Department of Tumor Pathology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi Ward, Hamamatsu, Japan.
Department of Tumor Pathology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi Ward, Hamamatsu, Japan.
Free Radic Biol Med. 2019 Feb 1;131:264-273. doi: 10.1016/j.freeradbiomed.2018.12.010. Epub 2018 Dec 12.
The NTHL1 gene encodes DNA glycosylase, which is involved in base excision repair, and biallelic mutations of this gene result in NTHL1-associated polyposis (NAP), a hereditary disease characterized by colorectal polyposis and multiple types of carcinomas. However, no proper functional characterization of variant NTHL1 proteins has been done so far. Herein, we report functional evaluation of variant NTHL1 proteins to aid in the accurate diagnosis of NAP. First, we investigated whether it would be appropriate to use 5-hydroxyuracil (5OHU), an oxidation product of cytosine, for the evaluation. In the supF forward mutation assay, 5OHU caused an increase of the mutation frequency in human cells, and the C→T mutation was predominant among the 5OHU-induced mutations. In addition, in DNA cleavage activity assay, 5OHU was excised by NTHL1 as well as four other DNA glycosylases (SMUG1, NEIL1, TDG, and UNG2). When human cells overexpressing the five DNA glycosylases were established, it was found that each of the five DNA glycosylases, including NTHL1, had the ability to suppress 5OHU-induced mutations. Based on the above results, we performed functional evaluation of eight NTHL1 variants using 5OHU-containing DNA substrate or shuttle plasmid. The DNA cleavage activity assay showed that the variants of NTHL1, Q90X, Y130X, R153X, and Q287X, but not R19Q, V179I, V217F, or G286S, showed defective repair activity for 5OHU and two other oxidatively damaged bases. Moreover, the supF forward mutation assay showed that the four truncated-type NTHL1 variants showed a reduced ability to suppress 5OHU-induced mutations in human cells. These results suggest that the NTHL1 variants Q90X, Y130X, R153X, and Q287X, but not R19Q, V179I, V217F, or G286S, were defective in 5OHU repair and the alleles encoding them were considered to be pathogenic for NAP.
NTHL1 基因编码 DNA 糖基化酶，参与碱基切除修复，该基因的双等位基因突变导致 NTHL1 相关息肉病（NAP），这是一种以结直肠息肉和多种癌变为特征的遗传性疾病。然而，迄今为止，尚未对变异 NTHL1 蛋白进行适当的功能表征。在此，我们报告了变异 NTHL1 蛋白的功能评估，以帮助 NAP 的准确诊断。首先，我们研究了使用 5-羟尿嘧啶（5OHU）是否合适，5OHU 是胞嘧啶的氧化产物。在 supF 正向突变检测中，5OHU 导致人细胞中突变频率增加，并且在 5OHU 诱导的突变中 C→T 突变占优势。此外，在 DNA 切割活性测定中，5OHU 被 NTHL1 以及其他四种 DNA 糖苷酶（SMUG1、NEIL1、TDG 和 UNG2）切除。当建立过表达五种 DNA 糖苷酶的人细胞时，发现包括 NTHL1 在内的五种 DNA 糖苷酶均具有抑制 5OHU 诱导突变的能力。基于上述结果，我们使用含有 5OHU 的 DNA 底物或穿梭质粒对 8 种 NTHL1 变体进行了功能评估。DNA 切割活性测定表明，NTHL1 的变体 Q90X、Y130X、R153X 和 Q287X，但不是 R19Q、V179I、V217F 或 G286S，对 5OHU 和另外两种氧化损伤碱基的修复活性有缺陷。此外，supF 正向突变检测表明，四种截短型 NTHL1 变体在人细胞中抑制 5OHU 诱导突变的能力降低。这些结果表明，NTHL1 的变体 Q90X、Y130X、R153X 和 Q287X 但不是 R19Q、V179I、V217F 或 G286S 在 5OHU 修复中存在缺陷，并且编码它们的等位基因被认为对 NAP 具有致病性。
