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Human Molecular Genetics Advance Access originally published online on January 8, 2008
Human Molecular Genetics 2008 17(8):1147-1155; doi:10.1093/hmg/ddn004
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Genetic variation in the CRP promoter: association with systemic lupus erythematosus

Jeffrey C. Edberg1,*, Jianming Wu1, Carl D. Langefeld4, Elizabeth E. Brown2, Miranda C. Marion4, Gerald McGwin, Jr3, Michelle Petri5, Rosalind Ramsey-Goldman6, John D. Reveille7, Summer G. Frank8, Kenneth M. Kaufman8,9,10, John B. Harley8,9,10, Graciela S. Alarcón1 and Robert P. Kimberly1

1 Division of Clinical Immunology and Rheumatology, Department of Medicine 2 Department of Epidemiology and 3 Section of Trauma, Burns, and Critical Care, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA 4 Section on Statistical Genetics and Bioinformatics, Division of Public Health Sciences, Department of Biostatistical Sciences, Wake Forest University, Winston-Salem, NC, USA 5 Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 6 Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 7 Division of Rheumatology, Department of Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA 8 Oklahoma Medical Research Foundation, Oklahoma City, OK, USA 9 US Department of Veteran Affairs, Oklahoma City, OK, USA 10 Department of Medicine, University of Oklahoma, Oklahoma City, OK, USA

* To whom correspondence should be addressed at: Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, SHEL207, 1825 University Blvd, Birmingham, AL 35294-2182, USA. Tel: +1 2059340894; Fax: +1 2059966734; Email: jedberg{at}uab.edu

Received October 9, 2007; Revised December 22, 2007; Accepted January 4, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The pentraxin C-reactive protein (CRP), an innate immune system opsonin which binds nuclear debris and apoptotic bodies, may protect against autoimmunity. A relative deficiency of CRP levels in patients with systemic lupus erythematosus (SLE) might contribute to altered handling of self-antigens. We report that the proximal 5' promoter region of CRP contains several polymorphisms that exhibit association with SLE in multiple populations. Strongest association was observed at the proximal promoter single nucleotide polymorphism (SNP) rs3093061 (CRP-707) (P = 6.41 x 10–7 and P = 2.13 x 10–6 in African-American and Caucasian case–control samples respectively). This association remains after adjustment for admixture. Linkage disequilibrium exists between SNPs in the proximal promoter and association of functional haplotypes containing rs3091244/rs3093062 (CRP-409/-390) appear to be driven by the rs3093061 (CRP-707) association. These data demonstrate that rs3093061 at the -707 site within the CRP gene is an SLE susceptibility locus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the presence of autoantibodies primarily directed against nuclear autoantigens as well as cytoplasmic and circulating proteins (1,2). These autoantibodies, together with alterations in cells of both the acquired and innate immune system, lead to the development of a chronic and severe clinical phenotype that can affect many different organs and tissues including kidneys, skin and brain. The prevalence of SLE in the general population is on the order of 1:2000 individuals, but varies by gender, ethnicity, socioeconomic status, genetic and environmental backgrounds (1–3).

A significant amount of data support a role for host genetics as both predisposing factors and as important determinants of patient outcomes (4–13). SLE shows strong familial aggregation and extended families in which SLE occurs have a high propensity for clustering of other autoimmune phenotypes (11). In addition, both linkage and association studies have shown a role for multiple genetic factors influencing disease susceptibility including the recent findings regarding IRF5, complement components and TNFSF4 (4–9,13). Likewise, both linkage and association studies have shown the importance of genetic factors in disease outcomes. For example, linkage at 5q14.3–15 in multiplex SLE pedigrees with autoimmune thyroid disease has been observed (12). Similarly, we have recently shown a strong association of a polymorphism in FCGR3A (CD16A) with development of end-stage renal disease (ESRD) in patients with SLE (10).

The pathogenesis of SLE is typically characterized by autoantibody-mediated tissue damage. Work in both humans and in model systems has suggested that autoantigen processing and presentation is important in the development of autoantibodies and systemic autoimmune disease (14–17). In particular, the handling of apoptotic cell debris is thought to be important in the promotion and development of autoantibodies in patients with SLE.

