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Functional promoter haplotypes of the human FAS gene are associated with the phenotype of SLE characterized by thrombocytopenia

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies against intracellular antigens and tissue injury. Defective apoptosis of activated immune cells leads to the development of autoantibodies in SLE. FasL initiated apoptosis is central for peripheral tolerance. Fas deficiencies in humans and mice predispose toward systemic autoimmunity. SLE is conferred by many genes. The genetic effects may be concentrated by familial clustering or by stratifying of subphenotypes. We have tested polymorphisms and haplotypes in FAS and FASL for association to SLE or subphenotypes in 126 multiplex American SLE pedigrees and found association of the FAS codon214 ACC/T as well as the FAS−670G>A-codon214 ACC/T′ haplotype to thrombocytopenia in SLE. Furthermore we have functionally characterized the FAS/FASL promoter polymorphisms associated with SLE in other populations and demonstrate that the activity depends on the allelic variants as well as on the haplotype. The presence of FAS−670G, which affects STAT1 binding, leads to the highest activity. FASL−844C activity is modified by the cis acting −478A and, hence, the haplotype and not the individual variant, determines the promoter activity. We conclude that the FAS/FASL promoter haplotypes are functional and that polymorphisms in FAS may contribute to thrombocytopenia in SLE.

Introduction

Systemic lupus erythematosus (SLE), which is characterized by multiorgan involvement, autoantibody production and tissue injury, is a complex autoimmune disease where environmental as well as genetic factors are clearly important in disease development. The etiology of SLE has a definite genetic component as inferred from increased familial aggregation among siblings and an increased concordance rate among monozygotic twins compared to dizygotic twins.1, 2, 3 This is further supported by the identification of genetic associations of candidate gene polymorphisms4, 5 as well as the establishment of linkage to loci in genome-wide scans in SLE.6, 7, 8, 9, 10, 11, 12 In man, SLE is not often a single gene disorder; the genetic effect generally appears to be conferred by polymorphisms in multiple genes. However, the responsible genes have been difficult to identify since SLE is an unusually heterogeneous disease with a wide range of clinical manifestations among patients. In addition, a relatively large number of genetic effects appear to influence the process leading to the clinical expression and heterogeneity of SLE.

There is evidence for familial aggregation of the various clinical features of SLE.13, 14, 15 Familial subphenotypes of SLE may be the result of fewer genes compared with overall disease susceptibility, and stratifying by subphenotypes may reduce heterogeneity and facilitate the identification of gene polymorphisms associated with SLE. One such subphenotype, thrombocytopenia, is an independent marker for severe and deadly SLE,16, 17, 18, 19 and stratification for thrombocytopenia found evidence for linkage to 1q23, a region involving the FasL gene, as well as suggestive linkage to a region on 10q24 in close proximity to FAS.20

The Fas and FasL genes are considered possible candidate genes in human SLE, since evidence from animal models of lupus with defective Fas and FasL expression point to a role for FasL-mediated apoptosis in the pathogenesis of SLE. The naturally occurring MRL lpr (lymphoproliferative) mouse strain is considered a model for human SLE as these mice develop nephritis, hypergammaglobulinemia, and antinuclear antibodies in addition to lymphadenopathy. The genetic basis for the lpr mouse strain is an insertion of a transposon into intron 2 of the Fas gene that reduces the normal splicing of the FAS transcript leading to the virtual complete absence of Fas expression.21 The less severe lprcg strain expresses a nonfunctional Fas caused by a missense mutation within the death domain encoding part of the Fas gene.22 The gld mouse has a missense mutation in the extracellular domain of FasL, which abrogates its function.23 The phenotype of these mutations, when bred onto the autoimmune MRL background, is very similar. However, the autoimmune features of the lpr mutation vary on other strains, generating, for example, a less severe phenotype in C57BL6, 129 or C3H mice.24, 25

Fas is a membrane bound receptor belonging to the TNF-receptor superfamily of death receptors having three extracellular cystein-rich domains and a functional 80 amino-acid residue intracellular death domain. FasL is a type II transmembrane protein of 40 kDa, which belongs to the TNF family. Membrane bound FasL is proteolytically cleaved by metalloproteinases to produce a soluble form. Binding of Fas to its ligand, FasL, triggers the formation of the death-inducing signaling complex (DISC) by recruiting the adaptor molecule FADD, which in turn activates caspase 8. The death receptor initiated pathway then diverges depending on cell type. In type I cells (eg thymocytes, T cells), caspase 8 activates caspase 3 leading to apoptosis, while in type II cells (eg hepatocytes) the mitochondrial pathway releasing cytochrome c is activated followed by formation of the apoptosome, which together with procaspase 9 activates caspase 3. This latter pathway can be inhibited by Bcl-2.26, 27 The FasL–Fas mediated pathway plays a major role in maintaining immune homeostasis, particularly in activation induced cell death of activated lymphocytes in the periphery after the elimination of antigen, and in the negative selection of thymocytes in the thymus. B-cell homeostasis also seems to be regulated by FasL–Fas interaction.28 FASL is one of the major effectors of T-cell mediated cytotoxicity. Fas has also been implicated in costimulation and possibly proliferation of memory T cells.29

We previously scanned the human Fas and FasL genes and identified several sequence variants.30, 31 The aim of this study was to test the frequent polymorphisms in FAS and FASL for association to SLE, and, especially, to distinctive SLE phenotypes and to test the functional variation of haplotypes of the FAS and FASL promoters upon gene expression.

