Abstracts S24

Poster Group 7 - Immune Regulation 94 HUMORAL AND CELLULAR IMMUNE RESPONSE 93 MECHANISMS DIRECTED AGAINST THE FETUS SUPERIOR SUPPRESSION BY RAPAMYCIN+FK506, CONTRIBUTE TO PLACENTAL ABRUPTION. OVER RAPAMYCIN+CSA, DUE TO ABROGATED CTL INDUCTION, IMPAIRED MEMORY RESPONSES AND Andrea Steinborn, Cyrus Sayehli, Christoph Sohn, Erhard Seifried, PERSISTENT APOPTOSIS Edgar Schmitt, Manfred Kaufmann, Christian Seidl. Department of Obstetrics and Gynecology, University of Frankfurt; Hans JPM Koenen, Etienne CHJ Michielsen, Jochem Verstappen, Institute of Immunology, University of Mainz; Department of Esther Fasse and Irma Joosten. Department for Blood Transfusion Transplantation Immunology, RCBDS, and Transplantation Immunology, University Medical Center Placental abruption is an unpredictable severe complication in Nijmegen, The Netherlands. pregnancy. In order to investigate the possibility that the activation Immunosuppressive therapy is best achieved with a combination of agents of the fetal non- may be involved in the targeting multiple activation steps of T-cells. In transplantation, Cyclosporin-A pathogenesis of this disease, IL-6 release from cord blood (CsA) or Tacrolimus (FK506) are successfully combined with Rapamycin monocytes was examined by intracellular cytokine staining and (Rap). Rap and CsA were first considered for combination therapy because flow cytometric analysis. Our results demonstrate that preterm FK506 and Rap target the same intracellular protein and thus may act in an antagonistic way. However, in clinical studies FK506+Rap proved to be placental abruption (n=15) in contrast to uncontrollable preterm effective. To date, there is no in vitro data supporting these in vivo findings and labour (n=33) is associated with significantly (p<0.001) increased it is unclear whether the observed effects are T cell mediated. In a human release of IL-6 from the fetal monocytes. The same holds true for polyclonal allogeneic in vitro model, we found that although combined drug Rhesus disease that is characterized by a maternal production of treatment markedly reduced expansion of naive T cells, T cell activation against the rhesus-D antigen expressed by the fetal occurred irrespective of the drug combination used. The induction of cytotoxic effector T cells was reduced by CsA+Rap, but completely abolished by erythrocytes. This suggests that in the course of Rhesus disease IL- FK506+Rap. Importantly, combined immunosuppression allowed generation of 6 release of monocytes is induced by -mediated cross- memory CD4+ and CD8+ T cells and hence did not result in T cell anergy. linking of these cells to the erythrocytes in the fetal circulation. To However, FK506+Rap treatment resulted in a reduced number of allospecific elucidate this potential mechanism, the presence of anti-HLA- memory T cells showing a decreased cell cycle turnover and cytokine antibodies was assessed in the maternal circulation of patients with production. In contrast, CsA+Rap treatment led to increased memory T cell numbers responding with elevated kinetics. The ability of Rap to promote placental abruption (n=17) and patients with uncontrollable apoptosis, which contributes to T cell suppression, remained unaffected upon preterm labour (n=29). The percentage of women producing anti- combination with FK506 or CsA. These data support the combined use of paternal HLA-antibodies was significantly (p< 0.01) increased in FK506+Rap over CsA+Rap for immunosuppressive therapy the group of women with preterm placental abruption (47%) in comparison to women with uncontrollable preterm labour (14%). Our results suggest that an increased humoral immune response of the mother against the fetus is involved in the pathogenesis of placental abruption.

