A Prospective Approach to Investigating the Natural History of Pre-Clinical Rheumatoid Arthritis (RA) Using First-Degree Relatives of Probands with RA

Abstract

Objective

To describe a large, multi-center prospective cohort study of first-degree relatives (FDRs) of probands with rheumatoid arthritis (RA), and outline the utility of such a study in investigating the natural history of RA development.

Methods

1058 FDRs, none of whom met the American College of Rheumatology (ACR) criteria for RA, have been enrolled into a prospective study investigating genetic and environmental influences on the development of RA-related autoimmunity. Demographic, epidemiologic, genetic, autoantibody, and physical examination data from the initial study enrollment visit is described for these FDRs, and the relationship is examined between genetic factors, autoantibodies, inflammation, and joint disease.

Results

Fifty-five percent of FDRs have ≥1 copy of the shared epitope (SE); 20% have ≥1 copy of PTPN22 polymorphism; ~16% are positive for rheumatoid factor (RF, including isotypes), and/or anti-cyclic citrullinated peptide (anti-CCP) antibody. RF-IgM positivity is associated with ≥1 tender joint/s on examination (OR 2.50, 95% CI 1.27 to 4.89, p<0.01), and elevated levels of CRP (OR 5.31, 95% CI 1.45 to 19.52, p = 0.01).

Conclusion

FDRs without RA demonstrate high prevalence of genetic risk factors and RA-related autoantibodies. Additionally, RF association with tender joints and elevated CRP suggests autoantibodies are a valid intermediate marker of RA-related autoimmunity in this cohort. This prospective FDR cohort will be a valuable resource for evaluating the relationship between genetic, epidemiologic factors and the development of RA-related autoimmunity.

Introduction

Rheumatoid arthritis (RA) is a systemic inflammatory disease of unknown etiology characterized by destructive joint disease, significant morbidity and increased mortality (1). There are numerous genetic factors associated with increased risk for RA including HLA alleles containing the shared epitope (SE), PTPN22 polymorphism, and others (2–6). These genetic factors are thought to predispose individuals to the development of autoimmunity, likely due to genetic and environmental interactions (Figure 1). Epidemiologic studies have identified several potential risk factors for disease including cigarette smoke, hormones, and infections, but it is not known at what point during disease evolution these factors are important (7–13). However, elevated autoantibodies, cytokines, and inflammatory markers occur years prior to clinical onset of joint symptoms, suggesting that epidemiologic factors may play an early role in the development of RA (14–18). As such, the investigation of subjects during the pre-clinical stage is important to furthering our understanding of disease pathogenesis.

Figure 1.

Rheumatoid arthritis (RA) is proposed to develop in phases. In phase 1, genetic risk is present. Transition between Phase 1 and Phase 2 may occur due to genetic and environmental interactions. During Phase 2, autoimmunity is present, evidenced by the presence of RA-related autoantibodies. Transition from Phase 2 to Phase 3 may occur due to additional environmental or inflammatory effects leading to target organ injury and the development of symptoms of RA.

Studies of the Etiology of Rheumatoid Arthritis (SERA) - the First-Degree Relative (FDR) cohort

The basis for our study design comes from pre-clinical studies of Type 1 diabetes mellitus (T1DM) at the University of Colorado and elsewhere (19–22). By utilizing autoantibodies as surrogate markers for disease-related autoimmunity, these T1DM studies have identified potential environmental exposures contributing to disease pathogenesis and evolution (19, 20). We have created a prospective cohort using a model similar to that of T1DM studies, with the intent to investigate the natural history of RA development. Our cohort consists of FDRs of probands with RA and is designed to examine the role of genetic and environmental factors in the development of RA-related autoimmunity, and to explore pre-clinical immunological changes within this population. FDRs will be a valuable resource to the research community for following the natural history of pre-clinical RA, as they are 1) enriched with genetic and possibly environmental risk factors for RA, 2) have a higher prevalence of autoantibodies, and 3) have an increased risk for development of RA (23–31). Our discussion herein will provide a detailed description of 1058 FDRs from this unique cohort that have been enrolled as of September 2008.

