The role of non-coding genetic variants on transthyretin gene transcription in transthyretin amyloidosis

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

Impact of the Location of CpG Methylation within the GSTP1 Gene on Its Specificity as a DNA Marker for Hepatocellular Carcinoma

Hypermethylation of the glutathione S-transferase π 1 (GSTP1) gene promoter region has been reported to be a potential biomarker to distinguish hepatocellular carcinoma (HCC) from other liver diseases. However, reports regarding how specific a marker it is have ranged from 100% to 0%. We hypothesized that, to a large extent, the variation of specificity depends on the location of the CpG sites analyzed. To test this hypothesis, we compared the methylation status of the GSTP1 promoter region of the DNA isolated from HCC, cirrhosis, hepatitis, and normal liver tissues by bisulfite–PCR sequencing. We found that the 5′ region of the position −48 nt from the transcription start site of the GSTP1 gene is selectively methylated in HCC, whereas the 3′ region is methylated in all liver tissues examined, including normal liver and the HCC tissue. Interestingly, when DNA derived from fetal liver and 11 nonhepatic normal tissue was also examined by bisulfite-PCR sequencing, we found that methylation of the 3′ region of the promoter appeared to be liver-specific. A methylation-specific PCR assay targeting the 5′ region of the promoter was developed and used to quantify the methylated GSTP1 gene in various diseased liver tissues including HCC. When we used an assay targeting the 3′ region, we found that the methylation of the 5′-end of the GSTP1 promoter was significantly more specific than that of the 3′-end (97.1% vs. 60%, p<0.0001 by Fisher's exact test) for distinguishing HCC (n = 120) from hepatitis (n = 35) and cirrhosis (n = 35). Encouragingly, 33.8% of the AFP-negative HCC contained the methylated GSTP1 gene. This study clearly demonstrates the importance of the location of CpG site methylation for HCC specificity and how liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC

Aberrant methylation of the <i>GSTP1</i> in HCC stratified by clinicopathological characteristics.

1<p>Kruskal-Wallis test.</p><p>AFP, alpha-fetoprotein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus.</p

Comparison of the specificity of the 5′-end and the 3-end of the <i>mGSTP1</i> as a biomarker to distinguish HCC samples from tissue samples of other liver diseases, as determined by MSP assays.

<p>(A) Locations of forward (F) and reverse (R) primers and TaqMan probe (P) of the 5′-end MSP (5′-MSP) (including the TaqMan probe) and 3′-end MSP (3′-MSP) SybrGreen assays. The CpG sites (vertical bars) and the transcription start site (TSS) are indicated. Receiver operating characteristic (ROC) curves of the methylated <i>GSTP1</i> gene as a marker to discriminate HCC (n = 120) from non-HCC liver tissues including hepatitis (n = 35) and cirrhosis (n = 35) (B), or hepatitis, cirrhosis, and adjacent non-HCC (C), generated by 5′-end MSP and 3′-end MSP assays, respectively, as indicated. The amount of methylated DNA was the average of two duplicate MSP assays as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035789#s4" target="_blank">Materials and Methods</a>. The area under the curve of each ROC (AUROC) curve and the specificity and sensitivity determined by the cutoff of 10 copies per input of 300 copies of DNA are shown in the inserted table. Note that the CpG sites included in each primer and probe are as follows; 5′-end MSP (F: −27 to −24; P: −23 to −19; R: −11 to −10) and 3′-end MSP (F: −4 to −2; R: +4 to +7).</p

Summary of clinicopathological characteristics of the tissues analyzed using the MSP assays.

1<p>4 of the 6 normal livers are “normal” liver tissues with concomitant cholangiocarcinoma.</p>2<p>Across all subjects (n = 196), age was analyzed by the Student <i>t</i> test and gender by Fisher's exact test.</p><p>AFP, alpha-fetoprotein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; SD, standard deviation.</p

Summary of clinicopathological characteristics of the tissues analyzed by BSP sequencing.

1<p>4/6 of the 6 normal livers are “normal” liver tissues with concomitant cholangiocarcinoma.</p><p>AFP, alpha-fetoprotein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; SD, standard deviation.</p

Examples of the variable specificities of methylated <i>GSTP1</i> genes for HCC reported in previous studies.

1<p>The CpG site number is referred to the transcription start site.</p><p>HCC, hepatocellular carcinoma; MSP, methylation-specific PCR.</p

Comparison of the extent of CpG methylation of the GSTP1 sense strand promoter region among various normal and diseased liver tissues¹.

1<p>The extent of methylation at each CpG site was analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035789#pone-0035789-g001" target="_blank">Figure 1C</a>: no methylation (no methylation detected); low methylation (50% or less); high methylation (more than 50%).</p>2<p>The CpG sites included in the 5′-end and the 3′-end are described in the text.</p>3<p>The percent of CpG methylation was calculated as the number of methylated CpG sites per category/total CpG sites analyzed×100%.</p>4<p>p value was determined using Student's <i>t</i> test.</p><p>HCC, hepatocellular carcinoma.</p

Methylation profiles of the sense and antisense strands of the <i>GSTP1</i> gene by BSP sequencing of DNA isolated from normal liver and diseased liver tissues.

<p>(A) Diagram of the locations of bisulfite sequencing primers and the CpG sites, indicated by vertical bars, in the promoter and the first exon regions of the <i>GSTP1</i> gene (Genbank accession #M24485, nt. 999–1387). The transcription start site (TSS) is also indicated. The CpG sites are bracketed by the bisulfite sequencing primers for the forward (F) and reverse (R) sense strands (GSTP1_S_F and GSTP1_S_R) and the antisense strands (GSTP1_AS_F and GSTP1_AS_R). (B) Methylation status of each CpG site in both sense (S) and antisense (AS) strands of the promoter and the first exon regions of the <i>GSTP1</i> gene from −28 to +4 on the basis of the sense strand 5′ to 3′ direction relative to TSS in hepatocellular carcinoma (HCC, n = 20) tissue, matched adjacent non-HCC liver tissue (Adj Non-HCC, n = 20), and normal (n = 6), hepatitis (n = 5), and cirrhosis (n = 5) tissues. The filled boxes indicate methylation detected and open boxes indicate no methylation detected. (C) Analysis of the extent of methylation at each CpG site of the sense strand <i>GSTP1</i> gene by BS-PCR sequencing of DNA isolated from normal liver and diseased liver tissues. CpG site locations, BS-PCR sequencing assay, and DNA samples are the same as in panel B. The filled boxes indicate a high level of methylation detected (more than 50%); hatched boxes indicate a low level of methylation detected (50% or less); and open boxes indicate no methylation detected.</p