AKC CANINE HEALTH FOUNDATION

251 West Garfield Road Suite 160 Aurora, Ohio 44202

Ph:330.995.0807 Fax:330.995.0806

Email:akcchf@aol.com Web:www.akcchf.org

April 16, 2001

Kevin Welch

Miniature Bull Terrier Club of America

P.0. Box 9301

Reston, V A 20195

Re: Grant No.1867: Genetic Markers for Lens Luxation in Miniature Bull Terriers

Principal Investigator: Gary Johnson, DVM, PhD

Dear Mr. Welch:

We are pleased to forward to you the final progress report for the above referenced grant, which your club is co-sponsoring.

This progress report has been reviewed by our science officer, Dr. C. Richard Dorn, and has been approved. If you have any questions regarding the progress of this research please feel free to contact either Dr. Dorn at (614) 436-1101 or Deborah Lynch at (330) 995-0807.

We extend our thanks and appreciation to you and your club members for your support of canine health.

Sincerely,

Erika Werne

Genetic Causes for Lens Luxation in Miniature Bull Terriers:

Final report for Canine Health Foundation Grant #1867

(January 31, 2001 )

Gary S. Johnson DVM PhD

Department of Veterinary Pathobiology

College of Veterinary Medicine

University of Missouri

209A Connaway Hall, Columbia, MO 65211

Phone: 573-882-6723 Fax: 573-884-5415 Email: JohnsonGS@missouri.edu

1. Isolate genomic DNA from members of Miniature Bull Terrier families in which lens luxation is segregating.

2. Determine the normal nucleotide sequence for a segment of the canine fibrillin 1 gene which includes exon 59 and compare this sequence with corresponding sequences from DNA from Miniature Bull Terriers with lens luxation.

3. Place canine fibrillin 1 locus on the emerging canine genome linkage map.

4. Genotype Miniature Bull Terrier lens luxation family members with respect to an informative canine fibrillin gene marker and determine if the marker is genetically linked to lens luxation disease.

Accomplishments on each of these objectives have been as follows:

Objective 1. Isolate genomic DNA from members of Miniature Bull Terrier families in which lens luxation is segregating. In our preliminary work for this grant proposal we had collected samples from 36 Miniature Bull Terriers who were members of families where lens luxation had appeared. Three of these dogs were affected with lens luxation. Since that time we have received additional samples, for a current total of 78 Miniature Bull Terriers, including 8 who are affected with lens luxation. Some of these families are shown in Figure 1.

Most of these DNA samples were purified from EDT A blood by phenol/chloroform extraction. The DNA was ethanol precipitated, re-dissolved in a tris/EDTA buffer, then stored frozen. This DNA is kept as part of our collection 0! DNA samples from over 22,500 individuals, mostly dogs and cattle. The identification numbers and phenotype information, supplied by the owners, is compiled in a computer spreadsheet. In addition to the electronic copies of this data, all information supplied by owners is filed by breed for future reference.

Objective 2. Determine the normal nucleotide sequence for a segment of the canine fibrillin 1 gene which includes exon 59 and compare this sequence with corresponding sequences from DNA from Miniature Bull Terriers with lens luxation. Because a mutation in exon 59 of the human fibrillin gene has caused isolated lens luxation in human patients 1, we amplified and sequenced an exon 59-containing gene segment of the canine fibrillin gene from Miniature Bull Terriers with lens luxation and from dogs of other breeds with normal eyes. In each case we obtained identical sequences for the exon 59-containing segment.

Objective 3. Place canine fibrillin 1 locus on the emerging canine genome linkage map. Other segments of the fibrillin 1 gene from several dogs were amplified and sequenced. We found three polymorphic sites and devised marker assays for each of them. The assay developed for one of the polymorphic sites found on the fibrillin 1 gene was used to genotype the Cornell/Ralston Purina reference families, and this data submitted to Dr Elaine Ostrander at the Fred Hutchinson Cancer Research Institute. The fibrillin 1 gene mapped to canine chromosome 30 between flanking type 2 markers, CXX.204 and FH2050.

Objective 4. Genotype Miniature Bull Terrier lens luxation family members with respect to an informative canine fibrillin gene marker and determine if the marker is genetically linked to lens luxation disease. All three fibrillin markers were uninformative in the Miniature Bull Terrier families, as all family members had the same alleles. When the marker was mapped, there were 2 highly polymorphic flanking type 2 microsatellite markers identified. These also were found to be uninformative in the Miniature Bull Terrier pedigrees tested.

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 suggests that a founder mutation, occurring before the various terrier breeds became closed registries, might be responsible for the lens luxations 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.2 If this is true, 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 65 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, and have submitted a pre-proposal (Molecular-Genetic Causes for Canine Lens Luxation and Glaucoma -full proposal pending) to continue this investigation. 

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. Mutations in three genes, myocilin,3 cytochrome P4501 B14,5 and the forkhead transcription factor gene FKHL76 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.7 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.