Poster Group 8 - and Anthropology

95 96 THE ORIGIN OF MAYANS ACCORDING TO HLA GENES AND SNP SCREENING OF A KOREAN POPULATION USING THE UNIQUENESS OF AMERINDIANS. ALKALINE-MEDIATED DIFFERENTIAL INTERACTION (AMDI) Eduardo Gómez-Casado, Jorge Martínez-Laso, Juan Moscoso, Jorge Zamora, Patricia Lucas Gramajo#, Carlos Silvera-Redondo, Ernesto Lowy, and Antonio Susan. Tonks, Jin C. Kim and Walter.F. Bodmer Arnaiz-Villena. Cancer Research UK Department of Immunology and Molecular Biology, Hosp. 12 de Octubre, Universidad Complutense, 28040 Madrid, Spain. E-mail: The polymerase chain reaction (PCR) and the identification of SNPs (single nucleotide polymorphisms) throughout the genome have had a major impact on [email protected]. #Department of Laboratory, Hospital Regional the study of human population genetics at the DNA level. It is important, de Xela (Quetzaltenango, Guatemala). however, not to rely on a single set of markers to elucidate the demographic and The HLA allele frequency distribution of the Mayans from Guatemala has been migrational history of a given population, but to use a range of markers, studied and compared with those of other First American Natives and encompassing the non-recombinant portion of the , the worldwide populations (a total of 12,364 chromosomes and 6,182 individuals mitochondrial genome and autosomal markers, which may or may not be under from 60 different populations). The main conclusions are: 1)The closest selective pressure. Amerindian group to Mayans is the Arhuacs, who were the first recorded PCR has greatly enhanced the ability to type for SNPs. Until recently, Caribbean Islands inhabitants. 2)Mayans are not so close to Mesoamerican however, many detection methods have relied on the use of gels or specially Zapotec, Mixe and Mixtec Amerindians, who genetically cluster together. Mixe labelled probes. AMDI (Alkaline Mediated Differential Interaction) is a had been related to Mayans only on linguistic bases. 3)DRB1*0407 and detection system devised in our laboratory which avoids the use of gels, lends DRB1*0802 alleles are found in 50% of Mayans; these alleles are also found in itself to automation and high throughput screening, and is simple and other Amerindians ,but the Mayans high frequencies may be showing a founder inexpensive. effect for this Mesoamerican-Caribbean population. 4)Extended Mayan specific We now describe the typing of a Korean population for a number of markers, HLA are described for the first time. 5)Language and genes do not including HLA, KIR and SNPs in the non-recombining region of the Y completely correlate in microgoegraphical studies. 6)Peopling of the Americas chromosome using AMDI. The results obtained are direct fluorescence readings was probably more complex than postulated by Greenberg and others (three from a fluorimeter, from which DNA types can be automatically assigned. peopling waves). Significant genetic input from outside is not noticed in Meso These results have been compared with those using a gel-based detection system and South American Amerindians according to the genetic analyses; while all and show good concordance in signal and the presence or absence of a band. world populations (including Africans, Europeans, Asians, Australians, Polynesians, North American Na-Dene Indians and Eskimos) are genetically related. Meso and South American Amerindians tend to remain isolated in the neighbour joining, correspondence and plane genetic distances analyses.

Genes and Immunity Abstracts S25

97 98 GENE[VA] PROJECT: CHECKING AND TESTING HLA DATA WHAT IS A FREQUENT HLA ?

