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CD95 polymorphisms are associated with susceptibility to MS in women
Journal of Neuroimmunology 146 (2004) 162 – 170 www.elsevier.com/locate/jneuroim
CD95 polymorphisms are associated with susceptibility to MS in women A population-based study of CD95 and CD95L in MS Orhun H. Kantarci a, David D. Hebrink a, Sara J. Achenbach b, Elizabeth J. Atkinson b, Mariza de Andrade b, Cynthia T. McMurray c, Brian G. Weinshenker a,* a Department of Neurology, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, MN 55905, USA Department of Health Sciences Research, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, MN 55905, USA c Departments of Pharmacology, Biochemistry and Molecular Biology and Molecular Neuroscience Program, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, MN 55905, USA b
Received 15 July 2003; received in revised form 17 August 2003; accepted 1 October 2003
Abstract CD95/CD95L interaction results in activation-induced apoptosis thereby regulating clonal expansion of T cells outside the thymus. Genetic defects in this system result in autoimmune lymphoproliferation in mice and men. CD95-induced cell death may be defective in MS. We studied the association of CD95 and CD95L polymorphisms with MS in 221 unique patients representing 79% ascertainment in Olmsted County, MN, and 442 gender-, age- and ethnicity-matched controls. Being a homozygote for the G allele of CD95 5V( 670)*A ! G SNP ( p = 0.034; OR: 1.59, 95% CI: 1.06 – 2.38) and for the C allele of CD95 E7(74)*C ! T SNP ( p = 0.007; OR: 1.73, 95% CI: 1.17 – 2.56) increased susceptibility to MS exclusively in women. There was strong but incomplete linkage disequilibrium between the two markers ( p < 0.001; D V = 0.546). Homozygosity for 5V( 670)*A or E7(74)*C explained 28% of risk of MS in women but 0% of the risk in men in Olmsted County, MN. Our results agree with the previously published studies and highlight that the association of the polymorphisms is restricted to women with MS. We did not find an association between CD95L and susceptibility to MS nor CD95 or CD95L and age of onset, disease course and disease severity. D 2003 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; CD95; CD95L; Polymorphism; Susceptibility; Gender
1. Introduction Activation-induced apoptosis mediated through expression of CD95 (Fas/APO-1) and CD95-ligand (CD95L) is an important mechanism of regulating clonal expansion of mature T cells outside the thymus (Russell, 1995). In germinal centers, T cell-induced activation of CD95 on B cells also regulates peripheral tolerance of B cells (Elkon and Marshak-Rothstein, 1996). Defects in activation-induced apoptosis arising from naturally occurring mutations in CD95 and CD95L cause systemic autoimmunity and extensive lymphoproliferation resembling systemic lupus
* Corresponding author. Tel.: +1-507-284-4234; fax: +1-507-2664419. E-mail address: [email protected] (B.G. Weinshenker). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.10.002
erythematosus in mice with lymphoproliferation (lpr) and generalized lymphadenopathy (gld) syndromes (Cohen and Eisenberg, 1991; Nagata and Golstein, 1995; Nagata and Suda, 1995). Autoimmunity and lymphoproliferation are more prominent in females than in male lpr mice (Tornwall et al., 1999; Trune and Kempton, 2002). The human counterpart of lpr/gld is autoimmune lymphoproliferative syndrome (ALPS) (Canale and Smith, 1967), which is linked to mutations in CD95 (Fisher et al., 1995; Rieux-Laucat et al., 1995; Drappa et al., 1996) and rarely to mutations in CD95L (Wu et al., 1996) or caspase-10 (Wang et al., 1999). Most of the ALPS mutations are clustered in the intracellular death domain, specifically in exon 9 of CD95 (Rieux-Laucat et al., 2003). Varying degrees of autoimmunity involving multiple organ systems, CD3+/CD4 /CD8 T cell and B cell proliferation resembling lymphoma and hypergammaglobulinemia
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are the hallmarks of ALPS; these manifestations are the results of loss of function in the death domain caused by the mutations. Lymphoma also occurs in the some pedigrees (Fisher et al., 1995; Lim et al., 1998). The variation in the phenotype depends on the type and location of the mutations. Penetrance of individual mutations even within a given pedigree is variable, suggesting the presence of modifier genes (Rieux-Laucat et al., 2003). Hence, polymorphisms of CD95 and CD95L can influence susceptibility to complex autoimmune phenotypes but may not result in the full spectrum of ALPS. In patients with Dianzani’s autoimmune lymphoproliferative disease (DALD), an ALPS-like clinical pattern is present but CD3+/CD4 /CD8 T cells and CD95 or CD95L mutations are absent (Dianzani et al., 1997). Family members of DALD patients have increased frequency of autoimmune diseases inherited paternally, and women, but not men, have an increased frequency of cancer (Ramenghi et al., 2000). DALD patients have defective CD95 function as do those with multiple autoimmune syndrome (MAS) wherein first to second degree relatives have increased frequency of autoimmunity in the absence of ALPS phenotype (Ramenghi et al., 2000). In experimental allergic encephalomyelitis (EAE) induced in lpr and gld mice from C57Bl background, decreased CD95 or CD95L expression results in a limited disease phenotype compared to the wild-type mice (Waldner et al., 1997; Okuda et al., 1998; Sabelko-Downes et al., 1999). Despite histological evidence for EAE, these mice have decreased incidence and severity of symptoms, and their disease is usually limited to a monophasic episode rather than a relapsing – remitting course observed in the wild-type mice (Malipiero et al., 1997). In mice from SJL background, however, deficiency of CD95 is associated with increased severity of EAE and decreased apoptosis of inflammatory cells in the CNS (Suvannavejh et al., 2000). As in ALPS, other genetic factors probably determine the phenotype (Sabelko-Downes et al., 2000). CD95 expression is weak or absent in healthy oligodendroglia but is significantly enhanced in acute or chronic MS lesions (Dowling et al., 1996; D’Souza et al., 1996). Interferon gamma (IFNg) induces apoptotic cell death by upregulating CD95 and CD95L expression in microglia (Badie et al., 1999). Unlike the activated T cells, oligodendroglia do not undergo activation-induced apoptosis (Bonetti et al., 1997) but microglia and infiltrating CD4+ lymphocytes with increased expression of CD95L (D’Souza et al., 1996) may lyse the oligodendroglia expressing CD95 in a bystander fashion (Wang et al., 1996). This is similar to the B-cell apoptosis induced by activated T cells (Elkon and Marshak-Rothstein, 1996). In the CNS, elimination of autoreactive T cells could be through activation-induced apoptosis or via microglia expressing CD95L lysing the T cells expressing CD95 (Pender and Rist, 2001). Therefore, functional polymorphisms of CD95 or CD95L could influence the susceptibility to or persistence of MS either by
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impairing the elimination of autoreactive T or B cells or increasing susceptibility of oligodendroglia to T-cell or microglia-induced death. Patients with MS and their parents in some families have a higher frequency of resistance to CD95-induced T-cell death compared to controls (Comi et al., 2000). Earlier studies reported no association between CD95 and MS (Cascino et al., 1998; Huang et al., 2000) although a trend was reported by Huang et al. (2000). Recent studies found an association between polymorphisms of CD95 (Van Veen et al., 2002) and CD95L (Zayas et al., 2001) and MS. We studied the association of CD95 and CD95L with susceptibility to, with age of onset and with course and severity of MS, both in the overall population and after stratification for gender.
