Andrej Ficek

Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans

Glutathione S-transferase genotypes GSTT1, GSTM1, GSTP1 were characterised in 155 middle-aged men and compared with parameters of oxidative stress at the level of DNA and lipids, with antioxidant enzymes, and with plasma antioxidants in smokers and non-smokers. Smokers had on average significantly lower levels of Vitamin C, beta-carotene and beta-cryptoxanthin and higher amounts of oxidised purines and pyrimidines in lymphocyte DNA. The GSTM1 null genotype was associated with elevated glutathione as well as with higher Vitamin C concentration in plasma. Vitamin C was higher in GSTT1+ compared with GSTT1 null--as was glucose-6-phosphate dehydrogenase activity. The homozygous GSTP1 a/a genotype was associated with significantly higher levels of GST activity measured in lymphocytes, in comparison with the b/b genotype. Using multifactorial statistical analysis we found significant associations between smoking, GSTP1 genotype, plasma Vitamin C, and purine base damage in lymphocyte DNA. The difference in Vitamin C plasma levels between smokers and non-smokers was seen only with the GSTP1 b/b genotype. This group accounted also for most of the increase in purine oxidation in smokers. In contrast, the link between smoking and oxidised pyrimidines in DNA was seen only in the GSTT1 null group. It seems that polymorphisms in the phase II metabolising enzyme glutathione S-transferase may be important determinants of commonly measured biomarkers.

Polymorphisms of candidate genes in Slovak autistic patients.

Autism is one of the most genetically influenced neuropsychiatric disorders. However, its detailed genetic basis is far from being clear. Genome-wide association studies have revealed a number of candidate genes, mostly related to synaptogenesis and various neuroendocrine pathways. In our study we have focused on oxytocin (OT), oxytocin receptor (OXTR), GABA receptor gamma 3 (GABRG3), neuroligin (NLGN4X), and reelin (RELN). After signed consent, 90 autistic boys and 85 healthy controls were enrolled in the study. Polymorphisms of OT (rs2740204), OXTR (rs2228485), GABRG3 (rs28431127), and NLGN4X (rs5916338) were analyzed using restriction fragment length polymorphism. (GGC)n STR polymorphism in the 5′ UTR of the RELN gene was genotyped using fragment analysis. The only significant association in autistic boys in Slovakia was found with higher number of GGC repeats in the RELN gene (P=0.001) potentially explaining lower RELN levels in blood and brain of autistic patients.

Phenylalanine hydroxylase deficiency in the Slovak population: Genotype–phenotype correlations and genotype-based predictions of BH4-responsiveness

We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 135 Slovak PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed using high-resolution melting analysis, with subsequent sequencing analysis of the samples showing deviated melting profiles compared to control samples. The PAH gene was also screened for deletions and duplications using MLPA analysis. Forty-eight different disease causing mutations were identified in our patient group, including 30 missense, 8 splicing, 7 nonsense, 2 large deletions and 1 small deletion with frameshift; giving a detection rate of 97.6%. The most prevalent mutation was the p.R408W, occurring in 47% of all alleles, which concurs with results from neighboring and other Slavic countries. Other frequent mutations were: p.R158Q (5.3%), IVS12+1G>A (5.3%), p.R252W (5.1%), p.R261Q (3.9%) and p.A403V (3.6%). We also identified three novel missense mutations: p.F233I, p.R270I, p.F331S and one novel variant: c.-30A>T in the proximal part of the PAH gene promoter. A spectrum of 84 different genotypes was observed and a genotype based predictions of BH4-responsiveness were assessed. Among all genotypes, 36 were predicted to be BH4-responsive represented by 51 PKU families. In addition, genotype-phenotype correlations were performed.

Effect of unexpected sequence interruptions to conventional PCR and repeat primed PCR in myotonic dystrophy type 1 testing.

