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High resolution DRB1 typing by Sequencing Based Typing in 77 DR4 positive Rheumatoid Arthritis patients
77
Abstracts
P412
HLA-DRBI ALLELES AND RISK OF VASCULmS IN RHEUMATOID ARTHRITIS; GENETIC HETEROGENEITY BETWEEN CLINICAL SUBGROUPS OF VASCULITIS. Voskuyl AE, Hazes JMW, Verduyn W, Schreuder GMTh, Vries RRP de, Breedveld FC. Depts 01 Rheumalology, and Immunohematology and Bloodbank, University Hospjtal Leiden, The Netherlands A case control study was designed to study the distributionof HLA-DRBI
allelesina group of 31 rheumatoid vasculitisIRV) patients and comparison with a group or 76 RA patients without vasculttis. More specifically we studied the distribution of ORB1*04 subtypes, and of the shared epitope (SE) formed by M 70-74 in DRB! exon 2, which is shared among DR1 , DR10 and most DR4 molecules. HLA-DRBI typing of all patients was performed by PCR..sSO method. High resolution typing was used for DRB1*Dl and DRB1*04 subtyping. Overall, no associationswere found between the SE, DRB1*04 alleles and AV. The risk of developing RV with minor skin vasculitis (i.e. purpura or pe1echiae) was increased in patients wtth DRB1*D401 {Odds ralio(OR) = 14.1 (95% CI:l.6·625)} and DRB1*04 homozygous palients {OR=20.0 (95%CI: 1.6-970)}. Nosuch associations were found for patients wtthmajor organ lesions. Those data are suggestive for gene1icheterogeneity among the clinical subgroups of RV.
P414
P413 HLA ASSOCIATIONS
IN PATIENTS WITH LATE ONSET RHEUMATOID
:.~::.~, K BoIsf, K Tarass ll, A. Drossos3, C. papasteri.de.', H.M. Moutsop oulos".
1. Dept of Immunology - Histocompatibility, 'Evangelismos' Hosptal, Athens - Greece . 2. Dept of Pathophysiology, School of Medicine, National University of Athens. 3. Dept of InternalMedicine, loannina Medical School, Greece, Patients with late onset (after age 60 yrs) rheumatoid arthritis (LO-RA) compose a unique SUbgroup. which has clinical features and a clinical course distinct from thai generally observed in younger RA patients. Previous studies in Greeks have been revealed associations of RA with HLA-ORB1· 0405 as weD as with DR1 and DR10. The aim of the present study was to assess the HLA correlations with lo-RA
PW::;~';;.,;ng g roups of oubjecm were studied : a) 46 LC-RA patients (24 males, 22 females), b) 105 healthy individuals (50 males and 55 females ) age d 18-65
In o ur
years (co ntrols I), c) 171 healthy individuals (57 males and 114 females ) aged > 75 years (contro ls II). HLA class I Md II typing was performed using dassical ....oIogicai
technlq.Jes.