Results

Association studies

In a screening panel of 23 SLE patients and 23 matched controls, we initially determined the frequency of six variants in FAS and two in FASL, which we identified earlier in a complete mutational scan of these genes.30, 31 From these we selected three single-nucleotide polymorphisms (SNPs) in FAS and two in FASL, and genotyped the 681 family members of 126 American multiplex SLE pedigrees for these as well as a FASL microsatellite in the 3′UTR.32 Of the FAS polymorphisms evaluated, two were located to the promoter: −690C>T (dbSNP rs 2234758) in a possible mutational hotspot and −670G>A (dbSNP rs 1800682) in a GAS (gamma activation site) binding site.21 The third is a silent mutation in codon214 (ACC to ACT) (dbSNP rs 2234978) in exon 7, which does not lead to an amino-acid change.

Two FASL polymorphisms, −844C>T and −478A>T, were in the promoter. The −844C>T (dbSNP rs 763110) has been shown to affect the binding site of the transcription factor enhancer-binding protein beta element (EBPβ) and the promoter activity of FASL.26 The microsatellite is a dinucleotide repeat (GT) in 3′UTR of FASL (dbSNP rs10640513) very close to exon 4 and might have an effect upon mRNA stability. The distribution and frequencies of the alleles of these FAS and FASL polymorphisms from the founders in the 126 pedigrees evaluated are shown in Table 1.

Table 1 Distribution of the FAS and FASL polymorphism alleles in the 126 lupus pedigrees from 147 founders

Polymorphisms in FAS and FASL genes were tested individually for association to SLE in the 126 American multiplex SLE pedigrees using the Transmission Disequilibrium Test (TDT) and Pedigree Disequilibrium Test (PDT). There were between 26 and 87 informative transmission with odds ratios from 1.03 to 1.58, obtaining χ2 values of 0.6 to 1.6. The χ27df value obtained for the FASL microsatellite was 4.503 (P-value=0.7203). Haplotypes were established from the same polymorphisms and tested for association to SLE by the GENEHUNTER-TDT. In FAS we tested haplotypes comprising the −690T>C, −670G>A and codon214ACC/T in exon 7 as well as haplotypes comprising only the −670G>A and codon214ACC/T for association to SLE by the PDT. Pairwise LD between SNPs were: −690′ −670′: 0.645 (All) 1.0 (EA) 1.0 (AA), −670′ codon214ACC/T′: 0.579 (All) 0.588 (EA) 0.181 (AA) and −690′ codon214ACC/T′: 1.00 (All), 1.0 (EA), 0.996 (AA). We found no significant association of the −690T>C, −670G>A, codon214ACC/T haplotype to SLE (χ26df=4.133, P-value 0.659). In FASL we tested haplotypes from the −844′ FASL microsatellite′. Pairwise LD between markers were: −844′−478′: 0.60 (All), 0.840 (EA), 0.194 (AA); −478′ microsatellite′: 0.742 (All), 0.879 (EA), 1.0 (AA); and −844′ microsatellite′: 0.259 (All), 0.410 (EA), 0.519 (AA). Thus, we found no significant association of any of the FAS or FASL haplotypes to SLE in the 126 SLE pedigrees (data not shown).

Association with SLE subphenotypes

As certain SLE traits show familial aggregation, we stratified the 126 pedigrees by the presence of different clinical manifestations of SLE (thrombocytopenia, nephritis, oral ulcers, photosensitivity, and antibodies to double-stranded DNA, Sm, nuclear ribonuclear protein (nRNP), Ro and La) in any affected in the pedigree. We then tested these stratified pedigrees separately for association to the same FAS and FASL polymorphisms and haplotypes.

We found significant association of the codon214ACC/T polymorphism in FAS with the severe phenotype of SLE characterized by thrombocytopenia (P=0.006, uncorrected) in all pedigrees (Table 2). The definition criteria of thrombocytopenia is <100 000 platelets per microliter. We then constructed a mild SLE subphenotype using only affected individuals with antibodies to nRNP and tested the codon214ACC/T polymorphism for association to this phenotype. We found no association of codon214ACC/T with the mild phenotype (Table 2).33

Table 2 TDT results using thrombocytopenia and nRNP as SLE severe and mild subphenotypes, respectively

To determine whether an FAS haplotype spanning a large part of the gene would show the same association in the SLE pedigrees with thrombocytopenia, we established haplotypes from the two frequent polymorphisms, codon214ACC/T in exon 7, and −670G>A affecting a GAS binding site in the promoter, and analyzed haplotypes for association. We found significant evidence for association for the −670–codon214′ haplotype in these pedigrees (genehunter TDT P<0.005) (Table 3). Haplotypes 1 and 3 were preferentially transmitted to affected subjects (Table 4). The −670–codon214′ haplotype was, however, not associated with the general SLE phenotype (data not shown).

Table 3 Association results for the FAS −670′ −codon214′ haplotype in the thrombocytopenia subgroup of families
Table 4 GENEHUNTER-TDT results for the FAS −670′ −codon214′ haplotype using thrombocytopenia as the SLE phenotype

