Comprehensive Linkage and Association Analyses
Identify Haplotype, Near to the TNFSF15 Gene,
Significantly Associated with Spondyloarthritis
Elena Zinovieva1,2, Catherine Bourgain3, Amir Kadi1,2, Franck Letourneur1,2, Brigitte Izac1,2,
Roula Said-Nahal4, Nicolas Lebrun1,2, Nicolas Cagnard5, Agathe Vigier1,2, Se ´bastien Jacques1,2,
Corinne Miceli-Richard6, Henri-Jean Garchon1,2, Simon Heath7, Ce ´line Charon7, Delphine Bacq7,
Anne Boland7, Diana Zelenika7, Gilles Chiocchia1,2,4., Maxime Breban1,2,4.*
1Institut Cochin, Universite ´ Paris Descartes, CNRS (UMR 8104), Paris, France, 2INSERM U567, Paris, France, 3INSERM U535, Universite ´ Paris Sud – Paul Brousse Hospital,
Villejuif, France, 4Rheumatology Division, Ambroise Pare ´ Hospital (AP-HP), and Versailles Saint Quentin en Yvelines University, Boulogne-Billancourt, France,
5Bioinformatics Platform, Faculty of Medicine Paris Descartes, Necker Hospital, Paris, France, 6Rheumatology Division, Kremlin-Bice ˆtre Hospital (AP-HP), Kremlin-Bice ˆtre,
France, 7National Genotyping Center (CNG), Evry, France
Spondyloarthritis (SpA) is a chronic inflammatory disorder with a strong genetic predisposition dominated by the role of
HLA-B27. However, the contribution of other genes to the disease susceptibility has been clearly demonstrated. We
previously reported significant evidence of linkage of SpA to chromosome 9q31–34. The current study aimed to
characterize this locus, named SPA2. First, we performed a fine linkage mapping of SPA2 (24 cM) with 28 microsatellite
markers in 149 multiplex families, which allowed us to reduce the area of investigation to an 18 cM (13 Mb) locus delimited
by the markers D9S279 and D9S112. Second, we constructed a linkage disequilibrium (LD) map of this region with 1,536 tag
single-nucleotide polymorphisms (SNPs) in 136 families (263 patients). The association was assessed using a transmission
disequilibrium test. One tag SNP, rs4979459, yielded a significant P-value (4.961025). Third, we performed an extension
association study with rs4979459 and 30 surrounding SNPs in LD with it, in 287 families (668 patients), and in a sample of
139 cases and 163 controls. Strong association was observed in both familial and case/control datasets for several SNPs. In
the replication study, carried with 8 SNPs in an independent sample of 232 cases and 149 controls, one SNP, rs6478105,
yielded a nominal P-value,361022. Pooled case/control study (371 cases and 312 controls) as well as combined analysis of
extension and replication data showed very significant association (P,561024) for 6 of the 8 latter markers (rs7849556,
rs10817669, rs10759734, rs6478105, rs10982396, and rs10733612). Finally, haplotype association investigations identified a
strongly associated haplotype (P,8.861025) consisting of these 6 SNPs and located in the direct vicinity of the TNFSF15
gene. In conclusion, we have identified within the SPA2 locus a haplotype strongly associated with predisposition to SpA
which is located near to TNFSF15, one of the major candidate genes in this region.
Citation: Zinovieva E, Bourgain C, Kadi A, Letourneur F, Izac B, et al. (2009) Comprehensive Linkage and Association Analyses Identify Haplotype, Near to the
TNFSF15 Gene, Significantly Associated with Spondyloarthritis. PLoS Genet 5(6): e1000528. doi:10.1371/journal.pgen.1000528
Editor: Kathleen Kerr, University of Washington, United States of America
Received August 21, 2008; Accepted May 19, 2009; Published June 19, 2009
Copyright: ? 2009 Zinovieva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was financially supported by grants from the French Rheumatology Society (http://www.rhumatologie.asso.fr), from the National Research
Program on Oseo-Articular Diseases (PRO-A), from the ‘‘Arthritis-Courtin’’ foundation (http://www.fondation-arthritis.org/), and by unrestricted grant from the
Schering-Plough company. Elena Zinovieva benefited for three years from a fellowship grant from the ‘‘Arthritis-Courtin’’ foundation (http://www.
fondation-arthritis.org/).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Spondyloarthritis (SpA) is one of the most frequent varieties of
articular inflammatory disorders with an estimated prevalence of
0.3% in the western European adult population . It is
characterized by a predominant axial skeleton inflammation, by
a frequent occurrence of enthesitis and peripheral arthritis, and
also by a high rate of extra-articular features, the most
characteristic of which are acute anterior uveitis, psoriasis, and
inflammatory bowel diseases (such as ulcerative colitis or Crohn’s
disease (CD)) . Depending on its clinical features, SpA is
classically subdivided into the following subsets: ankylosing
spondylitis (AS), which is the prototypical form characterized by
predominant axial skeletal involvement and advanced radiograph-
ic sacroiliitis, psoriatic arthritis (PsA), arthritis associated with
inflammatory bowel disease (AIBD), reactive arthritis (ReA), and
undifferentiated SpA (uSpA). Familial aggregation among these
conditions has been well established. Notably, we have previously
shown, by analyzing a large number of pedigrees with multiple
cases of SpA, that all subtypes are likely to be determined by a core
set of predisposing factors and may therefore be studied together
in genetic studies [3–5].