2. Materials and methods 2.1. Study population We studied 221 unique patients representing 79% ascertainment in Olmsted County, MN, from two overlapping population-based prevalence cohorts of 122 patients from 1991 (Rodriguez et al., 1994) and 188 patients from 2000 (Pittock et al., 2003). Within the 10-year period, 15 patients died and 18 moved out of the prevalence area. Eighty-nine patients were ascertained in both time points and 99 patients were ascertained only in 2000. Patients in the 1991 cohort had previously been typed for MHC class II alleles (Weinshenker et al., 1998). One hundred fifty-seven patients were women and 64 were men. Information was unavailable regarding disease course in 6 of 221 patients. Relapsing – remitting and secondary progressive cases were classified together as bout-onset (N = 198). There were 17 primary progressive cases. Patients with disease duration of 5 years or longer were ranked for disease severity by stratifying for progression index (EDSS/duration) within 5-year cohorts as previously described (Weinshenker et al., 1997). Four hundred forty-two controls matched for ethnicity, age and gender were selected from 4000 Mayo Clinic patients from whom DNA samples had been obtained. The controls did not have MS or another inflammatorydemyelinating disease. Matching for ethnicity was based on the country of descent of each grandparent as previously described elsewhere (Kantarci et al., 2000). Most commonly, the grandparents of the cases were of Dutch or German descent (39%), followed by British, Scottish or Irish (22%) and Scandinavian (21%). The remaining 18% comprised of all the other nationalities. Matching efficiency was 100% (i.e. all four grandparents match) for 29% of cases, equal or greater than 75% in 38% and equal or greater than 50% in 87% of the cases. Except for partial Native American descent (0.6% of the grandparents of cases and 0.4% of controls) and east Indian descent (0.6% of the grandparents
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Fig. 1. The structure of the CD95 located in chromosome 10q24.1 and location of polymorphisms are shown. 5V( 670)*G ! A was genotyped using an RFLP with BstN1 [PCR oligonucleotides: E1( 957)25D and E1( 642)19U], E3(26)*A ! G was genotyped using an RFLP with BsrGI [PCR oligonucleotides: I2( 5)30D and I3(175)25U] and E7(74)*C ! T was genotyped using a Dra1 RFLP [PCR oligonucleotides: E7( 316)22D and I7(606)24U]. I = intron; E = exon; 5V = 5V relative to exon 1.
of cases and 0.7% of the grandparents of the controls), all patients and controls were Caucasian. Controls were, on average, 6.7 years older than the cases.
orientation. For brevity, the PCR conditions for these reactions are not shown (Figs. 1 and 2). 2.3. Statistical analysis
2.2. Genotyping single nucleotide polymorphisms We studied three previously known single nucleotide polymorphisms (SNPs) for each gene to establish haplotypes that span the genes (Figs. 1 and 2). We selected SNPs that were studied by others (Cascino et al., 1998; Huang et al., 2000; Van Veen et al., 2002) or selected from the public SNP database (www.ncbi.nih.gov) based on location and heterozygosity rates for optimal informational content. Polymorphisms are named according to the position of the allele relative to an exon or intron (e.g., E3(26)*A ! G stands for a single nucleotide change from A to G 26 nucleotides from the beginning of exon 3). Oligonucleotides used in the PCR reactions for RFLP are named according the location of the first base in their sequence, the number of bases and their orientation (downstream, D, or upstream, U). For example E1( 957)25D refers to an oligonucleotide placed at 957th base upstream of exon 1, extending over 25 bases in a downstream
We initially studied all three polymorphisms for individual genes in the 1991 cohort. We then genotyped additional individuals ascertained in 2000 to further address positive findings in the 1991 cohort in the combined cohort. Association between a marker and susceptibility to disease was tested by Cochran – Armitage trend test. Differences in carrier frequencies between sporadic cases and controls, women and men, and bout-onset and primary progressive disease course were assessed using Fisher’s Exact statistics for contingency tables. Odds ratios and 95% exact confidence intervals were also calculated. Attributable risk was estimated using information about disease prevalence and the risk of carrying a given allele (Bruzzi et al., 1985). We analyzed the polymorphisms both as independent markers, and as two- or three-marker haplotypes when applicable to enhance the polymorphic informational content in evaluating the association with susceptibility to MS. Haplotype frequencies were imputed for individuals with ambiguous
Fig. 2. The structure of the CD95L located in chromosome 1q23 and location of polymorphisms are shown. 5V( 659)*C ! T was genotyped using an RFLP with [PCR oligonucleotides: E1( 686)27D and E1( 460)25U], I2(1174)*C ! T was genotyped using a NlaIII RFLP [PCR oligonucleotides: I2(919)21D and I2(1567)29U]and I2(2777)*A ! G was genotyped using an RFLP with Mn1I [PCR oligonucleotides: I2(2650)24D and I2(3026)24U]. I = intron; E = exon; 5V = 5V relative to exon 1.
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1.17 – 2.56) and being a carrier of E7(74)*T decreased the risk of MS ( p = 0.007; OR: 0.58, 95% CI: 0.39– 0.85) (Fig. 3). For the 5V( 670) SNP, being a homozygote for 5V( 670)*A increased the risk of MS in women ( p = 0.034; OR: 1.59, 95% CI: 1.06 – 2.38) and being a carrier of 5V( 670)*G decreased the risk ( p = 0.034; OR: 0.63, 95% CI: 0.42 – 0.95) (Fig. 3). The genotype distribution did not differ between men and women in the control population for both 5V( 670) ( p = 0.528) and E7(74) ( p = 0.308) SNPs. There was significant but incomplete linkage disequilibrium (LD) between 5V( 670) and E7(74) SNPs overall ( p < 0.001; DV = 0.546). LD occurred in both men ( p < 0.001; DV = 0.533) and women ( p < 0.001; DV = 0.553). The allele 5V( 670)*A was in LD with E7(74)*C (Table 1). Haplotype analysis showed a trend for association with MS in the overall group (simulated p = 0.092) (Table 1). This association was marginally significant in women (simulated p = 0.046). Specifically, the 5V( 670)*G/E7(74)*T haplotype was less frequent in women with MS compared to controls ( p = 0.064; OR: 0.62, 95% CI: 0.39 – 1.01) whereas it was more frequent, although not significantly so, in men with MS compared to controls ( p = 0.400; OR: 1.41, 95% CI: 0.70 –2.84) (Fig. 3). Given the increased risk of MS was associated with being a homozygote for the common allele of each of these polymorphisms, the decreased risk associated with the haplotype defined by the uncommon alleles was expected given the strong linkage disequlibrium between these markers. The difference between men and women was not due a lower power to detect an association in men; indeed, there was an opposite trend in genotype distribution in men. Since each of the polymorphisms was associated with disease susceptibility and LD was incomplete, we used logistic regression analysis to determine whether each polymorphism had an independent effect. Being a homozygote for 5V( 670)*A did not have an independent effect when controlled for being a homozygote for E7(74)*C in
haplotypes by an expectation-maximization algorithm and compared between cases and controls using a test for associations between traits and haplotypes where phase is ambiguous (Schaid et al., 2002). Simulated p-values were estimated to help eliminate spurious significant results that may result from multiple testing. We compared the findings of this study with those of previously published studies and conducted a pooled analysis of data using a logistic regression model, adjusting for study differences. The mean ranked severity scores and age at onset were compared according to genotype and carrier status using the Wilcoxon rank sum statistics.
3. Results 3.1. CD95 In the 1991 cohort, 7.4% (9 of 122) of the patients and 8.6% (21 of 244) of the controls were carriers of E3(26)*G allele. The difference was not significant both in the overall group and after stratification by gender. Hence, this polymorphism was not considered for further analysis. The commoner 5V( 670) and the E7(74) SNPs were associated with susceptibility to MS in women ( p = 0.042 and p = 0.026, respectively) but not in men ( p = 0.363 and p = 1.000, respectively). We studied 5V( 670) and E7(74) SNPs and their corresponding imputed two-marker haplotype distribution in the combined cohort (Table 1). E7(74) SNP was associated with susceptibility to MS in the overall group ( p = 0.015). Specifically, being a homozygote for E7(74)*C was associated with increased risk of MS ( p = 0.016; OR: 1.52, 95% CI: 1.09– 2.11) and being a carrier of E7(74)*T was associated with decreased risk of MS ( p = 0.016; OR: 0.66, 95% CI: 0.47– 0.92). This association, however, was due to the effect in women ( p = 0.006). In women, being a homozygote for E7(74)*C increased the risk of MS ( p = 0.007; OR: 1.73, 95% CI:
Table 1 Genotype and haplotype distribution in CD95 in the combined cohort, stratified by gender Marker
Total
Women
MS N 5V( 670)
E7(74)
5V( 670)/E7(74)
GG GA AA CC CT TT G/C G/T A/C A/T
37 108 73 119 79 17 – ** – – –
Control %
N
%
17.0 49.5 33.5 55.3 36.7 7.9 21.5 20.2 52.2 6.1
86 234 121 197 193 48 – – – –
19.5 53.1 27.4 45.0 44.1 11.0 21.7 24.3 45.4 8.6
Men
p*
MS N
%
N
%
0.131
25 72 59 86 57 10 – – – –
16.0 46.2 37.8 56.2 37.3 6.5 21.5 17.6 53.2 7.6
58 169 87 132 145 33 – – – –
18.5 53.8 27.7 42.6 46.8 10.7 19.5 25.9 46.6 8.0
0.015
0.092
Control
* Cochran – Armitage trend test for overall distribution of genotypes. ** Due to imputation of haplotypes only frequencies are presented and simulated p-values are reported.