C-reactive protein (CRP), a pentraxin, is an important innate immune modulator that facilitates the clearance and handling of cellular debris and apoptotic bodies (18,19). CRP is an acute phase protein whose levels rise with inflammatory responses. The functions of CRP are not necessarily limited to the opsonization of cell debris and apoptotic bodies. CRP can also activate complement and it can bind to certain Fc receptors (20). Thus, CRP can have important immunoregulatory functions and has been shown to have protective effects in mouse models of autoimmune disease. Interestingly, some studies have suggested that SLE is characterized by lower CRP levels than would be predicted (21,22). Heritability estimates suggest that as much as 60% of the variance in base line CRP levels is attributable to genetic variation (23–25). Recent studies by us and others have shown that CRP levels are influenced by genetic variation in the CRP promoter (24–30). A functionally important tri-allelic single nucleotide polymorphism (SNP) (rs3091244) at position -390 relative to the start codon, a second SNP (rs3093062) at position -409 and an intronic microsatellite are associated with CRP levels (29).

In SLE, two studies have found evidence for an association between CRP variants and SLE or SLE nephritis in Caucasians (31,32). However, the SNP found to associate with the SLE phenotype has no known functional role in CRP production (basal or induced) and it remains unknown which if any promoter SNPs associate with disease. The tri-allelic rs3091244 variant was the only promoter SNP examined and was found not to associate with disease (31). Thus, it remains undetermined if there are other proximal CRP promoter variants that associate with SLE and it remains unknown what role genetic variants in the CRP locus have in SLE in African-Americans. In this study, we show in two independent African-American populations that a CRP promoter variant rs3093061 at position -707, or a promoter SNP haplotype containing this SNP, associates with the SLE phenotype. This finding is further replicated in a Caucasian population with SLE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
We initially determined the extent of genetic variation in the 1 kb proximal promoter of the CRP locus through a combination of direct sequencing of genomic DNA from 50 Caucasian and 50 African-American healthy control donors and through assessment of prior re-sequencing data (23,25). A total of six promoter SNPs were identified (Table 1). We also included a coding region synonymous SNP and an SNP in the 3'-untranslated region (3'-UTR) that have been previously examined by Russell et al. (31) in patients with SLE. For analysis of genetic association with SLE, patients were derived from the case–control study (CASSLE) constituted at the University of Alabama at Birmingham (UAB) that comprises Caucasians and African-Americans, healthy control participants and the African-American families enrolled in the Lupus Multiplex Research Repository (LMRR) based at Oklahoma Medical Research Foundation (12,33). Clinical characteristics of patients have been published (3,10,12,33,34). Baseline demographic features for the CASSLE study participants are summarized in Table 2.


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  		Table 1. C-reactive protein (CRP) variants

 


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  		Table 2. Demographics of the CASSLE case–control study

 
All markers were initially genotyped in the CASSLE case–control study. Because of the differences in the minor allele frequency (MAF) of the SNPs among the African-American and Caucasian controls, these groups were analyzed separately. Summary statistics are shown in Table 3. All markers were in Hardy–Weinberg equilibrium except for slight deviations at the uncommon -861 and -390 among Caucasians (P = 0.01). The CRP-860 SNP (rs3093060) was invariant in the Caucasian population and was not included in further analysis of this group. Patterns of linkage disequilibrium (LD) between the SNPs in the controls for each ethnic group were determined (Fig. 1). As is evident, there are significantly different LD patterns between the two ethnic groups. In particular, the LD pattern of -707 was distinct with high LD across the locus among African-Americans that was largely absent in the Caucasians. Of note is the high LD between the functionally important SNP -390 and -409 with all markers except -821 in Caucasians. Despite the strong LD between these SNPs, the predictive power between SNPs was low except for -707/-490 in African-Americans (r2 = 0.71). For initial analysis of the -390 tri-allelic variant (rs3091244), we collapsed the T and A alleles because of their similar functional properties (29).


