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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

To assess the copy number variation of complement C4A and C4B genes in patients with rheumatoid arthritis (RA).

Methods

DNA samples were obtained from 299 patients and controls and analyzed for copy number variation of total complement C4, C4A, and C4B genes. The results were compared by chi-square analysis, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated.

Results

Chi-square analysis revealed similar distribution patterns of total C4 alleles in RA patients (n = 160), non-RA patients (n = 88), and healthy controls (n = 51). There was no trend toward C4A deficiency as in lupus. Significant differences in C4B distribution were observed in RA patients, in whom an ∼2-fold increase in the frequency of homozygous and/or heterozygous C4B deficiency (0 or 1 allele) (40%) was present relative to non-RA patients or healthy controls (both 21.6%). C4B deficiency was more frequent in seropositive RA patients than in seronegative RA patients (44% versus 31%). The odds of C4B deficiency were 2.99 (95% CI 1.58–5.65) (P = 0.0006) in seropositive RA patients relative to non-RA controls. These findings were confirmed in a larger healthy control cohort, yielding an OR of 1.83 (95% CI 1.21–2.76) (P = 0.0056). The association of the shared epitope with C4B deficiency was significantly greater in seropositive RA patients than in non–seropositive RA controls (96% versus 54.5%) (P < 0.0001), suggesting that C4B deficiency interacts with the shared epitope in the development of seropositive RA.

Conclusion

Our findings indicate a relationship between C4B copy number variation and RA that approximates that seen between C4A copy number variation and lupus. The concurrence of C4B deficiency and the shared epitope in seropositive RA may have broad implications for our understanding of RA pathogenesis.

Rheumatoid arthritis (RA) is a systemic inflammatory disease that involves the synovium of affected joints, with a prevalence of 0.1–1% worldwide (1). RA patients are classified as seronegative or seropositive based on the results of blood tests for rheumatoid factor (RF) or anti–citrullinated protein antibodies (ACPAs) (2–5). Seropositive patients typically express both RF and ACPAs. Most ACPA-positive RA patients (85%) have the shared epitope, a 5–amino acid sequence ([R/Q][K/R]RAA) at positions 70–74 in the HLA–DR β-chain (2, 3). The shared epitope sequence appears to confer a risk of RA, since HLA–DRB1 alleles not associated with RA differ at this site (for review, see ref.6). Both seropositivity and the presence of the shared epitope are predictors of increased morbidity and mortality from RA (4, 5). The association of RF with the shared epitope was observed only in the presence of anti–cyclic citrullinated antibodies (3). Given the role of RF in immune complex clearance, this suggests that RF is induced in response to ACPA-associated immune complexes (7, 8). RF is associated with pathology through immune complex formation in the joint and at extraarticular sites (1).

The complement system is a group of plasma and membrane proteins involved in immunity as well as protection against autoimmunity. The proximal components of the classical pathway (C1, C4, and C2) are important for the clearance of apoptotic cells and immune complexes (9–11). In humans, 2 classes of polymorphic C4 proteins, C4A and C4B, exist. They are distinguished by the nature of their covalent linkage with target cells or immune complexes through the highly reactive thioester carbonyl group. Activated C4A tends to form an amide bond with amino groups on antigens, and activated C4B is strongly reactive with hydroxyl groups on glycerols or glycosylated antigens (12–14). In in vitro hemolytic assays, purified C4B reacted ∼4 times more efficiently than purified C4A (15). Although mice also have 2 classes of C4-like proteins, C4 and Slp (sex-limited protein), the functionality of Slp in the mouse complement pathways remains unclear (16, 17). Activated mouse C4 reacts biochemically like human C4B because it also consists of the orthologous histidine-1106 residue that facilitates the nucleophilic attack of the thioester carbonyl group to form a covalent ester bond with substrate (14, 18).

