# Genetic Variability of RXRB, PPARA, and  PPARG in Wegener's Granulomatosis

Authors: Stefan Wieczorek; Silvia Knaup; Wolfgang L. Gross; Jörg T. Epplen
Journal: PPAR Research
 (2009)

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## Abstract

A major genomic region involved in Wegener's granulomatosis includes the gene for retinoid receptor beta (RXRB) which forms heterodimers with peroxisome proliferator-activated receptors (PPARs). It is unclear whether this association directly arises from the RXRB allele(s) or via a linked variation.
In order to reveal any hitherto unknown and potentially disease-relevant variation of the RXRB gene, we have genotyped four tagging SNPs of this genomic region and have directly sequenced selected WG patients and controls representing disease-associated haplotypes. Additionally, we have genotyped 2 SNPs each in the genes for PPARα and PPARγ (PPARA and PPARG). Hence, we confirmed the strong association of the RXRB locus with WG but could not reveal any novel variation in RXRB. None of the PPARA and PPARG SNPs showed association with WG. Moreover, no epistatic effect was seen between RXRB and PPARA/PPARG alleles.
These results do not support an etiopathological role of PPAR in WG. Analyses of further genes functionally linked to RXRB may provide additional data useful to evaluate the RXRB association found in WG.


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## Body

## 1. Introduction

Wegener's
granulomatosis (WG) is a form of small vessel vasculitis belonging to the group
antineutrophil cytoplasmic antibody- (ANCA-) associated vasculitides (AAV). WG
usually presents with granulomatous lesions of the upper respiratory tract and
often develops into generalized vasculitis with multiple organ involvement in
later disease stages. In WG ANCA typically recognize proteinase 3, delineating this
diagnostic entity from other AAV forms like Churg Strauss syndrome or
microscopic polyangiitis, in which ANCA are usually directed against
myeloperoxidase (reviewed in [1]). Current concepts of its etiopathology are
incomplete, but WG is accounted to the large and heterogenous group of complex
disorders arise from a mostly elusive interplay of environmental and genetic
factors.A major genomic
locus for WG was identified on chromosome 6p21.3, including (among others) the
genes forHLA-DPB1, RXRB, and RING1 [2]. The linkage disequilibrium (LD) pattern of this genomic
region is complex with larger blocks of strong LD alternating with regions of
high recombination rates. There is evidence that the association of this locus
with the disorder may arise from more than one variation, making 6p21.3 a
quantitative trait locus for WG. While a strong association is demonstrable
with the HLA-DPB1*0104 allele, a
(partly) independent association was detected for the defined region including
potential WG candidate genes RING1 and RXRB [3]. In addition certain
alleles of RXRB, the gene encoding retinoid
X receptor beta, are highly significantly associated with WG susceptibility [4].Peroxisome proliferator-activated receptors (PPAR) are
group of nuclear receptors mediating the effects of peroxisome proliferators on
gene transcription. Binding of PPAR to regulatory DNA sequences requires the
heterodimerization with RXR. PPAR mediated effects are complex and include
modulation of inflammatory processes. PPARα
is a ligand for leukotriene B4, and PPARα deficient mice show a prolonged
response to inflammatory stimuli [5]. PPARα also appears to be involved in downregulation
of the activity of cyclooxigenase 2 (COX2) and nuclear factor kappa-b (NFκB). Likewise,
PPARγ
exhibits anti-inflammatory properties. It is upregulated in activated
macrophages and inhibits the expression,  for
example, of the inducible nitric oxide synthase [6].Genetic polymorphisms of PPAR
have been investigated in numerous conditions, predominantly in metabolic
disorders like type 2 diabetes or atherosclerosis [7–9]. There is evidence for accelerated
atherosclerosis in WG patients [10], suggesting that both conditions may share
some (genetic) risk factors.Given the
pleiotropic role of PPAR in the regulation of (vascular) inflammation, we have
hypothesised that genetic predisposition to WG may arise from the interaction
of certain genetic variations of RXRB and PPAR. We have, therefore, screened
theRXRB gene for novel, potentially
WG-specific variations. Moreover, we have genotyped two single nucleotide
polymorphisms in each of the genes for PPARα and PPARγ in large panels of WG cases and healthy
controls. Finally, we searched for epistatic effects of certain alleles of RXRB and PPARA or PPARG.


