Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on November 25, 2003

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Human Molecular Genetics, 2004, Vol. 13, No. 1 137-147
DOI: 10.1093/hmg/ddh021
© 2004 Oxford University Press

Polymorphism at the C-reactive protein locus influences gene expression and predisposes to systemic lupus erythematosus

Andrew I. Russell¹, Deborah S. Cunninghame Graham¹, Christopher Shepherd¹, Cheri A. Roberton¹, John Whittaker², John Meeks³, Richard J. Powell⁴, David A. Isenberg⁵, Mark J. Walport^1, and Timothy J. Vyse^1,*

¹Rheumatology Section, Imperial College Faculty of Medicine, Hammersmith Hospital, London W12 0NN, UK, ²Department of Epidemiology and Public Health, Imperial College Faculty of Medicine, St Mary's Campus, Norfolk Place, London W2 1PG, UK, ³Department of Pathology, Imperial College Faculty of Medicine, Hammersmith Hospital, London W12 0NN, UK, ⁴Clinical Immunology Unit, Queen's Medical Centre, Nottingham NG7 2UH, UK and ⁵The Middlesex Hospital, University College London, Arthur Stanley House, Tottenham Street, London W1T 4NJ, UK

Received September 29, 2003; Accepted November 6, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Relative deficiency of pentraxin proteins is implicated in the pathogenesis of systemic lupus erythematosus. The C-reactive protein (CRP) response is defective in patients with acute flares of disease, and mice with targeted deletions of the serum amyloid P component gene (Sap) develop a lupus-like illness. In humans, the genes for CRP (CRP) and SAP (APCS) map to 1q23.2 within an interval linked with SLE. We have investigated the candidate genes CRP and APCS in two cohorts totalling 586 UK simplex SLE families. The inheritance of an intronic dinucleotide repeat and seven single nucleotide polymorphisms in the CRP and APCS genes was examined by application of family-based tests of association and linkage. Basal levels of CRP were influenced independently by two polymorphisms at the CRP locus, CRP 2 and CRP 4. Furthermore, the latter polymorphism was linked/associated with SLE and antinuclear autoantibody production. Thus, the polymorphism associated with reduced basal CRP was also associated with the development of SLE. These data support the hypothesis that defective disposal of potentially immunogenic material is a contributory factor in lupus pathogenesis. The identification of polymorphisms that determine basal CRP levels has implications in ischaemic heart disease, where CRP level is an important predictor of risk.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with highly variable clinical features including glomerulonephritis, neuropsychiatric disease and cutaneous manifestations. SLE is characterized by autoantibodies directed against a number of constituents of the cell nucleus. These include double-stranded DNA (dsDNA), chromatin and small nuclear ribonucleoproteins (snRNPs), and antibodies to negatively charged phospholipids (reviewed in 1,2).

The aetiology of SLE remains incompletely understood; it includes hormonal, environmental and genetic factors (3, reviewed in 4). Genetic susceptibility to SLE is inherited as a complex trait, and recent independent genome-wide searches, in ethnically diverse multiplex families, have identified multiple intervals linked with SLE (5–9). When these data sets are compared, an interval on the long arm of chromosome 1, 1q23–24, is linked with SLE in multiple populations (5,8). This interval is orthologous to a region of distal chromosome 1 in the mouse (10), which is known to harbour susceptibility loci for murine lupus in several mouse strains (reviewed in 4). The genes for the pentraxin proteins C-reactive protein (CRP) and serum amyloid P component (SAP) (11) map to these intervals in both species. They are candidates as disease susceptibility genes for lupus by virtue of their position and the physiological activity of their products.

The genes for CRP (CRP) and SAP (APCS) have been physically mapped to 1q23.2 on an Ensembl contig AL445528 (www.ensembl.org). They are paralogues sharing at least 50% nucleotide identity and are separated by approximately 130 kb (chromosome 1 working draft contig NT_004406). Both genes consist of two exons, and have disproportionately long 3' untranslated regions (3'-UTR) (12,13), suggesting a possible regulatory role for this sequence.

Defective processing of cellular waste has been proposed as a contributory factor in SLE (reviewed in 14). There is evidence that the innate immune system, particularly the complement system, may be protective against autoimmunity. CRP and SAP are highly conserved mammalian acute phase reactants that could play a role in this protection (reviewed in 15). These pentraxins bind to specific membrane constituents and nuclear material, the major autoantigens in SLE, released during cell death or exposed on the cell surface during apoptosis (16). CRP binds to small nuclear ribonucleoproteins (17) and polar phospholipids (18) and SAP binds to and solubilizes native long chromatin (19). Characteristically, in human SLE there is relative failure of the acute phase CRP response during active disease despite evident tissue inflammation (20). Similarly, the inducible SAP response is defective in the New Zealand NZB/W F₁ murine model (21). Treatment of NZB/W F₁ mice with CRP ameliorates the accelerated phenotype induced by immunization with chromatin (22). Furthermore, mice rendered null for Sap by targeted gene deletion (in the context of a permissive genetic background) developed spontaneous antinuclear autoimmunity and a lupus-like glomerulonephritis (23). It has recently been shown that NZB/W F₁ mice carrying a human transgene for CRP exhibit less severe disease (24).