DNA Luciferase assay

FAS promoter haplotypes

There are four naturally occurring haplotypes in the −1372 to +1 Fas promoter region comprising three polymorphisms: −1194 A>T, −690T>C and −670G>A. The FAS promoter −1194A−690T−670G haplotype, which lacks the consensus binding site for GAS (STAT1) in position −670, showed the highest constitutive as well as interferon gamma (IFNγ) and phorbol myristic acid (PMA) stimulated activity when compared to the three other haplotypes that all have the consensus binding site for STAT1 (Figure 1). The expression level of the haplotypes with the preserved STAT1 consensus binding site was more than 40% reduced under all three conditions. There was no significant difference in the promoter activity of the −1194A−690T−670A and −1194A−690C−670A haplotypes, while the −1194T−690T−670A haplotype, which has a mutation of the c-Myb binding site, showed further reduction of the constitutive, IFNγ, and PMA stimulated activities. The FAS −1194A>T SNP, which abolishes a c-Myb binding site and thus is interesting from a functional point of view, is too rare (T allele frequency 0.006) to be tested for association to SLE or SLE subphenotypes. In Figure 1 we have combined the results from the −1194A−690T−670A, −1194T−690T−670A and −1194A−690C−670A haplotypes (here denoted −670A) and compared them to the−1194A−690T−670G haplotype (denoted −670G). The difference in the promoter activity between the −670G haplotype and the −670A haplotype is highly significant when the individual haplotypes or the combined haplotypes are compared. The activity of the insert harboring the −670G allele compared to the combined activities from the three inserts harboring the −670A allele are impressively different, P=0.000022, 0.000039, and 0.003 under unstimulated, PMA and IFNγ stimulated conditions, respectively.

Figure 1
figure1

Constitutive, PMA and IFNγ stimulated activity of the Fas 1372-pGL3E reporter constructs with the four −1194A>T-690T>C670G>A haplotypes transfected into Jurkat cells. Jurkat cells were transiently transfected with the Fas 1372-pGL3E reporter containing either the −1194A690T670G, the −1194A690T670A, −1194A690C670A or the −1194T690T670A haplotype. The Luciferase activities of the three inserts harboring the −670A allele, which binds STAT1, have been combined and are denoted as −670A. The construct with the −1194A−690T−670G haplotype is referred to as −670G. Cells were left unstimulated or stimulated for 17 h with either PMA (25 pg/ml) or IFNγ (200 ng/ml). Activity is expressed as the Firefly/Renilla ratio. Columns are average promoter activity (N=6) and error bars are s.e.m. ANOVA P-value=4.9E-38. P-value (T-test) when comparing the activity of the insert harboring the −670G allele with the combined activities from the three inserts harboring the −670A allele, P=0.000022, 0.000039, and 0.003 under unstimulated, PMA and IFNγ stimulated conditions, respectively.

FASL promoter haplotypes

The FASL promoter activity depends on the haplotype and not the individual polymorphisms (Figure 2). The activity of the −844C allele, which has a consensus binding site for EBPβ, depends on the cis allele of the −478 polymorphism. The −844C−478A haplotype shows the highest activity compared to all other haplotypes. The constitutively and PMA stimulated activities of the −844C−478A and −844T−478A haplotypes are different, P=0.003 and 0.03. The activities of the of the −844C−478T and −844T−478T haplotypes of the constitutively and PMA stimulated activities are also different, P=0.0004 and 0.002, respectively (Figures 3 and 4).

Figure 2
figure2

Effect of the naturally occurring −844C>T478A>T haplotypes on FASL promoter activity in Jurkat cells. Jurkat cells were transiently transfected with the Fasl pGL3E reporter containing either the −844C478A, the −844T478A, the −844C478T or the −844T478T haplotype. Haplotypes harboring the −844C allelic variant (shaded columns) can bind EBPβ. Cells were left unstimulated or stimulated for 17 h with PMA (25 ng/ml). The activity of the individual haplotypes is expressed as the average Firefly/Renilla ratio of activities (N=6). Error bars are s.e.m. P-value ANOVA=7.1E-16. The constitutively and PMA stimulated activities of the −844C478A and −844T478A haplotypes are different, P (T-test)=0.003 and P (T-test)=0.03. The activities of the −844C478T and −844T478T haplotypes of the constitutively and PMA stimulated activities are also different, P (T-test)=0.0004 and P (T-test)=0.002, respectively.

Figure 3
figure3

Diagram of FAS with locations of SNPs: −690C>T and −670G>A in the promoter and 214ACC/T in exon 7. Haplotypes −690′−670′ codon214ACC/T′ and −670′ codon214ACC/T′ and are indicated. Pairwise LD between markers were: −690′, −670′: 0.645, −670′ codon214ACC/T′: 0.579 and −690′ codon214ACC/T′: 1.00.

Figure 4
figure4

Diagram of FASL with locations of markers: −844C>T and −478A>T in the promoter and FASL microsatellite in 3′UTR. Haplotypes −844′ −478′ microsatellite′ and −844′ microsatellite′ are indicated. Pairwise LD between markers were: −844′ −478′: 0.60, −478′ microsatellite′: 0.742 and −844′ microsatellite′: 0.259.

There is no significant difference in the basal or PMA stimulated promoter activity of the −844T−478T and −844T−478A haplotypes.

Discussion

We demonstrate for the first time that the presence of the FAS −670A allele in a consensus binding site for the transcription factor GAS lowers the activity of the human FAS promoter. This −670G>A polymorphism in the FAS promoter is part of an extensive (21884 base pairs) haplotype, spanning a substantial part of the human Fas gene and associated with the phenotype of SLE characterized by thrombocytopenia. We find a highly significant association of a silent codon214 ACC/T polymorphism in exon 7 included in this haplotype with the SLE subphenotype, thrombocytopenia. We also show that the activity of the human FASL promoter depends on a −844C>T−478A>T haplotype and not on the individual alleles of either polymorphism. The higher promoter activity of the −844C allele compared to the −844T allele is only present if the −478A allele is in cis. If −478A is in trans this relationship is reversed.

