Feng Xiao, Xiaoran Zhang, Sarah U. Morton, Seong Won Kim, Youfei Fan et al.
Rare coding mutations cause ~45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases, but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity. A majority increased enhancer activity, often at regions with undetectable reference sequence activity. Of ten DNVs tested by introduction into their native genomic context, four altered the expression of neighboring genes and iPSC-CM transcriptional state. To prioritize future DNVs for functional testing, we used the MPRA data to develop a regression model, EpiCard. Analysis of an independent CHD cohort by EpiCard found enrichment of DNVs. Together, we developed a scalable system to measure the effect of ncDNVs on CRE activity and deployed it to systematically assess the contribution of ncDNVs to CHD.
Feng Xiao, Xiaoran Zhang, Sarah U. Morton, Seong Won Kim, Youfei Fan, Joshua M. Gorham, Huan Zhang, Paul J. Berkson, Neil Mazumdar, Yangpo Cao, Jian Chen, Jacob Hagen, Xujie Liu, Pingzhu Zhou, Felix Richter, Yufeng Shen, Tarsha Ward, Bruce D. Gelb, Jonathan G. Seidman, Christine E. Seidman, William T. Pu. Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease. 2024-02-20. https://doi.org/10.1038/s41588-024-01669-y
A 400-bp lentiSTARR-seq library containing candidate cardiac regulatory regions and negative controls was introduced into human iPSC-derived cardiomyocytes at differentiation day 17 and collected at day 24. Six biological DNA and RNA libraries were sequenced to measure enhancer activity as RNA relative to genomic DNA.
A 400-bp lentiSTARR-seq library containing 2,891 candidate cardiac regulatory regions and 859 negative controls was introduced into human iPSC-derived cardiomyocytes at differentiation day 10 and collected at day 17. Six biological DNA and RNA libraries were sequenced to measure enhancer activity as RNA relative to genomic DNA.
A lentiviral MPRA tested 6,590 prioritized congenital-heart-disease noncoding de novo variants as matched 171-bp reference and alternate allele oligos in human iPSC-derived cardiomyocytes. The library was applied at differentiation day 17 and assayed on day 24 with four biological DNA and RNA replicates; the processed table contains the 4,210 intact REF–ALT pairs that passed the published coverage filter.
The top 123 cardiac enhancers from the cardiac enhancer screen were split into three overlapping 171-bp fragments, and each fragment was tested as a wild-type sequence plus 17 tiled 10-bp deletions. The barcoded lentiviral MPRA library was applied to iPSC-CMs on differentiation day 17 and assayed on day 24 with four biological DNA and RNA replicates.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 12 files (ZIP)GSE208284_family.soft.gzGSE208284_Mutagenesis_raw_counts_DNA1-4_RNA1-4.txt.gzGSE208285_CHD_raw_counts_DNA1-4_RNA1-4.txt.gzGSE208285_family.soft.gzGSE211417_CM_MPRA_rawcounts_d17.txt.gzGSE211417_CM_MPRA_rawcounts_d24.txt.gzGSE211417_family.soft.gzREADME.txtSupplemental_Data_1_Cardiac_enhancer_MPRA.xlsxSupplemental_Data_2_Cardiac_enhancer_mutagenesis.xlsxSupplemental_Data_3_CHD_ncDNV_MPRA.xlsxSupplemental_Tables_1_7_MPRA_validation.xlsx