Resultl: A) In compa rison to contro ls I: aJ increased hquency of HLA-A1 (38.88% vs 12.38%, x' ~ 7. 1 00, p<'-4.125, p <'=6.616, p<0.Q25, RR=3.54) and b) decreased frequency of -DR7 )12.78% vs 38.33%, x' - 8.720, p
P415
ASSOCIATION OF RHEUMATOID ARTHRITIS Wl11I HLADRBl*0401 Al"'D DRBl*0408 ALLELES BUT NOT Wl11I THE
BIGB R1!SOLlITION ORBI TYPING BY SBQUENCING BASED TYPING IN 77 0R4 POSITIVE RHEUMATOID ARTIIIlITIS PATIENTS
DQBl*Q301
Daisy d e Bruyn, Christ ien Voo rter, Jud ith Lie and El l a v an d en
ALLELEIN GERMAN CAUCASIANS
Berg-Loo nen
Boehm Bernhard 0 I), Loeliger Cornelius 2) , Kuehn! Pele r 2) , Manfras Burkhard I), Weiss Uta I) , Scherbaura Werner I) I) Department of Internal Medicine, Uni versity of VIm, UIm, Germany, 2) Department of Transplantation Immunology , University of Hamburg , Hamburg Germany. The association of HLA-DRBI"'04 alleles and the corresponding HLADQB I alleles was analyzed in 134 patients with rheumatoid arthritis (RA) and in 92 HLA~DRB I *04 positive healthy controls from a homogenous population in Germany . The gece frequency (gl) of the HLA-DRBI "'040t all ele was 0.68 in RA versus 0.6 in co ntrols. Gf of the DRBI"'0404 all ele was found be significantly decreased (0. 03 versus 0.1 ; p
P416
Tissue Typi ng Lab oratory, Un i v e r s i t y Maastricht, The Netherlands
Hos p ital
aaaar r-Lcht ,
Accurate allele assiI;Jnment for HLA-DRBI alleles is ra ther
~~~~~exi.tTh~n~~~~~n~fl:J.fJ:f;r~f;let~st;m{~OW;~2 q~~;z;~ a~~ necessary SSP reactions for high resolution typing. In a blind study on 77 serologically DR4-positive Rheumatoid Arthritis patient s we investigated ORBl high resolut i on Sequencing Based Typing a n d comp ared th is to the typing obtained by peR-SSP. By SBT b oth DRBl a lleles could be typed for unambiguou sl y in
~io~u~a~fn:~:9a::rlye~·0 Fi:e~fi~;mi~:Bi~Jt:t~u:r~tf~~eC:::P~~~i~~;
samples no u nique a ll el e pat.t. ecn cou l d be o b t a i n e d . ThesQ s amp l e s we re hete ro zy g ous f o r t wo ORBI *04 alleles and i nv olv ed t wo DRB! *04 c ombinat i ons ; DRBl*0 40 1, *04 0 2 whi c h could not be distingU ished from DRB! -04 13,*0414 and DRB1-0401 , -0404 wh i ch c ould not be d istingui s hed fr om DRBI- 0408, -04 13. These a ll e l e comb ina t ions coul d a ls o not be di st i ng u i s he d by PCR-SSP . By SS ~
~~i~;048a:fleete.~~e:~~n:het~~:ib~~~:~~~~a~~~~~U~rea6e~~~o~1
DRBI *0408 next to DRBI-0401 cou l d not be de t.erm f ned , By 581' these patients were type d tru e l y homcayqc ua for the oRBI *0401 allele. High resolution DRBI t yping by SBT is achieved for 71 o ut of 77 seroloCii cally DR4-pos i tive p a t i e nt s samples, demonstrat i nq its c a pa.c.l.t y as a typing technique. Typi n9 was performed b y
(6~~~ ~~~~~~i:e~;r~N~o~~aU~~C~eg ~~B~*~~~lm~~~i:ilvf:,XPf:S(i6~~
were DRB1"'0404, 6 (8\) were DRBI *0405 and 2 ( 3\) was DRB! *0408 .
-
~~~mu~ i:~~ ~~e~~t:~~~ig~l ie1:t~~~~n;~~ o~~q~i~ieo~~~t~~n:e: ~f~
ved afte r furthe r opt i misat ion of the seque nce pr imers u s e d.