Induction of the acute phase CRP response in hepatocytes is promoted by the synergistic action of IL-1ß and IL-6. The proximal 250 nucleotides of 5' flanking sequence of CRP contain response elements for multiple transcription factors, including members of several HNF and C/EBP families (25–29). The intron of CRP contains a polymorphic GT dinucleotide repeat sequence (30), length polymorphism of which has been correlated with basal CRP levels (31).

We propose that genetic polymorphism at the pentraxin locus contributes to susceptibility to human SLE. We have tested this hypothesis by identifying single-nucleotide polymorphisms (SNPs) across the CRP and APCS genes and then conducting a family-based study of linkage and association in two large cohorts of SLE families. Finally, we provide data supporting a mechanism that underlies the genetic association with SLE.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
SNP identification
Direct sequencing across the CRP gene identified four SNPs and confirmed the intronic GT repeat (Fig. 1 and Tables 1 and 2). The proximal promoter (extending to -250 nucleotides) was not polymorphic; CRP 1, a tri-allelic SNP at position -286, did not alter known transcription factor motifs. No coding polymorphisms were evident in CRP; a synonymous mutation in the third nucleotide of codon 188 (leucine), resulting from a guanine to cytosine transition at nucleotide 641, was named CRP 2. In the 3' portion of CRP two SNPs were present with high frequency: CRP 3 and CRP 4. The latter SNP is present in only the longer of the two CRP transcripts described. Neither of these polymorphisms disrupted known consensus sequences associated with altered mRNA stability. A low frequency cytosine to adenine exchange eight nucleotides upstream of CRP 3 was in strong linkage disequilibrium with this SNP, which did not add to the association and haplotype analysis. The APCS coding region was not polymorphic apart from a synonymous mutation in the third nucleotide of the valine-specifying codon at residue 144 (APCS 2). APCS 1 and APCS 3 were identified within the 5' and 3' flanking sequence respectively. APCS 1 lies upstream of the transcription initiation site but not within known transcription factor binding motifs. APCS 3 is located 16 nucleotides downstream of the 3' limit of known APCS mRNA.

View larger version (15K): [in this window] [in a new window]   Figure 1. The structure and orientation of the pentraxin genes. The pentraxin genes, CRP and APCS are paralogues sharing approximately 60% nucleotide identity. They both consist of two exons separated by a single intron. The first exon encodes the leader peptide and first two amino acids of the mature peptide and the second exon encodes the remaining 202 (SAP) and 204 (CRP) amino acids. The orientation of these genes in centromeric (Cen) and telomeric (Tel) directions is marked by arrows. Exons are represented by boxes, and introns and intergenic regions by lines. Filled boxes denote translated regions; open boxes untranslated regions. The region boxed in grey represents a portion of the gene downstream from the limit of the 3'-UTR, but which contains a possible polyadenylation signal. Arrows mark the locations of SNPs within the CRP and APCS genes. The GT dinucleotide repeat located within the intron of CRP is represented by CRP (GT)n.

 

View this table: [in this window] [in a new window]   Table 1. Pentraxin SNPs. SNPs were denoted numerically from 5' to 3' within each gene. The nucleotide position within mRNA and location within human chromosome 1 working draft sequence NT_04406 are specified. Allele frequencies for the minor alleles at each SNP are listed in the three major ethnic groups comprising our study cohorts: European-Caucasian (E), Indo-Asian (I) and Afro-Caribbean (AF)

 

View this table: [in this window] [in a new window]   Table 2. Length polymorphism in the CRP dinucleotide repeat. Allele frequency was calculated in the parental chromosomes in the major ethnic groups [European-Caucasian, Afro-Caribbean (AF), Indian-Caucasian] in our study. The alleles are named according to the number of GT repeats as described in Szalai et al. (31)

 
Transmission disequilibrium test for single markers
Ethnicity influenced allele frequency at a number of SNPs (Tables 1 and 2). The transmission disequilibrium test (TDT) was therefore performed using genotype from the 299 European-Caucasian families from cohort 1 (Table 3), and also in supplementary analysis by inclusion all 344 families. There was a trend to over-transmission of CRP (GT)₁₆, most frequent allele of the CRP dinucleotide repeat (²=3.82, P=0.05); there was compensatory under-transmission of (GT)₂₁ allele. We also demonstrated that patients with SLE were more likely to inherit the minor allele of SNP CRP 4 (P=0.05). The inclusion of data from the 45 non-European families increased the transmission distortion for both markers (P=0.03 and P=0.02, respectively). Application of the pedigree disequilibrium test (PDT) confirmed the association of CRP 4 with SLE in European-Caucasian families (P=0.007). Using PDT the trend to over-transmission of CRP (GT)₁₆ did not reach significance (Z=1.69, P=0.09). Segregation of APCS 3 alleles was distorted in European families, but this did not reach statistical significance. Other SNPs segregated at random with no association/linkage to SLE.