Johan Renquin, David Roessli, Alicia Sanchez-Mazas Pierre-Antoine Gourraud1, Henk Van Der Zanden2, Colette Laboratory of Genetics and Biometry, Department of Anthropology Raffoux3, Jean-François Eliaou4, Anne Cambon-Thomsen1 on behalf and Ecology, University of Geneva, Switzerland of the MADO consortium www.euromado.org 1- INSERM U558 Investigating HLA genetic diversity in human populations requires to follow Toulouse, 2- Europdonor Leiden, The Netherlands 3-FGM several important steps in data analysis, among which checking for Hardy- Paris, France 4-CHU Saint Eloi Monptellier, France Weinberg equilibrium, or testing linkage disequilibrium and associations with diseases. Although a number of computer programs are currently available for In 2001, around 14 out of 100 HLA A B DR split phenotypes of the new population genetics analyses, they do not always apply to HLA data which haematopoietic stem cell (HSC) potential donors were not already present in the exhibit both numerous loci and high numbers of alleles per locus, including a file of Bone Marrow Donor Worldwide: four times less than ten years ago. The possible blank recessive. recruitment of unrelated HSC donor needs to be optimised, trying to avoid One of the GeneVA (« Gene Visual Access ») project’s aim is to help frequent HLA types. To go on increasing the probability to find a donor for a immunogeneticists in differents steps of investigation after their laboratory patient, here we aim at defining which HLA are frequent in . work. How to format and handle HLA data, and how to choose statistical Methods: We adopted the haplotype frequency estimation viewpoint as it methods for specific research purposes are often not straightforward, leading to gives an estimation of the HLA distribution in the population from where the very heterogeneous data sets. We propose here some interactive tools to donors came. Haplotype estimations were obtained by Maximum likelihood standardise HLA data formats for their use in different computer programs, and method implemented within an Expectation Maximisation algorithm. Counting to perform online several preliminary steps in HLA data analysis in relation to phenotypes performed validations of results. Criterion for inclusion in the list of anthropology and HLA-disease studies. Among these tools, Fisher tests frequent haplotype was: frequency >=1% in at least one of the 5 considered specifically adapted to two-locus associations as well as HLA and disease countries (NL ENG FRA HUN IT) associations are available. As an application, we present here the results of some Result: A list of 16 HLA frequent haplotypes at 4-digit resolution typing level of these tests on a large HLA data base of worldwide populations. and the corresponding list of alleles has been set from the study of frequent haplotypes in NL ENG FRA HUN IT. Examples: A*0101 B*0801 DRB1*0301; A*0301 B*0702 DRB1*1501; A*0201 B*4402 DRB1*0401; A*2902 B*44031 DRB1*07011. Discussion: Within an EU consortium these lists are used to choose techniques (SNP genotyping, Microsatellites Genotyping, Micro arrays) in order to target out the frequent HLA profiles. Hence such procedure could be tested as part of recruitment strategies for the Registries. Such analysis shows how taking into account HLA distribution could be a key element of Registry strategy design.

99 100 USE OF HAPLOTYPE INFORMATION IN THE SEARCH FOR ASSOCIATION OF HAPLOTYPE ESTIMATES WITH COMPATIBLE DONOR: EXAMPLE IN FRANCE. QUANTITATIVE PHENOTYPES: VARIATIONS IN THE INTERLEUKIN-21 RECEPTOR GENE AND IGE-LEVEL Pierre-Antoine Gourraud1, Amar Baouz2, Colette Raffoux2, Anne Cambon-Thomsen1 1- INSERM U558 Toulouse, France 2-FGM Inke R. König1, Gregor Bein2, Ulrich Mansmann3, Matthias Paris, France Hecker2, Anette Bohnert2, Holger Hackstein2, Andreas Ziegler1 1Institute of Medical Biometry and Statistics, University at Lübeck; Typing the relatives and studying the HLA segregation within the family, the actual level of HLA information used in familial HSC transplantation is 2Institute of Clinical Immunology and Transfusion Medicine, Justus- HLA haplotypes. In unrelated situation, the matching for compatible donor Liebig-University Giessen; 3Institute of Medical Biometry and is based on phenotype information. However haplotype frequency Informatics, Ruprecht-Karls-Unviersity Heidelberg information can be estimated at population level. Here we aim at showing Analyzing the association between total serum IgE levels and variations in several aspects of this population information at individual level. the interleukin-21 receptor gene (IL21R), recent results have revealed a Methods: 1- Haplotype estimation based on likelihood methods significant association of carrying at least one IL21R polymorphism (T- implemented within an Expectation Maximisation algorithm, which is 83C) with high IgE levels (> 100 kU/l) in females (Hecker et al., 2003). adapted to missing values. 2- Haplotype prediction software using Furthermore, haplotypes consisting of the four single nucleotide population haplotype frequencies. French donors (N= 108 508) and patients polymorphisms (SNPs) were estimated separately in probands with high and Registries (N= 6 022) are considered. low IgE levels; the respective frequencies were found to be different in the Result: The likelihood of the various haplotypes combinations proposed two groups. for an individual based on his phenotype and on the population haplotype To increase the overall power of the study, the next step in the analysis frequencies depends on the combination of frequent or rare possible was not to categorize the IgE level but to use the quantitative information haplotypes. They vary between 90% according to cases. Ex : A(2;32) itself. However, we are not aware of any approach allowing the estimation B(7;18) DR(1;15) results from the association of A-B-DR haplotype (2-7- of haplotype frequencies taking into account a quantitative phenotype. We 15) and (32-18-01) in 92,1% of the cases. Haplotype prediction might be therefore compared two different approaches: In the first, we estimated systematically given to clinicians within the FGM intranet. It would evaluate haplotype probabilities using an Expectation-Maximization algorithm across the chances for a phenotype match to be a haplotype match too. Haplotype all individuals, regardless of their IgE level. Secondly, haplotypes were analysis can thus be applied at both population and individual level. estimated depending on the specific IgE level of an individual. Discussion: In the general framework of haplotype analysis, it also In the subsequent Monte-Carlo simulation, each person was assigned a contributes to show that individual haplotype prediction could be relevant. combination of two haplotypes based on the estimated probabilities and a The haplotype point of view is powerful as it reduces the number theoretical random number. To receive stable estimates, the number of replications was possible phenotypes analysis. At individual level the genetic background of set to 100,000. Mean IgE levels were then compared between different the donors limits such approach. Different population frequency estimations groups of haplotypes and haplotype combinations. should be used if a particular genetic background is suspected. The investigated methods will be compared with regard to availability of Performed within the MADO EU consortium www.euromado.org ; in implementations and power. collaboration with LERIA, le Kremlin-Bicêtre France Reference: Hecker, M, Bohnert, A., König, I.R., Bein, G., Hackstein, H. (2003) Novel genetic variation of human interleukin-21 receptor is association with elevated IgE levels in females. Genes and Immunity, in press.

