A Comparative Approach to Physical and Linkage Mapping of Genes ...

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 P. Werner, M. G. Raducha, U. Prociuk, P. S. Henthorn, and D. F. Patterson

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.

From the Center for Comparative Medical Genetics and Section of Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. This research was supported by National Institutes of Health grants HL18848 from the National Heart Lung and Blood Institute and RR02512 from the Division of Research Resources, as well as a grant from the Mrs. Cheever Porter Foundation. P.W. was supported by a fellowship from the Robert J. Kleberg and Helen C. Kleberg Foundation. Address correspondence to Donald F. Patterson, Section of Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, VHUP 4030, 3900 Delancey Street, Philadelphia, PA 19104–6010. This paper was delivered at the International Workshop on Canine Genetics at the College of Veterinary Medicine, Cornell University, Ithaca, New York, July 12–13, 1997. q 1999 The American Genetic Association 90:39–42

Through the medium of clinical veterinary medicine, the domestic dog receives the highest degree of medical scrutiny at the level of individual animals of any species other than man. More than 350 genetic diseases are currently known to occur among the more than 200 breeds of dogs worldwide and the number of newly recognized genetic disorders in dogs is presently increasing by about 5–10 per year (Patterson, in press). In time, as the diagnostic armamentarium of clinical veterinary medicine continues to increase in parallel with human medicine, it can be expected that the number of genetic disorders in the dog will approach the number known in humans. Over 1,000 human genetic disorders have been mapped to specific gene loci in the human genome (McKusick and Amberger 1997). A more complete understanding of the genes underlying canine diseases, includ-

ing their locations in the dog genome is important for two reasons. First, the information will provide a more effective basis for diagnosis and control of these disorders in dog populations. Second, many known as well as yet-to-be-discovered genetic diseases of dogs are “models” of human genetic diseases and can be extremely useful in advancing the understanding and treatment of genetic diseases in human patients. The comparative approach to gene mapping, as well as to other aspects of genetics, is based on the now well-recognized fact that all species of animals, as well as lower organisms, share genes that have been conserved in their structure and function during evolution and are thus true homologs of each other. Likewise, animal genetic diseases that are similar in their clinical and pathologic features in different mammalian species are likely to

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be due to defects in homologous genes, and thus are often not just “models” but true homologs of each other. The benefits of studying gene-homologous diseases tend to be reciprocal across species (Patterson et al. 1988). For example, knowledge regarding the immunologic properties and the clinical and pathologic signs in children with X-linked severe combined immunodeficiency ( XSCID) made it possible to recognize this same disorder in dogs (Jezyk et al. 1989). Later, the canine XSCID gene was mapped to the same gene region of the dog X chromosome as had been shown in humans ( Deschenes et al. 1994). Subsequently, canine XSCID was shown to be due to a defect in the same gene as implicated in humans—the gene encoding the gamma (common) chain of the interleukin-2 receptor ( Henthorn et al. 1994). In a reciprocal fashion, the homologous canine disorder is now providing knowledge regarding the detailed pathophysiology of the immune system and the potential treatment of XSCID in children by transplantation and gene therapy ( Felsburg et al. 1997). Multiple chromosomal rearrangements have occurred during evolution of the many species of mammals, resulting in differences between species in the numbers and morphology of chromosomes. However, homology between species exists at the level of syntenic blocks of genes (O’Brien 1991). Although there has been “chromosomal shuffling,” at the subchromosomal level synteny has been conserved for blocks of contiguous genes, and the location of even one gene from a syntenically conserved group can often provide a landmark from which to deduce the chromosomal locations of other genes in the group. O’Brien et al (1993), based on the principle of conservation of synteny, proposed a group of “anchor reference loci” for comparative genome mapping in mammals, with the idea that these can serve as landmarks with which to orient gene maps across species. In this article we illustrate a comparative approach to gene mapping in the dog. The method provides a means to produce physical and linkage maps of genes on dog chromosomes, at the same time providing “gene-associated simple sequence repeat polymorphisms” by which the genes can be integrated in a linkage map, including anonymous canine microsatellite markers such as those described by Ostrander et al. (1993) and recently reported to occur in linkage groups by Mellersh et al. (1997). Integration of the microsatellite and gene

