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Structural insertiondeletion variation in IRF5 is associated with a risk haplotype and defines the precise IRF5 isoforms expressed in systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 4, April 2007, pp 1234–1241
DOI 10.1002/art.22497
© 2007, American College of Rheumatology
Structural Insertion/Deletion Variation in
IRF5 Is Associated With a Risk Haplotype and
Defines the Precise IRF5 Isoforms Expressed in
Systemic Lupus Erythematosus
Sergey V. Kozyrev,1 Susanna Lewén,1 Prasad M. V. Linga Reddy,1 Bernardo Pons-Estel2 and
the Argentine Collaborative Group, Torsten Witte3 and the German Collaborative Group,
Peter Junker,4 Helle Laustrup,4 Carmen Gutiérrez,5 Ana Suárez,5
Maria Francisca González-Escribano,6 Javier Mart´ın7 and the Spanish Collaborative Group,
and Marta E. Alarcón-Riquelme1
Objective. To determine whether specific isoforms
of IRF5 are transcribed in patients with systemic lupus
erythematosus (SLE) who have risk genotypes in the
exon 1B donor splice site at single-nucleotide polymorphism (SNP) no. rs2004640.
Methods. Peripheral blood mononuclear cells
were obtained from SLE patients and healthy controls
from Argentina, Spain, and Germany and from trio
families from Spain and Denmark. A reporter assay was
used to investigate the role of SNP no. rs2004640. IRF5
expression in relation to the genotypes of functional
SNPs was analyzed using quantitative polymerase chain
reaction. Sequencing and genotyping of the IRF5 gene
was performed.
Results. Sequencing of complementary DNA from
individuals with different genotypes showed 4 basic
isoforms transcribed from all 5ⴕ-untranslated regions
(5ⴕ-UTRs), suggesting no preferential isoform transcription based on rs2004640 genotypes. Analysis of
translation efficiency showed that exon 1A was the most
efficient in initiating protein synthesis. We identified a
novel polymorphic insertion/deletion that defines the
pattern of expression of isoforms of IRF5. The insertion
consists of 4 repeats in exon 6 affecting the protein
interaction domain. The insertion segregates in the risk
haplotype with the high expression allele of a poly(A)
site SNP no. rs10954213 and the exon 1B donor splice
allele of the 5ⴕ-UTR SNP no. rs2004640. The poly(A)
polymorphism correlated with levels of IRF5 in cells
stimulated with interferon-␣. The SNP most strongly
associated with SLE was SNP no. rs2070197 (P ⴝ 5.2 ⴛ
10ⴚ11), which is a proxy of the risk haplotype, but does
not appear to be functional.
Conclusion. None of the functional variants investigated in this study is strongly associated with SLE,
with the exception of the exon 1B donor splice site, and
its functional importance appears to be small. Our
results suggest that there may be other functional
polymorphisms, yet to be identified, in IRF5. We did not
observe evidence of epistatic interaction between the
functional SNPs.
Supported by the Swedish Research Council, the Swedish
Association Against Rheumatism, the 80-Year Foundation of King
Gustaf V, the Marcus Borgströms Foundation, the Magnus Bergvalls
Foundation, the Torsten and Ragnar Söderbergs Foundation, and the
Danish Rheumatism Association. Dr. Kozyrev’s work was supported
by the Gurli and Edward Brunnbergs Foundation for Rheumatic
Research. The German Collaborative Group’s work was supported by
a grant from BMBF Kompetenznetz Rheuma C2.12. Dr. AlarcónRiquelme is supported by the Knut and Alice Wallenberg Foundation
through a Royal Swedish Academy of Sciences award.
1
Sergey V. Kozyrev, PhD, Susanna Lewén, MSc, Prasad M. V.
Linga Reddy, MSc, Marta E. Alarcón-Riquelme, MD, PhD: Rudbeck
Laboratory, Uppsala University, Uppsala, Sweden; 2Bernardo PonsEstel, MD: Sanatorio Parque, Rosario, Argentina; 3Torsten Witte,
MD, PhD: Medical School Hannover, Hannover, Germany; 4Peter
Junker, MD, Helle Laustrup, MD: Odense University Hospital,
Odense, Denmark; 5Carmen Gutiérrez, MD, PhD, Ana Suárez, PhD:
Hospital Universitario Central de Asturias, Universidad de Oviedo,
Oviedo, Spain; 6Maria Francisca González-Escribano, PhD: Hospital
Virgen del Rocı́o, Sevilla, Spain; 7Javier Martı́n, MD, PhD: Instituto
de Biomedicina López Neyra, CSIC, Granada, Spain.
