Polymorphism within the telomeric MHC and expression of rheumatoid arthritis candidate genes

Several recent studies have indicated a second susceptibility locus for rheumatoid arthritis (RA) in the telomeric MHC close to the tumour necrosis factor locus, a region containing a number of genes of unknown function including HLA-B associated transcripts 1 and 2 (BAT1 and BAT2) and lymphotoxin alpha (LTA). Several single-nucleotide polymorphisms have been described in this region; however, the influence of each on susceptibility is not known. In this study we sought to examine the association of individual single-nucleotide polymorphisms with expression of each mRNA transcript in response to an inflammatory stimulus.

Peripheral blood mononuclear cells were collected from healthy blood donors and adherent cells were allowed to differentiate into macrophages by culture in the presence of 5% serum for 7 days. Cells were then stimulated with 100 ng/ml lipopolysaccharide and harvested at 4-hour and 18-hour timepoints. Total RNA was prepared using standard methods and reverse transcription performed with ImpromII™ (Promega, Madison, WI, USA). The resulting cDNA was used as a template for real-time PCR using gene-specific primers for each gene. Each PCR product was detected with the double-stranded DNA-binding dye SYBR Green^® I (Applied Biosystems, Foster City, CA, USA) and the ABI 7900HT sequence detection system (Applied Biosystems). Quantitation was performed using standard curves generated from plasmid DNA containing each cloned PCR product and 18S was used as a loading control. Levels of mRNA transcripts were expressed in relative units (RU). Genotyping was performed for the BAT1 -991 (1751673), BAT2 (1688185), LTA (1720807) and TNF -308 (1718089) polymorphisms using TaqMan allelic discrimination assays. Expression levels of each mRNA transcript were compared between individuals of genotype 1.1, 1.2 and 2.2 using Kruskal–Wallis tests.

Expression of BAT1 mRNA transcripts was significantly less in BAT1 -991 2.2 individuals (n = 6) compared with both 1.1 (n = 6) and 1.2 (n = 5) (median levels 0.22 RU compared with 0.76 RU and 1.1 RU, respectively) (P = 0.02). Expression of BAT2 transcripts was lower in 1.1 individuals (n = 9) compared with both 1.2 (n = 8) and 2.2 (n = 4) genotypes (median levels 0.43 RU compared with 0.77 RU and 0.82 RU, respectively) (P = not significant). For LTA expression, individuals of genotype 2.2 (n = 4) produced the highest levels, those of genotype 1.1 (n = 6) the lowest, with those of 1.2 genotype (n = 6) intermediate (median levels 6.1 RU compared with 0.81 RU and 2.1 RU, respectively) (P = 0.02). Expression of all three transcripts had fallen to low levels by the 18-hour timepoint and no differences were apparent between individuals of different genotype.

Polymorphism in the BAT1 -991, BAT2 and LTA genes are associated with levels of each mRNA transcript produced in response to an inflammatory stimulus. This suggests the possibility that they may be able to influence susceptibility to RA, and that the protein products they encode may play a role in inflammatory signalling. We are currently extending this genotype–phenotype analysis to the other members of this gene cluster particularly 1C7, LST1 and AIF1.

Affiliations

1. Division of Genomic Medicine, University of Sheffield, UK
   • D Mewar
   • , I Marinou
   • , JM Timms
   • , R Kilding
   •  & AG Wilson

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