p
MS N
%
N
%
0.061
12 36 14 33 22 7 – – – –
19.4 58.1 22.6 53.2 35.5 11.3 21.4 27.0 49.6 2.0
28 65 34 65 48 15 – – – –
22.1 51.2 26.8 50.8 37.5 11.7 27.0 20.6 42.5 10.0
0.006
0.046
Control
p
0.887
0.787
0.042
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Fig. 3. Association of CD95 with susceptibility to MS stratified by gender. 95% confidence intervals (solid horizontal bars), odds ratios (numbers over each bar), and p-values are given. The hatched Y-axis marks an odds ratio of 1.
of MS for the year 1995 – 2000 in Olmsted County is 2.3fold higher for women than men (10.4 and 4.5 per 100,000, respectively). Assuming that the association of CD95 polymorphisms with susceptibility to MS in women but not in men may contribute in part to higher predilection for MS in women, we calculated the attributable effect of these polymorphisms. The projected incidence rate would have been 2.0-fold higher for women than men had patients not been exposed to the risk of being homozygous for 5V( 670)*A and 1.9-fold higher if not exposed to the risk of being homozygote for E7(74)*C. Other potential explanations for gender bias exist, including nongenetic explanations. The calculation presents the theoretical effect on differing incidence in men and women assuming that the genetic asso-
women ( p>0.10). When compared to being homozygous for neither SNP, being homozygous for both 5V( 670)*A and E7(74)*C ( p = 0.015; OR: 1.73, 95% CI: 1.11– 2.69) had a similar risk of MS in women as being homozygous for either 5V( 670)*A or E7(74)*C ( p = 0.006; OR: 1.77, 95% CI: 1.19 –2.64). These results suggested that the effects of these polymorphisms were independent and not additive. We estimated the risk of MS in the Olmsted County population attributable to the alleles based on the odds ratio and the frequency of the allele, as well as the prevalence of MS in Olmsted county. Being homozygous for 5V( 670)*A explained 14%, for E7(74)*C explained 24%, and for 5V( 670)*A or E7(74)*C explained 28% of risk of MS in women but 0% of the risk in men. The 5-year incidence rate
Table 2 Pooled analysis of association of CD95 with susceptibility to MS Study population
N*
5V( 670) GG N
Cascino et al., 1998
Italy
Huang et al., 2000
Australia (Caucasian)
Van Veen et al., 2002
Holland
Kantarci et al. (current study)
Olmsted
Total
E7(74) GA
%
N
AA %
N
CC %
MS C MS
62 111 124
22
17.7
58
46.8
44
35.5
C MS C MS C MS C
183 383 206 221 442 790 942
40 80 42 37 86 139 168
21.9 20.9 20.4 17.0 19.5 18.6 20.2
97 185 118 108 234 351 449
53.0 48.3 57.3 49.5 53.1 49.9 54.0
46 118 46 73 121 235** 213
25.1 30.8 22.3 33.5 27.4 31.5 25.8
N 33 59
MS = multiple sclerosis; C = control. * Frequencies may not add up to total numbers due to missing values. ** 5V( 670)*AA vs. 5V( 670)*GA + 5V( 670)*GG, p = 0.004; OR: 1.39, 95% CI: 1.11 – 1.73. *** E7(74)*CC vs. E7(74)*CT + E7(74)TT, p=0.025; OR: 1.39, 95%CI: 1.04-1.86.
119 197 152*** 256
CT %
N
TT %
N
%
53.2 53.1
18 42
29.1 37.8
11 10
17.7 9.0
55.4 45.0 54.9 46.7
79 193 97 235
36.7 44.1 35.0 42.8
17 48 28 58
7.9 11.0 10.1 10.6
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( p = 0.364) and the association between the 5V( 659)*T allele and increased risk of MS became nonsignificant ( p = 0.072; OR: 1.38, 95% CI: 0.98 – 1.94). Hence, typing of further markers and haplotype analysis in the combined cohort was not performed. There was no association between any of the markers in CD95L and gender, age of onset, disease course or disease severity in MS ( p>0.05, data not shown).
ciation in women that we described contributes to gender bias in MS. There was no association between any of the markers in CD95 and age of onset, disease course or disease severity in MS (data not shown). Table 2 shows the results of a pooled analysis from multiple published studies on association of CD95 with susceptibility to MS. Being a homozygote for 5V( 670)*A was associated with increased risk of MS in the overall population ( p = 0.004; OR: 1.39, 95% CI: 1.11– 1.73).
4. Discussion 3.2. CD95L We found that homozygotes for the common polymorphisms of CD95 and their corresponding haplotypes (recessive effect) are associated with increased susceptibility to MS in women. In a previous study, no association was found between E7(74) SNP and MS (Cascino et al., 1998). The population studied was small (62 patient with MS and 111 controls); no information was provided for the source of patients and controls; gender stratification was not performed. Our results are in agreement with two of the previously published studies (Van Veen et al., 2002; Huang et al., 2000). Van Veen et al. (2002) reported that being a carrier for the 5V( 670)*G allele decreases risk of MS and being a homozygote for 5V( 670)*A increases risk of MS. The authors did not comment on the specificity of the association to women but their published work provides the relevant data. When reanalyzed, their study also supports an association of this SNP in women (OR: 0.53, 95% CI:
In the 1991 cohort, there was a trend for association with susceptibility to MS for the 5V( 659) SNP ( p = 0.072). Specifically, carriers of the 5V( 659)*T allele had increased risk of MS ( p = 0.006; OR: 2.02, 95% CI: 1.23 – 3.32), primarily due to a difference in the frequency of heterozygotes (Table 3). Despite the strong linkage disequilibrium between the three markers in CD95L ( p < 0.0001), none of the other markers was associated with MS ( p>0.10). The overall distribution of haplotypes was significantly different between cases and controls (simulated p = 0.016) (Table 3). The 5V( 659)*T/I2(1174)*T/I2(2777)*A haplotype was associated with increased risk of MS (OR: 2.63, 95% CI: 1.01 – 6.86). The haplotypes including 5V( 659)*C were more frequent in controls and the haplotypes including 5V( 659)*T were more frequent in cases. (Table 3). The 5V( 659) SNP of CD95L was further studied in the combined cohort. The overall trend was lost
Table 3 Genotype and haplotype distribution in CD95L in the 1991 and combined cohorts Marker
1991
Combined***
MS
5V( 659)
I2(1174)
I2(2777)
5V( 659)/I2(1174)/ I2(2777)
CC CT TT CC CT TT AA AG GG C/C/A C/C/G C/T/A C/T/G T/C/A T/C/G T/T/A T/T/G
Control
p*
N
%
N
%
28 77 17 18 61 43 27 62 33 – **
23.0 63.1 13.9 14.8 50.0 35.2 22.1 50.8 27.0 0.0
91 114 37 41 121 82 56 110 77 –
37.6 47.1 15.3 16.8 49.6 33.6 23.0 45.3 31.7 0.7
– – – – – – –
3.7 39.2 11.6 0.0 36.1 8.4 1.1
– – – – – – –
7.0 40.8 12.7 1.0 33.0 3.4 1.6
0.072
MS
Control
p
N
%
N
%
70 124 26
31.8 56.4 11.8
172 206 62
39.1 46.8 14.1
0.364
0.628
0.643
0.016
* Cochran – Armitage trend test. ** Due to imputation of haplotypes only frequencies are presented and simulated p is reported. *** Only the 5V( 659) polymorphism was studied in the combined cohort due to lack of association between the other polymorphisms and susceptibility to MS in the 1991 cohort.
