[Phenotype and genotype analysis of 55 children patients with Wilson's disease].

Objective: To understand the clinical phenotype and spectrum of ATP7B gene mutation in children with Wilson's disease (WD). Methods: A total of 55 cases diagnosed with WD at the Children's Hospital Affiliated to Nanjing Medical University from June 2012 to June 2018 were taken as the research subject. ATP7B gene point mutation was detected by direct sequencing after PCR amplification. Heterozygous mutation in children was discovered by sequencing. Furthermore, the long segment mutation of exon was analyzed by multiplex ligation-dependent probe amplification (MLPA). Results: All 55 WD children had varying degree of liver damage symptoms. Among them, 2 cases had combined neurological symptoms. The positive rates of K-F ring (21%), 24-hour urine copper (97.7%), and ceruloplasmin were all abnormal. The results of ATP7B gene had identified 8 homozygous, 41 compound heterozygous and 6 heterozygous in 55 cases. Direct sequencing method had detected ten cases of ATP7B heterozygotes. In addition, MLPA analysis showed that other allele in four cases had a deletion of the ATP7B gene exon. In all cases, 35 different ATP7B gene mutations were detected, including 23 missense mutations, 3 frameshift mutations, 4 nonsense mutations, 3 exon deletions and 2 splicing changes. The most common allele mutation was c.2333G > T/p.R778L in exon 8, with an allele frequency of 36.54%, followed by c.2975C > T/p.P992L in exon 13, with an allele frequency of 14.42%. Conclusion:ATP7B gene c.2333G > T/p.R778L and c.2975C > T/p.P992L mutations are the most common mutations in children with WD in China. WD patients report shows that there are three long deletion mutations in the exon of the ATP7B gene. For WD children whose DNA sequencing is heterozygous ATP7B gene, it is suggested to further use MLPA method to detect deletion mutations of exons.目的： 了解肝豆状核变性（Wilson disease，WD）患儿的临床表型与ATP7B基因突变谱。 方法： 以2012年6月至2018年6月期间在南京医科大学附属儿童医院确诊为WD的55例患儿为研究对象，采用PCR扩增后直接测序法检测ATP7B基因点突变，测序仅发现杂合突变患儿；进一步采用多重连接依赖的探针扩增法（MLPA）进行外显子长片段突变分析。 结果： 55例WD患儿均有不同程度的肝损害症状，其中2例合并神经系统症状。K-F环阳性率（21%）、24小时尿铜阳性率（97.7%）、铜蓝蛋白均异常。55例患者中ATP7B基因鉴定结果：纯合子8例，复合杂合子41例和杂合子6例。直接测序法检测到10例ATP7B基因杂合子，进一步采用MLPA分析发现，其中4例患者另一个等位基因携带ATP7B基因外显子缺失。在所有患者中，共检测到35种不同的ATP7B基因突变，包括23种错义突变，3种移码突变，4种无义突变，3种外显子缺失和2种剪接改变。最常见的等位基因突变为外显子8的c.2333G > T/p.R778L，等位基因频率为36.54%，其次是外显子13的c.2975C > T/p.P992L，等位基因频率为14.42%。 结论：ATP7B基因c.2333G > T/p.R778L和c.2975C > T/p.P992L变异为中国WD患儿中最常见的变异。WD患者中报道3种ATP7B基因外显子大片段缺失变异，对于DNA测序为ATP7B基因杂合子的WD患儿，建议进一步采用MLPA方法检测外显子缺失变异。.