The HLA-B27 allele is the first genetic factor which was
demonstrated to be associated with AS [6,7] and other SpA
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[4,5,8]. Although about 80% of Caucasian patients are HLA-B27
positive, as compared to only 6–8% in the general population, the
exact mechanism for this association remains poorly understood
Family and twin studies have demonstrated additional non-
MHC susceptibility regions elsewhere in the genome . For
example, concordance rates for HLA-B27 positive monozygotic
twins are twice as high as the concordance rates for HLA-B27
positive dizygotic twins . Furthermore, the involvement of
genetic factors arising from outside the HLA region is suggested by
the large non-HLA component of the relative recurrence risk for
the SpA estimated in sib-pairs (lnon-HLA). Indeed, if the overall
relative recurrence risk in sibling (ls) has been estimated to be 40
, estimates of the ls component attributable to the HLA
region (lHLA), based on previous affected sib-pairs linkage
analyses, ranges from 5.2 to 6.25 [12,13]. Variants in several
genes such as the IL-1 family gene cluster [14,15], IL-23R ,
and ARTS1/ERAP1 , have recently been reported to be
associated with AS based on a candidate-gene approach [14,15] or
a non-synonymous single-nucleotide polymorphisms (SNPs) ge-
nome-wide association study .
Our team has previously reported results of the first genome-
wide linkage screen and its extension study performed in SpA .
Overall, 893 individuals from 120 multiplex families (families with
several patients) comprising 336 affected relative pairs have been
genotyped in this study. Non parametric multipoint linkage
analysis of the whole dataset yielded evidence for significant
linkage to the chromosomal region 9q31–34 (NPLmax=4.87,
P=261025). This locus overlapped with one of those identified by
the genome-wide linkage screen performed in AS by a group from
Oxford . We named this new susceptibility location SPA2, in
reference to the MHC locus, which we considered as SPA1 .
SPA2 encompasses a 23.95 cM region (17.44 Mb) containing 85
genes and predicted coding sequences as well as 110 pseudogenes.
This locus is very appealing with regard to SpA susceptibility. First
of all, it is one of three genomic regions paralogous to the MHC,
which is the major SpA susceptibility region [17,18]. Furthermore,
it is syntenic to the Pgis2 susceptibility locus mapped in a murine
model of SpA . Within its borders SPA2 contains both the
TNFSF15 gene found to be associated with CD a condition
belonging to the SpA spectrum [20,21], and the TRAF1-C5 locus
associated with rheumatoid arthritis another inflammatory
rheumatic disease [22,23].
The goal of the present study was to identify variants associated
with the disease and located in the SPA2 locus. Using a
comprehensive four-step linkage and association study in a total
of 287 families including 668 affected individuals, followed by an
independent case/control analysis (2 samples including a total of
371 cases and 312 controls), we identified a strongly associated six-
SNPs haplotype, located at 28.6 kb from the TNFSF15 gene.
Linkage fine mapping
The initial step of our study aimed to refine the linkage signal in
the 23.95 cM (17.44 Mb) SPA2 locus. To realise this investigation
we selected a fine-grained set of 28 microsatellite markers (more
than one marker per cM). These markers were genotyped in 149
independent multiplex families (including the 120 families studied
in our initial genome-screen)  (Figure 1B) consisting of 1,065
individuals including 458 affected with SpA (Figure 1A, Table 1).
Non parametric multipoint linkage analysis allowed us to
identify two prominent linkage peaks yielding a significant Zlr
value.2.91 (nominal P,1.7961023) corresponding to a P,0.05
after correction for multiple testing (Table 2, Figure 2A). The
highest peak of linkage was found for the marker D9S1824
at 120.1 cMfromthe p-telomere
P=6.9461024). At this stage of the study it was not possible to
discriminate between these two peaks, thus we decided to pursue
our investigations in the 13.1 Mb region surrounding them
between D9S279 and D9S112 (Figure 2A).
Linkage disequilibrium (LD) mapping
In the second part of our study we performed a linkage
disequilibrium (LD) mapping of the 13.1 Mb region selected after
the linkage fine mapping, using a family-based association test. We
employed a tag SNP strategy that consisted of genotyping a set of
1,536 markers, extensively representative of genetic variability at
the chromosomal region, in a sample of 136 families (Table 1).
The sample was composed of 36 families with the highest linkage
values in the selected 13.1 Mb region, and 100 novel families
never tested before for either linkage or association (Figure 1).
Among the 1,536 tag SNPs genotyped, 1,489 (96.9%) were
suitable for family-based association testing. The remaining 47 tag
SNPs were discarded from the analysis for genotyping failure or
lack of polymorphism across the sample. Association analysis was
performed using a transmission disequilibrium test (TDT) adapted
for families larger than trios, and suitable for testing of association
in the area of known linkage . Considering the number of tag
SNPs tested, applying crude Bonferroni correction would set the
nominal P-value corresponding to a global type I error of 5% at
3.461025. However such threshold is overly conservative, since
some of the 1,489 tag SNPs presented a weak level of LD and were
therefore not totally independent. To date, there is no consensus
on the best method to take into account the non independence
between SNPs. A method such as that proposed by Nyholt ,
would set the nominal 5% threshold at P=5.5661025. Alterna-
tively, accepting a corrected global type I error of 7.5% with
Bonferroni would set the nominal threshold at P=561025. Using
such criteria, one single intergenic tag SNP, rs4979459, was found
to be significantly associated, with SpA (P=4.961025, Figure 2A,
Table S1). Suggestive association was also found for several
Spondyloarthritis (SpA) is a common variety of articular
inflammatory disorder characterized by axial and/or
peripheral arthritis, frequently associated with extra-
articular manifestations such as psoriasis, uveitis, and
inflammatory bowel diseases (ulcerative colitis or Crohn’s
disease (CD)). SpA is a complex disorder with high
heritability. The MHC class I HLA-B27 allele is a very strong
risk factor for its development, but other genetic factors
located outside the MHC also play a role in disease
investigation, we have demonstrated that a region located
on the chromosome 9q31–34 was involved in SpA
susceptibility. The present study aimed to further charac-
terize this locus. Using a stepwise linkage and association
approach, we identified a haplotype spanning 6 single-
nucleotide polymorphisms strongly associated with SpA
and located in a genomic region paralogous to the MHC,
near to the TNFSF15 gene. Interestingly, polymorphisms of
this gene have previously been shown to be associated
with CD. This original finding offers a new research track
for the understanding of SpA pathophysiology, which is
still poorly understood, as well as new hope for diagnostic
and therapeutic innovation.