METHODS

Proband recruitment

Probands with RA are identified from academic centers, Veterans’ hospitals, and private and public sector rheumatology clinics at sites based in New York, Chicago, Omaha (as center of the Rheumatoid Arthritis Investigational Network [RAIN]), Denver, Seattle, and Los Angeles. For inclusion, probands must meet ≥4 ACR classification criteria based on clinical and laboratory findings present on chart review, or have a diagnosis of RA from a board-certified rheumatologist (32). The latter definition is included to prevent exclusion of patients who no longer fulfill the ACR criteria due to well-controlled disease. Additionally, probands must be diagnosed with RA after the age of 16 to avoid inclusion of those with juvenile inflammatory arthritis, which likely has different genetic factors and autoantibody profiles than adult RA. Probands are excluded if no FDRs are alive or interested in participation.

FDR recruitment

For this study the definition of FDR includes parent, full sibling, or offspring of a proband. FDRs are recruited through their probands or through responses to advertising. An FDR is eligible to participate in the study if they do not have a diagnosis of RA and are ≥18 years-old. If an FDR self-reports a diagnosis of RA, their medical record is reviewed and they are excluded if ≥4 ACR classification criteria are met, or if diagnosed with RA by a board certified rheumatologist. Once enrolled, FDRs come to a study site to undergo the initial clinical research visit. At the initial visit, two FDRs were found to have previously undiagnosed RA after evaluation by a study physician and were excluded from entry.

Ethical considerations

Research protocols and consent process for this study are approved by the Institutional Review Boards at each participating site. In addition, an observational study monitoring board (OSMB) has been created to provide oversight for this study. The board consists of an individual with RA, an FDR, a geneticist, an ethicist, and a community-based rheumatologist to assist with ongoing discussion regarding the ethics of obtaining and reporting genetic and autoantibody data.

Proband and FDR data collection

Probands

Each proband is evaluated at a single clinical research visit where the following data is obtained: 1) demographic information, 2) detailed history of RA (including treatment and severity of disease), and 3) blood and urine collection (see below). Probands with verified RA who decline such evaluation are still permitted to contribute FDRs to the cohort.

FDR initial visit

Once enrolled, FDRs are evaluated in a clinical research visit and the following data is obtained: 1) demographic information, 2) medical history including prior diagnoses of autoimmune or infectious diseases and current medications and supplements, 3) epidemiologic questionnaires with assessment of hormonal and environmental exposures, 4) the connective tissue disease screening questionnaire (CSQ): a 30-item questionnaire that can assess for RA or other connective tissue diseases (33), 5) a standardized interview and 68-count joint examination by a trained study physician or nurse, and 6) blood and urine collection for testing for genetic factors, autoantibodies, inflammatory markers, nutritional factors, measurements of oxidative stress, and assessment of other biomarkers. Additionally, samples are stored for future studies. FDRs that cannot come to a study site are evaluated with mailed questionnaires, joint symptoms are ascertained via phone interview, and blood samples are collected at local laboratories.

FDR follow-up

All FDRs are invited for longitudinal follow-up; FDRs that are positive for any RA-related autoantibody are seen annually, and autoantibody negative FDRs are seen every other year. At these follow-up visits, FDRs complete interval-assessment questionnaires, undergo joint interview and examination, and have blood drawn for studies as above. Additionally, FDRs are instructed to contact study personnel if they develop signs/symptoms of RA in the intervening periods. FDRs with an abnormal joint evaluation at their initial or follow-up visit return six weeks later for hand and wrist radiographs, as well as a repeat interview, examination, and blood draw.

