儿童肝豆状核变性临床表型及ATP7B基因突变关联性分析

Correlation of clinical features of Wilson's disease with ATP7B gene mutation in children

目的了解汉族儿童肝豆状核变性(WD)患儿的临床表型与ATP7B基因突变的相关性。方法以2005年7月至2012年12月就诊于复旦大学附属儿科医院确诊WD患儿为研究对象,按起病部位分为肝病型和神经型,以临床表现分为临床型和亚临床型。采用PCR技术扩增ATP7B基因全部外显子并直接测序。提取临床表型和基因型的信息;分析两者之间的相关性。结果 52个无亲缘关系家庭的53例WD患儿进行分析。肝脏损害52例(98.5%)。肝病型41例,神经型12例;临床型19例,亚临床型34例。①肝病型ALT升高明显;神经型24 h尿铜水平、胆汁酸水平和K-F环阳性率均显著高于肝病型;亚临床型起病年龄、ALT或AST异常率显著高于临床型;24 h尿铜水平、胆汁酸升高比例和K-F环阳性率显著低于临床型。②53例WD患儿ATP7B基因外显子序列分析发现致病性突变等位基因97个,突变频率为91.5%。纯合突变8例,复合杂合36例,杂合突变9例。错义突变23种,插入/缺失突变8种,无义突变2种,剪接突变3种。③错义突变与非错义突变,纯合突变与杂合突变患儿在起病年龄、K-F环阳性率、铜蓝蛋白水平、24 h尿铜水平和ALT、AST异常率等方面差异无统计学意义。④3种高发突变p.Arg778Leu(35.0%,34/97)、p.Pro992Leu(15/97,15.5%)和p.Ala874Val/Pro(5/97,5.2%)在临床型和亚临床型、肝病型和神经型间的分布差异无统计学意义。⑤纯合、错义和错义+剪接突变在临床型和亚临床型、肝病型和神经型间的分布差异无统计学意义。结论 WD患儿几乎均有肝脏受累,多以肝病表现起病。ATP7B常见突变为p.Arg778Leu、p.Pro992Leu和p.Ala874Val,常见基因型和临床表型间未发现显著相关性。p.Ala874Val/Pro突变患儿起病年龄较低,剪接突变对血清铜蓝蛋白影响较小。

Objective Wilsong disease （WD） is a rare children （52 families） from the Chinese Han population to Methods Cases of children diagnosed as WD in Jul 2005 - autosomal recessive disorder. We have evaluated 53 WD cases in expand our knowledge of ATP7B mutations and clinical features. Feb 2012 were analyzed retrospectively with clinical symptoms and laboratory test results. The cases were categorized into hepatic group and neurologic group by disease region and categorized into clinical type and subclinical type by presentation. The coding and promoter regions of the ATPTB gene were analyzed by direct sequencing of genomic DNA. Then the genotype-phenotype correlations were analyzed. Results 53 cases of WD （52 probands） were collected （male, n =37; female, n = 19; age range, 2.5 - 13.5 years）. 52 cases were with liver disease（98.5% ）, 41 cases with diagnosed hepatic presentation （6.0 ＋ 2.3 ）years and 12 cases as neurologic presentation （9.5 ＋ 3.0） years. 19 and 34 cases were clinical type and subclinical type, respectively. Alanine transpeptidase（ALT） level was higher in the hepatic group however the level of 24 h urinary copper, serum bile acid （ SBA）, and positive rate of K-F ring were higher in neurology group. ALT/AST ratio and ALT or AST level were higher in the subclinical type than that in the clinical type but the age at onset, 24 h urinary copper, positive rate of K-F ring and SBA were lower in subclinical type than that in clinical type. 97 pathogenicity alleles （91.5%） with thirty six different mutations were identified in these patients. The frequency of homozygotes, combined heterozygotes and heterozygotes were 8 36 and 9, respectively. 23 types of missense mutation, 8 types of frameshift mutations, 2 types of nonsense mutation and 3 types of splice mutation were found. Frequency of missense mutation, synonymous mutation, homozygotes or heterozygotes were not associated with the age of onset, K-F rings, or the level of ALT, AST, 24 h urinary copper, serum ceruloplasmin（SCp）. Common mutations included p. Arg778Leu （34/97）, p. Pro992Leu （15/97） and p. Ala874Val （ 5/ 97 ） , their frequencies did not significantly differ in hepatic and neurologic groups, clinical and subclinical types. Patients with p.Ala874Va/Pro mutation had smaller age of onset （P = 0. 017, 0. 000）. 6 cases with splicing mutations were also associated with high serum ceruloplasmin （ P = 0. 000）. Conclusion Wilson ~s disease in Chinese children almost all had liver damages and dominant hepatic presentations. Therefore exons （8, 11, and 13） and mutations （p. Arg778Leu, p. Pro992Leu, p. Ala874Va/ Pro） should be considered high priority for cost-effective testing in China. The relationship between phenotype and genetype was not found. But patients with p. Ala874Va/Pro mutation develop disease early and patients with splicing mutations were associated with high serum ceruloplasmin.