View this table: [in this window] [in a new window]   Table 3. TDT of CRP and APCS polymorphisms in Cohort 1 European families. Results of single marker transmission disequilibrium analysis, using TRANSMIT, in cohort 1 (299 families). SNP alleles at a given locus are referred to as 1 (major allele) and 2 (minor allele). Although transmission of all alleles of the dinucleotide repeat was analysed, only the three most frequent CRP (GT)n alleles (16, 20 and 21) are presented. Values for observed and expected transmissions and the resulting chi square value (2) with 1 degree of freedom are given

 
CRP 4 was also associated/linked with the presence of antinuclear autoantibodies (ANA) in probands and seropositive siblings (²=4.13, P=0.04). However no associations were observed between other autoantibodies (anti-dsDNA, anti-Ro, anti-C1q, IgG ACA and IgM ACA) and pentraxin polymorphisms (data not shown). Stratification for renal involvement in the probands (128 families in cohort 1) was negative for association/linkage between renal lupus and any pentraxin polymorphism.

Linkage disequilibrium and haplotype analyses
Linkage disequilibrium (LD) analysis in Europeans identified strong LD across CRP (D' 0.91–0.98). A separate haplotype block was observed spanning the APCS locus. The degree of LD between these two genes was weak (D' 0.21–0.70; Fig. 2). The strength of LD within each pentraxin gene was reflected by restricted haplotype diversity. Five CRP haplotypes represented at least 85% all haplotypes present (Table 4). Using SNP genotypes only and the GENEHUNTER package to conduct TDT in trios there was strong support for association with CRP haplotype 2, carrying the rare allele at CRP 4 (P=0.0008). This represents a statistically significant association even if the conservative Bonferroni correction of five is applied reflecting the five common haplotypes across CRP. The microsatellite CRP (GT)₁₆ allele contributed to three common CRP haplotypes, of which only haplotype 2 (1.16.1.1.2) was associated with SLE when trios and single-parent families were studied (TRANSMIT P=0.007). Since the association with SLE was observed with haplotype 2 and not with haplotype 1 (Table 4), the association between CRP (GT)₁₆ and SLE observed in cohort 1 was the result of its presence on a haplotype defined by the minor allele of CRP 4. Three APCS haplotypes accounted for nearly 100% of genotypes; there was a weak association of SLE with a haplotype carrying the rare allele at APCS 3 (P=0.004). Using TDT analysis of all seven SNPs across the pentraxin locus, no haplotypes were associated with lupus or related phenotypes.

View larger version (20K): [in this window] [in a new window]   Figure 2. LD across the pentraxin locus. The results of linkage disequilibrium mapping using GENEHUNTER 2 are presented in this matrix. The values for D' between each SNP are presented in each box. Black boxes denote strong LD (D'>0.8), dark grey boxes intermediate LD (D' 0.7–0.8), pale grey boxes denote weak LD (D' 0.5–0.7) and white boxes very weak or negligible LD (0.2>D'<0.5).

 

View this table: [in this window] [in a new window]   Table 4. TRANSMIT TDT analysis for haplotypes across the CRP genes in the European families of cohort 1. Haplotypes are numbered according to their frequency, and the allelic variants that make up each haplotype are listed from 5' to 3' in columns 2–6. Observed (O) and expected (E) transmissions are listed in columns 8 and 9. This analysis has been constrained to those haplotypes with frequencies of at least 5%. The CRP 1.16.1.1.2 haplotype (haplotype 2) was over-transmitted to affected offspring (European families P=0.017, all families P=0.07).

 
Cohort 2 and combined analysis
The rare allele at CRP 4 is a unique marker for the CRP haplotype that showed association with SLE. Thus, the inheritance of CRP 4 was studied in further families. As we describe later, CRP 4 and CRP 2 were shown to be associated with basal CRP serum levels (see below). Thus, CRP 2 and CRP 4 were tested for association in a second cohort of 242 families (cohort 2, Table 5). Genotyping of CRP 2 and CRP 4 defines three haplotypes corresponding to haplotypes 1, 2 and 5 (Table 4). The second cohort was slightly smaller than the first cohort with fewer transmissions subject to analysis. However, there was a suggestion of transmission distortion at CRP 4 towards lupus probands (analysis of 175 trios P=0.03, analysis of all families TRANSMIT P=0.08). The transmissions of CRP 2 and CRP 4 were then studied in all families from cohort 1 and cohort 2 together. This analysis increased the strength of association of the haplotype carrying the rare allele of CRP 4 with SLE was increased (TRANSMIT ²=7.4, P=0.006), in 498 European-Caucasian families. This association was strengthened when all 586 families (independent of ethnicity) were analysed (TRANSMIT P=0.003). When trios only were studied the association with CRP 4 was stronger (143 transmissions compared with 92 non-transmissions P=0.0008) in all 394 trios. This result may reflect bias introduced by inferring missing parental haplotypes. There was no over-transmission of CRP 2 observed, but this SNP is at low frequency (0.07). As with cohort 1, no particular subset of SLE appeared to be particularly associated with CRP 4. However, we did run the TDT examining for association with ANA production in the combined cohorts, again there was a significant over-transmission of the rare allele of CRP 4 to ANA positive individuals (median of 25 TRANSMIT iterations ²=12, P=0.002).