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0.30 – 0.92 for carriers of the 5V( 670)*G allele; OR: 1.89, 95% CI: 1.08 –3.30 for homozygotes of the 5V( 670)*A allele) but not in men. No gender information is provided in the study by Huang et al. (2000). They found a trend in their unstratified population for the same genotypes with odds ratios (OR: 0.61, 95% CI: 0.37– 1.00 and OR: 1.64, 95% CI: 1.00 –2.69, respectively) similar to both our results and those of Van Veen et al. (2002). When all three studies are combined, being a homozygote for 5V( 670)*A is associated with increased risk of MS in the overall population (Table 2). We did not find a convincing association between CD95L and MS. The initial trend in the 1991 cohort became nonsignificant when reanalyzed in the combined cohort. Zayas et al. (2001) have reported an association between a CA repeat marker 46 kb upstream of CD95L and MS. The association was stronger in familial cases. Given the distance between the marker and CD95L, the association detected by Zayas et al. (2001) may not extend into CD95L. Alternatively, the differences between our studies may represent population heterogeneity. CD95 mutations are associated with a lymphoproliferative syndrome in mice and the human counterpart, ALPS (Rieux-Laucat et al., 2003). Our study does not address the question of presence or absence of mutations in MS patients compared to controls but rather establishes an association between common polymorphisms and MS. Given the extent of strong linkage disequilibrium between the promoter and the exon 7 polymorphisms, our findings may be due to functional polymorphism(s) or mutations in CD95 that are in linkage disequilibrium with the two polymorphisms studied here. Most ALPS mutations are found in the 3Vend of CD95, in exon 9 coding for the intracellular death domain, resulting in a failure to bind the FADD/MORT1 signaling protein and decreased caspase recruitment and activation (Martin et al., 1999). We found a stronger effect for E7(74) SNP in the 3V end of the gene, but the significance of this observation is uncertain given the relatively small number of markers we studied. ALPS mutations have been found in extracellular domains as well, but these are less penetrant than the intracellular mutations and hence are potentially more relevant to sporadic disease (Jackson et al., 1999). Alternatively, our findings could be due to linkage disequilibrium with another gene at a distance from CD95. In a similar effort to ours, the 5V( 670) SNP was recently found to be associated with early onset sporadic Alzheimer’s disease (Feuk et al., 2000) but not associated with late-onset or familial cases (Theuns et al., 2001). Same authors also studied extended haplotypes around CD95 and demonstrated that the effect was indeed limited to the promoter polymorphism (Feuk et al., 2002). Hence, direct functional effects of the polymorphisms studied here is possible. There is no functional effect know for the silent E7(75) SNP, but the promoter polymorphism modifies the consensus sequence of a transcriptional element GAS (gam-
ma activation site) from TTC(N)2 – 4GAA to TTC(N)2 – 4GG A. GAS is the binding site for IFNg-activated janus kinase– signal transducer and activator of transcription 1 (JAK – STAT1) signaling pathway (Huang et al., 1997; Aaronson and Horvath, 2002). Hence, one can speculate that this polymorphism may modify IFNg-induced CD95 expression and apoptosis of the target cell. In the case of oligodendroglia, the variant sequence with reduced function would reduce cell death and be favorable for patients who are destined to develop MS, while the allele with preserved function would be detrimental. In the case of autoreactive T or B cells, however, the variant sequence would result in decreased IFNg-induced apoptotic elimination of these cells that might predispose to or aggravate existing MS. Current evidence indicates that once MS develops IFNg administration is detrimental (Panitch et al., 1987). The effect of IFNg-induced CD95-mediated apoptosis may have different effects on different stages of induction and maintenance of MS. Further understanding of the allelic effects of the polymorphisms and correlation with apoptosis markers will be necessary to better appreciate the significance of our genetic data. It is possible that the resistance to CD95induced T-cell death in MS patients, as reported by Comi et al. (2000), could be related to CD95 polymorphisms as well as other defects in the CD95-mediated apoptosis pathway; comprehensive surveys for mutations in CD95 have not yet been conducted in MS. Depending on the genetic background of the mouse strain, lpr mutations may lead to decreased susceptibility to and severity of disease in EAE compared to the wild type (Suvannavejh et al., 2000). Likewise, in NOD mice with a mutation in the CD95 death domain, onset of diabetes is significantly delayed (Savinov et al., 2003). Despite the widespread SLE-like systemic autoimmunity, CNS inflammatory-demyelinating disease is not common in the ALPS, DALD or MAS pedigrees (Waishnaw et al., 1999; Ramenghi et al., 2000; Rieux-Laucat et al., 2003). These results suggest that the effect of CD95 on the autoimmune phenotype may differ depending on the presence or absence of other modifier genes and is more complex than defined for the prototypical ALPS families. Indeed, this is the case in Alzheimer’s disease where the effect of 5V( 670) SNP was strongest in patients who are also carriers of the ApoE*e4 allele (Feuk et al., 2000). Our study independently highlights that the association of the alleles of CD95 and susceptibility to MS are confined to women. This association may be one factor that explains why women have a higher risk of MS than men do. Our results suggest that having either one of the CD95 polymorphisms explains 28% of the risk of MS in women in Olmsted County, MN. Interestingly, the autoimmunity observed in lpr mice is also gender specific. Autoimmune hearing loss is more common in female lpr mice compared to male lpr mice (Trune and Kempton, 2002) and autoantibody production is higher in female lpr mice than male lpr mice (Tornwall et al., 1999). Estrogen receptor beta is
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expressed in low levels in thymic CD4/CD8 T cells in wild-type mice and at high levels in the peripheral CD4 / CD8 T cells in lpr mice (Tornwall et al., 1999). The inheritance of defective CD95-induced T-cell death and the associated increase in the frequency of autoimmune diseases and cancer in DALD pedigrees also appears to vary by gender and by lineage (paternal or maternal) (Ramenghi et al., 2000). Our results suggest that the role of CD95 mutations in determining increased susceptibility to women for MS and potentially other autoimmune disease is worthy of further study.
Acknowledgements This study has been supported by the Mayo Foundation and National MS Society, Grant no. RG-2870-A-2. Part of these results have been presented in part in the ECTRIMS/ ACTRIMS Meeting in Baltimore, 2002.