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Figure 1. Flow diagram showing a linkage strategy using gene-associated SSRPs. cDNA probes were used to isolate clones from dog genomic libraries. Gene-verified clones were screened for SSRPs and also used for FISH to identify a chromosomal assignment. Polymorphic SSRs were used to genotype the dogs and multipoint linkage analysis was conducted to create a linkage map.

maps is an essential means to realize the reciprocal advantages of comparative medical genetics. If an anonymous microsatellite in the dog is found to be linked to a canine disease, but its location in the canine genome relative to nearby genes is not known, it is not possible to take advantage of the relatively dense maps of the human or mouse genomes to deduce the possible candidate genes in the region. Nor is it possible to transfer the canine mapping information to the human genome to discover the location of an as yet unmapped homologous disease gene in the human genome. We have used a comparative approach to create physical and linkage maps of dog chromosome 9 (CFA9), the telomeric twothirds of which was found to be highly homologous to human chromosome 17q ( HSA17q). Details of the methods and information about the SSRPs are reported in Werner et al. (1997). The overall approach is illustrated in the flow diagram shown in Figure 1. In summary, human or mouse cDNAs or coding sequence PCR products from specific genes found on HSA17 and mouse chromosome 11 (MMU11) were used to screen a dog cosmid or phage library. Positive clones were gene-verified

by fragment isolation, back hybridization to the cDNA probe, and sequencing. Verifed clones were then screened with simple sequence repeat probes and, in those that were positive, the sequences flanking the repeats were determined and used to design PCR primers. The PCR products were then tested in our canine pedigrees to determine if they were polymorphic and informative for linkage analysis. The same cosmid or phage clones were used as probes for fluorescence in situ hybridization ( FISH) studies to identify the physical site of the locus in question on a dog chromosome.

Results Using human or mouse cDNA probes (see Werner et al. 1997) we were able to isolate canine clones for 13 genes on HSA17. Two genes from HSA17p—GLUT4 and PMP22— mapped by FISH to CFA 5. Eleven genes from HSA17q mapped to the centromeric two-thirds of CFA9: P4HB, GALK1, TK1, GH1, MYL4, BRCA1, RARA, THRA1, MPO, and NF1 ( Figure 2). A linkage map of CFA9 was constructed using a large panel of F1 backcross offspring produced by outcrossing members of a partially inbred

Figure 2. Physical and linkage maps of CFA9 and CFA5 compared to the homologous regions on HSA17 and MMU11. The “consensus” genetic map of the MMU11 (MGDB) shows the distances in centiMorgans on the left, the idiogram of CFA5 and CFA9 shows the physical localization of the genes mapped compared to the idiogram of HSA17 on the right. The linkage map of CFA9 is shown in the middle, displaying the distances between the loci in centiMorgans on the left.

line of keeshonds to a series of beagles and backcrossing the F1 hybrids to the keeshond line (described in Patterson et al. 1993). As shown in Figure 2, the syntenic group of 11 genes conserved between HSA17q and CFA9 is also conserved on MMU11 ( Kurtz and Zimmer 1995; Lossie et al. 1994). Conservation of synteny for a number of these genes has also been reported on bovine chromosome 19 (Solinas-Toldo et al. 1995; Yang and Womack 1995). The gene order from the centromere in the dog—P4HB, GALK1, TK1, MYL4, GH1, BRCA1, RARA, THRA1, MPO, NF1, CRYBA1—based on combined information from FISH and linkage, is very similar to that on HSA17q and MMU11. However, the most telomeric genes on HSA17 are located near the centromere of CFA9, indicating an inversion of the entire group in relation to the centromere. On the centromeric one-third of CFA9, GALK1, TK1, MYL4, GH1, RARA, and THRA1 constitute a closely linked group spanning 4.7 cM, whereas the corresponding interval in the mouse is given as 21 cM [Mouse Genome Database (MGDB): http:// www. informatics. jax. org/