View this table: [in this window] [in a new window]   Table 5. Features of the study cohorts. Details of the demographic features of the 45 SLE patients whose DNA was used for SNP screening (SNP screening cohort) and the two cohorts of SLE families examined in this study. Glomerulonephritis refers to the documented involvement of the kidneys in the proband

 
Comparison of basal CRP in lupus probands and healthy siblings
Having demonstrated evidence for association of the rare allele of CRP 4 with SLE we next sought to determine whether this polymorphism (and other allelic variants of CRP) influenced serum CRP levels. Thus, CRP levels were measured in the serum of 344 probands and 452 siblings. This analysis was constrained to those individuals without an apparent acute phase response in whom CRP levels were measured within the normal range, below 10 mg/l. We chose to investigate the potential effect of polymorphisms on basal CRP expression rather than acute phase expression, which most likely involves different regulatory elements at the CRP locus. Therefore, 27 probands and 31 siblings were excluded because of an evident acute phase response. Basal CRP levels were not significantly different between siblings and probands (P=0.24, two-tailed Mann–Whitney test).

The effect of CRP haplotype on serum CRP level
European-Caucasian individuals with unambiguous haplotype assignment by TRANSMIT were included in this analysis; 49 probands and 58 siblings were therefore excluded. Probands (n=234) and siblings (n=302) were analysed separately, but since the groups were not statistically different, the data sets were also combined (Fig. 3). Haplotype 2 (1.16.1.1.2) and haplotype 5 (1.16.2.1.2) were associated with low levels of CRP in serum (mean 2.05 and 1.27 mg/l, respectively) compared with the common haplotype 1 (2.16.1.2.1), mean basal CRP 2.82 mg/l; these comparisons were significant, P<0.0001 (haplotypes 2 and 5 versus haplotype 1), and P=0.02 (haplotype 2 versus haplotype 5), in the combined analysis of 536 individuals. Haplotype 5 is likely to have arisen on the background of an ancestral haplotype 2, since these two haplotypes differ only at CRP 2, implicating both CRP 2 and CRP 4 in the association with low basal CRP levels.

View larger version (29K): [in this window] [in a new window]   Figure 3. The association of CRP haplotype with basal serum CRP levels. The left-hand part of the figure shows the five common CRP haplotypes with corresponding allelic composition at the five markers across the CRP locus. To the right of this the mean serum basal CRP levels are shown for each haplotype. Significant differences (P<0.001) are indicated by double asterisks.

 
The effect of CRP SNP alleles on serum CRP level
Analysis of basal CRP levels in individuals with differing CRP 4 genotype demonstrated a gene dose-specific effect (Fig. 4A); homozygosity for allele 2 was associated with the lowest mean level of CRP. Furthermore, an additional effect of CRP 2 genotype was also apparent (Fig. 4B). The effect of CRP 2 and CRP 4 allelic variants on basal CRP levels was also studied using modified linear regression. These analyses indicated correlation with CRP 4 genotype (P=0.003) and an independent effect from CRP 2 (P=0.01). The weaker association with CRP 2 reflects the lower allele frequency of this SNP. When affected status was added to the model, it was evident that the association of CRP 4 and CRP 2 with basal CRP levels was present in both SLE patients and in their siblings. We used the parameters derived from the regression model to predict basal CRP expression based on genotype at CRP 2 and CRP 4; inheritance of minor alleles at both SNPs predicted a CRP level of 0.9 mg/l, whereas inheritance of the common alleles at both SNP predicted a value of 3.4 mg/l.

View larger version (12K): [in this window] [in a new window]   Figure 4. (A) CRP 4 genotype influences CRP levels. This bar chart illustrates the effect on CRP 4 genotype on serum CRP levels in the combined analysis of Caucasian probands and siblings. Median CRP levels in the heterozygous and 2/2 homozygous groups were both significantly lower than in 1/1 homozygotes (P<0.0001, double asterisk). (B) The interaction of CRP 2 and CRP 4 genotype on basal CRP. In this analysis Caucasian probands and siblings were divided by their CRP 2 and CRP 4 genotypes. The results are graphed on this bar chart. Individuals homozygous for the major (1) allele at box loci are represented by the white bar, individuals homozygous for 1 at CRP 2 but heterozygous at CRP 4 by a pale grey bar. The dark grey bar represents double heterozygotes and the black bar, those individuals homozygous for 2 at CRP 4, and heterozygous at CRP 2. There were too few double homozygotes for meaningful analysis.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
We tested the hypotheses that polymorphism at the pentraxin locus might contribute to genetic susceptibility to human SLE, and that genetic variation in CRP could specify basal levels of C-reactive protein. The pentraxin proteins are important in innate immunity and in addition bind certain constituents of the cell nucleus, promoting their clearance and reducing their immunogenicity. Structural variants of the proteins have not been described; consistent with our observation that the coding sequences of both genes were monomorphic. Thus the aetiological effect of pentraxin gene polymorphism would be expected to be quantitative rather than qualitative.

