Molecular cloning and functional analysis of gene responsible for multiple ocular defect of cattle

牛多发性眼缺陷（MOD）是一种常染色体隐性遗传疾病，其特征是晶状体发育不良、视网膜脱离、玻璃状动脉持续存在和小眼。该位点定位于牛18号染色体的近端区域。通过单倍型分析，我们进一步将MOD位点定位在牛18号染色体上1.1 Mb的范围内，并确定了该区域所有基因的核苷酸序列。在患病动物和正常动物之间对这些基因的核苷酸序列进行比较，发现WFDCl基因外显子2上有一个核苷酸插入，导致帧移位突变。WFDCl是一种含有wap型四二硫核心结构域的小分泌蛋白。通过免疫组化染色和原位杂交，观察Wfdcl在胚胎和成年小鼠眼晶状体、视网膜和视神经中的特异性表达。尽管WFDCl在哺乳动物发育中的确切功能尚不清楚，但目前的研究结果表明，WFDCl的这种突变导致多种眼睛缺陷，并且该基因在胚胎和成人眼睛中特异性表达，这表明WFDCl在哺乳动物眼睛发育中的重要作用。这是第一份直接证明WFDCl参与哺乳动物特定发育过程的报告。

Multiple ocular defects (MOD) in cattle is an autosomal recessive hereditary disorder characterized by dysplasia of the lens, retinal detachment, persistence of the hyaloid artery, and microphthalmia. The locus responsible for MOD was mapped to the proximal region of bovine chromosome 18. We further refined the localization of the MOD locus to within a 1.1 Mb interval on bovine chromosome 18 by haplotype analysis and determined the nucleotide sequences of all genes in this region. A comparison of the nucleotide sequences of these genes between affected and normal animals revealed a one-nucleotide insertion in exon 2 of the WFDCl gene, which resulted in a frame shift mutation. WFDCl is a small secretory protein containing a WAP-type four disulfide core domain. Specific expression of Wfdcl was observed in the lens, retina, and optic nerves of embryonic and adult mouse eyes by immunohistochemical staining and in situ hybridization. Although the exact function of WFDCl in mammalian development remains unknown, the present findings showing that this mutation in WFDCl resulted in multiple eye defects and that the gene is specifically expressed in embryonic and adult eyes demonstrated the essential role of WFDCl in mammalian eye development. This is the first report providing direct evidence for the involvement of WFDCl in a particular process of mammalian development.