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HLA-B27 alleles and Ankylosing Spondylitis in a mediterranean population
76
P406 DIFFERENT HLA-B27 SUBTYPES ASSOCIATE WITH ANKYLOSING SPONDYLITIS AND UNDIFFERENTIATED SPONDYLOARTHROPATHIES 1N ASIAN INDIANS. Kanga U and Mehra NK. All India Institute of Medical Sciences, New Delhi, India. The presence of ankylosing spondylitis (AS) or related spondyloarthropathies (SpA) has been documented in individuals with all molecular subtypes ofHLA-B27. In the present study, 60 HLA B27+ve SSA patients and 17 healthy controls belonging to North India were analyzed to ascertain heterogeneity of the B27 molecule in this population. ID-IEF and PCR-SSOP technologies were used to analyze polymorphism in exon 2 and 3 of the HLA-B27 gene. Four different subtypes viz B*2702, 04,05 and 07 were encountered. Other subtypes i.e. B*2701, 03, 06 and 08 were not encountered. B*2704 (common oriental subtype) and B*2705 (common caucasian subtype) were the most common subtypes in the control and patient groups. B*2704 has not been described in western caucasians in which 90-95% of the B27 gene is represented by B*2705. This finding is important particularly since Asian Indians have ancestral affinity with caucasoids. B*2704 was the predominant subtype in the AS group (70:8o/k) co~pared t.o Its frequency of 47% in healthy controls (RR=2.73) while 10 undifferentiated SpA group, B*2705 occurred most frequently (73%, RR=3.05). B27 subtypes segregated differently in males and females. 12 of the 17 male AS patients carried B*2704 as compared to I of 8 healthy males (X 2=3.9, P<.05). On the other hand, in undifferentiated SpA, B*2705 was significantly raised in female patients (100%) as compared to bealthy females (22.2%, X2=4.9, P<.05). Subtype distribution in indicative of racial admixture in Asian Indian population.
P408
NATURAL LIGAND MOTIFS OF CLOSELY RELATED HLA-DR4 MOLECULES PREDICT FEATURES OF RHEUMATOID ARTHRmS ASSOCIATED PEPTIDES Thomas Friede*, Volker Gnau#, Gunther Jung#, Wieland Keilholz*, Stefan Stevanovic" and Hans-Georg Rammensee* *German Cancer Research Center, Heidelberg, Germany #oepartrnent of Organic Chemistry.University of Tubingen, Germany Rheumatoid arthritis (RA) is strongly associated with certain HLA-DR4 alleles (DRB I *0401, 0404, 0405), whereas DRBI*0402 and 0407 are not. We have analysed the ligand motifs by sequencing of individual ligands and poolsequencing. We were able to resolve sequence information for 70 individual natural ligands (60 of them not reported before). Taken together, we found that the RA-associated DR4 alleles differ from the nonassociated ones in their peptide binding specificity at relative position 4 of the bound peptide. The RA-associated DR4 alleles prefer negative charged amino acids (Asp or Glu). However, the nonassociated ones favour positive charged amino acids (Lys or Arg). Comparing the two most strongly RA-associated DR4 molecules, DRBI*0401 in the Caucasoid population and DRBI*0405 in the Japanese population, we found that DRBI*0405 shows a unique specificity for negative charged amino acids at relative position 9, which is not the case for DRB 1*0401. The large set of natural ligand sequences and the detailed ligand motifs should be helpful for the identification of RAspecific DR4-restricted T-cell eoirones.
