric localization of this locus to NF1 on CFA9. A linkage map developed for the distal region of CFA9 included: NF1-(2·7 CM )-CRYBA1-(6·5 CM )-RXRA-(22 CM )-HSPA5. The next best order, RXRA-NF1-CRYBA1-HSPA5 with a difference in the log odds of 1·43 does not correspond to our findings with FISH. The most probable map order places HSPA5 distal to RXRA on CFA9 whereas in humans it lies centromeric of RXRA on HSA9q34.     

A high-resolution comparative map of canine Chromosome 5q14.3–q33 constructed utilizing the 1.5× canine genomesequence

A high-density map of the region of canine Chromosome 5 (CFA5) surrounding the evolutionary breakpoint between human Chromosomes 1p32 and 17p11 was constructed by integrating a radiation hybrid map including 41 microsatellites, 10 BACs, and 59 genes and a linkage map including 18 markers. A collection of canine genomic survey sequences providing 1.5× coverage was used to identify dog orthologs of human genes, proving instrumental in the development of this map. Of particular interest is the canine BHD gene, within which we have previously described a single nucleotide polymorphism associated with Hereditary Multifocal Renal Cystadenocarcinoma and Nodular Dermatofibrosis (RCND) in German Shepherd dogs. The corresponding region of the human genome is particularly gene rich, containing genes involved in development, metabolism, and cancer that are likely to be of interest in future mapping studies. This current mapping effort on CFA5 expands the degree to which initial findings of linkage in canine families can be followed by successful positional cloning efforts and increases the value of the human genome sequence for defining candidate genes. Moreover, this study demonstrates the utility of genomic survey sequences when combined with accurate genome maps for rapid mapping of disease susceptibility loci.     

Chromosomal assignment of canine TSC2, PKD1 and CLN3 genes by radiation hybrid- and linkage analyses

The canine tuberous sclerosis 2 (TSC2) gene has been mapped to canine chromosome 6 using a canine whole genome radiation hybrid panel. There is close linkage between canine TSC2 and the polycystic kidney disease 1 gene (PKD1), as has been observed in humans and other mammalian species. The gene responsible for the human juvenile form of neuronal ceroid lipofuscinosis (CLN3), maps close to TSC2 and PKD1 in humans, and is also syntenic in the dog. We further demonstrate linkage to a group of polymorphic markers assigned to canine chromosome 6 (CFA6).     

Refined genetic and comparative physical mapping of the canine copper toxicosis locus

Recently, the copper toxicosis (CT) locus in Bedlington terriers was assigned to canine chromosome region CFA10q26, which is homologous to human chromosome region HSA2p13-21. A comparative map between CFA10q21-26 and HSA2p13-21 was constructed by using genes already localized to HSA2p13-21. A high-resolution radiation map of CFA10q21-26 was constructed to facilitate positional cloning of the CT gene. For this map, seven Type I and eleven Type II markers were mapped. Using homozygosity mapping, the CT locus could be confined to a 42.3 cR₃₀₀₀ region, between the FH2523 and C10.602 markers. On the basis of a partial BAC contig, it was estimated that 1-cR₃₀₀₀ is equivalent to approximately 210 kb, implying that the CT candidate region is therefore estimated to be about 9 Mb. Received: 16 December 1999 / Accepted: 23 February 2000     

A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps

Cross-species reciprocal chromosome painting was used to delineate homologous chromosomal segments between domestic dog, red fox, and human. Whole sets of chromosome-specific painting probes for the red fox and dog were made by PCR amplification of flow-sorted chromosomes from established cell cultures. Based on their hybridization patterns, a complete comparative chromosome map of the three species has been built. Thirty-nine of the 44 synteny groups from the published radiation hybrid map and 33 of the 40 linkage groups in the linkage map of the dog have been assigned to specific chromosomes by fluorescence in situ hybridization and PCR-based genotyping. Each canine chromosome has at least one DNA marker assigned to it. The human-canid map shows that the canid karyotypes are among the most extensively rearranged karyotypes in mammals. Twenty-two human autosomal paints delineated 73 homologous regions on 38 canine autosomes, while paints from 38 dog autosomes detected 90 homologous segments in the human genome. Of the 22 human autosomes, only the syntenies of three chromosomes (14, 20, and 21) have been maintained intact in the canid genome. The dog-fox map and DAPI banding comparison demonstrate that the remarkable karyotype differences between fox (2n = 34 + 0-8 Bs) and dog (2n = 78) are due to 26 chromosomal fusion events and 4 fission events. It is proposed that the more easily karyotyped fox chromosomes can be used as a common reference and control system for future gene mapping in the DogMap project and CGH analysis of canine tumor DNA.     

