Abstract
Purpose: :
Blind sterile locus 2 (bs2) is a spontaneous autosomal recessive mouse mutation exhibiting a phenotype of cataracts as well as male sterility. The goal of this study was to identify the gene and mutation responsible for the bs2 phenotypes.
Methods: :
Mouse eyes were examined with a slit lamp biomicroscope with a Nikon SLR-based Photo Slit Lamp imaging system following mydriasis with 1% Atropine Sulfate. For wild type and bs2 histology, tissues were fixed in Zinc-formalin, or Davidson’s solution, embedded in paraffin and sectioned to 4 microns. Following H&E staining, sections were mounted and photographed.For linkage studies, given that bs2/bs2 male mice are sterile, bs2/bs2 female mice were outcrossed to CAST/EiJ; the resulting F1 progeny were subsequently intercrossed to generate 262 F2 progeny. At four weeks of age F2 progeny were clinically evaluated for the presence of cataract, genotyped with polymorphic microsatellites and evaluated for linkage. Exons of candidate genes were PCR amplified and sequenced.
Results: :
Detailed clinical and histological evaluation revealed that bs2 mice have smaller eyes. Cataracts noted at P28 were due to severely disrupted lens fiber cells. Linkage analysis mapped bs2 to mouse chromosome 2, approximately 45cM distal from the centromere. Fine mapping established a 3.1Mb bs2 critical region containing 19 candidate genes. Sequencing of alkylglycerone-phosphate synthase (Agps), a gene within the bs2 critical region, revealed a G to A substitution at the +5 position of intron 14. This mutation, located within the intron 14 splice-donor site, is most likely responsible for the aberrant splicing of Agps mRNA which ultimately results in two abundantly expressed alternatively-spliced Agps transcripts in addition to low levels of full-length Agps transcript. Agps encodes a ubiquitously expressed peroxisomal enzyme which catalyzes formation of the ether bond in the synthesis of ether lipids.
Conclusions: :
We present evidence that bs2 mice have significantly decreased levels of ether lipids due to a mutation in Agps. Human mutations in AGPS result in rhizomelic chondrodysplasia punctata type 3 (RCDP3), a disease for which bs2 is the only animal model. The bs2 mouse represents a useful model for elucidating the mechanism of cataract formation resulting from ether lipid depletion.
Keywords: linkage analysis • candidate gene analysis • cataract