Address correspondence and reprint requests to Marta E.
Alarcón-Riquelme, MD, PhD, Department of Genetics and Pathology,
Rudbeck Laboratory, Uppsala University, Dag Hammarskjolds vag 20,
751 85 Uppsala, Sweden. E-mail: marta.alarcon@genpat.uu.se.
Submitted for publication December 11, 2006; accepted in
revised form December 21, 2006.
1234
SLE AND STRUCTURAL INSERTION/DELETION VARIATION IN IRF5
IRF5, a key component of the type I interferon
pathway, has a strong genetic association with systemic
lupus erythematosus (SLE). IRF5 is a transcription
factor involved in the transcriptional activation of various proinflammatory cytokines and interferon-␣ (IFN␣)
(1) and is primarily involved in host defense against
viruses. The various isoforms of IRF5 have different
effects on the interferon system (2).
The genetic association of IRF5 with SLE was
recently identified (3), and we have previously described
a mechanism through which IRF5 contributes to genetic
susceptibility to SLE (4). The IRF5 gene has 4 alternative exons in the 5⬘-untranslated region (5⬘-UTR),
named exon 1A, exon 1B, exon 1C, and exon 1D. The T
allele of the single-nucleotide polymorphism (SNP) no.
rs2004640 introduces a donor splice site leading to the
expression of exon 1B transcripts and the reduction of
exon 1C–derived transcripts (4). A second SNP located
5 kb downstream of IRF5, rs2280714, has a T allele that
is strongly associated with high levels of IRF5 (4).
Alleles at both SNP positions comprise a haplotype
associated with SLE (4).
Our primary hypothesis was that the 5⬘-UTRs
affected which of the alternative isoforms would be
expressed, and that the splice donor mutation represented by SNP no. rs2004640 changed their pattern of
expression. Identification of the major isoforms of IRF5
in patients with SLE is of major importance in understanding the mechanisms through which IRF5 leads to
the development of SLE. In the present study, we found
that a previously unknown structural insertion/deletion
in exon 6 of the IRF5 gene leads to the precise expression of specific isoforms in the risk haplotype associated
with SLE.
PATIENTS AND METHODS
Patients and controls. Peripheral blood mononuclear
cells (PBMCs) were obtained from SLE patients and healthy
controls from Argentina, Spain, and Germany. The groups of
subjects from Argentina and Spain have been previously
described (4). The group of subjects from Germany consisted
of 288 SLE patients and 245 healthy controls from various
centers in northern Germany. Samples from the German
subjects were collected at the University of Hannover. The
study was conducted with the participation of the members of
the Argentine, German, and Spanish Collaborative Groups
(Appendix A).
Trio families were from 2 separate populations. Complete trios consisted of both parents and an affected child, and
incomplete trios consisted of one parent and an affected child.
One set of 89 families was from Asturias in northern Spain and
the Madrid region (49 complete trios), and the second group of
1235
79 families was from Denmark (22 complete trios). All individuals fulfilled the American College of Rheumatology 1982
revised criteria for SLE (5). Control samples for expression
analysis were obtained from healthy donors at the Uppsala
Academic Hospital Blood Bank, as previously described (4).
Stimulation of PBMCs. Freshly isolated PBMCs were
stimulated with 1,000 units of IFN␣ (RayBiotech, Norcross,
GA) for 6 hours in RPMI 1640 medium supplemented with
penicillin/streptomycin and 10% fetal calf serum.
Complementary DNA (cDNA) synthesis and quantitative polymerase chain reaction (PCR) of IRF5 UTR–specific
transcripts. Total RNA from individuals with SLE carrying the
various genotypes was purified from PBMCs with TRIzol
(Invitrogen, San Diego, CA). We reverse-transcribed 2 ␮g of
total RNA with 2 units of MultiScribe reverse transcriptase in
PCR buffer II containing 5 mM MgCl2, 1 mM dNTPs, 0.4 units
of RNase inhibitor, and 5 ␮M oligo(dT). All reagents were
from Applied Biosystems (Foster City, CA). Synthesis was
performed at 42°C for 45 minutes, followed by 95°C for 5
minutes.
