Table 1. Primers, their sequence, annealing temperature and extension time

F: Forward, R: Reverse

Polymerase Chain Reaction and Sequencing

DNA was extracted from whole blood using proteinase K-phenol-chloroform method. Eight exons (6,8,9,10,11,12,13,14) of ATP7B gene were amplified by PCR in a 25µl reaction (H2O 19.4µl, dNTP 10mMol 0.5µl, MgCl2 50mMol 0.75µl, 0.3µl of 12.5pMol primers (as listed in table 1), and Taq DNA polymerase 1unit). Amplification was performed in a three step reaction for 40 cycles (for exons 6 and 14) and 45 cycles for the other exons, 95°C 30s, 72°C 30s, 95°C 5min, and 72°C 10min as initial denaturation and final extension.

Bi-directional sequencing was performed using the ABI 3130xl, Genetic analyzer (Applied-Biosystem, USA) in accordance with supplier's instructions. Forward and reverse sequences were aligned and compared with reference sequence using computer software Bioedit and clustalX.

To assess the possibility that the novel mutations detected in this study might be polymorphisms that do not alter the function of the gene, we tested 50 control chromosomes in our population. We have classified sequence changes in the WD gene as polymorphisms if they do not modify the amino acid sequence of the protein product or result in nonconservative changes in nonessential residues of the protein or are detected in normal chromosomes of the same population (control group) or in chromosomes with defined disease causing mutation.

Mutations are named according to the guidelines from http://www.HGMD.org/, using the reference sequence with the GenBank accession number NM_00053.1. The nucleotide +1 is the A of the ATG translation initiation codon. The ATG translation initiation codon is also the first codon.

RESULTS

We screened 70 WD patients from 54 unrelated families for nucleotide variants in seven exons of the ATP7B gene. The study population included 40 males and 30 females with their age ranged 5-40 years.

Of 70 patients, 18 (25.7%) had at least one mutation, while 52 patients failed to show any mutation in the studied exons. Altogether, 12 different mutations including 9 missense and 3 deletion (one of which is nonsense) have been found. According to the literature and the “Wilson disease mutation database” (http://www.uofa-medical-genetics.org/wilson/ index.html), the following 4 mutations are being reported for the first time: T778I, 2532delA, 2803-2805ACG>-TG, and H1069N (table 2).

Totally, 15 (21.4%) patients were homozygous, one (1.4%) was compound heterozygous with different mutations identified on both alleles and 2 (2.9%) were compound heterozygous for whom the second mutation was not identified. The most common mutation is H1069Q point mutation, presents in 4 of 70 patients (5.7%), all of whom are homozygous. Table 2 summarizes the various identified genotypes and the corresponding age at disease onset, presence of KF ring plus the involved organ liver, brain or both.

Interestingly, D642H mutation has been detected in one family which leads to both hepatic and mixed Wilson's disease presentations. In the heterozygous form of this mutation, our patients were two sisters with hepatic WD, and their brother who was homozygous for D642H and presented with rather neurological WD.

Table 2. Characteristics of 27 Iranian patients with Wilson’s disease with ATP7B mutations

H: Hepatic, N: Neurologic, CuBD: Copper biding domain, TM: Transmembrane Domain, TD: Transduction Domain

Table 3. Comparison of the observed polymorphisms in

WD patients and controls

  Exon 14 has been identified as a hot spot for mutations. We detected two disease causing mutations in this exon, one of which was novel. Totally, 4 patients revealed to have H1069Q, of whom one has another disease causing mutation (V890M) in exon 11. Surprisingly, all of these patients presented with hepatic involvement. H1069N mutation was detected in a 19-year old girl, presented with acute hepatic failure and neurological manifestations.

One of the novel deletions found in this study involved one nucleotide in exon 10 (2532delA). It occurred in a 9-year old boy presented with hepatic failure.

2803-2805ACG>-TG mutation was detected in a member of a relatively large family. Homozygous patient had hepatic WD and we could not detect any neurological signs in the affected patient.

In addition to 12 different mutations, 4 different sequence variations with no clinical significance were identified (table 3). These variants have been defined in previous studies as a polymorphism detected in a normal population or not altering the amino acid composition in the protein.

DISCUSSION

Wilson's disease is an autosomal recessive disorder, which is generally described by recurrence fulminant hepatitis and chronic liver cirrhosis (15). In most of the cases, neurological symptoms, including depression, Parkinson symptom, pseudosclerosis, distonia and akinetic rigid syndrome will be appeared in the third decade of life (16-18).

This study was our preliminary attempt to describe the mutation spectrum of seven exons of ATP7B gene among Iranian population. Besides, the data presented herein point out the prevalence of the mutations and their associated phenotype. Our study is being continued to evaluate all other exons of ATP7B gene.

We found 12 different mutations, including 4 novel disease causing mutations, in 18 individuals out of 70 referred patients. In the rest of the patients we could not detect any mutation in these exons, however, they may have mutations in other exons. Also we identified several amino acid changes or non-change mutation that were defined as the single nucleotide polymorphisms (SNP) due to their high prevalence in either healthy populations or patients. We used them as the polymorphic markers to confirm our results and perform the segregation analysis of ATP7B mutation screening in the patients.

Exon 14 has been introduced as a hot spot for mutations; we detected three different mutations in this exon, of which 2 were novel. H1069Q, located in exon14, is the most frequent mutation in ATP7B gene responsible for WD in US, and central European population (19,20). We observed this mutation in four patients all of whom had only hepatic presentation and aged 5 to 40 years. Moreover, we found a novel causative mutation, H1069N. We screened 50 unrelated individuals for those mutations and none of the tested subjects were heterozygote or homozygote. H1069N mutation has been occurred in a conserved region. Alteration happens in non-polar amino acid “histidin” to hydrophilic “asparagines” within the highly conserved SEHPL domain that is highly deleterious (21-23).

2803-2805ACG>-TG mutation was detected in a relatively large family. Homozygous patients had hepatic WD and we could not detect any neurological signs in these patients. However, with a quick glimpse to all observed phenotype presented by frameshift mutations in our patients, we can confirm the role of recognized frameshift mutations, 1639delC and 2803-2805ACG>-TG in presentation of hepatic signs of WD without any neurological symptoms.

Mutations in exon 6 of WND gene, including 1639delAT and D642H, result to WD in either heterozygous or homozygous state. We can explain this debate through the previously described predominant effect of causative mutations in exon6 of ATP7B gene. D642H was observed in 3 siblings of one family. Two sisters with heterozygous mutation had hepatic WD, and their brother who was homozygous for the so-called mutation had mixed phenotype including liver involvement in addition to neurological symptoms.

R778L is one of the most prevalent mutations in Eastern Asian countries, as reported in Chinese, Korean, Japanese, and Hungarian and leads to liver involvement in WD patients. We found this mutation in the compound heterozygous state with R969Q in one patient who had hepatic failure. It represents the mixed ethnic background of Iranian population.

Needless to say, analysis of other ATP7B exons should be carried out in order to draw a better conclusion.