Wu Z, Wang N, Lin M, Fang L, Murong S, Yu L. Mutation Analysis and the Correlation Between Genotype and Phenotype of Arg778Leu Mutation in Chinese Patients With Wilson Disease. Arch Neurol. 2001;58(6):971-976. doi:10.1001/archneur.58.6.971
Copyright 2001 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.2001
The defective gene (ATP7B) that causes Wilson disease (WD) codes for a putative copper-transporting P-type adenosine triphosphatase. After cloning of ATP7B, the spectrum of mutations and their clinical consequences have been investigated in patients with WD in different ethnic populations. However, the spectrum of mutations and the correlation of genotype-phenotype in the Chinese population have not been extensively studied.
To investigate the characterization of mutations of ATP7B and the correlation between genotype and phenotype in the Chinese population.
We studied 60 unrelated healthy Chinese and 65 unrelated Chinese families, including 84 patients with WD and 126 parents. Genomic DNA was prepared from peripheral blood leukocytes using a salt-precipitation method. Polymerase chain reaction single-strand conformation polymorphism and subsequent direct sequencing were used to identify the mutations and polymorphisms of ATP7B. Statistical analysis was performed using t test or χ2 test.
We identified 18 mutations (7 novel) and 11 polymorphisms (3 novel). The novel mutations are −36C→T, Trp650ter, Gln914ter, 2810delT, Thr935Met, Arg1041Pro, and Glu1173Lys. The novel polymorphisms are 1168A→G (Ile390Val), 2785A→G (Ile929Val), and 3316G→A (Val1106Ile). Two mutations, Arg778Leu and Thr935Met, are relatively frequent, representing 37.7% and 10.0% of patients, respectively. To our knowledge, we are the first to report the correlation between the genotype and phenotype of Arg778Leu. The result shows that Arg778Leu homozygotes are associated with the early onset of WD with hepatic presentation.
The Arg778Leu and Thr935Met mutations are hot spots in the Chinese population. The features of mutations of ATP7B differ between the Chinese and Western ethnic populations. The Arg778Leu mutation has severe effects on the function of ATP7B. These findings are valuable for developing a fast and effective method to diagnose the presence of the WD gene.
WILSON DISEASE (WD) is an autosomal recessive disorder of copper transport. Toxic accumulation of copper causes tissue damage, primarily in the liver, brain, and kidneys. The disease phenotype includes progressive liver degeneration and/or neurologic impairment and, frequently, kidney malfunction.1 The worldwide prevalence of the disease is estimated to be 30 per million, with a corresponding gene frequency of 0.56% and a carrier frequency of 1/90.2 Treatment involves the removal of excess copper by means of chelating agents, such as penicillamine,3 or the blocking of intestinal copper absorption with zinc salts.4
The defective gene in WD encodes a putative copper-transporting P-type adenosine triphosphatase (ATP7B) highly homologous to the protein encoded by the Menkes syndrome gene.5- 8 After the cloning of ATP7B, the repertoire of mutations and some clinical consequences have been described in affected family members of different ethnic backgrounds, and certain mutations are found to be relatively frequent in patients of European origin.9- 14 In Chinese patients with WD, the arginine-to-leucine substitution at codon 778 (Arg778Leu) and arginine-to-glutamine substitution at codon 778 (Arg778Gln) occurring at a higher frequency have been reported.15 Herein we report the identification of 18 disease mutations and 11 polymorphisms in the Chinese population, the genotypes of ATP7B, and the clinical phenotypes of WD.
