RESULTADOS PARA EL OBJETIVO GENERAL

The RFLP studies with genomic probes for human pi,4-GalTase showed that, like many other gene loci, this gene locus is also polymorphic in man. The use of the enzyme Sad revealed that the pl,4-GalTase locus exists as different polymorphic alleles, as shown by the homozygosity and heterozygosity of the 11-kb and/or 8-kb bands. Further investigation showed that the variation lies outside the coding region, in intron 2 of the gene. Whether this region of the locus is involved in regulation of gene expression remains to be determined. The Sad polymorphism was found both in normal individuals and in patients with RA, with no significant difference in frequency between these two groups. The enzyme EcoRI revealed an additional polymorphism with some individuals missing a 5.5-kb band and possessing a faint band of 5.0-kb size, although this weak band was only visible in lanes containing higher amounts of DNA. The other two enzymes, Hindin and Eg/n, did not detect any polymorphism at all.

In another study (Dalziel and Axford 1994), possible polymorphism in the pl,4-GalTase gene was investigated using 10 different restriction enzymes, in combination with two probes; a 1.28-kb cDNA probe specific for the coding region and a 0.2-kb probe specific for the 5' untranslated region. Only one restriction enzyme, Psfl, was reported to detect a polymorphism in one out of ten controls and one out of ten patients with RA. Therefore, although the pi,4-GalTase gene is present in different allelic forms in human, none of the reported polymorphisms are specifically associated with RA.

Possible structural alterations in the human p58°^^ protein kinase gene was investigated previously by Delves and colleagues. It was reported that the gross structure of the p58°^^ protein kinase gene locus remains intact in patients with RA. This gene locus is also polymorphic both in normal individuals and in patients with RA, although no polymorphisms unique to RA patients were observed (Delves eta! 1990).

In murine studies, the (31,4-GalTase gene was found to have a similar structure in each of 11 different inbred strains using a cDNA probe containing 1.6 kb of the murine (31,4-GalTase locus. MRL Ipr/lpr mice, which possess high levels of agalactosyl IgG in serum, showed no gross structural alterations of the (31,4-GalTase gene. The p58°^^ gene locus, however, showed an allelic variation in two autoimmune-prone strains of mice. Although apparently encoding a full length mRNA, the overall structure of the p58°^^ protein kinase gene locus appeared to be very different in NOD and SJL mice compared to the other strains tested. It is not known if these differences in any way affect putative regulatory regions of the gene. NOD strain mice spontaneously develop both a Sjogrens-like syndrome and a polyendocrine autoimmunity which includes pancreatic and thyroid autoimmune disease (Kikutani et al 1992, Bernard et al 1992). SJL strain mice, whilst not developing spontaneous autoimmune disease, are particularly susceptible to induced autoimmune diseases, possibly due to defective suppression of autoantibody responses (Elliot and Cooke 1992). It is also of note that SJL mice develop a non-autoimmune insulin-dependent diabetes mellitus after inoculation with encephalomyocarditis EMC-D virus or reovirus type 1 (Bae et al 1989, Onodera et al 1978). Although our data indicates that the different structure of p58°^^ protein kinase gene does not abolish IgG galactosylation in NOD and SJL mice, any alteration in the regulation of the p58°^^ gene could have other implications for cellular dysfunction in these autoimmune-prone strains. In addition to pi,4-GalTase activation, this protein kinase is able to phosphorylate histone HI and casein, suggesting a range of substrate specificities, and its over-expression leads to a prolonged late telophase/early G1 phase of the cell cycle (Bunnell et al 1990). The p58°^^ gene locus in the non-diabetic but genetically related CTS and NON strains (Kikutani and Makino 1992) shows the same structure to that found in the majority of strains tested. Therefore, even if it turns out to be of no functional significance, the polymorphism of this gene locus in SJL and

NOD mice provides an additional marker for genetic analysis of these autoimmune-prone strains.

Rheumatoid arthritis and most other autoimmune diseases have multifactorial and/or polygenic aetiology. Unravelling the molecular genetics of such complex multifactorial diseases is much more difficult than for single gene disorders. Identification of disease associations in the general population, linkage analysis in affected families, and the study of animal models are usually the approaches taken in trying to understand multifactorial disorders. Studies on the structural analysis of both the pi,4-GalTase (present study and Dalziel et al 1994) and p58°^^ (Delves at al 1990) genes in human and also in an animal model of RA, MRL Ipr/lpr (present study) have not found any associations between p1,4-GalTase and p58^^^ genetic polymorphisms and RA. Sequencing the polymorphic region(s) of the p1,4-GalTase and the p58®^^ protein kinase gene loci will reveal the structural basis for the polymorphisms and show if the RFLPs affect the binding sites of the enzyme or the regulatory regions of the gene. More detailed analysis of the human Sad polymorphism in the present study showed that this RFLP did not lie in the coding region of the gene. Whether this part of the second intron of pi,4- GalTase is involved in the regulation of gene expression remains to be shown.

In conclusion, the results of the RFLP analyses presented here, taken together with the fact that normal galactosylation is re-established during disease remission in patients with RA, would argue against a defective allelic variant of either pi,4-GalTase or the regulatory p58°^^ protein kinase being associated with the reduced IgG galactosylation seen in rheumatoid arthritis.