الفهرس 
Rheumatoid ArthritisOnly 14 pages are availabe for public view
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Abstract
Rheumatoid arthritis (RA) has been associated with a group of human leukocyte antigen (HLA)-DRB1 alleles that share a common amino-acid sequence at residues 70-74 called the shared epitope (SE). Recent studies showed significant association between Anti CCP and RF, as well as between Anti CCP and SE positivity in RA. Shared epitope allele are*0101,*0102, *0401,*0404,*0405,*0408,*1001, and* 1402. Interestingly, there are differences in the strength of the association between different shared epitope alleles and RA.
HLADRB04 alleles represent considerably stronger susceptibility factor than the other SE alleles. More over these alleles are associated with higher level of joint destruction and the HLA*0401 alleles are particularly associated with bone erosions (Xue et al., 2008).
Our aim was to evaluate HLA-DRB1 01, 04 & 10 genotypes in Egyptian RA patients and to determine their relation to the clinical and radiological parameters especially RA severity and activity, and to laboratory parameters especially anti-CCP 3antibodies.
Our study involved 40 RA patients and 20 control subjects.
The RA patients were diagnosed according to the American College of Rheumatology (ACR) revised criteria for the classification of RA (Arnett et al., 1988).
All patients were subjected to the following:
Full medical history taking and thorough clinical examinations.
The disease activity which was assessed using the simplified disease activity index (SDAI) (Smolen et al., 2003).
The clinical disease severity which was assessed using the mechanical joint score (MJS) (Johnson et al.,2002).
The radiological severity which was assessed using the modified sharp/van Der heijde method; also called simple erosion narrowing score (SENS) (Van der heidge et al., 1999).
Laboratory investigations:
Blood was taken from each subject of the study group and serum was separated for the following laboratory investigations according to the manufacturer’s instructions:
C-reactive protein (CRP).
Rheumatoid factor (RF) by nephelometry using Turbox RF-PAIA KIT.
Anticycliccitrullinated peptide antibodies (anti-CCP3) by semiquantitative enzyme-linked immunosorbent assay (ELISA)
The remaining blood was collected on vaccutainer tube containing EDTA and part of it was used for determination of erythrocyte sedimentation rate (ESR) and the rest was divided into 2 eppendorf tubes then stored at -80°C for later HLA-DRB1 genotyping:
HLA-DRB1 Genotyping
DNA extraction: Genomic DNA was extracted from 200 µl of whole blood using QIAamp DNA blood minikits
PCR: HLA-DRB1 alleles (DRB1 01, DRB1 04, DRB1 10) were identified by conventional PCR using specific primers,
Amplification was done using Taq PCR Master Mix Kit supplied by Qiagen.
Agarose gel electrophoresis: 10 µl of each amplified DNA & 1000 bp ladder (molecular weight marker) were separated on 2% agarose gel containing 0.3 µg/ml of ethidiumbromide , photographed & analyzed.
Statistical analysis:
The collected data were computed and statistically analyzed and revealed that
The frequency of HLA-DRB1 04 was found to be significantly higher in the RA patients compared to the normal controls (P< 0.05). Also, our results show that it was significantly associated with RA. However none of the other HLA-DRB1 alleles was significantly higher in the RA group (P >0.05) and they were non significantly associated with RA.
The frequencies of positive anti-CCP antibodies, RF and CRP and ESR >32 were found to be significantly higher in the rheumatoid arthritis patients than in the normal controls (P < 0.001).
Relation of the Studied HLA-DRB1 Alleles Status to different demographic and Clinical Parameters :
(a) HLA-DRB1 01:
Regarding the activity of disease (SDAI), the study revealed that the frequency of positive HLA-DRB1 01 was significantly higher, non significantly higher and significantly lower than negative HLA-DRB1 01 in the severe, moderate and mild activity of RA respectively. It was also revealed that the mean value of the SDAI was significantly higher in the RA patients with positive than with those with negative HLA-DRB1 01. These results on qualitative and quantitative data indicate the association of positive HLA-DRB1 01 with more disease activity.
As regards the clinical severity of disease (MJS), it was found that the mean value of the MJS was significantly higher in the RA patients with positive than with those with negative HLA-DRB1 01. However, when the radiological severity of disease (SENS) was evaluated, it was found that there was non-significant difference in the mean value of SENS.
(b) HLA-DRB1 04:
Similar results to HLA-DRB1 04 were observed as regards disease activity, clinical and radiological severity.
(c) HLA-DRB1 10:
Regarding the activity of disease (SDAI), the study revealed that the frequency of positive compared to the negative HLA-DRB1 10 was non significantly lower and non significantly higher in the severely and moderately active RA respectively and significantly lower in the mildly active RA. It was also revealed that the mean value of the SDAI was non significantly higher in the RA patients with positive than with those with negative HLA-DRB1 10. These results on qualitative and quantitative data indicate no association of positive HLA-DRB1 10 with more severe disease activity and that only negative HLA-DRB1 10 was associated with mildly active RA.
As regards the clinical severity of disease (MJS), similar result to HLA-DRB1 01 and HLA-DRB1 04 was obtained. However, when the radiological severity of disease (SENS) was assessed, it was found that mean value of the SENS was significantly higher in the RA patients with positive than with those with negative HLA-DRB1 10. These data indicate the association of HLA- DRB1 10 with clinical and radiological severe RA disease.
Relation of the Studied HLA-DRB1 Alleles Status To The Different Laboratory Parameters:
Positive HLA-DRB1 04 allele was the only HLA-DRB1 allele that was significantly associated with anti-CCP positive than with anti-CCP negative RA (P< 0.001 with OR>1). Moreover, the presence of one or more of the alleles studied, was significantly associated with anti-CCP positive RA and the absence of HLA-DRB1 alleles were significantly associated with anti-CCP negative RA (P< 0.001). However when we calculated the frequency of one, two or three HLA-DRB1 alleles individually, they were non significantly associated with anti-CCP positive RA. However the OR describing the association of three HLA-DRB1 alleles with anti-CCP positivity (3.0) was higher than that of two HLA-DRB1 alleles (1.0) which was also higher than that of one HLA-DRB1 01 allele (0.5) but this was no significant relation. In addition to these findings, there was a positive significant correlation between the anti-CCP antibodies level and the positive one or more of the alleles tested or positive HLA-DRB1 04, HLA-DRB1 10 but not with positive HLA-DRB1 01.
The frequency of positive HLA-DRB1 alleles (each one individually or two or three) showed no significant difference between seropositive and seronegative RA and the frequency of positive anti–CCP was no significantly lower in the seropositive RA.
Sensitivity, Specificity, PPV and NPV of the Different Laboratory parameters In the Diagnosis of Rheumatoid arthritis were assessed and showed that the best sensitivity and specificity for RA diagnosis were achieved using RF or anti-CCP antibodies for the diagnosis of the disease (considering any one of them positive).
Conclusion
HLA-DRB1 SE+ alleles (*01, *04) were highly expressed in the group of 40 Egyptian RA patients studied.
They were strongly associated with the production of the highly specific anti-CCP 3Ab, which could be involved in the disease process. The presence of high anti-CCP3 Ab titers was associated with more active, aggressive and erosive disease as determined by SDAI for activity, MJS and SENS for clinical and radiological severity respectively.