| Background and ObjectiveWilson disease (WD) [MIM #277900] is an autosomal recessive disorder with copper metabolism defect, including deficient incorporation of copper into ceruloplasmin and impaired biliary copper excretion. The metabolism lesions lead to toxic accumulation of copper in various tissues, principally the liver and brain, which could cause complex clinical manifestation, such as hepatic, neurological or psychiatric impairment and Kayser-Fleischer (K-F) ring. The worldwide prevalence of WD is estimated to be 1 in 30 000, with a corresponding gene frequency of 0.56% and a carrier frequency of about 1 in 90. And in China, the incidence of Wilson disease is up to 1 in 10 000, which is the first in single-gene genetic disorders of neurology. Early diagnosis and treatment are essential in decreasing morbidity and mortality for Wilson disease. However, the varied clinical manifestations of Wilson disease are insidious and nonspecific except the K-F ring, and some patients may not be biochemically distinguishable from healthy carries, since ceruloplasmin levels may be normal in 5～10% of patients and low in 15～25% of carriers. It is easy to delay the diagnosis. Therefore, in addition to the clinical characteristics, such as liver disease, neurological symptoms and K-F ring in corneal and family history, it is necessary for WD patients, especially some atypical cases, to diagnosis through molecular diagnosis. As one of means to make a definite diagnosis, mutation analysis of the disease causing gene of Wilson disease is also able to detect the asymptoms patients and heterozygote and make genetic screening and prenatal diagnosis which can prevent the born of children with WD.As the only disease-causing gene of Wilson disease, WD gene maps at chromosome 13q14.3 and encodes a copper transporting P-type ATPase composed of 1465 amino acid residues that form copper-binding domains, functional domains of ATP7B and transmembrane domains. The mutant forms are various, which is characteristic of a compound heterozygous mutation and a rare homozygous mutation, and a few common mutations with a wide range of rare mutations. Mutation analysis has been investigated in many WD patients in different ethnic populations and more than 500 disfferent disease-causing mutations have been identified. More than 100 of these mutations have been identified in Chinese populations. These mutations are distributed in the functional domains of ATP7B gene. In addition, the mutations of ATP7B gene are plenty of genetic heterogeneity. The hot spot mutations were various in different coutries. For example, in Europe, the most common mutations are p.His1069Gln in exon 14 and p.Gly1266Lys in exon 18. But the dominant mutation in Asia is p.Arg778Leu in exon 8. Therefore, it is important to establish the mutation spectrum in different regions and ethnic for early diagnosis and treatment, which has great significance to improve the quality of patients with WD in China.Denaturing high-performance liquid chromatography (DHPLC) has been proved to be a reliable, rapid, and highly sensitive method for detecting point mutations based on the different melting characteristics of hetroduplexes and homoduplexes by ion pair reverse-phase HPLC under partially denaturing conditions. Its sensitivity and specificity are higher than commonly used denatured gradient gel electrophoresis (DGGE), chemical cleavage of mismatch (CCM), conformation sensitive gel electrophoresis (CSGE) and single-strand conformation polymorphisms (SSCP) technologies and has been widely used in mutational screening and molecular diagnosis of hereditary diseases. According to these adavantages of DHPLC assay, we attempted to study on mutations of ATP7B gene by combination of PCR/DHPLC and DNA sequencing directly and establish the mutation spectrum in southern Chinese population, which can provide useful informations for early diagnosis and genetic consulting of Wilson disease.Materials and MethodsA total of 169 blood samples from 70 unrelated families were enrolled in this study, including 73 WD patients (2 affected siblings in one family) and 96 family members. All were ethnic Han Chinese originating from Guandong and Guangxi Province. The onset age was from 3 to 48 years. And the 73 patients consisted of 41 males (56.16%) and 32 females (43.84%). The diagnosis of WD was primary based on clinical symptoms- the presence of Kayser-Fleischer (K-F) ring in the cornea, acute or chronic liver failure and/or typical neurological symptoms; and biochemical data- low serum ceruloplasim (<0.2 g/L), high excretion of 24h urinary copper (>100μg/24h). In addition,200 healthy Chinese Han individuals were enrolled as controls. Genomic DNA was extracted from peripheral blood using standard phenol-chloroform extraction procedure. All the coding exons of ATP7B gene and their associated splice-site junctions were amplified by the polymerase chain reaction (PCR). The PCR products of all samples were analyzed by DHPLC and samples with abnormal peaks were sequenced directly.Results and discussionWe investigated the whole exons of ATP7B gene in 73 patients (146 chromosomes) with WD from 70 families, and identified 50 different peaks. Through DNA directly