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Mutation Analysis and the Correlation Between Genotype and Phenotype of Arg778Leu Mutation in Chinese Patients With Wilson Disease
Zhi-Ying Wu, MD, PhD;
Ning Wang, MD, PhD;
Min-Ting Lin, MD;
Ling Fang, MD, PhD;
Shen-Xing Murong, MD;
Long Yu, MD, PhD
Arch Neurol. 2001;58:971-976.
ABSTRACT
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Background 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.
Objective To investigate the characterization of mutations of ATP7B
and the correlation between genotype and phenotype in the Chinese population.
Methods 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  2 test.
Results 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 1168AG
(Ile390Val), 2785AG (Ile929Val), and 3316GA (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.
Conclusions 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.
INTRODUCTION
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, 6, 7, 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, 10, 11, 12, 13, 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.
PATIENTS AND METHODS
SAMPLE
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
POLYMERASE CHAIN REACTION AMPLIFICATION AND SINGLE-STRAND CONFORMATION
POLYMORPHISM ANALYSIS
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.
EXON AMPLIFICATION AND DNA SEQUENCING
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.
STATISTICAL ANALYSIS
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 2 test. The criterion for significant difference
was P<.05.
RESULTS
DETECTION AND ANALYSIS OF MUTATIONS
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, 22, 23
The novel mutations include cytosine-to-thymine substitution at nucleotide
36 (-36CT), 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 -36CT 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.
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Table 1. Mutation Detected in ATP7B Gene*
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DETECTION AND ANALYSIS OF POLYMORPHISMS
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.
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Table 2. Probable Polymorphisms in ATP7B Gene*
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Three novel polymorphisms, adenylic acid–to–guanylic acid
transition at nucleotide 1168 (1168AG) (isoleucine-to-valine substitution
at codon 390 [Ile390Val]), adenylic acid–to–guanylic acid transition
at nucleotide 2785 (2785AG) (isoleucine-to-valine substitution at codon
929 [Ile929Val]), and adenylic acid–to–guanylic acid transition
at nucleotide 3316 (3316GA) (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 GENOTYPES AND PHENOTYPES
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: -36CT,
thymine-to–guanylic acid transversion at 3' nucleotide 5 of intron
4 (1078-5TG), 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.
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Table 3. Clinical Presentation of WD Patients With Arg778Leu Mutation*
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Table 4. Evaluation of Correlation Between Genotype and Phenotype for
Arg778Leu*
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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.
COMMENT
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—1708-5TG, 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-GC), 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 -36CT
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 (2
= 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.
AUTHOR INFORMATION
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.
From the Department of Neurology (Drs Wu, Wang, Fang, and Murong) and
the Institute of Neurological Sciences (Dr Lin), First Affiliated Hospital,
Fujian Medical University, Fuzhou, and the Institute of Genetics, Fudan University,
Shanghai (Dr Yu), People's Republic of 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: zhiyingwu67{at}yahoo.com).
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