Amount Requested: Up to $35,000/yr for two years.

Current Funding Sources for project: We currently have an $8,000 AKC/Canine Health Foundation grant (Active Grant #1867) to study lens luxation in Miniature Bull Terriers. We are requesting funds to extend the study to include breeds and organizations mentioned in the following summary.

Summary: Heritable lens luxation, if not treated promptly, will induce secondary glaucoma. In addition, heritable primary glaucoma can cause secondary lens luxation. Since it is not always known whether lens luxation or glaucoma is the primary disease, we believe it is rational to study both diseases together. Primary lens luxation is thought to be an autosomal recessive trait in several breeds of true terriers (including Miniature Bull Terriers, Jack Russell Terriers and Sealyham Terriers), in Tibetan Terriers, and in Petit Basset Griffon Vendeens. Glaucoma is thought to be the primary disease in Welsh Terriers, Basset Hounds and Welsh Springer Spaniels. The identity of the mutant genes responsible for primary canine lens luxations and primary canine glaucomas have not yet been determined. Our long-range goal is to identify the mutant genes and devise DNA markers for the respective disease loci to assist dog breeders in producing offspring that will remain free of lens luxation and glaucoma. This will be accomplished by obtaining DNA samples from affected dogs and their close relatives. We will then test for cosegregation (genetic linkage) between the disease phenotypes and marker alleles either for candidate genes or for strategically placed loci from the canine genome linkage map.

Significance of Research: Both lens luxation and glaucoma can cause blindness. We continue to work on and expand this area of research because of requests from organizations and/or individual dog owners and breeders associated with the following breeds:

Background and Preliminary Work: Lens luxation is an autosomal recessive trait in several breeds of true terriers, in Tibetan Terriers and in Petit Basset Griffon Vendeens. Lens luxation is a medical emergency that unless promptly treated can lead to glaucoma and blindness. The gene responsible for canine lens luxation has not been identified. Identification of this locus would enable us to devise a DNA marker assay for identifying carriers and affected dogs before the luxations occur. Lens luxation occurs because of breakage of the zonules, the fibers that hold the lens in place. Genes for fibrillin 1 and microfibril-associated protein 3 code for two major components of these zonules and so are considered candidate genes.¹ The only human mutation known to cause isolated lens luxation is in exon 59 of the fibrillin 1 gene.² Mutations elsewhere in this gene cause Marfan syndrome, in which lens luxation is found in conjunction with skeletal and cardiovascular abnormalities.

We began these studies by investigating lens luxation in Miniature Bull Terriers. As part of that study we have been accumulating DNA samples from Miniature Bull Terrier families in which lens luxation is segregating. To date we have accumulated DNA samples from 77 Miniature Bull Terriers including eight with bilateral lens luxation. Pedigrees of some of the Miniature Bull Terrier families appear in Figure 1 on the next page. Since the fibrillin 1 gene is a candidate gene for isolated lens luxation, we amplified and sequenced segements of this gene from several dogs. We found polymorphic sites and devised marker assays; however, these markers were uninformative in the Miniature Bull Terrier pedigrees as only one allele was present. We therefore used one of these fibrillin 1 gene polymorphic sites to genotype the Cornell/Ralston Purina reference families and mapped the fibrillin 1 gene to canine chromosome 30. Flanking highly polymorphic type 2 microsatellite markers from canine chromosome 30 were also found to be uninformative in the Miniature Bull Terrier pedigrees. We next amplified and sequenced an exon 59-containing segment of the fibrillin 1 gene. The nucleotide sequences from affected Miniature Bull Terriers were identical to those from normal Miniature Bull Terriers and normal dogs from other breeds.

We concluded that because of intensive inbreeding and/or a narrow base of founders, many of the alleles segregating in most breeds were lost from Miniature Bull Terriers and, thus, global mapping studies would be difficult. We also noted that lens luxation occurred in many breeds of terriers originating in the British Isles, but not in most non-terrier breeds. This suggested that a founder mutation, occurring before the various terrier breeds became closed registries, might be responsible for the lens luxation in the terriers. Tibetan Terriers, a breed unrelated to the terriers from the British Isles, appear to be an exception. Some Tibetan Terrier breeders, however, believe that their lens luxation problem stems from a true terrier of English origin which was allowed into the Tibetan Terriers registry because it resembled a Tibetan Terrier.³ If this is true,

(pedigrees deleted from public version to preserve anonymity)

Tibetan Terriers may prove to be an ideal breed for global mapping of the lens luxation locus. We have therefore begun collecting DNA from Tibetan Terriers with lens luxation and their close relatives. To date we have 22 Tibetan Terriers in this study, including 4 affected dogs. So far, however, none of the families are extensive enough to support genome mapping. If lens luxation in the Tibetan Terriers does, indeed, stem from the same founding mutation responsible for lens luxation in the true terriers, discoveries made by studying Tibetan Terriers should be directly applicable to lens luxation in the true terrier breeds.