P410 PRONOSTIC FACTORS IN RHEUMATOID ARTHRITIS (RA) Eliaou Jean-Francois l, KhIat Myriam-, Babron Marie-Claude.', Sany Jacques", Clot Jacques",Clerget-Darpoux Francoise>, Combe Bernard". ILaboratoire d'Immunologie-INSERM U291 et 4Federation de Rhumatologie-INSERM U291, CHU Montpellier. 2INED, Paris. 3Unite de Genetique-Bpidemiologie, INSERM-U155, Paris, France. Clinical and biological profiles at the onset of RA and HLA typing were retrospectively studied in 2 groups of 94 patients with severe (group I) or limited (group 2) articular damage based on radiological abnormalities. The two groups were matched according to the disease duration (mean: 8.1 yr). In a previous pilot analysis we demonstrated that ESR, CRP and HLA markers were the most relevant independent variables and could indicate the outcome in early RA. Particularly, 96% of group I patients were DRBI*0401, 0404 or 01 as compared to 55% in group 2 and 37% in controls. Genotypes with double dose of RA-susceptible DRB I alleles were detected in 34% group I patients compared to 8.5% in group 2 and 8% in controls. In the present analysis, combining epidemiological, biostatistical and genetic approaches, we first categorized patients according to clinical and biological parameters. We then estimate the genetic risk for each category by comparing the distribution of the HLA genotypes. Using MASC analysis, we propose a model for the impact of the HLA component in the progression of RA. Thus specific forms of RA are associated with particular clinical, biological and genetic profiles, some of these factors being able to Serve as pronostic tools for outcome at the onset of RA.
Abstracts
P407
HLA·B27 ALLELES AND ANKYLOSING SPONDYLITIS MEDITERRANEAN POPULATION
IN A
Reviron 0. 1, Andre M.l, Monlagnie R.',Roudier J.2, Raux H.2, Mercier p.l Laboratoire histocompatibilite, CRTS . Marseille, France. 2: Immuno-Rhumatologie, hopita! Conception. Marseille, France. HLA-827 alleles distributiondiffers among various ethnic groups. To identify the HLA-827 alleles in healthy controls and Ankylosing Spondylitis (AS) patients from Marseille, we used the 12th Workshop 827 PCR-SSOP method improved by a probe labelling using digoxigenin instead of 32P. 49 827 -positive individuals were subtyped. After 827 specific amplificationof exons 2 and 3, the amplified products were hybridized with 11 sequence-specific oligonucleotide probes. Frequencies, Odd Ratio (O.R.) and confidence interval (C.I.) are summarized below' 1:
6'2701 6'2702 6'2703 6'2704 6'2705 6'2706 6'2707 6'2708
11 AS
38 controls
0%
0%
45%
11% 0% 0% 82%
0% 0%
55% 0%
0%
3% 8%
0%
0%
O.R.
C.1.
7,1
1,5 -34,2
8'2702 allele seems to be overrepresented in patients : 45 % versus 11% in controls (p < 0,02). 8'2705 also exists with a substantial frequency in the patients (55 %). At present, more patients and controls are beeing typed and will be compared to 12th Workshop data.
P409 HEAT SHOCK PROTEIN 70 (HSP7D-I) PROMOTER REGION POLYMORPmSM AND RHEUMATOID ARTHRITIS S. Joseph, N.K. Mehra, V. Taneja, S. Quadri, D.P. Singal Department of Pathology, McMaster University, Hamilton, ON, Canada A number of studies have reported associations between HLA-DR4 and RA. These associations are however incomplete, it is likely therefore that additional gene(s) contribute to the development of RA. Since HSPs have been postulated to playa role in autoimmune diseases, we examined the role of HSP70-1 promoter alleles in susceptibility to RA. Genomic DNA, obtained from 90 adult Caucasian (65 European and 25 Asian Indian) patients with seropositive RA and 113 normal (60 European and 53 Asian Indian) healthy subjects, was PCR-amplified for promoter and 5' untranslated regions (-273 to +215) of HSP70-1 gene. Three HSP7()..1 promoter alleles (A,B,C) were identified by typing of PCR-amplified DNA. The prevalence of HSP70-1 promoter allele B was significantly (RR=5.2; p<0.OO8) higher in European patients (21.5%) as compared to normal controls (5.0%). Similar results were obtained in Asian Indians (patients=24%; controls=5.7%; RR=5.3; p<0.018). Further analysis of data demonstrated that HSP7()..1 and DR4 independently contribute to RA, e.g. HSP7()..1 B was significantly (RR=9.2; p<0.OO3) increased in DR4negative patients. The present results from two ethnic groups show that HSP70-1 protein may cause susceptibility to RA.