Genes and Immunity Abstracts S26

101 102 HIGH PREVALENCE OF MULTIPLE SCLEROSIS AFFECTS SEARCHING FOR RARE HLA ALLELES - A STRATEGY THAT FREQUENCIES OF DQ2 AND DR2-DQ6 HAPLOTYPE IN A WORKS SMALL POPULATION OF KOCEVJE IN SOUTH SLOVENIA Jonathan Downing and Christopher Darke Blanka Vidan-Jeas, Andreja Rako, Beatrika Kon an-Vra ko, Matja Jeras, Welsh Transplantation and Immunogenetics Laboratory, Welsh Borut Peterlin Blood Transfusion Center of Slovenia, Ljubljana Blood Service, Pontyclun, Cardiff, CF72 9WB Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central The HLA genes are the most polymorphic genes known in man with novel nervous system in which an autoimmune response probably contribute to the alleles discovered regularly. Approximately 7.4 million bone marrow pathogenesis. While the HLA-DRB1*1501,DQA1*0102,DQB1*0602 (DR2- volunteers (BMV) world wide, have been HLA class I typed using serology DQ6 haplotype) is associated to MS mostly in populations of Northern alone. However, some rare HLA gene products are undetectable, or difficult to European origin, the DRB1*0301,DQB1*0201,DQA*0501 haplotype was detect by serology. This will have resulted in the miss typing of some BMV, found associated to MS in Sardinia, and to relapsing/remitting form of MS in which will have implications in the selection of donor/patient pairs. Also, in Sweden and USA. We carried out a population based case-control association order to furnish intelligent HLA typing services it is essential to be fully aware study involving 30 patients suffering from MS and a control group of 30 healthy of the immunogenetics of the local population. individuals. The samples were collected in the South Slovenia, in relatively We have applied a simple strategy to search the Welsh Bone Marrow Donor isolated region of Kocevje occupying 920km2. The population of 31.258 Registry (WBMDR) for rare alleles that were previously undetected by our individuals is historically distinct from Slovenian population. Very high serological typing. This was based on determining the alleles' principle carrying prevalence of MS (134,4/100000) is a hallmark of the population of Kocevje. In haplotype, from family or population studies, and searching for phenotypes that our group of patients 35% had relapsing/remitting-MS, 23,5% had primarily possessing the likely haplotype. progressive-MS and 40,7% had secondary progressive form of MS. We found As an example, the HLA-B*2723 product is not easily detected by serology. the DR2-DQ6 haplotype over-represented (p=0,008; RR=8,69) and We found the B*2723-bearing haplotype to be A*26; DRB1*12; DQB1*0301. DQB1*0201/2 allele(s) under-represented (p=0.037, RR=0,33) in the group of A total of 47 out of 9,185 WBMDR BMV, typed several years ago by serology alone, were identified possessing HLA-A26 and/or DR12 and with a single patients compared to controls. Comparisons between the control group of Kocevje and Slovenian population revealed decreased frequency of the HLA-B specificity. Two of these were found to possess B*2723. This strategy has also been used successfully to find examples in the WBMDR haplotype DR2-DQ6 (3%vs14%) and increased frequency of DQB1*0201/2 ( 53% vs 35%) in the control group of Kocevje. These differences may arise due BMV, of the first HLA-C null allele, Cw*0409N, previously undifferentiated to the genetic drift and selection influenced by the high prevalence of the MS in from other Cw*04 alleles. Kocevje.