P417
GENEITCSUSCEPTIBILITY TO RHEUMATOID ARTHRfTIS : STIJDYOF HLA-DRA/I,'D TNF LOCI USINGTHE MASC METHOD
ROLE OF HJA·DR·DR AND DR-DP INTERACTIONS GENETIC SUSCEPTIBILITY TO RHEUMATOID ARTHRmS
Genin Emmanuellel, BabronMarie-Claude1, McDennott Michael2, Mulcahy Brian2, Waldron-LynchFrank2, Adams ClaW, Shanahan Fergus2, Molloy Michael-, O'Gara Fergal-,
Perdriger Alethl , Guggenbuhl PascalI, Chales Gerard', Le Dantee Philippe', Yaouang Jacqueline'', Genetet Bernard' , Pawlotsky Yves', Semana Gilbere 1 - Rhumatologie, CHRU. 2 - Laboratoire Universitaire d'Immunologie, ETS Bretagne-Est.3 - BpidemiologieCHRU, Rennes, France
Clerget-Darpoux Francoise! INSERM U155, Paris, France! ; University College, Cork, Ireland2 We have investigated the HLA component involved in rheumatoid arthritis (RA) susceptibility with the MASC method. The informationon the distribution of the marker genotypes in unrelatedaffectedindex cases and of haplotypesharing with one affected sib is used simultaneously. A sample of 50 families each having at least two affected sibs and typed for the HLA-DR and TNF loci was available. Strong associations were observed between alleles of these two markers and RA. The MASC method was successively applied to data from these two markers. We tested the involvement of a locus closely linked to the marker versus direct involvementof the marker locus itself. We' also tested the shared epitope hypothesis which assumes the involvement of amino acid sequences in position 70-74 on the 1\ chain of DR molecules in RA susceptibility. Finally we used information simultaneously on both ALA-DR and TNI' markers in order to determine if the TNF marker providedevidence for a susceptibilitylocus in additionto HLA-OR.
IN
10 order to aoalyse the relationship between DR and DP loci in the genetic susceptibility to rheumatoid arthritis (RA), ALA-ORB1 and DPBl polymorphism was studied in 155 RA patieots compared to 150 cootrols. Our data were consistent with the involvementof amino-acid at position 71 of the DRBl chain in the disease susceptibility.The higher risk for RA was observed in subjects who carried both a lysine (K) and an arginine(R) at position 71 : 21.9 % of patieots carried such an heterozygosity (KIR) compared to 0.6 % of controls (pc < ]0-6, OR = 42). In the abseoce of arginine, the presence of lysine was still associated with the disease (33 % vs 19 %, pc < 0.003. OR 2). On another hand, the analysis of the HLAOPBIlocus showed that the DPBl *0401 allele frequency was significantly increased in the RA patient group wbo expressed only arginine at position 71 (0 = 47) (82 % vs 56 % in controls, p < 0.008). 10 conclusion, our results strongly suggest that HLA-DR-DR and HLA-DR-DP interactions may play an important role in the genetic susceptibility to RA
=
Abstracts
P412
HLA-DRBI ALLELES AND RISK OF VASCULmS IN RHEUMATOID ARTHRITIS; GENETIC HETEROGENEITY BETWEEN CLINICAL SUBGROUPS OF VASCULITIS. Voskuyl AE, Hazes JMW, Verduyn W, Schreuder GMTh, Vries RRP de, Breedveld FC. Depts 01 Rheumalology, and Immunohematology and Bloodbank, University Hospjtal Leiden, The Netherlands A case control study was designed to study the distributionof HLA-DRBI
allelesina group of 31 rheumatoid vasculitisIRV) patients and comparison with a group or 76 RA patients without vasculttis. More specifically we studied the distribution of ORB1*04 subtypes, and of the shared epitope (SE) formed by M 70-74 in DRB! exon 2, which is shared among DR1 , DR10 and most DR4 molecules. HLA-DRBI typing of all patients was performed by PCR..sSO method. High resolution typing was used for DRB1*Dl and DRB1*04 subtyping. Overall, no associationswere found between the SE, DRB1*04 alleles and AV. The risk of developing RV with minor skin vasculitis (i.e. purpura or pe1echiae) was increased in patients wtth DRB1*D401 {Odds ralio(OR) = 14.1 (95% CI:l.6·625)} and DRB1*04 homozygous palients {OR=20.0 (95%CI: 1.6-970)}. Nosuch associations were found for patients wtthmajor organ lesions. Those data are suggestive for gene1icheterogeneity among the clinical subgroups of RV.
P414
P413 HLA ASSOCIATIONS
IN PATIENTS WITH LATE ONSET RHEUMATOID
:.~::.~, K BoIsf, K Tarass ll, A. Drossos3, C. papasteri.de.', H.M. Moutsop oulos".