Myotonic dystrophy type 1 (DM1) is caused by expansion of the CTG trinucleotide repeat in the DMPK gene. Our study focuses on the effect of recently described unusual sequence interruptions inside the CTG tract on conventional polymerase chain reaction (PCR) and triplet repeat primed PCR (TP-PCR) amplifications, which are the methods now widely used in molecular testing for DM1. For molecular characterization of the CTG repeat tract, we used conventional fluorescent PCR with bidirectional labeling and both forward and reverse direction TP-PCR. Though the results of the methods are still unambiguous for most alleles, mistyping and false results may occur in the typing of some unordinary alleles carrying sequence interruptions. The presence of these interruptions may lead not only to altered TP-PCR profiles, as can be expected, but also to abnormal electrophoretic mobility of complementary strands produced by conventional amplification of such alleles. Our findings suggest that the simultaneous combination of bidirectionally labeled conventional PCR with TP-PCR performed in both directions may be necessary for increasing the reliability and accuracy of the TP-PCR-based assay for DM1 testing.

High-resolution melting analysis for genotyping of the myotonic dystrophy type 1 associated Alu insertion/deletion polymorphism

Since its introduction, high-resolution melting (HRM) analysis has been used for genotyping of various types of sequence alterations. In this study, we report the use of HRM for genotyping of the 1-kb insertion/deletion polymorphism, involving a problematic region of five consecutive Alu elements, that is associated with myotonic dystrophy type 1. We combined a three-primer polymerase chain reaction (PCR) amplification approach with HRM using two primer sets. Analyses based on curve shapes are sensitive enough to differentiate between genotypes with both primer sets. In addition, the newly designed insertion-specific primer from the second primer set equalizes the allele-specific amplicon lengths, thereby reducing the possibility of preferential amplification of shorter fragments.

GJB2 gene mutations in Slovak hearing-impaired patients of Caucasian origin: spectrum, frequencies and SNP analysis.

To the Editor: In developed countries, due to genetic factors, more than 60% of patients suffer from prelingual, moderate to profound hearing impairment, and more than 70% of hereditary hearing impairment cases are non-syndromic (nonsyndromic hearing impairment, NSHI). Of these, approximately 80% are estimated to be autosomal recessive, with 22 recessive genes identified so far (http://webhost.ua.ac.be/hhh/). Among them, most causative mutations are in the gene GJB2 (1). This gene is located at 13q12 and belongs to the connexin gene family. Connexin genes are simple in organization, composed of two exons. The first exon encodes the 50-untranslated region, while the second exon encodes the complete open reading frame and the 30-untranslated region. This makes connexin genes relatively easy targets for screening of mutations by direct sequencing. Thus far, more than 100 different NSHIcausing mutations and 24 polymorphic variants have been identified worldwide within the GJB2 gene (http://davinci.crg.es/deafness/). Although the majority of mutations are recessive, some of them are dominant [W44C (2); R75W (3)]. As it is the case in many recessive disorders, the mutational spectra often vary at this locus between different populations, with a single mutation being sometimes prevalent and thus specific for a particular population. In Caucasians, the 35delG mutation accounts for up to 85% of the GJB2 mutant alleles (4–6). However, it is less frequent in populations of non-European origin, where other GJB2 mutations prevail. Thus, the mutation 235delC is prevalent in Koreans (7), Japanese (8), Chinese (9) and Taiwanese (10); 167delT prevails in Ashkenazi Jews (11), R143W in Africans (12, 13) and W24X in NSHI patients of Indian origin (14–16). The exact frequency of congenital hearing impairment or deafness in the Slovak population is unknown, but it is estimated to be approximately 3–4 in 1000 newborns with impaired hearing and 1 in 1000 with deafness (personal communication with Dr Zuzana Kabatova, First Department of Otolaryngology, Comenius University Faculty of Medicine). This study is aimed to determine the spectrum and frequencies of GBJ2 gene (coding region) mutations in Slovak NSHI patients of Caucasian origin.

Genetic Polymorphisms Related to Testosterone Metabolism in Intellectually Gifted Boys

Prepubertal testosterone levels are lower in intellectually gifted boys. The aim of this pilot study was to analyze potential genetic factors related to testosterone metabolism in control and gifted boys. Intellectually gifted (IQ>130; n = 95) and control (n = 67) boys were genotyped. Polymorphisms of interests were chosen in genes including androgen and estrogen receptors, 5-alpha reductase, aromatase and sex hormone binding globulin. Significant differences between control and gifted boys in genotype distributions were found for ESR2 (rs928554) and SHBG (rs1799941). A significantly lower number of CAG repeats in the AR gene were found in gifted boys. Our results support the role of genetic factors related to testosterone metabolism in intellectual giftedness. Increased androgen signaling might explain previous results of lower testosterone levels in intellectually gifted boys and add to the understanding of variability in cognitive abilities.

