Polymorphism of the human alpha1 immunoglobulin gene 3' enhancer hs1,2 and its relation to gene expression

Abstract

We studied the hs1,2 transcriptional enhancer identified downstream of the human alpha1 gene of the immunoglobulin H (IgH) locus, for which two different allelic configurations (a and b) were previously reported by Southern blotting. By using a polymerase chain reaction (PCR) method we amplified minisatellites within the hs1,2 core enhancer, with variable numbers of tandem repeats (VNTR) defining three 'PCR alleles' alpha1A, alpha1B and alpha1C (including one, two and three repeats, respectively). Five different alpha1 h1,2 genotypes were encountered in a population of 513 donors, representing 13.8, 34.5, 49.7, 1.3 and 0.6% for the AA, BB, AB, AC and BC genotypes, respectively. Luciferase assays showed that increasing the number of minisatellites increased the transcriptional strength of the alpha1 hs1,2 enhancer. Simultaneous determination of Southern blot alleles and VNTR alleles only showed a partial linkage between both types of polymorphism, altogether defining at least six different allelic forms of the 3'alpha1 region. In conclusion, the present study further demonstrates the genetic instability of the 3'alpha region, for which multiple alleles have been generated through inversions and internal deletions and/or duplications. This study also strengthens the hypothesis that the polymorphism at the IgH 3' regulatory region of the alpha1 gene could play a role in the outcome of diseases involving immunoglobulin secretion.

Figure 1

Map of the IgH locus showing the hs1,2 enhancer downstream the α1 genes (a) and PCR product amplification (b). (a) Orientation and numbers of tandem repeats downstream the α1 genes: α1A allele (one repeat), α1B allele (two repeats) and α1C allele (three repeats). (b) Patterns of PCR amplification: AA genotype (α1A allele fragment); AB genotype (α1A and α1B allele fragments), BB genotype (α1B allele fragments), AC genotype (α1A and α1C allele fragments) and BC genotype (α1B and α1C allele fragments).

Figure 2

(a) Hybridization of HindIII-restricted human DNA from individuals with the α membrane exon probe showing either homozygocity (aa and bb) or heterozygocity (ab) for the α1 gene Southern blot alleles. Sizes are indicated in kb. (b) Schematization of the Southern blot α1 and α2 alleles and their concordance with PCR alleles. H: HindIII sites. The total enhancer hs1,2 is depicted by the filled circle. The number of repeats in the hs1,2 enhancer is depicted by rectangles.

Figure 3

Comparison of sequences of hs1,2 variants.[n] refers to the appropriate reference. Potential transcription factor binding sites are indicated in the invariant region (see and 15). The EMBL accession numbers were AJ298015, AJ298016, AJ298017 and AJ298018 for α1A, α1B, α1C and α1C2 alleles, respectively.

Figure 4

Transcriptional strength of the three PCR alleles of the α1 h1,2 enhancer and the α2 h1,2 allele in a luciferase gene reporter assay. (a) Construction: hs1,2 elements were cloned as BamHI–XhoI fragments in BamHI–SalI sites of the pGL3-pVH vector. (b) Estimation of luciferase activity for the four fragments. For each assay, luciferase light units were divided by β galactosidase light units to normalise the cell transfection efficiency. Results are reported as fold-activation as compared to the pVH promoter basal activity. Mean ±SEM of four independent experiments with six assays in parallel. *P = 0·005, **P = 0·024 and ***P = 0·019 as compared to α1A, α1B and α1C, respectively (t-test for paired samples).