ABSTRACT
PURPOSE: Cataracts are an important cause of blindness in humans but there are few large animal models available. One of these animal models is Ovine Heritable Cataract, a bilateral cortical cataract which develops after birth. This cataract has been used as a model for human cataracts in drug trials, but the gene responsible for the cataract trait is unknown. A genetic test for cataract would improve the efficiency of the model by predicting which animals would develop cataracts. Identifying the genetic basis of the cataract would indicate its relevance to human cataract. METHODS: A genome scan was performed on 20 sheep chromosomes, representing 86% of the genome, to determine the position of the cataract locus. Additional microsatellite markers were tested on chromosome 6 using a larger pedigree. Fine mapping was performed using a breakpoint panel of 36 animals and novel microsatellite markers taken from the bovine genome assembly. All exons of the candidate gene nudix (nucleoside diphosphate linked moiety X)-type motif 9 (NUDT9) were sequenced in normal and affected sheep. RESULTS: Significant linkage was found between cataract status and markers on chromosome 6. Linkage analysis on the larger pedigree showed the most likely position of the cataract locus was between 112.3 and 132.9 cM from the centromere. During fine mapping, NUDT9 was considered as a positional candidate for the cataract gene because it was located within the linked interval and is expressed in the lens. The gene was ruled out as the cataract gene after extensive genotype analysis, but a single nucleotide polymorphism (SNP) inside it provided a useful restriction fragment length polymorphism (RFLP) marker for further fine mapping. Twelve new markers were found and used to map the cataract locus to between 131.1 and 131.8 cM from the centromere. CONCLUSIONS: A region of ovine chromosome 6 strongly linked to cataract has been identified, and a genetic test for cataract based on a SNP within this region has been developed. The best candidate gene within this region is AF4/FMR2 family, member 1 (AFF1), the mouse equivalent of which is associated with an inherited cataract.
Subject(s)
Cataract , Chromosomes, Mammalian/genetics , DNA-Binding Proteins/genetics , Pyrophosphatases/genetics , Sheep Diseases , Sheep, Domestic/genetics , Animals , Cataract/genetics , Cataract/veterinary , Cattle , Chromosome Mapping , Disease Models, Animal , Genetic Linkage , Genetic Loci , Genetic Testing , Genotype , Humans , Microsatellite Repeats , Pedigree , Phenotype , Polymorphism, Restriction Fragment Length , Polymorphism, Single Nucleotide , SheepABSTRACT
A series of N-heterocyclic dipeptide aldehydes 4-13 have been synthesised and evaluated as inhibitors of ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC(50) values of 290 and 25nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor, displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been determined. Because of the lack of available structural information on the ovine calpains, in silico homology models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed SAR for compounds 4-13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9 (CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens from calcium-induced opacification.
