From its initiation in 1997 and as previously described (4), a rheumatological pre-clinical evaluation and a questionnaire regarding socio-demographic and individual behavior factors are proposed to FDR from RA patients (i.e., kids, siblings, and parents), and such evaluation has been extended to second-degree relatives (SDR: grandparents, aunts, uncles, nieces, and nephews) in multicase RA families. Among them, 144 pre-RA individuals were selected based on two criteria: (i) first, fulfilling the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria at diagnosis (7), and before 2010, a consensus diagnosis was made by three experienced rheumatologists; and (ii) second, an evaluation conducted ≥ 2 years before the onset of RA.

Control groups were composed of familial RA individuals who had not progressed to RA (FDR, n = 328) after ≥ 2 years of follow-up and healthy controls (HC, n = 355). HC included subjects without any signs of chronic disease and no autoinflammatory/autoimmune diseases among first- and second-degree relatives. The study was approved by the Ethical Committee of the Kazan State Medical Academy, Kazan, Russia (Permit no. 1/2002). Consent to conduct studies and to allow publication of the results was received from all the individuals involved in the study according to the legal requirements in Russia.

At the inclusion visit, an evaluation of joint symptoms was performed by a rheumatologist, and the evaluation was completed with magnetic resonance imaging (MRI) in the case of joint symptoms (pain and morning stiffness) in the small joints of the feet and hands. The seven criteria that define CSA were collected from medical records to evaluate CSA status (positivity ≥ 3): arthralgia (≤1 year), metacarpophalangeal (MCP) arthralgia, morning stiffness ≥ 60 min, severe symptoms present in the morning, difficulty with making a fist by testing the strength and the ability to completely close the fist, positive MCP squeeze test, and the presence of FDR with RA (3). In some cases, due to the design of the study that included RA relatives, the “presence of FDR with RA” was not included as the criterion in the CSA score, and this was specified. Before the formulation of the CSA criteria in 2017 and as presented in Table 2, the non-standard assessment of pre-RA activity was performed using 11 criteria that were used a posteriori to evaluate CSA status or were referred to as non-CSA criteria to be included in the analysis (small/large/upper and lower/symmetrical/lower limb arthralgia) (8–12). Moreover, parameters collected at the inclusion included socio-demographic factors (age, sex, rural residence, childlessness, and education level) and individual behavior factors (fish/alcohol/coffee consumption and active/passive/no tobacco smoking). For statistical purposes, education was dichotomized into low educational level (secondary and high school graduates) and high educational level (university graduates). When serum was available at the time of the inclusion, anti-CCP and IgM RF were tested.

HLA typing was performed by DNA sequencing of loci A, B, C, and DQB1 and 2–4 exons of the DRB1 gene using the HLA holotype kit (Omixon, Budapest, Hungary). Briefly, the isolated DNA concentration was estimated using an Implen NP80 NanoPhotomer (Fisher Scientific, Pittsburgh, PA), and long-range PCR was performed next with the resulting amplicons of the five loci. Then, in the process of library preparation, the amplicon pools were fragmented, ends were repaired, and barcodes were ligated. Finally, the sequencing was performed on a MiSeq next-generation sequencing (NGS; Illumina, San Diego, CA) instrument in the pair-terminal reading mode. The readings obtained (251 pb) were analyzed using the HLA twin software (Omixon) with two algorithms in a day-by-day assembly mode and by mapping against the global international ImMunoGeneTics (IMGT) database. The following alleles were considered as shared epitope (SE) alleles: DRB1^*01:01, ^*01:02, ^*04:01, ^*04:04, ^*04:05, ^*04:08, and ^*10:01 (13).

Quantitative results are expressed as the mean and interquartile (IQ) and compared by pairwise analysis. Categorical data were analyzed using Fisher's exact test, and when appropriate, a false discovery rate post-hoc correction was applied, and an odds ratio (OR) with a 95% confidence interval (CI) was determined. All tests and figures were built with Prism 9.4 (GraphPad Software, La Jolla, CA), with the exception of the radar plots (Microsoft Corp, Redmond, WA).

As presented in Table 1, 827 individuals included in this study were subdivided into three groups: pre-RA stage corresponding to 144 individuals having evolved to RA [median: −3 years (IQ: −2/−10 years) from diagnosis]; 328 FDR from RA and without RA evolution after ≥ 2 years follow-up; and 355 HC. Age at inclusion was similar between pre-RA and FDR, while older in HC. The three groups included more women than men, which is related to the design of the Tatarstan cohort that favors the inclusion of women. Pre-RA, FDR, and HC subgroups were similar with regard to ACPA positivity, and RF positivity was lower in HC.

At the RA stage, the 144 pre-RA individuals presented an elevated disease activity score (DAS28-ESR, median = 5.1), a functional impact of the disease (HAQ, median = 1.7), inflammation (ESR ≥ 30 mm/h, 58.2%; and CRP ≥ 5 mg/L, 68.3%), and the detection of ACPA (≥20 U/mL, 75.6%) and/or RF (≥14 IU/mL, 71.6%).

At the early phase of RA (3, 14), a CSA score of ≥3 was effective in distinguishing pre-RA from HC (p < 10⁻⁴). However, to distinguish pre-RA from FDR, the criteria “presence of FDR with RA” had to be removed from the CSA score (p = 0.0005).