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Trune, D.R., Kempton, J.B., 2002. Female MRL.MpJ-Fas(lpr) autoimmune mice have greater hearing loss than males. Hear. Res. 167 (1 – 2), 170 – 174. Van Veen, T., Kalkers, N.F., Crusius, J.B.A., Van Winsen, L., Barkhof, F., Jongen, P.J.H., Pena, A.S., Polman, C.H., Uitdehaag, B.M.J., 2002. The Fas 670 polymorphism influences susceptibility to multiple sclerosis. J. Neuroimmunol. 128, 95 – 100. Waishnaw, A.K., Toubi, E., Ohsako, S., Drappa, J., Buys, S., Estrada, J., Sitarz, A., Zemel, L., Chu, J.L., Elkon, K.B., 1999. The spectrum of apoptotic defects and clinical manifestations, including systemic lupus erythematosus, in humans with CD95 (Fas/APO-1) mutations. Arthritis Rheum. 42 (9), 1833 – 1842. Waldner, H., Sobel, R.A., Howard, E., Kuchroo, V.K., 1997. Fas- and FasL-deficient mice are resistant to induction of autoimmune encephalomyelitis. J. Immunol. 159, 3100 – 3103. Wang, R., Rogers, A.M., Ratliff, T.L., Russell, J.H., 1996. CD95-dependent bystander lysis caused by CD4+ T helper 1 effectors. J. Immunol. 157, 2961 – 2968. Wang, J., Zheng, L., Lobito, A., Chan, K., Dale, J., Sneller, M., Yao, X., Puck, J.M., Straus, S.E., Lenardo, M.J., 1999. Inherited human Caspase 10 mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome type II. Cell 98 (1), 47 – 58. Weinshenker, B.G., Wingerchuk, D.M., Liu, Q., Bissonet, A.S., Schaid, D.J., Sommer, S.S., 1997. Genetic variation in the tumor necrosis factor alpha gene and the outcome of multiple sclerosis. Neurology 49 (2), 378 – 385. Weinshenker, B.G., Santrach, P., Bissonet, A.S., McDonnell, S.K., Schaid, D., Moore, S.B., Rodriguez, M., 1998. Major histocompatibility complex class II alleles and the course and outcome of MS: a populationbased study. Neurology 51 (3), 742 – 747. Wu, J., Wilson, J., He, J., Xiang, L., Schur, P.H., Mountz, J.D., 1996. Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease. J. Clin. Invest. 98 (5), 1107 – 1113. Zayas, M.D., Lucas, M., Solano, F., Fernandez-Perez, M.J., Izquerdo, G., 2001. Association of a CA repeat polymorphism upstream of the Fas ligand gene in multiple sclerosis. J. Neuroimmunol. 116, 238 – 241.
CD95 polymorphisms are associated with susceptibility to MS in women A population-based study of CD95 and CD95L in MS Orhun H. Kantarci a, David D. Hebrink a, Sara J. Achenbach b, Elizabeth J. Atkinson b, Mariza de Andrade b, Cynthia T. McMurray c, Brian G. Weinshenker a,* a Department of Neurology, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, MN 55905, USA Department of Health Sciences Research, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, MN 55905, USA c Departments of Pharmacology, Biochemistry and Molecular Biology and Molecular Neuroscience Program, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, MN 55905, USA b
Received 15 July 2003; received in revised form 17 August 2003; accepted 1 October 2003
Abstract CD95/CD95L interaction results in activation-induced apoptosis thereby regulating clonal expansion of T cells outside the thymus. Genetic defects in this system result in autoimmune lymphoproliferation in mice and men. CD95-induced cell death may be defective in MS. We studied the association of CD95 and CD95L polymorphisms with MS in 221 unique patients representing 79% ascertainment in Olmsted County, MN, and 442 gender-, age- and ethnicity-matched controls. Being a homozygote for the G allele of CD95 5V( 670)*A ! G SNP ( p = 0.034; OR: 1.59, 95% CI: 1.06 – 2.38) and for the C allele of CD95 E7(74)*C ! T SNP ( p = 0.007; OR: 1.73, 95% CI: 1.17 – 2.56) increased susceptibility to MS exclusively in women. There was strong but incomplete linkage disequilibrium between the two markers ( p < 0.001; D V = 0.546). Homozygosity for 5V( 670)*A or E7(74)*C explained 28% of risk of MS in women but 0% of the risk in men in Olmsted County, MN. Our results agree with the previously published studies and highlight that the association of the polymorphisms is restricted to women with MS. We did not find an association between CD95L and susceptibility to MS nor CD95 or CD95L and age of onset, disease course and disease severity. D 2003 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; CD95; CD95L; Polymorphism; Susceptibility; Gender
1. Introduction Activation-induced apoptosis mediated through expression of CD95 (Fas/APO-1) and CD95-ligand (CD95L) is an important mechanism of regulating clonal expansion of mature T cells outside the thymus (Russell, 1995). In germinal centers, T cell-induced activation of CD95 on B cells also regulates peripheral tolerance of B cells (Elkon and Marshak-Rothstein, 1996). Defects in activation-induced apoptosis arising from naturally occurring mutations in CD95 and CD95L cause systemic autoimmunity and extensive lymphoproliferation resembling systemic lupus
* Corresponding author. Tel.: +1-507-284-4234; fax: +1-507-2664419. E-mail address: [email protected] (B.G. Weinshenker). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.10.002
erythematosus in mice with lymphoproliferation (lpr) and generalized lymphadenopathy (gld) syndromes (Cohen and Eisenberg, 1991; Nagata and Golstein, 1995; Nagata and Suda, 1995). Autoimmunity and lymphoproliferation are more prominent in females than in male lpr mice (Tornwall et al., 1999; Trune and Kempton, 2002). The human counterpart of lpr/gld is autoimmune lymphoproliferative syndrome (ALPS) (Canale and Smith, 1967), which is linked to mutations in CD95 (Fisher et al., 1995; Rieux-Laucat et al., 1995; Drappa et al., 1996) and rarely to mutations in CD95L (Wu et al., 1996) or caspase-10 (Wang et al., 1999). Most of the ALPS mutations are clustered in the intracellular death domain, specifically in exon 9 of CD95 (Rieux-Laucat et al., 2003). Varying degrees of autoimmunity involving multiple organ systems, CD3+/CD4 /CD8 T cell and B cell proliferation resembling lymphoma and hypergammaglobulinemia
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are the hallmarks of ALPS; these manifestations are the results of loss of function in the death domain caused by the mutations. Lymphoma also occurs in the some pedigrees (Fisher et al., 1995; Lim et al., 1998). The variation in the phenotype depends on the type and location of the mutations. Penetrance of individual mutations even within a given pedigree is variable, suggesting the presence of modifier genes (Rieux-Laucat et al., 2003). Hence, polymorphisms of CD95 and CD95L can influence susceptibility to complex autoimmune phenotypes but may not result in the full spectrum of ALPS. In patients with Dianzani’s autoimmune lymphoproliferative disease (DALD), an ALPS-like clinical pattern is present but CD3+/CD4 /CD8 T cells and CD95 or CD95L mutations are absent (Dianzani et al., 1997). Family members of DALD patients have increased frequency of autoimmune diseases inherited paternally, and women, but not men, have an increased frequency of cancer (Ramenghi et al., 2000). DALD patients have defective CD95 function as do those with multiple autoimmune syndrome (MAS) wherein first to second degree relatives have increased frequency of autoimmunity in the absence of ALPS phenotype (Ramenghi et al., 2000). In experimental allergic encephalomyelitis (EAE) induced in lpr and gld mice from C57Bl background, decreased CD95 or CD95L expression results in a limited disease phenotype compared to the wild-type mice (Waldner et al., 1997; Okuda et al., 1998; Sabelko-Downes et al., 1999). Despite histological evidence for EAE, these mice have decreased incidence and severity of symptoms, and their disease is usually limited to a monophasic episode rather than a relapsing – remitting course observed in the wild-type mice (Malipiero et al., 1997). In mice from SJL background, however, deficiency of CD95 is associated with increased severity of EAE and decreased apoptosis of inflammatory cells in the CNS (Suvannavejh et al., 2000). As in ALPS, other genetic factors probably determine the phenotype (Sabelko-Downes et al., 2000). CD95 expression is weak or absent in healthy oligodendroglia but is significantly enhanced in acute or chronic MS lesions (Dowling et al., 1996; D’Souza et al., 1996). Interferon gamma (IFNg) induces apoptotic cell death by upregulating CD95 and CD95L expression in microglia (Badie et al., 1999). Unlike the activated T cells, oligodendroglia do not undergo activation-induced apoptosis (Bonetti et al., 1997) but microglia and infiltrating CD4+ lymphocytes with increased expression of CD95L (D’Souza et al., 1996) may lyse the oligodendroglia expressing CD95 in a bystander fashion (Wang et al., 1996). This is similar to the B-cell apoptosis induced by activated T cells (Elkon and Marshak-Rothstein, 1996). In the CNS, elimination of autoreactive T cells could be through activation-induced apoptosis or via microglia expressing CD95L lysing the T cells expressing CD95 (Pender and Rist, 2001). Therefore, functional polymorphisms of CD95 or CD95L could influence the susceptibility to or persistence of MS either by
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impairing the elimination of autoreactive T or B cells or increasing susceptibility of oligodendroglia to T-cell or microglia-induced death. Patients with MS and their parents in some families have a higher frequency of resistance to CD95-induced T-cell death compared to controls (Comi et al., 2000). Earlier studies reported no association between CD95 and MS (Cascino et al., 1998; Huang et al., 2000) although a trend was reported by Huang et al. (2000). Recent studies found an association between polymorphisms of CD95 (Van Veen et al., 2002) and CD95L (Zayas et al., 2001) and MS. We studied the association of CD95 and CD95L with susceptibility to, with age of onset and with course and severity of MS, both in the overall population and after stratification for gender.