IRF5 isoforms with distinct 5⬘-UTRs were quantified
using TaqMan real-time PCR on an ABI Prism 7700 Sequence
Detector (Applied Biosystems) and SDS version 1.9.1 software. Primers used to distinguish PCR products with different
UTRs have been published previously (4). The exon 1D–
specific primer was 5⬘-GCTCAGCCCGGATCTGCAGTTGCCAG-3⬘. We used a common reverse primer lying in
exon 3 and a common TaqMan probe labeled with FAM and
TAMRA. We performed 45 cycles of 2-step PCR (95°C for 15
seconds and 63°C for 1 minute) in buffer containing 1.5 mM
MgCl2, 200 ␮M each dNTP, 0.5 units of Platinum Taq polymerase (Invitrogen), primer-probe mixture, and cDNA. Expression levels were normalized to levels of the human TBP
gene (Applied Biosystems).
Cloning and sequence analysis of IRF5 isoforms. PCR
amplification of diverse isoforms of IRF5 was performed with
the same forward primers used for the TaqMan assay. The
common reverse primer 5⬘-GCAGCCTTGTTATTGCATGCCAGCTG-3⬘ was designed to allow amplification of the
full-length isoforms. Cycle conditions were 95°C for 3 minutes,
followed by 40 cycles of 95°C for 15 seconds, 60°C for 15
seconds, and 72°C for 1.5 minutes. PCRs were performed in a
25-␮l reaction volume with 0.5 units of High Fidelity Platinum
Taq polymerase in the buffer supplied by the manufacturer
(Invitrogen). Electrophoresis was performed on PCR products
on a 1% agarose gel. PCR products were subcloned in
pCR4-TOPO vector (Invitrogen), and 50 random positive
clones were analyzed by sequencing. PCR was also used for
direct analysis of insertion/deletion patterns in cDNA prepared from PBMCs. The following primers were used: forward
5⬘-TGCAGGAGAGGAGGAGGAAGAAGAG-3⬘, reverse
5⬘-AACTTGATCTCCAGGTCGGTCA-3⬘.
In vitro translation efficiency. To determine the effect
of alternative 5⬘-UTRs on protein synthesis, we amplified
full-length UTRs using a common reverse primer, as for the
quantitative PCR, and primers Ap1 and Ap2 and human
spleen BD Marathon-Ready cDNA (Clontech, Palo Alto, CA).
PCR products were gel purified with a kit from Qiagen
(Chatsworth, CA) and subcloned in pCR4-TOPO vector for
sequence analysis. The longest fragments were subcloned in
pGL3 promoter vector. Plasmid DNA was purified with the
1236
KOZYREV ET AL
EndoFree plasmid Maxi kit (Qiagen) and transfected with
Lipofectamine 2000 into HEK 293 cells. To allow transfection
normalization, pRL-TK vector (Promega, Madison, WI) was
cotransfected with the reporter constructs. After 48 hours of
incubation, cells were harvested and lysed in a passive lysis
buffer, according to the recommendations of the manufacturer
(Promega). Firefly and Renilla luciferase activity levels were
measured in duplicate with the Dual Luciferase Reporter
Assay (Promega). Experiments were repeated 4 times with 2
independent DNA preparations.
Transterm (http://uther.otago.ac.nz/Transterm.html)
was used to analyze the 3⬘-UTR of IRF5 (6).
Genotyping. For genotyping, we used the TaqMan
SNP Genotyping Assay (Applied Biosystems), and detection
was performed using an ABI 9700 real-time PCR system. The
primers and probes were designed by the Applied Biosystems
Assays-on-Demand service for allele discrimination with the
5⬘-nuclease assay and fluorogenic probes. The average genotype completeness for SNP no. rs10954213 and no. rs2070197
was 98.8% for the samples from Argentine subjects, 99.6% for
the samples from German subjects, and 86% for the samples
from Spanish subjects. Genotyping for the insertion/deletion
was performed by PCR analysis under the conditions specified above, with the following primers: forward 5⬘GCCGTCCACACGCACTCTCTGTAG-3⬘, reverse 5⬘-CTGAAGCCAGCAGGGTTGCCAG-3⬘. PCR products were resolved on 2.5% agarose gels.
Accession numbers. Sequences for the full-length 5⬘UTRs of IRF5 have been deposited in GenBank under
accession numbers DQ995491, DQ995492, DQ995493, and
DQ995494. Accession numbers for the sequences of IRF5 with
insertion and deletion are DQ995495 and DQ995496, respectively.
Statistical analysis. Analyses of the genetic associations of the SNPs and haplotypes in patients and controls and
the family-based analysis were performed using Haploview,
version 3.2, which incorporates only complete trios. We used
Family Based Association Testing software (7), which estimates genotypes for the nongenotyped parents so that information from the incomplete trios can be used. The software
was used to analyze all Spanish and Danish trios jointly (n ⫽
168) and corroborate the association results, taking into consideration all genetic information. Results were comparable
with those obtained using Haploview. Five trios showed Mendelian inconsistencies and were excluded from the analysis.
