Data_Sheet_1_Characterization of Two Cases of Congenital Dyserythropoietic Anemia Type I Shed Light on the Uncharacterized C15orf41 Protein.PDF

CDA type I is a rare hereditary anemia, characterized by relative reticulocytopenia, and congenital anomalies. It is caused by biallelic mutations in one of the two genes: (i) CDAN1, encoding Codanin-1, which is implicated in nucleosome assembly and disassembly; (ii) C15orf41, which is predicted to encode a divalent metal ion-dependent restriction endonuclease with a yet unknown function. We described two cases of CDA type I, identifying the novel variant, Y94S, in the DNA binding domain of C15orf41, and the H230P mutation in the nuclease domain of the protein. We first analyzed the gene expression and the localization of C15orf41. We demonstrated that C15orf41 and CDAN1 gene expression is tightly correlated, suggesting a shared mechanism of regulation between the two genes. Moreover, we functionally characterized the two variants, establishing that the H230P leads to reduced gene expression and protein level, while Y94S induces a slight decrease of expression. We demonstrated that C15orf41 endogenous protein exhibits nuclear and cytosolic localization, being mostly in the nucleus. However, no altered nuclear-cytosolic compartmentalization of mutated C15orf41 was observed. Both mutants accounted for impaired erythroid differentiation in K562 cells, and H230P mutant also exhibits an increased S-phase of the cell cycle in these cells. Our functional characterization demonstrated that the two variants have different effects on the stability of the mutated mRNA, but both resulted in impaired erythroid maturation, suggesting the block of cell cycle dynamics as a putative pathogenic mechanism for C15orf41-related CDA I.

1型先天性红细胞生成异常性贫血（CDA type I）是一种罕见的遗传性贫血，以相对性网织红细胞减少及先天性畸形为主要特征。其致病原因为两个基因中的任意一个发生双等位基因突变：（i）CDAN1，编码Codanin-1蛋白，该蛋白参与核小体的组装与解离过程；（ii）C15orf41，预测编码一种二价金属离子依赖性限制性内切核酸酶，但其具体功能尚未明确。本研究报道了2例1型先天性红细胞生成异常性贫血病例，鉴定出C15orf41 DNA结合域中的新型变异体Y94S，以及该蛋白核酸酶结构域中的H230P突变。
我们首先对C15orf41的基因表达与蛋白定位进行了分析，结果显示C15orf41与CDAN1的基因表达水平呈紧密相关，提示二者存在共同的调控机制。此外，我们对上述两种变异体开展了功能表征，证实H230P突变可导致基因表达与蛋白水平降低，而Y94S变异仅引起表达水平轻度下降。
研究证实，内源性C15orf41蛋白主要定位于细胞核，同时也存在于胞质中，但未观察到突变型C15orf41的核质分区出现异常改变。两种突变体均可导致K562细胞的红细胞分化受损，且H230P突变还可使该细胞的细胞周期S期比例升高。
本研究的功能表征结果表明，两种变异体对突变mRNA的稳定性存在不同影响，但二者均会引发红细胞成熟障碍，提示细胞周期动态平衡紊乱可能是与C15orf41相关的1型先天性红细胞生成异常性贫血的潜在致病机制。