We studied 60 unrelated healthy Chinese subjects (controls) and 65 unrelated Chinese families that included 84 patients with WD and 126 parents. Of the 84 patients, 44 have been described previously.16 The patients with WD and controls are from the Han ethnic group of the same geographic area in China. Eight of these families were consanguineous. The age at onset for each patient ranged from 4 to 39 years. The diagnosis of WD was based on clinical symptoms, lowered plasma ceruloplasmin and copper serum concentrations, high urinary copper concentration, and the occurrence of the characteristic copper deposition of Kayser-Fleischer rings in the corneal periphery.1 All patients with WD had Kayser-Fleischer rings. Genomic DNA was extracted from the whole blood collected in sodium EDTA by a salt-precipitation method.17
Exons 1 through 21 of ATP7B were amplified using primers complementary to the DNA sequences flanking the exon-intron boundaries. The primers used for amplification of exon 1,18 exons 2 through 21, and the lengths of polymerase chain reaction (PCR) products have been described elsewhere.10 The PCR amplifications were performed in 50-µL total volume containing 50 mmol/L of potassium chloride, 10 mmol/L of Tris (pH 8.0), 1.5 mmol/L of magnesium dichloride, 0.20 µg of genomic DNA, 0.25 µmol/L of each primer, 200 µmol/L of each deoxynucleoside triphosphate, and 2.0 units of Taq polymerase (Sogon Inc, Shanghai, China). The reactions were performed at 94°C for 2 minutes followed by 30 cycles consisting of denaturation at 94°C for 45 seconds, annealing at 64°C to 56°C for 45 seconds, and elongation at 72°C for 1 minute, with the last elongation step for 5 minutes in a programmable thermal controller (PTC-100; MJ Research, Inc, Waltham, Mass).
For single-strand conformation polymorphism (SSCP) analysis, 5 µL of the PCR product was diluted with 5 µL of loading buffer containing 95% formamide, 20 mmol/L of EDTA, and 0.05% each of bromphenol blue and xylene cyanol FF. The samples were denatured at 95°C for 5 minutes, cooled on ice, and then applied on a 8% nondenaturing polyacrylamide gel containing 5% glycerol. Electrophoresis was performed at a constant temperature of 20°C or 4°C and at a constant voltage of 500 or 200 V for 7 to 16 hours. After electrophoresis, the gels were silver stained,19,20 air dried, and stored for documentation.
Patient samples exhibiting shifts relative to control samples on SSCP findings were subjected to direct sequencing for identification of the mutations. The patient DNA samples that did not exhibit a shift pattern of SSCP in exons 5, 8, 12, 14, and 18 relative to control samples were also sequenced to detect the mutation, since mutations are more often clustered in these exons. The DNA from patients and controls were amplified according to the conditions described above. The products were purified using a quick spin column (Qiagen Inc, Valencia, Calif). The DNA sequencing was performed on a 377 DNA automatic sequencer or 3700 DNA analyzer (both from PE Applied Biosystems, Foster City, Calif) using a commercially available sequencing kit (Big Dye Terminator Ready Reaction Mix Cycle; PE Applied Biosystems). Point mutation in heterozygotes was detected reliably by means of manual inspection of characteristic double peaks. If the height of a peak in the mutated allele was shorter than 50% of the wild-type allele at the same position, the opposite strands of corresponding regions were sequenced to confirm these mutations.
Data were analyzed using a commercially available statistical package (SPSS, Version 8; SPSS Inc, Chicago, Ill). Results were presented as mean ± SD. Statistical analysis was performed using t test or χ2 test. The criterion for significant difference was P<.05.
We investigated 84 patients with WD from 65 Chinese families, and identified a total of 18 mutations (Table 1). Seven mutations are novel, and 11 have been described elsewhere.10,15,21- 23 The novel mutations include cytosine-to-thymine substitution at nucleotide 36 (−36C→T), tryptophan termination at codon 650 (Trp650ter), glutamine termination at codon 914 (Gln914ter), thymine deletion at nucleotide 2810 (2810delT), threonine- to -methionine substitution at codon 935 (Thr935Met), arginine-to-proline substitution at codon 1041 (Arg1041Pro), and glutamic acid–to-lysine substitution at codon 1173 (Glu1173Lys). The 2810delT mutation, which causes a frame shift, is predicted to produce a shortened nonfunctional protein. The Trp650ter mutation, which is caused by a guanylic acid–to–adenylic acid transition at the third nucleotide of the codon, and Gln914ter, which is caused by a cytosine-to-thymine transition at the first nucleotide of the codon, would result in truncated proteins. The −36C→T mutation identified in the 5′ untranslated region may alter the expression or translation of ATP7B. The Thr935Met, Arg1041Pro, and Glu1173Lys mutations are all missense mutations. Among 65 unrelated patients, our screening method detected 2 mutations in 36 patients (17 homozygotes and 19 compound heterozygotes) and at least 1 mutation in 23 patients. The other 6 patients escaped the detection. The sensitivity of testing is 73.1% (95/130). No mutation was detected by means of direct sequencing in exons 5, 8, 12, 14, and 18 of the patients' ATP7B that did not exhibit shifts relative to healthy samples in the SSCP analysis. All nucleotide substitutions found in ATP7B of the patients are real point mutations, because none of them were seen in 120 healthy chromosomes analyzed or in chromosomes with defined disease-causing mutations.
