Background: There is an increasing understanding of the relationship between rare coding pathogenic variants and their clinical sequelae. However, for bleeding, platelet, and thrombotic disorders (BPD) a large portion of heritable cases remain without a molecular diagnosis. We have shown that some of these unexplained cases are caused by non-coding variants in the gene-regulatory space (Turro et al., 2020).

Aims: This study aims to identify the regulatory regions of 93 diagnostic-grade BPD genes (Megy et al. 2019) in relevant cell types to increase the heritable-BPD diagnostic yield.

Methods: We differentiated human induced pluripotent stem cells into megakaryocytes, endothelial cells and hepatocytes. A capture Hi-C approach was used to identify regulatory regions of the 93 BPD genes (BPD-regulome hereafter) in these cells. The BPD-regulome was further constrained using statistical and experimental methods. We overlaid epigenetic features, RedPop and applied the BeviMed statistical approach (Greene et al., 2016; Turro et al., 2020). We searched the whole-genome-sequencing (WGS) data of 838 unexplained heritable BPD cases for putative pathogenic variants in the BPD-regulome, and measured their effects on transcription in reporter assays.

Results: We produced the highest resolution interaction maps to date for the 93 BPD genes, identifying 62,027 interactions to putative regulatory elements. This framework identified 31 possible associations between rare variants in the BPD-regulome of unexplained cases at a posterior probability >0.7. Moreover, a visual review of structural variants overlapping the BPD-regulome identified three possibly explanatory aberrations, including the recently described pathogenic deletion in the HDAC6-GATA1 locus (Turro et al. 2020). Finally, we showed that seven of these variants in the BPD-regulome alter gene expression in reporter assays.

Conclusion(s): We generated a high-resolution map of the BPD-regulome in the relevant cell types. The regulatory regions defined in this study lay the foundation to explore WGS-data from unexplained BPD cases for causal variants.

Image

Fig.1 | A BPD regulatory region identified with the capture Hi-C approach, differential use of the regulatory space in the three cell types, and effect of variants in BPD-regulome. -A- RUNX1 chromatin structure in the three cell types relevant for human haemostasis -i.e. megakaryocytes, endothelial cells and hepatocytes, respectively orange, red and brown-. Each cell type has the chromatin interactions identified with the capture Hi-C -top track- and H3K27Ac to show regulatory regions -bottom track-. -B- All the interactions identified per gene, in the different cell types, have been used for the dimensionality reduction -i.e. PCA- and show the different patterns of promoter interactions for some of the diagnostic-grade genes. -C- Regulatory capacity of the regions identified with the capture Hi-C approach and the effect of rare regulatory variants identified in the NIHR Rare-Diseases cohort. The regulatory capacity of each region has been tested using the wild type sequence of the regulatory regions and the sequence harbouring the variant identified in the NIHR Rare-diseases cohort. The horizontal black line is the Luciferase/Renilla ratio for the empty vector.

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