肝豆状核变性基因类型与临床表型关系研究

目的 了解中国人肝豆状核变性（Wilson‘s disease,WD)患者的P型ATP7B基因突变的分布类型与发生情况,研究WD患者临床表现型和基因型之间的关系。方法 采用生化酶测定,聚合酶链反应,单链构象多态性分析,限制性内切酶图谱和DNA序列分析等分子生物学技术,对57例无亲缘关系家庭的60例WD患者进行ATP7B基因突变的检测。结果 60例中52例有不同程度的肝脏损害症状（87%）,其中30例临床表现为单纯肝脏损害症状,12例为肝脏合并神经系统症状,10例肝脏合并其他症状;7/60例表现为单纯神经系统症状;1例5岁,无临床症状,经DNA序列分析确认基因突变11种,其中错义突变类型5种（R778L,V1140A,G943S,V1106I和V1216M）,碱基缺失1种（1384de117),多态性变化5种（IVS4-5T/C,A2495G,C2310G,IVS18 6C/T和IVS20 5A/G）,WD患者52/114个778位点的精氨酸被置换为亮氨酸（R778L）,R778L基因突变发生率为45.6%,52例WD伴肝脏损害的患者中38例存在R778L基因突变（占73%）,其中14例为R778L纯合型,24例为杂合型。2例V1106I基因突变类型的携带者均为迟发型WD患者(1.7%)。3例已知ATP7B基因突变类型（R778L/V1106I,R778L/V1216M和R778L/R778L）的铜-ATP酶活性较正常对照分别下降44.55%,88.23%和69.49%。结论 1384de117和V1140A为ATP7B新基因突变类型,WD患者的最常见R778L基因突变类型在本组的发生率为45.6%。在临床表现有肝损害的患者中,73%的患者携带R778L基因突变的等位基因。

Genotype and phenotype correlation in Chinese patients with Wilson's Disease

OBJECTIVE: To determine distribution and mutation pattern of type P ATP7B gene mutation and to explore genotype and phenotype correlation in patients with Wilson's disease (WD). METHODS: Sixty patients with WD from 57 no kinship families, 37 male and 23 female, were enrolled in this study. The age of onset ranged from 4.6 - 39 years, < or = 16 years in 55 patients. Some exons of ATP7B gene mutation were analyzed in patients with WD by using biochemical methods, polymerase chain reaction-single strand configuration polymorphism (PCR-SSCP), restriction fragment and DNA sequence analysis. Totally 778 coding regions were identified with restriction enzyme Msp I. The activity of Cu-ATPase was assessed by measuring inorganic phosphorus in 3 patients with known genotype. RESULTS: Fifty-two of 60 patients (86%) had presented with hepatic manifestations, 30 of them had only hepatic manifestations, 12/52 patients had hepatic and neurological manifestations at the same time; 10/52 patients had hepatic and other symptom; 7/60 patients had only neurological symptom, one patient had no symptom. Eleven mutations were detected by DNA sequencing, including five missense mutations (R778L, V1140A,G943S, V1106I and V1216M), one deletion (1384del17) and five polymorphisms (IVS4-5T/C, A2495G, C2310G, IVS18 + 6C/T and IVS20 + 5A/G) were identified. R778L mutation was identified 52/114 alleles (45.6%). R778L occurred in 38/52 patients with hepatic manifestation (73%), homozygosis of R778L was demonstrated in 14 patients and heterozygosity of R778L in 24 patients. V1106I mutation was 1.7%, G943S, V1140A, and V1216M was 0.86% respectively in this study. Two patients with delayed onset of neurological symptoms occurred V1106I mutation of ATP7B. Cu-ATPase activity of 3 patients with known mutation (R778L/V1106I, R778L/V1216M and R778L/R778L) declined by 44.55%, 88.23% and 69.49%, respectively, compared with normal control. CONCLUSION: The 1384del17bp and V1106I are two novel mutations found in patients with WD. R778L was common mutation of ATP7B gene with frequency of 45.6% in this study. The mutation in exon 8 of WD gene may play an important role in pathogenesis of WD in Chinese. Carriage of R778L mutation seems to be correlated with hepatic manifestation.