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Identification of a High Frequency of Mutation at Exon 8 of the ATP7B Gene in a Chinese Population With Wilson Disease by Fluorescent PCR
Pingyi Xu, MD, PhD;
Xiuling Liang, MD;
Joseph Jankovic, MD;
Wei-dong Le, MD, PhD
Arch Neurol. 2001;58:1879-1882.
ABSTRACT
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Background Wilson disease (WD) is an autosomal recessive
disorder of copper transport. Mutation analysis has led to the
discovery of more than 100 mutations at ATP7B, and most of
them are population specific.
Objectives To verify the high frequency of mutation at exon 8
of ATP7B in Chinese patients with WD and to establish a DNA
diagnostic method for WD.
Setting University medical centers.
Patients and Methods Screening for mutations at exon 8 of
ATP7B by fluorescent polymerase chain reaction analysis and
restriction analysis was conducted in 106 unrelated Chinese patients
with WD and in 55 individuals from 10 Chinese families with WD.
Results Five homozygotes and 32 heterozygotes were identified.
Sequence analysis showed a missense mutation (2273G T) and a nonsense
mutation (2250CG) together at exon 8. The rate of gene
mutation in 106 patients was 35% (5% homozygous and 30%
heterozygous). Samples of DNA from 55 individuals from 10
Chinese families with WD were examined by fluorescent polymerase chain
reaction. We found that 13 siblings were carriers (24%).
Conclusions A high frequency of mutation at exon 8
of the ATP7B gene exists in the Chinese population,
and fluorescent polymerase chain reaction analysis may be an effective
and accurate assay in detection of the WD gene.
INTRODUCTION
WILSON DISEASE (WD) is an autosomal recessive disorder of copper transport. Because of
decreased biliary copper excretion and reduced copper incorporation
into ceruloplasmin, copper accumulates in tissues, particularly the
liver, basal ganglia, cornea, and kidney. Clinical presentation usually
occurs in the first or second decade of life and is characterized by
liver disease, extrapyramidal and psychiatric symptoms, renal
disturbance, or Kayser-Fleischer ring in the cornea.1 The
worldwide frequency of WD is 1 in 35 000 to 1 in 100 000
live births.2 The epidemiologic features vary considerably
in different countries and ethnic backgrounds. The prevalence of WD in
China seems to be much higher than that in Western
countries.3
The WD gene has been cloned and found to encode a
copper-transporting p-type adenosine triphosphatase (ATP7B).
To date, more than 100 mutations, including small insertions and
deletions and missense, nonsense, and splice site mutations, have been
described in ATP7B.4, 5, 6 Most of these mutations are
population specific.7, 8, 9, 10, 11 There have been several
studies12, 13, 14, 15, 16, 17 of mutations of the ATP7B gene in
mainland China, and all indicated that the mutation of
Arg778Leu at exon 8 is a hotspot in the Chinese population with WD.
Because of the limited number of patients with WD enrolled in these
studies, the frequencies reported varied considerably (range,
17%-30%).13, 14, 15, 16, 17 In this study, we present the
results of mutation screening by fluorescent polymerase chain reaction
(PCR) analysis and restriction enzyme digestion analysis of exon 8 of
the ATP7B gene in 106 Chinese patients with WD and
55 nonsymptomatic siblings from 10 Chinese families with WD. Our
objectives were (1) to verify the high frequency of mutation at exon 8
of ATP7B in Chinese patients with WD and (2) to establish a
DNA diagnostic method for WD using fluorescent PCR
analysis.
PARTICIPANTS AND METHODS
STUDY POPULATION
The study included 106 unrelated Chinese patients with WD and classical
symptoms and signs of WD (69 males and 37 females; age range, 6-25
years; mean ± SD age, 12.4 ± 3.8 years),
55 nonsymptomatic siblings from 10 Chinese families with WD (38 males
and 17 females; age range, 9-28 years; mean ± SD age,
16.4 ± 4.1 years), and 55 unrelated healthy individuals
(30 males and 25 females; age range, 10-25 years;
mean ± SD age, 14.7 ± 3.7 years)
(Table 1). In
haplotype data published on these families,18 no evidence
was found for locus heterogeneity in the linkage analysis. Of 106
patients with WD, 35 had a family history of WD. At the early stage of
disease, 40 patients (23 males and 17 females; age range, 5-21 years;
mean ± SD age, 11.3 ± 3.1 years) had the
primary symptom of hepatic dysfunction, 31 (18 males and 13 females;
age range, 7-24 years; mean ± SD age,
14.6 ± 4.7 years) had neurological manifestations, and
37 (23 males and 14 females; age range, 12-25 years;
mean ± SD age, 14.4 ± 2.8 years) had both
as their first complaint. All patients were treated customarily with
D-penicillamine, 0.75 to 1.50 g/d. Once improvement occurred
and decreased total body copper content was demonstrated, patients were
placed on maintenance therapy at half the initial dose. At 2-year
follow-up, 68 patients experienced significant improvement, 28 had
moderate improvement, and 10 showed no improvement after
D-penicillamine treatment.
