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Extended report

STAT4 associates with systemic lupus


erythematosus through two independent effects that
correlate with gene expression and act additively
with IRF5 to increase risk
A-K Abelson,1 A M Delgado-Vega,1 S V Kozyrev,1 E Sanchez,2 R Velazquez-Cruz,3
N Eriksson,4 J Wojcik,5 M V P Linga Reddy,1 G Lima,6 S DAlfonso,7 S Migliaresi,8
V Baca,9 L Orozco,3 T Witte,10 N Ortego-Centeno,11 the AADEA group, H Abderrahim,5
B A Pons-Estel,12 C Gutierrez,13 A Suarez,13 M F Gonzalez-Escribano,14 J Martin,2
M E Alarcon-Riquelme1,15

c Additional figures and tables ABSTRACT and most recently, BANK1.10 A genetic association
are published online only at Objectives: To confirm and define the genetic associa- with the signal transducer and activator of
http://ard.bmj.com/content/
tion of STAT4 and systemic lupus erythematosus (SLE), transcription 4 (STAT4) was identified in rheuma-
vol68/issue11
investigate the possibility of correlations with differential toid arthritis (RA) with SNP rs7574865 and this
For numbered affiliations see splicing and/or expression levels, and genetic interaction association was also found in SLE.11 From the RA
end of article
with IRF5. studies, the genetic association was defined to the
Correspondence to: Methods: 30 tag SNPs were genotyped in an third intron of STAT4. As has been shown for IRF5,
Dr M E Alarcon-Riquelme, independent set of Spanish cases and controls. SNPs several polymorphisms may contribute to the risk,
Uppsala University, The Rudbeck surviving correction for multiple tests were genotyped in and the risk might also differ among populations.5
Laboratory, Dag Hammarskjolds
vag 20, SE-75185, Uppsala, five new sets of cases and controls for replication. STAT4 Thus, our aim with this study was to revise the
Sweden; Marta.alarcon@ cDNA was analysed by 59-RACE PCR and sequencing. STAT4 genetic association using independent sets
genpat.uu.se Expression levels were measured by quantitative PCR. of Europeans and Latin American populations with
Results: In the fine mapping, four SNPs were significant a dense set of tag single nucleotide polymorphisms
A-KA, AMD-V, SVK, ES, JM and
MEA-R, at their respective after correction for multiple testing, with rs3821236 and (SNPs) to define if rs7574865 and thus, the third
positions, contributed equally to rs3024866 as the strongest signals, followed by the intron signal is the sole genetic contributor to
this work previously associated rs7574865, and by rs1467199. susceptibility in STAT4.
Accepted 29 October 2008 Association was replicated in all cohorts. After conditional STAT4 is a critical regulator of immune
Published Online First regression analyses, two major independent signals, responses, primarily induced by the dendritic cell-
9 December 2008 represented by SNPs rs3821236 and rs7574865, produced interleukin 12 (IL12), leading to the
remained significant across the sets. These SNPs belong development of Th1 cells, which have the ability
to separate haplotype blocks. High levels of STAT4 to secrete high levels of interferon c (IFNc). STAT4
expression correlated with SNPs rs3821236, rs3024866 is activated after ligation of IL12 to its receptor,
(both in the same haplotype block) and rs7574865 but not which associates with the tyrosine kinases Tyk2
with other SNPs. Transcription of alternative tissue- and Jak2.12 13 These are expressed in activated T
specific exons 1, indicating the presence of tissue-specific and B cells and, particularly, NK cells. Activation of
promoters of potential importance in the expression of STAT4 leads to the formation of homodimers of
STAT4, was also detected. No interaction with associated STAT4 that translocate into the nucleus and induce
SNPs of IRF5 was observed using regression analysis. transcription of IFNc. In addition, STAT4 activa-
Conclusions: These data confirm STAT4 as a suscept- tion is also induced by IFNa/b stimulation. This
ibility gene for SLE and suggest the presence of at least stimulation does not appear to lead to Th1
two functional variants affecting levels of STAT4. The development, but only to an acute IFNc secretion
results also indicate that the genes STAT4 and IRF5 act by CD4+ T and NK cells where IL18 is also
additively to increase the risk for SLE. required. IFNa/b induces STAT4 phosphorylation
through direct interaction of STAT4 with the
IFNaR2 subunit.
Here, we fine mapped a Spanish set of cases and
controls. We found evidence for another peak of
Systemic lupus erythematosus (SLE) has a strong association beyond the intron 3 SNP rs7574865.
genetic component supported by high familial This association was replicated in four indepen-
aggregation and twin and family studies.1 Like dent sets of cases and controls. We also found
most autoimmune diseases, the HLA has an evidence for a correlation between the associated
important contribution24 and we recently showed SNPs and expression levels of STAT4 in peripheral
that the HLA is the strongest genetic factor in blood mononuclear cells (PBMCs). When we
people of European ancestry followed by IRF5 and analysed the possibility of genetic interaction
ITGAM.25 Other somewhat weaker but well- between STAT4 and IRF5, we found no inter-
established associations have been found with action, but rather an additive increase in the risk
FCGRIIA,6 PTPN22,7 PDCD1,8 TNFSF4,9 BLK2 3 for SLE.