There was no overall association to SLE for any of the FAS or FASL polymorphisms or haplotypes. However, in animal models of SLE, mutations in FAS and FASL are not considered to be sufficient to cause SLE, but rather to contribute to autoimmunity depending on the general genetic background. In the phenotype of SLE characterized by thrombocytopenia, suggestive linkage in areas including the FAS and FASL genes has been established,20 and we therefore tested the a priori hypothesis that FAS and FASL might contribute to SLE subphenotypes rather than the SLE phenotype itself.

The FAS −670G>A polymorphism has been tested in SLE in three case–control studies in three different ethnic groups, namely Japanese, Korean and Australian SLE patients and healthy controls. The −670A allele was found to be associated with SLE in a case–control study comprising 109 Japanese patients and 140 controls.34 The authors demonstrated in an electromobility shift assay that only the −670A allele was able to bind the transcription factor STAT1, but they were unable to show a significant difference in promoter activity of the −670A and −670G alleles in a Luciferase reporter study. A possible explanation for this could be the use of the pGL3 basic vector instead of the pGL3 enhancer vector, used by us, and the transfection of an ovarian carcinoma cell line, HeLa, instead of the more relevant Jurkat T cell line. Also, their promoter insert was several hundred base pairs shorter and did not include the silencer region of the promoter between −1035 and −1008.35

The −670A allele as well as the −670AA genotype were found to be associated with the presence of anti-RNP antibodies in a case–control study comprising 87 Korean SLE patients and 87 controls and confirmed in second Korean SLE cohort of 85 patients.36 Interestingly, we found marginal significance of association (P<0.03) between the −670 A>G polymorphism and the presence of anti-RNP antibodies using the TDT. In a case–control study in 79 Australian SLE patients and 86 controls, the −670 AA genotype was found to be associated with photosensitivity or oral ulcers.37

When comparing these three studies with our results, it seems likely that the −670 A>G polymorphism is a genetic contributor to different SLE traits. In our study the −670A allele led to a significantly lower constitutive promoter activity, a difference that was sustained after PMA or IFNγ stimulation.

The FASL −844C>T promoter polymorphism has been tested in a case–control study of 211 African-American SLE patients and 150 controls,38 and the −844CC genotype was found to be associated with SLE. In the same study it was demonstrated that the −844C allele specifically binds the transcription factor EBPβ and that the −844C allele has a higher promoter activity compared to the −844T allele in a Luciferase reporter assay. We could not confirm the association to SLE using the TDT in our family collection, not even in the 40 African-American families. We further explored the functional consequences of this polymorphism as well as the −478A>T, and could demonstrate that the allele at −478 is crucial for which of the alleles at −844 is most active, and conclude that the haplotype and not the individual alleles determine promoter activity. Even so, the haplotype is not associated in our hands.

We think that FAS and FASL should be considered candidate contributory genes in human SLE. Polymorphisms in these genes do not seem to be sufficient to cause SLE as a phenotype, but may, depending on the genetic background of the individual, act as permissive genes important for the development of the phenotype of SLE characterized by thrombocytopenia. Fas and FasL may exert their effects through modulation of peripheral tolerance.

We found an association of the FAS −670–codon214′ haplotype as well as the codon214ACC/T polymorphism to the SLE phenotype characterized by thrombocytopenia, but no association of the functional −670A>G polymorphism to this phenotype. There are two possible explanations: (1) There might be an unknown functional polymorphism within the −670–codon214′ haplotype, which is in tight linkage disequilibrium with the codon214ACC/T, but not the −670G>A polymorphism and as intron 1 is very large, there might be hitherto unknown functional polymorphisms in this intron, which could explain the genetic association we observed. The HapMap shows strong linkage disequilibrium in FAS. (2) The silent codon214ACC/T polymorphism in exon 7 by itself might lead to the observed phenotypic variability by influencing exonic splicing accuracy or efficiency. The phenomenon of exonic splicing skipping is known from other diseases, such as the neuromuscular disease, Spinal muscular atrophy, as reviewed in Cartegni et al.39 However, the methods for predicting exonic splicing enhancers are still under development. Our results also need to be confirmed in another cohort, as our sample size is relatively small. Clearly, further genetic as well as in vivo studies are required to establish the possible explanations for the genetic association we describe here of the FAS polymorphism and haplotype associated with the SLE phenotype characterized by thrombocytopenia. We conclude that promoter variants in the FAS and FASL genes are functional. The functional promoter variant in FAS is included in a haplotype spanning a substantial part of FAS, which is associated with a severe form of SLE.

Methods

Subjects

SLE patients and their families were enrolled in the lupus genetics study as previously described.6 Medical records were obtained on each potential affected who also completed an extensive questionnaire and interview with a trained physician's assistant or registered nurse to determine clinical manifestations and establish diagnosis. A less comprehensive questionnaire was completed by unaffected family members, but they were also screened for the presence of SLE. The resulting sample contained 126 multiplex SLE pedigrees (77 European-American, 40 African-American and 9 other), including 681 individuals, 294 of which were classified as affected.

DNA isolation

Upon obtaining informed consent, blood samples and/or buccal swabs were collected from each of the participants. Genomic DNA was isolated from peripheral blood mononuclear cells, buccal cell swabs or EBV-transformed cell lines using standard methods.

Genotyping

In a screening panel of SLE patients and matched controls we initially tested the frequency of six FAS and two FASL SNPs, earlier identified by us.30, 31

PCR-restriction fragment length polymorphism (PCR-RFLP) genotyping of the −690T>C and the −670G>A polymorphisms in the FAS promoter and the codon214ACC/T polymorphism in exon 7 of FAS

The presence of a T at −690 and of a G at −670 of the FAS promoter creates an Nde1 and an Mva1 restriction enzyme site, respectively, and the presence of a T at nucleotide 154 in exon 7 creates a Dra1 restriction enzyme site.