Mapping a SpA Locus Near to TNFSF15
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Figure 1. Study design. (A) shows the different stages of the study displayed according to their chronological order from left to right. Frames in
blue describe the familial and case/control samples used for each different step. Frames in orange describe the sets of markers, the limits of the
intervals covered by these markers on chromosome 9 (in distance from the p-telomere), and the techniques used for single-nucleotide
polymorphisms initial genotyping. Frames in green describe the type of genetic analysis performed, the statistic, and the program used in each case.
(B) shows compositions of family and case/control samples used in each step of the study. The three first columns show for each step of the study
which familial, and case and/or control sample respectively, was used. The far right column displays, for each genotyped sample, the step of the study
in which it was involved. The overlay parts of the samples are matched. Ex: Of the 136 families included in the LD mapping association study, 36 were
also used in the fine mapping linkage study and in the initial whole genome and extension screen. NB: Both case/control samples were composed of
independent individuals not tested elsewhere. Abbreviations: LD: linkage disequilibrium, SpA: spondyloarthritis, SNP: single-nucleotide
Table 1. Pairwise distributions among first and second degree relative pairs included in family-based study designs.
Study design Linkage fine mapping Linkage disequilibrium mappingExtension study
Sib-pairs251 73 266
Parent/child 14294 211
Total556 210 606
aNiece/aunt, niece/uncle, nephew/aunt, nephew/uncle pairs.
For each family-based study design, the number of each affected relative pairs type included in the study is displayed.
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additional markers in LD with rs4979459 (Figure 2B, Table S1).
The evidence of association for these SNPs was also supported by
the comparison of observed and expected distributions of
association P-values (Figure 3A). The distribution of observed P-
values was very suggestively skewed from the null distribution with
23 SNPs having P,0.01, versus 14 expected under the null
hypothesis. All SNPs demonstrating significant or suggestive
association were located within an 80 kb LD block downstream
from the TNFSF15 gene (Figure 2A and 2C). There was no other
region in the SPA2 locus presenting significant or even suggestive
association with SpA. Notably the TRAF1-C5 locus previously
identified as associated with rheumatoid arthritis did not show any
association with SpA in our investigation (Figure 2A).
Extension family-based and case/control association
To refine the association signal identified by the LD mapping
stage we genotyped the tag SNP rs4979459 and an additional
panel of 30 surrounding SNPs in an extended sample of 287
families comprising 668 SpA patients (including the 149 families of
the linkage fine mapping, the 100 families added for the LD
mapping, and 38 additional families (Table 1, Figure 1B)), as well
as in an independent set of 139 cases and 163 controls (Figure 1A
and 1B). Association was investigated by the TDT described above
for the family sample and by an allelic chi-square test for the case/
control set. In keeping with the LD mapping stage, we used the
Bonferroni correction for multiple testing. The nominal P-values
to achieve global type I errors of 5% and 7.5% significance were
1.6161023and 2.4261023respectively. Of note, the Nyholt
correction set the nominal 5% threshold at P=3.9361023.
In the family-based association study, the 5% Bonferroni
corrected significance threshold was reached for 3 SNPs:
rs10817669, rs10739427, and rs10759734, and the 7.5% signif-
icance threshold for 2 additional SNPs: rs10733612, and
rs7849556 (Table 3, Table S2). Several other markers including
the tag SNP rs4979459 yielded low P-values (Table S2). For all
these SNPs the major allele was overtransmitted to affected
In the case/control study two markers, in strong LD with each
other, reached a Bonferroni corrected 0.005 significance thresh-
old: rs6478105 and rs10982396 (Table 3, Figure 4). Odds ratios
(ORs),1 were observed for both of them, indicating that the
Table 2. Results of linkage analysis of 28 microsatellite markers spanning the SPA2 locus.
Marker Genetic location (cM) Physical location (bp) Zlrnominal P Information content
D9S1677112.9110,977,411–110,977,668 2.38 8.6861023
D9S160 113.2 111,418,819–111,418,9762.476.8461023
D9S1675114.4 112,128,955–112,129,173 2.349.6861023
D9S1854 114.4112,149,837–112,150,087 2.349.6961023
D9S1683 115.1 112,903,157–112,903,3312.19 1.4161022
D9S279 118.8 115,267,469–115,267,7142.78 2.6861023
D9S1824120.1115,931,926–115,932,043 3.20 6.9461024
D9S1855121.3 116,550,769–116,550,9892.90 1.8961023
D9S155 121.4116,938,283–116,938,4062.65 3.9861023
D9S177121.5 116,999,255–116,999,3732.79 2.6361023
D9S170123.0 118,107,200–118,107,3152.51 6.1161023
D9S1802 123.3118,663,193–118,663,409 2.673.8061023
D9S1811 123.9 119,261,235–119,261,616 2.673.7961023
D9S1864125.4 119,526,784–119,527,0532.52 5.8161023
D9S1881 134.5 126,019,300–126,019,5293.07 1.0961023
D9S1825 135.1126,927,953–126,928,083 3.03 1.2461023
D9S1829 135.2 127,814,464–127,814,679 3.031.2361023
D9S112 136.8128,413,136–128,413,2662.91 1.8061023
Analysis was carried out in 149 independent families with multiple cases of spondyloarthritis. Three markers previously studied in a subset of 120 of these families
appear in bold . Markers were placed according to ENSEMBL database. Zlr statistics were calculated by multipoint analysis with ALLEGRO program. Markers within
frames are those for which positive linkage was detected with P,0.05, after correction for multiple testing.
Mapping a SpA Locus Near to TNFSF15
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Mapping a SpA Locus Near to TNFSF15
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major allele was more frequent in cases than in controls. Other
markers yielded non-significant low P-values (Table S3).
The evidence of association for these SNPs was also supported
by the comparison of the observed and expected distributions of P-
values for association (Figure 3B). The distribution of observed P-
values was very suggestively skewed from the null distribution with
7 SNPs having P,0.01 in the family-based study and 2 in the
case/control study, versus 0 expected under the null hypothesis.
