Zuzana Hrubá

Chimeric CYP21A1P/CYP21A2 genes identified in Czech patients with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in the majority of severe cases, also the biosynthesis of aldosterone. Approximately 95% of all CAH cases are caused by mutations in the steroid 21-hydroxylase gene (CYP21A2). The CYP21A2 gene and its inactive pseudogene (CYP21A1P) are located within the HLA class III region of the major histocompatibility complex (MHC) locus on chromosome 6p21.3. In this study, we describe chimeric CYP21A1P/CYP21A2 genes detected in our patients with 21-hydroxylase deficiency (21OHD). Chimeric CYP21A1P/CYP21A2 genes were present in 171 out of 508 mutated CYP21A2 alleles (33.8%). We detected four types of chimeric CYP21A1P/CYP21A2 genes: three of them have been described previously as CH-1, CH-3, CH-4, and one type is novel. The novel chimeric gene, termed CH-7, was detected in 21.4% of the mutant alleles. Possible causes of CYP21A1P/CYP21A2 formation are associated with 1) high recombination rate in the MHC locus, 2) high recombination rate between highly homologous genes and pseudogenes in the CYP21 gene area, and 3) the existence of chi-like sequences and repetitive minisatellite consensus sequences in CYP21A2 and CYP21A1P which play a role in promoting genetic recombination.

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.

A p.(Glu809Lys) Mutation in the WFS1 Gene Associated with Wolfram-like Syndrome: A Case Report

Wolfram-like syndrome (WFSL) is a rare autosomal dominant disease characterised by congenital progressive hearing loss, diabetes mellitus, and optic atrophy. The patient was a boy with the juvenile form of diabetes mellitus and findings which clinically matched the symptoms of Wolfram syndrome. At the age of 3 1/4 years, diabetes mellitus was diagnosed in this boy who also had severe psychomotor retardation, failure to thrive, a dysmorphic face with Peters anomaly type 3 (i.e. posterior central defect with stromal opacity of the cornea, adhering stripes of the iris, and cataract with corneolenticular adhesion), congenital glaucoma, megalocornea, severe hearing impairment, a one-sided deformity of the auricle with atresia of the bony and soft external auditory canal, non-differentiable eardrum, missing os incus, hypothyreosis, and nephrocalcinosis. Molecular-genetic examinations revealed a de novo mutation p.(Glu809Lys) in the WFS1 gene. No mutations were detected in the biological parents. The mutation p.(Glu809Lys) in the WFS1 gene is associated with WFSL.

G.P.222

Limb-girdle muscular dystrophy (LGMD) is defined as a muscular dystrophy with predominantly proximal distribution of muscle weakness. It includes a number of disorders with heterogeneous etiology. We determined the frequency of recessive LGMD subtypes (LGMD2A, LGMD2D, LGMD2I and LGMD2L) within a cohort of Czech LGMD2 patients using mutation analysis of the calpain3 (CAPN3), fukutin-related protein (FKRP), alpha-sarcoglycan (SGCA), and anoctamin5 (ANO5) genes. Last year we introduced next generation sequencing to accelerate patient diagnosis and to widen spectrum of analysed genes. We designed capture library to target the coding exons of genes responsible for all known types of LGMD and genes responsible for muscular dystrophy with similar phenotype to LGMD. We observed that mutations of the CAPN3 gene are the most common cause of LGMD2. The frequency of particular forms of LGMD2 was 32.6% for LGMD2A, 4.1% for LGMD2I, 2.8% for LGMD2D, and 1.4% for LGMD2L. Using next-generation sequencing, we identified two patients with mutations in the gene encoding dysferlin (DYSF) – LGMD2B and a patient with mutations in the gene encoding beta-sarcoglycan (SGCB) – LGMD2E. In total, we determined mutations in 41% of Czech LGMD2 patients. This work was funded by the project of the Internal Grant Agency of the Czech Ministry of Health (NT/14574-3); the projects of the Czech Ministry of Education “CEITEC – Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) and SuPReMMe (CZ.1.07/2.3.00/20.0045).