## 2. Patients and Methods

### 2.1. Subjects

All
patients included in this study were diagnosed with WG according to the
criteria of theAmerican College of
Rheumatology and the Chapel Hill consensus conference [11]. They were selected at the
interdisciplinary vasculitis centre at the University of Luebeck/Rheumaklinik
Bad Bramstedt. All patients were asked for their ancestry and reported German
decent for at least two generations. Healthy German blood donors were used as
controls and ancestry was evaluated equal to the patient group.For
theRXRB locus we genotyped the same
patients (n=282) and controls (n=380) that were previously analyzed for the
6p21.3 locus [3]. In order to increase statistical power, the SNP in the PPAR
genes were analyzed in expanded sample comprising 462 WG cases and 701 controls.Ethical
principles for medical research involving human subjects as defined in theDeclaration of Helsinki have been
followed. The study design was approved by the local ethics committee at the University of Luebeck,
Germany
(No. AZ 06-087).


### 2.2. Genotyping

Four types SNPs rs9277935,
rs2072915, rs2744537, and rs1547387 (see Figure1) were selected from the
HapMap database to serve as tagSNPs for a region of 10 kb including the RXRB gene. With this selection all
HapMap SNPs within this region are efficiently tagged (r2>0.9).
rs2072915
and rs2744537 are located within the 3′ untranslated region (UTR) of RXRB, while rs9277935 is located
approximately 1 kb 3′ of the last RXRB exon. rs1547387 is located 5′ of RXRB, in exon 5 of the SLC39A7 gene, in which it constitutes a silent SNP (Ser209Ser).Figure 1
Location of the
four tagging SNPs of theRXRB gene
and LD structure. The LD is plotted as D′ and intensity coded (darker shades
corresponding to higher degree of LD). The RXRB gene is transcribed from centromer to telomer, that is, from right to left. RXRB exons are depicted as black boxes, and translation start and stop points are
marked by white vertical lines.rs9277935,
rs2072915, and rs2744537 were genotyped via PCR-RFLP techniques and a
commercially available TaqMan genotyping assay (Applied Biosystems) was used
for rs1547387.For rs1800206
(Val162Leu) inPPARA also a
commercially available TaqMan assay was used, while rs6008259
(3′ UTR of PPARA), rs1801282 (PPARG, Ala12Pro), and rs3856806 (PPARG, His449His) were genotyped using newly designed primers and
probes (see Table 1).Table 1
Oligonucleotides and restriction
enzymes used for genotyping.
GeneSNPForward (F) and reverse (R) primer (5′>3′)Allele specific probesRestriction enzymeFragment lengthsRXRBrs9277935F: TGCCCCTTGTAGGTCTCCAC(1)—BtgIT-Allel: 208 bpT/GR: CCTCCCACTGTGCCCTAAG-Allel: 34 + 174 bprs2072915F:
ACATCTCCACCAGCCCCTTC—MboIT-Allel:
325 bpT/AR:
GTCCTTCCCCCAGCACAAAGA-Allel: 224 + 101 bprs2744537F: TCTTCAAGCTCATTGGTGAC—BsaIT-Allel: 451 bpT/GR: CCCATTTCCACTCTTCAGATG-Allel: 326 + 125 bprs1547387Information not provided by the manufacturer——C/GPPARArs1800206Information not provided by the manufacturer——G/Crs6008259F:
CCCCTGTGTCAACAAGATCCAG:
Fam-CTGTGTTGTCCCCAGCGACCC——G/AR:
CTTGAATGGCACAGGGTACATCA:
YY-CCTGTGTTGTCCCCAACGACCCPPARGrs1801282F:
TTATGGGTGAAACTCTGGGAGATTG:
Fam-TCCTATTGACGCAGAAAGCGATTCC——G/CR:
TTGTGATATGTTTGCAGACAGTGTATCC:
YY-TCCTATTGACCCAGAAAGCGATTCCTTrs3856806F:
CCAGAAAATGACAGACCTCAGACAT:
Fam-TCACGGAACATGTGCAGCTACTGC——T/CR:
GGAGCGGGTGAAGACTCATGC:
YY-ATTGTCACGGAACACGTGCAGCTAC(1)  A 17 mer nucleotide (GTAAAACGACGGCCAGT) was added 5′ to the forward primer increase fragment length differences between the two alleles,
FAM: 6-carboxy-fluorescein; YY: Yakima Yellow.