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Extended report

PATIENTS AND METHODS determine independence of the SNPs from rs7574865. All
Patients and controls logistic regression analyses were done with R.
In total, 1581 cases and 1851 controls were used in this study, Multiple logistic regression was used to evaluate if additive or
all with complete data for the SNPs analysed: 390 cases and 620 interaction effects were present between SNPs within STAT4 and
controls from Spain, 247 cases and 220 controls from Germany, IRF5. To measure the ability to discriminate between SLE cases
221 patients and 207 controls from Italy, 171 patients and 171 and controls, the area under the receiver operating characteristic
controls from Argentina, and 231 cases and 250 controls from curve (C statistic) was calculated. To statistically compare the C
Mexico (adults) along with a set of 321 paediatric patients and statistics we applied the method of DeLong et al.21
383 adult controls. The patient and control sets studied here
have been described previously.10 14 All patients fulfilled the 1982 59-RACE and 39-RACE PCR
American College of Rheumatology criteria for the classification Marathon-ready cDNA from different tissues (Clontech,
of SLE. Mountain View, California, USA) was used as template for
amplification of tissue-specific 59- and 39-UTRs. The following
Selection of tag SNPs pairs of nested gene-specific primers were used for 59-RACE
Twenty-nine tag SNPs covering the STAT1 and STAT4 genes, PCR: 59-GAAATTCTACTGAGAGACTCCCATTG-39 and 59-
and the intergenic region, were selected using Haploview GAATCGTTGCCATGGTTTCATTGTTAG-39 and for 39-
version 3.32 from the HapMap-CEU population genotype data. RACE PCR: 59-CTAAACTATCAGGTAAAGGTTAAGGCATC-
Aggressive tagging mode was used to select tag SNPs with a 39 and 59-GGTAAACACTACAGCTCTCAGCCTTG-39. Adapter
minor allele frequency >5%, with an r2 threshold >0.8. primers Ap1 and Ap2 were provided with cDNA. Nested PCR was
rs7574865 was added to the tag list after being reported as carried out using 1/30 of the first-round PCR products. Thirty-five
associated with RA.11 SNPs associated in the Spanish fine cycles of PCR (95uC for 20 s, 60uC for 15 s and 72uC for 3 min)
mapping, after quality control and correction for multiple were performed after initial denaturation at 95uC for 5 min in
testing, were typed in the German, Italian, Argentinian, and buffer containing 1.5 mM MgCl2, 200 mM of each of dNTPs,
both Mexican sets. 0.4 mM of each of the corresponding primers and 0.5 U of
Platinum Taq high fidelity enzyme (Invitrogen, Carlsbad,
California, USA). PCR products were analysed by sequencing.
Genotyping
Spanish samples were genotyped in Granada using TaqMan 59
exonuclease assay (ABI, Foster City, California, USA). German, RNA purification and STAT4 expression analysis
Italian and Latin American samples were genotyped at Uppsala Total RNA was purified as described elsewhere10 from PBMCs
University. Mexican paediatric samples were genotyped at obtained with agreed consent from 73 healthy volunteers. RNA
Instituto Nacional de Medicina Geno mica using the same (2 mg ) was reverse transcribed using oligo-dT primers according
method. Genotyping consistency between the centres was to the manufacturers protocol (Applied Biosystems, Foster
established to be near to 100%.15 City, California, USA).
STAT4 expression was determined by real-time PCR using
SYBR Green detection. Cycling conditions were as follows:
Statistical analyses 95uC for 5 min, 45 cycles of PCR (95uC for 15 s, 60uC for 10 s
The Spanish genotype data was processed using Haploview and 72uC for 20 s). a Isoform was detected with the following
version 4.0,16 PLINK version 1.0217 and R. Quality control filters primers: forward 59-CATCTCAACAATCCGAAGTGATTCA-39
were applied to remove SNPs with .10% missing data in cases and common reverse primer 59-GTCAGAGTTTATCCTGT-
or controls (one SNP excluded), deviations from Hardy CATTCAGCAG-39. b-Isoform-specific forward primer was 59-
Weinberg equilibrium (p,0.001, two SNPs excluded), or minor TGACCTTGTTATCTCTTTAAGCCGA-39. Expression levels
allele frequency ,5% in controls (two SNPs excluded). Twenty- were normalised against TATA-binding protein gene expression
five SNPs remained. Subjects with an individual missing amplified with commercial reagents (Applied Biosystems). All
genotyping rate .10% were also removed (n = 42). experiments were run in triplicate.
Genotyping rate in the remaining subjects was 97.4%.
Pairwise linkage disequilibrium (LD) measures (D9 and r2)
between SNPs and maximum-likelihood haplotype frequencies Statistical analysis of gene expression
were estimated with the EM algorithm. Multiple testing was Analysis of variance and F test were used to determine the
corrected by Bonferroni and false discovery rate methods.18 difference in the mRNA expression level in relation to each of
Statistical analysis of the replication sets included only the SNPs, taking the three possible genotypes as factor levels.
subjects with 100% individual genotyping calls. DerSimonian Significance of the gene expression was also tested with linear
Laird and CochranMantelHaenszel methods implemented in regression.22
StatsDirect and PLINK were used to estimate the meta-analysis
odds ratio for all populations assuming random and fixed effects RESULTS
models on the allelic association, respectively. The hetero- Association was detected for several SNPs across STAT4 and the
geneity test based on partitioning the x2 statistic implemen- STAT1STAT4 intergenic region, with the strongest association
ted in PLINK, was used to test between-population with rs3821236 (p = 7.0761028), followed by rs3024866
differences. Univariate genotypic odds ratios were estimated (p = 3.8361027), rs7574865 (p = 9.3761026) and rs1467199
by logistic regression.20 These were adjusted by adding the (p = 761025), all of which remained significant after correction
stratification variable population to the logistic regression for multiple tests (table 1). The region LD structure defined six
model containing the genotype as the exposure variable. This haplotype blocks, two located in STAT1 (blocks 1 and 2), one in
test is identical to the one degree of freedom Mantel the intergenic region (block 3) and three in STAT4 (blocks 46)
Haenszel test of the hypothesis that the stratum-specific (fig 1 and online supplementary fig 1). Our results indicate
odds ratios are 1.19 Conditional logistic regression was used to association of two additional haplotype blocks than that