Figure 1
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  		Figure 1. Linkage disequilibrium (LD) patterns in the C-reactive protein (CRP) locus for the single nucleotide polymorphisms (SNPs) examined in this study. Measurements of LD were calculated by the software Dprime. The statistics D' (top right triangle) and r2 (lower left triangle) are shown for African-American controls (A) and Caucasian controls (B). Dark shading: D' > 0.75/r2 > 0.75, intermediate shading: 0.50<D'≤0.75/0.502<r2<0.752, light shading: 0.25 < D' ≤ 0.50/0.252 < r2 < 0.502.

 


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  		Table 3. Single site genetic association of C-reactive protein (CRP) single nucleotide polymorphisms (SNPs) with systemic lupus erythematosus (SLE) in the CASSLE case–control study

 
Analysis of genotypic distribution in the African-American participants in the CASSLE study showed strong association of the CRP-707 SNP (rs3093061) with the SLE phenotype (P = 3.32 x 10–6, Table 3). Indeed, in this population, the level of significance was greater under a dominant model of inheritance (P = 6.41 x 10–7) where CRP-707GG homozygotes had an approximately 2-fold reduction in risk of SLE compared with wild-type homozygotes [odds ratio (OR) = 0.49, 95% confidence interval (CI) 0.37–0.65]. In addition, suggestive association was observed at CRP-409, CRP-390 and CRP4 among African-American enrolled in CASSLE. The CRP-409, CRP-390 and CRP4 variant allele associations were slightly enhanced under the additive model (Table 3). However, because of the higher LD between CRP-707 and these other CRP SNPs, it is highly plausible that these weaker associations were due to the strong association at the CRP-707 site with SLE susceptibility. Because of the multi-center enrollment of the patients and controls, we tested for center effects but none were found (data not shown).

We replicated the association between the CRP-707 variant allele (rs3093061) and SLE among African-Americans in Caucasians with SLE in the same CASSLE population (P = 1.25 x 10–5, Table 3). The association with CRP-707GG among Caucasians showed a similar protective effect (OR = 0.37, 95% C.I. 0.24–0.56; P = 2.13 x 10–6 under the dominant model). Suggestive association at CRP2 (rs1800947) with the variant allele and SLE was also observed. This effect is unlikely to be due to the association of variant alleles at CRP-707 and CRP2 because of the low LD between these two sites in the Caucasian population.

Bonferroni multiple comparisons adjustment for the number of genetic markers does not dampen the evidence for -707 association (Caucasian corrected P-value = 8.75 x 10–5; African-American corrected P-value = 2.66x10–5). The association between CRP-390 and SLE in the African-Americans also remains significant (corrected P-value = 0.0320). We also considered the issue of population stratification in our cases and controls. A total of 909 Caucasian samples in this study were also part of the recent Lupus Large Association Study (LLAS), a large case–control replication sample for our genome wide association study in SLE (see Materials and Methods) (35). As part of the LLAS a principal component analysis on over 8000 SNPs were computed to allow adjustment for potential admixture in tests of association. In the subset of 909 Caucasians individuals that overlapped these two studies we computed these above association tests with the principal component loadings as covariates to adjust for potential admixture. The significant -707 association remained [P = 1.17 x 10–6, OR = 0.30 (0.19–0.49)]. Details of these analyses are reported in Supplementary Material, Table S1.

We performed a logistic regression analysis to determine if the CRP-707 SNP (rs3093061) was sufficient to explain all of the association with SLE. Under a dominant model, we found that the CRP-409 SNP (rs3093062) (presence/absence of the A allele) was associated with SLE after adjusting for the -707 SNP (presence/absence of the G allele) in both the African-American and Caucasian populations (African-Americans, P = 0.024, OR = 1.91, 95% CI 1.09–3.36; Caucasians, P = 0.030, OR = 5.04, 95% CI 1.18–21.60). We note that the MAF for CRP-409 in Caucasians is <1% resulting in an unreliably high OR in that group.

