While genome-wide association studies (GWAS) and positive selection scans identify genomic loci driving human phenotypic diversity, functional validation is required to discover the variant(s) responsible. We dissected the IVD gene locus—which encodes the isovaleryl-CoA dehydrogenase enzyme—implicated by selection statistics, multiple GWAS, and clinical genetics as important to function and fitness. We combined luciferase assays, CRISPR/Cas9 genome-editing, massively parallel reporter assays (MPRA), and a deletion tiling MPRA strategy across regulatory loci. We identified three regulatory variants, including an indel, that may underpin GWAS signals for pulmonary fibrosis and testosterone, and that are linked on a positively selected haplotype in the Japanese population. These regulatory variants exhibit synergistic and opposing effects on IVD expression experimentally. Alleles at these variants lie on a haplotype tagged by the variant most strongly associated with IVD expression and metabolites, but with no functional evidence itself. This work demonstrates how comprehensive functional investigation and multiple technologies are needed to discover the true genetic drivers of phenotypic diversity.

中文翻译：

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三个连锁变体对异戊酰辅酶A脱氢酶基因表达具有相反的调节作用

虽然全基因组关联研究 (GWAS) 和正选择扫描可以识别驱动人类表型多样性的基因组位点，但仍需要进行功能验证来发现负责的变异。我们剖析了 IVD 基因位点（编码异戊酰辅酶 A 脱氢酶），通过选择统计数据、多重 GWAS 和临床遗传学对功能和健康具有重要意义。我们结合了荧光素酶测定、CRISPR/Cas9 基因组编辑、大规模并行报告基因测定 (MPRA) 以及跨调控位点的删除平铺 MPRA 策略。我们鉴定了三种调控变异，包括插入缺失，它们可能支持肺纤维化和睾酮的 GWAS 信号，并且与日本人群中积极选择的单倍型相关。这些调控变异在实验上对 IVD 表达表现出协同和相反的作用。这些变体的等位基因位于由与 IVD 表达和代谢物最密切相关的变体标记的单倍型上，但本身没有功能证据。这项工作展示了如何需要全面的功能研究和多种技术来发现表型多样性的真正遗传驱动因素。