Genome-wide enhancer-associated tandem repeats are expanded in cardiomyopathy

Abstract

Background: Cardiomyopathy is a clinically and genetically heterogeneous heart condition that can lead to heart failure and sudden cardiac death in childhood. While it has a strong genetic basis, the genetic aetiology for over 50% of cardiomyopathy cases remains unknown.

Methods: In this study, we analyse the characteristics of tandem repeats from genome sequence data of unrelated individuals diagnosed with cardiomyopathy from Canada and the United Kingdom (n = 1216) and compare them to those found in the general population. We perform burden analysis to identify genomic and epigenomic features that are impacted by rare tandem repeat expansions (TREs), and enrichment analysis to identify functional pathways that are involved in the TRE-associated genes in cardiomyopathy. We use Oxford Nanopore targeted long-read sequencing to validate repeat size and methylation status of one of the most recurrent TREs. We also compare the TRE-associated genes to those that are dysregulated in the heart tissues of individuals with cardiomyopathy.

Findings: We demonstrate that tandem repeats that are rarely expanded in the general population are predominantly expanded in cardiomyopathy. We find that rare TREs are disproportionately present in constrained genes near transcriptional start sites, have high GC content, and frequently overlap active enhancer H3K27ac marks, where expansion-related DNA methylation may reduce gene expression. We demonstrate the gene silencing effect of expanded CGG tandem repeats in DIP2B through promoter hypermethylation. We show that the enhancer-associated loci are found in genes that are highly expressed in human cardiomyocytes and are differentially expressed in the left ventricle of the heart in individuals with cardiomyopathy.

Interpretation: Our findings highlight the underrecognized contribution of rare tandem repeat expansions to the risk of cardiomyopathy and suggest that rare TREs contribute to ∼4% of cardiomyopathy risk.

Funding: Government of Ontario (RKCY), The Canadian Institutes of Health Research PJT 175329 (RKCY), The Azrieli Foundation (RKCY), SickKids Catalyst Scholar in Genetics (RKCY), The University of Toronto McLaughlin Centre (RKCY, SM), Ted Rogers Centre for Heart Research (SM), Data Sciences Institute at the University of Toronto (SM), The Canadian Institutes of Health Research PJT 175034 (SM), The Canadian Institutes of Health Research ENP 161429 under the frame of ERA PerMed (SM, RL), Heart and Stroke Foundation of Ontario & Robert M Freedom Chair in Cardiovascular Science (SM), Bitove Family Professorship of Adult Congenital Heart Disease (EO), Canada Foundation for Innovation (SWS, JR), Canada Research Chair (PS), Genome Canada (PS, JR), The Canadian Institutes of Health Research (PS).

Keywords: Cardiomyopathy (CMP); Long-read sequencing; Tandem repeat expansions (TREs); Transcription regulation.

Fig. 1

Sequence characteristics and genomic locations of rare tandem repeat expansions. a, b The distribution of the repeat unit (motif) GC-composition in rare tandem repeats in the CA (a) and UK (b) CMP cohorts. Blue bars represent the percent of the regions in individuals from the CA CMP cohort; light blue bars represent the percent of the regions in individuals from the UK CMP cohort; grey bars represent the percent of the regions in individuals from the control cohort. c, d The distribution of the dominant motif length of repeat units in the CA (c) and UK (d) CMP cohorts. One-sided Wilcoxon's rank sum test p-value is labelled inside the box. e Association analysis between the number of rare TREs in individuals from the CA (n = 305 regions) and UK (n = 280 regions) CMP cohorts, and different genomic features (x-axis) based on 1 kb segments. Odds ratios (y-axis) were derived from logistic regression coefficients of the genomic features with 95% confidence intervals. f The distribution of repeat size in probands and their parents (i.e., parent-proband duos), in the regions where the repeat is expanded in the proband (n = 9 regions from 9 parent-proband duos).