To provide further support for the association of the CRP promoter with SLE, we genotyped the CRP promoter using African-American multiplex SLE families in LMRR (12,33). Pedigree disequilibrium test (PDT) analysis of the CRP-707 variant alone, while not reaching statistical significance, did trend towards under transmission of the minor allele consistent with the under representation of this allele in the case–control study. However, in this independent family-based study, association of a CRP-707 containing haplotype (CRP-861/-821/-707) was confirmed by PDT analysis (global P = 0.046) (Table 4). Thus, we have provided evidence of a genetic association between the CRP-707 promoter SNP (rs3093061) and SLE in three independent studies utilizing both case–control and family-based approaches.


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  		Table 4. Replication of association of CRP(C-reactive protein)-707 haplotype with systemic lupus erythematosus (SLE) in a family-based African-American studya

 
Promoter SNPs would not be expected to be functional in isolation of other nearby SNPs, especially under situations in which high LD is observed between closely arrayed SNPs. Accordingly, haplotypes were determined and analyzed for association with SLE in the CASSLE case–control study. Using rolling 2- and 3-marker haplotypes, strong association with SLE was observed around the CRP-707 site (Table 5). All 2- and 3-marker haplotypes containing the CRP-707G allele in separate evaluations of the African-American and the Caucasian populations demonstrated significant association with levels of significance ranging from P = 3.3 x 10–5 to P = 3.9 x 10–7. However, they do not provide strong additional evidence of association beyond the individual -707 association.


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  		Table 5. Haplotypic genetic association of C-reactive protein (CRP) single nucleotide polymorphisms (SNPs) with systemic lupus erythematosus (SLE) in the CASSLE case–control study

 
To identify protective and risk haplotypes, we restricted our analysis to the CRP-707 site together with the known function sites, CRP-409/-390 (Table 6). Our prior work has shown that the 11 and 22 -409/-390 haplotypes result in lower constitutive and stimulated CRP levels than the 12 and 13 haplotypes in healthy control donors and reporter construct studies respectively (29). Among African-Americans the -707/-409/-390 haplotype 111 was associated with increased risk of SLE (OR = 1.47, 95% CI: 1.22–1.77). Haplotype 122 was observed in <1% of cases and controls. In contrast, variation at the -707 site to the less common allele changed the risk haplotypes to protective haplotypes (211 and 222) among the African-Americans. A further protective haplotype, 213 but not 113, was also observed further extending the role of the -707 site in generating protective CRP promoter haplotypes. In the Caucasians, similar trends were found. The 111 haplotype trended towards increased risk but did not reach statistical significance (OR = 1.15, 95% CI 0.98–1.35). The 122 haplotype was not observed among Caucasians. The minor allele of CRP-707 again resulted in haplotypes (122 and 212) associated with decreased risk of SLE. The other minor alleles of CRP-707 containing haplotypes were observed in <1% of the cases and controls limiting our ability to define protective haplotypes.


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  		Table 6. Analysis of CRP Promoter haplotypes in the CASSLE case–control study

 
Analysis of extended promoter haplotypes, including all analyzed promoter variants, was performed (Table 7). While many additional haplotypes were defined in the African-American group, definition of at-risk and protective haplotypes generally aligned with the common and minor alleles of CRP-707 respectively. The most frequent CRP-707 common allele containing haplotype (111111, Table 7) defined an at-risk haplotype (P = 0.039, OR = 1.36, 95% C.I. 1.11–1.68) whereas the most frequent CRP-707 minor allele haplotype (111222) defined a protective haplotype (P = 0.073, OR = 0.73, 95% CI 0.58–0.93) for SLE susceptibility.