We examined the inheritance of polymorphisms at the pentraxin locus in a family-based association study of SLE. Strong LD was present within each of the CRP and APCS genes, although minimal LD exists between the two genes. There are no functional genes that lie between CRP and APCS. We identified five common CRP haplotypes by characterizing the inheritance of four SNPs and one polymorphic microsatellite. By application of transmission testing we demonstrated that inheritance of CRP haplotype 2 by affected probands with SLE from their parents was not random. Considering the composition of the CRP haplotypes, it was apparent that the specific allelic variant associated with SLE was an allele of CRP 4. We demonstrated using both haplotype analysis and single marker inheritance that the less common A allele of the single nucleotide polymorphism CRP 4 in the 3' region of the CRP gene is associated with the development of SLE. The association of this allele with SLE was detected in all 586 families (P=0.006) using an analysis package TRANSMIT to infer missing parental genotypes. When only trios were examined, that is family units comprising both parents and an affected offspring, strong statistical support was obtained for the association in 394 families (P=0.0008). The A allele at CRP 4 had a relatively high frequency in European and Asian-Indian populations (0.3) and was present in Afro-Caribbean families too, albeit at a lower frequency (0.14). The association was present when all families were studied and when only European families were selected; there were insufficient numbers of non-European families to study separately.

SLE is a clinically heterogeneous disorder. Basic clinical and serological data were obtained on all affected family members and their siblings. We repeated the family-based association tests in SLE patients that had been categorized according to the presence or absence of renal disease, or by their autoantibody profiles. There was no evidence that any particular subgroup of SLE patients preferentially inherited the rare allele of CRP 4, although there is an inherent loss of power in these subgroup analyses as the number of families analysed falls. Previous studies have indicated an elevated frequency of ANA in SLE families (32). Approximately 30% of the siblings in the SLE families had elevated antinuclear antibodies in the cohort investigated in this study and there was evidence of familial clustering of antinuclear antibody production (manuscript in preparation). Using the production of ANA to define affection status, there was evidence for distorted transmission of CRP 4 alleles to offspring that produce antinuclear antibodies (P=0.002), suggesting that this allele may act to promote susceptibility to antinuclear autoimmunity.

Having shown that the rare allele of CRP 4 was associated with the development of SLE, we undertook a study of basal CRP expression in the SLE patients and their siblings to determine whether this polymorphism had any effect on serum CRP levels. Basal CRP levels as measured by high-sensitivity CRP assay were compared between individuals to whom unambiguous CRP haplotypes could be assigned. Two haplotypes were defined that were significantly associated with reduced basal CRP expression. The SNPs that demarcated these haplotypes were CRP 2 and CRP 4. Linear regression analyses corroborated the conclusion that these two SNPs made an independent contribution to basal CRP levels and that their influence was independent of SLE status.

On the basis of these findings we suggest that reduced basal CRP expression predisposes to the development of SLE. However this would predict an association between the CRP 2 locus and SLE, since the rare allele of CRP 2 was associated with reduced basal CRP expression. The failure to detect an association between this SNP (frequency 0.07) and SLE may be explained by consideration of power. Simple power calculations indicate that for genotypic relative risk (GRR) of 1.75, 80% power (with P=10^-3) is achieved using 740 trios for a disease allele frequency of 0.07 (33). Thus our study is currently underpowered to reliably detect a weak effect from CRP 2, if it exists. The numbers of families examined in this study are sufficient to produce 80% power to detect disease susceptibility alleles with frequency of 0.09 or greater with a modest effect (GRR=1.75). Therefore, single marker TDT and haplotype analysis should exclude the APCS 1 and APCS 2 polymorphisms examined here in genetic susceptibility to human SLE.

An allele of CRP 4 was associated with ANA production. This observation has parallels in murine models since mouse strains deficient in production of the pentraxin SAP express antinuclear autoimmunity (23). It could also be relevant that autoantibody production in lupus-prone mice maps to an orthologous locus on distal mouse chromosome 1 (34,35). The association of the rare allele of CRP 4 with antinuclear autoantibody production supports the ‘waste disposal’ hypothesis of SLE (16). Since there is evidence supporting the concept that CRP participates in the removal of apoptotic cells (36), our data support the view that low basal CRP expression impairs the ‘physiological’ removal of cellular debris. The data presented in this paper support the concept that this is a factor contributing to the development of SLE and antinuclear autoimmunity. Data from Dnase 1 and Sap knockout mice have been cited to support this hypothesis (reviewed in 14), but with the exception of rare mutations in DNASE1 within the Japanese population (37) there has been little direct genetic evidence to support this concept in human SLE.

