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Technical Briefs |
1 Service d’Hématologie-Oncologie, Centre de Recherche, Hôpital Sainte-Justine, Montréal (Québec), H3T 1C5 Canada
2 Département de Pédiatrie, Université de Montréal, Montreal (Quebec), H3T 1C5 Canada
aaddress correspondence to this author at: Centre de Recherche, Hôpital Sainte-Justine, 3175 Côte Ste-Catherine, Montréal (Québec), H3T 1C5 Canada; fax 514-345-4731, e-mail maja.krajinovic{at}umontreal.ca
Glucocorticoids (GCs) have a major antiproliferative effect, which has led to the use of their synthetic homologs for immunosuppression, treatment of inflammation, and induction of cytotoxicity (1)(2). GCs exert their effect by binding to an intracellular GC receptor (GR), forming a complex that translocates to the nucleus, where GCs then regulate the expression of target genes interacting with promoter GC-responsive elements (1). The different GR forms, resulting from GR gene variability, can affect the regulation of many biological functions, such as hypothalamic-pituitary-adrenal axis regulation and GC responsiveness, thereby underlying susceptibility to many diseases. Indeed, GR mutations have been associated with altered cardiovascular function, metabolic disturbances, and hematologic malignancies (3)(4)(5). Likewise, functional GR variability might affect the therapeutic response to corticosteroid drugs (5). Identification of different GR gene variants may thus be helpful in assessing the role of the GR gene in disease susceptibility or in adjudging predisposition to corticosteroid-associated adverse drug reactions.
Several polymorphisms of the GR gene, which might have an impact on GC sensitivity, have been reported (6)(7)(8). Among these, the BclI polymorphism was identified by Southern blotting using human GR cDNA-specific probes (9) that identified two alleles with fragment lengths of 4.5 and 2.3 kb. Several clinical investigations have subsequently suggested that this GR polymorphism is linked to altered GR function (6)(10)(11)(12)(13)(14)(15). An association between the BclI polymorphism and changes in tissue-specific corticosteroid sensitivity, as well as with poor feedback regulation of the hypothalamic-pituitary-adrenal axis, has been reported (6)(10). This was further documented by association of the BclI polymorphism with abdominal obesity (11)(12), insulin resistance (6)(13), and development of an atherogenic profile (6)(14). Similarly, the larger allele of BclI is more frequent in a group of individuals genetically predisposed to develop hypertension (15). The molecular identity of this polymorphism, however, is still unclear, and its analysis has been based on the laborious and time-consuming Southern blot approach, which requires large quantities of DNA and is difficult to apply to large-scale genotyping (6)(10)(11)(12)(13)(14)(15).
Here we present the characterization of the BclI polymorphism, as well as the application of two simple genotyping assays for its detection: PCR with restriction fragment length polymorphism (RFLP) analysis and allele-specific oligonucleotide (ASO) hybridization. Using these approaches, we assessed the frequency of this polymorphism in populations of different origin.
The BclI site was suggested to be situated in either the first or second intron of the GR gene (6). To search for BclI sites in these gene segments, we used the genomic sequence derived from human chromosome 5 contig (GenBank accession no. NT_029289), on which the exon/intron boundaries were positioned according to the GR mRNA sequence information (GenBank accession no. NM_000176.1) and BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/). The genomic sequence corresponding to the first two introns was analyzed for BclI restriction sites using WebCutter 2.0 software (http://www.firstmarket.com/cutter/cut2.html). We found three BclI sites, one in intron 1 and two in intron 2, whose relative positions to the exon 2/intron 2 boundary are indicated in Fig. 1A
. Only the polymorphic BclI site in intron 2, 647 bp from exon/intron junction, produced the fragment sizes corresponding to those obtained by Southern blot experiments (i.e., fragments of 2.2 kb generated in the presence and 3.9 kb in the absence of the BclI site; Fig. 1A
). Although the size of the larger allele would be slightly different from that reported previously (9), no other BclI site produced a fragment of similar size.
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Using this information, we designed primers flanking the BclI site at position +647 (forward, 5'-AAATTGAAGCTTAACAATTTTGGC-3'; reverse, 5'-GCAGTGAACAGTGTACCAGACC-3') and amplified genomic DNA samples from the Institutional DNA bank of healthy volunteers recruited for the study of human genomic sequences variability. (The study was approved by the Institutional Ethical Committee, and informed consent was obtained from all participating individuals.) The PCR 206-bp product was amplified in 20 µL containing 20 ng of genomic DNA, 0.5 µM each amplimer, 100 µM deoxynucleotide triphosphates, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, and 0.5 U of Taq polymerase (Platinum; Invitrogen) with the following conditions: initial denaturation at 94 °C for 5 min; 40 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, and extension at 72 °C for 45 s; followed by a final extension at 72 °C for 7 min.