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  		Table 7. Analysis of all C-reactive protein (CRP) promoter haplotypes in the CASSLE case–control study

 
Analysis of the extended promoter haplotypes in CASSLE among Caucasians revealed a potential role for CRP-821 in defining both at-risk and protective haplotypes (Table 7). An at-risk haplotype containing the minor allele of CRP-821 together with the CRP-707/-409/-390 111 haplotype was associated with increased risk of SLE (OR = 1.29, 95% CI: 1.07–1.54). In contrast, the same minor allele of CRP-821 together with the CRP-707/-409/-390 211 haplotype, while rare, resulted in decreased risk of SLE (OR = 0.15, 95% CI: 0.06–0.41). Thus, the CRP promoter in Caucasians appears to have additional influences beyond the putative core CRP-707/-409/-390 SNPs that appear most important for SLE susceptibility among African-Americans.

Given SLE susceptibility, we examined the influence of common variation in CRP with clinical manifestations associated with SLE progression [renal involvement, cardiovascular disease (CVD), stroke, hypertension and diabetes] in the subset of patients enrolled in the longitudinal portion of CASSLE (PROFILE). At the single locus marker level, the tri-allelic variant at position -390TA (alleles 2 and 3) was associated with a 1.3 to 2.3-fold decreased risk of CVD, stroke, hypertension and diabetes among African-American SLE patients compared with the -390C allele following additive or dominant models (data not shown). A similar trend was observed among Caucasians, albeit not significantly. However, at the individual level, putative CRP-390TA-containing haplotypes (390TA/409G/707A) were over-represented among Caucasian SLE patients with hypertension compared with 390C/409G/707A carriers (OR = 0.66, 95% CI 0.49–0.89) suggesting that the -390TA variant may be more important for disease progression, as determined by these indices, than for SLE susceptibility. No significant associations were observed among SLE patients with renal involvement (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
This is the first study to comprehensively analyze CRP proximal promoter SNPs in relation to the SLE phenotype and we present evidence for a role of CRP promoter genetic variants in susceptibility to SLE. In multiple independent study populations, including both African-Americans and Caucasians, the variation in the CRP promoter at CRP-707 (rs3093061), and haplotypes containing CRP-707, strongly and reproducibly associates with the SLE phenotype. Principal component analysis indicates that this association is not due to population stratification. In our African-American population, we do observe association between CRP4 and SLE and a suggestive association between CRP2 and SLE in the Caucasian population was observed. We note the high degree of LD between the promoter SNPs and CRP2 and CRP4 (Fig. 1) which may explain prior reported association between CRP4 and SLE and of CRP2 and CRP levels in which only 1 promoter SNP was analyzed (31).

Our results provide a biological link between SLE and genetic variants in the CRP promoter. Our prior work, and the work of others, has provided evidence of association between the CRP-409/-390 variants which alter functional E-box transcription factor binding sites (29) and CRP levels in healthy individuals (24–29). Additional sites in the promoter predicted to be functional are the -821 site that alters a glucocorticoid receptor GR transcription factor and the -861 site which alters another E-box site. The most strongly associating SNP, CRP-707, is not predicted to directly lie in a transcription factor binding site (TRANSFAC 7.0 Web Site, http://www.gene-regulation.com/pub/databases.html). However, it is important to note that while the regression analysis suggests a modest independent genetic effect at CRP-409, our haplotype analysis did not identify any specific haplotypes that associated with disease beyond the effects seen at CRP-707 alone.

The identification of LD blocks in the CRP promoter containing known functionally important transcription factor binding sites with SLE suggests a role for genetic variation in the promoter in SLE and in CRP transcriptional efficiency. While we have examined all SNPs within 1 kb of the transcriptional start site, it is likely that there are additional variants that will be important determinants of both basal and induced CRP levels. For example, CRP4 is in the 3'-untranslated region of the CRP gene and could alter message stability (31). CRP2, while a synonymous SNP, could also alter translational efficiency and protein folding as recently reported in MDR (36). Thus, there are multiple levels at which CRP levels can be affected by host genetics.