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1748
Extended report

Table 1 Results from the fine mapping conducted in Spanish patients with systemic lupus erythematosus and matched controls
Multiple test correction
Cases Controls Assoc Frequency Frequency Genotypic Allelic
Chr. Position SNP aa/Aa/AA aa/Aa/AA allele cases controls test p value test p value OR L95 U95 Bonferroni FDR
2 191546759 rs13395505 83/189/110 90/222/160 A 0.46 0.43 0.252 0.108 1.17 0.97 1.42 1.000 0.574
2 191553970 rs1547550 70/165/145 52/225/195 G 0.40 0.35 9.5561023 2.5061022 1.25 1.03 1.53 0.625 0.184
2 191554382 rs4327257 8/81/291 15/111/346 C 0.13 0.15 0.459 0.198 0.83 0.63 1.10 1.000 0.946
2 191558344 rs2280234 88/169/124 75/224/171 A 0.45 0.40 3.0761022 2.2661022 1.25 1.03 1.52 0.566 0.184
2 191558811 rs2280233 68/161/141 92/216/148 C 0.40 0.44 0.243 0.127 0.86 0.70 1.05 1.000 0.640
2 191559011 rs2280232 29/131/219 28/168/240 C 0.25 0.26 0.450 0.727 0.96 0.77 1.20 1.000 1.000
2 191568747 rs12693591 5/102/268 14/143/311 A 0.15 0.18 0.137 0.069 0.79 0.61 1.02 1.000 0.385
2 191574903 rs13029247 56/138/139 48/197/216 C 0.38 0.32 2.7661022 1.7061022 1.29 1.05 1.59 0.424 0.180
2 191577408 rs2030171 69/172/133 58/202/194 C 0.41 0.35 2.9961022 7.3561023 1.31 1.08 1.60 0.184 0.088
2 191588747 rs1467199 47/146/167 24/183/265 G 0.33 0.24 7.4561025 7.0061025 1.54 1.25 1.91 1.7561023 1.6761023
2 191604299 rs3024935 3/48/323 3/69/385 T 0.07 0.08 0.647 0.455 0.87 0.60 1.25 1.000 1.000
2 191605785 rs925847 50/171/158 46/196/230 T 0.36 0.31 0.076 2.2161022 1.27 1.04 1.55 0.552 0.184
2 191611003 rs3821236 42/151/183 18/151/302 A 0.31 0.20 5.9061027 7.0761028 1.84 1.47 2.29 1.7761026 6.7461026
2 191613134 rs3024877 66/181/132 49/219/203 T 0.41 0.34 3.5061023 1.1961023 1.39 1.14 1.69 2.9661022 2.2661022
2 191625589 rs16833220 8/77/254 10/134/293 G 0.14 0.18 4.3961022 3.7161022 0.74 0.56 0.98 0.927 0.236
2 191631086 rs3024866 60/171/151 31/189/252 C 0.38 0.27 1.9561026 3.8361027 1.70 1.38 2.09 9.5861026 1.8361025
2 191637523 rs932169 3/55/321 5/59/382 C 0.08 0.08 0.781 0.815 1.04 0.73 1.50 1.000 1.000
2 191639709 rs1517352 102/159/103 75/241/137 A 0.50 0.43 3.1261024 6.8861023 1.31 1.08 1.59 0.172 0.088
2 191646845 rs7594501 0/37/335 3/65/403 A 0.05 0.08 0.057 3.2861022 0.64 0.43 0.97 0.819 0.223
2 191651517 rs3024921 1/37/338 3/43/423 T 0.05 0.05 0.752 0.972 0.99 0.64 1.53 1.000 1.000
2 191662292 rs10931480 14/95/270 21/151/298 G 0.16 0.21 0.054 2.3561022 0.75 0.58 0.96 0.586 0.184
2 191663097 rs10931481 58/174/150 42/207/221 G 0.38 0.31 6.9561023 2.4361023 1.36 1.12 1.67 0.061 3.8761022
2 191664494 rs13011805 1/63/314 6/96/367 T 0.09 0.12 0.100 4.88E61022 0.72 0.52 1.00 1.000 0.291
2 191672878 rs7574865 41/153/181 18/170/284 T 0.31 0.22 1.7461025 9.3761026 1.64 1.31 2.03 2.3461024 2.9861024
2 191743288 rs10176621 29/146/205 33/148/262 C 0.27 0.24 0.294 0.212 1.15 0.92 1.44 1.000 0.961
The table shows the genotypes counts for the 25 SNPs that passed frequency and genotyping pruning, as well as the results for both allelic and genotypic association tests. The four SNPs italicised were chosen to be replicated in independent sets of
cases and controls since they had a significant p value ,1.0061025 and remained associated after multiple tests correction and were independent.
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FDR, false discovery rate; L95, lower limit of the 95% confidence interval; OR, odds ratio; SNP, single nucleotide polymorphism; U95, upper limit of the 95% confidence interval.

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Extended report

Figure 1 Fine mapping of the STAT1STAT4 region. The physical position (top panel) of the SNPs typed in 390 patients and 480 controls from Spain
covering the ,200 kb STAT1 STAT4 region is shown. The region linkage disequilibrium (LD) structure defined six haplotype blocks (middle panel) of
which three were associated with disease susceptibility (see block structure and R2 values in supplementary fig 1). Risk haplotypes are shown in red
and main single-marker hits replicated are underlined (see table 2 and supplementary table 1). The bottom panel shows the significance of the
association data presented as the log10 (p value) for 25 tag SNPs passing genotyping quality control. The blocks have been defined using the solid
spine of the LD method in Haploview version 4.0. The p values of the risk and protective haplotypes at each block in the Spanish population are as
follows: 1block 3 risk haplotype p value = 0.0037; 2block 3 protective haplotype p value = 761024; 3block 4 risk haplotype p value = 2.6461026; 4block
4 protective haplotype p value = 2.2461025; 5block 6 risk haplotype p value = 9.5261026; 6block 6 protective haplotype p value = 0.0331.

