Mario Lovicu

Molecular characterization of Wilson disease in the Sardinian population—Evidence of a founder effect

Wilson disease (WD) in the Sardinian population has an approximate incidence of 1:7,000 live births. Mutation analysis of the WD gene in this population reported in our previous articles led us to the characterization of two common mutations and a group of 13 rare mutations accounting for the molecular defect of 8.5, 7.9, and 15.1% of the WD chromosomes. However, molecular analysis of the WD chromosomes containing the most common haplotype, which accounts for 60.5% of the WD chromosomes, failed to define the disease‐causing mutation. In this study, we characterized the promoter and the 5′ UTR of the WD gene sequence and carried out a mutation analysis in this DNA region from patients with the most common haplotype. The promoter is contained in a GC‐rich island and shows a TATA and a CAAT consensus sequence as well as potential binding sites for transcription factors and metal response elements. In all the analyzed 92 chromosomes with this haplotype, we detected a single mutation consisting of a 15‐nt deletion from position –441 to position –427 relative to the translation start site. Expression assays demonstrated a 75% reduction in the transcriptional activity of the mutated sequence compared to the normal control. By adding this mutation to those that have been already characterized, we have now defined the molecular defect in 92% of the WD chromosomes in Sardinians. The high frequency, the expected prevention by preclinical diagnosis and early treatment of the devastating effect of WD on the nervous system and liver tissue, and the feasibility to detect most of molecular defects by DNA analysis indicate that WD in the Sardinian population should be added to the list of diseases currently detected by newborn screening. Hum Mutat 14:294–303, 1999.

Mutation analysis in patients of Mediterranean descent with Wilson disease: identification of 19 novel mutations.

In this study, we report further results of mutation analysis of the ATP7B gene in Wilson disease (WD) patients of Mediterranean origin. A total of 136 WD chromosomes, 73 of which were of Italian, 43 of Turkish, 18 of Sardinian, and two of Spanish origin, were analysed and the mutation characterised in 84.5% of them. We found 50 different mutations of which 19 are novel, including three nonsense, one frameshift, and 15 missense mutations. The mutations detected were rare and mostly found in the compound heterozygous state together with other mutations and only rarely in homozygosity. Most of these mutations lie in the transmembrane and ATP binding loop regions. These data expand our knowledge of both the structure-function relationships of the WD protein and the molecular pathology of WD, thus improving our capability of prevention and genetic counselling.

Further delineation of the molecular pathology of Wilson disease in the Mediterranean population

This study presents the update results of an ongoing project on the delineation of the spectrum of mutations at the Wilson disease (WD) gene in WD patients of Mediterranean origin. In studying 59 patients, of whom were 26 Continental Italians, 22 Sardinians, 9 Turkish, and 2 Albanians, we have found 31 novel and three known mutations. Of the novel mutations, 3 are deletions, two nonsense, 2 splice or consensus splice site, and 24 missense. The large majority of the missense mutations lie in evolutionary conserved regions of the WD gene of documented functional importance. Most of our patients were compound heterozygotes, and only a few were homozygotes. In addition, three polymorphisms were detected. By adding the new data to those previously reported by our group, we have to date detected 85% of mutations in the WD chromosomes from Continental Italians, 30% from Sardinians, 81.7% from Turkish and 66.7% from Albanians. Most of the mutations characterized are rare, and only a limited number are common. Of the common mutations 5 were found in Continental Italians, two in Sardinians and a single one in Turkish. Because there are so many causative mutations of the disease, the preclinical and prenatal diagnosis of WD should be carried out by a combination of mutation and linkage analysis. Hum Mutat 12:89–94, 1998.

Wilson disease mutations associated with uncommon haplotypes in mediterranean patients

Abstract This study reports 12 novel mutations of the Wilson disease (WD) gene which have been detected by the molecular analysis of 29 patients of Mediterranean descent carrying uncommon chromosomal haplotypes at the WD locus. These mutations include two nonsense, one splice site and nine missense. The missense mutations lie in regions of the WD gene critical for its function, such as the transmembrane region, the transduction domain and the ATP loop and ATP-binding domain, indicating that they are disease-causing mutations. These new findings improve our knowledge for the role played by functional domains on the ATP7B function.

Haplotype and mutation analysis in Greek patients with Wilson disease

In this study, we report the results of haplotype and mutation analysis of the ATP7B gene in Wilson disease (WD) patients of Greek origin. We have analysed 25 WD families and two single patients and characterised 94% of the WD chromosomes investigated. We have found 12 different molecular defects (three frameshifts, two splice site, two nonsense, five missense mutations), four of which are novel. Five of the mutations are widely prevalent accounting for 74% of the WD chromosomes analysed. These results may enable preclinical diagnosis in the large majority of WD patients of Greek descent, thereby improving genetic counselling and disease management.