All the markers significantly associated with SpA in all of our
association studies belong to the same LD block (Figure 4).
The 5 SNPs located within the TNFSF15 gene, which were
previously found to be associated with CD [20,21], did not reach a
level of significant association in both the family-based design and
the case/control study (Table S2 and Table S3).
Replication case/control association study
To replicate our results in an independent sample, we
genotyped a set of eight SNPs consisting of the most strongly
associated markers in the foregoing extension study (rs7849556,
rs10817669, rs10759734, rs6478105, rs1982396, and rs10733612),
the tag SNP (rs4979459) and one SNP located in the neighboring
TNFSF15 gene (rs4246905) in an additional independent set of
232 cases and 149 controls (Figure 1, Table 3). Association was
assessed using a chi-square test. After correction for multiple
testing, one SNP, rs6478105, reached a suggestive association level
(nominal P=0.029), the Bonferroni corrected for multiple testing
thresholds being 0.0063 (5%) and 0.0094 (7.5%).
Of note, OR,1 was observed for all eight SNPs. This trend was
similar to that observed in the whole extension step of the study,
for both family-based and case/control approaches (see above),
suggesting that even if the P-value for significance was not reached
here, the direction of association was consistent between both
studies for all markers.
Pooled case/control analyses
The entire case/control sample, comprising the ‘‘extension’’
and the ‘‘replication’’ part (371 cases and 312 controls) was
actually an independent set, as compared to the family sample
(Figure 1). Thus, it made sense to perform an association analysis
on this ‘‘pooled’’ sample. Results of this analysis are displayed in
the Table 3. Significant association level was reached for two of
the eight tested SNPs: rs6478105 (P=361025; OR=0.5) and
rs10982396 (P=261024; OR=0.53). Furthermore, three other
SNPs presented suggestive association
(rs7849556, rs10759734, and rs10733612). Notably, HLA-B27
conditioned exploratory analyses showed exactly the same trend of
allelic distribution between cases and controls, suggesting that the
detected association signal was independent of the presence of
HLA-B27 (Table S4 and Table S5).
We also performed combined analysis of the genotyping issued
from both the family extension sample and either the pooled case/
control set or the extension case/control set for the same eight
SNPs. The tests were performed using the Cochran-Mantel-
Haenszel method  (Table 3 and Table S6, respectively). These
investigations led us to confirm the strong association with SpA of
the whole LD block containing these SNPs (Figure 4).
Of note the non-synonymous SNP, rs4246905, which changes a
histidine into arginine, located in the fourth exon of TNFSF15
gene and previously identified as strongly associated with CD
[20,21] reached a suggestive P-value of less than 0.01 in both
combined analyses. Nonetheless, this was by far the weakest
association of the eight tested markers.
Figure 3. Quantile-quantile (Q-Q) plots comparing the distri-
butions of observed versus expected P-values. (A) shows the LD
mapping Q-Q plot. The blue squares represent the observed P-values.
The grey line represents the null hypothesis of no true association. (B)
shows the extension study mapping Q-Q plot. The green circles and the
red triangles represent the observed P-values for the family-based and
case/control studies respectively. The grey line represents the null
hypothesis of no true association.
Figure 2. Results of linkage disequilibrium mapping association study. (A) shows results of both fine mapping linkage step (right y axis) and
LD mapping step (left y axis). Linkage results are shown in red, with the dashed straight line symbolizing the 0.05 Bonferroni corrected significance
threshold. Family-based association results are displayed in blue, with each diamond corresponding to a studied tag SNP. The dotted straight line
shows the 0.075 Bonferroni corrected significance threshold. In green are represented the locations of the TNFSF15 gene, associated with Crohn’s
disease and of the TRAF1-C5 locus associated with rheumatoid arthritis. (B) shows a zoomed view of the 135 kb region of interest delineated by
frames in (A). Each diamond, corresponding to a studied tag SNP, appears coded by color and size, according to its linkage disequilibrium correlation
coefficient (r2) with the most significantly associated tag SNP (rs4979459), as displayed in the legend. (C) shows linkage disequilibrium structure
across the 135 kb region zoomed in (B), based on r2coefficient calculated with the CEU HapMap database. The intron exon structure of the TNFSF15
gene lying within this locus is represented at the top of (C). The genotyped tag SNPs shown in (B) are indicated with red bars. Three of these tag SNPs
lie within the TNFSF15 gene.
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Family-based and case/control haplotypic analyses
The six SNPs presenting the lowest P-values after the combined
analysis were located in the same strong LD block (Figure 4). It is
known that in the presence of multiple tightly linked markers a
haplotype test may be more powerful to detect association.
Association results for the haplotypes comprising these six SNPs
are displayed in the Table 4, together with their estimated
The family-based haplotype association analysis identified the
most frequent allele H1 as overtransmitted to affected children
with a high significance level (Table 4; P=8.8161026), the other
three alleles being undertransmitted. The significance threshold
corrected for the multiplicity of tested haplotypes and extrapolated
with Bonferroni method was set to 0.013.
The case/control haplotype association analysis conversely
showed that the rare haplotype H2 was very significantly more
frequent in controls than in cases (Table 4; P=8.7561025), with
all the other haplotypes being more frequent in cases than in
controls. Case/control HLA-B27 conditioned analyses showed
that the frequency of H2 haplotype was significantly decreased in
patients, independently of the presence of HLA-B27 (Table S4 and
A significant omnibus haplotype association was detected in
family-based sample (P=4.561025at 4 degrees-of-freedom (df)),
as well as in the case/control one (P=7.061024at 3 df).
We report for the first time an association between several SNPs
determining a haplotype near the TNFSF15 gene (9q32) and SpA.
This was achieved by a comprehensive study with several linkage
and association steps.
