Georgios Loudianos

Wilson's disease (WD) is a rare genetic disorder of copper transport due to mutations in the ATP7B gene. This results in copper overload and tissue damage that is most evident in the liver and brain. Over 1000 pathogenic mutations have been found in WD patients, and of these, mutations within the ATP7B coding region predominate. In this study, we perform functional analyses of 7 rare sequence variations in the promoter region of the ATP7B gene. We performed dual luciferase reporter assays in HepG2 and SH-SY5Y cell lines under basal conditions and after the addition of 10 μM or 40 μM concentrations of CuSO₄. The results showed that promoter activity varied both according to the haplotype and the cell line used. Our results suggest a potential role of promoter polymorphisms in the variation of ATP7B gene expression with probable implications in copper accumulation in the organism and consequent phenotype variation in WD. In addition, given the association of copper concentration and ATP7B expression in the brain, promoter polymorphisms could act as susceptibility determinants of neurodegeneration in the most common late-onset nervous system diseases. Finally, promoter involvement in ATP7B overexpression and platinum resistance presents new mechanisms of great importance that need to be better understood. Further experimental and clinical studies are necessary to further explore the role of the ATP7B promoter in copper and platinum management. These studies will offer new insights for the development of molecular therapies for brain degenerative and tumor diseases.

Background and Objectives: Wilson's disease (WD) in children and adolescents is predominantly asymptomatic or oligo‐symptomatic. The symptoms are nonspecific and difficult to distinguish from other hepatic or neuropsychiatric disorders. In this study, we present the experience of a pediatric referral center for WD diagnosis and treatment.Patients and Methods: We retrospectively analyzed clinical and laboratory data from 99 patients with WD of Sardinian origin, including physical examination, laboratory biochemical testing, liver biopsy, and genetic analysis.Results: Patients were prevalently oligo‐symptomatic or asymptomatic. The median age of diagnosis was 8.78 years. Ceruloplasmin values were lower than normal values in all analyzed patients. Twenty‐four‐hour urinary copper levels were higher than 40 μg/24‐h in 92/96 patients. In all analyzed patients with the exception of one, liver copper was higher than 250 μg/g of dry weight but all had >75 μg/g of dry weight. Statistical analysis showed correlation between the age at diagnosis, serum copper, and 24‐h urinary copper. Correlation was also found between serum copper and 24‐h urinary copper. Molecular analysis of ATP7B gene allowed complete characterization in all the analyzed patients.Conclusion: A high index of clinical suspicion and biochemical tests including liver tests, serum ceruloplasmin, and basal 24‐h urinary copper excretion and genotype determination are key to WD diagnosis. The long experience that a referral center for WD possesses is an important factor in making WD diagnosis a more accurate process. Studies in animal models on WD could be used as a guide to further investigate the molecular mechanisms that regulate copper metabolism and influence the natural history of WD.

We describe epidemiological, genetic, and clinical data of the 1124-2del mutation in the G6PT gene, detected in homozygosity in three glycogen storage disease type Ib patients of Sardinian origin. This mutation was found to be associated with four sequence variations: c.593 A>T (p.N198I), c.625+19 C>T, c.1062 C>T (N354N), and c.1224 G>A (p.T408T) in the G6PT gene. RNA studies were performed for c.1124-2del and c.625+19 C>T. The c.1124-1del2 acceptor splicing mutation showed skipping of 31 nucleotides of exon 9 due to the activation of a downstream cryptic acceptor splice site in 1154-1155 nucleotide positions, resulting in a downstream stop codon at aa position 402. RNA analysis of c.625+19 C>T variation showed a small amount of alternative splicing with skipping of exon 4, resulting in a stop codon at aa position 211. Our cases present most of features of the severe form of disease, including early onset with chronic neutropenia, frequent infections, and inflammatory bowel disease. Our results suggest a founder effect for glycogen storage disease type Ib that facilitates diagnosis using mutation analysis, sparing patients from liver biopsy. DNA-based diagnosis will enable us to make accurate determination of carrier status and prenatal diagnosis, thus improving genetic counseling.

Objectives: Herein we report the results of mutation‐based screening for Wilson disease (WD) in 2 isolated populations of Sardinia and the Greek island of Kalymnos.Patients and Methods: Mutation analysis was performed in 110 and 9 WD families originating respectively from Sardinia and Kalymons using single‐strand conformation polymorphism and sequencing methods. In Sardinia, a limited screening was performed for −441/−427del in 5290 newborns, whereas in Kalymnos 397 newborns underwent mutation screening for H1069Q and R969Q using appropriate methods.Results: In Sardinia, mutation analysis showed the presence of 6 mutations accounting for 85% of chromosomes, 1 of which (−441/−427del) is present in 61.7% of alleles. The screening for −441/−427del in 5290 newborns revealed the presence of 122 heterozygotes, which is equal to an allelic frequency of 1.15%. Assuming the same distribution of WD mutations in the general Sardinian population, we also inferred an allelic frequency of 0.77% for mutations other than −441/−427del, which accounts for an overall frequency of any WD mutation of 1.92%. Assuming Hardy‐Weinberg equilibrium, these data could be translated into a WD incidence of 1 in 2707 live births. In Kalymnos, mutation analysis in 9 WD families revealed the presence of only 2 mutations. The screening of 397 newborns revealed the presence of 18 heterozygotes for H1069Q, 9 for R969Q, and 1 compound heterozygote for these mutations, which is equal to an allele frequency of 3.7%. Assuming Hardy‐Weinberg equilibrium, the expected carrier rate is 7%.Conclusions: These data indicate the need for health education for WD prevention in these isolated populations.