الفهرس 
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Abstract
Rheumatoid arthritis (RA) is a chronic autoimmune disease that commonly leads to progressive joint damage, impaired function, and progressive disability. Having a family history of RA increases the risk of RA by 3- to 9-fold.
Given the presence of auto-antibodies, such as rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA (tested as anti-cyclic citrullinated peptide (Anti-CCP), which can precede the clinical manifestations of RA by many years, RA is considered an autoimmune disease. Although structural changes, which can be visualized by conventional radiography or other imaging techniques, best distinguish RA from other arthritic disorders, joint damage is rarely apparent in the very early stages of disease, but rather accumulates consistently over time.
Gene-gene and gene-environment interactions are key features in the development of RA and other complex diseases. To date, one of the main pathogenetic hypothesis would be an interaction between the HLA-DRB1-shared epitope alleles (HLA-DRB1*SE) and tobacco smoking as risk factor for the production of anti-citrullinated protein/peptide antibodies (ACPAs), and then the development of RA. Other genetic and environmental factors could play a role in the pathogenesis of RA, as protein tyrosine phosphatase non-receptor 22 (PTPN-22) rs2476601 single nucleotide polymorphism (SNP). This association has been restricted to the rheumatoid factor (RF) and/or the ACPA positive subset.
Musculoskeletal ultrasound (MSUS) is a reliable, cost effective and safe imaging modality used as a complement to other diagnostic methods in rheumatology. It is superior to clinical examination to identify synovitis when there is diagnostic doubt, and can be used to improve the certainty of diagnosis above clinical signs and symptoms alone. Although gray-scale MSUS has an important role in synovitis identification, power Doppler (PD) imaging are more beneficial in identifying active inflammation by detecting hyper-vascularisation and hyperemia in synovial inflammation, so we can use this modality for early detection and identification of inflammatory arthritis in suspected symptomatic subjects.
The gut microbiome is known to influence development of the innate and adaptive immune system, and may also play a role in the development of autoimmunity.
Systemic inflammation and autoimmunity in RA begin long before the onset of detectable joint inflammation. Data suggest that RA-related autoimmunity may be initiated at a mucosal site years before the onset of joint symptoms, as in the oral, lung and gastrointestinal mucosa. Gut microbiota exert a profound effect on the host immune system, both locally and at distant sites. Mechanisms through which the microbiota may be involved in the pathogenesis include altered epithelial and mucosal permeability, loss of immune tolerance to components of the indigenous microbiota, and trafficking of both activated immune cells and antigenic material to the joints. The potential to manipulate the microbiome, by intake of probiotics and fecal microbial transplant (FMT) is recently investigated.
This can be useful to be looked for in first degree relatives of RA patients, early detecting those at a risk for future susceptibly to RA.
Our current study was done to early detect RA among first degree relatives (FDRs) of patients. We evaluated the clinical and laboratory data of the FDRs including serum Anti-CCP, and if there was a genetic role for PTPN22 -rs2476601 gene. We also identified the predominant gut microbiota species and correlated them with clinical, serological and genetic findings. Also, we did musculoskeletal ultrasonography (MSUS) for FDRs for early detection of inflammatory arthritis among them.
This study was conducted on 105 Egyptian subjects recruited from Rheumatology outpatient clinics & Rheumatology unit of Alexandria Main University Hospital. They were divided into three groups: group A: 25 RA patients diagnosed according to 2010 ACR/ EULAR criteria, group B: 40 FDRs of RA patients, and group C: 40 healthy controls, matched for age and sex.
All cases were subjected to the following:
Full history taking with special emphasis on joint symptoms, complete clinical examination, clinical assessment of disease activity in RA patients using SDAI and HAQ.
Laboratory investigations included:
Routine laboratory investigations (CBC, AST, ALT, blood urea and serum creatinine), acute phase reactants (ESR and quantitative CRP) and serological tests: ANA, RF, anti-CCP. PCR for PTPN 22 gene and gut microbiota detection by Matrix-Assisted Laser Desorption-Ionization, Time of Flight, Mass Spectrometry (MALDI-TOF MS).
Imaging included:
Joint ultrasonography using 7 joint scoring system which was conducted on wrist, 2nd MCP, 3rd MCP, 2nd PIP and 3rd PIP joints of the dominant hand and 2nd and 5th MTP joints of foot.