Gene copy number variation constitutes a major source of genetic variation but its genetic relevance was only recently recognized (19, 20). Many inherent multiallele copy number variations are highly complex, including continuous gene copy number variations, secondary sequence polymorphisms, and the integration of mobile genetic elements (21–23). Genome-wide association studies (GWAS) relying on single-nucleotide polymorphisms and array comparative genome hybridizations usually do not have the ability to detect complex diversities of multiallele copy number variations (19). The human complement C4 gene complex is located within the central region of the major histocompatibility complex (MHC; also known as the HLA) on the short arm of chromosome 6 and is a major genomic site of gene copy number variations. In different individuals, 2–8 copies of C4 genes in a diploid genome are frequently detectable, and each of these C4 genes can code for a C4A protein or a C4B protein. One-half to two-thirds of the general population have 2 copies of C4A and 2 copies of C4B in a genome. The remainder have variable combinations of 0–6 copies of C4A and C4B genes (22, 24–26). Such variations in C4A and C4B copy number are not accurately detectable by GWAS and array comparative genome hybridization approaches, and thus are missed in most large-scale disease-association studies, including those for RA (20, 27).

C4 gene copy number influences biosynthesis, as serum levels of C4 proteins parallel their gene copy number (23, 25). Thus, an individual can completely lack either C4A or C4B but have a normal total number of C4 alleles encoding the other C4 isotype. This finding has led to a refined understanding of the relationship between systemic lupus erythematosus (SLE) and C4A. A selective deficiency in C4A alleles has been demonstrated in SLE patients, without an association with C4B gene copy number (e.g., a compensatory increase in C4B gene copy number) (22). In this study, we demonstrate that the reverse relationship exists in RA, with a selective C4B deficiency occurring at an increased frequency in seropositive RA patients. These data suggest that C4B deficiency may play a role in RA pathogenesis and/or phenotype.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Patients.

The RA patients (n = 160) in this study were followed up at the Dartmouth-Hitchcock Medical Center. Each of the patients met the American College of Rheumatology 1987 revised criteria for RA (28) and were followed up by a board-certified rheumatologist. Patients with seropositive RA (n = 115) had a record of a positive test result for RF or ACPAs. These patients were 20–92 years old, and 74% were women. Patients with seronegative RA (n = 45) were comparable in age to the seropositive patients (range 25–82 years), and 69% were women. The non-RA patients (n = 88) were 19–80 years old, and 62% were women. Non-RA patients were recruited from the Rheumatology Clinic at Dartmouth-Hitchcock Medical Center and included patients with a number of different diseases. The most common diseases (affecting >3 patients) were psoriatic arthritis (n = 13), antineutrophil cytoplasmic antibody–associated vasculitis (n = 11), polymyalgia rheumatica (n = 8), giant cell arteritis (n = 8), myositis (n = 7), Sjögren's syndrome (n = 5), osteoarthritis (n = 5), and SLE (n = 4). A cohort of healthy male volunteers (n = 51) ages 19–59 years was used as the healthy control group. All patients and controls were of European ancestry.

Complement C4A and C4B gene copy number variations and HLA–DRB1 typing.

After informed consent was obtained from the subjects, peripheral blood was collected in PAXgene Blood DNA tubes (Qiagen), and DNA was isolated according to the recommendations of the manufacturer. Total C4, C4A, and C4B gene copy numbers were determined by Southern blot analyses of Taq I and of Psh AI plus Pvu II restriction enzymes, as previously described (29, 30). Briefly, genomic DNA samples were digested with Taq I, subjected to agarose gel electrophoresis, and transferred to nylon hybridization membrane for Southern blot analyses. Using a probe corresponding to the RP-C4 genomic region, Taq I restriction fragment length polymorphism (RFLP) analysis yields the total copy number of C4 genes. Using a C4d-specific probe, Psh AI–Pvu II RFLP analysis yields the relative copy numbers of C4A and C4B. In the case of a low quantity of genomic DNA and ambiguous results, quantitative real-time polymerase chain reaction experiments for copy numbers of total C4, C4A, and C4B were performed to obtain the missing data or to independently validate Southern blot analysis results (31). HLA–DRB1 typing was performed by the American Red Cross, Penn-Jersey Blood Services Region.