FAS promoter primers, which yield a PCR product of 234 bp, were: forward 5′ IndexTermAGCTTTGTTTTCCTCTTGAGA and reverse 5′ IndexTermCAGAGCGCAGAGGTCC. FAS exon 7 primers were: forward 5′ IndexTermTCTCACATGCATTCTACAAGG and reverse 5′ IndexTermGCAAGACTCCATCTCAAACAA and give a PCR product of 261 bp.

PCR conditions

Genomic DNA (3 ng) was amplified in a 20 μl reaction volume containing in final concentrations: 0.8 μM forward and reverse primers, 50 μM dNTPs, 1 × reaction buffer with 1.0–1.5 mM MgCl2 and 0.5 U of Taq DNA Polymerase (Promega Corporation, Madison, WI, USA). The reactions were carried out in 96-well microtiter plates on a thermal cycler. PCR cycling conditions were 95°C (5 min) for one cycle followed by 95°C (30 s), 52–56°C for different primers (30 s), and 72°C (30 s) for 35 cycles. A final cycle of 72°C for 10 min completed the reaction.

Digest conditions

The amplified PCR products from the FAS promoter and from exon 7(10 μl) were digested with restriction enzymes in a 20 μl final reaction volume using 2 μl of the accompanying reaction buffer. The PCR amplicon from the FAS promoter was digested with 5 U of Nde1 (Promega Corporation, Madison, WI, USA) for 4 h in Buffer D and with 4 U of Mva1 (Boehringer Mannheim Biochemica, Mannheim, Germany) for 16 h in Buffer H. The PCR amplicon from FAS exon 7 was digested with 5 U of Dra1 (Promega Corporation, Madison, WI, USA) for 16 h in Buffer B. All digestions were carried out at 37°C. Controls of known sequenced genotypes were included for every set of digestions carried out. The digested products were resolved on a 2% agarose gel stained with ethidium bromide and visualized using UV transillumination.

Typing for the −844C>T FASL promoter polymorphism

We typed for the −844C>T polymorphism by mutagenically separated PCR (MS-PCR).40

Primer sequences were: forward primer (T allele): 5′IndexTermAATGAAAATGAAAACATTGT, forward primer (C allele): 5′IndexTermTAAATAAGTAAATAAATAAACTGGGCAAACCTTGAAAATGAAAACAACGC and reverse primer: 5′IndexTermCCCACTTTAGAAATTAGATC and yield PCR products of 114 and 84 bp for the C and T allele, respectively. The PCR conditions with 3 ng of genomic DNA per sample are described in detail in Nolsoe et al.30

Typing for the −478A>T FASL promoter polymorphism

We genotyped for the −478A>T promoter variant by analysis of PCR-generated single base primer extension products using the ABI Prism® SNaPshot™ Multiplex kit from Applied Biosystems. The sequences of the amplification primers and extension primer, respectively, are 5′IndexTermGGGAAGAGATGATGGCAACA (forward primer), 5′IndexTermGAGAATGGTCAGTGGGGCTAT (reverse primer) and 5′ IndexTermTATGAATTATAATTGCATGCT (extension primer).

FASL microsatellite

Using biotinylated primers we typed the FASL (GT)n repeat by PCR of 3 ng genomic DNA followed by PAGE, blotting and visualization utilizing a streptavidin-peroxidase (Sigma, St Louis, MO, USA) catalyzed ECL reaction (Amersham Life Science, Cleveland, OH, USA) as described in detail in Nolsoe et al.31 Forward primer 5′IndexTermBiotACTTCTAAATGCATATCCTGAGCC and reverse primer 5′IndexTermATCTTGACCAAATGCAACCC yield PCR amplicons varying between 211 and 225 basepairs corresponding to 13–20 GT repeats. Samples homozygous for the different alleles were confirmed by sequencing on an ABI Prism 310 DNA sequencer system (Perkin Elmer, Foster City, CA, USA).

Analysis

Prior to any analysis, sibling, half-sibling, and parent–offspring relationships were confirmed using statistics generated by RELTEST, a feature of SAGE (Statistical Solutions Ltd, Cork, Ireland).

In addition, we stratified pedigrees on the presence of certain clinical manifestations (thrombocytopenia, nRNP, nephritis, oral ulcers, photosensitivity, antibodies to double-stranded DNA, antibodies to Sm, antibodies to Ro and antibodies to La) and analyzed them separately.

Association analysis

The TDT41 and PDT42 were used to assess possible association of the FAS and FASL polymorphisms with SLE. Haplotypes were generated and analyzed from GENEHUNTER-PLUS.43

Linkage disequilibrium values were calculated as follows: Pairwise linkage disequilibrium. Hardy–Weinberg equilibrium was assessed by the simple chi-square test at each locus. Linkage disequilibrium estimates for marker pairs were obtained by first estimating two-locus haplotype frequencies from genotypic data using a likelihood-based approach. Estimated haplotype frequencies from the product of relevant allele frequencies (ie the expected haplotype frequencies under linkage equilibrium) were then subtracted to arrive at the standard measure of linkage disequilibrium.44 Linkage disequilibrium was then standardized to vary between −1.0 and 1.0 using traditional allele frequency corrections to arrive at the standardized measure of linkage disequilibrium.45 Since the focus was on the strength of linkage disequilibrium, the sign was ignored. Statistical significance was measured by the χ2 test. All pairwise LD were statistically significant at the α=0.05 level.

