Wojciech Niewiarowski

Arylamine N‐acetyltransferase (NAT2) gene mutations in children with allergic diseases

The overrepresentation of phenotypically slow acetylators among patients with atopic allergy has been reported in previous studies. The N‐acetyltransferase coding gene has not yet been investigated in allergic diseases. This study was designed to determine the differences in the distribution of mutation frequency and genotypes that encode normal and defective activity of N‐acetyltransferase in children with atopic allergies compared with healthy children. In 56 children with documented inhalational, food, or mixed allergies and in 100 healthy control children with no clinical or laboratory signs of allergy, the genotype coding for N‐acetyltransferase was identified by means of the polymerase chain reaction followed by analysis of restriction fragment length polymorphism. Nucleotide transitions in the following positions were investigated: 481 C⇒T, 590 G⇒A, 803 A⇒G, and 857 G⇒A, which enabled the identification of six genotypes, including the wild‐type (wt) allele, and 16 genotypes, including mutated alleles (homozygotic and herterozygotic). The statistical analysis showed significant differences in the distribution of the frequency of the occurrence of mutated alleles and genotypes between the two groups of children. In 51 children (91%) with allergy, genotypes that encode acetylation defect were found; genotypes that code for normal N‐acetyltransferase were observed in only five allergic children (9%). In the control group the distribution of genotypes coding for normal and defective N‐acetyltransferase activity is 38% and 62%, respectively. Thus study enabled the conclusion that the slow acetylation genotype is a genetic marker of predisposition to atopy.

The arylamine N-acetyltransferase (NAT2) polymorphism and the risk of adverse reactions to co-trimoxazole in children

AbstractObjective: The evaluation of the importance of the genetically determined acetylation defect for the development of adverse reactions to co-trimoxazole in children. Methods: The study comprised 48 children aged 3 months to 3 years, who were being treated for interstitial pneumonia with co-trimoxazole. During the treatment, daily clinical examinations and biochemical tests to monitor the functions in various organs enabled us to detect adverse reactions to the drug. The therapy was continued or discontinued according to the results of these examinations. In all children we identified the genotype coding for N-acetyltransferase (NAT2). For this purpose, DNA was isolated from peripheral blood. Polymerase chain reaction (PCR) was then carried out, followed by restriction mapping with the KpnI, DdeI, TaqI, and BamHI endonucleases in order to identify the four mutations at the NAT2 gene locus: 481C → T; 803A → G; 590G → A and 857G → A, respectively. Results: In 29 children (60%) various adverse effects occurred and in 19 children (40%) no adverse reactions to treatment occurred. We found statistically significant differences in the occurrence of the identified wt alleles, and alleles with 590A and 857A mutations between the two groups of children studied. In the group with adverse effects, 87% of children had genotype coding for slow acetylation and only 13% had genotypes containing the wt allele. In the group without adverse effects the results were reversed: 89% had genotypes with the wt allele, and only 2 children (10%) were found to have the homozygotic mutation (slow acetylation). Conclusion: The results show that the occurrence of adverse effects from co-trimoxazole is closely connected with the genotype coding for slow acetylation.

The chemical synthesis of the RP and SP diastereomers of thymidyl(3′-5′)thymidyl 0,0-phosphorothioate

The synthesis and separation of diastereomere of protected thymidyl(3′–5′)thymidyl 0,0-phosphoranilidate (4) allowed to obtain in the stereospecific manner title compounds 5, whose absolute configuration at P atom was assigned enzymatically. TP(S)T diastereomers (5) were obtained independently via “phosphite” procedure.

Comparison of acetylation phenotype with genotype coding for N-acetyltransferase (NAT2) in children

The present study focused on evaluation of the extent to which genotype coding for N-acetyltransferase agrees with acetylation phenotype in children at various ages. In 82 Caucasian children aged from 1 mo to 17 y (57 boys and 25 girls) and including 37 infants, the acetylation phenotype was evaluated from the urinary metabolic ratio of 5-acetylamino-6-formylamino-3-methyluracil (AFMU) to 1-methylxanthine (1X) after oral administration of caffeine. At the same time, by use of PCR and restriction analysis of amplified fragments of the N-acetyltransferase gene, four nucleotide transitions were identified: 481C→T (KpnI), 590 G→A (TaqI), 803 A→G (DdeI), and 857 G→A (BamHI). The wild-type allele was detected in 27 (33%) children, and the slow acetylation genotype was found in 55 (67%) children. The results of the study show that the metabolic ratio AFMU/1X could be calculated only in 72 children, because in 10 (14%) infants <20 wk of age, AFMU was not detected. Determination of the relation between the acetylation phenotype and genotype revealed that 18 children (23%) containing at least one wild-type allele had AFMU/1X <0.4 (slow acetylation activity) and 7 (8%) of genotypically slow acetylators presented high metabolic ratio (high acetylation activity). We concluded that the disagreement between the acetylation phenotype and genotype is more often found in the group of children characterized by low AFMU/1X and that in small children only N-acetyltransferase genotype studies enable the detection of genetic acetylation defect.

REMOTE CONTROL OF DIASTEREOSELECTIVITY OF DN-ASE II AND ENDONUCLEASE ECO RI TOWARDS PHOSPHOROTHIOATE ANALOGUES OF OLIGONUCLEOTIDES

Abstract (Rp)-Thymidyl 3′-(4-nitrophenyl phosphorothioate) (TP(S)NP) is converted in the presence of DN-ase II into (Sp,Rp)-thymidyl (3′–5′)-thymidyl phosphorothioate 3′-(4-nitrophenyl phosphorothioate) (TP(S)TP(S)NP), while (Sp)-TP(S)NP under analogous conditions gives (Rp,Sp)-TP(S)TP(S)NP, but as a minor product only. The preponderant product was recognized as (Sp,Rp)-thymidyl (3′–5′) thymidyl phosphorothioate 5′–(4-nitrophenyl phosphorothioate) (NPP(S)TP(S)T). All phosphorothioyl transfer reactions occur with retention of configuration at phosphorus atoms, which speaks for a double-displacement process and involvement of phosphorothioylated enzymes as reactive intermediates. Nucleolytic activity of DN-ase II, characteristic for this enzyme with respect to natural oligonucleotides, is not observed if TP(S)NP are used as the substrates. However, the transferase activity is even extended for the (4-nitrophenyl phosphorothioate)-shift from 3′ to 5′-position of dinucleotide, but for diastereoisomer (Rp,Sp)-...