Characterization of the TNFa microsatellite's reliability, MHC associations and occurrence in two ethnically different SLE populations

The major histocompatibility complex (MHC) encodes a diverse selection of genes, many of which have immunological functions. Several of these genes are polymorphic but are maintained in strong linkage disequilibrium as conserved extended haplotypes. Particular alleles at these loci or particular haplotypes have been found to be associated with increased susceptibility to various diseases. The TNFa microsatellite, with a variable number of dinucleotide repeats, is found 3.5 kb upstream of the tumour necrosis factor loci in the central region of the MHC. This project examined the reliability of the TNFa typing assay adapted for use in this laboratory. The TNFa microsatellite was then used to -- a) further characterize extended haplotypes -- b) investigate possible associations of alleles of TNFa with null alleles of the fourth component of complement (C4) -- c) examine the possibility of disease susceptibility genes for systemic lupus erythematosus (SLE) in the TNFa region in two separate ethnic groups. -- Fourteen TNFa alleles were identified and named in accordance with published literature. Repeat typings of samples and analysis of a large kindred were used to test reliability of results obtained using TNFa microsatellite typing. A high level of consistency was demonstrated. The only errors in typing were in distinguishing heterozygosity with two consecutive alleles from homozygosity of the larger allele. -- The presence of extended haplotypes, i.e. haplotypes sharing alleles at HLA-B,Bf,C4A,C4B and TNFa, was demonstrated. Although several extended haplotypes did exist, the most common extended haplotype in the population studied was HLAB8;TNFa2;BfS;C4AQ0;C4B1. It occurred in 15 of 75 unrelated chromosomes. There were examples of specific haplotypes sharing alleles at all loci except TNFa. There were five separate instances where this occurred in this data set. In the absence of HLA-DR data, the reasons for this are difficult to ascertain but possible explanations include multiple historic recombinations, rare extended haplotypes, and mutations of the original TNFa alleles occurring on the extended haplotypes. -- The possible association of TNFa alleles with null alleles of C4 was investigated. If a strong association could be demonstrated, the TNFa allele could serve as a useful marker of the null alleles of C4 which are sometimes difficult to ascertain. Although TNFa2 occurred frequently with C4AQ0 on the extended haplotype HLA-B8;BfS;C4AQ0;C4B1 known to contain a deleted C4A gene, no other clear association between particular TNFa alleles and C4 null alleles was observed. Similarly, in a population with null alleles identified by densitometry, there was no one TNFa variant occurring in all people with low C4A levels (relative to C4B). None of these "nulls" represented the 30 kb deletion commonly observed in Caucasians carrying the HLA-B8 extended haplotype. -- The frequency of alleles of the TNFa microsatellite in Caucasian and Korean SLE patients was examined and compared to the appropriate ethnically matched controls. Both ethnic groups are known to have MHC associations with SLE, although not necessarily the same associations. We wished to use the TNFa microsatellite as a marker for the central region of the MHC to investigate whether there may be genes in the vicinity of the microsatellite, and in linkage disequilibrium with it, that may influence susceptibility to this disease. Our results demonstrated a significantly increased frequency of the TNFa2 allele in both ethnic groups. Although other genes exist in this region, the tumour necrosis factor loci, located 3.5 kb from this microsatellite, may be good candidate genes for susceptibility to SLE and are worthy of further study