1. Dept of Immunology - Histocompatibility, 'Evangelismos' Hosptal, Athens - Greece . 2. Dept of Pathophysiology, School of Medicine, National University of Athens. 3. Dept of InternalMedicine, loannina Medical School, Greece, Patients with late onset (after age 60 yrs) rheumatoid arthritis (LO-RA) compose a unique SUbgroup. which has clinical features and a clinical course distinct from thai generally observed in younger RA patients. Previous studies in Greeks have been revealed associations of RA with HLA-ORB1· 0405 as weD as with DR1 and DR10. The aim of the present study was to assess the HLA correlations with lo-RA
PW::;~';;.,;ng g roups of oubjecm were studied : a) 46 LC-RA patients (24 males, 22 females), b) 105 healthy individuals (50 males and 55 females ) age d 18-65
In o ur
years (co ntrols I), c) 171 healthy individuals (57 males and 114 females ) aged > 75 years (contro ls II). HLA class I Md II typing was performed using dassical ....oIogicai
technlq.Jes.
Resultl: A) In compa rison to contro ls I: aJ increased hquency of HLA-A1 (38.88% vs 12.38%, x' ~ 7. 1 00, p
P415
ASSOCIATION OF RHEUMATOID ARTHRITIS Wl11I HLADRBl*0401 Al"'D DRBl*0408 ALLELES BUT NOT Wl11I THE
BIGB R1!SOLlITION ORBI TYPING BY SBQUENCING BASED TYPING IN 77 0R4 POSITIVE RHEUMATOID ARTIIIlITIS PATIENTS
DQBl*Q301
Daisy d e Bruyn, Christ ien Voo rter, Jud ith Lie and El l a v an d en
ALLELEIN GERMAN CAUCASIANS
Berg-Loo nen
Boehm Bernhard 0 I), Loeliger Cornelius 2) , Kuehn! Pele r 2) , Manfras Burkhard I), Weiss Uta I) , Scherbaura Werner I) I) Department of Internal Medicine, Uni versity of VIm, UIm, Germany, 2) Department of Transplantation Immunology , University of Hamburg , Hamburg Germany. The association of HLA-DRBI"'04 alleles and the corresponding HLADQB I alleles was analyzed in 134 patients with rheumatoid arthritis (RA) and in 92 HLA~DRB I *04 positive healthy controls from a homogenous population in Germany . The gece frequency (gl) of the HLA-DRBI "'040t all ele was 0.68 in RA versus 0.6 in co ntrols. Gf of the DRBI"'0404 all ele was found be significantly decreased (0. 03 versus 0.1 ; p
P416
Tissue Typi ng Lab oratory, Un i v e r s i t y Maastricht, The Netherlands
Hos p ital
aaaar r-Lcht ,
Accurate allele assiI;Jnment for HLA-DRBI alleles is ra ther
~~~~~exi.tTh~n~~~~~n~fl:J.fJ:f;r~f;let~st;m{~OW;~2 q~~;z;~ a~~ necessary SSP reactions for high resolution typing. In a blind study on 77 serologically DR4-positive Rheumatoid Arthritis patient s we investigated ORBl high resolut i on Sequencing Based Typing a n d comp ared th is to the typing obtained by peR-SSP. By SBT b oth DRBl a lleles could be typed for unambiguou sl y in
~io~u~a~fn:~:9a::rlye~·0 Fi:e~fi~;mi~:Bi~Jt:t~u:r~tf~~eC:::P~~~i~~;
samples no u nique a ll el e pat.t. ecn cou l d be o b t a i n e d . ThesQ s amp l e s we re hete ro zy g ous f o r t wo ORBI *04 alleles and i nv olv ed t wo DRB! *04 c ombinat i ons ; DRBl*0 40 1, *04 0 2 whi c h could not be distingU ished from DRB! -04 13,*0414 and DRB1-0401 , -0404 wh i ch c ould not be d istingui s hed fr om DRBI- 0408, -04 13. These a ll e l e comb ina t ions coul d a ls o not be di st i ng u i s he d by PCR-SSP . By SS ~
~~i~;048a:fleete.~~e:~~n:het~~:ib~~~:~~~~a~~~~~U~rea6e~~~o~1
DRBI *0408 next to DRBI-0401 cou l d not be de t.erm f ned , By 581' these patients were type d tru e l y homcayqc ua for the oRBI *0401 allele. High resolution DRBI t yping by SBT is achieved for 71 o ut of 77 seroloCii cally DR4-pos i tive p a t i e nt s samples, demonstrat i nq its c a pa.c.l.t y as a typing technique. Typi n9 was performed b y
(6~~~ ~~~~~~i:e~;r~N~o~~aU~~C~eg ~~B~*~~~lm~~~i:ilvf:,XPf:S(i6~~
were DRB1"'0404, 6 (8\) were DRBI *0405 and 2 ( 3\) was DRB! *0408 .
