A novel c.-22T>C mutation in GALK1 promoter is associated with elevated galactokinase phenotype.
A novel c.-22T>C mutation in GALK1 promoter is associated with elevated galactokinase phenotype.
复制标题
DOI：
10.1186/1471-2350-10-29
复制
发表时间：
2009-03-24
影响因子：
--
通讯作者：
Song J
中科院分区：
文献类型：
--
作者：
Park HD;Kim YK;Park KU;Kim JQ;Song YH;Song J
在GALK1缺乏症患者中发现了许多GALK1基因变异。然而，在GALK1活性升高的个体中GALK1的分子特征相对未知。我们研究了GALK1活性升高与分子GALK1基因变异的关系，以及GALK1活性升高的分子机制。对63名受试者的聚合酶链式反应产物进行测序，以筛选GALK1启动子的核苷酸变化。发现c.-179A和gt；G、c.-27A和gt；C和c.-22T>；C三个核苷酸替换。在c.-22T和gt；C突变中，13个T/C或C/C基因型者的GALK1活性显著高于50个T/T基因型者(p<；0.001)。在Hep3B细胞中的双荧光素酶报告实验表明，带有c.-22C突变等位基因的GALK1启动子的荧光素酶活性比c.-22T突变等位基因的荧光素酶活性增加了约2.5倍。在电泳迁移率改变分析中观察到一种特异的DNA-蛋白质复合体，与c-22C的亲和力略高于与c-22T的亲和力。在GALK1活性升高的个体中经常观察到的c.-22T和GT；C突变，通过增强与一种目前未知的核蛋白的结合，增加了报告基因的表达。这些结果表明，GALK1活性的提高是由于GALK1启动子内的核苷酸变异导致基因表达增强所致。
Many genetic variations of GALK1 have been identified in the patients with galactokinase (GALK1) deficiency. However, the molecular characteristics of GALK1 in individuals with elevated GALK1 activity are relatively unknown. We investigated the relationship between elevated GALK1 activity and the molecular GALK1 gene variations, and the molecular mechanism underlying elevated GALK1 activity. PCR products from 63 subjects, without any attenuation of galactose degradation enzymes, were sequenced to screen for nucleotide alterations in the GALK1 promoter. Three nucleotide substitutions were identified: c.-179A>G, c.-27A>C, and c.-22T>C. With respect to the c.-22T>C mutation, GALK1 activity in 13 subjects with the T/C or C/C genotype was significantly higher than those in 50 subjects with the T/T genotype (p < 0.001). The dual luciferase reporter assay in Hep3B cells showed that the luciferase activity with the GALK1 promoter with the c.-22C mutant allele increased approximately 2.5-fold, compared to that with the c.-22T. A specific DNA-protein complex was observed in an electrophoretic mobility shift assay, with slightly higher affinity to c.-22C than to c.-22T. The c.-22T>C mutation, which was observed frequently in individuals with elevated GALK1 activity, increased the expression of a reporter gene through enhanced binding of a currently unidentified nuclear protein. These results suggest that the elevated GALK1 activity resulted from enhanced gene expression, due to nucleotide variation within GALK1 promoter.
登录
查看更多内容
影响因子：
3.6
作者：
Yang, F;Agulian, T;Levitsky, LL
通讯作者：
Levitsky, LL
影响因子：
5.8
作者：
Cartharius, K;Frech, K;Werner, T
通讯作者：
Werner, T
影响因子：
8.8
作者：
Park, HD;Park, KU;Song, JH
通讯作者：
Song, JH
影响因子：
4.2
作者：
Nishimura, Y;Tajima, G;Saheki, T
通讯作者：
Saheki, T
影响因子：
2.8
作者：
Roder, K;Schweizer, M
通讯作者：
Schweizer, M