Autoantibody testing

Testing for RA-related autoantibodies is performed at the University of Colorado Division of Rheumatology Clinical Research Laboratory (Clinical Laboratory Improvements Amendments [CLIA]-certified). Testing is performed for the RF isotypes IgM, IgG, and IgA by ELISA assays using QUANTA Lite^™ kits, and results are reported in units per milliliter (U/mL). RF is also measured by nephelometry (RF-Neph) according to manufacturer’s specifications (Dade Behring, Newark, Delaware, USA). A positive RF (ELISA isotypes or by nephelometry) is defined as the level present in 5% or less of healthy controls according to ACR RA criteria (32). Cut-offs for RF positivity have been established using 490 randomly selected healthy blood donors from the Denver area.

Antibodies against citrullinated peptides are tested by ELISA using the anti-cyclic citrullinated peptide (anti-CCP)-2 kit (Diastat, Axis-Shield, Dundee, Scotland, UK). Per the manufacturer’s specifications, a positive test is defined as ≥5 U/mL. For all autoantibody assays, 5% of antibody negative samples as well as all positive results are re-tested and confirmed by blinded duplicate analysis.

Genetic testing

Genetic testing in FDRs to date has been limited to the shared epitope (SE) and PTPN22 polymorphism, performed at the Benaroya Research Institute at Virginia Mason, in Seattle, Washington, although DNA and RNA are stored for future analyses. All probands are tested for the SE as well, however PTPN22 testing has been performed only in a limited number of subjects during early enrollment (N = 80), as well as ongoing evaluation at the Seattle site (N = 144). Complete subtyping for HLA-DR4 alleles is done via a modification of a real-time PCR approach, as described previously (34). In addition to the primers and probes described, one additional probe has been added to allow resolution of DRB1*0403 and *0406, allowing for identification of the major DRB1 polymorphisms and accurate resolution of DRB1*0401 to *0421. DR4 subtypes that are considered SE positive include DRB1*0401, 0404, 0405, 0408, 0409, 0410, 0413, 0416, 0419, and 0421. A real-time low resolution PCR analysis is also performed to identify the presence of SE-containing DR1 alleles, including *0101, 0102, 0104, 0105, 0107, 0108, and 0111.

The single nucleotide polymorphism within the PTPN22 gene (1858C→T) is tested for with fluorescently-labeled MGB Eclipse probe (Nanogen), with follow-up PCR utilizing Applied Biosystems 7900HT.

C-reactive protein (CRP)

High-sensitivity CRP testing is performed by nephelometry by the Clinical Translational Research Center (CTRC) at The Children’s Hospital in Denver, Colorado. CRP results are divided into the following categories: 0–3 mg/L, 3–10 mg/L, 10–20 mg/L, and > 20 mg/L.

Data analysis

Cross-sectional analysis has been performed using data obtained at the FDR initial visit (N=1058) from July 2003 through September 2008. The relationship between autoantibodies and 1) genetic factors (SE and PTPN22), 2) joint findings, and 3) CRP have been evaluated using Chi-square test of proportions. Sparse data categories were addressed with Fishers exact testing. For analysis with outcome variables consisting of multiple categories such as the CRP analysis, logistic regression was performed using a reference cell design model (35).

In addition to assessing the relationship between single autoantibodies and various factors listed above, several combinations of antibodies were also evaluated: 1) ‘Any antibody positive’, and 2) ‘≥ two RF isotypes and/or anti-CCP antibody positive’. The latter grouping was decided upon as prior research has suggested that positivity for two or more RF assays and/or anti-CCP antibody is highly specific for RA, and may be predictive of future disease (14, 17, 36–38).

For analysis of association between autoantibodies and joint disease on exam, our definition of joint involvement did not include isolated knee tenderness or swelling given the potential for a non-inflammatory etiology. These subjects were considered not to have an inflammatory arthritis, and were retained in the analysis.

All statistical analyses were performed using SAS 9.2.

RESULTS

Proband characteristics (Table 1)

Table 1.