Statistical analysis.

Statistical analysis was performed using JMP, version 8.0 (SAS Institute). Chi-square analyses were used to determine the differences in total C4, C4A, and C4B gene copy numbers among groups. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by analysis of 2 × 2 tables, using Fisher's exact test for comparisons.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Increased frequency of C4B deficiency in RA patients.

We examined the gene copy number variations of complement C4A and C4B by Southern blotting in a population of healthy volunteers (n = 51), non-RA patients (n = 88), and RA patients (n = 160) (Table 1). The distribution of total C4 genes was examined by chi-square analysis (Figure 1). There was no statistically significant difference in total C4 gene copy numbers between the 3 groups (healthy controls, non-RA patients, and RA patients). The distribution of C4A and C4B gene copy numbers was analyzed. A difference in C4A gene copy number was observed between healthy controls and RA patients (P = 0.0117). This difference resulted from an increased frequency of the presence of 2 copies of C4A in healthy controls compared to RA patients (67% versus 46%), and was not seen between RA patients and non-RA patients. There was no obvious trend toward C4A deficiency (0–1 allele) in RA, as has been reported for SLE patients (22).

Table 1. Distribution of C4A and C4B gene copy number variations*
 C4A/C4B gene copy number
012345
  • *

    Values are the percent of patients with each number of C4A/C4B gene copies. All subjects were of European descent. No patient had more than 5 copies of either C4A or C4B. RA = rheumatoid arthritis.

  • The Ohio cohort is a separate, unrelated cohort of healthy controls consisting of 389 women with a mean ± SD age of 38.6 ± 11.1 years and 128 men with a mean ± SD age of 34.3 ± 12.1 years (22).

All RA patients (n = 160)1.3/4.419.4/35.646.3/56.329.4/3.12.5/0.61.3/0
 Seropositive RA patients (n = 115)1.7/6.118.3/37.440.0/53.935.7/1.72.6/0.91.7/0
 Seronegative RA patients (n = 45)0/022.2/31.162.2/62.213.3/6.72.2/00/0
Non-RA patients (n = 88)1.1/1.121.6/20.551.1/73.922.7/2.33.4/2.30/0
Healthy controls (n = 51)0/2.017.6/19.666.7/70.615.7/5.90/2.00/0
Ohio cohort (n = 517)0/2.717.3/26.956.3/63.321.6/6.83.3/0.20.4/0
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Figure 1. Distribution patterns of C4, C4A, and C4B gene copy number (GCN) in healthy volunteers (vol), patients with rheumatic diseases other than rheumatoid arthritis (non-RA), and RA patients. Groups were compared by chi-square analysis. Values shown in boldface are significant. The distributions of C4A and C4B were significantly different between the healthy volunteers and the RA patients. A significant difference between the non-RA patients and the RA patients was seen for C4B only.

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In contrast, chi-square analysis of the frequency distribution of C4B gene copy numbers demonstrated significant differences between RA patients and healthy controls (P = 0.0364) and between RA patients and non-RA patients (P = 0.0058). The most apparent difference was the 2-fold increase in C4B homozygous or heterozygous deficiency in RA patients (copy number 0 or 1 in 64 of 160 patients [40%]) relative to non-RA patients and healthy controls (both 21.6%). In the healthy control, non-RA, and RA groups, there were 1, 1, and 7 cases of homozygous deficiency and 10, 18, and 57 cases of heterozygous C4B deficiency, respectively.

Increased frequency of C4B deficiency in seropositive RA patients.