Odds ratios were calculated as previously described (4). Correlations of IRF5 messenger RNA (mRNA) levels were analyzed using the t-test included in GraphPad software (GraphPad Software, San Diego, CA), as previously described (4). R
language was used for logistic regression analysis using the case
control material.
RESULTS
Low contribution of exon 1B transcripts to the
overall levels of IRF5. We performed quantitative analysis of IRF5 gene expression in human PBMCs upon
stimulation with IFN␣. These studies clearly showed
that exon 1A transcripts were expressed at higher levels
than other exon 1 transcripts in both stimulated and
unstimulated cells, while transcripts derived from exon
1B or exon 1C were produced at levels 100 times lower
(Figure 1a). Exon 1D transcripts initially cloned from
human spleen cDNA were also found at very low levels.
We reasoned that despite the low levels of
mRNA for some isoforms, the corresponding protein
levels might be different and dependent on 5⬘-UTR–
mediated translation efficiency. Therefore, we cloned
cDNA for the 4 full-length 5⬘-UTRs in the luciferase
reporter vector and analyzed their effect on in vitro
protein expression. The most potent was the 5⬘-UTR
encoded by exon 1A. Exon 1B and exon 1D did not show
any effect compared with the control (pGL3 promoter
vector), while the presence of exon 1C significantly
inhibited the efficiency of protein translation (Figure
1b). Thus, mRNA for exons 1B, 1C, and 1D all have
poor translation efficiency as compared with that for
exon 1A.
We then selected 18 individuals carrying the
various genotypes of rs2004640. Using primers specific
for each 5⬘-UTR, we performed PCR amplification and
cloned cDNA from those individuals and sequenced 50
clones for each. We observed that all 4 promoters and
5⬘-UTRs led to expression of the same set of isoforms
(V1, V4, V5, and V6 transcripts; results not shown).
Hence, the effect of exons 1B, 1C, and 1D on the
production of IRF5 protein isoforms, as compared with
exon 1A, is negligible, since they code for the same
isoform set as does exon 1A, which is expressed at far
higher levels.
Determination of isoform pattern by structural
insertion/deletion and alternative 3ⴕ acceptor splice
sites. Interestingly and unexpectedly, based on the sequences found in different samples, we could classify the
individuals into 3 separate groups: those who had V1
and V4 isoforms, those who had V5 and V6 isoforms,
and those who expressed all 4 isoforms (Figure 1c),
suggesting Mendelian segregation and the effect of new
structural genetic variation. We sequenced the complete
IRF5 gene in 6 individuals from each of the 3 groups.
We identified a novel insertion/deletion, which
lacked 2 of 4 tandem repeats in exon 6 encoding for a
proline-rich region within the putative PEST and interaction domains. The presence or absence of the repeats
determined the isoforms to be expressed, such that
individuals with 4 repeats (insertion) expressed isoforms
V5 and V6, while individuals with 2 repeats (deletion)
expressed isoforms V1 and V4 (Figure 1c). An alternative 3⬘ acceptor splice site in exon 6 defined the expression of V1 or V4 and V5 or V6 isoforms, respectively
SLE AND STRUCTURAL INSERTION/DELETION VARIATION IN IRF5
1237
Figure 1. a, Expression levels of exon 1A, 1B, and 1C transcripts in unstimulated human peripheral blood mononuclear cells (PBMCs) and PBMCs
stimulated with interferon-␣ (IFN␣). The data are typical of findings in individuals who have the T allele of rs2004640 and the A allele of rs10954213.
Expression levels were normalized to levels of TBP. The expression level of the exon 1D transcript was detected with nested polymerase chain
reaction (PCR) and is not shown on the plot. Values are the mean relative x-fold expression. b, Relative translation efficiency of the 4 alternative
5⬘-untranslated regions (5⬘-UTRs). HEK 293 cells were transfected with either pGL3 promoter vector (pGL3-prom) or constructs with alternative
5⬘-UTRs preceding luciferase gene. Firefly luciferase activity was measured with the Dual Luciferase Reporter Assay, and values were normalized
to Renilla luciferase activity. Values are the mean ⫾ SD of the relative luciferase activity in 4 experiments. c, IRF5 gene structure. Solid boxes are
protein coding exons. Open boxes are noncoding exons. Top, Location of the single-nucleotide polymorphisms and insertion/deletion (in-del)
variation. Bottom, Two alternative 3⬘ acceptor splice sites in exon 6, giving rise either to long or short isoforms with 2 repeats, V1 or V4, or to long
or short isoforms with 4 repeats, V5 or V6. The PCR gel shows the cDNA expression pattern obtained with PCR amplification of the segment from
exon 4 to exon 7, in 6 individuals with insertion/deletion genotypes having 2 repeats (2R), 4 repeats (4R), or both. Each lane represents 1 individual.