### 2.3. Direct Sequencing of RXRB

The entireRXRB gene (all 10 exons and exon/intron boundaries) was directly sequenced in 5 WG
patients typed homozygous for the associated RXRB haplotype (see below; primer sequences available on request).
For comparison 5 healthy controls homozygous for the inversely associated (i.e., protective) haplotype were also sequenced.


### 2.4. Statistical Analysis

Genotypes were
recorded in linkage format. Association for each single marker was tested by
using chi square tests on contingency tables. AP value <.05 was
considered significant. LD between each SNP of a respective locus, and
haplotype block frequencies were calculated by using Haploview 4.1 [12].Interaction betweenRXRB and
PPARA/PPARG SNPs were
calculated with GAIA (http://gump.qimr.edu.au/GAIA/gaia.html; [13]). This application uses a logistic regression
model which tests for pairwise locus/locus interactions between genes. We
applied an additive interaction model for each pair of SNPs testing the
significance of the interaction model terms over and above any main effects.


## 2.1. Subjects

All
patients included in this study were diagnosed with WG according to the
criteria of theAmerican College of
Rheumatology and the Chapel Hill consensus conference [11]. They were selected at the
interdisciplinary vasculitis centre at the University of Luebeck/Rheumaklinik
Bad Bramstedt. All patients were asked for their ancestry and reported German
decent for at least two generations. Healthy German blood donors were used as
controls and ancestry was evaluated equal to the patient group.For
theRXRB locus we genotyped the same
patients (n=282) and controls (n=380) that were previously analyzed for the
6p21.3 locus [3]. In order to increase statistical power, the SNP in the PPAR
genes were analyzed in expanded sample comprising 462 WG cases and 701 controls.Ethical
principles for medical research involving human subjects as defined in theDeclaration of Helsinki have been
followed. The study design was approved by the local ethics committee at the University of Luebeck,
Germany
(No. AZ 06-087).