Measurement of the functional activity of FAS and FASL promoter haplotypes by Luciferase activity

Reporter gene constructs

FAS: The four different naturally occurring haplotypes of the FAS promoter region −1372 to +1 containing the −1194 A>T, −690T>C and −670G>A polymorphic sites were cloned into the pGL3 Enhancer Luciferase Vector (Promega). Upper primer 5′ IndexTermCAGGTACCGGGTCTTCCTCATGGCACTA and lower primer 5′ IndexTermTACAAGCTTGAGCAATCCTCCGAAGTGAAA introducing a KpnI site at the 5′ and a HindIII site at the 3′ of the fragments were used to PCR genomic DNA from individuals with different haplotypes of the FAS promoter. The resultant KpnI–HindIII fragments were cloned into the KpnI–HindIII sites of the pGL3 Enhancer Luciferase Vector using the Original TA cloning® kit (Invitrogen, Carlsbad, CA, USA). Promoter positive clones were identified by PCR using the vector specific forward (5200) and reverse (5201) primers as well as forward primer 5′ IndexTermAGCTTTGTTTTCCTCTTGAGA and reverse primer 5′ IndexTermCAGAGCGCAGAGGTCC, which amplifies the FAS promoter around the −690T>C and −670G>A polymorphisms. The clones were sequenced to verify the presence of the various polymorphic sites. The four naturally occurring haplotypes of the FAS promoter were:

1194A −690T −670G, −1194A −690T −670A, −1194A −690C −670A and −1194T −690T −670A.

The −1194A −690T −670G haplotype lacks the (GAS) STAT1 binding site in −670, and the −1194T −690T −670A haplotype lacks the c-Myb binding site in −1194.

FASL: The four different naturally occurring haplotypes of the FASL promoter region −870 to +100 comprising the −844C>T −478A>T polymorphic sites were cloned into the pGL3 Enhancer Luciferase Vector (Promega) as described above for the FAS promoter constructs. The −200 to +100 region of the promoter region is essential for TCR-mediated FasL activation.46 Upper primer 5′ IndexTermTAGGTACCTAAATAAACTGGGCAAACAATG and lower primer 5′ IndexTermCTGAAGCTTGGCAGCTGGTGAGTCAGG introducing a KpnI site at the 5′ and a HindIII site at the 3′ of the fragments were used to PCR genomic DNA from individuals with different haplotypes of the FASL promoter. The resultant KpnI–HindIII fragments were cloned into the KpnI–HindIII sites of the pGL3 Enhancer Luciferase Vector using the Original TA cloning® kit (Invitrogen, Carlsbad, CA, USA). Promoter positive clones were identified by PCR using the vector specific forward (5200) and reverse (5201) primers as well as forward primer 5′IndexTermAAATAAACTGGGCAAACAATG and reverse primer 5′ IndexTermGGCCAGAGAAGTCACTCC, which amplifies the FASL promoter around the −844C>T −478A>T polymorphisms. The clones were sequenced to verify the presence of the various polymorphic sites. The four naturally occurring haplotypes of the FASL promoter were: −844C −478A, −844T −478A, −844C −478T and −844T −478T.

Luciferase-reporter assay

The human Jurkat Thelper cell line was cultured in RPMI supplemented with 10% FCS (Gibco), penicillin and streptomycin. Cells were grown at 37°C in 5% CO2.The cells were transfected during the log phase of their growth.

Using Superfect™ Jurkat T cells at a concentration of 0.8 × 106 cells/ml were transfected with 2.5 μg/ml plasmid DNA (85% Fas or FaslpGL3 enhancer vector construct and 15% Tyrosine Kinase-Promoter Renilla Luciferase internal vector control (Promega) TK-pRL vector) for 2 h.

The cells were then centrifuged and resuspended in RPMI and then left unstimulated or stimulated for 17 h with either PMA (25 pg/ml) or IFNγ (200 ng/ml). These concentrations were found after titrations. In each experiment the individual constructs variants were included in triplicates. Cells were lysed and quantitation of luminescence was performed using the Dual-Luciferase™ kit and a Lumat LB 9507 luminometer.

The activity of the individual constructs is expressed as

Firefly activity is normalized by the Renilla activity, and the results are expressed as the Firefly/Renilla ratio of activities.

Statistical analysis

Initially, the ANOVA (one-sided analysis of variance) was used to compare differences between groups and when significant at P<0.05, the t-test (paired) was used to compare experimental ratios between different clones. Results with a P<0.05 were considered significant. Results are shown as averages±standard error of the mean (s.e.m.).

References

  1. 1

    Harley JB, Moser KL, Gaffney PM, Behrens TW . The genetics of human systemic lupus erythematosus. Curr Opin Immunol 1998; 10: 690–696.

    CAS  Article  Google Scholar 

  2. 2

    Deapen D, Escalante A, Weinrib L et al. A revised estimate of twin concordance in systemic lupus erythematosus. Arthritis Rheum 1992; 35: 311–318.

    CAS  Article  Google Scholar 

  3. 3

    Kelly JA, Moser KL, Harley JB . The genetics of systemic lupus erythematosus: putting the pieces together. Genes Immun 2002; 3 (Suppl 1): S71–S85.

    CAS  Article  Google Scholar 

  4. 4

    Salmon JE, Millard S, Schachter LA et al. Fc gamma RIIA alleles are heritable risk factors for lupus nephritis in African Americans. J Clin Invest 1996; 97: 1348–1354.

