PDF(Pubmed)
Abstract：
CAD is a large, 2225 amino acid multienzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are nonspecific, there is no biomarker, and the protein has over 1000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in seven affected individuals; they would benefit from uridine treatment. We also tested nine variants previously reported as pathogenic and confirmed the damaging effect of seven. However, we reclassified two variants as likely benign based on our assay, which is consistent with their long-term follow-up with uridine. We found that several computational methods are unreliable predictors of pathogenic CAD variants, so we extended the functional assay results by studying the impact of pathogenic variants at the protein level. We focused on CAD\'s dihydroorotase (DHO) domain because it accumulates the largest density of damaging missense changes. The atomic-resolution structures of eight DHO pathogenic variants, combined with functional and molecular dynamics analyses, provided a comprehensive structural and functional understanding of the activity, stability, and oligomerization of CAD\'s DHO domain. Combining our functional and protein structural analysis can help refine clinical diagnostic workflow for CAD variants in the genomics era.
摘要：
CAD是一个很大的，从头嘧啶生物合成所需的2,225个氨基酸多酶蛋白。病理性CAD变异导致发育性和癫痫性脑病，对尿苷补充剂高度敏感。CAD缺乏很难诊断，因为症状是非特异性的，没有生物标志物，这种蛋白质有1000多种已知的变体。为了改善诊断，我们使用生长互补试验评估了20个未报告的错义CAD变异体的致病性,该试验在7个患病个体中鉴定出11个致病变异体;他们将受益于尿苷治疗.我们还测试了先前报道为致病性的9种变体，并证实了7种的破坏作用。然而,根据我们的分析，我们将两个变异体重新分类为可能是良性的，这与他们使用尿苷的长期随访一致。我们发现几种计算方法是致病性CAD变异的不可靠预测因子，因此，我们通过研究致病变异在蛋白质水平上的影响来扩展功能测定结果。我们专注于CAD的二氢乳清酶（DHO）域，因为它积累了最大的破坏性错义变化密度。8种DHO致病变体的原子分辨率结构，结合功能和分子动力学分析，提供了对活动的全面结构和功能理解，稳定性,和CAD的DHO域的寡聚化。结合我们的功能和蛋白质结构分析可以帮助完善基因组学时代CAD变异的临床诊断工作流程。本文受版权保护。保留所有权利。
