Possible control of paternal imprinting of polymorphisms of the ADAM33 gene by epigenetic mechanisms and association with level of airway hyperresponsiveness in asthmatic children.

INTRODUCTION: ADAM33 is the candidate gene most commonly associated with asthma and airway hyperreactivity (AHR). AIM: The aim of this study was to determine whether level of AHR is associated with certain alleles or haplotypes of the ADAM33 gene in asthmatic children. METHODS: One hundred and nine asthmatic children and 46 controls from the general population were examined with spirometry before and after histamine and methacholine inhalation. All subjects were genotyped for single-nucleotide polymorphisms (SNPs) of the ADAM33 gene. Haplotypes were determined according to genotypes of the patient's parents. RESULTS: We found the three most frequent ADAM33 haplotypes (a1-3) were associated with the highest level of AHR to methacholine and histamine in 66% of asthmatic children. The paternally transmitted GGGCTTTCGCA haplotype was seen in 73.3% asthmatic children with serious AHR to methacholine challenge (paternal and maternal origin of haplotype 73.3% to 37.5, P=0.046) Significant differences in the relative frequency of paternal haplotypes with high levels of AHR to histamine were found (P=0.013). CONCLUSION: ADAM33 haplotypes (a1, a2, a3) are associated with severity of AHR and are significantly more often transmitted in the paternal line.

Association of STAT6 and ADAM33 single nucleotide polymorphisms with asthma bronchiale and IgE level and its possible epigenetic background.

BACKGROUND: ADAM33 and STAT6 belong to the candidate genes that have been commonly associated with asthma, bronchial hyperresponsiveness or IgE levels. Our objective was to assess the association of 11 SNPs of the ADAM33 and 6 of the STAT6 and their haplotypes with IgE levels and asthma. We also evaluated the possible role of parental origin of haplotypes on IgE levels. METHODS: We enrolled 109 children with asthma and 45 healthy controls. Genotyping was performed by TaqMan probes and confirmed by sequencing. Haplotype construction was based on the knowledge of parental genotypes and also inferred by using the EM algorithm and Bayes' theorem. RESULTS: None of the SNPs were associated with elevated IgE level or asthma. We found that the most frequent STAT6 haplotype ATTCAA (built from rs324012, rs324011, rs841718, rs3024974, rs3024974, rs4559 SNPs, respectively) was associated with elevated total IgE levels (P=0.01) and this haplotype was predominantly transmitted paternally (P<0.001). We compared our results with those of studies performed on German and Australian Caucasian populations and found that rs324011, rs3024974 and rs4559 SNPs in STAT6 should have a major effect on IgE levels. Therefore, we suggest the TCA haplotype alone (built from rs324011, rs3024974 and rs4559 SNPs, respectively) in STAT6 is associated with total IgE elevation. CONCLUSIONS: The influence of paternal origin of the STAT6 haplotype on IgE levels is surprising but the exact role of possible paternal imprinting in STAT6 regulation should be investigated and confirmed in future studies.

Refined fluorescent STR quantification of cell-free fetal DNA during pregnancy in physiological and Down syndrome fetuses.

BACKGROUND: Cell-free fetal (cff) DNA analysis by short tandem repeats (STR) has the advantage of better recognizing the different genotypes. However, quantitative examination by quantitative fluorescent (QF) polymerase chain reaction (PCR) by STRs is limited to only a rough approximation. This project focuses on a more precise calculation of the relative cff DNA amount tested in the STRs' loci. METHODS: The cff DNA was analyzed in 363 samples from 258 pregnant women with physiological fetuses in different stages of pregnancy (from 4-37 gestational weeks) separately in three STRs [D21S1435, D21S1446 and PentaD (pD)] and also by gonosomal sequences amelogenin gene, X/Y-linked/testis specific protein, Y-linked (AMELX/Y/TSPY). Seventeen samples of cff DNA from fetuses with Down syndrome (DS) were compared. We optimized the refined quantitative fluorescent (RQF) PCR for STRs in a particular locus. RESULTS AND CONCLUSIONS: The cff DNA detection rate was 74% in at least one of the STRs. The efficiency decreased from shorter to longer PCR fragments. All three STR and gonosomal loci proved an increase in cff DNA during pregnancy. The stutter variability rate is greatest in short STR fragments and decreases as the STR fragments increase in length. Results showed that DS samples had a significantly higher amount of cff DNA.