The basis of our investigations aimed to narrow down from our
previous whole genome linkage screen the susceptibility region for
SpA on chromosome 9q31–34 called SPA2 . Since initial
linkage in this locus was spread over a long distance of 23.95 cM,
our first attempt was to refine it using a fine-grained set of 28
microsatellite markers in an extended set of families. The results
revealed two areas of statistically significant linkage, with the
highest linkage peak located on the marker D9S1824 at 120.1 cM
(115.9 Mb) from the p-telomere, only 1.3 cM apart from
D9S1776, which corresponded to the linkage peak in our former
screen. The second statistically significant area was located near a
suggestive linkage peak reported in AS (D9S1682, P=661024)
, supporting the validity of linkage between this region and
SpA. Since non parametric linkage analyses usually do not
characterize linkage localization with high precision , we
decided to continue our investigations on the 13.1 Mb region
between markers D9S279 and D9S112 comprising both signifi-
cantly linked areas.
To identify whether one or several loci of the refined SPA2
region were associated with SpA we performed a family-based
dense LD mapping of this 13.1 Mb area, using a tag SNP strategy.
Our sample set was enriched in families with strong evidence of
linkage, in order to minimize the risk of false negative results. After
correction for multiple testing, significant association was observed
for one single tag SNP, rs4979459 (P=461025).
Several arguments support the assumption that this is a true
positive finding. Firstly, suggestive association was also found for
several additional markers in LD with rs4979459, indicating that
the identified significant association was unlikely to be explained
by systematic genotyping error. Secondly, the use of a family-
based design rules out the possible confounding effect of
population stratification. Finally, rs4979459 was located in the
Table 3. Results of family-based and case/control association studies for eight SPA2 single-nucleotide polymorphisms (SNPs).a
Family Extension Results
Case/Control Extension Results (139Cases/163 Controls)
Case/Control Replication Results
(232 cases/149 controls)
Pooled Case/Control Results
(371 cases/312 controls)
OR (95% CI)
aRefer to Figure 1 for the study design. OT: overtransmitted to affected children allele; OR: odds ratio; CI: confidence interval.
bAsymptotic P-values of the chi-square test for allele frequencies comparison between cases and controls.
cAsymptotic P-values for combined family and case/control samples computed with the Cochran-Mantel-Haenszel test.
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Figure 4. Linkage disequilibrium (LD) plot for the eight single-nucleotide polymorphisms genotyped in the replication study. LD
calculations were based on pairwise r2values of our entire case/control dataset. These values are displayed, in percentage, in the grey boxes. The red
triangle indicates the LD block, computed by the program HAPLOVIEW, which was further used for haplotypic association with the program PLINK.
Table 4. Results of family-based and case/control haplotype association analyses for the most associated LD block.a
Extension family-based study (287 families 1,578
individuals, 668 SpA patients)
Pooled case/control study (371 SpA
pedigreesZ FBAT P-value
- OMNIBUS -/ 12725.26 4.561025
H1AAAACC 0.714116 4.458.8161026
0.75 0.71 0.095
21.11 0.2670.08 0.158.7561025
23.15 0.0020.10 0.090.613
21.840.066 0.06 0.040.110
aSNP: single nucleotide polymorphism.
bThe tested haplotype consisted of the 6 following SNPs: rs7849556 (1); rs10817669 (2); rs10759734 (3); rs6478105 (4); rs10982396 (5); rs10733612 (6).
Results are shown for the omnibus haplotype tests and for the four individual haplotypes called H1 to H4, with an allele frequency high enough to allow meaningful
statistical tests. These represented 99% of the alleles. For the family-based study, the number of analyzed families, large sample FBAT statistic (Z), and the association P-
value are shown. A positive Z means that the concerned allele was found as overtransmitted to affected offspring; a negative Z conversely indicates an
undertransmission. When the omnibus haplotype association is concerned the ‘‘Z’’ symbolizes the chi-square statistic. For this type of test the number of degrees-of-
freedom (df) was 4. For case/control analyses the frequency of each haplotype-allele in cases and in controls, as well as the association P-value are displayed. The
number of df for the omnibus association was 3.
Mapping a SpA Locus Near to TNFSF15
PLoS Genetics | www.plosgenetics.org8 June 2009 | Volume 5 | Issue 6 | e1000528
direct vicinity of our highest linkage peak, between the
microsatellite markers D9S279 and D9S1855.
None of the genes having a counterpart in the MHC, i.e. those
that are theoretically good candidates for disease susceptibility,
were shown to be associated with the disease in this study. Notably,
the TRAF1-C5 locus which lies at 122.7 Mb from the p-telomere
and was recently described as associated with rheumatoid arthritis
[22,23], did not show any association with SpA in our study.
To refine the association pointed out by our LD map, we
performed an extension study with 31 SNPs. Characteristically,
the initial strong association observed with rs4979459, was not as
strong in the extension stage, suggesting that rs4979459 is not the
causal variant. Its effect was probably overestimated in the LD
mapping step, since initial positive reports tend to overestimate
found effects, while subsequent studies regress to the true value
. Moreover, SpA is a complex disease with known genetic
heterogeneity, and therefore enriching the LD mapping family set
with strongly linked families is also likely to have contributed to
After extension and replication steps of our study, strong
association was seen in the family sample as well as in the entire
case/control set with SNPs located in the strong 40.3 kb LD block
showed in the Figure 4. The markers that were found to be
associated by these two approaches were not exactly the same.
Several reasons could readily explain such an apparent discrep-
ancy. First of all, the alleles of rs6478105 and rs10982396, which
were found to be associated with SpA in the pooled case/control
sample, were highly frequent (.0.89). Therefore there were a
relatively modest number of informative families for these markers
in the family-based study, since at least one parent has to be
heterozygous to render the family suitable for association analysis.
On the other hand, the power of our case/control study was much
lower to demonstrate association with rs10817669 (the SNP
significantly associated in the family-based study (OR of 0.82)),
than with rs6478105 and rs10982396 (OR of 0.50, and 0.53,
Nevertheless, even if the significance level was not reached in
every study for every marker (probably attributable to a lack of
power), we can see that in three totally independent samples trends
for all SNPs were exactly the same: the frequent allele of each
marker was overtransmitted to affected children and noticeably
more present in cases rather than in controls. Moreover the results
of combined analysis of family and case/control samples also
strongly support the association with all six SNPs, composing the
40.3 kb LD block. Finally, our haplotype investigations also
confirmed this trend and showed that haplotypes composed of
markers of this block were very significantly associated with the
disease, for both family-based and case/control investigations.
