Allele-specific transcription factor binding in a cellular model of orofacial clefting

Katharina L M Ruff,Julia Welzenbach,Elisabeth Mangold,Michael Knapp,Kerstin U Ludwig,Julia Fazaal,Frederic Thieme,Ronja Hollstein,Jan Gehlen

doi:10.1038/s41598-022-05876-7

Abstract

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a frequent congenital malformation with multifactorial etiology. While recent genome-wide association studies (GWAS) have identified several nsCL/P risk loci, the functional effects of the associated non-coding variants are largely unknown. Furthermore, additional risk loci remain undetected due to lack of power. As genetic variants might alter binding of transcription factors (TF), we here hypothesized that the integration of data from TF binding sites, expression analyses and nsCL/P GWAS might help to (i) identify functionally relevant variants at GWAS loci, and (ii) highlight novel risk variants that have been previously undetected. Analysing the craniofacial TF TFAP2A in human embryonic palatal mesenchyme (HEPM) cells, we identified 2845 TFAP2A ChIP-seq peaks, several of which were located near nsCL/P candidate genes (e.g. MSX1 and SPRY2). Comparison with independent data suggest that 802 of them might be specific to craniofacial development, and genes near these peaks are enriched in processes relevant to nsCL/P. Integration with nsCL/P GWAS data, however, did not show robust evidence for co-localization of common nsCL/P risk variants with TFAP2A ChIP-seq peaks. This data set represents a new resource for the analyses of craniofacial processes, and similar approaches with additional cell lines and TFs could be applied to generate further insights into nsCL/P etiology.

Highlights

Non-syndromic cleft lip with/without cleft palate is a frequent congenital malformation with multifactorial etiology
While the results of our analyses provide only limited evidence for a role of genetically mediated effect of Non-syndromic cleft lip with/without cleft palate (nsCL/P) risk variants at TFAP2A ChIP-seq peaks in human embryonic palatal mesenchyme (HEPM), the map of binding sites as well as the framework described here can be used as blueprint for further integrative analyses of epigenetic and genetic data in nsCL/P
For 2 out of these 11 transcription factors (TF) (TFAP2A and MSX1), additional support for an involvement in nsCL/P etiology was available through their location at previously identified nsCL/P risk loci (i.e., 6p24 and 4p168)

Summary

Introduction

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a frequent congenital malformation with multifactorial etiology. Integration with nsCL/P GWAS data, did not show robust evidence for co-localization of common nsCL/P risk variants with TFAP2A ChIP-seq peaks This data set represents a new resource for the analyses of craniofacial processes, and similar approaches with additional cell lines and TFs could be applied to generate further insights into nsCL/P etiology. Heritability estimates from twin studies and multiplex pedigrees are high, with reported values of up to 90%5,6 This suggests that genetic factors make a substantial contribution to nsCL/P etiology. Multiple investigations have been performed to identify the causal variants, risk genes, and functional mechanisms that contribute to nsCL/P7 These have included several genome-wide association studies (GWAS) and meta-analyses in diverse populations.

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Conclusion