containing rs7574865 (block 6): block 3, which contains pairwise correlation coefficients (R2) suggest that the individual
rs1467199, and block 4 harbouring rs3821236 and rs3024866 SNP associations reflect independent effects, except for
(fig 1). rs3024866, which has a relatively high R2 with rs3821236
To replicate the genetic associations and increase statistical (R2 = 0.64) (fig 1). This was confirmed by conditional logistic
power, we genotyped the associated SNPs in five independent regression analysis: rs3821236 remained significant when con-
sets from Italy, Germany, Argentina, and Mexico (one adult and ditioning on rs7574865, and vice versa (supplementary table 3).
one paediatric set). Homogeneity test showed that the odds Thus, rs3821236 and rs7574865 represent two independent
ratios for the SNPs could be combined, except rs3024866 and genetic effects within STAT4. rs3821236 is located in the 16th
rs7574865, which had some heterogeneity across the strata. intron, ,60 kb downstream from the third intron where the
None of the SNPs provided association with the German association has been confined in previous studies.11 SNPs
population except for a borderline association with rs7574865 rs3821236 and rs3024866 are tagging a 26 kb haplotype block
(table 2 and supplementary table 1). The Mexican paediatric set (block 4) covering part of the gene between intron 8 and 16 (fig 1
showed association only with rs1467199 (p = 0.008, supple- and supplementary fig 1) that contain three of the six markers
mentary table 2). Despite the low heterogeneity introduced by associated with SLE in the Spanish fine mapping after multiple
the German set (homogeneity p = 0.01), it was included in the test correction (table 1). After a conditional haplotype-based
meta-analysis. The Mexican paediatric set displayed the highest association test the haplotype block 4 (SNPs rs3821236 and
heterogeneity and it could not be combined (compare supple- rs932169) remain significant (p = 0.002277) after controlling for
mentary table 2 with table 2). In the meta-analysis, SNPs rs7574865, confirming its independency.
rs3821236 (p = 5.96610220) and rs7574865 (p = 4.44610223) Using the receiver operating characteristic curve and C
showed the strongest association across all strata in the allelic statistic, we observed that rs7574865 was a somewhat better
and genotypic tests, but rs3024866 was also associated risk predictor (C = 0.590) than rs3821236 (C = 0.577), but the
(p = 2.31610212) (table 2). Although rs1467199 reached significant difference did not reach statistical significance (supplementary
association in the meta-analysis, at the individual population level table 4). Thus, each SNP alone provides the same level of
it was only replicated in the Argentine set (rs1467199-CG
prediction of the risk for SLE, independently.
p = 1.6361022, rs1467199-GG p = 0.053) (supplementary table 1).
We tested whether the associated SNPs constituted indepen-
dent effects. rs7574865 is not in strong LD with rs3024866 Statistical analysis of the interaction with IRF5
(R2 = 0.29) or rs3821236 (R2 = 0.42), which are located 62 kb IRF5 was found as a well-established non-major histocompat-
and 42 kb from rs7574865, respectively, and therefore they are ibility complex association with SLE. To study if the effect of
not proxies. rs1467199 has R2 = 0.30 with rs3821236, R2 = 0.18 STAT4 was independent of IRF5, or if there were epistatic
with rs3024866 and R2 = 0.13 with rs7574865 (fig 1). The low effects between SNPs in these genes we performed multiple

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Extended report

Table 2 Population-specific replication and general stratified allelic association analysis of the main associated single nucleotide polymorphisms
(SNPs) in the fine mapping
SNP Risk allele Population/test Frequency cases Frequency controls p Value OR

rs1467199 G Germany 0.229 0.231 0.941 0.99


Italy 0.235 0.229 0.840 1.03
Spain 0.333 0.250 7.7361025 1.50
Argentina 0.345 0.284 0.084 1.33
Mexico* 0.171 0.134 0.121 1.33
Pooled (fixed effects) 1.8261024 1.27
Pooled (random effects) 1.4161022 1.24
Homogeneity test 0.138

rs3821236 A Germany 0.213 0.186 0.302 1.18


Italy 0.301 0.174 1.3661025 2.04
Spain 0.317 0.197 2.0761029 1.89
Argentina 0.506 0.354 5.9161025 1.87
Mexico 0.458 0.318 1.3061025 1.81
Pooled (fixed effects) 5.96610220 1.77
Pooled (random effects) 8.98610211 1.75
Homogeneity test 0.124

rs3024866 C Germany 0.251 0.258 0.809 0.96


Italy 0.355 0.249 7.1661024 1.66
Spain 0.383 0.268 9.6461028 1.70
Argentina 0.477 0.383 1.3461022 1.47
Mexico 0.520 0.395 1.3861024 1.66
Pooled (fixed effects) 2.31610212 1.51
Pooled (random effects) 1.3061024 1.48
Homogeneity test 0.024

rs7574865 T Germany 0.265 0.210 0.050 1.35


Italy 0.394 0.193 1.41610210 2.70
Spain 0.327 0.221 1.8161027 1.72
Argentina 0.415 0.319 8.8561023 1.52
Mexico 0.547 0.364 2.4161028 2.10
Pooled (fixed effects) 4.44610223 1.82
Pooled (random effects) 7.8161028 1.82
Homogeneity test 0.013
Fixed effects: CochranMantelHaenszel meta-analysis controlling for strata under fixed effects model; random effects: DerSimonianLaird meta-analysis controlling for strata under
random effects model; homogeneity test: between-strata homogeneity test.
*Only Mexican adult samples were used in this analysis.

