Identification of three new single nucleotide polymorphisms in the human tumor necrosis factor-alpha gene promoter

Abstract

We have identified three new human tumor necrosis factor-alpha (TNF-alpha) promoter polymorphisms with single nucleotide (nt) substitutions at -862, -856, and -574 nt relative to the TNF-alpha transcription start site. The -862 and -856 nt TNF-alpha promoter polymorphisms occur with high frequency in Caucasian and Cambodian individuals and are each non-randomly associated with three extended HLA haplotypes. This study, in which 61 independent TNF-alpha promoters were analyzed spanning from -977 to +93 nt relative to the TNF-alpha mRNA cap site, establishes a new canonical TNF-alpha promoter sequence. Furthermore, we show that none of the three novel polymorphisms at -862, -856 and -574 nt or polymorphisms previously described at positions -238, -308 and +70 have an effect upon TNF-alpha gene expression in activated lymphocytes. Thus, these TNF-alpha promoter polymorphisms likely serve as markers for neighboring genes encoding HLA or other undefined molecules in the MHC that may influence disease susceptibility.

Fig. 1. The human TNF-α gene promoter from−977 to +93 nt relative to the TNF-α transcription start site

The three new polymorphisms detected at −862, −856, and −574 nt; the previously detected Polymorphisms, marked by an asterisk (*), at −237, −243, −307, and −375 nt; and the +70 polymorphic addition are noted in bold. The more uncommon polymorphic allele is shown below. The start site of transcription is at +1. Regulatory motifs in the human TNF-α promoter which have been shown to have a functional role in TNF-α gene regulation (–16) are underlined.

Fig. 2. Testing the functional effect of the−862, −856/+70, −574, −307, and −237 polymorphisms upon TNF-α gene expression in A20 B cells (A) and Ar-5 T cells (B)

Cells were transfected with the wild type (′977 TNF-α promoter) CAT (WT) and polymorphic TNF-α promoter CAT plasmids tarrying the variants noted in the figure. Twenty-four hours later the cells were mock stimulated (Uninduced) or induced with PMA plus ionomycin (P+I) as descibed in Material and methods. Quantification of the conversion of ¹⁴C-chloramphenicol to its acetylated forms was obtained using a Betagen Betascope (Waltham, MA) and in A and B three independent transfections were averaged.

Fig. 3. Testing the functional effect of the−862, −856/+70, −307, and −237 polymorphisms upon TNF-α gene expression in Raji B cells. An autoradiogram of a CAT assay of Raji cells transfected with the wild type (−977 TNF-α promoter) CAT (WT) and polymorphic TNF-α promoter CAT plasmids carrying the variants noted in the figure

Cells were transfected using lipofectamine as described in the methods section Twenty-four hours later the cells were mock stimulated (UN) or induced with PMA as descibed in Material and methods. The figure shows a representative experiment. Raji cells were co-transfected with a CMV-βgal plasmid (a gift from D. Thanos) and, prior to performing CAT assays, extracts were normalized to β-gal activity.