Our study revealed that:
Comparative data analysis of the three studied groups:
There was no statistically significant difference between the three studied groups regarding their age, sex and residence.
14 (35%) out of 40 relatives (group B) were symptomatic, while 21 patients (84%) (group A) had arthralgia ± arthritis. 65% of the first-degree relatives (26 relatives) were asymptomatic, whereas only 4 patients (16%) were asymptomatic.
The mean values of all ESR, CRP, ANA, RF and ANA were higher in group A than group B, and were higher in FDRs than healthy controls (without a statistical difference between group B and C, except for CRP, p=0.001). There was a statistically significant difference in the levels of all serological markers between the different studied groups (P<0.001).
4 patients (16%) out of 25 patients were heterozygous (CT) for PTPN 22 gene, 2 first degree relatives (5%) were heterozygous (CT) and only 1 healthy subject (2.5%) as well.
Coliforms’ variations found in the three studied groups were: E-coli, klebsiella, proteus, morganella, Citrobacter and Enterobacter. There was a statistically significant difference in the number of candidates with normal gut microbiota and those with positive findings between the three studies groups (i.e. between group A and B, between group A and C, and between group B and C) (p<0.001, p1=0.002, p2<0.001, p3=0.001).
The difference in number of coliforms between the three studied groups was statistically significant (p=0.006, p1=0.800, p2=0.006, p3=0.012 ). Only one relative with Bifidobacteria, 4 relatives with Bacteroides (p=0.040), and 7 patients with Prevotella copri (p<0.001) were detected.
There was a statistically significant difference between the number of patients (7) and relatives (3) with Clostridium baratii in their stool samples (p= 0.036), and number of patients (7) and healthy controls (3) with the same variations (p= 0.036). The difference in number of candidates with Clostridium baratii between the three studied groups is statistically significant (p=0.044).
Relations and correlations in group A (RA patients) (n=25):
Four out of 25 RA patients were heterozygous for PTPN22 gene (CT), whereas 21 patients were homozygous (CC). 3 out of 4 heterozygous patients had moderate disease activity according to SDAI, only one patient was highly active. Most of the homozygous patients were moderately active (11), 7 were highly active, and 3 had low activity. None of them was in remission. There was no statistically significant difference between the numbers of patients under the two categories.
Mean value of ANA in heterozygous RA patients (4 patients) (12.50 ± 1.91 u/ml) was significantly higher than the mean value in homozygous patients (21 patients) (8.76 ± 2.0 u/ml) (p=0.007). All heterozygous patients were ANA positive. The mean levels of ESR, CRP, RF and Anti-CCP weren’t significantly different between the two different subgroups. (p>0.05). The 4 heterozygous patients tested high for ESR, CRP and positive for RF, ANA and Anti-CCP.
There was no statistically significant relation between positive stool findings in patients and being symptomatic or not, urban vs rural residence, gene heterozygosity, SDAI. No significant relation was found with disease duration as well.
Nineteen patients out of 25 were found to have positive stool findings in their guts. 14 (73.7%) were high in ESR level, 15 (78.9%) were high in CRP level, 11 (57.9%) had positive RF value and 10 (52.6%) had positive Anti-CCP. The number of patients with positive stool results and high/positive serological tests was greater than the number of patients with normal results, but this difference was statistically non-significant.
There was no statistically significant difference in the mean values of all serological tests done between patients with positive gut microbiota results and those without (p>0.05). Also, there was no statistically significant relation between positive gut microbiota results and gene heterozygosity in patients. Most of patients positive for gut microbiota results (84.2%) were homozygous for the gene.
Relations and correlations in group B (First degree relatives of RA patients) (FDRs) (n=40):
Fourteen (35%) relatives out of 40 were symptomatic. The mean value of all serological tests done for relatives was statistically non-Significant between relatives who complained of some arthralgia, and those who didn’t. (p>0.05)
Regarding the asymptomatic FDRs, 4 of them tested high for ESR, 3 tested high for CRP, only one tested positive for both ANA and RF (the same relative), and 2 tested positive for Anti-CCP.
Twenty-six (65%) out of 40 FDRs did show some findings in their MSUS, 18 of them (69.2%) were asymptomatic, only 8 relatives (30.8%) had arthralgia.