We examined the copy number variations of C4, C4A, and C4B in our RA patient groups as a function of ACPA or RF seropositivity, relative to non-RA patients (Figure 2). Chi-square analysis demonstrated no significant differences in the C4 and C4A allele frequency distributions in seropositive RA patients or seronegative RA patients versus non-RA patients. Interestingly, homozygous or heterozygous C4B deficiency was concentrated in the seropositive RA population; 50 of the 115 seropositive patients (44%) had 0 or 1 copy of the C4B gene in a diploid genome. Of these patients, 7 were completely C4B deficient, while 43 had 1 copy of C4B. Of the 45 seronegative patients, 14 (31%) had C4B deficiency (all had heterozygous C4B deficiency). Chi-square analysis of C4B distribution in the RA patient groups relative to the non-RA patient group showed a statistically significant difference only between the non-RA patients and the seropositive RA patients (P = 0.0018).

thumbnail image

Figure 2. Distribution patterns of C4, C4A, and C4B gene copy number in non-RA patients (n = 88), seropositive RA patients (RAsero[+]) (n = 115), and seronegative RA patients (RAsero[−]) (n = 45). Groups were compared by chi-square analysis. The value shown in boldface is significant. The distribution of C4B was significantly different between the non-RA patients and the seropositive RA patients only. Statistically significant differences for C4A gene copy number were not seen for any of the comparisons, and a significant difference was not observed for C4B gene copy number between the non-RA patients and the seronegative RA patients. See Figure 1 for other definitions.

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OR analysis of the presence of C4B deficiency in seropositive RA.

We examined the odds of having C4B deficiency (0–1 copy) in the non-RA and RA populations relative to healthy controls. While there was no increase for C4A, the odds of having C4B deficiency were significantly increased in the total RA population (OR 2.42 [95% CI 1.16–5.07], P = 0.0187) and seropositive RA population (OR 2.80 [95% CI 1.31–6.00], P = 0.0086), but not in the non-RA or seronegative RA populations.

Using the non-RA population as the control group yielded a slightly stronger relationship. The OR for C4B deficiency in the total RA group was 2.59 (95% CI 1.41–4.76) (P = 0.0019). In the seropositive RA group, the observed OR was 2.99 (95% CI 1.58–5.65) (P = 0.0006). In each of these analyses, there were trends toward an increased OR in the seronegative RA patient population, but these did not reach statistical significance (P = 0.35 and P = 0.20 versus healthy controls and non-RA controls, respectively).

Finally, we examined these relationships using a different and larger Caucasian control cohort, the Ohio control cohort (n = 513) (24) (Table 2). The percentages of men in the Ohio control cohort, the entire RA group, and the seropositive RA group were nearly identical (25%, 27%, and 25%, respectively). This analysis confirmed our finding of statistically significant increases in the frequency of C4B deficiency relative to a distinct control Caucasian population from Ohio.

Table 2.  OR of having C4B isotype deficiency relative to the Ohio control cohort*
Patient groupOR (95% CI)P
  • *

    C4B deficiency refers to having 0 or 1 copy of the C4B gene. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by analysis of 2 × 2 tables, using Fisher's exact test for comparisons. RA = rheumatoid arthritis.

Non-RA patients0.61 (0.35–1.06)0.0953
All RA patients1.58 (1.10–2.29)0.0155
 Seropositive RA patients1.83 (1.21–2.76)0.0056
 Seronegative RA patients1.07 (0.55–2.07)0.87

Association between C4B deficiency and the shared epitope in seropositive RA.