mgd.html; October 1996]. The lower rate of recombination for this gene region in the dog than in the mouse may be the result of the inversion in the dog which has brought the group of genes near the centromere. Recombination has been reported to be decreased in regions near the centromere in other species ( Doggett et al. 1996). NF1 and CRYBA1 are closely linked to each other at a distance of 2.7 cM but loosely linked to the more centromeric group (interval of 31.2 cM between NF1 and RARA). The more distal localization of NF1 was apparent in FISH studies. FISH of PMP22 and GLUT4, located on HSA17p, localized them to the centromeric half of CFA5, indicating disruption of the HSA17 synteny at the centromere. Accordingly, no linkage was found between GLUT4 and the loci on CFA9. The twochromosome localization on CFA9 and CFA5 identified by single-locus probes was also seen using a total human chromosome probe for HSA17 (data not shown). In these FISH studies, there was hybridization of the probe (Coatsome 17, Oncor) to both the region of CFA5 occupied by the HSA17p genes, GLUT4 and PMP22, and the

centromeric two-thirds of CFA9, the latter representing the region where single-locus probes identified the group of 11 genes from HSA17q. There was no hybridization to the distal one-third of CFA9, indicating that this chromosomal region in the dog is homologous to a portion of one or more other human/mouse chromosomes.

Note Added in Proof Following submission of this article, we were able to show that the telomeric end of canine chromosome 9, in the region that did not hybridize to the human chromosome 17 painting probe, consists at least in part of a segment homologous to human chromosome 9q. Two gene loci, both found on HSA9q34, are located in this region: Retinoic Acid Receptor Alpha (RXRA) and Heat Shock Protein 5 ( HSPA5). RXRA is linked to CRYBA1 at a distance of 6.5 cM and HSPA5 is linked distally to RXRA at a distance of 21.9 cM (Werner et al. 1998). The positions of these loci on CFA9 suggest that the more telomeric end of this canine chromosome

Werner et al • Comparative Physical and Linkage Mapping of Genes on Canine Chromosomes 41

may contain additional gene loci that are located centromeric to RXRA on HSA9q.

Discussion The major advantage of the approach presented for comparative mapping of canine disease genes lies in the development of physical and linkage maps of genes in previously unmapped genomes such as that of the dog. The value of this approach has been demonstrated recently by the report of Acland et al. (1998). These authors describe studies in which a form of hereditary retinal degeneration in the dog, progressive rod-cone dysplasia (prcd), was found to be linked to anonymous microsatellite markers reported by Mellersh et al. (1997). Through the generation and characterization of canine/rodent hybrid cells ( Langston et al. 1997), Acland et al. initially found homology to HSA17 and they were then able to link their anonymous microsatellite markers to the region of the SSRP map of CFA9 we had previously reported (Werner et al. 1997). It is homologous to the region of HSA17q which contains a candidate gene, the RP17 locus, mutations at which are responsible for a form of retinitis pigmentosa in humans ( Bardien et al. 1995, 1997). Since the human RP17 gene has not yet been cloned, the dog “model,” which is hypothesized to be a true homolog of RP17, could lead to the identification of the human gene defect. This method facilitates mapping by providing access to the extensive data from well-mapped species like human and mouse, thus allowing the use of information on conserved syntenic groups to infer the identity of neighboring genes. As expressed sequence tags ( ESTs) are placed on the human gene map and as the human genome project nears completion, canine gene-associated SSRPs will provide pre-

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cise anchors between human and canine genomes, allowing the utilization of ESTs for mapping endeavors in the dog. The gene-associated SSRPs are simultaneously physical, linkage, and comparative mapping markers and thus provide a means to integrate the gene maps with anonymous microsatellite markers which are an important tool for fine-mapping of disease genes.

References Acland GM, Ray K, Mellersh CS, Gu W, Langston AA, Rine J, Ostrander EA, and Aguirre GD, 1998. Linkage analysis and comparative mapping of canine progressive rod-cone degeneration (prcd) establishes potential locus homology with retinitis pigmentosa (RP17) in humans. Proc Natl Acad Sci USA 95:3048–3053. Bardien S, Ebenezer N, Greenberg J, Inglehearn CF, Bartmann L, Goliath R, Beighton P, Ramesar R, and Bhattacharya SS, 1995. An eighth locus for autosomal dominant retinitis pigmentosa is linked to chromosome 17q. Hum Mol Genet 4:1459–1462. Bardien S, Ramesar R, Bhattacharya S, and Greenberg J, 1997. Retinitis pigmentosa locus on 17q (RP17): fine localization to 17q22 and exclusion of the PDEG and TIMP2 genes. Hum Genet 101:13–17.