Characterization of the Molecular Defect in the ATP7B Gene in Wilson Disease Patients from Yugoslavia

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism resulting from the absence or dysfunction of a copper transporting P-type ATPase (ATP7B). Approximately 150 mutations of the ATP7B have been identified to date. In this paper, we report the results of molecular characterization and genotype-phenotype analysis, which we have carried out on 35 patients from Yugoslavia affected by WD. Using single-strand conformational polymorphism (SSCP) followed by direct sequencing, we characterized the molecular defect in 80% of WD chromosomes and found 11 different mutations, three of which are novel. The most common mutations that accounted for the molecular defect in 71.3% of WD chromosomes were H1069Q (48.9%), 2304-2305insC (11.4%), R616Q (5.7%), and A1003T (5.7%). The results produced in this paper indicate that the best strategy for mutation detection in Yugoslavian patients with WD is an SSCP analysis of exons 14, 8, 5, and 13, where most of the defects (73.1%) lie, followed by mutation analysis of the remaining exons in ATP7B in patients in whom the mutation was not detected by the finitial screening. These data can be used to develop straightforward genetic testing in this population or in other countries composed of a genetically mixed population like the United States, where a significant number of immigrants came from Central and Eastern Europe.

Delineation of the spectrum of Wilson disease mutations in the Greek population and the identification of six novel mutations.

In this study, we report the further results of an ongoing project on the delineation of the spectrum of mutations on the ATP7B gene in Wilson disease (WD) patients of Greek origin. We have analyzed 24 additional families and detected 16 mutations (five frameshifts, two splice site, two nonsense, and seven missense), of which six are novel. On adding these results to the ones already published by us, we conclude that WD shows a marked allelic heterogeneity in the Greek population. Of the total number of mutations so far detected, the most common eight account for the molecular defect in 72.8% of the WD chromosomes. The most frequent mutation is the His0169Gln, which has a frequency of 28.5%, similar to those reported in North European populations. Using these data, an efficient strategy of mutation screening for WD is possible in this population, thus improving the possibility of preclinical diagnosis.

Efficient strategy for molecular diagnosis of wilson disease in the Sardinian population

Wilson disease (WD) is an autosomal recessive disorder of copper transport resulting from the defective function of a copper-transporting ATPase (ATP7B) (1)(2)(3). More than 200 disease-causing mutations have been identified (4). In the Sardinian population, WD has an incidence of ∼1 in 7000 live births (5). Using single-strand conformation polymorphism (SSCP) and sequencing methods for mutation analysis, we have characterized 92% of the chromosomes analyzed and identified 16 different WD-causing mutations, 6 of which (−441/−427del, 213–214delAT, 1512–1513insT, R778W, 2463delC, and V1146M) are relatively common and account for 85% of chromosomes (6). On the basis of these data, we developed a reverse dot-blot (RDB) method as a practical solution to mutation screening in this population. DNA samples from Sardinian WD patients carrying different combinations of the most common mutations (−441/−427del, 2463delC, V1146M, 213–214delAT, 1512–1513insT, and R778W) were used as controls. Our aim was to obtain the same PCR conditions for all six pairs of primers that were used to amplify the regions containing the six most common mutations. We therefore designed primers with an identical melting temperatures and tested their specificity first in single and then in multiplex PCRs. We also wanted to obtain relatively equal yields for all PCR products to obtain comparable signals using the RDB method. We therefore tested different concentrations for each pair of primers and finally established primer concentrations that allowed us to obtain approximately …

The canine copper toxicosis gene MURR1 is not implicated in the pathogenesis of Wilson disease

BackgroundIt has recently been demonstrated that the Wilson disease (WD) protein directly interacts with the human homolog of the MURR1 protein in vitro and in vivo, and that this interaction is specific for the copper transporter. The aim of the present study was to clarify the role of MURR1 in the pathogenesis of WD as well as in other WD-like disorders of hepatic copper metabolism of unknown origin.MethodsUsing the single-strand conformation polymorphism (SSCP) method followed by sequencing, we analyzed the 5′ untranslated region (UTR) and three exons of the MURR1 gene in three groups of patients: 19 wd patients in whom no mutations were detected in the ATP7B gene, 53 wd patients in whom only one mutation in the ATP7B gene was found, and 34 patients in whom clinical and laboratory data suggested a WD-like disorder of hepatic copper metabolism of unknown origin.ResultsWe  detected in these patients six rare nucleotide substitutions, namely one splice-site consensus sequence and one missense and four silent nucleotide substitutions. All substitutions except one were found in the heterozygous state. No difference in the frequencies of the various substitutions was observed between patients and controls.ConclusionsThese data suggest that the MURR1 gene and its protein product are unlikely to play a primary role in the pathogenesis of Wilson disease. More extensive studies with larger numbers of clinically homogeneous patients should be carried out to establish whether nucleotide alterations in the MURR1 gene may have a role in causing WD or WD-like disorders or act as modifying factors in the phenotype variability in WD.

Early and severe neurological features in a Wilson disease patient compound heterozygous for two frameshift mutations

Abstract We describe a patient with Wilson disease who presented at 11 years of age with neurological symptoms and subsequent rapid progression of neurological impairment but absent hepatic manifestations. Molecular analysis showed compound heterozygosity for two frameshift mutations, 2299insC and 214delAT, which most likely result in an absent or inactive protein product. Mutation-phenotypic analysis indicates that this genotype does not explain the severe phenotype, suggesting the presence of modifying factors. Conclusion Wilson disease may present even in childhood or adolescence with neurological abnormalities in the absence of hepatic manifestations.