Numerous studies have identified variation in CRP levels and CPR genetic variants as powerful and specific predictor of CVD risk in healthy individuals (26,27,30). We were able to demonstrate that alleles at the functionally important CRP-390 site were associated with an increased risk of CVD in African-Americans and hypertension in Caucasians. While these results are consistent with the established associations between CRP and CVD, further work in phenotypically characterized populations of patients with SLE will be necessary to fully explore the importance of CRP variants in CVD in patients with SLE as well as other clinical manifestations of SLE. Our inability to replicate the association between CRP4 and SLE nephritis (32) highlights the difficulty and importance of replication of genetic findings in multiple studies.

Our studies provide support and rationale for more detailed functional characterization of the CRP promoter region. The influence of multiple promoter SNPs, based on our haplotype determinations, should be tested for differences in promoter activity and transcription factor binding. Genetically determined lower levels of CRP could allow for alterations in the presentation of autoantigens to the immune system resulting in the generation of autoantibody production and autoimmunity. Indeed, either infusion of CRP or introduction of a CRP transgene into mice with experimental autoimmune disease results in amelioration of disease (37–40). However, it is important to emphasize that analysis to date of proximal promoter SNPs and extended CRP SNP haplotypes has been shown to explain <5% of the total variance for CRP expression (24). Clearly, with the high heritability of basal CRP expression, additional genetic factors involved in regulating CRP expression are yet to be described. These factors could include additional CRP variants and variants in other genes such as the transcriptional regulators IL6 and IL1B that are associated with CRP levels (24,41,42).

The CRP gene is encoded on human chromosome 1q21-23, a region that has shown multiple linkage and association signals with the SLE phenotype (33,35). Further genetic characterization of this region, including the known CRP receptors FCGR1 and FCGR2, will likely yield additional insights into the genetic component of SLE susceptibility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Participants
Patients with SLE were derived from the case–control study (CASSLE) constituted at the UAB that is comprised of Caucasians and African-Americans with longitudinal follow-up data available on a subset of participants [the longitudinal PROFILE study (10,34)] and healthy control participants. Patients and controls were from UAB, Johns Hopkins University, Northwestern University and University of Texas Health Science Center. The second study population included 830 individuals in 100 multiplex SLE African-American families enrolled in the LMRR based at Oklahoma Medical Research Foundation (12,33). All participants provided written informed consent and these studies were approved by the Institutional Review Board of each participating institution. Clinical characteristics of patients in the PROFILE and LMRR studies have been published (3,10,12,33,34). Baseline demographic features for the CASSLE study participants are summarized in Table 2. All patients enrolled into the CASSLE case–control study were seen by a study physician and had the diagnosis of SLE based on the presence of at least four ACR criteria (43,44). Ethnicity was self-reported and verified by the ethnicity of the participants' four grandparents, when known. All participants had the cumulative presence/absence of the revised ACR criteria documented at the time of enrollment. Characteristics of the LMRR participants have been previously published (12,33).

Genotyping
Genotyping of the CRP variants was performed by Pyrosequencing. Using a nested polymerase chain reaction (PCR) strategy, we initially amplified two regions of the CRP gene. One reaction encompassed the proximal promoter and the second spanned from the CRP coding region to the 3'-UTR. Second round PCR reactions were then performed around groups of closely spaced SNPs (Table 8). Pyrosequencing primers used for each SNP (or group of SNPs) are also shown in Table 8. First round PCR reactions contained 25–50 ng of template DNA, 1.5U Taq polymerase, 0.10 µM of each primer, 0.2 mM dNTP, 1.5 mM of MgCl2 and 20 mM of Tris–HCl (pH 8.4) and 50 mM KCl in a 25 µl volume. Cycling conditions were 5 min at 95°C, 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 45 s, and then a final extension step at 72°C for 7 min. Second round PCR conditions were identical except 0.25 µl of first round PCR product was used as template and 40 cycles of amplification were performed. All PCR reactions were run in ABI9700 PCR machines (Applied Biosystems). Following PCR, single stranded PCR products were purified from 10 µl of second round PCR reaction using a biotinylated primer and immobilization to streptavidin beads with the PyroMark Vacuum Prep Workstation (Biotage, Charlottesville, VA, USA), denatured with NaOH and annealed to the sequencing primer by heating to 80°C for 2 min. Pyrosequencing reactions were performed according to the manufacturer's instructions on a PSQ-HS96A system (Biotage). Genotyping of CRP variants was successful in >98% of samples. 1% of samples failed at multiple sites and were omitted from analyses. Quality control measures included replication of one DNA plate (95 samples) with identical genotypes at each variant from two independent PCR amplifications/Pyrosequencing reactions. Within the family-based study, five families with unexplained Mendelian errors were omitted from the study. Genotyping reliability was confirmed by comparison of Pyrosequencing results to direct sequence based analysis in 100 healthy controls (50 Caucasian and 50 African-American) and 97 patients with SLE (47 Caucasian and 50 African-American) that had been previously sequenced (29).