sequencing, we found they were consisted by 38 different disease-causing mutations and 13 polymophisms. DHPLC peaks had great repeatability. That means the peak shape was similar among thedifferent samples with same mutations in the same segment. The PCR/DHPLC technology was a reliable, rapid, semi-automatic, high-throughtput and highly sensitive method for detecting mutations, and it was not only applicable for clinical/prenatal diagnosis of genetic disease, but also for the large-scale epidemiological study.In this study, we identified 38 different ATP7B mutations that included 24 missense mutations,3 splice-site mutations,5 nonsense mutations and 6 frameshift mutations. All of them were distrubuted in exon 3,7,8,11,12,13 and 16. Five mutations were identified on exon 8 or 12, four on exon 16, three on exon 3,7,11 or 13, while none was found in exons 1,4,5,9,20 and 21. Mutations in ATP7B were identified in 69 of the 73 patients (2 with three mutations,31 with 2 mutations and 36 with only one mutation) and no mutation was detected in 4 patients. The mutation detection rate achieved 69.86%(102 disease alleles found in 146 chromosomes). We recommend screening exons 8,16 and 13, which cover 70.19% of mutations.Only 3 among 73 patients were homozygotes, indicating that ATP7B mutation is characteristic of a compound heterozygous mutation and a rare homozygous mutation. Only 1 mutation was found in 36 patients and none was identified in 4 patients, suggesting that the other unknown mutation might be located on the outside exons and the flanking regions, such as the promoter, intron or other DNA control regions. The results were confirmed that the genetic background of ATP7B mutant was very heterogenenous. And we can not speculate the phenotype by genotype. In addition, we observed two patients carried three mutations. According to the family study, we found that c.3426G>C co-segregated in cis with c.3443T>C and the parental genotypes confirmed compound heterozygosity of c.3426G>C and c.3443T>C on the same chromosomes. This phenomenon had been described only in WD patients in Hong Kong Chinese, which suggested that this cis-regulation might be specific for southern Chinese WD patients.The most common mutation in our patient was c.2333G>T (p.Arg778Leu), which was detected in 32 patients (two Homozygous,16 compound heterozygous and 14 single heterozygotes) and accounting for 23.29% of alleles studied (34/146). It was consistant with the views previous reported. We comprised the clinical feature of patients with Arg778Leu among two homozygotes and sixteen compound heterozygotes. And statistical results indicated that the average age of onset in p.Arg778Leu homozygotes was significantly older than compound heterozygoted (P=0.002), and there was no statistical difference in serum copper and ceruloplasmin between these two groups. These results indicated that p.Arg778Leu mutation was a mild mutation which had no relation on the phenotype. As the number of samples was small, the further research was dependant on the accumulation of cases and the study on the protein expression. The second common mutation was still controversial in China. And in here, the second most frequent mutation was c.3443T>C (p.Ile1148Thr) located in exon 16, which accounted for 9.59% of alleles studied (14/146). The p.His1069Gln mutaion is the most common mutation in European populations but it was not found in our patient. In addition to the mutations,13 polymorphisms were identified. Among the 13 polymorphisms,11 were found in the present study and two of them are novel. The novel mutations C.1839C＞T (p.Ile613Ile) in exon 5 and c.2922G>A (p.Thr974Thr) in exon13 are synonymous and, although they were not detected in the 200 heathy controls, we still consider them to be polymorphisms.Ten novel mutations were identified in our study including 6 missense mutations (c.2120A>G, C.2564C＞A, c.2620G>C, c.2761A>C, c.3236G>T and c.3446G>A),2 splice-site mutations (c.2121+3A>T and c.3244-2A>G),1 nonsense mutation (c.3682A>T) and 1 frameshift mutation (c.18751876insAATT). All the novel mutations were classified as disease-causing mutations and none was found in the 200 healthy controls. And after retrieving in PubMed and online database of Wilson disease (http://www.wilsondisease.med.ualberta.ca/database.asp) and human mutation (http://www.hgmd.cf.ac.uk/ac/validate.php), they were confirmed to be novel ATP7B mutations, which had never been reported. Meanwhile, they expanded the range of mutations in Chinese population and enriched the mutation spectrum of the ATP7B gene in worldwide.In a conclusion, our study established a rapid and accurate molecular diagnostic method for Wilson disease. Through the mutation analysis for southern Chinese patients, we also established the mutation spectrum. In addition, we had identified ten novel mutations. It expanded the range of mutations in Chinese population and enriched the mutation spectrum of the ATP7B gene in worldwide. These findings were useful for the further study of the genetic characteristic and for early diagnosis and treatment, which has great significance to improve the quality of patients with WD in China. |
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