We have detected 11 other base substitutions in the ATP7B gene that are silent mutations or exist in healthy chromosomes and in chromosomes with defined disease-causing mutations as well as in intronic sequence. These variants should be considered polymorphisms (Table 2). We also detected 5 base substitutions, 4 of which were reported previously12,13 as polymorphisms. However, they were detected in all healthy and all WD chromosomes. Therefore, we do not consider them to be polymorphisms.
Three novel polymorphisms, adenylic acid–to–guanylic acid transition at nucleotide 1168 (1168A→G) (isoleucine-to-valine substitution at codon 390 [Ile390Val]), adenylic acid–to–guanylic acid transition at nucleotide 2785 (2785A→G) (isoleucine-to-valine substitution at codon 929 [Ile929Val]), and adenylic acid–to–guanylic acid transition at nucleotide 3316 (3316G→A) (valine-to-isoleucine substitution at codon 1106 [Val1106Ile]), that occur at highly conserved amino acid residues were found to exist in chromosomes with defined disease-causing mutations. They should be considered polymorphisms, although they were not observed in 120 healthy chromosomes. We suppose that these conservative changes may affect the clinical expressivity of disease-causing mutations, and the finding needs further investigation.
Evaluation of the correlation between genotype and phenotype is possible only in homozygotes. As so many different mutations are detected, and as most patients are compound heterozygotes, it is difficult to investigate the potential correlation between genotypes and phenotypes. In this study, 21 symptomatic patients from 17 families have been identified to be homozygous for a single mutation. The homozygotes were detected only for the following mutations: −36C→T, thymine-to–guanylic acid transversion at 3′ nucleotide 5 of intron 4 (1078-5T→G), Arg778Leu, and Arg1041Pro. The common mutation, Arg778Leu, is the only mutation that occurs at sufficient frequency to allow the evaluation in multiple individuals who are homozygous for the mutation. To evaluate the functional significance of Arg778Leu, we searched for correlation between genotypes and several phenotypic manifestations of the illness, including age at onset, neurologic vs hepatic onset of the illness, and the level of ceruloplasmin activity (Table 3 and Table 4). The remaining 3 patients were homozygous for 3 different mutations, and we could not draw meaningful conclusions because of the diverse amino acid changes.
The mutation Arg778Leu was homozygous in 18 patients from 14 families whose ages at onset ranged from 10 to 17 years. As shown in Table 4, their average age at onset was 14.2 years. Their ceruloplasmin levels ranged from 6 to 80 mg/L, with a mean value of 31.2 mg/L. Thirteen patients presented with hepatic symptoms, whereas 3 presented with neurologic symptoms at onset. Five patients died without receiving regular chelation therapy. This mutation was also found to be heterozygous in 11 patients from 9 families with WD. Each of them carried another missense mutation in the other chromosome. Their ages at onset ranged from 15 to 29 years, with the average age at onset of 22.0 years. Ceruloplasmin levels ranged from 40 to 100 mg/L, with a mean value of 54.6 mg/L. All patients except 1 presented with neurologic symptoms at onset.
Our data are compatible with the hypothesis that the mutations tend to occur in a population-specific manner. Some mutations appear to be population specific, whereas others are common in many populations. Four mutations—adenylic acid insertion at nucleotide 523 (523insA), Arg778Leu, Arg778Gln, and glutamine-to-threonine substitution at codon 1142 (Gln1142Thr)—have been found in Chinese pedigrees from Taiwan,15,23 whereas the other 3 mutations—1708-5T→G, alanine-to-valine substitution at codon 874 (Ala874Val), and arginine-to-glycine substitution at codon 919 (Arg919Gly)—have been found in Japanese pedigrees.21,22 Those 7 mutations, however, also have been identified in the Chinese patients of our investigation. The results suggest that those mutations are most likely specific for Oriental origin. Two mutations, including guanylic acid–to-cytosine transversion at 3′ nucleotide 1 of intron 4 (1708-G→C), which was found in an Indian patient,10 and aspergine-to-serine substitution at codon 1270 (Asn1270Ser), which was identified in Sicilian,7 continental Italian and Turkish,9 and Costa Rican populations,12 were also detected in this study. Among the mutations published to date, several amino acid residues are the targets of multiple mutations. In particular, the arginine residue at position 778 was reported to be substituted with leucine,10 glycine,9 glutamine,15 or tryptophan,12 which indicates the critical role of arginine at position 778 for the function of transmembrane 4. Similarly, the glycine residue at position 943 was reported to be replaced with serine10 or asparagine.23 Regions of messenger RNA sequence between translation and transcription start sites are known to be crucial for ribosome binding. Therefore, the −36C→T mutation identified in the 5′ untranslated region may alter the expression of the WD gene. This possibility needs further investigation, and the analysis of the effects of sequence changes on the expression of a transfected reporter gene will be a useful way to do so. These new findings expand our knowledge of the spectrum of mutations in ATP7B in patients of Chinese descent and provide new information about critical DNA sequences for gene function.
