Massive-parallel reporter assays (MPRA) for functional characterization of non-coding common risk variants for nonsyndromic cleft lip with or without cleft palate.

• Applicant: Dr. Kerstin Ludwig
• Subject Area: Human Genetics
• Term: from 2018 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 396961086

Genome-wide association studies (GWAS) have generated comprehensive insights into genetic risk factors that contribute to complex genetic disorders. However, the translation of these findings into biologically relevant processes is still very poor, as is our understanding of how exactly the associated risk variants influence molecular processes. Reasons for this are, among others, (i) the localization of the signals in non-protein-coding regions, (ii) the haplotype structure of the human genome, and (iii) the limited availability of high-throughput methods for functional analyses. Due to the linkage disequilibrium, statistical methods alone are not sufficient to separate causal (driver) variants from the benign (passenger) variants. Previous methods for functional analyses include indirect (e.g., ChIPSeq) and direct (e.g., reporter assays) methods: Indirect methods describe regulatory states of a DNA region in a cell system, but are not able to analyze the effect of individual genetic variants. Direct methods can be used for allele-specific assays, but they are extremely inefficient for the analysis of multiple variants in parallel. Also, they have limited sensitivity for the low effect sizes expected for risk alleles of complex genetic phenotypes.These constraints also apply to nonsyndromic orofacial cleft (nsOFC), one of the most common human malformations: although GWAS identified about 40 genetic risk regions (mostly in non-coding regions), it is not yet clear how these regions mediate the biological effect for nsOFC. Through the recent implementation of high-throughput technologies (e.g., Next-Generation Sequencing (NGS)), quantitative methods for the characterization of high-throughput regulatory effects have recently become availalbe. One of these methods, so-called massive-parallel reporter assay (MPRA), allows the analysis of thousands of variants and/or multiple risk regions in a single experiment. The MPRA principle is based on: (i) the synthesis of oligonucleotides with potential regulatory elements and their allelic manifestations, (ii) the introduction of reporter libraries equipped with molecular barcodes into human cells, and (iii) the quantification of barcodes using NGS. In the present project, MPRA is to be implemented in the laboratory and first applied to the already known nsOFC risk loci in human embryonic mesenchyme cells of the palatal shelves (HEPM cells). In addition to the systematic insights gained in the pathogenesis of nsOFC, this method can also be applied to other phenotypes in the future using different cell types and will help to understand the functional and molecular basis of complex genetic disorders.

• DFG Programme: Research Grants