Characteristics of probands with rheumatoid arthritis (RA), July 2003 – September 2008 (N=674)^*

Proband Data	N (%), except where otherwise indicated
Mean age at diagnosis of RA	45
Sex (female)	556 (82)
Race (NHW)**	559 (83)
Shared epitope present*** (N=642 with testing performed)
≥1 allele	411 (64)
1 allele	296 (46)
2 alleles	115 (18)
PTPN22 polymorphism present (N = 211 with testing performed)****
≥1 allele	54 (25.5)
1 allele	47 (22)
2 alleles	7 (3)
Autoantibody positivity (% total)
Anti-CCP	448 (66.5)
RF-IgM	380 (62.0)
RF-IgG	322 (52.4)
RF-IgA	341 (55.5)
Anti-CCP and any RF assay	406 (60.2)
DMARD therapy (including prednisone)	650 (96.4)
Erosive disease at time of diagnosis of RA (N = 509 with report of radiographs at diagnosis)	253 (49.7)
Mean HAQ score at time of enrollment	0.79

^*All probands met ≥4 American College of Rheumatology criteria for RA or had been diagnosed with RA by a board-certified rheumatologist. 33 probands contributing FDRs to the cohort did not undergo an evaluation and therefore are not included in this table.

^**NHW: non-Hispanic white. Other race categories: African American, American Indian, Asian, Biracial, Hispanic.

^***Alleles considered to contain the shared epitope include: DR4: DRB1*0401, 0404, 0405, 0408, 0409, 0410, 0413, 0416, 0419, and 0421; DR1: *0101, 0102, 0104, 0105, 0107, 0108, and 0111.

^****Testing limited to probands enlisted during early enrollment, and continued at Seattle site only

Abbreviations: CCP=anti-cyclic citrullinated peptide antibody; RF-IgM, IgG, IgA=rheumatoid factor isotypes; DMARD=disease modifiying anti-rheumatic drug therapy.

674 probands with RA are included in the current description, with a mean age at diagnosis of 45 years. Based on laboratory testing done at their enrollment visit, 69.6% were positive for at least 1 RF isotype (IgM, IgG, IgA), 66.5% were positive for anti-CCP, and 60.2% were positive for anti-CCP and RF (by any assay). A significant association exists between the SE and CCP positive RA (OR 3.84, 95% CI: 2.75–5.35; p-value 0.0001), replicating the findings of previous studies (39). Additional information on probands can be found in Table 1.

FDR characteristics (Tables 2 and 3)

Table 2.

First-degree relatives (FDRs) of probands with RA: characteristics at initial study visit, July 2003–September 2008 (N=1058)

FDR Characteristics	N (%) unless otherwise indicated
Study sites
Chicago	4 (0.3)
Denver	388 (36.7)
Los Angeles	248 (23.4)
New York	75 (7.1)
RAIN*	157 (14.8)
Seattle	186 (17.6)
Age
Mean age (standard deviation)	47 (16)
Ages by intervals
18–29	153 (14)
30–39	220 (21)
40–49	246 (23)
50–59	218 (21)
60–79	198 (19)
≥80	21 (2)
Sex (female)	753 (71)
Race (NHW)**	864 (81.7)
Income level
< $20,000/yr	103 (9.7)
$20,000–$75,000/yr	449 (42.5)
> $75,000/yr	421 (39.8)
Missing data	84 (7.9)
Educational level***
High school	182 (17.4)
College	593 (56.6)
Post-graduate	245 (23.4)
Missing data	28 (2.6)
Cigarette use
Non-smoker	607 (58)
0.5–19.5 pack-years	349 (33)
> 20 pack-years	97 (9)
Oral contraceptive, ever use (amongst female FDRs)	619 (81)
FDR type  Siblings  Parents  Offspring	327 (30.9%) 178 (16.8%) 553 (52.3%)
Number of FDRs per family (families N=696)  Families with 1 FDRs per family  Families with 2 FDRs per family  Families with ≥3 FDRs per family	1.5 459 (66.0%) 145 (20.8%) 92 (13.2%)

^*RAIN=Rheumatoid Arthritis Investigators Network, based at the University of Nebraska Medical Center

^**NHW: non-Hispanic white. Other race categories: African American, American Indian, Asian, Biracial, Hispanic.