In addition to the Miniature Bull Terriers and Tibetan Terriers, we have individuals with lens luxation and partial families in Sealyham Terriers, Bassett Hounds, and Petit Basset Griffon Vendeens. In some of these breeds the lens luxation may be secondary to glaucoma. Thus, we have also begun to focus on glaucoma as a primary inherited disease and are collecting DNA from affected dogs and their close relatives. In the glaucoma studies we have samples from 17 Basset Hounds including 5 affected individuals, 28 Petit Basset Griffon Vendeens including 2 affected individuals, and 46 Welsh Terriers including 6 affected individuals. In addition, we have DNA from one Welsh Springer Spaniel with glaucoma. Examples of collected Basset Hound families are shown in Figure 2. Mutations in three genes, myocilin,⁴ cytochrome P4501B1^5,6 and the forkhead transcription factor gene FKHL7⁷ have been shown to be responsible for glaucoma in people. In addition, mouse studies indicate that mutations in the tyrosinase related protein 1 gene can contribute to development of glaucoma.⁸ We have developed markers for the canine myocillin gene and the canine tyrosinase related protein 1 gene and are currently testing these markers in the Basset Hound families.

(pedigrees deleted from public version to preserve anonymity)

1. To accumulate DNA from individual dogs with lens luxation and/or glaucoma and their relatives.

2. To examine individual dogs with lens luxation and/or glaucoma to define the phenotype as closely as possible.

3. To establish a canine glaucoma/lens luxation internet web site similar to our canine epilepsy web site.

4. To devise informative DNA marker assays for the canine genes for microfibril-associated protein 3, cytochrome P4501B1 and FKHL7, and any other candidate genes that may appear in the scientific literature.

5. To use the new marker assays from Objective 5 along with our already existing marker assays for fibrillin, myocillin and tyrosinase related protein 1 to test for linkage between these markers and lens luxation or glaucoma.

6. To begin global mapping with markers from the canine genome linkage map in canine lens luxation and glaucoma families.

Specific Objective 1. To accumulate DNA from individual dogs with lens luxation and/or glaucoma and their relatives. As indicated in the above section, we have already begun to collect the DNA samples necessary for this project. Typically, canine blood is drawn into a tube with EDTA anticoagulant and shipped to us via overnight delivery in a cooler with ice. Each sample is assigned a number and logged into a spreadsheet along with information about the donor (species, breed, gender, age, pedigree information, medical history, etc). The spreadsheet currently has information on over 22,000 individual donors. We isolate white cell DNA by routine phenol/chloroform extraction and store the DNA in labeled vials in the freezer.

Ms Liz Hansen is an Information Specialist with major responsibilities in sample recruitment. She is a dog breeder, trainer and exhibitor. She excels at explaining our projects to dog owners and has been very effective in recruiting key samples for other ongoing projects. She will contact Breed Club Representatives and Newsletter Editors to publicize our need for samples. She will also make direct contacts with owners of key samples. In addition, we expect that the internet web site from Specific Objective 3 will be helpful in recruiting samples.

Specific Objective 2. To examine individual dogs with lens luxation and/or glaucoma to define the phenotype as closely as possible. We have been reluctant to undertake projects involving eye diseases since the time that Dr Keith Collins left the University of Missouri. The other Veterinary Ophthalmologists at the University of Missouri have been so heavily committed to clinical duties and/or to administration that they could not make substantial contributions to our research efforts. We are now fortunate that Dr Kristina Narfstrom, a Veterinary Ophthalmologist from Sweden, has agreed to accept an Endowed Chair at the University of Missouri. Dr Narfstrom has expressed an interest in collaborating on this project but we have not yet had an opportunity to discuss the details. I will be meeting with Dr Narfstrom next week and she will be starting work at the University of Missouri in December, 2000. The details for phenotype evaluation will be included in the full proposal.

Specific Objective 3. To establish a canine glaucoma/lens luxation internet web site similar to our canine epilepsy web site. The web site will be managed by Liz Hansen. It will be patterned after our canine epilepsy web site (www.canine-epilepsy.net) which has been visited over 21,000 times. The new web site will present basic information on the diseases being studied, the objectives of our research, participation forms and instructions, and links to other sources of information, such as the AKC/Canine Health Foundation and the CERF web site. We will also include a discussion forum, where visitors to the site can post questions and discuss topics related to the diseases being studied. We expect that this site will provide a useful source of information for owners, breeders, and others dealing with dogs that have these eye defects. As with the epilepsy site, we also expect this to encourage participation in our research by providing easy access to forms and procedures.

Specific Objective 4. To devise informative DNA marker assays for the canine genes for microfibril-associated protein 3, cytochrome P4501B1 and FKHL7, and any other candidate genes that may appear in the scientific literature. PCR primers will be designed from GenBank sequences and used to amplify 1 kb segments of canine DNA. The amplicons will be sequenced as previously described.⁹ Sequences from five unrelated dogs will be compared to reveal polymorphic sites. It is expected that most of these polymorphic sites will be single nucleotide polymorphisms. PCR/RFLP assays will be designed so that we can genotype individual samples with respect to these polymorphic sites.