103 104 CONSTRUCTION OF HAPLOTYPE BLOCKS AND LINKAGE- THE CONTRIBUTION OF DIFFERENT GENES TO GENETIC DISEQULIBRIUM MAPS DIFFERENTIATION BETWEEN ABORIGINAL POPULATIONS OF NORTHERN RUSSIA Inke R. König, Andreas Ziegler Institute of Medical Biometry and Statistics, University at Lübeck Irina Evseeva, Susan Tonks, Spencer Wells, Oleg Balanovsky, Victor In recent years, the investigation of the genetic background of complex Spitsin, Leonid Alexeev, Margatita Boldyreva, Walter F. Bodmer. diseases using genome-wide linkage disequilibrium (LD) designs has Cancer Research UK gained increasing interest. These designs require knowledge of the Three aboriginal populations from Northern Russia (Nentsy, Saami and Pomors) underlying pattern of LD throughout the and the were genotyped for HLA (A, B, C, DRB1, DQA1 and DQB1 loci) and for a availability of extensive LD maps. With these, the appropriate density of number of SNPs within NRY. These populations were also phenotyped for four polymorphic markers in a given region can be selected, which will in erythrocyte enzymes (ACP1, GLO1, PGM1, ESD). turn increase the power of a study. Analysis of molecular variance was carried out based on the allele frequencies However, different methods have been suggested to construct maps of of the loci studied. The major contribution to the genetic distinction between LD. Some rely on the definition of haplotype blocks with high levels of these populations was due to GLO1 and HLA-C (FST 0.056 and 0.042) whilst LD with the subsequent estimation of haplotypes within blocks (e.g., the mean (10 loci) FST was 0.021, typical of the populations in the North- European part of Eurasia. The FST range was more stable for the HLA loci Gabriel et al., 2002). Others take into account the decline of LD with (0.015-0.042 with the mean of 0.025) compared to the classical erythrocyte distance (e.g., Maniatis et al., 2000). Furthermore, some map definitions markers (0-0.056 with the mean of 0.015). use data from independent probands, whereas others require family- FST, obtained from NRY-haplotype frequencies was several times higher based data. As a consequence, application of different procedures will (0.139) than that of the autosomal genes, which is usual for Y-chromosome lead to the selection of different markers for mapping. markers and may reflect a major contribution of the male lineage to the genetic To facilitate the application and the selection of an appropriate method, pattern of the population. an overview over the different methods will be given. The special focus FST in total northern population (N=342) will be on the practical advantages and disadvantages of the various Locus FST Locus FST procedures as well as on the ready availability of implementations. HLA-A 0.030 ESD 0 References: Gabriel, S.B., Schaffner, S.F., Nguyen, H., Moore, J.M., HLA-B 0.015 GLO1 0.056 Roy, J., Blumenstiel, B., Higgins, J., DeFelice, M., Lockner, A., Faggart, HLA-Cw 0.042 PGM1 0.003 M., Liu-Cordero, S.N., Rotimi, C., Adeyemo, A., Cooper, R., Ward, R., HLA-DRB1 0.025 ACP1 0.001 Lander, E.S., Daly, M.J., Altshuler, D. (2002) The structure of haplotype HLA-DQA1 0.026 4 classical loci 0.015 blocks in the human genome. Science, 296, 2225-9. HLA-DQB1 0.016 10 autosomal loci 0.021 Maniatis, N., Collins, A., Xu, C.F., McCarthy, L.C., Hewett, D.R., 6 HLA loci 0.025 NRY (14 SNPs) 0.139 Tapper, W., Ennis, S., Ke, X., Morton, N.E. (2002) The first linkage disequilibrium (LD) maps: delineation of hot and cold blocks by diplotype analysis. Proc Natl Acad Sci U S A., 99, 2228-33.

