Molecular detection of the G(-248)A BAX promoter nucleotide change in B cell chronic lymphocytic leukaemia

Abstract

Background: A novel single nucleotide polymorphism (SNP), G(-248)A, in the 5' untranslated region of the BAX promoter and its association with reduced protein expression, progression beyond Rai stage 0, and treatment resistance in chronic lymphocytic leukaemia (CLL) has been reported previously.

Aim: To develop a restriction enzyme analysis (REA) based method for routine detection of BAX promoter SNP in a clinical laboratory.

Methods: The BAX promoter was analysed in duplicate by REA and sequencing in 90 samples (from 45 patients with CLL, 43 controls, and two cell lines). The promoter region was amplified, digested with restriction endonucleases (Aci I and Tau I), and separated by gel electrophoresis.

Results: After digestion, the normal GG genotype samples produced three distinct bands. The homozygous AA replacement abolished the cleavage site, resulting in a single band. Although the heterozygous samples produced three bands, the two smaller visible bands were reduced in intensity (> 50%). The test characteristics of Aci I REA were better than those of Tau I REA, in terms of sensitivity (100% v 77.8%), specificity (98.6% v 92.3%), positive predictive value (95.03% v 87.4%), and negative predictive value (100% v 85.83%).

Conclusions: REA using Aci I is a highly sensitive and specific method for detecting the BAX G(-248)A SNP in CLL.

Figure 1

(A) Aci I and (B) Tau I restriction enzyme maps for the BAX promoter region.

Figure 2

Restriction enzyme analysis and parallel direct sequencing of polymerase chain reaction (PCR) products. (A) Aci I and (B) Tau I endonuclease cleavage followed by 10% polyacrylamide gel analysis. M, molecular weight marker, 100 bp DNA ladder (Gibco BRL, Burlington, Ontario, Canada). (A) Lane 1, chronic lymphocytic leukaemia (CLL) sample 1, amplified BAX promoter segment before digestion; lanes 2–5, PCR products digested with Aci I; lane 2, CLL sample 2 with a heterozygous single nucleotide polymorphism (SNP); lanes 3 and 4, CLL samples 3 and 4 with no SNP; lane 5, RL cell line with homozygous SNP. (B) PCR products digested with Tau I; lane 2, CLL sample 2 with a heterozygous SNP; lane 3, CLL sample 3 with no SNP; lane 5, RL cell line with homozygous SNP. (C) Left hand column: direct sequence of the BAX promoter region. The control sample (I) shows no alteration, whereas a CLL sample (II) shows a heterozygous SNP, and the RL cell line (III) has a homozygous SNP. Middle and right hand columns: corresponding samples digested with the Aci I and Tau I enzymes, respectively

Figure 3

Restriction enzyme analysis (with Aci I ) for the G(−248)A single nucleotide polymorphism (SNP) in the BAX gene. M, molecular weight marker (100 bp DNA ladder; Gibco BRL); N, control sample showing three distinct bands, 352 256 bp, and 96 bp; P1, positive control with heterozygous SNP showing the 352 bp (major) and 256 bp (minor) bands and an almost invisible 96 bp band; P2, positive control with a homozygous SNP, which abolishes a restriction enzyme site, resulting in a single 352 bp band; lanes 1–5, chronic lymphocytic leukaemia cases; lanes 1, 4, and 5, heterozygous SNP; lanes 2 and 3, no SNP; lane 6, RL showing the homozygous SNP; lanes 7–12, controls without the G(−248)A SNP.