No US findings were detected in 14 relatives, 6 of them (42.9%) were complaining.
There was no statistically significant difference in the results of MSUS between symptomatic and asymptomatic relatives. (p>0.05).
The mean value of total MSUS score in asymptomatic relatives (26 relatives) (2.42 ± 2.67) wasn’t statistically significantly different from the mean value in relatives with arthralgia (14 relatives) (1.79 ± 1.97). (p>0.05)
Right hand was affected in 42.5% of relatives, with RT MCP2 joint was the most affected one (11 relatives) (27.5%), followed by RT. MCP3 joint (7 relatives) (17.5%), then comes the wrist joint with 4 affected relatives (10%). RT PIP2 and PIP3 joints were affected once for each joint (2.5%). Other findings were detected in RT hand US, with one relative having swollen RT. Median nerve, one relative with Tenosynovitis in the LT. wrist joint and one relative with osteophytes in 2nd and 3rd PIPs.
Right Foot was involved in 67.5% of relatives, with Right MTP2 joint affected in 55% of them (22 relatives), while Right MTP5 joint was affected in 9 relatives (22.5%). Aside from joints involved in the 7- ultrasonographic score; Right MTP1 was affected in 27.5% of relatives (11 relatives), Right MTP3 was involved in 4 relatives (10%). Left MTP2 and MTP5 joints were affected once for each joint (2.5% for each joint).
There was no statistically significant correlation between different MSUS scores and ESR, CRP, ANA, Anti-CCP and RF in FDRs.
There were 6 relatives showing high ESR level (>15 mm/hr), and the rest (34 relatives) were normal. The mean value of total MSUS score in relatives with high ESR level (5.33 ± 3.50) was statistically significantly higher than in those with normal ESR (1.65 ± 1.76). (p=0.002)
There were 7 relatives showing high CRP level (>3 mg/L), and the rest (33 relatives) were normal. The mean value of total MSUS score in relatives with high CRP level (2.57 ± 1.99) was not statistically significantly higher than in those with normal CRP (2.12 ± 2.55).
There were 2 relatives with positive ANA (>10 u/ml), and the rest (38 relatives) were negative. The mean value of total MSUS score in relatives with positive ANA (9.0 ± 4.24) was statistically significantly higher than in those with negative result (1.84 ± 1.78). (p=0.018)
There were 3 relatives with positive RF (>6 IU/ml), and the rest (37 relatives) were negative. The mean value of total MSUS score in relatives with positive RF (7.33 ± 4.16) was statistically significantly higher than in those with negative result (1.78 ± 1.77). (p=0.008)
There were 3 relatives with positive Anti-CCP (>5 u/ml), and the rest (37 relatives) were negative. The mean value of total MSUS score in relatives with positive ANTI-CCP (6.0 ± 5.29) was not statistically significantly higher than in those with negative result (1.89 ± 1.90).
Two out of 40 relatives were heterozygous for PCR PTPN 22 Gene. The mean value of ANA level in heterozygous relatives (10.60 ± 0.42) was statistically significantly higher than in those with homozygous PCR (4.94 ± 2.21). (p=0.018). The mean value of Anti-CCP in heterozygous relatives (5.50 ± 0.71 u/ml) was statistically significantly higher than in those with homozygous result (2.16 ± 1.34 u/ml) (p=0.030). There was no statistically significant difference in the levels of ESR, CRP and RF between the two subgroups. All heterozygous relatives tested high for ESR, positive for ANA and RF. Only one of them tested positive for Anti-CCP and was high in CRP level.
Regarding the homozygous relatives (38 relatives), none of them tested positive for ANA, only one tested positive for RF (2.6%), two tested positive for Anti-CCP (5.3%) and six of them showed high CRP level (15.8%). There was a statistically significant difference in the number of relatives under each subgroup regarding high ESR level (p=0.019), positive ANA (p=0.001) and positive RF value (p=0.004).
Both relatives with heterozygous gene PCR showed normal gut microbiota.
The mean value of synovitis GS score in the two heterozygous relatives (8.50 ± 4.95) was statistically significantly higher than the mean value in homozygous ones (1.84 ± 1.78). (p=0.018). Also, the mean value of tenosynovitis GS score in the two heterozygous relatives (0.50 ± 0.71) was statistically significantly higher than the mean value in homozygous ones (0.0 ± 0.0). (p=<0.001). The mean value of total MSUS score in the two heterozygous relatives (9.0 ± 4.24) was statistically significantly higher than the mean value in homozygous ones (1.84 ± 1.78). (p=0.018).