ssa
P411 HLA-DRB1' GENOTYPES IN GREEK RHEYMATOID ARTHRITIS PATIENTS: ASSOCIATION OF CERTAIN GENOTYPES WITH DISEASE SEVERITY, AGE AT ONSET SEX. 1
C. Stavropqulos-Giokas , M. spy..ropoulou-VlachOu', Kaklamanis 2, A. Unos2, E. Giziaki2, E. Kaklaman.i z
Y. Koumantaki2 ,
I. t
P.
10epartment of Immunology and National Tissue Typing Center, General Hospital of
Athens ~G. Gennlmatas
ft ,
'Department of Hygiene and Epidemiology. Univef$ity of Athens Medical Sct100l and
3Rheumatology Clinic, Evangelismos Gen8fil1 Hospital, Athens. Greece The aim of this study was to examine the association of certain HLA-ORB1· genotypes in Greek RA petients and investigate whether the sl16ngth of association is influenced by sevaral features of the disease, as well as the sex and the age of onset. A case control stUdy was undertaken comparing the frequendes of certain HLA·DRB1' genotypes between 66 Greek RA petients and 130 controls. DNA HLA typing was perfDml8d by PCR-SSO including DR1, DR4 subtyping analysis. HLADR4 and DR10 were significantly Increesed in RA patienls, while HLA-DRl was increased bu1 not slgnlflcently so. The DRB1' alleles "0101. '0401. '0405 and '1001 were associated with higher risk for the deve!Qpment of RA. RA patients cony the predisposing ~shared epi1ope. (E) in a proportion of 64% versus 26.9% of controls. The risk at developing RA. for individuate carrying a single SE allele was 3 times higher and for those canying 2 SE a _ 8.8 _ higher then in SE negetive individuals. The Increased risk was higher in thOO
P406 DIFFERENT HLA-B27 SUBTYPES ASSOCIATE WITH ANKYLOSING SPONDYLITIS AND UNDIFFERENTIATED SPONDYLOARTHROPATHIES 1N ASIAN INDIANS. Kanga U and Mehra NK. All India Institute of Medical Sciences, New Delhi, India. The presence of ankylosing spondylitis (AS) or related spondyloarthropathies (SpA) has been documented in individuals with all molecular subtypes ofHLA-B27. In the present study, 60 HLA B27+ve SSA patients and 17 healthy controls belonging to North India were analyzed to ascertain heterogeneity of the B27 molecule in this population. ID-IEF and PCR-SSOP technologies were used to analyze polymorphism in exon 2 and 3 of the HLA-B27 gene. Four different subtypes viz B*2702, 04,05 and 07 were encountered. Other subtypes i.e. B*2701, 03, 06 and 08 were not encountered. B*2704 (common oriental subtype) and B*2705 (common caucasian subtype) were the most common subtypes in the control and patient groups. B*2704 has not been described in western caucasians in which 90-95% of the B27 gene is represented by B*2705. This finding is important particularly since Asian Indians have ancestral affinity with caucasoids. B*2704 was the predominant subtype in the AS group (70:8o/k) co~pared t.o Its frequency of 47% in healthy controls (RR=2.73) while 10 undifferentiated SpA group, B*2705 occurred most frequently (73%, RR=3.05). B27 subtypes segregated differently in males and females. 12 of the 17 male AS patients carried B*2704 as compared to I of 8 healthy males (X 2=3.9, P<.05). On the other hand, in undifferentiated SpA, B*2705 was significantly raised in female patients (100%) as compared to bealthy females (22.2%, X2=4.9, P<.05). Subtype distribution in indicative of racial admixture in Asian Indian population.