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Table 1. Summary of Clinical and Laboratory Data in 106 Patients With Wilson Disease,
55 Heterozygous Carriers, and 55 Control Subjects*
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EXAMPLE CASE REPORT
A 12-year-old schoolboy had "infectious hepatitis" at age 5 years
but no symptoms or signs of liver disease or any other serious illness
since then. He started with intentional tremor in the right arm at age
10 years, and then developed a progressive "wing beating" in 2
arms. Physical examination showed that his liver and spleen were
palpable, his facial expression was fixed, and his smile was turned to a grin. Kayser-Fleischer rings were detected in his
cornea by the naked eye. He had severe rigidity in all limbs, but his
mind remained clear. His sensation and reflexes were normal,
and pyramidal signs were absent. Laboratory findings showed that his
serum copper level was elevated to 30 µg/dL (4.86 µmol/L) and his
serum ceruloplasmin concentration was less than 1 mg/dL, but 24-hour
urine copper values were greater than 1105 µg. Computed tomographic
examination demonstrated hypodense lesions in bilateral basal ganglia.
The patient was previously treated with a short course of zinc acetate,
followed by D-penicillamine, 1.0 g/d for 6 months and 0.5
g/d for 1 year. His clinical symptoms were remarkably improved after
D-penicillamine treatment.
DNA ANALYSIS
Genomic DNA samples were extracted from blood cells using standard
techniques. Two fluorescent probes were synthesized based on the
wild-type and mutant-type sequences at exon 8 of the ATP7B
gene. The wild fluorescent probe sequence was 5'-CAGCCACCGGCCCAGGG-3'
and the mutant fluorescent probe sequence was
5'-AGCCACAGGCCCAGGG-3'. Fluorescent PCR analysis was carried out in the
ABI PRISM 7700 Sequence Detection System (Perkin-Elmer Instruments,
Wellesley, Mass). After 20-minute denaturation at 95°C, the
PCR consisted of 40 cycles at 93°C for 45 seconds and 65.5°C for 2
minutes. Using the 5' nuclease assay, a specific fluorescent signal was
generated and measured at every cycle during a run. Fluorescent
readings were quantitatively analyzed in the system: the normal
sequence of exon 8 of the ATP7B gene was marked as green and
the mutant as red. Two curve reaction lines were automatically drawn
according to the numbers of amplified specific gene.
Direct DNA sequencing was conducted in forward and reverse
directions using an automated sequencer. The accuracy of fluorescent
PCR analysis of exon 8 of ATP7B was confirmed by restriction
enzyme digestion of MspI (Promega, Madison, Wis) according to
nucleotide sequence analysis.
RESULTS
In 55 healthy individuals, analysis of fluorescent PCR amplification
showed only an increment of green fluorescent line in the
sequence detection system, indicating that there
were no mutations at exon 8 in this group. In 106 patients with WD, 5
mutant homozygotes (5%) and 32 heterozygotes (30%) of exon 8 were
found by fluorescent PCR analysis (Figure
1). We examined 55 siblings of
10 families with WD; 13 siblings (24%) were carriers and the others
were normal (Figure 1A, C). Direct DNA sequencing for 1 case
of homozygous, 2 cases of heterozygous, and 1 normal sample showed 2
different mutations—a missense (2273GT) and a nonsense
(2250CG)—together in these abnormal DNA samples. Sequence analysis
of exon 8 in homozygotes showed a 2273GT mutation and a 2250CG
mutation together in forward and reverse sequencing. There were 2273N
and 2250N mutations in one heterozygote and two 2273GT and 2250CG
mutations in another heterozygote. The data suggest that the
heterozygote is a partial homozygote (eg, a duplex zygote of homozygote
combined with a heterozygote of gene mutation). No mutations
of 2273GT and 2250CG were found in any controls (Figure
2).
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Figure 1.