logistic regression analysis and C statistic including the now, two isoforms have been described for STAT4, a and b.23
genotype data for three SNPs of IRF5 (rs2004640, rs2070197 Only the known a and b isoforms were detected when we
and rs10954213) from our previous studies,5 14 The analyses tested spleen testis, kidney, lung, pancreas, intestine and uterus
revealed no significant interaction effects between STAT4 and for new isoforms.
IRF5 SNPs, but close to complete independent effects on SLE Since 59- and 39-UTRs may substantially affect gene expres-
risk as shown by a slight change in estimates when combined in sion,14 we performed detailed analysis of non-coding 59-exons and
a multivariate model (data not shown). 39-UTRs. The pattern of 59-UTRs was diverse in different tissues
The three SNPs of IRF5 had C statistics of 0.587 0.585 and (table 3), but all of them led to the known isoforms of STAT4.
0.565, respectively. (supplementary table 5). rs2070197 tags the
major risk haplotype and has the highest predictive ability.14
Analysis of STAT4 expression levels
Addition of rs7574865 to the IRF5 SNPs increased the predictive
Levels of STAT4 gene expression in human mononuclear cells
value of the models significantly (rs7574865 with rs2004640: C
were assessed by quantitative real-time PCR. Since expression
statistic = 0.632, p = 1.6661025; rs7574865 with rs2070197: C
of the b transcript was much lower than of the a transcript and
statistic = 0.636, p = 3.28610211 and rs7574865 with
its expression followed the trend of the a transcript in all
rs10954213: C statistic = 0.624, p = 9.6261027). The results
samples, it was excluded from the analysis. By using analysis of
support an additive effect of SNPs of both genes, particularly
variance to test if differences in gene expression correlated with
rs7574865 and rs2070197, to increase risk for SLE (fig 2).
genotypes, we found a modest upregulation of STAT4 mRNA
with the CC risk genotype of rs3024866 (p = 0.0281). By
Tissue-specific alternative transcripts of STAT4 regression analysis, risk alleles of SNPs rs3821236 and
In order to investigate if differential splicing could be related to rs7574865 correlated with higher STAT4 expression.
the genetic association of STAT4, functional annotation of gene Importantly, SNP rs1467199 did not correlate with STAT4
transcripts expressed in different tissues was performed. Until expression (fig 3).

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Extended report

mapping effort was continuing, and after having identified a


strong signal for this gene in a 100k scan in Argentine subjects
(data not shown). The data from the Spanish mapping
suggested the presence of several peaks independent of
rs7574865. Analyses using a larger set of joined samples
confirmed the independent effect of rs3821236 in intron 16.
Conditional SNP and haplotype regression analysis supported
this result.
We observed differences between north and south European
samples: the German set contributed, albeit weakly, to the
association only at rs7574865, whereas the association of
rs3821236 was contributed particularly by the Spanish, Italian
and Latin American sets. In our view it is highly plausible that
several independent risk haplotypes are involved in disease
susceptibility, with some having stronger effects in some
populations than others.
The weak result of the German set could not be due to a lack
of power, as the size of the other sets is comparable.
We are approaching a phase in complex disease genetics
where identification of the genes involved in disease suscept-
ibility is becoming a reality. Therefore, it is of interest that we
Figure 2 Predictive ability of STAT4 and IRF5 single nucleotide understand the relationship between the various genetic effects.
polymorphisms (SNPs) for systemic lupus erythematosus (SLE). The Here we examined the possibility of genetic interaction between
predictive ability of the two genes for SLE was investigated using the C IRF5 and STAT4. We observed no genetic interaction but we
statistic. The test to compare two dependent receiver operating observed a significant increase in the predictive ability for SLE
characteristic curves is used. Within STAT4, the SNP rs7574865 is the when STAT4 and IRF5 SNPs were added. These results suggest
strongest predictor for SLE and adds a significant fraction to the that the IRF5 and STAT4 SNPs act additively to increase the risk
predictive ability of the IRF5 SNP rs2070197. Between genes the best for SLE. While this work was under revision, an independent
combination is rs7574865+rs2070197 having an overall C statistic of
study corroborated this additive effect.24
0.636.
Identification of the functional variants for STAT4 might
prove relatively difficult considering the large size of this gene
and the location of the associated SNPs. Complete resequencing
DISCUSSION
of the gene and genotyping may be needed to reveal the true
We confirmed the genetic association between STAT4 and SLE
functional variants. We observed no differences in splicing of
and through a fine mapping effort we identified a second effect
the gene in PBMCs. Instead we found a correlation between
independent of rs7574865 located in the vicinity of intron 16.
expression levels of STAT4 in PBMCs and the STAT4 associated
The publication of Remmers, et al,11 identifying STAT4 as a
SNPs, rs7574865, rs3024866 and rs3821236 but not rs1467199. It
susceptibility gene for RA and SLE was published while our fine
should be noted that PBMC samples are expected to have
varying numbers of T and NK cells, high variation in gene
expression and a weak correlation. Further, in complex diseases
Table 3 Usage and splicing of 59- and 39-UTRs in STAT4
we also expect effects caused by functional variants to be
Tissue 59-Terminal exons Acc Number modest and difficult to define.14
Kidney exon1B-exon2-exon3 NM_003151 How does a modest increase in STAT4 expression contribute
exon1C-exon1D-exon2-exon3 EU304788 to the risk for SLE? STAT4 is a transcription factor through
which the functional effects of IL12 are conducted, leading to
Spleen exon1B-exon2-exon3 IFNc production. The STAT4 pathway has been studied in mice
exon1A-intron-exon1B-exon2-exon3 EU304789 and patients with SLE, with contradictory results. In two
exon1C-D-E-F-exon2-exon3 EU304790 studies, mice deficient for STAT4 show increased development
exon1D-E-G-H-exon2-exon3 EU304791
of glomerulonephritis,25 26 whereas a third study reported
opposite findings, in line with our observations.27
Testis exon1B-exon2-exon3
We detected only two isoforms of STAT4, one of which was
exon1A-exon2-exon3 BC031212
exon1A-alt.spliced exon1B last 68 bp-exon2-exon3 EU304792
expressed at extremely low levels (b), and tissue-specific
promoters as observed by the presence of alternative, tissue-
Lung exon1B-exon2-exon3 specific 59-UTRs (table 3). Given that such promoters could
adjust transcription of the gene in a particular tissue, this poses
Pancreas exon1B-exon2-exon3 an additional obstacle for defining the role of genetic variation.
exon1A-intron-exon1B-297nt intron1-exon2-exon3 EU304793 Other authors showed that the SLE risk haplotype of STAT4 is
overexpressed in mesenchymal cells,24 supporting a tissue-
Small intestine exon1B-exon2-exon3 specific component involved in the regulation of STAT4
expression. Using purified cell populations (eg, NK cells, which
Uterus exon1B-exon2-exon3 constitute only 5% of the blood leukocytes but have high basal
exon1A-intron-exon1B-297nt intron1-exon2-exon3 EU304793 level of STAT4, kidney mesangial cells, etc) may be critical for
Accession numbers for previously described a and b isoforms are shown in italics. correct assessment of the altered levels of STAT4 gene