The high risk haplotype (H1) identified in our study was too
frequent to explain the strong linkage signal detected in SPA2
, and was likely only a surrogate for the causal variant(s). As
the LD block containing this haplotype is located 28.6 kb from the
TNFSF15 gene, one of the best candidate genes in the region, in
particular because of its implication in CD [20,21], it made sense
to test this gene directly. However, our LD mapping stage data did
not implicate the TNFSF15 ‘‘strictly genic’’ region (introns and
exons). Indeed, no tag SNP association was identified in this region
and none of the five SNPs described as associated with CD were
associated with SpA in our analyses. A full re-sequencing of all
exons, 1st and 3rd introns, and additional intronic boundaries of
the TNFSF15 gene in several patients and controls did not reveal
unmatched variations either (data not shown).
We were also well aware of the limits presented by our
extension/replication approach, which was focused only on the
‘‘LD mapping’’ of an association peak. Thus, we tested by a classic
candidate-gene approach several genes located within the region
surrounding the linkage peaks. We first performed variants
screening in a group of independent SpA patients from families
presenting a high linkage signal within the studied region, and
unrelated controls. The most suggestive polymorphisms were
subsequently genotyped. The association with the disease was
therefore either assessed with TDT in a sample of independent
trios or with a chi-square test in a large case/control sample. In
this way, we tested the implication of Tenascine-Cytoactine gene
(TNC), coding for an extracellular matrix glycoprotein and
presenting a paralogous counterpart in the MHC class III locus.
No association was found between polymorphisms in this gene and
SpA . We also performed a more systematic candidate-gene
approach, testing among others Tumor necrosis factor ligand
superfamily member 8 - CD30 ligand (TNFSF8), Zinc finger
protein 618 (ZNF618), Kinesin-like protein (KIF12), Alpha-1-acid
glycoprotein 1 Precursor (ORM1), Alpha-1-acid glycoprotein 2
Precursor (ORM2) and alpha-1-microglobulin/bikunin precursor
(AMBP) genes. The implication in the disease of polymorphisms
tested within these genes was excluded by this approach,
corroborating the results of our LD mapping, presented in this
article (Zinovieva E et al. manuscript in preparation).
Despite the fact that SNPs within the TNFSF15 coding region
were excluded by our combined tag SNP and candidate-gene
approach, it is still possible that polymorphisms identified by the
H1 haplotype play a role in the regulation of this gene. TNFSF15
belongs to the TNF superfamily of genes, otherwise implicated in
SpA  and is specifically implicated in gut inflammation, a
frequent SpA manifestation [32,33]. Its product, TNFSF15, also
called TLA1, is implicated in the modulation of T-helper 17
lymphocytes activation , the number of which has been shown
to be increased in patients with SpA . Finally a recent study
reported that polymorphisms within TNFSF15 to be associated
with CD are playing a role in the transcriptional regulation of the
gene . This report compounds with our hypothesis, since our
findings could be consistent with an indirect role of TNFSF15 in
SpA. However, whether the causal variant(s) tagged by the six
SNPs haplotype is(are) related to TNFSF15 function and/or
regulation or to any other gene in the SPA2 region will require
Materials and Methods
This study was approved by the institutional ethics committee of
Cochin Hospital (Paris, France) and of Ambroise Pare ´ Hospital
(Boulogne-Billancourt, France), and written informed consent was
obtained from each participant.
Caucasian families consisting of one or several cases of SpA and
additional parents were recruited throughout France by the
‘‘Groupe Franc ¸ais d’Etude Ge ´ne ´tique des Spondylarthropathies’’
(GFEGS). In case/control panels, independent cases were
recruited through the Rheumatology clinic of Ambroise Pare ´
Hospital (Boulogne-Billancourt), or through the national self-help
patients’ organization: ‘‘Association Franc ¸aise des Spondylarthri-
tiques’’. Independent controls were obtained from the ‘‘Centre
d’Etude du Polymorphisme Humain’’, or were recruited as healthy
spouses of cases with either no children or no affected children.
The phenotypic description of patients from familial and case/
control samples is shown in Table 5. The diagnosis of SpA was
made according to the classification criteria of Amor et al 
Mapping a SpA Locus Near to TNFSF15
PLoS Genetics | www.plosgenetics.org9 June 2009 | Volume 5 | Issue 6 | e1000528
and/or the European Spondylarthropathy Study Group (ESSG)
. Within the group of SpA, AS was diagnosed according to the
modified New York criteria . Regarding extra-articular manifes-
tations, the diagnosis of psoriasis required the presence of typical
diagnosis of anterior uveitis required examination by an ophthal-
mologist. Inflammatory bowel disease diagnosis (including CD and
ulcerative colitis) was based on endoscopic and histological
examination of the gut. ReA was diagnosed according to the criteria
published by Willkens . Finally, uSpA was diagnosed when SpA
criteria were fulfilled, without any of the foregoing diagnosis.
DNA isolation and HLA-B typing
Genomic DNA was extracted from peripheral blood using
standard methods. HLA-B typing was routinely performed by a
polymerase chain reaction (PCR) - based sequence-specific
method . For individuals already typed as positive for HLA-
B27, retyping was not routinely performed.
Linkage fine mapping.
heterozygosity, regularly spaced over the SPA2 region, between
D9S1677 and D9S112 (average marker spacing 0.89 cM and
mean heterozygosity of 0.76). Markers were selected from the
University of California Santa Cruz (UCSC) public database.
They included 3 of the 5 microsatellites previously studied in our
former screen , and 25 additional markers (Table 2, Figure 1).