    CAS  Article  Google Scholar 

  5. 5

    Arnett FC . The genetics of human lupus. In: Wallace DJ, Hahn BH (eds). Dubois' Lupus Erythematosus. Williams & Wilkins: Baltimore, 1997, pp 77–117.

    Google Scholar 

  6. 6

    Moser KL, Neas BR, Salmon JE et al. Genome scan of human systemic lupus erythematosus: evidence for linkage on chromosome 1q in African-American pedigrees. Proc Natl Acad Sci USA 1998; 95: 14869–14874.

    CAS  Article  Google Scholar 

  7. 7

    Gaffney PM, Kearns GM, Shark KB et al. A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families. Proc Natl Acad Sci USA 1998; 95: 14875–14879.

    CAS  Article  Google Scholar 

  8. 8

    Lindqvist AKB, Steinsson K, Johanneson B et al. A susceptibility locus for human systemic lupus erythematosus (hSLE1) on chromosome 2q. J Autoimmun 2000; 14: 169–178.

    CAS  Article  Google Scholar 

  9. 9

    Gaffney PM, Ortmann WA, Selby SA et al. Genome screening in human systemic lupus erythematosus: results from a second Minnesota cohort and combined analyses of 187 sib-pair families. Am J Hum Genet 2000; 66: 547–556.

    CAS  Article  Google Scholar 

  10. 10

    Shai R, Quismorio FP, Li L et al. Genome-wide screen for systemic lupus erythematosus susceptibility genes in multiplex families. Hum Mol Genet 1999; 8: 639–644.

    CAS  Article  Google Scholar 

  11. 11

    Gray-Mcguire C, Moser KL, Gaffney PM et al. Genome scan of human systemic lupus erythematosus by regression modeling: evidence of linkage and epistasis at 4p16–15.2. Am J Hum Genet 2000; 67: 1460–1469.

    CAS  Article  Google Scholar 

  12. 12

    Koskenmies S, Lahermo P, Julkunen H, Ollikainen V, Kere J, Widen E . Linkage mapping of systemic lupus erythematosus (SLE) in Finnish families multiply affected by SLE. J Med Genet 2004; 41: e2–e5.

    CAS  Article  Google Scholar 

  13. 13

    Tsao BP, Grossman JA, Riemekasten G et al. Familiality and co-occurrence of clinical features of systemic lupus erythematosus. Arthritis Rheum 2002; 46: 2678–2685.

    Article  Google Scholar 

  14. 14

    Rao S, Olson JM, Moser KL et al. Linkage analysis of human systemic lupus erythematosus-related traits: a principal component approach. Arthritis Rheum 2001; 44: 2807–2818.

    CAS  Article  Google Scholar 

  15. 15

    Olson JM, Song Y, Dudek DM et al. A genome screen of systemic lupus erythematosus using affected-relative-pair linkage analysis with covariates demonstrates genetic heterogeneity. Genes Immun 2002; 3 (Suppl 1): S5–S12.

    CAS  Article  Google Scholar 

  16. 16

    Drenkard C, Villa AR, Alarcon-Segovia D, Perez-Vazquez ME . Influence of the antiphospholipid syndrome in the survival of patients with systemic lupus erythematosus. J Rheumatol 1994; 21: 1067–1072.

    CAS  PubMed  Google Scholar 

  17. 17

    Mok CC, Lee KW, Ho CT, Lau CS, Wong RW . A prospective study of survival and prognostic indicators of systemic lupus erythematosus in a southern Chinese population. Rheumatology (Oxford) 2000; 39: 399–406.

    CAS  Article  Google Scholar 

  18. 18

    Reveille JD, Bartolucci A, Alarcon GS . Prognosis in systemic lupus erythematosus. Negative impact of increasing age at onset, black race, and thrombocytopenia, as well as causes of death. Arthritis Rheum 1990; 33: 37–48.

    CAS  Article  Google Scholar 

  19. 19

    Ward MM, Pyun E, Studenski S . Mortality risks associated with specific clinical manifestations of systemic lupus erythematosus. Arch Intern Med 1996; 156: 1337–1344.

    CAS  Article  Google Scholar 

  20. 20

    Scofield RH, Bruner GR, Kelly JA et al. Thrombocytopenia identifies a severe familial phenotype of systemic lupus erythematosus and reveals genetic linkages at 1q22 and 11p13. Blood 2003; 101: 992.

    CAS  Article  Google Scholar 

  21. 21

    Adachi M, Watanabe-Fukunaga R, Nagata S . Aberrant transcription caused by the insertion of an early transposable element in an intron of the Fas antigen gene of lpr mice. Proc Natl Acad Sci USA 1993; 90: 1756–1760.

    CAS  Article  Google Scholar 

  22. 22

    Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S . Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 1992; 356: 314–317.

    CAS  Article  Google Scholar 

  23. 23

    Takahashi T, Tanaka M, Brannan CI et al. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 1994; 76: 969–976.

    CAS  Article  Google Scholar 

  24. 24

    Nagata S, Golstein P . The Fas death factor. Science 1995; 267: 1449–1456.

    CAS  Article  Google Scholar 

  25. 25

    Giese T, Davidson WF . Chronic treatment of C3H-lpr/lpr and C3H-gld/gld mice with anti-CD8 monoclonal antibody prevents the accumulation of double negative T cells but not autoantibody production. J immunol 1994; 152: 2000.

    CAS  PubMed  Google Scholar 

  26. 26

    Budihardjo I, Oliver H, Lutter M, Luo X, Wang X . Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 1999; 15: 269–290.