Ann Rheum Dis 2009;68:17461753. doi:10.1136/ard.2008.097642 1751


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Extended report

Figure 3 Association between STAT4 polymorphism and expression levels. Analysis of variance and F test were used to determine difference in the
mRNA relative expression levels between subjects carrying different genotypes, taking the three possible genotypes as factor levels and the major
allele homozygous as reference. Multiple comparisons of means revealed that subjects with rs3024866-CC genotype have higher STAT4 expression
levels than those with rs3024866-TT (p value = 0.0281)

expression as well as definition of the splicing isoforms that de Asturias, Universidad de Oviedo, Oviedo, Spain; 14 Department of Immunology,
Hospital Virgen del Roco, Sevilla, Spain; 15 Oklahoma Medical Research Foundation,
could be involved.
Oklahoma City, USA
Major effects of the expression of human STAT4 may be
important in the kidney. Activation of STAT4 leads to increased Acknowledgements: We express our sincere gratitude to Susanna Lewen for help
expression of IFNc. A study has shown that increased with purification of PBMCs and total RNA, and Hong Yin for help with the preparation
expression of IL12 and IFNc in the kidneys of MRL-lpr/lpr mice of DNA samples. We also thank Adriana I Scollo, Armando M Perichon and Mariano
CR Tenaglia, CEDIM, Diagnostico Molecular y Forense SRL, Rosario, Argentina, for their
precedes the development of glomerulonephritis.28 29. Thus, the
help in DNA preparation of the Argentinian samples. We thank particularly the Lupus
localised action of the various genes in specific tissues may be of Patient Association of Asturias for their help in the collection of samples and all the
importance. This is also important to consider in view of the patients for their contribution.
recent results showing a strong correlation between rs7574865 The Argentine collaborative group participants are: Hugo R Scherbarth MD, Pilar C
and end-organ disease, in particular, kidney disease.30 Marino MD, Estela L Motta MD Servicio de Reumatologa, Hospital Interzonal General
de Agudos Dr Oscar Alende, Mar del Plata, Argentina; Susana Gamron MD, Cristina
Our results do support an important role of STAT4 in SLE Drenkard MD, Emilia Menso MD Servicio de Reumatologa de la UHMI 1, Hospital
susceptibility, a role that appears to vary between different Nacional de Clnicas, Universidad Nacional de Cordoba, Cordoba, Argentina; Alberto
populations and derives from two different and independent Allievi MD, Guillermo A Tate MD Organizacion Medica de Investigacion, Buenos Aires,
risk variants, whose functional nature needs to be examined. Argentina; Jose L Presas MD Hospital General de Agudos Dr Juan A Fernandez,
Buenos Aires, Argentina; Simon A Palatnik MD, Marcelo Abdala MD, Mariela Bearzotti
PhD Facultad de Ciencias Medicas, Universidad Nacional de Rosario y Hospital
Author affiliations: 1 Department of Genetics and Pathology, Rudbeck Laboratory, Provincial del Centenario, Rosario, Argentina; Alejandro Alvarellos MD, Francisco
University of Uppsala, Uppsala, Sweden; 2 Instituto de Biomedicina y Parasitologa Caeiro MD, Ana Bertoli MD Servicio de Reumatologa, Hospital Privado, Centro Medico
Lopez-Neyra, Consejo Superior de Investigaciones Cientficas, Granada, Spain; de Cordoba, Cordoba, Argentina; Sergio Paira MD, Susana Roverano MD, Hospital
3
Instituto Nacional de Medicina Genomica, Mexico City, Mexico; 4 Uppsala Clinical Jose M Cullen, Santa Fe, Argentina; Cesar E Graf MD, Estela Bertero PhD Hospital San
Research Centre, Uppsala University, Uppsala, Sweden; 5 Merck Serono, Chemin des Martn, Parana; Cesar Caprarulo MD, Griselda Buchanan PhD Hospital Felipe Heras,
Mines, Geneva, Switzerland; 6 Department of Immunology and Rheumatology, Instituto Concordia, Entre Ros, Argentina; Carolina Guilleron MD, Sebastian Grimaudo PhD,
Nacional de Ciencias Medicas y Nutricion Salvador Zubiran Mexico City, Mexico; Jorge Manni MD Departamento de Inmunologa, Instituto de Investigaciones Medicas
7
Department of Medical Sciences and IRCAD, University of Eastern Piedmont, Novara, Alfredo Lanari, Buenos Aires, Argentina; Luis J Catoggio MD, Enrique R Soriano MD,
Italy; 8 Rheumatology Unit, Second University of Naples, Naples, Italy; 9 Department of Carlos D Santos MD Seccion Reumatologa, Servicio de Clnica Medica, Hospital
Rheumatology, Pediatric Hospital, Centro Medico Nacional Siglo XXI, IMSS, Mexico Italiano de Buenos Aires y Fundacion Dr Pedro M Catoggio para el Progreso de la
City, Mexico; 10 Medical School Hannover, Hannover, Germany; 11 Department of Reumatologa, Buenos Aires, Argentina; Cristina Prigione MD, Fernando A Ramos MD,
Internal Medicine, Hospital Clnico San Cecilio, Granada, Spain; 12 Sanatorio Parque, Sandra M Navarro MD Servicio de Reumatologa, Hospital Provincial de Rosario,
Rosario, Argentina; 13 Department of Functional Biology, Hospital Universitario Central Rosario, Argentina; Guillermo A Berbotto MD, Marisa Jorfen MD, Elisa J Romero PhD

