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.

Anchoring of canine linkage groups with chromosome-specific markers.

A high-resolution genetic map with polymorphic markers spaced frequently throughout the genome is a key resource for identifying genes that control specific traits or diseases. The lack of rigorous selection against genetic disorders has resulted in many breeds of dog suffering from a very high frequency of genetic diseases, which tend to be breed-specific and usually inherited as autosomal recessive or apparently complex genetic traits. Many of these closely resemble human genetic disorders in their clinical and pathologic features and are likely to be caused by mutations in homologous genes. To identify loci important in canine disease genes, as well as traits associated with morphological and behavioral variation, we are developing a genetic map of the canine genome. Here we report on an updated version of the canine linkage map, which includes 341 mapped markers distributed over the X and 37 autosomal linkage groups. The average distance between markers on the map is 9.0 cM, and the linkage groups provide estimated coverage of over 95% of the genome. Fourteen linkage groups contain either gene-associated or anonymous markers localized to cosmids that have been assigned to specific canine chromosomes by FISH. These 14 linkage groups contain 150 microsatellite markers and allow us to assign 40% of the linkage groups to specific canine chromosomes. This new version of the map is of sufficient density and characterization to initiate mapping of traits of interest.

A comparative approach to physical and linkage mapping of genes on canine chromosomes using gene-associated simple sequence repeat polymorphisms illustrated by studies of dog chromosome 9.

We describe and illustrate a comparative approach to creating physical and linkage maps of genes on dog chromosomes. The approach is particularly useful in species, like the dog, which have a rudimentary gene map not integrated with microsatellite loci. Human or mouse cDNAs for genes to be mapped are used to isolate cosmid or phage clones from dog genomic libraries. Clones verified to contain the homologous canine gene coding sequences are screened for "gene-associated" simple sequence repeat polymorphisms (SSRPs). The unique sequences flanking the repeats are used to design PCR primers to amplify the repeat and gene-associated SSR length differences that are informative for linkage analysis used in canine pedigrees to study linkage between loci or with diseases. The same canine clones are employed as probes in fluorescence in situ hybridization (FISH) studies to physically map the loci to specific sites on dog chromosomes. This approach creates a combined gene and gene-associated microsatellite anchor locus framework map. In this article we review our recent use of this approach to map a series of genes found on human chromosome 17 (HSA17) to two dog chromosomes. Canine chromosome 9 (CFA9) contains 11 loci found on HSA17q, while two genes from HSA17p map to CFA5, demonstrating disruption of HSA17 synteny at the centromere. The order of 11 HSA17q genes on CFA9 was conserved in the dog, but the entire group is inverted with respect to the centromere when compared to human and mouse. Maps created by this approach can be used to advantage for integrating anonymous microsatellites with gene maps, including microsatellites found in genome scans to be linked to canine diseases. This makes it possible to identify the homologous chromosomal region in the human or mouse genome and to make use of this information in formulating hypotheses regarding candidate genes, as has recently been illustrated by other investigators.

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-(22CM)-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.

Physical and linkage mapping of human chromosome 17 loci to dog chromosomes 9 and 5.

Genome mapping in the dog is in its early stages. Here we illustrate an approach to combined physical and linkage mapping of type 1 anchor (gene) loci in the dog using information on syntenic homology from human and mouse, an interbreed cross/backcross, and a strategy for isolation of dog genomic clones containing both gene-specific sequences and simple sequence repeat polymorphisms. Eleven gene loci from human chromosome 17q (HSA17q) were mapped to the centromeric two-thirds of dog chromosome 9 (CFA9), an acrocentric chromosome of medium size: P4HB, GALK1, TK1, GH1, MYL4, BRCA1, RARA, THRA1, MPO, NF1, and CRYBA1. Eight of these were also positioned on a linkage map spanning 38.6 cM. Based on combined fluorescence in situ hybridization and linkage mapping, the gene order on CFA9 is similar to that of the homologous genes on HSA17q and mouse chromosome 11 (MMU11), but in the dog the gene order is inverted with respect to the centromere. Canine loci, GALK1, TK1, GH1, MYL4, THRA1, and RARA constitute a closely linked group near the centromeric end of CFA9, spanning a genetic distance of only 4.7 cM. Canine NF1 and CRYBA1 lie distally, near the lower border of the Giemsa band adjacent to the distal one-third of CFA9. NF1 and CRYBA1 are loosely linked to the more centromeric group (31.2 cM). No HSA17 genes were found on the telomeric one-third of CFA9. Painting of dog chromosomes with a human whole chromosome 17 probe showed hybridization with only the proximal two-thirds of CFA9, consistent with the conclusion that the distal one-third corresponds to a segment or segments of other human chromosomes. Two loci, GLUT4 and PMP22, located on HSA17p, were mapped by FISH to dog chromosome 5 in a region also identified by the whole human chromosome 17 paint, indicating disruption of HSA17 syntenic homology at the centromere.

Production of a monoclonal antibody to human liver alkaline phosphatase.

A monoclonal antibody to human liver alkaline phosphatase (ALP) has been produced by the mouse-hybridoma method using a partially purified enzyme preparation as antigen. The particular hybridoma secreting the antibody was detected by a screening procedure based on the retention of enzyme activity by the enzyme/antibody complex. The antibody cross-reacts strongly with human kidney and bone ALPs but not with human placental or intestinal ALPs. It also cross-reacts with liver and kidney ALPs from gorilla, chimpanzee and orangutan. It shows no significant reaction, under the conditions used, with liver or kidney ALPs from several lower primates. An antibody affinity column was prepared and shown to be effective for the final stages of liver ALP purification.