HLA–DRB1 typing demonstrated that 95 of the 113 seropositive RA patients (84%) had the shared epitope (Table 3), consistent with the findings of previous studies (3). Of the 50 seropositive RA patients with C4B deficiencies (0–1 allele), 48 (96%) were shared epitope positive. In the 63 seropositive RA patients with ≥2 C4B alleles, shared epitope positivity was significantly less frequent (present in 46 patients [73%]; P < 0.0009). Of the 44 individuals with heterozygous C4B deficiency who did not have seropositive RA (including healthy controls, non-RA patients, and seronegative RA patients), 24 (54.5%) had the shared epitope. A similar frequency of the shared epitope was found in the combined group of healthy controls and non-RA controls with C4B deficiency (17 of 30 [56.7%]). Thus, the frequency of the shared epitope in subjects with C4B deficiency who did not have seropositive RA was no greater than the frequency of the shared epitope in an unselected general Caucasian population (32, 33).

Table 3. Association of C4B deficiency and presence of the shared epitope in seropositive RA*
 Shared epitope positivityP
  • *

    Values are the number (%) of subjects with the shared epitope. In the non–seropositive rheumatoid arthritis (RA) group, which was made up of healthy volunteers, non-RA patients, and seronegative RA patients, the frequency of the shared epitope was similar to that seen in the general population (32,33). Of 14 seronegative RA patients with C4B deficiency, 7 had the shared epitope, which is consistent with the frequency seen in the general population (32,33).

  • Versus seropositive RA patients with C4B deficiency.

Seropositive RA (n = 113)95 (84) 
 C4B <2 (n = 50)48 (96) 
 C4B ≥2 (n = 63)46 (73)<0.0009
Non–seropositive RA  
 C4B <2 (n = 44)24 (55)<0.0001
Non-RA patients and healthy controls (n = 30)17 (57)<0.0001

The frequency of concurrence of the presence of the shared epitope with C4B deficiency was significantly greater in the seropositive RA patients than in the non–seropositive RA controls (healthy controls, non-RA patients, and seronegative RA patients) (96% versus 54.5%; P < 0.0001). The same level of statistical significance was seen using the combined group of non-RA patients and healthy volunteers as a comparator (P < 0.0001). The almost absolute coexistence of C4B deficiency and the shared epitope in seropositive RA far exceeded the frequency of their associations in the non–seropositive RA and control groups. One probable interpretation for this phenomenon is that C4B deficiency and the shared epitope interact in the induction of seropositive RA.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We demonstrated that the frequency of homozygous and/or heterozygous C4B deficiency was increased in the Caucasian RA patients in this study and was restricted to seropositive RA patients. The frequency of C4B deficiency (0–1 allele) in the seropositive RA patients (44%) was twice that seen in ethnically matched controls and non-RA patients from northern New England, yielding a statistically significant OR, ranging from 2.8 to 2.99. This OR was not influenced by abnormal distributions in our control populations, since the frequencies of C4B deficiency in our healthy control and non-RA patient populations were identical (both 21.6%). Using a different Caucasian control population from Ohio, in which a greater frequency (29.6%) of C4B deficiency was observed, we found that the OR for heterozygous C4B deficiency was significantly greater in both the total RA group (OR 1.58) and seropositive RA group (OR 1.83). No significant differences in total C4 or C4A distribution were seen between the seropositive patients and other groups.

The magnitude of the relationship between C4B deficiency and seropositive RA (OR 1.83) parallels that of the relationship between C4A deficiency and SLE, where a very similar OR (2.024) was seen using the same Ohio controls (22). In each disease, the presence of C4 deficiency might shape disease induction as well as phenotype, given the role of C4 in B cell tolerance (11) in addition to its established activity in immune complex clearance. Immune complex clearance might be particularly important for the association with C4B deficiency, given previous findings suggesting that ACPAs appear prior to the development of RF (8). The different natures of the covalent linkage of C4A and C4B (i.e., amino for C4A versus hydroxyl for C4B) (12, 13, 18) with the target cell/immune complex may play a role in this association. In this regard, certain citrullinated proteins in immune complexes associated with ACPAs may have a reduced availability of amino groups from arginine residues, since certain autoantigens undergo extensive citrullination (34, 35). This would reduce the binding of C4A, potentially leading to a greater dependence on C4B in the process of immune complex clearance. This model would predict a greater role for C4B relative to C4A in maintaining immune tolerance and in the removal of citrullinated antigens (11). Delayed or defective clearance of citrullinated antigens in C4B-deficient subjects would predispose to the generation of ACPAs and subsequent IgM RF induction, leading to RA (7, 8).