1752 Ann Rheum Dis 2009;68:17461753. doi:10.1136/ard.2008.097642


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Extended report

Servicio de Reumatologa Hospital Escuela Eva Peron Granadero Baigorria, Rosario, 5. Graham RR, Kozyrev SV, Baechler EC, et al. A common haplotype of interferon
Argentina; Mercedes A Garcia MD, Juan C Marcos MD, Ana I Marcos MD Servicio de regulatory factor 5 (IRF5) regulates splicing and expression and is associated with
Reumatologa, Hospital Interzonal General de Agudos General San Martn, La Plata; increased risk of systemic lupus erythematosus. Nat Genet 2006;38:5505.
Carlos E Perandones MD, Alicia Eimon MD Centro de Educacion Medica e 6. Magnusson V, Johanneson B, Lima G, et al. Both risk alleles for FcgammaRIIA and
Investigaciones Clnicas (CEMIC), Buenos Aires, Argentina; Cristina G Battagliotti MD FcgammaRIIIA are susceptibility factors for SLE: a unifying hypothesis. Genes Immun
Hospital de Ninos Dr Orlando Alassia, Santa Fe, Argentina. 2004;5:1307.
Bernardo Pons-Estel is the coordinator of the Argentine collaborative group. 7. Kyogoku C, Langefeld CD, Ortmann WA, et al. Genetic association of the R620W
The German collaborative group participants are: K Armadi-Simab, MD, Wolfgang L polymorphism of protein tyrosine phosphatase PTPN22 with human SLE. Am J Hum
Gross, MD, Abteilung Rheumatologie, University Hospital of Schleswig-Holstein, Genet 2004;75:5047.
Campus Luebeck, Rheumaklinik Bad Bramstedt, Luebeck, Germany, Erika Gromnica- 8. Prokunina L, Castillejo-Lopez C, Oberg F, et al. A regulatory polymorphism in PDCD1
Ihle, MD, Rheumaklinik Berlin-Buch, Berlin, Germany, Hans-Hartmut Peter, MD, is associated with susceptibility to systemic lupus erythematosus in humans. Nat
Medizinische Universitaetsklinik, Abteilung Rheumatologie und Klinische Immunologie, Genet 2002;32:6669.
Freiburg, Germany, Karin Manger, MD, Medizinische Klinik III derFAU Erlangen- 9. Graham DS, Graham RR, Manku H, et al. Polymorphism at the TNF superfamily gene
Nuernberg, Erlangen, Germany, Sebastian Schnarr, MD, Henning Zeidler, MD, TNFSF4 confers susceptibility to systemic lupus erythematosus. Nat Genet
Abteilung Rheumatologie, Medizinische Hochschule Hannover, Hannover, Germany, 2008;40:839.
10. Kozyrev SV, Abelson AK, Wojcik J, et al. Functional variants in the B-cell gene BANK1
Reinhold E Schmidt, MD, Abteilung Klinische Immunologie, Medizinische Hochschule
are associated with systemic lupus erythematosus. Nat Genet 2008;40:2116.
Hannover, Hannover, Germany.
11. Remmers EF, Plenge RM, Lee AT, et al. STAT4 and the risk of rheumatoid arthritis
The Italian collaborative participants are: Gian Domenico Sebastiani (UOC di
and systemic lupus erythematosus. N Engl J Med 2007;357:97786.
Reumatologia Ospedale San Camillo, Roma Italy), Enrica Bozzolo (IRCCS San Raffaele
12. Kaplan MH. STAT4: a critical regulator of inflammation in vivo. Immunol Res
Hospital, Milan, Italy), Mauro Galeazzi, (Department of Clinical Medicine and
2005;31:23142.
Immunology Sciences, Section of Rheumatology, Siena University, Siena, Italy), Nadia 13. Yao BB, Niu P, Surowy CS, et al. Direct interaction of STAT4 with the IL-12 receptor.
Barizzone (Department of Medical Sciences, University of Eastern Piedmont, Novara, Arch Biochem Biophys 1999;368:14755.
Italy) and Maria Giovanna Danieli and Professor Armando Gabrielli (Dipartimento di 14. Kozyrev SV, Lewen S, Linga Reddy MVP, et al, The German Collaborative Group,
Scienze Mediche e Chirurgiche, Universita` Politecnica delle Marche, Ancona, Italy). Junker P, Laustrup H, Gutierrez C, Suarez A, Gonzalez-Escribano MF, Martn J, The
The AADEA (Andalusian Association of Autoimmune Diseases) group participants are: Spanish Collaborative Group, Alarcon-Riquelme ME. Structural insertion/deletion
Jose L Callejas-Rubio, Servicio de Medicina Interna, Hospital Clnico San Cecilio, variation in IRF5 is associated with a risk haplotype and defines the precise isoforms
Granada; Juan Jimenez-Alonso and Mario Sabio, Servicio de Medicina Interna, expressed in SLE. Arthritis Rheum 2007;56:123441.
Hospital Virgen de las Nieves, Granada; Julio Sanchez-Roman and Francisco J Garca- 15. Sanchez E, Abelson AK, Sabio JM, et al. Association of a CD24 gene polymorphism
Hernandez, Servicio de Medicina Interna, Hospital Virgen del Rocio, Sevilla; Enrique De- with susceptibility to systemic lupus erythematosus. Arthritis Rheum 2007;56:30806.
Ramon and Mayte Camps, Servicio Medicina Interna, Hospital Carlos Haya, Malaga; 16. Barrett JC, Fry B, Maller J, et al. Haploview: analysis and visualization of LD and
Rosa Garca-Portales, Servicio Reumatologa, Hospital Virgen de la Victoria, Malaga; haplotype maps. Bioinformatics 2005;21:2635.