between mouse chromosome 11 and human chromosome 17. Mamm Genome 6:379–380. Langston AA, Mellersh CS, Neal CL, Ray K, Acland GM, Gibbs M, Aguirre GD, Fournier REK, and Ostrander EA, 1997. Construction of a panel of canine-rodent hybrid cell lines for use in partitioning of the canine genome. Genomics 46:317–325. Lossie AC, MacPhee M, Buchberg AM, and Camper SA, 1994. Mouse chromosome 11. Mamm Genome 5:64–80. McKusick VA and Amberger JS, 1997. Morbid anatomy of the human genome. In: Emery and Rimoin’s principles and practice of medical genetics (Rimoin DL, O’Connor JM, and Pyeritz RE, eds). New York: Churchill Livingstone; 137–234. Mellersh ES, Langston AA, Acland AF, Fleming MA, Ray K, Wiegand NA, Francisco LV, Gibbs M, Aguirre GD, and Ostrander EA, 1997. A linkage map of the canine genome. Genomics 46:326–336. O’Brien SJ, 1991. Mammalian genome mapping: lessons and prospects. Curr Opin Genet Dev 1:105–111. O’Brien SJ, Womack JE, Lyons LA, Moore KJ, Jenkins NA, and Copeland NG, 1993. Anchored reference loci for comparative genome mapping in mammals. Nat Genet 3:103–110. Ostrander EA, Sprague GF, and Rine J, 1993. Identification and characterization of dinucleotide repeat (CA)n markers for genetic mapping in dog. Genomics 16:207– 213. Patterson DF, in press. Canine genetic disease information system—a computerized knowledgebase of genetic diseases of the dog. St. Louis: Mosby-Yearbook.

Deschenes SM, Puck JM, Dutra AS, Somberg RL, Felsburg and Henthorn PS, 1994. Comparative mapping of canine and human proximal Xq and genetic analysis of canine X-linked severe combined immunodeficiency. Genomics 23:62–68.

Patterson DF, Haskins ME, Jezyk PF, Giger U, MeyersWallen VN, Aguirre G, Fyfe JC, and Wolfe J, 1988. Research on genetic diseases: reciprocal benefits to animals and man. J Am Vet Med Assoc 193:1131–1144.

Doggett NA, Sutherland RD, Altherr MR, Torney DC, Knill EH, Deaven LL, Callen DF, and Moyzis RK, 1996. Features of human chromosome organization revealed with the completed physical map of human chromosome 16. Am J Hum Genet 59(suppl):A26.

Patterson DF, Pexieder T, Schnarr WR, Navratil T, and Alaili R, 1993. A single major-gene defect underlying cardiac conotruncal malformations interferes with myocardial growth during embryonic development: studies in the CTD line of keeshond dogs. Am J Hum Genet 52:388–397.

Felsburg PJ, Somberg RL, Hartnett BJ, Suter SF, Henthorn PS, Moore PF, Weinberg KI, and Ochs HD, 1997. Full immunologic reconstitution following nonconditioned bone marrow transplantation for canine Xlinked severe combined immunodeficiency. Blood 90: 3214–3221. Henthorn PS, Somberg RL, Fimiani VM, Puck JM, Patterson DF, and Felsburg PJ, 1994. IL-2R gamma gene microdeletion demonstrates that canine X-linked severe combined immunodeficiency is a homologue of the human disease. Genomics 23:69–74. Jezyk PF, Felsburg PJ, Haskins ME, and Patterson DF, 1989. X-linked severe combined immunodeficiency in the dog. Clin. Immunol Immunopathol 52:173–189. Kurtz A and Zimmer A, 1995. Interspecies fluorescence in situ hybridization further defines synteny homology

Solinas-Toldo S, Lengauer C, and Fries R, 1995. Comparative genome map of human and cattle. Genomics 27:489–496. Werner P, Raducha MG, Prociuk U, Henthorn PS, and Patterson DF, 1997. Physical and linkage mapping of human chromosome 17 loci to dog chromosome 9 and 5. Genomics 42:74–82. Werner P, Raducha MG, Prociuk U, Lyons LA, Kehler JS, Henthorn PS, and Patterson DF, 1998. RXRA and HSPA5 map to the telomeric end of dog chromosome 9. Anim Genet 29:220–223. Yang Y-P and Womack JE, 1995. Human chromosome 17 comparative anchor loci are conserved on bovine chromosome 19. Genomics 27:293–297. Corresponding Editor: Gustavo Aguirre