    CAS  Article  Google Scholar 

  27. 27

    Cohen GM . Caspases: the executioners of apoptosis. Biochem J 1997; 326 (Part 1): 1–16.

    CAS  Article  Google Scholar 

  28. 28

    Fukuyama H, Adachi M, Suematsu S et al. Requirement of Fas expression in B cells for tolerance induction. Eur J Immunol 2002; 32: 223–230.

    CAS  Article  Google Scholar 

  29. 29

    Inaba M, Kurasawa K, Mamura M, Kumano K, Saito Y, Iwamoto I . Primed T cells are more resistant to Fas-mediated activation- induced cell death than naive T cells. J Immunol 1999; 163: 1315–1320.

    CAS  Google Scholar 

  30. 30

    Nolsoe RL, Kristiansen OP, Sangthongpitag K et al. Complete molecular scanning of the human Fas gene: mutational analysis and linkage studies in families with type I diabetes mellitus. The Danish Study Group of Diabetes in Childhood and The Danish IDDM Epidemiology and Genetics Group. Diabetologia 2000; 43: 800.

    CAS  Article  Google Scholar 

  31. 31

    Nolsoe RL, Kristiansen OP, Larsen ZM, Johannesen J, Pociot F, Mandrup-Poulsen T . Complete mutation scan of the human Fas ligand gene: linkage studies in Type I diabetes mellitus families. Diabetologia 2002; 45: 134.

    CAS  Article  Google Scholar 

  32. 32

    Sunden SL, Alward WL, Nichols BE et al. Fine mapping of the autosomal dominant juvenile open angle glaucoma (GLC1A) region and evaluation of candidate genes. Genome Res 1996; 6: 862.

    CAS  Article  Google Scholar 

  33. 33

    Reichlin M, Mattioli M . Correlation of a precipitin reaction to an RNAprotein antigen and a low prevalence of nephritis in patients with systemic lupus erythematosus. N Engl J Med 1972; 286: 908.

    CAS  Article  Google Scholar 

  34. 34

    Kanemitsu S, Ihara K, Saifddin A et al. A functional polymorphism in Fas (CD95/APO-1) gene promoter associated with systemic lupus erythematosus. J Rheumatol 2002; 29: 1183–1188.

    CAS  PubMed  Google Scholar 

  35. 35

    Rudert F, Lindridge E, Lasham A, Wang Y, Grandison P, Watson J . Silencer and enhancer regions in the human CD95 (Fas/APO-1) gene with sequence similarity to the granulocyte–macrophage colony-stimulating factor promoter: binding of single strand-specific silencer factors and AP-1 and NF-AT-like enhancer factors. DNA Cell Biol 1998; 17: 991.

    CAS  Article  Google Scholar 

  36. 36

    Lee YH, Kim YR, Ji JD, Sohn J, Song GG . Fas promoter −670 polymorphism is associated with development of anti-RNP antibodies in systemic lupus erythematosus. J Rheumatol 2001; 28: 2008–2011.

    CAS  PubMed  Google Scholar 

  37. 37

    Huang QR, Danis V, Lassere M, Edmonds J, Manolios N . Evaluation of a new Apo-1/Fas promoter polymorphism in rheumatoid arthritis and systemic lupus erythematosus patients. Rheumatol 1999; 38: 645–651.

    CAS  Article  Google Scholar 

  38. 38

    Wu J, Metz C, Xu X et al. A novel polymorphic CAAT/enhancer-binding protein beta element in the FasL gene promoter alters Fas ligand expression: a candidate background gene in African American systemic lupus erythematosus patients. J Immunol 2003; 170: 132–138.

    CAS  Article  Google Scholar 

  39. 39

    Cartegni L, Chew SL, Krainer AR . Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 2002; 3: 285–298.

    CAS  Article  Google Scholar 

  40. 40

    Rust S, Funke H, Assmann G . Mutagenically separated PCR (MS-PCR): a highly specific one step procedure for easy mutation detection. Nucleic Acids Res 1993; 21: 3623–3629.

    CAS  Article  Google Scholar 

  41. 41

    Spielman RS, McGinnis RE, Ewens WJ . Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 1993; 52: 506–516.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Martin ER, Monks SA, Warren LL, Kaplan NL . A test for linkage and association in general pedigrees: the Pedigree Disequilibrium Test. Am J Hum Genet 2000; 67: 146–154.

    CAS  Article  Google Scholar 

  43. 43

    Kong A, Cox NJ . Allele-sharing models: LOD scores and accurate linkage tests. Am J Hum Genet 1997; 61: 1179–1188.

    CAS  Article  Google Scholar 

  44. 44

    Kaplan NL, Hill WG, Weir BS . Likelihood methods for locating disease genes in nonequilibrium populations. Am J Hum Genet 1995; 56: 18–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Lewontin RC . On measures of gametic disequilibrium. Genetics 1988; 120: 849–852.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Holtz-Heppelmann CJ, Algeciras A, Badley AD, Paya CV . Transcriptional regulation of the human FasL promoter-enhancer region. J Biol Chem 1998; 273: 4416.

    CAS  Article  Google Scholar 

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Acknowledgements

We thank Marja Deckert for expert technical assistance.

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Correspondence to T Mandrup-Poulsen.

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Nolsøe, R., Kelly, J., Pociot, F. et al. Functional promoter haplotypes of the human FAS gene are associated with the phenotype of SLE characterized by thrombocytopenia. Genes Immun 6, 699–706 (2005). https://doi.org/10.1038/sj.gene.6364259

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Keywords

  • lupus
  • FAS
  • FASL
  • functional variants
  • thrombocytopenia

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