2. Materials and methods 2.1. Study population We studied 221 unique patients representing 79% ascertainment in Olmsted County, MN, from two overlapping population-based prevalence cohorts of 122 patients from 1991 (Rodriguez et al., 1994) and 188 patients from 2000 (Pittock et al., 2003). Within the 10-year period, 15 patients died and 18 moved out of the prevalence area. Eighty-nine patients were ascertained in both time points and 99 patients were ascertained only in 2000. Patients in the 1991 cohort had previously been typed for MHC class II alleles (Weinshenker et al., 1998). One hundred fifty-seven patients were women and 64 were men. Information was unavailable regarding disease course in 6 of 221 patients. Relapsing – remitting and secondary progressive cases were classified together as bout-onset (N = 198). There were 17 primary progressive cases. Patients with disease duration of 5 years or longer were ranked for disease severity by stratifying for progression index (EDSS/duration) within 5-year cohorts as previously described (Weinshenker et al., 1997). Four hundred forty-two controls matched for ethnicity, age and gender were selected from 4000 Mayo Clinic patients from whom DNA samples had been obtained. The controls did not have MS or another inflammatorydemyelinating disease. Matching for ethnicity was based on the country of descent of each grandparent as previously described elsewhere (Kantarci et al., 2000). Most commonly, the grandparents of the cases were of Dutch or German descent (39%), followed by British, Scottish or Irish (22%) and Scandinavian (21%). The remaining 18% comprised of all the other nationalities. Matching efficiency was 100% (i.e. all four grandparents match) for 29% of cases, equal or greater than 75% in 38% and equal or greater than 50% in 87% of the cases. Except for partial Native American descent (0.6% of the grandparents of cases and 0.4% of controls) and east Indian descent (0.6% of the grandparents
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Fig. 1. The structure of the CD95 located in chromosome 10q24.1 and location of polymorphisms are shown. 5V( 670)*G ! A was genotyped using an RFLP with BstN1 [PCR oligonucleotides: E1( 957)25D and E1( 642)19U], E3(26)*A ! G was genotyped using an RFLP with BsrGI [PCR oligonucleotides: I2( 5)30D and I3(175)25U] and E7(74)*C ! T was genotyped using a Dra1 RFLP [PCR oligonucleotides: E7( 316)22D and I7(606)24U]. I = intron; E = exon; 5V = 5V relative to exon 1.
of cases and 0.7% of the grandparents of the controls), all patients and controls were Caucasian. Controls were, on average, 6.7 years older than the cases.
orientation. For brevity, the PCR conditions for these reactions are not shown (Figs. 1 and 2). 2.3. Statistical analysis
2.2. Genotyping single nucleotide polymorphisms We studied three previously known single nucleotide polymorphisms (SNPs) for each gene to establish haplotypes that span the genes (Figs. 1 and 2). We selected SNPs that were studied by others (Cascino et al., 1998; Huang et al., 2000; Van Veen et al., 2002) or selected from the public SNP database (www.ncbi.nih.gov) based on location and heterozygosity rates for optimal informational content. Polymorphisms are named according to the position of the allele relative to an exon or intron (e.g., E3(26)*A ! G stands for a single nucleotide change from A to G 26 nucleotides from the beginning of exon 3). Oligonucleotides used in the PCR reactions for RFLP are named according the location of the first base in their sequence, the number of bases and their orientation (downstream, D, or upstream, U). For example E1( 957)25D refers to an oligonucleotide placed at 957th base upstream of exon 1, extending over 25 bases in a downstream
We initially studied all three polymorphisms for individual genes in the 1991 cohort. We then genotyped additional individuals ascertained in 2000 to further address positive findings in the 1991 cohort in the combined cohort. Association between a marker and susceptibility to disease was tested by Cochran – Armitage trend test. Differences in carrier frequencies between sporadic cases and controls, women and men, and bout-onset and primary progressive disease course were assessed using Fisher’s Exact statistics for contingency tables. Odds ratios and 95% exact confidence intervals were also calculated. Attributable risk was estimated using information about disease prevalence and the risk of carrying a given allele (Bruzzi et al., 1985). We analyzed the polymorphisms both as independent markers, and as two- or three-marker haplotypes when applicable to enhance the polymorphic informational content in evaluating the association with susceptibility to MS. Haplotype frequencies were imputed for individuals with ambiguous
Fig. 2. The structure of the CD95L located in chromosome 1q23 and location of polymorphisms are shown. 5V( 659)*C ! T was genotyped using an RFLP with [PCR oligonucleotides: E1( 686)27D and E1( 460)25U], I2(1174)*C ! T was genotyped using a NlaIII RFLP [PCR oligonucleotides: I2(919)21D and I2(1567)29U]and I2(2777)*A ! G was genotyped using an RFLP with Mn1I [PCR oligonucleotides: I2(2650)24D and I2(3026)24U]. I = intron; E = exon; 5V = 5V relative to exon 1.
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1.17 – 2.56) and being a carrier of E7(74)*T decreased the risk of MS ( p = 0.007; OR: 0.58, 95% CI: 0.39– 0.85) (Fig. 3). For the 5V( 670) SNP, being a homozygote for 5V( 670)*A increased the risk of MS in women ( p = 0.034; OR: 1.59, 95% CI: 1.06 – 2.38) and being a carrier of 5V( 670)*G decreased the risk ( p = 0.034; OR: 0.63, 95% CI: 0.42 – 0.95) (Fig. 3). The genotype distribution did not differ between men and women in the control population for both 5V( 670) ( p = 0.528) and E7(74) ( p = 0.308) SNPs. There was significant but incomplete linkage disequilibrium (LD) between 5V( 670) and E7(74) SNPs overall ( p < 0.001; DV = 0.546). LD occurred in both men ( p < 0.001; DV = 0.533) and women ( p < 0.001; DV = 0.553). The allele 5V( 670)*A was in LD with E7(74)*C (Table 1). Haplotype analysis showed a trend for association with MS in the overall group (simulated p = 0.092) (Table 1). This association was marginally significant in women (simulated p = 0.046). Specifically, the 5V( 670)*G/E7(74)*T haplotype was less frequent in women with MS compared to controls ( p = 0.064; OR: 0.62, 95% CI: 0.39 – 1.01) whereas it was more frequent, although not significantly so, in men with MS compared to controls ( p = 0.400; OR: 1.41, 95% CI: 0.70 –2.84) (Fig. 3). Given the increased risk of MS was associated with being a homozygote for the common allele of each of these polymorphisms, the decreased risk associated with the haplotype defined by the uncommon alleles was expected given the strong linkage disequlibrium between these markers. The difference between men and women was not due a lower power to detect an association in men; indeed, there was an opposite trend in genotype distribution in men. Since each of the polymorphisms was associated with disease susceptibility and LD was incomplete, we used logistic regression analysis to determine whether each polymorphism had an independent effect. Being a homozygote for 5V( 670)*A did not have an independent effect when controlled for being a homozygote for E7(74)*C in
haplotypes by an expectation-maximization algorithm and compared between cases and controls using a test for associations between traits and haplotypes where phase is ambiguous (Schaid et al., 2002). Simulated p-values were estimated to help eliminate spurious significant results that may result from multiple testing. We compared the findings of this study with those of previously published studies and conducted a pooled analysis of data using a logistic regression model, adjusting for study differences. The mean ranked severity scores and age at onset were compared according to genotype and carrier status using the Wilcoxon rank sum statistics.