^***Ever attended; may not indicate completion

Table 3.

FDRs: prevalence of genetic factors, autoantibodies, inflammatory markers, and findings on joint examination, July 2003–September 2008 (N=1058)

FDR characteristics	N (%)
Shared epitope present* (N=982 with testing performed)
≥1 allele	538 (55)
1 allele	451 (46)
2 alleles	87 (9)
PTPN22 polymorphism present (N=980 with testing performed)
≥1 allele	196 (20)
1 allele	183 (19)
2 alleles	13 (1)
Autoantibody positivity
Any autoantibody	167 (15.9)
Anti-CCP and/or ≥2 RF assays	51 (4.9)
Anti-CCP	18 (1.7)
RF-IgM	50 (4.7)
RF-IgG	65 (6.2)
RF-IgA	40 (3.8)
CRP quartiles (reference range 0–3mg/L)
0–3 mg/L	753 (71.2)
3–10 mg/L	250 (23.7)
10–20 mg/L	36 (3.4)
> 20 mg/L	18 (1.7)
Swollen joint count ≥ 1** Excluding knees	120 (14.8) 98 (12.1)
Tender joint count ≥ 1** Excluding knees	203 (24.1) 177 (21.8)

^*Alleles considered to contain the shared epitope include: DR4: DRB1*0401, 0404, 0405, 0408, 0409, 0410, 0413, 0416, 0419, and 0421; DR1: *0101, 0102, 0104, 0105, 0107, 0108, and 0111.

^**PIPs, MCPs, wrists, elbows, knees, ankles, and MTPs

Abbreviations: FDR=first-degree relative (parent, sibling, offspring) of a proband with rheumatoid arthritis; HLA=human leukocyte antigen; RA=rheumatoid arthritis; anti-CCP=anti-cyclic citrullinated peptide antibodies; RF-IgM, IgG, IgA= rheumatoid factor isotypes; CRP=C-reactive protein

1058 FDRs are included in this analysis, with a mean age of 47 years (standard deviation 16). Seventy-one percent are female, and 81.7% are non-Hispanic White. Forty-two percent reported ever smoking, with 9% reporting ≥20 pack-years of use. Of females, 81.0% reported ever use of oral contraceptives.

Of the 1058 FDRs, 813 underwent a joint examination: 14.8% of these FDRs had ≥1 swollen joint, and 24.1% had ≥1 tender joints (Table 3). The remaining subjects were evaluated off-site and therefore had no examination.

Fifty-five percent of FDRs had at least one allele containing the SE, with 9% having 2 copies. PTPN22 polymorphism (1 or 2 copies) was present in 20% of FDRs. In comparison, the general population prevalence of the SE and PTPN22 polymorphism (1 or 2 copies) is 43% and 16% respectively (40).

The presence of any RA-related autoantibody (RF assays and/or anti-CCP) was present in 15.9% of FDRs, with 1.7% positive for anti-CCP. Further analysis of FDR autoantibody positivity is reported in Table 3.

Association between autoantibodies and examination and CRP results (Table 4)

Table 4.

RA-related autoantibody associations with clinical findings in FDRs, July 2003–September 2008

Clinical Parameter (outcome)	Selected Definitions of RA-Related Autoimmunity (OR, 95% CI)
	Any Autoantibody** (N=167))	Anti-CCP (N=18)	RF-IgM (N=50)	RF-IgG (N=65)	RF-IgA (N=40)
CRP (mg/L)
0–3	1.00 (ref)	1.00	1.00	1.00	1.00
3–10	1.32 (0.91–1.92)	0.93 (0.3–2.88)	1.70 (0.91–3.19)	1.24 (0.7–2.19)	1.93 (1.00–3.75)
10–20	0.91 (0.35–2.4)	1.63 (0.21–12.79)	1.46 (0.33–6.37)	0.94 (0.22–4.02)	0.86 (0.11–6.56)
> 20	1.22 (0.34–4.30)	***	5.31 (1.45–19.52)	0.99 (0.13–7.67)	***
≥ 1 swollen joint* (N=98)	1.42 (0.84–2.43)	0.56 (0.07–4.30)	0.87 (0.3–2.51)	1.14 (0.5–2.60)	1.51 (0.57–4.05)
≥ 1 tender joint* (N=177)	1.29 (0.83–1.99)	0.59 (0.13–2.68)	2.50 (1.27–4.89)	0.84 (0.41–1.70)	0.90 (0.36–2.24)

