Patients and controls. The study group comprised 43 children (age range 10.2 ± 3.1 years) selected on the basis of ABA, patients from the Institute of Pulmonology and St. Olga’s Hospital in St. Petersburg, Russia. The characteristics of the recruited subjects are summarized in Table 1. The control group consisted of 51 healthy, unrelated subjects from St. Petersburg. Blood spots, collected on filter paper from each patient, were used directly for polymerase chain reaction (PCR), with or without prior DNA extraction.

PCR and PCR-RFLP (restriction fragment length polymorphism) analyses. GSTM1 and GSTT1 gene poly­morphism analyses were carried out, with slight modifica­tions as described elsewhere [10]. Co-amplification of a 192 bp DNA fragment of the CYPA1 gene (A1R: 5'-GAACTGCCACTTCAGCTGTCT-3'; A1F: 5'-GAAAGA CTCCCAGCGGTCA-3') was used throughout the study as a standard positive internal control for precise identification of the GSTM10/0 and GSTT10/0 genotypes. These were identified by the absence of the normal 271 bp DNA fragment typical for the GSTM1 active genotypes (GSTM1+/+ or GSTM1+/0), or a 315 bp DNA fragment typical for the GSTT1 active genotypes (GSTT1+/+ or GSTT1+/0).

A single PCR amplification was run with the following primers (GSTM1 F: 5'-GAACTCCCTGAAAA GCTAAAGC -3'; GSTM1 R: 5'-GTTGGGCTCAAATAT ACGGTGG-3'; and GST T1 F: 5'-TTCCTTACTGGTCC TCACATCTC -3'; GST T1 R: 5'-TCACCGGATCATGG CCAGCA-3'; 15 nM each in 25 mL amplification mixture plus 67 mM Tris-HCl (pH 8.8), 16.6 mM ammonium sulphate, 6.7 mM MgCl₂, 6.7 mM EDTA, 10 mM mercapto­ethanol, 170 mg BSA, 1.0 mM of each dNTP, 1 U of Taq polymerase (Bion, Moscow, Russia). Denaturation (7 minutes at 94°C) was followed by 32 cycles of amplification: 1 minute at 94°C, 1 minute at 53°C, 1 minute 20 seconds at 72°C, and final extension for 7 minutes at 72°C. The products were subjected to electrophoresis on a 7% polyacrylamide gel, stained with ethidium bromide and visualized under UV light.

Three polymorphic alleles of the GSTP1 gene were identified: GSTP1*A (wild type Ile105/Ala114), GSTP1*B (Val105/Ala114), and GSTP1*C (Val105/ Val114) [11]. The Ile105/Val105 gene polymorphism in GSTP1 was examined after PCR amplification through the use of primers to exon 5 (GST p1 F: 5'-CTCTATGGGAA GGACCAGCAGGAG-3' and GST p2 R: 5'-CAAGCCAC CTGAGGGGTAAGG-3'). The PCR conditions were as follows: denaturation (7 minutes at 94°C) was followed by 32 cycles of amplification: 1 minute at 94°C, 1 minute at 60°C, 1 minute 20 seconds at 72°C, and final extension for 7 minutes at 72°C. The resulting 192 bp fragment was digested with BsoMoI1 to identify the A/G transition at nucleotied 1313. The undigested PCR product has the molecular length of 192 bp. The amplification product of the Val/Val type is cut into 80 and 112 bp fragments, respectively.

A PCR-based genotyping test was subsequently devel­oped for the detection of the Ala114/Val114 gene poly­morphism. This test used a reverse PCR primer with a 1 bp mismatch (GST p4 R: 5'-GCCTTCACATAGTCATCCT TGCGC-3' from exon 6, and a forward primer (GST p3 F: 5'-GTTGTGGGGAGCAAGCAGAGG-3') from intron 5. The mismatched primer introduced a BstFN1 site in the Ala114 allele but not in the Val114 allele.