(Figure 1c). Further, all isoforms within each group (i.e.,
V1 and V4 in 1 group, V5 and V6 in 1 group, and V1,
V4, V5, and V6 in the heterozygous group) were equally
expressed, according to our comprehensive sequencing
analysis of transcripts in various individuals (results not
shown).
Role of the length of the 3ⴕ-UTR and SNP no.
rs10954213 in determining the level of IRF5. Through
bioinformatic analysis of the IRF5 gene, we found
that SNP no. rs10954213 is located in the 3⬘-UTR
polyadenylation site AAT(A/G)AA. Thus, SNP no.
rs10954213 potentially determines IRF5 expression levels, with the A allele predicting mRNA with a short
3⬘-UTR, and the G allele disrupting the poly(A) site and
thus causing transcription to continue. Computational
prediction with Transterm software revealed 2 strong
AU-rich elements (AREs) located within the long 3⬘UTR (6).
We tested whether any of the SNPs of IRF5
correlated with levels of IRF5 mRNA, and observed
that, as expected, rs10954213, the 3⬘-UTR SNP, correlated with levels of IRF5 mRNA, in particular when cells
were stimulated with IFN␣ (Figure 2). The highest
correlations were observed with the A allele, but these
did not reach statistical significance due to the number
of samples used. No correlation was observed for
rs2004640, insertion/deletion, or the risk haplotype (data
not shown).
1238
KOZYREV ET AL
Figure 2. Effect of rs10954213 on expression of the IRF5 gene in unstimulated PBMCs (non-stim) and PBMCs stimulated with 1,000 units IFN␣.
Diamonds represent individual samples, and bars represent the mean level of expression. The y-axis shows the expression levels normalized to TBP.
See Figure 1 for other definitions.
Insertion in the risk haplotype. The insertion was
found in the risk haplotype defined by the nonfunctional
SNP no. rs2070197, which is the SNP in IRF5 with the
highest level of association with SLE. SNP no. rs2070197
Table 1.
divided the risk haplotype identified previously (4) into
smaller haplotypes, with one (TCA) clearly segregating
in patients and one (GTG) clearly protective (Table 1).
The 2 novel SNPs, SNP no. rs10954213 and no.
Genetic association of the risk haplotype of IRF5*
Haplotype
Spanish samples
TTA
GTG†
GTA
TCA‡
Argentine samples
TTA
GTG†
GTA
TCA‡
German samples
TTA
GTG†
GTA
TCA‡
Combined samples
TTA
GTG†
GTA
TCA‡
TICA‡
Case
Frequency
Control
Frequency
␹2
P
OR (95% CI)
0.432
0.217
0.142
0.146
0.402
0.288
0.152
0.106
1.55
16.344
0.522
9.163
0.28
5.2 ⫻ 10⫺5
0.4698
0.002
–
0.68 (0.57–0.82)
–
1.44 (1.13–1.84)
0.309
0.363
0.094
0.178
0.303
0.431
0.129
0.091
0.042
5.616
3.636
19.026
0.836
0.0178
0.0565
1.28 ⫻ 10⫺5
–
0.75 (0.59–0.95)
–
2.18 (1.53–3.12)
0.374
0.274
0.135
0.165
0.343
0.331
0.168
0.091
1.073
3.979
2.244
12.466
0.300
0.046
0.134
0.0004
–
0.76 (0.58–0.99)
–
1.99 (1.35–2.93)
0.381
0.271
0.125
0.159
0.151
0.363
0.334
0.147
0.102
0.099
1.599
22.279
5.058
34.189
24.456
0.206
2.35 ⫻ 10⫺6
0.02
5.0 ⫻ 10⫺9
5.72 ⫻ 10⫺8
–
0.73 (0.65–0.83)
–
1.67 (1.40–1.99)
–
* The order of the single-nucleotide polymorphisms is rs2004640, rs2070197, and rs10954213. Odds ratios
(ORs) and 95% confidence intervals (95% CIs) are shown for the protective and risk haplotypes only.
† Protective haplotype.
‡ Risk haplotype. I ⫽ insertion.
SLE AND STRUCTURAL INSERTION/DELETION VARIATION IN IRF5
Table 2.
