胱硫醚合成酶基因突变与同型胱氨酸尿症临床表型关系的探讨

Relationship between cystathionine β synthase mutation and phenotypes in patients with homocystinuria

目的 了解由胱硫醚合成酶 (CBS)基因缺陷引起的同型胱氨酸尿症的基因突变类型 ,探讨其基因突变与临床表型的关系 ,确立同型胱氨酸尿症基因诊断方法。方法 对 2 0 0 0年至 2 0 0 2年深圳市儿童医院收集的 6例 ,来自 5个家系的同型胱氨酸尿症患儿进行了分子生物学研究。培养患儿的皮肤成纤维细胞 ,提取总的RNA和genomicDNA ,用PCR法、反转录PCR法、质粒扩增、DNA序列分析、限制性内切酶图谱等技术检测CBS基因的突变 ,并用基因表达和酶活性测定等技术确定致病变异。结果 我们发现了 8个变异 (H6 5R ,G116R ,E14 4K ,G2 5 9S ,K4 4 1X ,Ivs9+1g to a ,Ivs10 +3to +8aagaca to tc ,和 15 90del4 ) ,所有患儿均为杂合子。把这些变异在大肠杆菌细胞中进行基因表达 ,证实它们为致病变异。结论 CBS基因的变异是多样性的。在杂合子状态下 ,基因突变与维生素B6不反应性之间的关系难以确定。即使基因突变相同 ,临床表型也有差异。

Objective To investigate the mutations of CBS gene in patients with homocystinuria,and study the relationship between the mutations and phenotypes.Methods Six patients from five unrelated families were enrolled in this study.Total RNA and genomic DNA were extracted from cultured skin fibroblasts.The mutations on CBS gene were detected in patients with homocystinuria by using biochemical methods,such as polymerase chain reaction,reverse transcription PCR,subcloning,DNA sequence analysis and restriction fragment analysis.All of the mutations were expressed in E.coli and the CBS enzyme activity was measured by using autoaminoacid analysis.Results The detected mutations included 8 mutations(H65R,G116R,E144K,G259S,K441X,Ivs9+1g-to-a,Ivs10+3to+8aagaca-to-tc,and 1590del 4).All patients were compound heterozygous for each mutation.Expression studies in E.coli confirmed that all of the mutations were pathogenic.Conclusion Mutations in CBS gene are quite diverse.It seems difficult to determine the correlation between mutations and pyridoxine-nonresponsive in heterozygote for each mutation.Even if they have identical mutations for the CBS gene,their phenotypes can be different.