Reichardt J K, Woo S L
Howard Hughes Medical Institute, Department of Cell Biology, Houston, TX.
Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2633-7. doi: 10.1073/pnas.88.7.2633.
We describe the molecular characterization of two mutations responsible for galactosemia, an inherited disorder of galatose metabolism that causes jaundice, cataracts, and mental retardation in humans. The coding region of galactose-1-phosphate uridylyltransferase (GALT; UDPglucose:alpha-D-galactose-1-phosphate uridylyltransferase, EC 2.7.7.12) was amplified by the polymerase chain reaction from total cDNA of a classic galactosemic individual and was characterized by direct sequencing of the products. Two missense mutations were identified: (i) replacement of valine-44 by methionine and (ii) replacement of methionine-142 by lysine. These mutations led to a drastic reduction in GALT activity when individual mutant cDNAs were overexpressed in a mammalian cell system, although full-length protein is synthesized in this assay. The two galactosemia mutations account for 3 of the 15 galactosemia alleles analyzed. These results suggest that galactosemia is caused by a variety of mutations, which might be responsible for the observed clinical heterogeneity of this disorder. We also present the molecular characterization of two GALT polymorphisms: (i) replacement of leucine-62 by methionine and (ii) replacement of asparagine-314 by aspartate. It appears that galactosemia mutations tend to occur in regions that are highly conserved throughout evolution while the polymorphisms change variable residues.
我们描述了导致半乳糖血症的两种突变的分子特征，半乳糖血症是一种遗传性半乳糖代谢紊乱疾病，可导致人类出现黄疸、白内障和智力发育迟缓。通过聚合酶链反应从一名典型半乳糖血症患者的总cDNA中扩增出1-磷酸半乳糖尿苷酰转移酶（GALT；UDP葡萄糖：α-D-1-磷酸半乳糖尿苷酰转移酶，EC 2.7.7.12）的编码区，并对产物进行直接测序以进行特征分析。鉴定出两个错义突变：（i）缬氨酸-44被甲硫氨酸取代，（ii）甲硫氨酸-142被赖氨酸取代。当单个突变cDNA在哺乳动物细胞系统中过表达时，这些突变导致GALT活性急剧降低，尽管在该检测中合成了全长蛋白。这两个半乳糖血症突变占所分析的15个半乳糖血症等位基因中的3个。这些结果表明，半乳糖血症是由多种突变引起的，这可能是导致该疾病临床异质性的原因。我们还展示了两个GALT多态性的分子特征：（i）亮氨酸-62被甲硫氨酸取代，（ii）天冬酰胺-314被天冬氨酸取代。半乳糖血症突变似乎倾向于发生在整个进化过程中高度保守的区域，而多态性则改变可变残基。