1239
Genetic association of the individual SNP risk alleles of IRF5 in combined samples*
SNP
Associated
allele
Case
frequency
Control
frequency
␹2
P
rs2004640
rs2070197
Insertion/deletion
rs10954213
T
C
I
A
0.602
0.166
0.507
0.660
0.516
0.096
0.515
0.606
34.857
43.083
0.23
11.263
3.5 ⫻ 10⫺9
5.2 ⫻ 10⫺11
0.626
0.0008
* SNP ⫽ single-nucleotide polymorphism.
rs2070197, and the insertion/deletion were genotyped in
trios, patients, and controls. The risk haplotype (TCA)
was formed by the T allele of rs2004640, the C allele of
rs2070197 (T⬎C), the insertion, and the A allele of
rs10954213 (G⬎A) (i.e., TICA).
As shown in Table 1, the same risk haplotype was
found in 3 separate sets of patients and controls, from
Spain, Germany, and Argentina, with a combined P
value of 5 ⫻ 10⫺9. SNP no. rs2070197 was by itself
strongly associated with SLE (P ⫽ 5.2 ⫻ 10⫺11) (Table
2), but bioinformatic analysis of the sequence did not
provide any potential evidence that this SNP was functional. SNP no. rs10954213 was weakly associated (P ⫽
0.0008), while the insertion/deletion was not by itself
associated with SLE (Table 2). Of the functional SNPs,
rs2004640 was the most strongly associated with SLE
(P ⫽ 3.5 ⫻ 10⫺9). Using a set of trio families, we
confirmed the haplotype and the presence of the insertion in the risk haplotype (Table 3). Thus, individuals
with the risk haplotype (TICA) expressed V5 and V6
isoforms. The insertion was also present in a protective
haplotype (GITG).
Using multivariate logistic regression, we tested
whether the 3 functional polymorphisms increased the
risk of developing SLE. We did not find evidence of this.
The 3 SNPs together did not improve the association
found for rs2004640 alone (data not shown). Most
Table 3. Genetic association of the risk haplotype of IRF5 in trio
families*
Haplotype†
TDTA
GITG
GDTA
TICA‡
Transmitted/
untransmitted
Frequency
␹2
P
21.6/29.1
17.8/29.9
9.1/13.6
30.0/8.0
36.1
26.8
13.5
16.4
1.101
3.118
0.887
12.737
0.5491
0.0189
0.4028
0.000137
* The order of the single-nucleotide polymorphisms is rs2004640,
rs2070197, and rs10954213. The trios from Spain and Denmark were
from a total of 168 families with 71 complete trios. Transmitted alleles
are expressed as absolute numbers. Haplotype frequencies and P
values were obtained using Family Based Association Testing software.
† D ⫽ deletion; I ⫽ insertion.
‡ Risk haplotype.
individuals who were homozygous for rs2004640 and
rs10954213 were heterozygous for the insertion (data
not shown).
DISCUSSION
We have identified a novel structural variation
determining the expression of the IRF5 isoforms. It was
previously thought that isoforms in IRF5 resulted from
the 5⬘-UTR and splicing at exon 6 (2). However, in the
present study we found that a polymorphic insertion/
deletion in exon 6 and the alternative 3⬘ acceptor splice
site in this exon determine the 2 major isoform groups in
IRF5. Further, we showed that all 5⬘-UTRs of the IRF5
gene can lead to equal transcription of the 4 main
isoform types, V1, V4, V5, and V6. Thus, the particular
exon 1 used makes no difference in terms of the protein
identity, and only exon 1A transcripts are present at high
amounts in unstimulated cells and cells stimulated with
IFN␣. It is well known that 5⬘-UTRs play an important
role in the regulation of gene expression (8,9).
Alternative UTRs with different indexes of translation efficiency can influence the amount of protein
expressed. Some structural elements in the 5⬘-UTR
determine the efficiency with which the protein synthesis
will be initiated and elongated. These include the length
and composition of the UTR, secondary structure, presence of upstream initiation codons, and the conformity
of the sequence surrounding the initiation codon, with
Kozak consensus defining the strength of the latter. In
vitro analysis of the alternative 5⬘-UTRs of the IRF5
gene showed that the exon 1A–encoded UTR had a
much higher translation potential than the other 3
UTRs, thus further supporting the hypothesis that the
IRF5 protein isoform pool is made up of mainly exon 1A
transcripts and very little or no exon 1B that is used
when the associated T allele is present. Taken together,
these data support the conclusion that SNP no.
rs2004640 is not functionally important in determining
the isoforms of IRF5.
