Epigenomic Profiles of African-American Transthyretin Val122Ile Carriers Reveals Putatively Dysregulated Amyloid Mechanisms

Abstract

Background: The Val122Ile mutation in Transthyretin (TTR) gene causes a rare, difficult to diagnose hereditary form of cardiac amyloidosis. This mutation is most common in the United States and mainly present in people of African descent. The carriers have an increased risk of congestive heart failure, peripheral edema, and several other noncardiac phenotypes such as carpal tunnel syndrome, and arthroplasty which are top reasons for ambulatory/outpatient surgeries (OSs) in the country.

Methods: We conducted first-ever epigenome-wide association study using the Illumina's EPIC array, in Val122Ile carriers of African descent for heart disease and multiple OSs-an early disease indicator. Differential methylation across genome wide cytosine-phosphate guanine (CpG) sites was tested between carriers with and without heart disease and OS. Significant CpG sites were investigated for cis-mQTLs loci, followed by gene ontology and protein-protein interaction network. We also investigated the significant CpG sites in a secondary cohort of carriers for replication.

Results: Five differentially methylated sites (P≤2.1×10^-8) in genes-FAM129B, SKI, WDR27, GLS, and an intergenic site near RP11-550A5.2, and one differentially methylated region containing KCNA6 and GALNT3 (P=1.1×10^-12) were associated with heart disease. For OS, we observe 4 sites-2 sites in UBE2E3 and SEC14L5, and other 2 in intergenic regions (P≤1.8×10^-7) and 3 regions overlapping SH3D21, EVA1B, LTB4R2, and CIDEB (P≤3.9×10^-7). Functional protein-interaction module analysis identified ABCA1 (P=0.001) for heart disease. Six cis-mQTLs were associated with one of the significant CpG sites (FAM129B; P=4.1×10^-24). We replicated 2 CpG sites (cg18546846 and cg06641417; P<0.05) in an external cohort of biopsy-confirmed cases of TTR (transthyretin) amyloidosis. The genes identified are involved in transport and clearance of amyloid deposits (GLS, ABCA1, FAM129B); cardiac fibrosis (SKI); and muscle tissue regulation (SKI, FAM129B).

Conclusions: These findings highlight the link between a complex amyloid circuit and diverse symptoms of Val122Ile.

Keywords: amyloid; edema; methylation; mutation.

Figure 1:

Differentially methylated sites in African American TTR-Val122Ile carriers. (A) Methylation sites that were significantly associated with medical history of heart disease. (B) Methylation sites that were significantly associated for having had 10 or more outpatient surgeries. Each CpG site is represented as a data point, with the x-axis being the genomic location, grouped by chromosome and wherein colors represent alternating chromosomes. The y-axis is the −log10 of the p-value of the CpG site. Significant sites are shown as triangles and labelled with CpG probe name and genic annotation in parentheses, triangles pointing upwards signify hypermethylation, whereas triangles pointing downwards signify hypomethylation.

Figure 2:

Differentially methylated regions in TTR-Val122Ile carriers. (A) Regional association with heart disease (B-D) Regional association with 10 or more outpatient surgeries. Each panel displays the association of sites within each region, followed by genomic location, ENSEMBL gene name, DNAse, regulation, SNP tracks (from UCSC browser) and correlation of CpG sites shown as a heatmap.

Figure 3:

Enriched gene ontology (GO) biological processes. The dendrogram shows the FDRpvalue of the pathway associations and are grouped by similarity of function. The genes involved in each of the processes are highlighted in orange bars.

Figure 4:

Functional Protein-Protein Interaction (PPI) Networks. The differentially methylated modules consist of a network of genes based on their functional connectivity using protein-protein interaction. Each module has primary gene which is connected to other target genes in the network. Each module was significant p<0.05 using the FEM method (see methods). The genes in blue show hypermethylation and yellow represents hypomethylation. A) ABCA1 module was associated with heart disease and the significant target genes in addition to ABCA1 were SNTB2, BLOC1S2 and LIN7B. B) EXOSC4 module was associated with outpatient surgeries and also was the only significant gene in the network.

Figure 5:

Local-mQTL associated with site – cg06641417 mapped to the FAM129B gene. The top panel displays single nucleotide polymorphisms (SNPs) associated with CpG site – cg06641417 as a data point and color coded based on linkage disequilibrium with the top lead SNP in purple. The x-axis shows gene annotation (hg19) of the region and the y-axis displays the −log10 of p-value. The following panels present various annotations of the reported SNPs i.e. CADD - Combined Annotation Dependent Depletion, and RegulomeDB – score to identify regulatory variants. The bottom panel highlights the chromatin states of various regulatory features being putatively affected from chromatin markers observed in aorta tissue cell line. Visualization made in FUMA.

Figure 6:

Gene network of significant genic hits and their relationship with TTR. The genes circled in red are the query genes that were identified from the reported analysis. The network shows intermediate genes that connect the query genes based on different interactions as shown in the legend. The lines are colored based on the type of network domain shown in the figure.