Genes and Immunity Abstracts S27

105 106 HLA-A*24020102L IN THE UK POPULATION ASSOCIATION BETWEEN THE HLA-DQB1 ALLELES AND LONGEVITY: A STUDY IN SARDINIAN POPULATION PPJ Dunn, Jr Turton, S Williams, J Downing, S Day, N Bendukidze, CV Navarrete and C Darke. BBMR DNA Reference Laboratory, Letizia Scola, Giovanni Mario Pes, Domenico Lio, Ciriaco Carru, National Blood Service, Bristol UK and Welsh Transplantation and Antonio Crivello, Giuseppina Candore, Giuseppina Colonna- Immunogenetics Laboratory, Welsh Blood Service, Cardiff, UK Romano, Luigi Ferrucci, Luca Deiana, Giovannella Baggio, Claudio Franceschi, Calogero Caruso. Gruppo di Studio dell’ During the 3-year period to December 2002 tissue typing laboratories within the Immunosenescenza, Dipartimento di Biopatologia e Metodologie UK referred 12 cases with a problematic definition of the HLA-A24 allele to the NBS DNA Reference Laboratory in Bristol. In each case the A24 specificity Biomediche, Università di Palermo, Corso Tukory 211, 90134 was not detected by serology but A*24 was identified by DNA-based typing. Palermo, Italy.

DNA sequencing showed in all cases the presence of a mutation (G A) in Longevity is the result of numerous interacting factors including genetic, intron 2 characteristic of the ‘low’ expression allele HLA-A*24020102L. environmental and behavioural components. Some studies suggest that genetic An examination of possible haplotypes associated with this allele showed that determinants of longevity might reside in the polymorphisms for genes that A*24020102L; B*55; Cw*01 was present in 5 of the 12 cases. regulate immune responses as HLA genes. Longevity has been shown to be A search of this possible haplotype among 54,000 HLA-A,B,C,DR-typed associated with a positive selection of HLA alleles or haplotypes that confer donors registered with the British Bone Marrow Registry (BBMR) and 10,000 resistance to infectious diseases, respectively via peptide presentation or via A,B,C,DR,DQ-typed donors on the Welsh Bone Marrow Donor Registry antigen non-specific control of immune response. Some well planned and (WBMDR) revealed 41 donors in the BBMR and 7 in the WBMDR. SBT or designed association studies performed in Caucasians suggest that longevity is SSP were then used to test these donors for the presence of A*24020102L. The associated with HLA-DR11 allele or HLA-B8,DR3 haplotype, whereas other results showed that the BBMR had 9 A*24020102L donors while the WBMDR studies do not confirm these observations. Association studies, indeed, are had 2. subjected to a number of possible confounding factors, the homogeneity of the We have not yet examined the other possible A*24020102L-bearing population in term of geographical origin among others. Because of the lack of haplotypes therefore there are likely to be more donors carrying this allele. large-scale heterogeneity, the Sardinians represent a suitable population for However, our current data suggests that A*24020102L; B*55; Cw*01 is the association studies addressed to dissect the complex traits as longevity. The principal HLA-A*24020102L-bearing haplotype in the UK. >95% of the donors relatively homogeneous Sardinian population is indeed an ancient genetic on both Registries are white north-western European Caucasoids indicating that isolate with a gene pool characterized by a relative little genetic flow from the the frequency of A*24020102L in the Caucasoid population of the UK is various populations that have invaded the island during the last 3000 years. In a approximately 1 in 6,000.