## 2.2. Genotyping

Four types SNPs rs9277935,
rs2072915, rs2744537, and rs1547387 (see Figure1) were selected from the
HapMap database to serve as tagSNPs for a region of 10 kb including the RXRB gene. With this selection all
HapMap SNPs within this region are efficiently tagged (r2>0.9).
rs2072915
and rs2744537 are located within the 3′ untranslated region (UTR) of RXRB, while rs9277935 is located
approximately 1 kb 3′ of the last RXRB exon. rs1547387 is located 5′ of RXRB, in exon 5 of the SLC39A7 gene, in which it constitutes a silent SNP (Ser209Ser).Figure 1
Location of the
four tagging SNPs of theRXRB gene
and LD structure. The LD is plotted as D′ and intensity coded (darker shades
corresponding to higher degree of LD). The RXRB gene is transcribed from centromer to telomer, that is, from right to left. RXRB exons are depicted as black boxes, and translation start and stop points are
marked by white vertical lines.rs9277935,
rs2072915, and rs2744537 were genotyped via PCR-RFLP techniques and a
commercially available TaqMan genotyping assay (Applied Biosystems) was used
for rs1547387.For rs1800206
(Val162Leu) inPPARA also a
commercially available TaqMan assay was used, while rs6008259
(3′ UTR of PPARA), rs1801282 (PPARG, Ala12Pro), and rs3856806 (PPARG, His449His) were genotyped using newly designed primers and
probes (see Table 1).Table 1
Oligonucleotides and restriction
enzymes used for genotyping.
GeneSNPForward (F) and reverse (R) primer (5′>3′)Allele specific probesRestriction enzymeFragment lengthsRXRBrs9277935F: TGCCCCTTGTAGGTCTCCAC(1)—BtgIT-Allel: 208 bpT/GR: CCTCCCACTGTGCCCTAAG-Allel: 34 + 174 bprs2072915F:
ACATCTCCACCAGCCCCTTC—MboIT-Allel:
325 bpT/AR:
GTCCTTCCCCCAGCACAAAGA-Allel: 224 + 101 bprs2744537F: TCTTCAAGCTCATTGGTGAC—BsaIT-Allel: 451 bpT/GR: CCCATTTCCACTCTTCAGATG-Allel: 326 + 125 bprs1547387Information not provided by the manufacturer——C/GPPARArs1800206Information not provided by the manufacturer——G/Crs6008259F:
CCCCTGTGTCAACAAGATCCAG:
Fam-CTGTGTTGTCCCCAGCGACCC——G/AR:
CTTGAATGGCACAGGGTACATCA:
YY-CCTGTGTTGTCCCCAACGACCCPPARGrs1801282F:
TTATGGGTGAAACTCTGGGAGATTG:
Fam-TCCTATTGACGCAGAAAGCGATTCC——G/CR:
TTGTGATATGTTTGCAGACAGTGTATCC:
YY-TCCTATTGACCCAGAAAGCGATTCCTTrs3856806F:
CCAGAAAATGACAGACCTCAGACAT:
Fam-TCACGGAACATGTGCAGCTACTGC——T/CR:
GGAGCGGGTGAAGACTCATGC:
YY-ATTGTCACGGAACACGTGCAGCTAC(1)  A 17 mer nucleotide (GTAAAACGACGGCCAGT) was added 5′ to the forward primer increase fragment length differences between the two alleles,
FAM: 6-carboxy-fluorescein; YY: Yakima Yellow.


## 2.3. Direct Sequencing of RXRB

The entireRXRB gene (all 10 exons and exon/intron boundaries) was directly sequenced in 5 WG
patients typed homozygous for the associated RXRB haplotype (see below; primer sequences available on request).
For comparison 5 healthy controls homozygous for the inversely associated (i.e., protective) haplotype were also sequenced.


## 2.4. Statistical Analysis

Genotypes were
recorded in linkage format. Association for each single marker was tested by
using chi square tests on contingency tables. AP value <.05 was
considered significant. LD between each SNP of a respective locus, and
haplotype block frequencies were calculated by using Haploview 4.1 [12].Interaction betweenRXRB and
PPARA/PPARG SNPs were
calculated with GAIA (http://gump.qimr.edu.au/GAIA/gaia.html; [13]). This application uses a logistic regression
model which tests for pairwise locus/locus interactions between genes. We
applied an additive interaction model for each pair of SNPs testing the
significance of the interaction model terms over and above any main effects.