We also found, in the polyadenylation site of
IRF5, an SNP (G⬎A) determining the length of the
1240
3⬘-UTR. The A allele leads to a short 3⬘-UTR, while the
G allele encodes for a 1.5-kb long 3⬘-UTR and has 2
strong AREs known to be responsible for short half-life
and rapid RNA degradation (10). Messenger RNA with
a shorter 3⬘-UTR bearing no such signals should be
more stable and present at high levels. Indeed, we found
that the A allele of rs10954213 correlated with high
levels of IRF5, particularly when cells were stimulated
with IFN␣. AREs are abundant in cytokines and growth
factor genes, for which rapid mRNA turnover is crucial
for gene function (for review, see refs. 10 and 11). In
mice, mutations disrupting AREs in the tumor necrosis
factor ␣ (TNF␣) gene lead to elevated levels of circulating TNF␣ and hypersensitivity to stimulation with
lipopolysaccharide (12,13). Thus, it is possible that SNP
no. rs10954213 in the 3⬘-UTR poly(A) site of IRF5 could
be the functional mutation responsible for the level of
IRF5. The previously identified SNP no. rs2280714 is a
perfect proxy for rs10954213, but is located 5 kb 3⬘ of
IRF5 and therefore cannot clearly account for the high
levels of IRF5.
The isoforms of IRF5 do not differ in their DNA
binding sites, but differ in the PEST and interaction
domains (2). This may lead to the use of different
coactivator or inhibitor proteins acting in specific cells or
tissue, leading in turn to the promotion of a particular
set of IRF5 targets. Exon 6 repeats forming the
insertion/deletion polymorphism encode for the prolinerich region. Such structures, often present in transcription factors, may define the variety and affinity of other
cofactors during transcription activity (14–16). Whether
and how the insertion/deletion in the IRF5 gene influences gene function remain to be shown.
IRF5 is clearly an SLE susceptibility gene, and in
the present study we identified a risk haplotype, using
samples from several populations. However, neither the
insertion nor the highly expressed allele of rs10954213
were associated with SLE or appear to explain the
genetic effect of IRF5 on susceptibility. Also, rs2004640,
the functional polymorphism with the strongest association with SLE identified to date for IRF5, has a small
functional impact. Therefore, it is highly conceivable
that we have not identified all functional variation in
IRF5 contributing to disease susceptibility and that one
or more additional risk haplotypes in linkage disequilibrium with rs2004640 are yet to be found. Alternatively,
despite its lower effect on genetic risk, rs10954213 may
have a stronger functional impact on disease expression.
This remains to be shown.
Our results may explain why apparently functional polymorphisms may not be replicated in some
KOZYREV ET AL
studies, in which haplotypes have not been unambiguously defined. Defining the risk haplotypes in disease
will be necessary for understanding of the functional
genetics behind disease susceptibility.
Addendum. Since the time this paper was submitted
for publication, an article describing the polyadenylation site
SNP of IRF5, no. rs10954213, has been published (Graham
DS, Manku H, Wagner S, Reid J, Timms K, Gutin A, et al.
Association of IRF5 in UK SLE families identifies a variant
involved in polyadenylation. Hum Mol Genet 2006. E-pub
ahead of print).
ACKNOWLEDGMENTS
We would like to thank Hong Yin for technical assistance with the samples, and the Uppsala Genome Center for
sequencing. We acknowledge Rob Graham and David Altshuler for sharing information on the typing of SNP no.
rs10954213 and SNP no. rs2070197 prior to publication. SNP
no. rs10954213 was partly typed at the Broad Institute. We
thank Adriana I. Scollo, Armando M. Perichon, and Mariano
C. R. Tenaglia of CEDIM Diagnóstico Molecular y Forense
SRL (Rosario, Argentina) for their help in DNA preparation
of the Argentine samples, and Dr. Anne Voss for clinical help
with the Danish samples. We would like to particularly thank
the Lupus Patient Association of Asturias for help in the
collection of family samples.
AUTHOR CONTRIBUTIONS
Dr. Alarcón-Riquelme had full access to all of the data in the
study and takes responsibility for the integrity of the data and the
accuracy of the data analysis.
Study design. Kozyrev, Pons-Estel, Junker, Laustrup, AlarcónRiquelme.
Acquisition of data. Kozyrev, Lewén, Reddy, Witte, Junker, Laustrup,
Gutiérrez, Suárez, González-Escribano, Martı́n.
Analysis and interpretation of data. Kozyrev, Lewén, Reddy, Junker,
Laustrup, Martı́n, Alarcón-Riquelme.