P408
NATURAL LIGAND MOTIFS OF CLOSELY RELATED HLA-DR4 MOLECULES PREDICT FEATURES OF RHEUMATOID ARTHRmS ASSOCIATED PEPTIDES Thomas Friede*, Volker Gnau#, Gunther Jung#, Wieland Keilholz*, Stefan Stevanovic" and Hans-Georg Rammensee* *German Cancer Research Center, Heidelberg, Germany #oepartrnent of Organic Chemistry.University of Tubingen, Germany Rheumatoid arthritis (RA) is strongly associated with certain HLA-DR4 alleles (DRB I *0401, 0404, 0405), whereas DRBI*0402 and 0407 are not. We have analysed the ligand motifs by sequencing of individual ligands and poolsequencing. We were able to resolve sequence information for 70 individual natural ligands (60 of them not reported before). Taken together, we found that the RA-associated DR4 alleles differ from the nonassociated ones in their peptide binding specificity at relative position 4 of the bound peptide. The RA-associated DR4 alleles prefer negative charged amino acids (Asp or Glu). However, the nonassociated ones favour positive charged amino acids (Lys or Arg). Comparing the two most strongly RA-associated DR4 molecules, DRBI*0401 in the Caucasoid population and DRBI*0405 in the Japanese population, we found that DRBI*0405 shows a unique specificity for negative charged amino acids at relative position 9, which is not the case for DRB 1*0401. The large set of natural ligand sequences and the detailed ligand motifs should be helpful for the identification of RAspecific DR4-restricted T-cell eoirones.
P410 PRONOSTIC FACTORS IN RHEUMATOID ARTHRITIS (RA) Eliaou Jean-Francois l, KhIat Myriam-, Babron Marie-Claude.', Sany Jacques", Clot Jacques",Clerget-Darpoux Francoise>, Combe Bernard". ILaboratoire d'Immunologie-INSERM U291 et 4Federation de Rhumatologie-INSERM U291, CHU Montpellier. 2INED, Paris. 3Unite de Genetique-Bpidemiologie, INSERM-U155, Paris, France. Clinical and biological profiles at the onset of RA and HLA typing were retrospectively studied in 2 groups of 94 patients with severe (group I) or limited (group 2) articular damage based on radiological abnormalities. The two groups were matched according to the disease duration (mean: 8.1 yr). In a previous pilot analysis we demonstrated that ESR, CRP and HLA markers were the most relevant independent variables and could indicate the outcome in early RA. Particularly, 96% of group I patients were DRBI*0401, 0404 or 01 as compared to 55% in group 2 and 37% in controls. Genotypes with double dose of RA-susceptible DRB I alleles were detected in 34% group I patients compared to 8.5% in group 2 and 8% in controls. In the present analysis, combining epidemiological, biostatistical and genetic approaches, we first categorized patients according to clinical and biological parameters. We then estimate the genetic risk for each category by comparing the distribution of the HLA genotypes. Using MASC analysis, we propose a model for the impact of the HLA component in the progression of RA. Thus specific forms of RA are associated with particular clinical, biological and genetic profiles, some of these factors being able to Serve as pronostic tools for outcome at the onset of RA.
Abstracts
P407
HLA·B27 ALLELES AND ANKYLOSING SPONDYLITIS MEDITERRANEAN POPULATION
IN A
Reviron 0. 1, Andre M.l, Monlagnie R.',Roudier J.2, Raux H.2, Mercier p.l Laboratoire histocompatibilite, CRTS . Marseille, France. 2: Immuno-Rhumatologie, hopita! Conception. Marseille, France. HLA-827 alleles distributiondiffers among various ethnic groups. To identify the HLA-827 alleles in healthy controls and Ankylosing Spondylitis (AS) patients from Marseille, we used the 12th Workshop 827 PCR-SSOP method improved by a probe labelling using digoxigenin instead of 32P. 49 827 -positive individuals were subtyped. After 827 specific amplificationof exons 2 and 3, the amplified products were hybridized with 11 sequence-specific oligonucleotide probes. Frequencies, Odd Ratio (O.R.) and confidence interval (C.I.) are summarized below' 1:
6'2701 6'2702 6'2703 6'2704 6'2705 6'2706 6'2707 6'2708
11 AS
38 controls
0%
0%
45%
11% 0% 0% 82%
0% 0%
55% 0%
0%
3% 8%
0%
0%
O.R.