Fig. 2

Functional characteristics of rare tandem repeat expansions. a Burden of rare tandem repeat expansions in different genomic features in individuals with CMP compared to controls. Empirical p-values from 10,000 permutations are labelled for the features significantly enriched in individuals with CMP. Blue bars represent burden in individuals from the CA CMP cohort; light blue bars represent burden in individuals from the UK CMP cohort. The horizontal dashed line represents odds ratio = 1. b The distributions of gnomAD LOEUF (constraint score) in genes with rare TREs in the CA CMP (n = 127) and all other genes from the genome (n = 19,588), and in the UK CMP (n = 142) and all other genes in the genome (n = 19,705) (Wilcoxon's rank sum test p-value is labelled on top). c A map of genes and functions enriched in genes associated with rare TREs. Nodes represent genes harbouring rare TREs (circles) and their directly interacting partners (diamonds); colour indicates if a gene belongs (red) or does not belong (grey) to the enriched gene sets. Edge colour indicates the type of interaction: physical (pink) or through the pathway (blue); the width of the edge is proportional to the normalized maximum weight.

Fig. 3

Regulatory and clinical features of rare TSS-proximal tandem repeat expansions. a Distance from all detected tandem repeat expansions (grey) and rare TREs in individuals from the CA CMP cohort (blue) to the nearest transcription start site (TSS). b Burden of rare TREs in regulatory features in individuals with CMP compared to controls. Empirical p-values from 10,000 permutations are labelled for the features significantly enriched in individuals with CMP. Blue bars represent burden in individuals from the CA CMP cohort, light blue bars represent burden in individuals from the UK CMP cohort. The horizontal dashed line represents odds ratio = 1. c Comparison of the number of rare TREs (y-axis, 95% confidence interval) with respect to five variables (x-axis) in individuals with (orange) or without (magenta) the indicated feature. Two-sided Wilcoxon's rank sum test p-value is labelled for the variables with significant difference between the two groups.

Fig. 4

Tandem repeat expansion at the promoter region of DIP2B in three families. a Allele repeat size distribution for CGG-repeat expansion in DIP2B based on ExpansionHunter in individuals from the CA CMP cohort and their family members (n = 361). Each dot represents samples with the corresponding allele repeat size; the size of the dot is proportional to the number of samples. Samples from the families, where probands have tandem repeat expansions, are coloured in red (Family-1), orange (Family-2) and yellow (Family-3). b Prevalence of DIP2B expansion carriers among patients with neurological and cardiovascular phenotypes. p-values are labelled for the phenotypes significantly enriched in individuals with DIP2B expansion. Colour indicates significance level (red ≤0.05; grey >0.05). The horizontal dashed line represents odds ratio = 1. c Distribution of RNA-seq FPKM values for selected probands in DIP2B. Coloured dots represent affected probands with DIP2B expansion from Family-1 (red) and Family-3 (yellow). d, e Scatterplot of the allele size estimated by long-read sequencing (LRS, x-axis) and short-read sequencing (SRS, y-axis) for smaller (d) and larger (e) alleles. Pearson's correlation coefficient and p-value are labelled inside the plot. f, g Methylation level of DNA regions flanking repeat in DIP2B in the probands from Family-1 (f) and Family-3 (g). The left panel represents a wild type allele with unexpanded repeat, and the right panel represents an allele with an expanded repeat. The x-axis represents genomic coordinates, the y-axis represents DNA methylation level. The red arrow marks the repeat region, and the dashed red line marks the TSS.

Fig. 5

Differential expression of genes with TSS proximal rare tandem repeat expansions. a Enrichment analysis of genes with TSS-proximal rare TREs for the genes that are differentially expressed in patients with HCM or DCM as compared to individuals with non-failing hearts. The y-axis represents Fisher's exact test odds ratio. Blue bars represent burden in individuals from the CA CMP cohort, light blue bars represent burden in individuals from the UK CMP cohort. Error bars represent the 95% confidence interval. The horizontal dashed line represents odds ratio = 1. b hdWGCNA hierarchical clustering dendrogram of genes in the co-expression network of the left ventricle of the heart. Each colour represents one specific co-expression module, branches represent genes. c Average expression and percentage of cells expressed between the cell types and the five modules obtained from hdWGCNA analysis across different cell types in the left ventricle of the heart. For each dot, the size represents the percent expressed, and the colour represents the average expression level of genes within a specific module (y-axis) in cells of a certain type (x-axis). d Enrichment in genes with TSS proximal rare TREs for modules identified in the hdWGCNA analysis. The y-axis represents Fisher's exact test odds ratio with 95% confidence interval. Blue bars represent the CA CMP cohort; light bars represent the UK CMP cohort. The horizontal dashed line represents odds ratio = 1.