FAS GENE POLYMORPHISMS ARE NOT ASSOCIATED WITH SYSTEMIC LUPUS ERYTHEMATOSUS , MULTIPLE SCLEROSIS AND HIV INFECTION

ISABELLA CASCINO*, CLARA BALLERINP, SANDRA AUDINO~, GIANNI ROMBOLA', LUCA MASSACESP, GUAL TIERO COLOMBO' , RAFFAELLA SCORZA SMERALDI', SANDRA D' ALFONSO', PATRICIA MOMIGLIANO RICHIARDF, ROBERTO TOSl'" AND GIOVINA RUBERTI* "Deparlll/enr of ImmunoiJiology, Ill.l'Iitute of Cell Biology, NaTional Research Council, R(I/na, lui/y "Deparlment of Neurologica l and Psychiatrica l Sciences, University of Firenze, Italy ' Service of ImmunogeneTics, Careggi Ulliversitr Hospital, Firen ze, Iwly 'In stitute of Internal Medicine, Infectious Diseases and Imll1unopathology, Uni ve rsity of Milano IRCCS Ospedale Magg iore of Milo no 'D epartmenT of Medical Sciences, University of Torino, Novara

polymorph isms have been used (Fisher et af .. 1995) to demonstrate the inheritance of Fas mutations in children with a rare human autoimmune Iymphoprofilerative syndrome (ALPS).
We report a case-control association study of the above Fas polymorphism with the following three diseases: Multiple Sclerosis (MS), Systemic Lupus Erythematosus (SLE), and Human Immunodeficiency Virus (HIY) infection .
MS is considered as an autoimmune disease, but the underlying mechanisms of tissue injury and cell death are poorly understood.Possible mechanisms of cytotoxicity in human MS brain and the animal model , experimental allergic encephalomyelitis (EAE), have been studied and several candidate death effectors have been proposed (Martin et af. , 1995).Recently Fas antigen expression has been investigated in MS lesions and it has been suggested that Fas-mediated signalling might contribute to immuno-mediated injury in MS (D 'Souza et af., 1996;Dowling et af., 1996).
The identification of Fas and FasL mutations respectively in fpr and gfd mice which accelerate lupus-like autoimmunity (Nagata and Goldstein, 1995) and in children with ALPS (Fisher et af., 1995;Rieux-Laucat et af. , 1995) suggest the possibility that genes involved in apoptosis may playa role in the genetic susceptibility to SLE.
Apoptosis of T lymphocytes has been proposed as a mechanism that plays an important role in the pathogenesis of HIY disease (Ameisen, 1992;Gougeon and Montagnier, 1993).An increased susceptibility to Fas-mediated apoptosis has been observed in HIY+ individuals (Baumler et aI. , 1996;Katsikis et af., 1995;Li et af., 1995 ;Westendorp et af., 1995;Badley et af., 1996) suggesting that the Fas/FasL system plays an important role in the disease progression.
Three groups of patients, affected by MS, SLE and HIY diseases were typed for Fas polymorphisms.DNA samples were obtained from 62 MS (according to the Poser diagnostic criteria, Poseretal., 1983); 98 SLE, 283 HIY + patients as well as from III ethnically matched healthy controls.They were amplified by PCR and typed with digoxigenin-Iabelled (Dig Lurrtinescent Detection Kit for Nucleic Acids; Boehringer, Mannheim, Germany) or 32p labelled oligonucleotide probes for the polymorphic sites in exon 3 nucleotide position 416 (oligonucleotides: AGGGACTGCACA(orG)GTCAATGGG) and in exon 7 nucleotide position 836 (oligonucleotides: GAA TCTCCAACC( orT)TT AAA TCCT)(nucleotide numbering is according to Itoh et af., 1991).The primers for PCR were GR49: CTTTTTTCCTTGGGCAGGTG and GR50: AGACTCTT ACCATGTCCTTCA for the position 416 polymorphism and GRI9: AACATGCAGAAAGCACAGAA and GR52: ATGGAGCAAGACTCCA TC for the position 836 polymorphism.
The three observed haplotypes are indicated as Fas-A, -B and -C based on the combinations of variations involving the third position of codon 416 (ACA to ACG) and codon 836 (ACC to ACT) .(Table I).Frequencies of patients and controls carrying the different haplotypes (Table I) and the corresponding genotypes (Table 2) were compared by Chi-square test including Yates correction.
Genotypes could be unequivocally assigned even without family information because most individuals were homozygotes at one of the two positions.Out of the 554 individuals typed only five were double heterozygotes with ambiguous genotypes.They were compatible either with a B/C genotype or with the presence of a "D" haplotype (position 416 G, position 836 T) which had not been detected in the normal population.Due to this uncertainty, since their families were not available, those cases (two SLE and three HIY + patients) were excluded from the analysis.Within each group of patients and controls the genotype distribution did not deviate significantly from the Hardy-Weinberg equilibrium.Both haplotype and genotype frequencies were similar in patients and controls.No significant differences were observed when the homosexual and drug user HIV+ patients were considered separately.SLE is the most clinically and serologically diverse of the autoimmune connective tissue diseases, thus the Fas haplotype and genotype frequencies were also analyzed in relationship to patients clinical and immunologic findings.However no significant differences were observed in those subgroups (data not shown).
The participation of the Fas/FasL system as such in the pathogenetic mechanisms of the diseases is not touched by the present study, which pertain to the relevance of the Fas gene polymorphisms.More precisely the data show that Fas polymorphic functional variations that may be transmitted in linkage disequilibrium with the tested intragenic "silent" variations either do not exist or do not confer a differential susceptibility to the three diseases .Another possibility is that some infrequent Fas mutations may be responsible for particular disease subsets but do not show up in a general association study.The power of detecting a true association by linkage disequilibrium is known to depend heavily on the similarity of the population frequency of the so-called aetiologic mutation and that of the marker with which it associates (Muller-Myhsok and Abel, 1997).Thus the effect of an infrequent disease-associated variation would be detected in our study only if it happened to be associated with the rarest allele B. but not with the two frequent A and C alleles.Consequently, the data we present should not discourage the search for rare Fas mutations in particular disease subgroups where there may be reason to suspect more specifically a Fas involvement.
Another possibility to be tested is the association with polymorphisms of genes that regulate Fas expression such as lymphokines, e.g.IL-l ~ and TNFa.In addition it might be of interest to search for polymorphic variations in the FasL gene as well as in view of a test of possible genetic association.In fact , increased expression of FasL has recently been found in MS lesions (D 'Souza et al., 1996) and a FasL gene mutation has been related to a Iymphoproliferative disease (Wu et a!., 1996).

Table I .
Fas polymoprhisms in MS, SLE, HIV+ and controls*

Table 2 .
Distribution of Fas genotypes in MS, SLE, HIV + and controls *The B/C genotype was not unequivocally attributable (see text).