Miguel A Lopez-Nevot, Servicio de Inmunologa, Hospital Virgen de las Nieves, 17. Purcell S, Neale B, Todd-Brown K, et al. PLINK: a tool set for whole-genome association
Granada. and population-based linkage analyses. Am J Hum Genet 2007;81:55975.
Funding: This work has been supported in part by grants from the European 18. Benjamini Y, Drai D, Elmer G, et al. Controlling the false discovery rate in behavior
CVDIMMUNE project from the European Commission LSHM-CT-2006-037227, the genetics research. Behav Brain Res 2001;125:27984.
19. Day NE, Byar DP. Testing hypotheses in case-control studiesequivalence of
Swedish Research Council (12673), the Torsten and Ragnar Soderbersstiftelse, the
Mantel-Haenszel statistics and logit score tests. Biometrics 1979;35:62330.
Swedish Association Against Rheumatism, the King Gustaf the Vth 80th-Jubilee
20. Sasieni PD. From genotypes to genes: doubling the sample size. Biometrics
Foundation and the Knut and Alice Wallenberg Foundation for supporting MEAR
1997;53:125361.
through the Royal Swedish Academy of Sciences. This study was also supported by
21. DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or
grant SAF2006-00398 from the Spanish Ministerio de Educacion y Ciencia, grant
more correlated receiver operating characteristic curves: a nonparametric approach.
PI052409 from the Fondo de Investigacion Sanitaria (Spain), C2.12 from BMBF Biometrics 1988;44:83745.
Kompetenznetz Rheuma in Germany and FISM, Regione Piemonte (CIPE) and the 22. Purcell S, Daly MJ, Sham PC. WHAP: haplotype-based association analysis.
Consejo Nacional de Ciencia y Tecnologa (CONACYT: SALUD-2004-01-153). MEAR is Bioinformatics 2007;23:2556.
a Greenberg Scholar at the OMRF. 23. Hoey T, Zhang S, Schmidt N, et al. Distinct requirements for the naturally
Competing interests: JW and HA are employees of Merck Serono Inc and produced occurring splice forms Stat4alpha and Stat4beta in IL-12 responses. EMBO J
the Argentine 100k data on which our investigation and search for STAT4 variants was 2003;22:423748.
first based. 24. Sigurdsson S, Nordmark G, Garnier S, et al. A risk haplotype of STAT4 for systemic
lupus erythematosus is over-expressed, correlates with anti-dsDNA and shows
Ethics approval: Ethics committee approval from each of the participating additive effects with two risk alleles of IRF5. Hum Mol Genet 2008;17:286876.
institutions. 25. Singh RR, Saxena V, Zang S, et al. Differential contribution of IL-4 and STAT6 vs
STAT4 to the development of lupus nephritis. J Immunol 2003;170:481825.
REFERENCES 26. Jacob CO, Zang S, Li L, et al. Pivotal role of Stat4 and Stat6 in the pathogenesis of
1. Alarcon-Segovia D, Alarcon-Riquelme ME, Cardiel MH, et al. Familial aggregation of the lupus-like disease in the New Zealand mixed 2328 mice. J Immunol
systemic lupus erythematosus, rheumatoid arthritis, and other autoimmune diseases 2003;171:156471.
in 1,177 lupus patients from the GLADEL cohort. Arthritis Rheum 2005;52:113847. 27. Xu Z, Duan B, Croker BP, Morel L. STAT4 deficiency reduces autoantibody production
2. Harley JB, Alarcon-Riquelme ME, Criswell LA, et al. Genome-wide association scan and glomerulonephritis in a mouse model of lupus. Clin Immunol 2006;120:18998.
in women with systemic lupus erythematosus identifies susceptibility variants in 28. Fan X, Oertli B, Wuthrich RP. Up-regulation of tubular epithelial interleukin-12 in
ITGAM, PXK, KIAA1542 and other loci. Nat Genet 2008;40:20410. autoimmune MRL-Fas(lpr) mice with renal injury. Kidney Int 1997;51:7986.
3. Hom G, Graham RR, Modrek B, et al. Association of systemic lupus erythematosus 29. Schwarting A, Tesch G, Kinoshita K, et al. IL-12 drives IFN-gamma-dependent
with C8orf13-BLK and ITGAM-ITGAX. N Engl J Med 2008;358:9009. autoimmune kidney disease in MRL-Fas(lpr) mice. J Immunol 1999;163:688491.
4. Fernando MM, Stevens CR, Sabeti PC, et al. Identification of two independent risk 30. Taylor KE, Remmers EF, Lee AT, et al. Specificity of the STAT4 genetic association
factors for lupus within the MHC in United Kingdom Families. PLoS Genet for severe disease manifestations of systemic lupus erythematosus. PLoS Genet
2007;3:e192. 2008;4:e1000084.

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STAT4 associates with systemic lupus


erythematosus through two independent
effects that correlate with gene expression
and act additively with IRF5 to increase risk
A-K Abelson, A M Delgado-Vega, S V Kozyrev, E Snchez, R
Velzquez-Cruz, N Eriksson, J Wojcik, M V P Linga Reddy, G Lima, S
D'Alfonso, S Migliaresi, V Baca, L Orozco, T Witte, N Ortego-Centeno,
the AADEA group, H Abderrahim, B A Pons-Estel, C Gutirrez, A Surez,
M F Gonzlez-Escribano, J Martin and M E Alarcn-Riquelme

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