Analysis of forensically used autosomal short tandem repeat markers in Polish and neighboring populations

The purpose of this study was to evaluate the homogeneity of Polish populations with respect to STRs chosen as core markers of the Polish Forensic National DNA Intelligence Database, and to provide reference allele frequencies and to explore the genetic interrelationship between Poland and neighboring countries. The allele frequency distribution of 10 STRs included in the SGMplus kit was analyzed among 2176 unrelated individuals from 6 regional Polish populations and among 4321 individuals from Germany (three samples), Austria, The Netherlands, Sweden, Czech Republic, Slovakia, Belarus, Ukraine and the Russian Federation (six samples). The statistical approach consisted of AMOVA, calculation of pairwise Rst values and analysis by multidimensional scaling. We found homogeneity of present day Poland and consistent differences between Polish and German populations which contrasted with relative similarities between Russian and German populations. These discrepancies between genetic and geographic distances were confirmed by analysis of an independent data set on Y chromosome STRs. Migrations of Goths, Viking influences, German settlements in the region of Volga river and/or forced population resettlements and other events related to World War II are the historic events which might have caused these finding.

Mental rotation in intellectually gifted boys is affected by the androgen receptor CAG repeat polymorphism

Testosterone was shown to organize brain and modulate cognitive functions. It is currently unknown whether mental rotation is also associated with prenatal testosterone exposure and testosterone-related genetic polymorphisms. The aim of our study was to analyze associations between mental rotation performance, the actual testosterone levels, the prenatal testosterone level (expressed as 2D:4D ratio) and the androgen receptor CAG repeat polymorphism in intellectually gifted boys. One hundred forty-seven boys aged 10-18 years with IQ>130 were enrolled. Saliva samples were collected and used for ELISA of actual levels of salivary testosterone. The 2D:4D finger length ratio as an indicator of prenatal testosterone was measured on both hands and averaged. Amthauer mental rotation test was used for the assessment of this spatial ability. The CAG repeat polymorphism in the androgen receptor gene was analyzed using PCR and capillary electrophoresis. Linear regression revealed that 2D:4D finger length ratio and the number of CAG repeats in the androgen receptor gene were associated with mental rotation. Actual levels of testosterone did not correlate significantly with mental rotation. Multivariate analysis of covariance revealed that after adjustment of age as a confounding variable, only the effect of the genetic polymorphism was significant. The results are in line with our previous genetic analysis of intellectually gifted boys showing the importance of CAG repeat polymorphism in the androgen receptor gene. Details of the interactions between androgen signaling, testosterone levels and its metabolism especially during the prenatal development of brain function remain to be elucidated.

Spatial Abilities in Prepubertal Intellectually Gifted Boys and Genetic Polymorphisms Related to Testosterone Metabolism

Spatial abilities are known to be related to testosterone levels in men. Polymorphisms of genes related to androgen metabolism, however, have not been previously analyzed in association with spatial abilities. Our study analyzes genetic polymorphisms of androgen receptor (AR), aromatase (CYP19), and 5-alpha reductase (SRD5A2) in relation to mental rotation and spatial visualization in prepubertal intellectually gifted boys. DNA samples of 36 boys with an average age of 10.0 ± 0.7 years and an IQ higher than 130 were isolated from buccal cells in saliva. DNA was subsequently used for amplification by PCR. The CYP19 C1558-T polymorphism and SRD5A2 A49T polymorphism were determined by RFLP analysis, and the AR (CAG)n polymorphism was determined by fragment analysis. Salivary testosterone levels were measured with radioimmunoassay. Spatial abilities (mental rotation and spatial visualization) were assessed using standard psychometric tests. AR and CYP19 polymorphisms were not associated with spatial abilities...