The transthyretin-associated amyloidoses are a group of protein-folding disorders caused by deposition of the liver-secreted plasma protein transthyretin (TTR) in various tissues of the body. The sporadic form of the disease is caused by deposition of wild-type TTR whereas the inherited form is caused by deposition of mutated TTR; there are over 100 known amyloidogenic mutations of the TTR gene. The transcriptional regulation of the mouse transthyretin gene has been well studied. Organ-specific modulation of TTR mRNA is achieved by coordinated binding of hepatocyte-specific and ubiquitously expressed transcription factors to regulatory regions in the proximal promoter and upstream enhancer region. The hypothesis of this dissertation is that non-coding genetic sequence variations in the promoter of the transthyretin gene situated upstream of the regulatory regions, alter its transcriptional regulation, contributing to the onset and expression of transthyretin-associated amyloidosis. Previously, we identified a significant association of a non-coding polymorphism of the TTR promoter, rs3764479, with age of onset and survival in patients with ATTRwt amyloid disease. In this dissertation, electrophoretic mobility shift assays (EMSA) were used to investigate transcription factor binding of HepG2 nuclear proteins to short DNA probes with and without rs3764479. These mobility shift studies revealed that HepG2 nuclear extract proteins showed higher affinity to the wild-type TTR sequence than to one containing the rs3764479 SNP. Competition EMSAs suggested SNP-related changes in the binding of transcription factors hepatocyte nuclear factor-1 (HNF1) and hepatocyte nuclear factor-3b may alter TTR gene transcription. To investigate transthyretin gene regulation in V122I ATTR amyloid, the most prevalent TTR gene variant in the United States, the proximal promoter region from patients with V122I ATTR amyloidosis was sequenced and analyzed. In total, 8 SNPs were identified; one (rs955705399) was significantly associated with disease between the two V122I genotype-positive cohorts studied with and without cardiomyopathy. It is postulated that the presence of SNPs could influence gene expression and ultimately disease pathogenesis. In summary, these studies suggest that presence of disease-associated non-coding genetic variations modify transthyretin gene expression by disrupting transcription factor binding and may, in part, explain the clinical heterogeneity seen in patients with transthyretin-associated amyloidoses.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/42221 |
| Date | 03 March 2021 |
| Creators | Boldbaatar, Batbold |
| Contributors | Berk, John L. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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