Access Type

Open Access Dissertation

Date of Award

1-1-2010

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Molecular Biology and Genetics

First Advisor

Wayne D. Lancaster

Abstract

Abdominal aortic aneurysm (AAA) is a common disease for which mechanisms of formation are still not well understood. Despite a strong genetic component to AAA risk, specific risk alleles are still largely unidentified. AAA is also a localized disease with a majority occurring in the infrarenal abdominal aorta and is six times more common than aneurysms of the thoracic aorta. To determine whether risk alleles are present in functional positional candidate genes. we: 1. performed a genetic association study using DNA from AAA cases and controls in ten candidate genes and 2. performed exon sequencing on three genes with evidence of nominal association. To identify genes with regional expression in the aorta of potential relevance to AAA: 1. Illumina microarrays were used to perform a gene expression profiling of entire baboon aortas and 2. protein expression of one candidate gene, HOXA4, was examined in further detail.

Single nucleotide polymorphisms in three positional candidate genes, CEBPG, PEPD and CD22, that had both functions relevant to AAA and expression in AAA tissue exhibited nominal association and further studied by DNA sequencing of exons. Sequence variants were identified in PEPD and CD22, but not CEPBG, but no sequence variants had predicted functional changes. Analysis of the gene expression profiles from whole length baboon aortas identified 26 genes that exhibited lengthwise expression changes along aorta, including three genes from the homeobox (HOX) family of transcription factors. Comparison of HOX gene transcripts between AAA and control abdominal aortic tissue identified ten HOX genes that were significantly downregulated in AAA tissue. The most significantly downregulated HOX gene, HOXA4, also exhibited spatial expression changes and was studied in human aortic tissue. IImmunohistochemical staining of sections of human aorta using an antibody directed against HOXA4 showed expression in all three histological layers of the aorta, primarily by endothelial cells and smooth muscle cells. Immunohistochemical analysis and immunofluorescent staining of cultured cells in vitro demonstrated protein expression localizing to the nucleus and perinuclear regions of endothelial and smooth muscle cells Quantitative analysis of HOXA4 protein by western blot in 24 paired human thoracic and abdominal aortic samples showed that, consistent with the microarray results, HOXA4 had significantly lower expression (p 〈 0.0001) in the abdominal aorta. A negative correlation was also shown between HOXA4 was demonstrated between HOXA4 protein levels and increasing in both thoracic and abdominal aorta.

In conclusion, several novel genes with potential roles in AAA etiology and pathogenesis have been identified using two different methods. Two plausible functional candidate genes from a region with evidence of genetic linkage, CD22 and PEPD, were shown to have evidence of nominal association, suggesting that variation in or around these genes may contribute to the risk of developing an AAA. HOXA4 is novel candidate gene with differential expression by aortic location and age. Lower levels of HOXA4 in the abdominal aorta and further decreases in expression with increasing age suggest that HOXA4 may have a protective function with respect to AAA.

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