^*after exclusion of isolated knee involvement

^**Any autoantibody includes: Any RF assay and/or anti-CCP.

^***for this cell, no subjects had these outcomes.

Abbreviations: CCP=anti-cyclic citrullinated peptide antibody; CRP=C-reactive protein; ACR criteria=American College of Rheumatology 1987 Revised Criteria for the classification of RA, Arnett FC et al, 1988.

In univariate analysis, there were no significant associations between autoantibody positivity and age, sex, or race. However, in univariate analysis, RF-IgM positivity was significantly associated with joint tenderness (p = 0.0078), and CRP levels >20 mg/L (p = 0.012). Additional results are reported in Table 4.

Association between genetic factors and autoantibodies (Table 5)

Table 5.

RA-related autoantibody association with the presence of the shared epitope and PTPN22 polymorphism in FDRs, July 2003–September 2008

RA-related Autoantibodies	N	SE ≥1 alleles (OR, 95% CI)	N	PTPN22 ≥1 alleles (OR, 95% CI)
Anti-CCP	11	1.83 (0.58–6.78)	5	1.99 (0.58–2.67)
RF-IgM	22	0.81 (0.42–1.56)	6	0.63 (0.21–1.54)
RF-IgG	27	0.73 (0.41–1.29)	12	1.06 (0.5–2.09)
RF-IgA	22	1.07 (0.53–2.17)	9	1.29 (0.52–2.86)
Any Autoantibody*	85	1.04 (0.72–1.49)	30	1.00 (0.63–1.57)

^*Any autoantibody = positivity for 1 or more of the following: RF (any assay), and anti-CCP.

Abbreviations: Anti-CCP=anti-cyclic citrullinated peptide antibody; RF-IgM, IgG, IgA=rheumatoid factor isotypes

No significant association between autoantibodies and presence of the SE or PTPN22 polymorphism was present in this interim analysis (Table 5).

DISCUSSION

Evaluation of the pre-clinical phase is crucial to a more thorough understanding of RA development. Epidemiologic research thus far has been largely retrospective, and therefore unable to identify the timing of the effects of etiologic factors during the prolonged phases of early disease development. In addition, many epidemiologic studies have been performed using subjects with established disease; therefore, the effects from recall bias, chronic immunological changes associated with advanced disease, and interval treatment are unknown. The utility of a pre-clinical cohort to address these issues has been successful in fields such as T1DM, and we believe that using a similar methodology in the study of RA development will result in higher quality data collection and a more complete understanding of early immunological changes.

The pre-clinical period of RA can be difficult to study prospectively in general populations due to the low prevalence of disease (~1%). In order to increase our yield for identifying individuals with pre-clinical RA we have targeted a cohort with a higher risk for disease based on family history, likely due to enrichment of genetic and environmental risk factors. Due to these factors, we expect autoantibody positivity and seroconversion rates as well as development of incident joint disease to be higher in FDRs than in the general population, increasing the yield of study end points. Supporting this, the prevalence of ≥1 allele containing the SE or PTPN22 polymorphism in FDRs (55% and 20%, respectively) is higher than that reported in North American historical controls (43% and 16%, respectively) (40). In addition, the prevalence of anti-CCP antibody positivity in Denver FDRs is 1.3%, compared to a control prevalence of 0% amongst 182 Denver blood donors. Of great interest are FDRs in this cohort with positivity for 2 or more RF isotypes and/or anti-CCP, who based on prior literature are likely to develop symptomatic RA in the future (14, 17).

