NTHL1基因编码DNA糖基化酶,涉及碱基切除修复,该基因的双等位基因突变导致NTHL1相关息肉病(NAP),一种以结肠直肠息肉病和多种癌症为特征的遗传性疾病。然而,到目前为止,尚未对NTHL1变体蛋白进行适当的功能表征。在这里,我们报告了NTHL1变异蛋白的功能评估,以帮助NAP的准确诊断.首先,我们调查了使用5-羟基尿嘧啶(5OHU)是否合适,胞嘧啶的氧化产物,为了评估。在supF正向突变分析中,5OHU引起人体细胞突变频率的增加,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糖基化酶(SMUG1,NEIL1,TDG,和UNG2)。当建立过表达五种DNA糖基化酶的人类细胞时,发现五种DNA糖基化酶中的每一种,包括NTHL1,具有抑制5OHU诱导的突变的能力。基于以上结果,我们使用含5OHU的DNA底物或穿梭质粒对8种NTHL1变体进行了功能评估.DNA切割活性分析表明,NTHL1、Q90X、Y130X,R153X,和Q287X,但不是R19Q,V179I,V217F,或G286S,对5OHU和其他两种氧化损伤的碱基显示出缺陷的修复活性。此外,supF正向突变分析显示,4种截短型NTHL1变异体抑制人细胞中5OHU诱导的突变的能力降低.这些结果表明,NTHL1变体Q90X,Y130X,R153X,和Q287X,但不是R19Q,V179I,V217F,或G286S,在5OHU修复中存在缺陷,编码它们的等位基因被认为是NAP的致病性。
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.