The fine mapping markers panel
microsatellites with28 maximallevel of
Typing of microsatellites was performed at the French National
Genotyping Center (CNG, Evry, France) on a set of 149 multiplex
families (Figure 1, Table 1). Multiplex PCRs were performed using
fluorescently-labeled primers (FAM, HEX and NED) (Applied
Biosystems, Foster City, CA). Amplimers generated by PCRs were
loaded on a MegaBACE1000 (GE Healthcare, Chalfont St. Giles,
UK) and allele calling of the fragments was performed with the
A customized chip containing 1,536 tag SNPs
was designed using resources provided by the HapMap project
. Tag SNP selection aimed at covering almost all the
common variations (r2.0.8; more than 1 tag SNP/10 kb) of the
13.23 Mb target region, which was selected following the linkage
fine mapping stage. High-throughput genotyping was performed
on a customized Illumina BeadChips using the GoldenGate assay
at the CNG. A detailed protocol for this assay is described by
Illumina on their web site. The GoldenGate reaction is based on
allele specific extension and universal PCRs at 1,536 targets .
After amplification the GoldenGate assay products were
hybridized on a Sentrix 96Array matrix , a fiber-optic gene
array [44,45], and washed prior to being analyzed by
fluorescence. The call rate obtained was $0.99. Patients DNA
genotypes in 136 families by the mean of this methodology were
investigated (Figure 1, Table 1).
Extension association study.
described above was achieved, 25 SNPs were further selected
through the HapMap and dbSNP NCBI databases according to
the following criteria:
After the tag SNP screen
Table 5. Clinical characteristics of patients with spondyloarthritis included in the whole study.a
CharacteristicsFamilial patients (n=711) Singleton cases (n=371)
Age in year, mean6SD 4460.564660.70
Age at onset, in year, mean6SD 2460.362660.60
Sex ratio, men:women 383:328 177:194
Back/buttock pain97% 97%
Inflammatory bowel disease 7%7%
aThe registered manifestations correspond to those present at time of examination, or retrieved from past-medical history. Inflammatory bowel disease: Crohn’s disease
or ulcerative colitis; AS: ankylosing spondylitis, uSpA: undifferentiated spondyloarthritis; PsA: psoriatic arthritis; AIBD: inflammatory bowel disease-associated arthritis;
ReA: reactive arthritis.
bThe number of patients evaluated in each group was 710 and 327 respectively.
cRefers to radiographic sacroiliitis$grade II bilateral or grade III unilateral. The number of patients evaluated in each group was 643 and 282 respectively.
Mapping a SpA Locus Near to TNFSF15
PLoS Genetics | www.plosgenetics.org10June 2009 | Volume 5 | Issue 6 | e1000528
– The selected markers had a minor allele frequency (MAF) $0.1
in Caucasian populations, and were located nearby the tag SNP
previously identified to be associated with SpA (up to a maximum
of 30 kb upstream and of 106 kb downstream). These markers
were also as far as possible in strong LD (D’=1; strong r2) with the
associated tag SNP. We genotyped 5 more additional SNPs within
the TNFSF15 gene (rs4246905, rs6478108, rs7030574, rs6478109,
and rs7848647), being the closest gene located in the vicinity of the
peak of association. Of note, these 5 SNPs were chosen because
they have been previously identified as associated with CD
[20,21]. In all, 31 SNPs were genotyped in this extension part of
the study, including the tag SNP previously identified as associated
with SpA. Genotyping was carried out at the CNG using TaqMan
(assay-by-design) according to the manufacturer’s recommenda-
tions with probes and Mastermix from Applied Biosystems
(Courtaboeuf, France). End point fluorescence was detected using
an ABI7900HT reader (Applied Biosystems). Genotypes were
assigned with the SDS 2.1 software. Investigations were carried
out on a set made of 287 families (Table 1) as well as on an
independent sample of 139 cases and 163 controls (Figure 1).
SNPs located in the reduced 71.6 kb region was performed on a
new case/control sample composed of 232 SpA patients and 149
controls. Genotyping was put through a production-scale 48-plex
(SNPlex) assay (Applied Biosystems, Courtaboeuf, France) [46,47].
Automatic allele assignment was achieved with the GeneMapper
software v4.0 (Applied Biosystems), with the rules-clustering
Verification of SNPs genotyping.
the study, several individuals have been genotyped by two or three
different technologies (Illumina, Taqman, or SNPlex) for several
SNPs. The level of genotyping concordance was set to 95%. For
SNPs that did not reach this threshold (rs4979459, rs10759734,
rs6478105, rs10982396, and rs4246905) an alternative method of
genotyping was used in order to resolve the correct genotype. In this
way, 436 individuals were genotyped using melting curve analyses
(LightCycler System, Roche, Meylan, France). Both PCR primers
and hybridization probes were synthesized by Tib MolBiol (Berlin,
Germany). All observed discrepancies were solved.
Finally, a replication study focused on 8
In the different stages of
For familial studies, Mendelian inheritance inconsistencies were
identified with the PEDCHECK program . PLINK program
 was used to assess the deviation from Hardy-Weinberg
equilibrium in unrelated subjects. Family pairwise distributions
among first and second degree relative pairs were accounted for
with the PEDSTATS program .
For the fine mapping linkage study, allele frequencies were
estimated using MENDEL software . Evidence for linkage was
assessed using Zlr statistic  based on Spairswith the exponential
model and multipoint identity-by-descent computation using
ALLEGRO program . P-values were computed on the basis
of large-sample theory; the distribution of Zlr statistic approxi-
mates a standard normal random variable under the null
hypothesis [52,53]. Whole fine map significance was extrapolated
using Bonferroni correction for 28 tests.