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  		Table 8. PCR and Pyrosequencing primers used in the analysis of the CRP variants

 
Data analysis
Analyses were done separately for Caucasian and African-American samples. Each of the eight genetic markers was tested for departures from Hardy–Weinberg equilibrium expectations using the exact test (45) in the control sample. Measures of LD (D' and r2) were computed to determine the block structure with the expectation-maximization algorithm-based program Dprime (http://www.phs.wfubmc.edu/web/public_bios/sec_gene/downloads.cfm). In the CASSLE case–control study, each marker was tested for association with SLE using the software SNPGWA and Dandelion (http://www.phs.wfubmc.edu/web/public_bios/sec_gene/downloads.cfm). Specifically, for each SNP we computed the following tests: overall genotypic association test, dominant, additive and recessive genetic model tests, departures from additively, and two- and three-marker haplotype analyses. The OR and corresponding 95% CI were computed relative to the major allele for each of the above tests.

Although multiple tests were computed for each SNP to provide context for any association, the primary statistical inference was based on the overall genotypic test of association. Specifically, if the overall genotypic test of association was significant the three a priori genetic models were examined for the most likely source of the departure from the null hypothesis. If the lack-of-fit test is not significant then the additive model is reported. If the lack-of-fit test is significant then the minimum of the dominant, additive and recessive genetic models is reported. The approach is consistent with the Fisher's least significant difference (LSD) multiple comparisons approach within an individual SNP. It maintains the correct experiment-wise type 1 error rate since if the overall genotypic test is not statistically significant no individual genetic model is deemed statistically significant. Finally, across the SNPs a simple Bonferroni correction is applied.

To test for association with SLE in the pedigree data a generalized estimating equations (GEE1) was computed using the sandwich estimator to account for the within family correlation. In addition, the PDT was used to test for LD (i.e. association and linkage).

To account for potential confounding substructure or admixture in these samples, principal component analyses (PCA) were computed using 8230 SNPs in 1840 cases and 1819 controls of self-reported European descent from the LLAS (35). A total of 906 individuals from LLAS overlapped the Caucasian samples reported here. The first principal component explained 85% of the genetic variation. The associated first principal component scores measuring the genetic variation were used as a covariate in a logistic regression model.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by NIH PO1 AR49084 Program Project in the Genetics of SLE, AR43727, AR42460, AI24717, AR048940 [GenBank] , RR020143, a Mary Kirkland Award, the US Department of Veteran Affairs and by General Clinical Research Centers: M01-RR00032 (UAB), M01-RR00052 (JHU), M01-RR00048 (NU), and M01-RR02558 (UTH).


   ACKNOWLEDGEMENTS
 
We thank Debbie McDuffie and LiFeng Zhang for assistance with genotyping and Dr Alex Szalai for helpful discussions about CRP in autoimmune diseases. We also thank the SLEGEN Consortium [JB Harley (Director), ME Alarcón-Riquelme, LA Criswell, CO Jacob, RP Kimberly, KL Moser, BP Tsao, TJ Vyse, and CD Langefeld (Co-Director)] (https://www.phsapps.wfubmc.edu/SLEGEN) for access to data for the Principal Component analysis. Materials and data from the Lupus Family Registry and Repository (NO1-AR6-22771) is acknowledged and appreciated.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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