## 3. Results and Discussion

The overall call rate
for all 8 SNPs in patients and controls was 93.60%. None of the SNPs revealed
significant deviation from Hardy-Weinberg equilibrium. Two of theRXRB SNPs (rs9277935 and rs2744537) showed highly significant association
with WG (see Table 2). Accordingly, the GTTC
haplotype (calculated from all four SNPs of this locus) was significantly more
common in WG while the TTGC haplotype was overrepresented in controls (see Table 2). Based on the SNP data we have then selected 5 WG patients homozygous for
the GTTC haplotype for direct sequencing of the RXRB gene. For comparison 5 control subjects homozygous for the
TTGC haplotype were also analyzed. Yet, no novel sequence variation was
detected, a finding which is consistent with previous results from Szyld et al. [4], who had screened the RXRB gene in
WG patients without consideration of the haplotype structure of this locus. Results
from the initial tagging SNP genotyping were confirmed in the ten included
individuals for the two exonic tagging SNPs (rs2744537 and rs2072915). Two
other exonic SNPs (rs1152296 in exon 10/5′UTR and rs6531 in exon 7) were
identified and perfectly segregated with the previously identified risk/nonrisk
haplotypes.Table 2
Allele and haplotype frequencies.
GeneSNP/haplotypeAlleleFrequency in controlsFrequency WG patientsP valueOR (95% CI)RXRBrs9277935G0.760.896.65  ×  10-92.55 (1.86–3.56)T0.240.11rs2072915T0.710.75.1571.20 (0.93–1.55)A0.290.25rs2744537T0.730.576.63  ×  10-90.49 (0.39–0.63)G0.270.43rs1547387C0.870.85.340.85 (0.62–1.18)G0.130.15RXRB*GTTC0.240.401.15  ×  10-92.11 (1.66–2.69)TTGC0.190.079.73  ×  10-100.32  (0.22–0.47)other0.430.47.1571.18 (0.94–1.47)PPARArs1800206C0.930.95.0761.40 (0.96–2.05)G0.070.05rs6008259G0.800.83.0690.81 (0.65–1.02)A0.200.17PPARGrs1801282C0.850.85.740.96 (0.75–1.23)G0.150.15rs3856806C0.860.85.700.95 (0.75–1.22)T0.140.15OR: Odds ratio,
CI: confidence interval,
*haplotype frequencies calculated from
all four RXRB SNPs.Both associatedRXRB SNPs (rs9277935
and rs2744537) are in strong LD with HLA-DPB1 as well as with rs3117228 and
rs213208 (see Figure 2) which had revealed the highest (and partly HLA-DPB1 independent) association with WG
in a previous study [3]. Moreover, according to the HapMap data rs2744537 is in strong LD with rs6531 (a synonymous SNP in
exon 7 of RXRB) which was
significantly associated with WG earlier [4]. As for the other
associated SNPs in this genomic region, it is therefore hard to differentiate
which of the SNPs (or even a yet unknown variation of this locus) constitutes
the primary WG risk factor. A potential approach to overcome this problem is
the analysis of factors related to the different proteins encoded in the 6p21.3
region. We have therefore investigated SNPs in the genes for PPARα and PPARγ  which are functionally closely linked to RXRB.Figure 2
LD between WG
associated variations of the 6p21.3 genomic region.HLA-DPB1 is modelled as a biallelic marker with the associated HLA-DPB1*0401 as one allele and all other
alleles joint into the other allelic category. LD is plotted as D′ and
intensity coded (darker shades corresponding to higher degree of LD). Both RXRB SNPs investigated in this study
(rs9277935 and rs2744537) are in high LD with HLA-DPB1 as well as with the markers analyzed by Heckmann et al. (2008): rs3117228 (3′ of HLA-DPB1)
and rs213208 (intronic SNP in RING1,
approx. 10 kb centromeric of RXRB).The two SNPs inPPARG were in weak to moderate LD (D′  =  0.64,
r2  =  0.40) while the two SNPs in PPARA were virtually unlinked (D′  =  0.47, r2  =  0.06). Therefore, no haplotype
frequencies were calculated for these genes. Both SNPs in PPARG showed very similar allele frequencies in cases and controls
not revealing any significant differences (see Table 2). The two SNPs in PPARA both revealed a decreased
frequency of the minor allele but these differences did not reach significance
level (see Table 2). No significant pairwise epistatic effect for any of the
investigated SNPs, that is,
neither between PPARA and PPARG nor between PPARA (or PPARG,
resp.) and RXRB or HLA-DPB1. Taken together, we cannot
provide evidence for a genetically based involvement of PPARα and PPARγ in
the etiopathology of WG. Moreover, the strong WG association with the 6p21.3
locus is unlikely to be based on any coding variation of the RXRB gene. Future studies will therefore
have to focus on regulatory elements of this area (e.g., cis acting elements or micro RNAs).


## 4. Conclusions

These
results do not support a direct etiopathological role of RXRB and/or PPAR in
WG. Analyses of further genes functionally linked to RXRB, for example, retinoic acid receptors or vitamin D receptors, may
provide additional data useful to evaluate theRXRB association found in WG.



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*Source: 786781-2009-02-11.xml*