Introduction of DNA microarray in molecular diagnostics of Wilson disease

Summary

Background.
Wilson disease (WD) is a serious autosomal recessive disorder caused by mutations in the ATP7Bgene which encodes a copper-specific ATPase. WD patients suffer from impaired biliary excretion of copper from organism and its´ accumulation in body organs. Molecular diagnostics of WD is an important part of a correct diagnosis statement. The aim of the study was to design and validate a genotyping DNA microarray which enables the analysis of 87 causal mutations and 17 polymorphisms in the ATP7B gene, simultaneously.

Methods and Results.
97 WD patients with known genotypes and 46 samples prepared by mutagenesis were tested in the first phase of chip validation. All analyzed sequence variants were detected with 100% accuracy. DNA samples from WD suspected patients were tested in the second phase of validation. In total, we have analyzed 58 unrelated patients so far. The diagnosis of WD was confirmed in 10 patients, 13 patients were heterozygous for some mutations and 35 had no mutation in the ATP7B gene. Samples with none or one mutation found by microarray analysis were subsequently sequenced with no further causal mutations identified.

Conclusions.
The Wilson chip seems to be a fast and reliable method for the first line screening of mutations in the ATP7B gene.

Key words:
ATP7B gene, APEX method, DNA microarray, molecular diagnostics, Wilson disease

Wilson disease (WD, MIM 277900) is a serious autosomal recessive disorder of copper metabolism caused by deficiency of copper specific ATPase (ATP7B). Incidence of WD is estimated to be 1:35000 of newborns. ATP7B protein is physiologically responsible for copper transport within humans – via incorporation of copper into ceruloplasmin and its´ biliary excretion (1). ATP7B protein is mainly expressed in liver, brain, kidney and placenta. The accumulation of copper could lead to irreversible liver damage, neurological failure and to death, if ATP7B protein is functionless (2,3). The disease manifests mostly in the second decade of life which makes early diagnosis more difficult. Wilson disease can be diagnosed on the basis of combinations of clinical symptoms, biochemical parameters and molecular analysis of ATP7B gene (4-6). A typical symptom of WD – Kaiser-Fleischer ring – which originates from copper storage in the cornea, is not obvious many times (7). Biochemical diagnostics of WD is based on increased urinary copper excretion after D-penicillamine test (> 5 μmol/24h), decreased serum ceruloplasmin (< 0,2g/L), decreased serum copper levels (< 14 μmol/L) and increased hepatic copper content (> 250 μg/g). Nevertheless, some affected individuals could be biochemically undistinguishable from healthy heterozygotes (8). The molecular genetic analysis of ATP7B gene seems to be the most specific approach for WD diagnosis. The ATP7B gene is localized on human chromosome 13q14.3 and consists of 21 exons with ORF size 4398 bp (9,10). It encodes a 157 kDa protein containing 1465 amino acids (11). To date more than 400 mutations in the ATP7B gene have been reported, most of them are ethnically specific (12). The prevalent mutation in Caucasian population is p.H1069Q (c.3207C>A) which leads to a deficiency of ATP binding (13). At present, the molecular analysis of ATP7B gene relies on the classical method of detecting the most frequent mutations in single populations by restriction digestion, followed by direct sequencing. This approach is relatively expensive and time consuming. We have designed a genotyping DNA microarray for detection of 87 selected causal mutations and 17 polymorphisms in ATP7B gene based on the APEX technology (Arrayed Primer Extension) to make the molecular diagnostics of WD more effective.

Patients and methods

Patients

97 unrelated WD patients with previously determined genotypes by another molecular-biological method and 58 unrelated patients suspected for WD were analyzed using the Wilson chip. All patients came from Czech Republic and Slovakia and were selected for molecular analysis of the ATP7B gene from various clinical departments. Informed consent was obtained from all patients included in this study. Genomic DNA was isolated from peripheral blood using a salt precipitation method.

DNA microarray

Wilson chip enables a simultaneous detection of 87 mutations and 17 polymorphisms in the ATP7B gene, all of which have been detected in WD patients in our laboratory (Czech population) and are frequent in other populations. The amino-modified oligonucleotide probes specifically designed for detection of all selected sequence variants of the ATP7B gene are covalently bound on a glass slide previously coated with 3-aminopropy-ltrimethoxysilane plus 1,4-phenylenedi-isothiocyanate. On the chip, each mutation/polymorphism is determined by two different probes (sense/antisense), except the most frequent mutation p.H1069Q which is determined by four probes. The probes are designed according to the wild-type sequence of the ATP7B gene with their 3’OH end immediately adjacent to an appropriate nucleotide change (mutation/polymorphism). The melting temperature, minimal length variability and the formation of secondary structures are other important parameters of designed probes. The principle of mutational analysis on the Wilson chip is based on the APEX (Arrayed Primer Extension) reaction. In APEX, the immobilized probes are extended by exactly 1 fluorescently labeled ddNTP terminator (site of mutation/polymorphism) complementary to the co-hybridized fragment of sample DNA. This method is suitable for analysis of concrete substitutions, small insertions and deletions in the ATP7B gene.