3. Results 3.1. CD95 In the 1991 cohort, 7.4% (9 of 122) of the patients and 8.6% (21 of 244) of the controls were carriers of E3(26)*G allele. The difference was not significant both in the overall group and after stratification by gender. Hence, this polymorphism was not considered for further analysis. The commoner 5V( 670) and the E7(74) SNPs were associated with susceptibility to MS in women ( p = 0.042 and p = 0.026, respectively) but not in men ( p = 0.363 and p = 1.000, respectively). We studied 5V( 670) and E7(74) SNPs and their corresponding imputed two-marker haplotype distribution in the combined cohort (Table 1). E7(74) SNP was associated with susceptibility to MS in the overall group ( p = 0.015). Specifically, being a homozygote for E7(74)*C was associated with increased risk of MS ( p = 0.016; OR: 1.52, 95% CI: 1.09– 2.11) and being a carrier of E7(74)*T was associated with decreased risk of MS ( p = 0.016; OR: 0.66, 95% CI: 0.47– 0.92). This association, however, was due to the effect in women ( p = 0.006). In women, being a homozygote for E7(74)*C increased the risk of MS ( p = 0.007; OR: 1.73, 95% CI:
Table 1 Genotype and haplotype distribution in CD95 in the combined cohort, stratified by gender Marker
Total
Women
MS N 5V( 670)
E7(74)
5V( 670)/E7(74)
GG GA AA CC CT TT G/C G/T A/C A/T
37 108 73 119 79 17 – ** – – –
Control %
N
%
17.0 49.5 33.5 55.3 36.7 7.9 21.5 20.2 52.2 6.1
86 234 121 197 193 48 – – – –
19.5 53.1 27.4 45.0 44.1 11.0 21.7 24.3 45.4 8.6
Men
p*
MS N
%
N
%
0.131
25 72 59 86 57 10 – – – –
16.0 46.2 37.8 56.2 37.3 6.5 21.5 17.6 53.2 7.6
58 169 87 132 145 33 – – – –
18.5 53.8 27.7 42.6 46.8 10.7 19.5 25.9 46.6 8.0
0.015
0.092
Control
* Cochran – Armitage trend test for overall distribution of genotypes. ** Due to imputation of haplotypes only frequencies are presented and simulated p-values are reported.
p
MS N
%
N
%
0.061
12 36 14 33 22 7 – – – –
19.4 58.1 22.6 53.2 35.5 11.3 21.4 27.0 49.6 2.0
28 65 34 65 48 15 – – – –
22.1 51.2 26.8 50.8 37.5 11.7 27.0 20.6 42.5 10.0
0.006
0.046
Control
p
0.887
0.787
0.042
166
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Fig. 3. Association of CD95 with susceptibility to MS stratified by gender. 95% confidence intervals (solid horizontal bars), odds ratios (numbers over each bar), and p-values are given. The hatched Y-axis marks an odds ratio of 1.
of MS for the year 1995 – 2000 in Olmsted County is 2.3fold higher for women than men (10.4 and 4.5 per 100,000, respectively). Assuming that the association of CD95 polymorphisms with susceptibility to MS in women but not in men may contribute in part to higher predilection for MS in women, we calculated the attributable effect of these polymorphisms. The projected incidence rate would have been 2.0-fold higher for women than men had patients not been exposed to the risk of being homozygous for 5V( 670)*A and 1.9-fold higher if not exposed to the risk of being homozygote for E7(74)*C. Other potential explanations for gender bias exist, including nongenetic explanations. The calculation presents the theoretical effect on differing incidence in men and women assuming that the genetic asso-
women ( p>0.10). When compared to being homozygous for neither SNP, being homozygous for both 5V( 670)*A and E7(74)*C ( p = 0.015; OR: 1.73, 95% CI: 1.11– 2.69) had a similar risk of MS in women as being homozygous for either 5V( 670)*A or E7(74)*C ( p = 0.006; OR: 1.77, 95% CI: 1.19 –2.64). These results suggested that the effects of these polymorphisms were independent and not additive. We estimated the risk of MS in the Olmsted County population attributable to the alleles based on the odds ratio and the frequency of the allele, as well as the prevalence of MS in Olmsted county. Being homozygous for 5V( 670)*A explained 14%, for E7(74)*C explained 24%, and for 5V( 670)*A or E7(74)*C explained 28% of risk of MS in women but 0% of the risk in men. The 5-year incidence rate
Table 2 Pooled analysis of association of CD95 with susceptibility to MS Study population
N*
5V( 670) GG N
Cascino et al., 1998
Italy
Huang et al., 2000
Australia (Caucasian)
Van Veen et al., 2002
Holland
Kantarci et al. (current study)
Olmsted
Total
E7(74) GA
%
N
AA %
N
CC %
MS C MS
62 111 124
22
17.7
58
46.8
44
35.5
C MS C MS C MS C
183 383 206 221 442 790 942
40 80 42 37 86 139 168
21.9 20.9 20.4 17.0 19.5 18.6 20.2
97 185 118 108 234 351 449
53.0 48.3 57.3 49.5 53.1 49.9 54.0
46 118 46 73 121 235** 213
25.1 30.8 22.3 33.5 27.4 31.5 25.8
N 33 59
MS = multiple sclerosis; C = control. * Frequencies may not add up to total numbers due to missing values. ** 5V( 670)*AA vs. 5V( 670)*GA + 5V( 670)*GG, p = 0.004; OR: 1.39, 95% CI: 1.11 – 1.73. *** E7(74)*CC vs. E7(74)*CT + E7(74)TT, p=0.025; OR: 1.39, 95%CI: 1.04-1.86.
119 197 152*** 256
CT %
N
TT %
N
%
53.2 53.1
18 42
29.1 37.8
11 10
17.7 9.0
55.4 45.0 54.9 46.7
79 193 97 235
36.7 44.1 35.0 42.8
17 48 28 58
7.9 11.0 10.1 10.6
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( p = 0.364) and the association between the 5V( 659)*T allele and increased risk of MS became nonsignificant ( p = 0.072; OR: 1.38, 95% CI: 0.98 – 1.94). Hence, typing of further markers and haplotype analysis in the combined cohort was not performed. There was no association between any of the markers in CD95L and gender, age of onset, disease course or disease severity in MS ( p>0.05, data not shown).
ciation in women that we described contributes to gender bias in MS. There was no association between any of the markers in CD95 and age of onset, disease course or disease severity in MS (data not shown). Table 2 shows the results of a pooled analysis from multiple published studies on association of CD95 with susceptibility to MS. Being a homozygote for 5V( 670)*A was associated with increased risk of MS in the overall population ( p = 0.004; OR: 1.39, 95% CI: 1.11– 1.73).