The C4A and C4B genes reside in the class III region of the MHC, in which linkage disequilibrium with HLA class II and class I polymorphic variants has been shown to exist. The results of this study raised the possibility that multiple genetic factors in the MHC, e.g., C4B deficiency and haplotypes coding for shared epitopes of HLA–DRB1, confer susceptibility to seropositive RA. HLA–DRB1 typing of the seropositive RA population demonstrated a high frequency (96%) of the shared epitope in patients with C4B deficiency. This association did not appear to be due to linkage disequilibrium, as it was not seen in C4B-deficient individuals without seropositive RA. Rather, these data are consistent with the interpretation that C4B deficiency and the shared epitope interact in the development of seropositive RA. Such an interaction would be consistent with the hypothesis that C4B deficiency favors either the breaking of tolerance to citrullinated antigens or the managing of ACPA immune complexes discussed above. As mentioned above, GWAS are unable to detect C4A and C4B copy number variations (27). Studies to confirm our results will require accurate genotyping of large cohorts of RA patients for C4B gene copy number variation and HLA–DRB1 alleles.

In addition to a potential role in RA pathogenesis, C4B deficiency might shape disease phenotype and severity in RA, potentially through its role in immune complex clearance. In this regard, allotyping demonstrated that 50% of patients with RA and accompanying Felty's syndrome (n = 24) had a C4B-null allele (36). There were no patients with Felty's syndrome in our RA cohort. Ultimately, examination of disease severity and/or phenotype as a function of C4B deficiency in an RA patient cohort will be the necessary first step in addressing this issue.

In conclusion, we report the novel finding that C4B copy number variation exhibits a clear relationship with RA. This copy number variation manifested itself as the presence of C4B deficiency in >40% of the seropositive RA patients in this study but did not appear to result from linkage disequilibrium. This association approximates that seen between C4A copy number variation and SLE (22). Given this frequency and the strength of this relationship, our finding has broad implications for the understanding of RA. Not only might C4B deficiency play a role in disease pathogenesis and phenotype, the functional role of C4B in host defense suggest potential contributions to the safety and efficacy of antibody-based therapies.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Rigby had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Rigby, Wu, Zan, Zhou, Carlson, Hilton, Yu.

Acquisition of data. Rigby, Wu, Zan, Zhou, Carlson, Hilton, Yu.