Chromosome 1 localization of the human alpha-L-fucosidase structural gene with a homologous site on chromosome 2

Two cDNA clones coding for human alpha-L-fucosidase, one from the coding region and the other primarily from the 3' untranslated region, were used to map the location of the alpha-L-fucosidase gene. Southern filter analysis of somatic cell hybrid lines mapped the structural gene to the short arm of human chromosome 1, and in situ hybridization to chromosomes of human leukocytes further localized the homologous area to the 1p36.1----p34.1 region, with the most likely location being the distal region of 1p34. Further Southern filter analysis detected a second site of homology on chromosome 2. This alpha-L-fucosidase-like site has been designated FUCA1L.     

Linkage disequilibrium mapping in domestic dog breeds narrows the progressive rod-cone degeneration interval and identifies ancestral disease-transmitting chromosome

Canine progressive rod-cone degeneration (prcd) is a retinal disease previously mapped to a broad, gene-rich centromeric region of canine chromosome 9. As allelic disorders are present in multiple breeds, we used linkage disequilibrium (LD) to narrow the approximately 6.4-Mb interval candidate region. Multiple dog breeds, each representing genetically isolated populations, were typed for SNPs and other polymorphisms identified from BACs. The candidate region was initially localized to a 1.5-Mb zero recombination interval between growth factor receptor-bound protein 2 (GRB2) and SEC14-like 1 (SEC14L). A fine-scale haplotype of the region was developed, which reduced the LD interval to 106 kb and identified a conserved haplotype of 98 polymorphisms present in all prcd-affected chromosomes from 14 different dog breeds. The findings strongly suggest that a common ancestor transmitted the prcd disease allele to many of the modern dog breeds and demonstrate the power of the LD approach in the canine model.     

Assignment of the gene coding for human β-glucocerebrosidase to the region q21-q31 of chromosome 1 using monoclonal antibodies

A series of man-Chinese hamster somatic cell hybrids with a variable content of human chromosomes was used to study the localization of the human gene coding for the lysosomal enzyme beta-glucocerebrosidase (EC 3.2.1.45). In lysates made from hybrid cells, the human enzyme was specifically recognized by a mouse monoclonal antibody raised against human placental beta-glucocerebrosidase. This monoclonal antibody did not cross-react with Chinese hamster beta-glucocerebrosidase. After reaction of the antibody with the enzyme, beta-glucocerebrosidase was precipitated by addition of Protein A-Sepharose beads, and was detected on the beads by its enzymatic activity. From the analysis of a series of man-Chinese hamster hybrids, among which were hybrids with specific segments of chromosome 1, we conclude that the gene coding for human beta-glucocerebrosidase is localized in the region q21-q31 of chromosome 1.     

Comparative mapping of the DiGeorge region in the dog and exclusion of linkage to inherited canine conotruncal heart defects.