Results

Validation of the Wilson chip

It was necessary to validate the Wilson chip for use in routine molecular diagnosis. The ability of designed oligonucleotide probes to detect individual mutations and polymorphisms correctly, was tested in the first phase of microarray validation. The set of 97 WD patients with already known genotypes was chosen for microarray analysis. 43 different mutations and 15 polymorphisms were detected either in homo- or heterozygous forms with 100% accuracy. By this approach, the functionality of 118 probes (56%) was tested. 13 probes (11%) provided weak or no signal. Nevertheless, every tested mutation/polymorphism was reliably determined by at least one oligonucleotide probe (sense or antisense) (14). Further, 46 DNA samples were prepared carrying a causal mutation or polymorphism introduced by mutagenesis to test the rest of probes. From the total number of 92 oligonucleotide probes designed for the detection of frequent mutations and polymorphisms in other populations, 10 probes (11%) revealed weak or no signal. This was due to either the sense or antisense strand failing. Despite this, all mutated samples were analyzed with 100% accuracy.

In the second phase of chip validation, 58 DNA samples from unrelated WCH suspected patients were analyzed. From the total number of 116 analyzed alleles, 33 were mutated. Microarray results were confirmed by direct sequencing of the whole coding region of the ATP7B gene and no other mutations were observed on the rest of the gene. Wilson disease was confirmed in 10 patients, 13 patients were heterozygous for one mutation and 35 had no mutations found in their ATP7B gene (Tab.1, Fig.1).

Discussion

Miniaturization of analyses and the onset of microarray technology are current trends not only in the field of molecular diagnostics. Genotyping microarrays represent a suitable tool for detection of a broad spectrum of gene sequence variants in a single analysis (15,16). The aim of our study was to facilitate and simplify the molecular diagnostics of Wilson disease using a genotyping microarray based on APEX for detection of selected sequence variants in the ATP7B gene. The principle of the APEX technology is based on Sanger’s dideoxysequencing. The probes immobilized on chip are extended by exactly 1 fluorescently labeled terminator – a dideoxynucleotide (ddATP, ddGTP, ddCTP, ddUTP) complementary to the sequence of co-hybridized DNA. Each terminator is labeled by a different fluorescent dye. Hetero/homozygote sequence variants are determined according to which ddNTP is linked to each DNA probe. The APEX technology is limited to the detection of only a selected spectrum of known mutations, not any new ones. Nevertheless, this method is suitable for detection of substitutions as well as deletions and insertions of one or more nucleotides. Further, it is also possible to analyze more sequence variants by one oligonucleotide probe. The accuracy, simplicity and time efficiency are advantages of testing with APEX (17-22).

87 mutations and 17 polymorphisms of the ATP7B gene were selected for analysis via Wilson chip detection. The selected panel comprised mutations frequent in the Czech population as well as mutations prevalent in other ethnicities and polymorphisms for haplotype analysis (23-29). There were control probes printed on every chip to assure a proper run of the APEX reaction. Validation of Wilson chip revealed 23 (11%) probes providing weak or no signal from the total number of 210 spotted oligonucleotide probes. Nevertheless, due to the sense/antisense redundancy of the Wilson chip, all selected mutations/polymorphisms were determined correctly. The fact that some oligonucleotide probes provided weak or none fluorescent signal is most likely due to their localization in the ATP7B gene sequence with a high content of AT or GC pairs or sequence repeats. The formation of secondary structures, their length and melting temperature may also have impaired hybridization. It is always necessary to confirm each mutation that is detected by microarray analysis with another independent molecular biological method (f.e. restriction analysis).

We have developed a genotyping microarray for the molecular diagnostics of Wilson disease, thus increasing the first line of screening in the ATP7B gene from 5 to 87 mutations (14). The whole procedure, DNA sample preparation by the multiplex polymerase chain reaction and the APEX reaction itself, takes a few hours. The course of WD molecular diagnostics depends on the results obtained from the Wilson chip analysis. In the case that two mutations have been detected on the microarray, the diagnosis of Wilson disease is confirmed at the molecular level. If only one mutation has been observed by the microarray analysis, it is necessary to continue in the molecular diagnostics of WD by direct sequencing of the ATP7B gene. In the last case that no mutation has been found by microarray screening, a consultation with the practitioner should follow to make a decision about further molecular analysis. It is necessary to sequence the whole coding region of the ATP7B gene when the patient has clinically confirmed WD diagnosis (Fig.2).

The results of our study indicate that the Wilson chip seems to be a suitable screening method of single nucleotide polymorphisms (SNPs), small deletions and insertions in the ATP7B gene.

Abbreviations:

APEX – Arrayed Primer Extension

ddATP – dideoxyadenosinetriphosphate

ddGTP -dideoxyguanosinetriphosphate

ddCTP – dideoxycytidinetriphosphate

ddUTP – dideoxyuridinetriphosphate

ORF – open reading frame

SNP – Single Nucleotide Polymorphism

WD – Wilson Disease

This work was supported by projects of Ministry of Education, Youth and Sports, Czech Republic MSMT LC06023 and 2B08060.

MSc. Lucie Gojova

Centre of Molecular Biology and Gene Therapy

University Hospital Brno

Cernopolni 9, 613 00, Brno

fax:+420 532 234 623, tel: +420 532 234 624

e-mail: lgojova@fnbrno.cz, luciegojova@seznam.cz