There are several potential mechanisms by which variants at CRP 2 and CRP 4 might influence basal CRP expression. CRP appears to generate two transcripts (12) [www.ensembl.org, designated ENST00000325798 (longer transcript), and ENST00000255030 (shorter transcript)]; the longer of these carries CRP 4 in the 3' untranslated region. The relative biological importance of these two transcripts is not known. The stability of mRNA species may depend on the sequences within the 3'-UTR, as exemplified by the AU-rich sequence motifs that are important in regulation of cytokine mRNA (38,39). It is of interest that both CRP and APCS have disproportionately long 3'-UTRs (Fig. 1), implying a regulatory role in pentraxin mRNA processing. In a second shorter CRP transcript, CRP 4 is located within 3' flanking sequence just upstream of a putative polyA signal, consistent with the reported influence of the 3' flanking sequence on CRP expression (40). The mechanism by which CRP 4 influences basal CRP expression remains to be established, but it may operate at the transcriptional or post-transcriptional stage.

Pro-inflammatory cytokines IL-1ß and IL-6 drive the acute phase response of CRP via response elements located within the proximal 250 nucleotides of 5' sequence (25–29). Since levels of these cytokines are known to be elevated in patients with active SLE (41), functional polymorphisms within the CRP promoter could explain the inappropriately normal pentraxin levels observed in lupus. However, we observed the sequence of this region to be non-polymorphic. Furthermore, polymorphism at CRP 1, located upstream of this sequence, did not alter consensus sequence of known motifs of transcription factors, was not associated with the presence of SLE, or influence basal CRP levels. It should be pointed out that we have not addressed the possible effect of the CRP 1 on the magnitude of the CRP rise during an acute phase reaction.

The GT dinucleotide repeat within the intron of CRP has been reported to influence CRP expression (31); the most frequent alleles, (GT)₁₆ and (GT)₂₁, were associated with low basal levels of the protein. The data presented in this paper refine the microsatellite association by considering the relation between CRP levels and CRP haplotypes (Table 4). The two CRP haplotypes associated with reduced basal CRP expression, haplotypes 2 and 5, also carry the (GT)₁₆ repeat. Thus our data are in part compatible with those previously reported (31). However, haplotype 3, which carries (GT)₂₁, was associated with intermediate levels of basal CRP (Fig. 3). Since the CRP SNPs described generate a more accurate definition of CRP haplotypes than the microsatellite alleles alone, it is unlikely that length polymorphism of this intronic repeat exerts a direct biological effect on CRP regulation.

The existence of genetic regulation of levels of CRP expression has implications beyond the predisposition to autoimmunity. Over the last five years there has been increasing evidence of the predictive value of basal CRP in atherosclerosis. An association between a SNP (rs1800947, or CRP 2 as defined in this paper) in CRP and basal CRP expression has been described (42). The data presented in this manuscript are therefore in agreement with this report, but provide a more comprehensive, haplotype-based approach to the issue of the genetics of basal CRP regulation. Our data implicate independent effects from CRP 2 and CRP 4. The rare allele frequency of CRP 2 in our study was 0.07, a similar frequency to that observed by Zee and Ridker (0.06). They did not find an association between the CRP 2 polymorphism and arterial thrombosis. However, polymorphism at CRP 2 is not the sole genetic cause at the CRP locus of variation in CRP basal levels. Recently it has been reported that the lower frequency allele a SNP in the 3'-UTR of CRP (rs1130864, our CRP 3) is associated with elevated basal CRP (43). The rare allele of this SNP is carried on haplotype 1, as defined in this study (Table 4). This SNP does not uniquely demarcate this haplotype, which is also characterized by a polymorphism in the CRP promoter, CRP 1. Our data are compatible, but amplify this finding and indicate that CRP 3 may not necessarily be the causal variant on this haplotype. Thus, it remains possible therefore that polymorphism at the CRP locus influences the development and/or progression of atherosclerosis and we are investigating this hypothesis.

We conclude that polymorphism that a polymorphism in the 3' flanking sequence of CRP gene is associated with SLE and antinuclear autoantibody production, perhaps by specifying low levels of the protein. Relative deficiency of the chaperoning properties of CRP therefore could predispose to antinuclear autoantibody production which in turn contributes to the development of human lupus. These findings have relevance in the study of other conditions characterised by variation of CRP responses, in particular ischaemic heart disease.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
SLE Families
We have established a large collection of UK SLE nuclear families with predominantly one affected offspring per family. The demographic details of these families are summarized in Table 5. Of the total of 586 families, samples from both parents were available in 69% of cases; samples were taken from siblings when available. Siblings were always sampled in single parent families. The mean number of siblings in two parent families was 1.81 and the mean number in single parent families was 1.97; 44% of the siblings were male. The collection comprises genomic DNA and serum from patients who satisfy the American College of Rheumatology revised criteria for SLE (44). Patients were categorized as having renal lupus using ACR criteria. Clinical information was obtained from all participants by interview and completion of a questionnaire. Genomic DNA was isolated by a standard phenol–chloroform extraction from peripheral blood mononuclear cells (45). The study protocol was approved by the London multi-centre research ethics committee (MREC).