Family-based allelic single-locus association analyses (1,536 tag
SNP in the 136 families of the LD mapping and 31 SNPs in the
287 families of the extension study (Figure 1)) were carried out
using FBAT . FBAT is a flexible program appropriate for
analyses of family data larger than trios, allowing association tests
that are robust to population cofounds in the case where parental
data are missing and/or other offspring are included in the
analysis . We specified the option to calculate the variance
empirically (‘‘-e’’ option) in order to provide valid tests of
association in the presence of linkage . The global significance
threshold for each set of SNPs was assessed using Bonferroni
It has been shown that in some situations (such as when r2factor
between the risk variant and a particular multi-SNP haplotype is
very strong) haplotypes may provide more information for
association than corresponding single-locus tests . HBAT is
an elaboration of FBAT that allows family-based association tests
of haplotypes, even when the phasing is ambiguous. Family-based
haplotype-specific association in the presence of known linkage
was assessed with the ‘‘hbat -e’’ option of FBAT program, for a set
of pre-selected tightly linked markers. This option allows one to
perform two types of haplotype tests. In the first type, each
haplotype allele is tested for association against all the others using
a one degree-of-freedom (df) test. Significance of these tests must
be extrapolated with a multiple testing correction; here we used
the Bonferroni method. The second type of haplotype tests is a
global multiallelic test with several df. In this case, there is no need
to correct for multiple testing.
Case/control association studies were carried out using the
standard chi-square test comparing allelic frequencies between
cases and controls and giving asymptotic P-values, implemented in
PLINK package . Allele frequencies, ORs, and their 95%
confidence intervals were also estimated using this software. When
needed, the adjustment for multiple testing was performed using
the Bonferroni correction.
The quantile-quantile (Q-Q) plots (Figure 3) were constructed
by ranking the sets of association P-values from the largest to the
smallest and plotting them against their expected values. Under
the null hypothesis the expected P-value for the ith SNP is i/n,
where n is the total number of tested markers.
Haplotype-specific association was assessed in case/control
samples for a set of pre-selected tightly linked SNPs (the same as
for the family investigation described above) with the ‘‘hap-assoc’’
option of PLINK. This procedure takes into account the
uncertainty of haplotype phase and performs both one df chi-
square haplotype-specific tests, which significance must be
extrapolated with a multiple testing correction, here Bonferroni
correction, and an omnibus association statistic considering all the
The explorative combined analysis of the whole association data
from the extension and replication studies (1 family sample and 2
case/control samples; Figure 1) was performed with the ‘‘dfam’’
option of PLINK. This particular test implements a sib-TDT 
for nuclear families, to include sibships without parents as well as
unrelated individuals and assesses the association via a clustered-
analysis using the Cochran-Mantel-Haenszel test . It does not
take into account the presence of linkage in the region.
LD plots were constructed using HAPLOVIEW program .
The URLs for data presented herein are as follows:
* Association Franc ¸aise de Spondylarthritiques (AFS): http://
* Centre d’Etude du Polymorphisme Humain (CEPH): http://
* University of California Santa Cruz (UCSC) public database:
* French National Genotyping Center (CNG): http://www.cng.fr/
* Detailed protocol for the Illumina BeadChips GoldenGate
Mapping a SpA Locus Near to TNFSF15
PLoS Genetics | www.plosgenetics.org 11June 2009 | Volume 5 | Issue 6 | e1000528
* Ensembl database: http://www.ensembl.org/index.html
* NCBI dbSNP database: http://www.ncbi.nlm.nih.gov/projects/
* HapMap database: http://www.hapmap.org/
* PEDCHECK program: http://watson.hgen.pitt.edu/register/
* MENDEL program: http://www.genetics.ucla.edu/software/
* ALLEGRO program: http://www.decode.com/software/
* FBAT program: http://www.biostat.harvard.edu/,fbat/fbat.
* PEDSTATSprogram: http://www.sph.umich.edu/csg/
* PLINK program: http://pngu.mgh.harvard.edu/,purcell/
* HAPLOVIEW program: http://www.broad.mit.edu/mpg/
linkage disequilibrium mapping study (Illumina BeadChip geno-
typing - 136 families).
Found at: doi:10.1371/journal.pgen.1000528.s001 (1.74 MB
Complete results of the family-based association
extension study (TaqMan genotyping - 287 families).
Found at: doi:10.1371/journal.pgen.1000528.s002 (0.08 MB
Complete results of the family-based association
study (TaqMan genotyping - 139 SpA cases/163 healthy controls).
Found at: doi:10.1371/journal.pgen.1000528.s003 (0.07 MB
Complete results of case/control association extension
results in HLA-B27 positive case/control sample (261 SpA cases/
27 healthy controls).
Found at: doi:10.1371/journal.pgen.1000528.s004 (0.05 MB
Single SNP (1), and haplotype-based (2) association
results in HLA-B27 negative case/control sample (73 SpA cases/
255 healthy controls).
Found at: doi:10.1371/journal.pgen.1000528.s005 (0.05 MB
Single SNP (1), and haplotype-based (2) association
association-extension studies for eight SPA2 single-nucleotide
Found at: doi:10.1371/journal.pgen.1000528.s006 (0.04 MB
Results of combined family-based and case/control
We acknowledge the contribution of the investigators of GFEGS (Groupe
Franc ¸ais d’Etude Ge ´ne ´tique des Spondylarthropathies) and of the AFS
(Association Franc ¸aise des Spondylarthrites) to patient recruitment. We are
indebted to the patients and their relatives for their dedication to our
We thank Dr. Franc ¸oise Clerget-Darpoux and Dr. Jean-Pierre Hugot for
fruitful discussions, as well as Dr. Joel D. Taurog and Miss Emma Walton
for the proofreading of the manuscript.
Conceived and designed the experiments: EZ CB AK FL BI RSN CMR
HJG SH DZ GC MB. Performed the experiments: EZ AK FL BI NL AV
SJ SH CC DB AB. Analyzed the data: EZ CB AK FL BI NL NC AV HJG
SH CC AB DZ GC MB. Contributed reagents/materials/analysis tools:
CB FL RSN NL NC MB. Wrote the paper: EZ CB CC GC MB.
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Mapping a SpA Locus Near to TNFSF15
PLoS Genetics | www.plosgenetics.org13 June 2009 | Volume 5 | Issue 6 | e1000528