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

全基因组关联研究(GWAS)对导致复杂遗传疾病的遗传风险因素产生了全面的见解。然而，将这些发现转化为生物学上相关的过程仍然非常糟糕，我们对相关风险变量如何确切地影响分子过程的理解也是如此。原因包括：(1)信号在非蛋白质编码区的定位；(2)人类基因组的单倍型结构；(3)用于功能分析的高通量方法有限。由于连锁不平衡，仅靠统计方法不足以区分因果(驱动)变异和良性(乘客)变异。以前的功能分析方法包括间接(如ChIPSeq)和直接(如记者分析)方法：间接方法描述细胞系统中DNA区域的调控状态，但不能分析单个遗传变异的影响。直接方法可以用于等位基因特异性分析，但对于并行分析多个变种来说效率极低。此外，它们对复杂遗传表型的风险等位基因预期的低效应大小的敏感性也有限。这些限制也适用于非综合征性口面部裂(NsOFC)，这是人类最常见的畸形之一：尽管Gwas发现了大约40个遗传风险区域(主要在非编码区)，但目前尚不清楚这些区域如何调节nsOFC的生物效应。通过最近实施的高通量技术(例如，下一代测序(NGS))，表征高通量调节效应的定量方法最近已经成为可能。其中一种方法，即所谓的大规模平行报告分析(MPRA)，允许在一次实验中分析数千个变异和/或多个风险区域。MPRA原理基于：(I)合成具有潜在调控元件的寡核苷酸及其等位基因表现，(Ii)将配备分子条形码的报告文库引入人类细胞，以及(Iii)使用NGS对条形码进行量化。在本项目中，MPRA将在实验室中实施，并首先应用于已知的人胚胎腭架间充质细胞(HEPM细胞)中的nsOFC风险基因座。除了在nsOFC的发病机制上获得系统的见解外，该方法还可以应用于未来使用不同细胞类型的其他表型，并将有助于了解复杂遗传疾病的功能和分子基础。

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.