107 previous study we have evaluated HLA-DRB1 frequencies in Sardinian HLA-A,B,DR PHENOTYPE AND HAPLOTYPE FREQUENCY centenarians and controls. No significant differences were obtained by analysing ANALYSIS OF BONE MARROW DONORS BORN IN REGION the differences between Centenarians and controls even if HLA-DRB1*15 was LOMBARDY (ITALY) increased in centenarians. On the other hand, HLA region contains a great number of genes forming some very stable haplotypes, characterised by a strong linkage disequilibrium, probably conferring a selection advantage (ancestral Luca Mascaretti*, Alberto Degiuli˚, Antonella Lisa#, Rosalba haplotypes). Some of the highly conserved haplotypes are composed by the Guglielmino§ for Region Lombardy HLA Laboratories HLA-DR–DQ alleles and in some instances HLA-DQ alleles play a central role *Transfusion Center S. Gerardo Hospital Monza, ˚Transfusion Center in the association to disease (e.g. Selective IgA deficiency or Celiac Disease). A.O. Lodi Province, #Molecular Genetics Institute CNR Pavia, So we have evaluated the HLA-DQB1 frequency in 80 centenarians and 100 §Genetics Department University of Pavia controls from Sardinia by amplification refractory mutation system/polymerase chain reaction. The results show that the DQB1*06 allele is significantly The bone marrow donor registry of Lombardy comprises over 68000 donors. increased (p< 0.001, after Bonferroni correction, by 2 analysis) in centenarians Our aim was to analyse HLA frequencies based on place of birth (province) of (20%) vs controls (5%). These data indicate that the statistically not significant donors born in Lombardy. Materials and methods: 20 of 21 laboratories DRB1*15 increase observed in the previous study may be due to the linkage participated in the study; only records with place of birth within the region were with the DQB1*06 allele that may be associated to longevity in the studied considered. HLA alleles were chosen according to suggestions of Schipper et. al population. On the other hand, our data confirm that some of the HLA-DR/DQ (1996). For frequency analysis, the maximum likelihood method according to haplotypes may be more important for association studies than the individual the expectation-maximization algorithm was used. Results: 62844 records were HLA-DR and -DQ phenotypes. Our present and previous data not allow to available for analysis, 33491(53.3%) of which were eligible. 11.1% of donors confirm the association of longevity with other class II alleles (e.g. HLA-DR11 was not born in the province of residence. Phenotype frequencies: of the 9623 or DR3) observed in other Caucasian populations. On the whole these findings HLA-A,B phenotypes, 4703 (50.77%) were expressed once. Of the 7693 clearly show that HLA/longevity associations are population-specific, being HLA,A,B,DR phenotypes, 6564(85.32%) were expressed once. The most heavily affected by the population-specific genetic and environmental history. frequent phenotypes were HLA-A2,3;B35,51 (carried by 139 donors) and HLA- As an example, it is well known that the HLA-DR2, DQ6 (i.e., HLA- A29,33;B14,44;DR1,7 (15 donors). Haplotype frequencies: HLA-A2, B51 and DRB1*1501, DQA1*0102, DQB1*0602) haplotype contributes to the risk of HLA-A1,B8,DR3 were the most frequent haplotypes. The minimum number of developing multiple sclerosis in Caucasoids of Northern European heritage but HLA-A,B haplotypes (% of total) whose frequency added to 50% varied from not in Sardinia population. So, in our opinion, HLA genes might be considered 6.1% to 16.4% in different provinces; for HLA-A,B,DR, it varied from 7.7% to survival genes not longevity genes. 25.9%. Conclusions: some diversity among the 11 provinces was evidenced. To improve the analysis from the province to the commune level, missing data must be completed. This will allow us to devise new strategies for donor recruitment together with our bone marrow donor association (ADMO).