The frequency of these mutations in the population confirms that the spectrum of ATP7B mutations consists of a small number of relatively frequent mutations and a large number of rare mutations. The most common ATP7B mutation, histidine-to-glutamine substitution at codon 1069 (His1069Gln), appears frequently (10%-40%) in diverse populations, including those of North America, Russia, Great Britain, Holland, Sweden, and continental Italy, and in several Mediterranean samples, but it was not detected in the 84 patients with WD in this study. As shown in Table 1, the Arg778Leu and Thr935Met mutations, which represent 37.7% and 10.0%, respectively, of the mutations in the WD chromosomes we studied, are the most common. The remaining mutations were rare and observed in only a few patients. It seems that the sites of Arg778Leu and Thr935Met mutations are the mutation hot spots in the Chinese population. The results show that the features of mutations of ATP7B are different between Chinese and Western ethnic populations. Therefore, we first should select exons 8 and 12 instead of exons 14 and 18 to detect mutations of ATP7B in Chinese patients. These findings are valuable for developing a fast and an effective genetic diagnosis of WD in Chinese patients. According to the data collected so far, WD seems to result from a limited kind of frequent mutation, common and population specific, and from a large number of rare mutations. Among the 65 unrelated patients (130 chromosomes) we investigated, there was no mutation identified in 35 chromosomes. This lack of detection may be, in part, a consequence of the limitations of SSCP analysis. Some of the undetected mutations may be located in noncoding regions such as introns, promoters, regulatory sequences, and other control regions.
We observed that the average age at onset in Arg778Leu homozygotes was significantly younger than that in compound heterozygotes (P<.001). Similarly, the ceruloplasmin levels of Arg778Leu homozygotes were lower than those of heterozygotes (P<.05). Neurologic vs hepatic onset of the illnesses showed significant difference between Arg778Leu homozygotes and compound heterozygotes (χ2 = 13.60; P<.001). This difference may be due to the combination of Arg778Leu with other mild WD missense mutations not leading to typical WD.
Functional data reported previously24 indicated that the Arg778Leu mutation had severe effects on the function of ATP7B, which confirmed it as a disease-causing mutation. Our data correlate well with these functional data. Our study on the genotypes vs phenotypes shows that Arg778Leu homozygotes are associated with the early onset of WD with hepatic symptoms at presentation. In addition, when the chromosome carries the Arg778Leu mutation in exon 8, there is a conservative change (cytosine-to–guanylic acid transversion at nucleotide 2310 [C2310G]), suggesting that a normal polymorphism is present in this region. The Arg778Leu mutation is a common one in patients of Asian descent,10,11,15 who may be regarded as a group derived from a common ancestor. These findings suggest that the Arg778Leu mutation may be useful in the prediction of disease severity. Other factors, such as dietary copper intake and the individual's capacity for dealing with copper stress, can also be expected to modulate phenotypic response.
Accepted for publication November 17, 2000.
This project was supported by grant 39740017 from the National Natural Science Foundation, Beijing, China; grant 97082 from the Ministry of Public Health, Beijing; and grant 96A032 from the Fujian Provincial Public Health Bureau, Fuzhou, China.
Corresponding author and reprints: Zhi-Ying Wu, MD, PhD, Department of Neurology, First Affiliated Hospital, Fujian Medical University, 20 Chazhong Rd, Fuzhou 350005, People's Republic of China (e-mail: email@example.com).