Analysis and interpretation of data. Rigby, Wu, Zan, Zhou, Rosengren, Carlson, Hilton, Yu.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The technical assistance of Emilie Shipman is acknowledged.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  • 1
    Firestein GS, Budd RC, Harris ED Jr, McInnes IB, Ruddy S, Sergent JS, editors. Kelley's textbook of rheumatology. 8th ed. Philadelphia: Saunders; 2008.
  • 2
    Van der Helm-van Mil AH, Verpoort KN, Breedveld FC, Huizinga TW, Toes RE, de Vries RR. The HLA–DRB1 shared epitope alleles are primarily a risk factor for anti–cyclic citrullinated peptide antibodies and are not an independent risk factor for development of rheumatoid arthritis. Arthritis Rheum 2006; 54: 111721.
  • 3
    Irigoyen P, Lee AT, Wener MH, Li W, Kern M, Batliwalla F, et al. Regulation of anti–cyclic citrullinated peptide antibodies in rheumatoid arthritis: contrasting effects of HLA–DR3 and the shared epitope alleles. Arthritis Rheum 2005; 52: 38138.
  • 4
    Farragher TM, Goodson NJ, Naseem H, Silman AJ, Thomson W, Symmons D, et al. Association of the HLA–DRB1 gene with premature death, particularly from cardiovascular disease, in patients with rheumatoid arthritis and inflammatory polyarthritis. Arthritis Rheum 2008; 58: 35969.
  • 5
    Mewar D, Coote A, Moore DJ, Marinou I, Keyworth J, Dickson MC, et al. Independent associations of anti-cyclic citrullinated peptide antibodies and rheumatoid factor with radiographic severity of rheumatoid arthritis. Arthritis Res Ther 2006; 8: R128.
  • 6
    Feitsma AL, van der Helm-van Mil AH, Huizinga TW, de Vries RR, Toes RE. Protection against rheumatoid arthritis by HLA: nature and nurture. Ann Rheum Dis 2008; 67 Suppl III: iii613.
  • 7
    Winchester R. A golden anniversary: recognition that rheumatoid arthritis sera contain autoantibodies specific for determinants on native IgG molecules. J Immunol 2007; 178: 12278.
  • 8
    Nielen MM, van Schaardenburg D, Reesink HW, van de Stadt RJ, van der Horst-Bruinsma IE, de Koning MH, et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis Rheum 2004; 50: 3806.
  • 9
    Atkinson JP, Yu CY. Genetic susceptibility and class III complement genes. In: Lahita RG, Buyon JP, Koike T, Tsokos GC, editors. Systemic lupus erythematosus. 5th ed. Amsterdam: Elsevier Academic Press; 2011. p. 2145.
  • 10
    Manderson AP, Botto M, Walport MJ. The role of complement in the development of systemic lupus erythematosus. Annu Rev Immunol 2004; 22: 43156.
  • 11
    Carroll MC. The role of complement and complement receptors in induction and regulation of immunity. Annu Rev Immunol 1998; 16: 54568.
  • 12
    Law SK, Dodds AW, Porter RR. A comparison of the properties of two classes, C4A and C4B, of the human complement component C4. EMBO J 1984; 3: 181923.
  • 13
    Isenman DE, Young JR. The molecular basis for the differences in immune hemolysis activity of the Chido and Rodgers isotypes of human complement component C4. J Immunol 1984; 132: 301927.
  • 14
    Yu CY, Belt KT, Giles CM, Campbell RD, Porter RR. Structural basis of the polymorphism of human complement component C4A and C4B: gene size, reactivity and antigenicity. EMBO J 1986; 5: 287381.
  • 15
    Awdeh ZL, Alper CA. Inherited structural polymorphism of the fourth component of human complement. Proc Natl Acad Sci U S A 1980; 77: 357680.
  • 16
    Nonaka M, Nakayama K, Yeul YD, Takahashi M. Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). J Biol Chem 1985; 260: 1093643.
  • 17
    Sepich DS, Noonan DJ, Ogata RT. Complete cDNA sequence of the fourth component of murine complement. Proc Natl Acad Sci U S A 1985; 82: 58959.
  • 18
    Dodds AW, Ren XD, Willis AC, Law SK. The reaction mechanism of the internal thioester in the human complement component C4. Nature 1996; 379: 1779.
  • 19
    McCarroll SA, Altshuler DM. Copy-number variation and association studies of human disease. Nat Genet 2007; 39: S3742.
  • 20
