Characterization of the mutation causative for autosomal recessive hereditary nephropathy in the english cocker spaniel and analysis of gene expression in multiple models of hereditary nephropathy
Davidson, Ashley Greene
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The domestic dog, Canis familiaris, has over 450 naturally occurring inherited diseases. Over half of these diseases are clinically similar to human diseases making the dog an excellent model in which to study human hereditary diseases. Alport syndrome (AS), a group of heterogeneous, hereditary renal diseases, is one example of such a human disease. The disease is transmitted in three fashions: X-linked, autosomal recessive, and autosomal dominant. AS is caused by mutations in COL4?3, COL4?4 or COL4?5, all members of the type IV collagen family. The proteins products of these genes along with those of the other type IV collagen family members (COL4?1, COL4?2, and COL4?6) are structural components of basement membranes throughout the body. This dissertation describes the measurement of mRNA transcripts in two canine models of AS: a mixed breed model of X-linked AS (XLAS) and the English Cocker Spaniel (ECS) model of autosomal recessive AS (ARAS). The work done revealed a decrease in COL4?4 transcripts. The similarity between the decrease of COL4?5 in the XLAS model and that for COL4?4 in the ARAS model lead to the investigation of COL4?4 as the gene harboring the mutation causative for ARAS in the ECS. Upon sequencing COL4?4, the causative mutation was determined to be an A to T transversion in exon 3. To provide an in vitro model to study type IV collagens, a protocol was designed and experimentally validated to isolate and culture canine Sertoli cells. Canine testes cells were isolated and cultured. Cells were verified as Sertoli cells through positive identification of both SOX9 and Clusterin B proteins, along with sequence verification of SOX9 transcripts. This in vitro model provides a tool to further study the type IV collagens. Overall, the research described herein lead to the identification of the mutation causative for ARAS in the ECS. With this knowledge a genetic test was developed to test for the disease. This research also provided valuable information about the transcript levels of type IV collagens in two models of AS, and provided a novel model in which to study the type IV collagens further.