Next, to gain further insight into the relevance of disabilities in assessing those familial RA individuals at risk of developing RA, 11 clinical parameters were selected from CSA criteria (n = 6) and from additional arthralgia characteristics (n = 5). As presented in Table 2 and Figures 1A, B, all these parameters, except isolated lower limb arthralgia and pain joints in the upper and lower extremities (black line), were effective in distinguishing pre-RA from HC with an OR ranging from 1.6 to infinity. Among them, significance was conserved when applying a post-hoc correction (red line) except for difficulties with making a fist (0.01 < p < 0.05, blue line).

The same analysis performed between pre-RA and FDR (Figure 1C) retrieved three groups of parameters: (i) those parameters that are ineffective in discriminating pre-RA from FDR (black line), which included MCP/lower limb arthralgia, morning stiffness of ≥60 min, and difficulty with making a fist; (ii) those parameters with low discriminating efficacy and not confirmed following post-hoc adjustment (0.01 < p < 0.05, blue line) such as small joints and a negative squeeze test in MCP joints that reflects local inflammation; and (iii) those parameters that are highly effective after post-hoc adjustment (red line) in discrimination including arthralgia (OR = 1.8, CI: 1.2–2.7), large joint pain (OR = 2.2, CI: 1.4–3.4), upper and lower joint pain (OR = 2.9, CI: 1.7–4.8), symmetrical arthralgia (OR = 4.4, CI: 2.6–7.1), and most severe symptoms occurring in the morning (OR = 3.7, CI: 1.7–8.2). We conclude from such analysis, and based on the radar plot comparison that considers the FDR group according to the CSA score (Figures 1A, D), that the most discriminating clinical criteria to distinguish pre-RA from FDR was symmetrical arthralgia.

Epidemiological studies and more recently the use of Mendelian randomization approaches have confirmed the direct relationship between RA development and environmental factors such as educational level and tobacco smoking (2). Accordingly, socio-demographic factors (rural residence, childlessness, and educational level) and individual behavior factors (fish/alcohol/coffee consumption and active/passive tobacco smoking) were considered (Table 2; Figure 2A). Of note, the rate of inclusion for environmental risk factors ranged from 77.8 to 88.9% in pre-RA, 35.4 to 45.4% in FDR, and 49.9 to 58.0% in HC. When pre-RA individuals were compared to HC (0.01 < p, red line), the risk of RA development was higher among those living in rural areas (OR = 4.2; CI: 2.6–6.9) and lower in those reporting alcohol consumption (OR = 0.6, CI: 0.35–0.90) (Figure 2B).

When replacing HC with FDR in the analysis (Figure 2C), three risk factors were associated with pre-RA individuals: living in a rural environment (OR = 4.1, CI: 2.4–6.7), a lower educational status (OR = 4.4, CI: 2.6–7.5), and passive tobacco smoking (OR = 2.0, CI: 1.15–3.3). On the other hand, alcohol consumption (OR = 0.5, CI: 0.30–0.85) and childlessness (OR = 0.33, CI: 0.17–0.63) were protective, and no associations were retrieved when considering active tobacco usage and fish/coffee consumption. Among these factors, the radar plot analysis further supports prominent roles for educational attainment, rural location, and passive tobacco usage to discriminate pre-RA from FDR (Figure 2D).

High-resolution HLA allele class I (A, B, and C) and class II (DQB1 and DRB1) distribution was assessed by NGS in pre-RA (n = 59), in FDR (n = 50), and HC (n = 78) in order to perform association studies between (i) pre-RA vs. HC; (ii) FDR vs. HC; and (iii) pre-RA vs. FDR (Figure 3).

Alleles with a conserved sequence at amino-acid residues 70–74 in the third hypervariable region of HLA-DRB1 and referred to as SE showed a significant association with both pre-RA individuals (OR = 2.7, CI: 1.3–5.6; p = 0.005) and FDR (OR = 2.8, CI: 1.3–5.7; p = 0.005) when compared to HC. On the other hand, a significant negative association was observed with DRB1^*07:01 when comparing pre-RA relatives with HC (OR = 0.28, CI: 0.12–0.64; p = 0.002) and with B^*35:01 when comparing FDR with HC (OR = 0.23, CI: 0.08–0.67; p = 0.008). As HLA B^*35:01 was retrieved associated with FDR but not with pre-RA individuals, this allele was not considered further.

Finally, to test whether or not clinical and individual RA-associated parameters may be influenced by HLA-DRB1-SE and HLA-DRB1^*07:01, the comparison between pre-RA and FDR was repeated based on the HLA-DRB1 status (Figure 4). Results from such analysis revealed that symmetrical arthralgia (OR = 2.8, CI: 1.2–6.6; p = 0.02) and passive exposure to tobacco smoking (OR = 3.6; CI: 1.18–10.5; p = 0.02) predominated in pre-RA individuals harboring HLA-DRB1-SE, while alcohol consumption (OR = 0.07, CI: 0.01–0.6; p = 0.01) and childlessness (OR = 0.0, CI: 0–0.59; p = 0.01) were protective factors associated with HLA-DRB1^*07:01. The education level attained was retrieved close to the limit of significance for both HLA-DRB1-SE (OR = 2.5, CI: 1.1–5.7; p = 0.04) and HLA-DRB1^*07:01 (OR = 9.8, CI: 1.1–123; p = 0.04), which suggests that the education level represents an independent factor from HLA-DRB1 status.