There were 6 relatives with high ESR and 34 with normal level. Mean value of synovitis GS in high ESR relatives (5.17 ± 3.49) was statistically significantly higher than in normal ESR relatives (1.65 ± 1.76). (p=0.002). Synovitis PD, Tenosynovitis PD and Erosions were not detected sonographically in both subgroups. Mean value of Tenosynovitis GS in high ESR relatives (0.17 ± 0.41) was statistically significantly higher than in normal ESR subgroup (0.0 ± 0.0). (p=0.017). Mean value of Total MSUS score in high ESR relatives (5.33 ± 3.50) was statistically significantly higher than in normal ESR subgroup (1.65 ± 1.76). (p=0.002)
Seven relatives tested high for CRP and 33 were normal. Mean value of Tenosynovitis GS in high CRP relatives (0.14 ± 0.38) was statistically significantly higher than in normal CRP subgroup (0.0 ± 0.0). (p=0.030). There was no statistically significant difference in the mean value of synovitis GS, synovitis PD, Tenosynovitis PD, Erosions and total MSUS score between the two studied groups.
Only two relatives tested positive for ANA and 38 were normal. The mean value of synovitis GS in positive ANA relatives (8.50 ± 4.95) was statistically significantly higher than in normal ANA candidates (1.84 ± 1.78). (p=0.018). Mean value of Tenosynovitis GS in positive ANA relatives (0.50 ± 0.71) was statistically significantly higher than in normal ANA subgroup (0.0 ± 0.0). (p=<0.001). Mean value of Total MSUS score in positive ANA subgroup (9.0 ± 4.24) was statistically significantly higher than those with normal ANA (1.84 ± 1.78). (p=0.018). There was no statistically significant difference in the mean value of synovitis PD, Tenosynovitis PD and Erosions between the two studied groups.
Only 3 relatives tested positive for RF and 37 were normal. The mean value of synovitis GS in positive RF relatives (7.0 ± 4.36) was statistically significantly higher than in normal RF candidates (1.78 ± 1.77). (p=0.008). Mean value of Tenosynovitis GS in positive RF relatives (0.33 ± 0.58) was statistically significantly higher than in normal RF subgroup (0.0 ± 0.0). (p=<0.001). Mean value of Total MSUS score in positive RF subgroup (7.33 ± 4.16) was statistically significantly higher than those with normal RF (1.78 ± 1.77). (p=0.008). There was no statistically significant difference in the mean value of synovitis PD, Tenosynovitis PD and Erosions between the two studied groups.
Three relatives tested positive for Anti-CCP and 37 were normal. There was no statistically significant difference in the mean value of synovitis GS, synovitis PD, Tenosynovitis GS, Tenosynovitis PD, Erosions and total MSUS score between the two studied groups.
There was no statistically significant difference in the number of relatives showing normal gut microbiota and those with positive findings, in relation to symptomatology, smoking, residence and gene heterozygosity.
15 relatives out of 40 did have findings in their gut microbiota. One of them (the same one) had high ESR level, positive RF and tested positive for Anti-CCP. Two of the fifteen relatives had high CRP value. There was no statistically significant difference in the number of relatives showing normal gut microbiota and those with positive findings, in relation to high or positive different serological tests done in our study. There was no statistically significant difference in the mean value of all serological tests between relatives with positive results in their gut and those without.
There was no statistically significant difference in the mean value of different MSUS scores between relatives showing normal gut microbiota and those with positive findings.
Relations and correlations in group C (healthy controls) (n=40):
Three healthy subjects out of 40 had Clostridium Baratii in their gut microbiota.
Mean values of ESR and CRP in controls with positive gut microbiota (9.10 ± 1.01 and 2.67 ± 0.29 respectively) were higher than in controls with normal gut flora (8.35 ± 3.68 and 2.31 ± 0.77 respectively), but this difference didn’t reach a statistical significance.
There was no statistically significant difference in the mean values of ANA, RF and Anti-CCP between healthy controls with positive results in their gut and those without.