Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy.
Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy.
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DOI：
10.1038/s41436-020-0833-2
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发表时间：
2020-10
期刊：
影响因子：
--
通讯作者：
Ramón-Maiques S
中科院分区：
文献类型：
--
作者：
Del Caño-Ochoa F;Ng BG;Abedalthagafi M;Almannai M;Cohn RD;Costain G;Elpeleg O;Houlden H;Karimiani EG;Liu P;Manzini MC;Maroofian R;Muriello M;Al-Otaibi A;Patel H;Shimon E;Sutton VR;Toosi MB;Wolfe LA;Rosenfeld JA;Freeze HH;Ramón-Maiques S
CAD中的致病性常染色体隐性变异，编码启动嘧啶从头生物合成的多酶蛋白，导致严重的先天性代谢紊乱，可通过饮食补充尿苷治疗。这种情况是很难诊断的大尺寸的CAD超过1000个错义变异和非特异性的临床表现。我们的目的是开发一种可靠的和有辨别力的检测方法，以评估CAD变异的致病性，并选择可能受益于尿苷治疗的受影响个体。使用CRISPR/Cas9，我们产生了一种需要尿苷补充剂才能生存的人CAD敲除细胞系。用重组CAD瞬时转染敲除细胞在缺乏尿苷的情况下恢复生长。该系统确定错义变体，其抑制CAD并且不拯救生长表型。我们确定了25个人的双等位基因变异的CAD和CAD赤字的表型一致。我们使用CAD敲除互补测定来测试总共34种变体，鉴定出16种对CAD活性有害。这些致病性变异的组合证实了11例CAD缺陷的受试者，我们描述了他们的临床表型。我们设计了一种基于细胞的检测方法来检测CAD变异体的致病性，确定了11个CAD缺陷个体，这些个体可以从尿苷治疗中获益。
Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.
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