C.1.
7,1
1,5 -34,2
8'2702 allele seems to be overrepresented in patients : 45 % versus 11% in controls (p < 0,02). 8'2705 also exists with a substantial frequency in the patients (55 %). At present, more patients and controls are beeing typed and will be compared to 12th Workshop data.
P409 HEAT SHOCK PROTEIN 70 (HSP7D-I) PROMOTER REGION POLYMORPmSM AND RHEUMATOID ARTHRITIS S. Joseph, N.K. Mehra, V. Taneja, S. Quadri, D.P. Singal Department of Pathology, McMaster University, Hamilton, ON, Canada A number of studies have reported associations between HLA-DR4 and RA. These associations are however incomplete, it is likely therefore that additional gene(s) contribute to the development of RA. Since HSPs have been postulated to playa role in autoimmune diseases, we examined the role of HSP70-1 promoter alleles in susceptibility to RA. Genomic DNA, obtained from 90 adult Caucasian (65 European and 25 Asian Indian) patients with seropositive RA and 113 normal (60 European and 53 Asian Indian) healthy subjects, was PCR-amplified for promoter and 5' untranslated regions (-273 to +215) of HSP70-1 gene. Three HSP7()..1 promoter alleles (A,B,C) were identified by typing of PCR-amplified DNA. The prevalence of HSP70-1 promoter allele B was significantly (RR=5.2; p<0.OO8) higher in European patients (21.5%) as compared to normal controls (5.0%). Similar results were obtained in Asian Indians (patients=24%; controls=5.7%; RR=5.3; p<0.018). Further analysis of data demonstrated that HSP7()..1 and DR4 independently contribute to RA, e.g. HSP7()..1 B was significantly (RR=9.2; p<0.OO3) increased in DR4negative patients. The present results from two ethnic groups show that HSP70-1 protein may cause susceptibility to RA.
ssa
P411 HLA-DRB1' GENOTYPES IN GREEK RHEYMATOID ARTHRITIS PATIENTS: ASSOCIATION OF CERTAIN GENOTYPES WITH DISEASE SEVERITY, AGE AT ONSET SEX. 1
C. Stavropqulos-Giokas , M. spy..ropoulou-VlachOu', Kaklamanis 2, A. Unos2, E. Giziaki2, E. Kaklaman.i z
Y. Koumantaki2 ,
I. t
P.
10epartment of Immunology and National Tissue Typing Center, General Hospital of
Athens ~G. Gennlmatas
ft ,
'Department of Hygiene and Epidemiology. Univef$ity of Athens Medical Sct100l and
3Rheumatology Clinic, Evangelismos Gen8fil1 Hospital, Athens. Greece The aim of this study was to examine the association of certain HLA-ORB1· genotypes in Greek RA petients and investigate whether the sl16ngth of association is influenced by sevaral features of the disease, as well as the sex and the age of onset. A case control stUdy was undertaken comparing the frequendes of certain HLA·DRB1' genotypes between 66 Greek RA petients and 130 controls. DNA HLA typing was perfDml8d by PCR-SSO including DR1, DR4 subtyping analysis. HLADR4 and DR10 were significantly Increesed in RA patienls, while HLA-DRl was increased bu1 not slgnlflcently so. The DRB1' alleles "0101. '0401. '0405 and '1001 were associated with higher risk for the deve!Qpment of RA. RA patients cony the predisposing ~shared epi1ope. (E) in a proportion of 64% versus 26.9% of controls. The risk at developing RA. for individuate carrying a single SE allele was 3 times higher and for those canying 2 SE a _ 8.8 _ higher then in SE negetive individuals. The Increased risk was higher in thOO