    Craddock N, Hurles ME, Cardin N, Pearson RD, Plagnol V, Robson S, et al. Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls. Nature 2010; 464: 71320.
  • 21
    McCarroll SA. Copy number variation and human genome maps. Nat Genet 2010; 42: 3656.
  • 22
    Yang Y, Chung EK, Wu YL, Savelli SL, Nagaraja HN, Zhou B, et al. Gene copy number variation and associated polymorphisms of complement component C4 in human systemic erythematosus (SLE): low copy number is a risk factor for and high copy number is a protective factor against European American SLE disease susceptibility. Am J Hum Genet 2007; 80: 103754.
  • 23
    Yang Y, Chung EK, Zhou B, Blanchong CA, Yu CY, Fust G, et al. Diversity in intrinsic strengths of the human complement system: serum C4 protein concentrations correlate with C4 gene size and polygenic variations, hemolytic activities and body mass index. J Immunol 2003; 171: 273445.
  • 24
    Wu YL, Yang Y, Chung EK, Zhou B, Kitzmiller KJ, Savelli SL, et al. Phenotypes, genotypes and disease susceptibility associated with gene copy number variations: complement C4 CNVs in European American healthy subjects and those with systemic lupus erythematosus. Cytogenet Genome Res 2008; 123: 13141.
  • 25
    Saxena K, Kitzmiller KJ, Wu YL, Zhou B, Esack N, Hiremath L, et al. Great genotypic and phenotypic diversities associated with copy-number variations of complement C4 and RP-C4-CYP21-TNX (RCCX) modules: a comparison of Asian-Indian and European American populations. Mol Immunol 2009; 46: 1289303.
  • 26
    Blanchong CA, Zhou B, Rupert KL, Chung EK, Jones KN, Sotos JF, et al. Deficiencies of human complement component C4A and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX (RCCX) modules in Caucasians: the load of RCCX genetic diversity on MHC-associated disease. J Exp Med 2000; 191: 218396.
  • 27
    Fernando MM, Boteva L, Morris DL, Zhou B, Wu YL, Lokki ML, et al. Assessment of complement C4 gene copy number using the paralog ratio test. Hum Mutat 2010; 31: 86674.
  • 28
    Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988; 31: 31524.
  • 29
    Chung EK, Wu YL, Yang Y, Zhou B, Yu CY. Human complement components C4A and C4B genetic diversities: complex genotypes and phenotypes. Curr Protoc Immunol 2005; Chapter 13:Unit 13.8.
  • 30
    Chung EK, Yang Y, Rupert KL, Jones KN, Rennebohm RM, Blanchong CA, et al. Determining the one, two, three or four long and short loci of human complement C4 in a major histocompatibility complex haplotype encoding for C4A or C4B proteins. Am J Hum Genet 2002; 71: 81022.
  • 31
    Wu YL, Savelli SL, Yang Y, Zhou B, Rovin BH, Birmingham DJ, et al. Sensitive and specific real-time polymerase chain reaction assays to accurately determine copy-number variations (CNVs) of human complement C4A, C4B, C4-long, C4-short and RCCX modules: elucidation of C4 CNVs in 50 consanguineous subjects with defined HLA genotypes. J Immunol 2007; 179: 301225.
  • 32
    Meyer JM, Evans TI, Small RE, Redford TW, Han J, Singh R, et al. HLA-DRB1 genotype influences risk for and severity of rheumatoid arthritis. J Rheumatol 1999; 26: 102434.
  • 33
    Korendowych E, Dixey J, Cox B, Jones S, McHugh N. The influence of the HLA-DRB1 rheumatoid arthritis shared epitope on the clinical characteristics and radiological outcome of psoriatic arthritis. J Rheumatol 2003; 30: 96101.
  • 34
    Raptopoulou A, Sidiropoulos P, Katsouraki M, Boumpas DT. Anti-citrulline antibodies in the diagnosis and prognosis of rheumatoid arthritis: evolving concepts. Crit Rev Clin Lab Sci 2007; 44: 33963.
  • 35
    Van Beers JJ, Raijmakers R, Alexander LE, Stammen-Vogelzangs J, Lokate AM, Heck AJ, et al. Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance. Arthritis Res Ther 2010; 12: R219.
  • 36
    Clarkson R, Bate AS, Grennan DM, Chattopadhyay C, Sanders P, Davis M, et al. DQw7 and the C4B null allele in rheumatoid arthritis and Felty's syndrome. Ann Rheum Dis 1990; 49: 9769.