To confirm the presence of the BclI polymorphism, we digested 10 µL of the PCR products with 4 U of BclI (New England BioLabs) for 6 h at 50 °C and separated the resulting digested fragments on a 3% agarose gel. Digestion of the PCR product gave the following predicted fragment sizes: 90 and 116 bp in the case of homozygotes for the smaller allele, an additional band of 206 bp for heterozygous individuals, and a single band of 206 bp for larger allele homozygotes (Fig. 1B
). To characterize the underlying base substitution of the BclI polymorphism, we directly sequenced the PCR product using the Thermo SequenaseTM Radiolabeled Terminator Cycle Sequencing Kit (USB Corporation) according to the manufacturer’s instructions. We found a G-to-C transition in the second intron, 646 bp from the exon 2/intron 2 junction (Fig. 1C
), that abolished the BclI restriction site (TGATCA to TCATCA, where the underlined bases indicate the transition), thus producing the larger allele. Similar efforts to replace Southern blot analysis by PCR-based methods in analysis of different polymorphisms have been reported in the literature (16)(17).
The identification of the underlying base substitution allowed us to apply a PCR-ASO genotyping assay that, in addition to PCR-RFLP, has been widely used to analyze gene mutations and variations in numerous genes (18). We previously reported the successful use of this approach for large-scale genotyping (19). For this method, PCR products were denatured in 0.33 mol/L NaOH and 16.5 mmol/L EDTA in a total volume of 200 µL and subsequently transferred, in duplicate, to a HYBOND-n+ membrane (Amersham Pharmacia Biotech) and cross-linked to the membrane by use of ultraviolet light. Blots were prehybridized for 30 min at 37 °C in 10 mL of a solution containing 1x saline–sodium phosphate–EDTA (SSPE; 150 mmol/L NaCl, 10 mmol/L NaH2PO4, 1.1 mmol/L EDTA, pH 7.4), 0.75 mol/L NaCl, 70 mmol/L Tris-HCl, pH 7.4), 10 g/L sodium dodecyl sulfate, and 200 mg/L heparin. ASO probes (50 pmoles) were 5'-labeled using [
-32P]ATP (6000 Ci/mmol) and T4 kinase (Life Technologies) to a specific activity of 1–3 x 106 cpm/pmol. Hybridization with the 5 pmol of ASO-specific probe was carried out for 30 min at 42 °C in an excess (10x) of the nonlabeled probe for the other variant allele of the same polymorphism. The membranes were then washed with 2x SSPE containing 1 g/L sodium dodecyl sulfate for 10 min at room temperature and exposed overnight at -80 °C with intensifying screens. Identical twin membranes were hybridized with the allelic probes specific for the G (gag att Gat cag cag) or C (gag att Cat cag cag) variant of the BclI polymorphism and read in parallel. DNA samples of known genotypes served as controls. Representative examples of genotypes obtained by the ASO approach are presented in Fig. 1D
.
Using these two genotyping assays, we analyzed DNA samples of individuals from several populations, including Africans, Asians (both Southeast and East Asia), Amerindians, Middle Easterners, and Europeans. Both methods generated concordant results, thus validating the usefulness of both assays. The number of individuals with different genotypes, as well as the frequency of allele C in the tested populations, is given in Table 1
; the highest frequency of allele C was observed in Asians [mean (SD), 32.8 (8.7)%], and the lowest was in Amerindians [15.2 (6.2)%]. The frequency observed here for Europeans was similar to the one reported for the larger allele in Sweden (6).
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We conclude that characterization of the BclI polymorphism, together with the availability of PCR-based genotyping approaches, could allow fast screening of this polymorphism for monitoring of both disease susceptibility and therapeutic response variability. Further studies clarifying the reason for an association of this polymorphism with altered GR function could provide additional insight into the variability of the GR locus and the role of the BclI polymorphism.
Acknowledgments
This work was supported by the Cancer Research Society, Inc. and the Centre de Recherche, Hôpital Ste-Justine. I.F. has a studentship and M.K. and D.S. have scholarships from the Fonds de la Recherche en Santé du Québec. We are grateful to Alan Lovell for critical reading of the manuscript.
References
and ß mRNA destabilize mRNA and decrease receptor protein expression. Steroids 2002;67:627-636.[CrossRef][ISI][Medline]
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