The Genetic basis of Ovine Heritable Cataract

Abstract

Sheep have been bred at Lincoln University which develop cortical opacities at 1-2 months due to a genetic defect. This condition is referred to as Ovine Heritable Cataract (OHC), and has been used as an animal model for human cataracts. Breeding records were studied to determine the manner of inheritance of the cataract trait. Offspring affected by cataracts were produced from matings between normal, unrelated animals and animals affected by cataract. Since cataracts are rare in sheep, the normal unrelated animals were probably not carriers for the OHC trait and the results suggested a dominant mode of inheritance for OHC. The cataract trait could be inherited from both sires and dams, indicating that the inheritance was autosomal dominant. Matings between affected animals, and also between affected animals and normal animals, produced significantly fewer cataract offspring than expected for an autosomal dominant trait with full penetrance. For offspring of two affected parents, the proportion of affected offspring was not significantly different from that expected from a lethal homozygote manner of inheritance, and no known homozygotes have been produced. However the changes in litter size or premature death rates expected from a lethal homozygote effect were not observed. Also, a lethal homozygote effect would not explain why fewer affected animals than expected were produced by mating affected animals with normal animals. Therefore it was concluded that OHC is inherited in an autosomal dominant manner with incomplete penetrance. From a weighted average of the results of both types of matings, a penetrance of 0.81±.04 was calculated. In order to localise the gene responsible for OHC, a genome scan for a locus associated with OHC was carried out. DNA was extracted from a ram affected by OHC and 41 of his offspring. Ten human and mouse chromosomes with genes or regions linked to cataract were selected. The ovine homologues of these chromosomes were analysed, and no significant linkage was found. When the next ten largest chromosomes were tested, markers on ovine chromosome 6 (OAR6) showed significant linkage to the OHC locus with the most probable position for the mutation between 112.3 and 135.5 cM from the centromere of OAR6. A higher-resolution genome scan, using more markers in the linked region and 296 animals, narrowed the most likely region to between 112.3 and 132.9 cM. A breakpoint panel of 36 sheep with recombinations in the linked region was genotyped for eight new markers, selected for informativeness and ease of scoring from 63 bovine markers. Because of errors in the genome assembly used to select the markers, only three were within the linked region. Linkage analysis on the combination of old and new genotypes showed that the most likely position for the OHC locus was between 114.8 and 132.9 cM. A multipoint analysis performed on the same data gave a 1-LOD support interval of 123.3 to 128.8 cM. The breakpoint panel was then genotyped for three new markers, selected from 83 bovine markers. Linkage analysis incorporating the new genotypes showed that the most likely position for the OHC locus was between 131.1 and 132.9 cM. This interval was used to select candidate genes for OHC, but errors in the marker order and pedigree were discovered that made this interval unreliable. The candidate gene NUDT9, which is found in this region of chromosome 6, was sequenced to find any possible cataract mutations. A polymorphism was found with a strong association to OHC, and a large number of animals were genotyped. Some animals had a genotype inconsistent with the polymorphism being the OHC mutation, and the amino acid residue changed was not conserved in other mammals. Therefore the polymorphism was ruled out as the OHC mutation but was a useful marker. The linkage analysis was repeated, including the NUDT9 polymorphism as an additional marker and with corrections to the pedigree and marker order. Linkage analysis showed the most likely position for the OHC locus was between 131.1 and 131.8 cM. multipoint analysis gave a 1-LOD support interval of 131.3 to 131.7 cM from the centromere. The gene AFF1, which causes autosomal dominant cataracts in mice, is also found in the linked region. Primers were designed to amplify the entire AFF1 coding sequence, but only half of the sequence was successfully amplified. This region was sequenced in both affected and normal animals, and no polymorphisms with an association with OHC were discovered. However, AFF1 remains the best candidate for the OHC gene.