We plan to continue our study of this cohort using autoantibody positivity as an intermediate outcome in the development of RA. Existing data supports the relationship of pre-clinical RA-related autoantibodies to the development of future disease, especially in relatives of probands with RA (14, 15, 17, 23). Furthermore, the association of RF-IgM with tender joints and CRP elevations reported here supports the biologic relevance of autoantibody positivity in subjects without overt RA. We must be careful, however, to not overlook the importance of antibody positive subjects without signs of joint disease or inflammation. Potential genetic and environmental factors associated with antibody formation may be easier to elucidate from this population compared to retrospective cohorts. Of note, our group has already demonstrated several interesting associations with RF positivity amongst asymptomatic cohorts: 1) a negative association with oral contraceptive use, 2) a positive association with cigarette exposure, and 3) lack of an association with vitamin D levels (41, 42).

In addition to epidemiologic analysis, the FDR cohort provides us the opportunity to study in detail individuals with high-risk autoantibody profiles for RA, and potential discovery of additional biomarkers, pathogenic mechanisms, and sites of inflammation important to the development of RA. For example, in preliminary studies we have shown an association between autoantibody positivity and elevations of several cytokines in members of our cohort, as well as identifying a potential link between autoantibody positivity and asymptomatic lung disease (43, 44). As we continue to move forward the prospective, longitudinal design of our study will prove to be its greatest asset. Data collection is ongoing with 237 FDRs to date seen in follow-up visits (mean follow-up 2.5 years, range 7 months to 5 years). Study personnel have maintained contact with research participants, with a follow-up rate of 84.5% across study sites. Four FDRs have developed incident RA from this cohort in ~600 person-years of follow-up (~6.7 cases per 1000-person years), similar to incidence rates for RA in previously evaluated FDR cohorts (23).

Study limitations

Our proband source is not derived from a true population-based sample, and as such, there may be concerns as to the applicability to the general RA population. However, comparison with other cohorts in the literature reveals that our probands are similar to the overall RA population in terms of age at diagnosis, proportion of females, prevalence of erosive disease, and rates of antibody and genetic marker positivity (6, 45–48). In addition, there is a possibility that FDRs enrolled in our cohort are healthier, on average, than the general FDR population by virtue of their participation in the study. Alternatively, it is possible that FDRs have chosen to enter the study based on existing joint complaints or concern for undiagnosed RA. We recognize that both of these issues represent potential participation bias, but the inability to collect information on non-enrolled family members prevents systematic comparison with our FDRs. While this may potentially affect the external validity of our results, we believe our study design, specifically the creation of an internal control population derived from the same pool of FDRs as our cases, will provide excellent internal validity and therefore high quality data.

Interestingly, RF-IgM positivity is associated with CRP elevation and joint tenderness despite being less prevalent than RF-IgG. This may be due to the biologic activity of RF-IgM in its ability to form immune complexes more readily than RF-IgG. Alternatively, as RF-IgM may be less specific for RA its association with CRP elevation and joint tenderness may reflect another inflammatory condition not detected in our evaluation (38). Two RF-positive FDRs reported concurrent hepatitis C, however neither had the RF-IgM isotype.

Our study is limited at this interim analysis by lack of power for several association studies between autoantibodies and clinical outcomes, as well as between the SE or PTPN22 polymorphism. This will need further exploration when the cohort reaches full enrollment (goal FDRs 2100 by 2010).

CONCLUSION

Pre-clinical studies are crucial to understanding the relationship between genetic and environmental factors in the development of RA. This FDR cohort is a valuable resource for such studies, allowing for 1) high quality prospective data collection, and 2) genetic and epidemiologic investigations utilizing autoantibody positivity as an intermediate outcome. The association of RF positivity with abnormal joint examination and elevated CRP lends validity to the hypothesis that autoantibodies represent part of a continuum of immunological changes leading to the development of symptomatic RA.