Fluorescent polymerase chain reaction analysis of exon 8 of the
ATP7B gene in patients with Wilson disease (homozygotes [A]
and heterozygotes [B]) and controls (C). The red line indicates increased numbers of the mutation at exon 8 of the ATP7B gene; green line, increased numbers of the normal gene.
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To verify the accuracy of fluorescent analysis, all the PCR products of
exon 8 of ATP7B were digested by the restrictive enzyme
MspI. Of 106 patients, 5 were homozygotes and 32 were
heterozygotes. The results of analysis by restriction digestion were
the same as those using fluorescent PCR analysis (Table
2).
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Table 2. Detection of the Arg778Leu (2273GT) Mutation in Chinese Patients With Wilson Disease and in Controls by Fluorescent Polymerase Chain Reaction Analysis
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Clinically, patients with WD who had homozygous or
heterozygous 2273GT and 2250CG mutations showed no significant
difference from the other 69 patients with a normal sequence of exon 8
in terms of onset age, primary symptoms, and laboratory findings.
COMMENT
A previous study13 showed that Arg778Leu might be a
hotspot of mutations in the Chinese population by PCR–single-stranded
conformational polymorphism analysis in 21 exons of the ATP7B
gene. But the study was based on a small number of Chinese patients
with WD, and the
mutant frequency of exon 8 of the ATP7B
gene was less than that in other studies reported in
China.15, 16, 17 Xu et al13 also did not find other
mutations of exon 7, exon 12, and exon 16 of the ATP7B gene in
the previous study. Obviously, these mutations were rare in Chinese
patients.15, 16, 17 In this study, we examined the exon 8
mutations instead of screening all 21 exons of ATP7B by
fluorescent PCR. We found that 2 different mutations, 2273GT
(missense) and 2250CG (nonsense), exist together in exon 8 of the
ATP7B gene in Chinese patients with WD. Clinical analysis in
patients with WD who had 2273GT and 2250CG did not find a
correlation of the genotype with phenotype compared with normal exon 8
in patients with WD. The frequency of 2273GT and 2250CG mutations
in 106 independent Chinese patients examined is 35%, which is higher
than that reported in Taiwan,12 Japan,10, 19
Korea,8 and China.15, 16, 17, 18 The 2273GT and
2250CG mutations have not been reported in whites; a high frequency
of mutations presenting in exons 14 and 18 represented 38% of the
mutations in patients of European origin,7, 20, 21 which
indicates a different genetic background in whites and Chinese.
Exon 8 is located in the transmembrane domain (Tm4) of the
ATP7B gene.4, 5, 6 The amino acid changes codon 778
because of the mutation at exon 8, which may disrupt
the appropriate anchorage of the transporter in
the membrane. The change from a basic (Arg) to a neutral (Leu) amino
acid found in this study predicts a dramatic change in the primary and
secondary structure of this protein, which could culminate in impaired
copper transport.22, 23
This is the first study of fluorescent PCR analysis used in the
clinic setting to screen the gene mutations of patients with WD. It is
a 2-temperature PCR cycle that enhances the speed and overall
sensitivity of the amplification procedure.24 Because the
fluorescent PCR assay was applied for the selective amplification of a
characteristic sequence within the exon 8 fragment of ATP7B,
this technique—designed for the direct mutation detection of exon 8 of
the ATP7B gene—has the advantage of being efficient,
sensitive, cost effective, and applicable to large-scale screening for
the ATP7B gene mutation. Screening for WD in the patient's
family is important for early diagnosis and treatment to improve the
patient's prognosis. Using this method, we identified 13 siblings as
WD gene carriers among 55 siblings from 10 Chinese WD pedigrees;
the accuracy of this method is proved by the results of restriction
enzyme MspI analysis and by DNA direct sequencing.
AUTHOR INFORMATION
Accepted for publication May 14, 2001.
This study was supported by grant 39670290 from the National
Nature Science Foundation, Beijing, People's Republic of China, and by
a grant (1999 International Research Grant Award) from the Parkinson's
Disease Foundation, New York, NY.
We thank C. S. Yang, PhD, and F. Huang, MD, of Sun Yat-sun
University of Medical Sciences, Guangzhou, People's Republic of China,
for referring Chinese families with WD.
From the Department of Neurology, Baylor College
of Medicine, Houston, Tex.
Corresponding author: Wei-dong Le, MD, PhD, Room NB205, Department of
Neurology, Baylor College of Medicine, 6501 Fannin St, Houston, TX
77030 (e-mail: Weidongl{at}bcm.tmc.edu).
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