Specific Objective 5. To use the new marker assays from Objective 5 along with our already existing marker assays for fibrillin, myocillin and tyrosinase related protein 1 to test for linkage between these markers and lens luxation or glaucoma. The PCR/RFLP assays from Specific Objective 4 will be used to genotype individuals in the lens luxation and glaucoma families to test for linkage. LOD scores of 3.0 or greater will be considered indicative of significant linkage.¹⁰

Specific Objective 6. To begin global mapping with markers from the canine genome linkage map in canine lens luxation and glaucoma families. If we determine that the candidate genes are not responsible for the lens luxation and glaucoma in the breeds under study, we will try to localize the disease loci in at least two families on the canine genome linkage map.¹¹ The most informative lens luxation or glaucoma families will be selected by modeling with the SIMLINK computer program. Microsatellite analyses will be based on the method of Weber and May¹² but instead of using radio-labeled deoxynucleotide triphosphates, we will prelabel one of the PCR primers with T4 polynucleotide kinase.

Expected Outcomes: We expect good cooperation from dog owners so that we will be able to assemble useful families in most or all the breeds discussed above. We expect to produce a web site that will be useful and informative for owners of affected dogs, that will help recruit DNA samples from affected dogs and their relatives, and that will inform the public about our research and about the work of the AKC/Canine Health Foundation. We expect to produce markers for all the known candidate genes for lens luxation and for glaucoma. We expect to determine whether or not any of these candidate genes contain the mutations responsible for lens luxation or glaucoma in breeds under study. We do not expect to be able to complete whole genome scans for all the families we assemble; however, we expect make substantial progress (at least 70 markers in at least two families).

1. WR Abrams, RI Ma, U Kucich, MM Bashir, S Decker, P Tsipouras, JD McPherson, JJ Wasmuth, J Rosenbloom. Molecular cloning of the microfibrillar protein MFAP3 and assignment of the gene to human chromosome 5q32-q33.2. Genomics 26(1999)47-54.

2. L Lonnqvist, A Child, K Kainulainen, R Davidson, L Puhakka, L Peltonen. A novel mutation of the fibrillin gene causing ectopia lentis. Genomics 19(1994)573-576.

3. MB Willis, KC Barnett, MW Tempest. Genetic aspects of lens luxation in the Tibetan terrier. Veterinary Record 104(1979)409-412.

4. MF Adam, A Belmouden, P Binisti, AP Brezin, F Valtot, A Bechetoille, JC Dascotte, B Copin, L Gomez, A Chaventre, JF Bach, HJ Garchon. Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin-homology domain of TIGR in familial open-angle glaucoma. Human Molecular Genetics 6(1997)2091-2097. 5. I Stoilov, AN Akarsu, M Sarfarazi. Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (buphthalmos) in families linked to the GLC3A locus on chromosome 2p21. Human Molecular Genetics 6(1997)641-647.

6. BA Bejjani, DW Stockton, RA Lewis, KF Tomey, DK Dueker, M Jabak, WF Astle, JR Lupski. Multiple CYP1B1 mutations and incomplete penetrance in an inbred population segregating primary congenital glaucoma suggest frequent de novo events and a dominant modifer locus. Human Molecular Genetics 9(2000)367-374.

7. DY Nishimura, RE Swiderski, WLM Alward, CC Searby, SR Patil, SR Bennet, AKB Kanis, JM Gastier, EM Stone, VC Sheffield. The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25. Nature Genetics 19(1998)140-147.

8. B Chang, RS Smith, NL Hawes, MG Anderson, A Zabaleta, O Savinova, TH Roderick, JR Heckenlively, MT Davisson, SWM John. Interacting loci cause severe iris atrophy and glaucoma in DBA/2J mice. Nature Genetics 21(1999)405-409.

9. H Shibuya, DJ Nonneman, THM Huang, VK Ganjam, FA Mann, GS Johnson. Two polymorphic microsatellites in a coding segment of the canine androgen receptor gene. Animal Genetics 24(1993)345-348.

10. J Ott. Analysis of Human Linkage. 3^rd Edition. Johns Hopkins University Press, Baltimore, 1999.

11. NW Neff, KW Broman, CS Mellersh, K Ray, GM Acland, GD Aguirre, JS Ziegle, EA Ostrander, J Rine. A Second-generation genetic linkage map of the domestic dog, Canis familiaris. Genetics 151(1999)803-820.

12. JL Weber, PE May. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Amer J Human Genetics 44(1989)388-396.

Timeline: We have already begun to collect DNA samples from affected dogs and their close relatives. We expect to continue adding individual samples to our collection thorough out the two-year funding period. We expect to have the web site up and running by the end of the first year of the study. DNA marker assays for the canine microfibril-associated protein 3 gene and the canine cytochrome P4501B1 gene should be developed and ready for use by the end of the first year. Markers for additional candidate genes will be devised as soon as is practical, after the necessary sequence information becomes available to the public. In some instances we may not be able to produce markers for newly discovered candidate genes until the second year of the grant. We will begin screening candidate genes during the first year of the grant and continue this work during the second. Most of the global mapping work will be done during the second year of funding.