Slávka Pouchlá

Genotyping microarray as a novel approach for the detection of ATP7B gene mutations in patients with Wilson disease.

Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism that is caused by mutations in the ATP7B gene. To date, more than 300 mutations have been described in this gene. Molecular diagnostics of WD utilizes restriction enzyme digestion, multiplex ligation‐dependent probe amplification or a direct sequencing of the whole gene. To simplify and speed up the screening of ATP7B mutations, we have developed a genotyping microarray for the simultaneous detection of 87 mutations and 17 polymorphisms in the ATP7B gene based on the arrayed primer extension reaction. The patient’s DNA is amplified in four multiplex polymerase chain reactions, fragmented products are annealed to arrayed primers spotted on a chip, which enables DNA polymerase extension reactions with fluorescently labeled dideoxynucleotides. The Wilson microarray was validated by screening 97 previously genetically confirmed WD patients. In total, we detected 43 mutations and 15 polymorphisms that represent a majority of the common mutations occurring in the Czech and Slovak populations. All screened sequence variants were detected with 100% accuracy. The Wilson chip appears to be a rapid, sensitive and cost‐effective tool, representing the prototype of a disease chip that facilitates and speeds up the screening of potential WD patients.

Hyperphenylalaninemia in the Czech Republic: genotype-phenotype correlations and in silico analysis of novel missense mutations.

BACKGROUND Hyperphenylalaninemia (HPA) is one of the most common inherited metabolic disorders caused by deficiency of the enzyme phenylalanine hydroxylase (PAH). HPA is associated with mutations in the PAH gene, which leads to reduced protein stability and/or impaired catalytic function. Currently, almost 700 different disease-causing mutations have been described. The impact of mutations on enzyme activity varies ranging from classical PKU, mild PKU, to non-PKU HPA phenotype. METHODS We provide results of molecular genetic diagnostics of 665 Czech unrelated HPA patients, structural analysis of missense mutations associated with classical PKU and non-PKU HPA phenotype, and prediction of effects of 6 newly discovered HPA missense mutations using bioinformatic approaches and Molecular Dynamics simulations. RESULTS Ninety-eight different types of mutations were indentified. Thirteen of these were novel (6 missense, 2 nonsense, 1 splicing, and 4 small gene rearrangements). Structural analysis revealed that classical PKU mutations are more non-conservative compared to non-PKU HPA mutations and that specific sequence and structural characteristics of a mutation might be critical when distinguishing between non-PKU HPA and classical PKU mutations. The greatest impact was predicted for the p.(Phe263Ser) mutation while other novel mutations p.(Asn167Tyr), p.(Thr200Asn), p.(Asp229Gly), p.(Leu358Phe), and p.(Ile406Met) were found to be less deleterious.

Wilson's Disease: Monocentric Experiences Over a Period of 16 Years

BACKGROUND Wilsons disease (WD) is an autosomal recessive disorder of copper metabolism. The objective of this study is to present diagnostic pitfalls and long time follow-up data in Wilson disease. PATIENTS/METHODS We studied 21 WD patients and 14 heterozygote carriers aged 2-43 years, retrospectively. 18 WD patients presented liver disease, three had mixed neurological and hepatic involvement and 9 patients underwent orthotopic liver transplantation (OLT). RESULTS The median age at diagnosis of WD children without OLT was 10.16+/-3.8 (range, 5-16). All of females and younger age categories of patients prevailed in acute liver failure group. Serum ceruloplasmine levels were below 0.2 g/l in about (1/3) of WD carriers (X =0.27+/-0.09 g/l) and nearly (2/3) of children with WD (X = 0.21+/-0.13 g/l). A statistically significant difference (p<0.05) in the 24-h excretion of copper in urine was noticed between healthy controls, children with WD and WD heterozygote carriers. As diagnostic important proved the copper content of more than 250 microg/g hepatic dry weight. The Kayser-Fleischer?s ring was not observed in children. Ceruloplasmine, haemoglobin, ALT, ALP and plasma albumin were significantly different between fulminant and non-fulminant WD and could be used as indirect markers in evaluation of urgent OLT. CONCLUSION Detection of WD in children remains very difficult. The most important investigation is liver biopsy with the assessment of liver copper. Genetic analysis may help in doubtful cases.