The principal study cohort represents a random sample of 354 SLE families. Ten families were excluded from analysis due to ex-paternity of the pedigree, incorrect diagnosis or withdrawal from the study. The remaining 344 families are hereafter referred to as cohort 1. When a preliminary positive finding was obtained for the inheritance of a marker in cohort 1 replication was sought in a second cohort of 242 families (cohort 2).

The stored serum of all probands and their siblings was assayed for antinuclear autoantibodies and CRP. CRP was measured using a commercially available, automated high sensitivity immuno-turbidimetric method (Olympus, UK). Briefly, 2 µl of serum sample was mixed with a 160 µl of a 0.05% suspension of latex particles coated with goat anti-human CRP antibodies, in the presence of MOPSO buffer (pH 7.5). The presence of the resultant insoluble aggregates was detected at 800 nm and is proportional to the CRP concentration of the sample. Samples were run on an Olympus AU640e Chemistry Immunoanalyser (Olympus, UK). Antinuclear antibodies (ANA) were assayed by indirect immnuofluorescence; anti-double stranded DNA antibodies (anti-dsDNA), antibodies to extractable nuclear antigens (Ro, La, Sm, RNP) and IgG and IgM isotypes of anticardiolipin antibodies were assayed by ELISA. The methods employed are described elsewhere (46–48).

SNP identification
SNPs were identified by direct sequencing of the complete genomic region of CRP and APCS in 45 SLE patients representative of the study population (Table 5). Direct sequencing was performed in both orientations in order exclude sequencing artefacts. SNPs were then checked for uniqueness by the computer program BLAST (www.ncbi.nlm.nih.gov/BLAST) and cross-referenced against publicly available SNP databases, the SNP Consortium (http://snp.cshl.org) and dbSNP (www.ncbi.nlm.nih.gov/SNP).

CRP was amplified in two overlapping 1154 and 1343 bp amplicons, using reference sequence reported by Woo et al. (accession M11880.1). Oligonucleotide primers designed from chromosome 1 working draft sequence NT_004406 were generated to amplify a further 1076 bp of 5' flanking sequence. APCS was amplified with flanking sequence as a single 1354 bp PCR product using primers based on NCBI reference sequence (accession D00097.1) (13). The oligonucleotide sequences used are available on request. Polymerase chain reaction (PCR) amplifications were performed on 50 ng of genomic DNA using standard conditions (available on request) and were subjected to thermal cycling using a Peltier Thermal Cycler (MJ Research). Resultant PCR product was purified using a 96-well format microplate vacuum system according to manufacturer's instructions (MultiScreen^®-PCR Plates, Millipore). A 30–90 ng aliquot of purified PCR product was subjected to a cycle sequencing reaction using a fluorescent-labelled dideoxynucleotide kit (Big Dye Terminator Cycle Sequencing Ready Reaction Kit, Applied Biosystems), and extension products purified by 75% isopropanol precipitation. Sequenced samples were electrophoresed through denaturing 5% polyacrylamide gels (Long Ranger^TM) on an ABI PRISM 377 DNA Sequencer. Sequence data was analysed using Sequence Analysis version 3.4.1 and the alignment program Autoassembler^TM (Applied Biosystems). SNPs with a minor allele frequency of more than 5% were then investigated in the study cohorts.

SNP genotyping
The preferred method of genotyping was polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Where this method was unsuitable an amplification refractory mutation system–polymerase chain reaction (ARMS-PCR) assay was used.

PCR-RFLP
The genotyping assays employed are described in Table 6. In general, restriction maps were generated for each allele of a given SNP using REMAP [EMBOSS sequencing software package, via UK HGMP Resource Centre (www.hgmp.mrc.ac.uk)]. Oligonucleotide primers (Sigma Genosys) were designed to amplify 100–300 bp flanking the SNP and genomic DNA from each individual was amplified as described above. PCR product was digested to completion in appropriate buffer according to the manufacturer's specifications. The resulting fragments were size separated on 3% agarose or 10% polyacrylamide gels and visualised by ethidium bromide staining. APCS 2 was genotyped by a reverse primer-determined PCR-RFLP using the enzyme Fok I. To enhance allelic discrimination a 20meric tail of random nucleotides (ACA ATC TTG GGC TGA GCA TC) was added to the reverse primer increasing the size of the smallest fragment.

View this table: [in this window] [in a new window]   Table 6. PCR-RFLP assays employed to genotype SNPs in the pentraxin genes. The oligonucleotide primer sequences used for each assay and the discriminating restriction enzyme are given in this table

 
ARMS-PCR
APCS 3 was genotyped using ARMS-PCR. Paired amplifications were performed using a common forward primer and one of each allele specific reverse primer (designed with an allele-specific 3' nucleotide and the insertion of a penultimate 3' base mismatch which increased allele specificity). PCR conditions included 1.5 mM MgCl₂, and an annealing temperature 61°C. An internal control primer pair was included in each reaction in order to ensure the efficiency of amplification. Primer sequences were as follows: common forward primer, 5'-GGA GTC CTC ATC AGG TAT TG-3'; G allele-specific reverse primer, 5'-GAT ATG GAC TAT GAC AAT C-3'; T allele-specific reverse primer, 5'-GAT ATG GAC TAT GAC AAT A-3'; control forward primer, 5'-GTC TCA GCT CAC GAT CAC CA-3'; control reverse primer, 5'-CCG TAG CTG CCT GTA CCA AT-3'. The resultant 542 bp (and 249 bp control) amplicons were electrophoresed through 1% agarose gels and visualized by ethidium bromide staining. The genotype of those individuals with a positive T allele PCR was confirmed by direct sequencing.