-
~~~mu~ i:~~ ~~e~~t:~~~ig~l ie1:t~~~~n;~~ o~~q~i~ieo~~~t~~n:e: ~f~
ved afte r furthe r opt i misat ion of the seque nce pr imers u s e d.
P417
GENEITCSUSCEPTIBILITY TO RHEUMATOID ARTHRfTIS : STIJDYOF HLA-DRA/I,'D TNF LOCI USINGTHE MASC METHOD
ROLE OF HJA·DR·DR AND DR-DP INTERACTIONS GENETIC SUSCEPTIBILITY TO RHEUMATOID ARTHRmS
Genin Emmanuellel, BabronMarie-Claude1, McDennott Michael2, Mulcahy Brian2, Waldron-LynchFrank2, Adams ClaW, Shanahan Fergus2, Molloy Michael-, O'Gara Fergal-,
Perdriger Alethl , Guggenbuhl PascalI, Chales Gerard', Le Dantee Philippe', Yaouang Jacqueline'', Genetet Bernard' , Pawlotsky Yves', Semana Gilbere 1 - Rhumatologie, CHRU. 2 - Laboratoire Universitaire d'Immunologie, ETS Bretagne-Est.3 - BpidemiologieCHRU, Rennes, France
Clerget-Darpoux Francoise! INSERM U155, Paris, France! ; University College, Cork, Ireland2 We have investigated the HLA component involved in rheumatoid arthritis (RA) susceptibility with the MASC method. The informationon the distribution of the marker genotypes in unrelatedaffectedindex cases and of haplotypesharing with one affected sib is used simultaneously. A sample of 50 families each having at least two affected sibs and typed for the HLA-DR and TNF loci was available. Strong associations were observed between alleles of these two markers and RA. The MASC method was successively applied to data from these two markers. We tested the involvement of a locus closely linked to the marker versus direct involvementof the marker locus itself. We' also tested the shared epitope hypothesis which assumes the involvement of amino acid sequences in position 70-74 on the 1\ chain of DR molecules in RA susceptibility. Finally we used information simultaneously on both ALA-DR and TNI' markers in order to determine if the TNF marker providedevidence for a susceptibilitylocus in additionto HLA-OR.
IN
10 order to aoalyse the relationship between DR and DP loci in the genetic susceptibility to rheumatoid arthritis (RA), ALA-ORB1 and DPBl polymorphism was studied in 155 RA patieots compared to 150 cootrols. Our data were consistent with the involvementof amino-acid at position 71 of the DRBl chain in the disease susceptibility.The higher risk for RA was observed in subjects who carried both a lysine (K) and an arginine(R) at position 71 : 21.9 % of patieots carried such an heterozygosity (KIR) compared to 0.6 % of controls (pc < ]0-6, OR = 42). In the abseoce of arginine, the presence of lysine was still associated with the disease (33 % vs 19 %, pc < 0.003. OR 2). On another hand, the analysis of the HLAOPBIlocus showed that the DPBl *0401 allele frequency was significantly increased in the RA patient group wbo expressed only arginine at position 71 (0 = 47) (82 % vs 56 % in controls, p < 0.008). 10 conclusion, our results strongly suggest that HLA-DR-DR and HLA-DR-DP interactions may play an important role in the genetic susceptibility to RA
=