Conotruncal defects (CTDs) of the heart are a frequent component of DiGeorge, velocardiofacial, or other syndromes caused by deletions of the human chromosome 22q11 region (HSA22q11). In addition, some human patients with isolated nonsyndromic CTDs have been reported to have deletions of this region. Taken together, these findings lead to the conclusion that deletions of an HSA22q11 locus or loci produce abnormalities in cardiac development leading to CTDs. A spontaneous model of isolated inherited conotruncal malformations occurs in the keeshond dog. We have previously shown in experimental matings that nonsyndromic CTDs in the keeshond are inherited in a manner consistent with a major underlying locus. In the studies described in this article we tested two hypotheses: (1) the region of HSA22q11 commonly deleted in DiGeorge and related syndromes is evolutionarily conserved in the dog, and (2) a locus in this region is linked to hereditary CTD in the keeshond. Two loci within the minimal DiGeorge critical region (MDGCR) and two loci that lie telomeric to the MDGCR, one of which is commonly deleted in DiGeorge patients, were mapped in the dog using a combination of linkage analysis and fluorescence in situ hybridization (FISH). The results confirm conserved synteny of the loci DGS-I, CTP, D22S788 (N41), and IGLC on the telomeric end of canine chromosome 26 (CFA26). The group of four syntenic gene loci, which spans a genetic distance of 2.5 cM is the first to be mapped to this small acrocentric canine chromosome and adds gene-associated polymorphic markers to the developing dog linkage map. Linkage of loci in this region to hereditary CTD in the keeshond was excluded.     

Mapping of EST- and STS-markers in the human genome using a panel fo radiation hybrids

Ten DNA markers were localized in the human genome by a screening procedure against the radiation hybrid somatic cell panel (GeneBridge 4 RH Panel) using polymerase chain reaction (RH mapping method). DNA markers were developed to nucleotide sequences adjacent to NotI sites of human chromosome 3 (NotI-STS markers) and also to nucleotide sequences of human cDNA (EST markers). Three EST markers mapped (B10164, S16R and 18F5R) were localized in the human genome for the first time. Marker B10164 was found to be homologous to the nucleotide sequence of the BASP1 gene coding a major receptor protein. Markers S16R and 18F5R presumably tagged new genes, because no homologies were revealed among the nucleotide sequences presented in the databases. For four NotI-STS, more precise localization on human chromosome 3 was determined. On the basis of the data obtained, the NotI map may be integrated with other types of physical maps of human chromosome 3. RH mapping with a standard commercial panel of radiation hybrid somatic cells provided a chance to integrate the data obtained into international databases and existing integrated human chromosomal maps.     

Characterization of three microsatellite loci linked to the canine RP3 interval

X-linked retinitis pigmentosa (XLRP) is one of the most prevalent forms of a genetically heterogeneous group of inherited retinal disorders of man; more than 70% of XLRP families map to the RP2 or RP3 loci on the human X chromosome. Canine X-linked progressive retinal atrophy (XLPRA), observed in the Siberian husky, is the locus homologue of human RP3, but the gene responsible for XLPRA has not yet been identified. To develop polymorphic markers in the RP3 interval in dogs we have isolated microsatellites from canine BAC clones. Three tightly linked microsatellite loci, CUX20001, CUX30001, and CUX40002, have been investigated in 17 dog breeds or breed varieties. Calculated parameters of variability correspond with the number of repeats at each locus. Pedigree analyses showed tight linkage between the canine t-complex-associated testis-expressed 1-like gene (TCTE1l) and the gene ornithine carbamoyltransferase (OTC). Each microsatellite shows conservation within Canidae, and CUX20001 also amplified in Mustelidae and URSIDAE: These markers represent an important tool in the fine mapping process for the canine region homologous to the RP3 disease interval and are valuable for evaluation of conservation and homology of this region among related species.     

Characterization and chromosome assignment of the canine gamma-sarcoglycan gene (SGCG) to CFA 25q21-->q23

Mutations in the gene for gamma-sarcoglycan (SGCG) located on HSA 13q12 are responsible for limb girdle muscular dystrophy (LGMD2C) in human. Here we report the cloning of the canine SGCG gene together with its genomic structure and several intragenic polymorphisms. The coding part of the canine SGCG contains seven exons spanning at least 70 kb of genomic DNA. The chromosome assignment of the canine SGCG gene to CFA 25q21-->q23 confirms that the canine syntenic group 10 corresponds to CFA 25 and also supports the findings of human-canine reciprocal chromosome painting.