CRP dinucleotide repeat genotyping
Fluorescent primer PCR was performed using the primer pair described in [30], with 5' fluorescent labelling of the forward primer: forward primer, 5'-FAM-GAT CTA TCC CCT CAC TTA CG-3'; reverse primer, 5'-TAT GAA CAG AAC AGT GGA GC-3'.

PCR was performed as described above and amplification products were size-separated on a 3700 ABI PRISM DNA Sequencer (Applied Biosystems) against the Genescan-ROX 400HD size standard. Data was collected using GENESCAN 3.1 software (Applied Biosystems) and allelic assignment was semi-automated, using Genotyper 2.0 software (Applied Biosystems).

Statistical analysis
Allele frequencies were calculated from parental chromosomes for each ethnic group and compared using the chi square statistic. The pattern of LD and the presence of haplotype blocks across both pentraxin genes and across the locus as a whole were defined in the predominant European-Caucasian group using haplotype frequencies obtained from Transmit analysis (49) and from an estimation of D' (50) obtained from an extension of Genehunter 2 (Courtesy of Dr Mark Daly, Whitehead Institute).

Genotype data from SLE families were analysed by application of two family-based tests of association. In the TDT, transmission of marker alleles (or haplotypes) from heterozygous parents to affected individuals is examined and significant distortion away from random segregation is used to infer linkage and association (51). TDT was performed using the computer program Transmit version 2.5 (49) which is allows the reconstruction of missing parental genotype (52). Transmission of single markers was examined in addition to haplotype analyses. In order to optimise the information provided by the SLE family genotype data, analysis including genotype data from siblings was also performed using the PDT version 3.12 (53). This test integrates information from pedigrees irrespective of structure and size and is based on a random variable measuring transmission distortion across the entire pedigree.

Cohort 1 was analysed using SLE as the definition of ‘affection’ status, and also with families stratified for ethnicity and the presence of renal involvement. Markers with putative association/linkage in cohort 1 were also tested in cohort 2 and by combination of both data sets. The presence of autoantibodies was observed to cluster in families (manuscript in preparation) with autoantibody-positive siblings present in 30% of families. The presence of different autoantibodies was also used to define affection status for TDT analysis. To achieve this, TRANSMIT was constrained to select a single affected individual from each family at random. Multiple iterations of TRANSMIT were performed and the chi square values obtained for each SNP and their distribution was tested using the PRISM software package. The arithmetic mean of the chi square values was then used to derive the point P value for this analysis. Power calculations for TDT were performed based on the methods described by Risch and Merikangas (33).

Regression analysis comparing basal CRP with CRP genotype was conducted on a linear model. Since the serum CRP concentrations were not normally distributed, Box–Cox analysis (54) indicated that the data be transformed using the function ([CRP]^0.17-1)/0.17. We assumed a random family effect to represent correlation between sibs that might result from shared environmental factors or other genetic influences.

	ACKNOWLEDGEMENTS

 
We would like to express our sincerest thanks to all the patients and their families for providing us with their blood samples. This gratitude extends to the nurses and primary care physicians who took the blood samples; without their help this study would not have been possible. We would like to thank Mr Paul Spencer and Ms Tanzila Kazmi who helped coordinate the collection of samples. Many physicians provided patients for this study, and we would like to express our thanks to them: Kevin Davies, Dorian Haskard, Steven Sacks, Simon Allard, David D'Cruz, Charles Mackworth-Young, Bernard Colaco, Martin Seifert, Costantino Pitzalis, Lee-Suan Teh, Donal O'Donoghue, Elizabeth Lightstone, Robert Bernstein, Neil McHugh, Robert Moots, Mohammed Akil, Neil Snowden, Bridget Griffiths, Ian Griffiths, John Feehally, Ashvin Samanta, John Main, Robert Mactier, Peter Andrews, Joe Unsworth, Timothy Goodship, Gavin Spickett, Robert Wilkinson, Paul Wordsworth. We would like to thank Drs John Rioux and Mark Daly, Whitehead Institute, MIT, MA for assistance with linkage disequilibrium analysis. The Wellcome Trust supported this research in the form of a Senior Clinical Research Fellowship (T.J.V.) and Prize studentship (C.A.R.). We are indebted to the Arthritis Research Campaign (ARC) who provided a Research Fellowship (A.I.R.). Lupus UK gave financial support for the collection of DNA and sera from the participants and also assisted in advertising the study.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +44 2083832339; Fax: +44 2087433109; Email: t.vyse{at}imperial.ac.uk

Present address:

The Wellcome Trust, Euston Road, London NW1 2BE, UK.

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