Manuscript preparation. Kozyrev, Pons-Estel, Witte, Junker, Laustrup, Alarcón-Riquelme.
Statistical analysis. Kozyrev, Reddy, Alarcón-Riquelme.
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APPENDIX A: THE ARGENTINE, GERMAN, AND
SPANISH COLLABORATIVE GROUPS
Members of the Argentine Collaborative Group are as follows: Hugo R. Scherbarth, MD, Pilar C. Marino, MD, Estela L. Motta,
1241
MD (Hospital Interzonal General de Agudos Dr. Oscar Alende, Mar
del Plata), Susana Gamron, MD, Cristina Drenkard, MD, Emilia
Menso, MD (UHMI 1, Hospital Nacional de Clı́nicas, Universidad
Nacional de Córdoba, Córdoba), Alberto Allievi, MD, Guillermo A.
Tate, MD (Organización Médica de Investigación, Buenos Aires), Jose
L. Presas, MD (Hospital General de Agudos Dr. Juán A. Fernandez,
Buenos Aires), Simon A. Palatnik, MD, Marcelo Abdala, MD, Mariela
Bearzotti, PhD (Universidad Nacional de Rosario y Hospital Provincial del Centenario, Rosario), Alejandro Alvarellos, MD, Francisco
Caeiro, MD, Ana Bertoli, MD (Hospital Privado, Centro Medico de
Córdoba, Córdoba), Sergio Paira, MD, Susana Roverano, MD (Hospital José M. Cullen, Santa Fe), Cesar E. Graf, MD, Estela Bertero,
PhD (Hospital San Martı́n, Paraná), Cesar Caprarulo, MD, Griselda
Buchanan, PhD (Hospital Felipe Heras, Concordia, Entre Rı́os),
Carolina Guillerón, MD, Sebastian Grimaudo, PhD, Jorge Manni, MD
(Instituto de Investigaciones Médicas Alfredo Lanari, Buenos Aires),
Luis J. Catoggio, MD, Enrique R. Soriano, MD, Carlos D. Santos, MD
(Hospital Italiano de Buenos Aires y Fundación Dr. Pedro M.
Catoggio para el Progreso de la Reumatologı́a, Buenos Aires), Cristina
Prigione, MD, Fernando A. Ramos, MD, Sandra M. Navarro, MD
(Hospital Provincial de Rosario, Rosario), Guillermo A. Berbotto,
MD, Marisa Jorfen, MD, Elisa J. Romero, PhD (Hospital Escuela Eva
Perón, Granadero Baigorria, Rosario), Mercedes A. Garcia, MD, Juan
C. Marcos, MD, Ana I. Marcos, MD (Hospital Interzonal General de
Agudos General San Martı́n, La Plata), Carlos E. Perandones, MD,
Alicia Eimon, MD (Centro de Educación Médica e Investigaciones
Clı́nicas, Buenos Aires), and Cristina G. Battagliotti, MD (Hospital de
Niños Dr. Orlando Alassia, Santa Fe).
Members of the German Collaborative Group are as follows:
K. Armadi-Simab, MD, Wolfgang L. Gross, MD (University Hospital
of Schleswig-Holstein, Campus Luebeck, Rheumaklinik Bad Bramstedt, Luebeck), Erika Gromnica-Ihle, MD (Rheumaklinik BerlinBuch, Berlin), Hans-Hartmut Peter, MD (Medizinische Universitaetsklinik, Abteilung Rheumatologie und Klinische Immunologie,
Freiburg), Karin Manger, MD (Medizinische Klinik III derFAU
Erlangen-Nuernberg, Erlangen), Sebastian Schnarr, MD, Henning
Zeidler, MD (Abteilung Rheumatologie, Medizinische Hochschule
Hannover, Hannover), and Reinhold E. Schmidt, MD (Abteilung
Klinische Immunologie, Medizinische Hochschule Hannover, Hannover).
Members of the Spanish Collaborative Group are as follows:
Norberto Ortego, MD (Hospital Clı́nico San Cecilio, Granada), Enrique de Ramón, MD (Hospital Carlos Haya, Malaga), Juan JiménezAlonso, MD (Hospital Virgen de las Nieves, Granada), Julio SánchezRomán, MD (Hospital Virgen del Rocio, Sevilla), and Miguel Angel
López-Nevot, MD (Hospital Virgen de las Nieves, Granada).
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associates, precise, variation, insertiondeletion, systemic, erythematosus, expressed, defined, irf5, lupus, structure, haplotype, isoforms, risk
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