OBJECTIVES: The aim of this study was to take the molecular-genetic methods for detection of the most frequent mutations in patients suspected for achondroplasia (ACH) and hypochondroplasia (HCH) into the routine practice. BACKGROUND: Both disorders are usually caused by de novo gain-of-function type mutations in FGFR3 gene encoding the fi broblast growth factor receptor 3, which plays an important role in the metabolism of connective tissues. More than 99 % of ACH cases are caused by the glycine-to-arginine substitution at codon 380 and about 70 % of HCH cases result from the asparagine-to-lysine/-serine/-threonine substitutions at codon 540 in the consequence of the four different possible nucleotide changes occurred at the same codon. METHODS: Exons 10 and 13 of theFGFR3 gene were analysed by PCR-RFLP and sequencing analysis. The exon 13 sequencing was necessary for mutation type specifi cation. RESULTS: We confi rmed the diagnosis of ACH due to 1138G→A transition in 7 patients and we identifi ed 1620C→A transversion responsible for HCH in 2 patients. CONCLUSION: Due to serious limitations in recently used methods, we had to modify the molecular-genetic di-agnostics approach. We developed the reliable diagnostics and made it available for achondroplasia and hypochondroplasia suspected patients (Tab. 1, Ref. 5, Ref. 17). Text in PDF www.elis.sk.

BACKGROUND: Pompe disease is an autosomal recessive disorder of glycogen metabolism caused by deficiency in lysosomal enzyme α-glucosidase. OBJECTIVES: We present fi rst two patients from Slovakia with confi rmed Pompe disease. METHODS: Activity of α-glucosidase was measured using 4-methylumbelliferyl-α -D-glucopyranoside with the presence of acarbose, inhibitor that eliminates isoenzyme interference of maltase-glucoamylase. This methodical approach is substantial for determination of lysosomal enzyme defi ciency. Using molecular genetic methods, PCR-RFLP and direct sequencing of coding region α-glucosidase gene (GAA) we have identifi ed causal mutations in our patients. RESULTS: Late-onset type of disease was confi rmed by measuring α-glucosidase activity in leukocytes isolated from blood. The presence of common Caucasian mutation c.-32-13T>G was proved by genetic testing in the fi rst patient in homozygous state. Second patient was a compound heterozygote, with mutation c.-32-13T>G on one allele and mutation A486P on the second allele. CONCLUSION: We present a diagnostic algorithm for diagnosing the Pompe disease in patients of European origin. Enzyme replacement therapy has been used as a treatment option for improving the quality of life of patients. Early diagnosis and treatment of Pompe disease are considered to be critical for maximum effi cacy of enzyme replacement therapy (Tab. 1, Fig. 3, Ref. 20). Text in PDF www.elis.sk.

Prader-Willi and Angelman syndromes are clinically distinct neurodevelopmental genetic disorders that map to 15q11.2-q13 locus. The common phenotypes are attributable to loss of expression of parentally specific imprinted genes inside this region, where the gene function is dependent on parental origin. Initial diagnosis was proved for the years by methylation pattern analyses of the SNRPN exon 1/promoter region within the PWS/AS critical domain. Apart from unifying methylation-specific PCR and allele specific realtime PCR with melt-curve analysis as the fundamental methods for suspected diagnosis confirmation, we combined several specifically methods used to clarify the molecular cause. In our study we had identified and genotyped 24 PWS and AS patients from 450 suspected. Applied cluster of methods-microsatellite analysis of SNPs within the chromosome 15, Methylation-specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) and UBE3A gene sequence analysis, enable us to determined atypical deletion that does not include common breakpoints, novel highly likely to be pathologic UBE3A mutation, uniparental heterodisomy together with partial isodisomy and epimutation without any deletions in the imprinting centre. We present genotype-phenotype correlation of all positive cases. In addition, we estimate the incidence for Slovakian population at 1 in 20,000 for PWS and 1 in 40,000 for AS.