4. Discussion 3.2. CD95L We found that homozygotes for the common polymorphisms of CD95 and their corresponding haplotypes (recessive effect) are associated with increased susceptibility to MS in women. In a previous study, no association was found between E7(74) SNP and MS (Cascino et al., 1998). The population studied was small (62 patient with MS and 111 controls); no information was provided for the source of patients and controls; gender stratification was not performed. Our results are in agreement with two of the previously published studies (Van Veen et al., 2002; Huang et al., 2000). Van Veen et al. (2002) reported that being a carrier for the 5V( 670)*G allele decreases risk of MS and being a homozygote for 5V( 670)*A increases risk of MS. The authors did not comment on the specificity of the association to women but their published work provides the relevant data. When reanalyzed, their study also supports an association of this SNP in women (OR: 0.53, 95% CI:
In the 1991 cohort, there was a trend for association with susceptibility to MS for the 5V( 659) SNP ( p = 0.072). Specifically, carriers of the 5V( 659)*T allele had increased risk of MS ( p = 0.006; OR: 2.02, 95% CI: 1.23 – 3.32), primarily due to a difference in the frequency of heterozygotes (Table 3). Despite the strong linkage disequilibrium between the three markers in CD95L ( p < 0.0001), none of the other markers was associated with MS ( p>0.10). The overall distribution of haplotypes was significantly different between cases and controls (simulated p = 0.016) (Table 3). The 5V( 659)*T/I2(1174)*T/I2(2777)*A haplotype was associated with increased risk of MS (OR: 2.63, 95% CI: 1.01 – 6.86). The haplotypes including 5V( 659)*C were more frequent in controls and the haplotypes including 5V( 659)*T were more frequent in cases. (Table 3). The 5V( 659) SNP of CD95L was further studied in the combined cohort. The overall trend was lost
Table 3 Genotype and haplotype distribution in CD95L in the 1991 and combined cohorts Marker
1991
Combined***
MS
5V( 659)
I2(1174)
I2(2777)
5V( 659)/I2(1174)/ I2(2777)
CC CT TT CC CT TT AA AG GG C/C/A C/C/G C/T/A C/T/G T/C/A T/C/G T/T/A T/T/G
Control
p*
N
%
N
%
28 77 17 18 61 43 27 62 33 – **
23.0 63.1 13.9 14.8 50.0 35.2 22.1 50.8 27.0 0.0
91 114 37 41 121 82 56 110 77 –
37.6 47.1 15.3 16.8 49.6 33.6 23.0 45.3 31.7 0.7
– – – – – – –
3.7 39.2 11.6 0.0 36.1 8.4 1.1
– – – – – – –
7.0 40.8 12.7 1.0 33.0 3.4 1.6
0.072
MS
Control
p
N
%
N
%
70 124 26
31.8 56.4 11.8
172 206 62
39.1 46.8 14.1
0.364
0.628
0.643
0.016
* Cochran – Armitage trend test. ** Due to imputation of haplotypes only frequencies are presented and simulated p is reported. *** Only the 5V( 659) polymorphism was studied in the combined cohort due to lack of association between the other polymorphisms and susceptibility to MS in the 1991 cohort.
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0.30 – 0.92 for carriers of the 5V( 670)*G allele; OR: 1.89, 95% CI: 1.08 –3.30 for homozygotes of the 5V( 670)*A allele) but not in men. No gender information is provided in the study by Huang et al. (2000). They found a trend in their unstratified population for the same genotypes with odds ratios (OR: 0.61, 95% CI: 0.37– 1.00 and OR: 1.64, 95% CI: 1.00 –2.69, respectively) similar to both our results and those of Van Veen et al. (2002). When all three studies are combined, being a homozygote for 5V( 670)*A is associated with increased risk of MS in the overall population (Table 2). We did not find a convincing association between CD95L and MS. The initial trend in the 1991 cohort became nonsignificant when reanalyzed in the combined cohort. Zayas et al. (2001) have reported an association between a CA repeat marker 46 kb upstream of CD95L and MS. The association was stronger in familial cases. Given the distance between the marker and CD95L, the association detected by Zayas et al. (2001) may not extend into CD95L. Alternatively, the differences between our studies may represent population heterogeneity. CD95 mutations are associated with a lymphoproliferative syndrome in mice and the human counterpart, ALPS (Rieux-Laucat et al., 2003). Our study does not address the question of presence or absence of mutations in MS patients compared to controls but rather establishes an association between common polymorphisms and MS. Given the extent of strong linkage disequilibrium between the promoter and the exon 7 polymorphisms, our findings may be due to functional polymorphism(s) or mutations in CD95 that are in linkage disequilibrium with the two polymorphisms studied here. Most ALPS mutations are found in the 3Vend of CD95, in exon 9 coding for the intracellular death domain, resulting in a failure to bind the FADD/MORT1 signaling protein and decreased caspase recruitment and activation (Martin et al., 1999). We found a stronger effect for E7(74) SNP in the 3V end of the gene, but the significance of this observation is uncertain given the relatively small number of markers we studied. ALPS mutations have been found in extracellular domains as well, but these are less penetrant than the intracellular mutations and hence are potentially more relevant to sporadic disease (Jackson et al., 1999). Alternatively, our findings could be due to linkage disequilibrium with another gene at a distance from CD95. In a similar effort to ours, the 5V( 670) SNP was recently found to be associated with early onset sporadic Alzheimer’s disease (Feuk et al., 2000) but not associated with late-onset or familial cases (Theuns et al., 2001). Same authors also studied extended haplotypes around CD95 and demonstrated that the effect was indeed limited to the promoter polymorphism (Feuk et al., 2002). Hence, direct functional effects of the polymorphisms studied here is possible. There is no functional effect know for the silent E7(75) SNP, but the promoter polymorphism modifies the consensus sequence of a transcriptional element GAS (gam-
ma activation site) from TTC(N)2 – 4GAA to TTC(N)2 – 4GG A. GAS is the binding site for IFNg-activated janus kinase– signal transducer and activator of transcription 1 (JAK – STAT1) signaling pathway (Huang et al., 1997; Aaronson and Horvath, 2002). Hence, one can speculate that this polymorphism may modify IFNg-induced CD95 expression and apoptosis of the target cell. In the case of oligodendroglia, the variant sequence with reduced function would reduce cell death and be favorable for patients who are destined to develop MS, while the allele with preserved function would be detrimental. In the case of autoreactive T or B cells, however, the variant sequence would result in decreased IFNg-induced apoptotic elimination of these cells that might predispose to or aggravate existing MS. Current evidence indicates that once MS develops IFNg administration is detrimental (Panitch et al., 1987). The effect of IFNg-induced CD95-mediated apoptosis may have different effects on different stages of induction and maintenance of MS. Further understanding of the allelic effects of the polymorphisms and correlation with apoptosis markers will be necessary to better appreciate the significance of our genetic data. It is possible that the resistance to CD95induced T-cell death in MS patients, as reported by Comi et al. (2000), could be related to CD95 polymorphisms as well as other defects in the CD95-mediated apoptosis pathway; comprehensive surveys for mutations in CD95 have not yet been conducted in MS. Depending on the genetic background of the mouse strain, lpr mutations may lead to decreased susceptibility to and severity of disease in EAE compared to the wild type (Suvannavejh et al., 2000). Likewise, in NOD mice with a mutation in the CD95 death domain, onset of diabetes is significantly delayed (Savinov et al., 2003). Despite the widespread SLE-like systemic autoimmunity, CNS inflammatory-demyelinating disease is not common in the ALPS, DALD or MAS pedigrees (Waishnaw et al., 1999; Ramenghi et al., 2000; Rieux-Laucat et al., 2003). These results suggest that the effect of CD95 on the autoimmune phenotype may differ depending on the presence or absence of other modifier genes and is more complex than defined for the prototypical ALPS families. Indeed, this is the case in Alzheimer’s disease where the effect of 5V( 670) SNP was strongest in patients who are also carriers of the ApoE*e4 allele (Feuk et al., 2000). Our study independently highlights that the association of the alleles of CD95 and susceptibility to MS are confined to women. This association may be one factor that explains why women have a higher risk of MS than men do. Our results suggest that having either one of the CD95 polymorphisms explains 28% of the risk of MS in women in Olmsted County, MN. Interestingly, the autoimmunity observed in lpr mice is also gender specific. Autoimmune hearing loss is more common in female lpr mice compared to male lpr mice (Trune and Kempton, 2002) and autoantibody production is higher in female lpr mice than male lpr mice (Tornwall et al., 1999). Estrogen receptor beta is
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expressed in low levels in thymic CD4/CD8 T cells in wild-type mice and at high levels in the peripheral CD4 / CD8 T cells in lpr mice (Tornwall et al., 1999). The inheritance of defective CD95-induced T-cell death and the associated increase in the frequency of autoimmune diseases and cancer in DALD pedigrees also appears to vary by gender and by lineage (paternal or maternal) (Ramenghi et al., 2000). Our results suggest that the role of CD95 mutations in determining increased susceptibility to women for MS and potentially other autoimmune disease is worthy of further study.
Acknowledgements This study has been supported by the Mayo Foundation and National MS Society, Grant no. RG-2870-A-2. Part of these results have been presented in part in the ECTRIMS/ ACTRIMS Meeting in Baltimore, 2002.
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