Genes and Immunity Abstracts S28

108 109 SEQUENCING ANALYSIS OF HLA-A,-B AND DRB1 CONFIRMATION OF HLA-B*8202 IN A NATIVE SOUTH POLYMORPHISMS IN CHAOUYA MOROCCAN POPULATION. AFRICAN FAMILY

Angelica Canossi (1), Daniela Piancatelli (1), Anna Aureli (1), A.Poles, P.Brookes, S.Davey, & N.Davey Khadija Oumhani (2), Marilena Di Rocco (1), Tiziana Del Beato (1), The sequence of HLA-B*8202 was derived from several cell lines of both Gabriella Liberatore (1), Carlo Umberto Casciani (1), Rajae El Aouad Oriental and Caucasoid ethnicity. There is little published information on this (2) and Domenico Adorno (1). (1) Istituto C.N.R. Trapianti particular allele. We have confirmed the presence and sequence of this allele in D’Organo e l’Immunocitologia, L'Aquila, Italy. (2) Immunology two haplotypes in a South African family. Laboratory, Institut National D'Hygiene, Rabat, Morocco. Five siblings were tissue typed, initially “serological level typing” by PCR- SSP. This resulted with one HLA-B specificity assigned but the second In order to provide genetic and anthropological information for the Chaouya specificity was ambiguous with primers positive for both HLA-B45, and -B56. (CH) population, an Arabic-speaking population from West Morocco, we When tested by RSCA for HLA Class-I, the undefined second allele produced a performed a sequence-based typing at the HLA-A, B (exons 2,3,4) and DRB1 novel mobility. This mobility value was close to the mobilty values for HLA- loci (exon 2) on blood samples from 99 healthy unrelated volunteers. This B*8101 and HLA-B*3504, when using FLRs HLA-B*3801, HLA-B*4201, and ethnical group was compared with other African and Mediterranean HLA-B*4402. SBT confirmed the presence of HLA-B*8202. The two populations. The population was in Hardy-Weinberg equilibrium and the haplotypes present were: operation of a balancing selection (heterozygote advantage) for all the HLA loci A*3004 B*8202 Cw*0304 DRB1*15 DRB5*01 DQB1*06 studied was observed. Gene diversity was elevated (A locus: 0.92, B locus: 0.97, A*6801 B*8202 Cw*0304 DRB1*15 DRB5*01 DQB1*06 DRB1 locus: 0.94), as described in many African populations. A total of 28 Using this information we have been able to update our PCR-SSP primer HLA-DRB1, 27 HLA-A and 40 HLA-B alleles were found. The novel A*3010 specificities and the RSCA mobility value for HLA-B*8202. The primer pattern allele (AF323494-6) and the B*1403 exon 4 sequence (AF279664) were by PCR-SSP resembles the serological profile of the HLA-B*8202 allele described. Overall, several alleles previously observed to be common in African described for the cell line VTIS68967, in that both HLA-B45, and HLA-B56 populations (DRB1*01021,*0806,A*3402,*6802,B*1503, *4102), some were detected. Further investigation of the serological profile of the HLA- variants identified in Spaniards (A*0101,*3001, B*0801) and some uncommon B*8202 allele is in progress. alleles (DRB1*03021,*1503, A*3004,*8001, B*2708,*3910,*5001) were evidenced in this group. The predominant A-B-DRB1 haplotypes (>3%) in the Chaouya were A*0201-B*5101-DRB1*1501 and A*0201-B*5001- DRB1*0701.This analysis highlights some unique genetic characteristics of CH within Morocco and has demonstrated the influence of intermingling with various populations.

110 111 IDENTIFICATION OF CYTOKINE GENE POLYMORPHISMS IN FIRST DETECTION OF THE HLA-A*24020102L ALLELE IN A GREEK POPULATION THE CZECH POPULATION

Nikki Constantinidou, Kleopatra Spanou, Olga Moraloglou, N. Bendukidze1, L.Zahlavova, L.Kupkova, P.Dunn1, S.Day1, M. Gecka, Maria Kanariou Tilanus2, E.Ivaskova. Department of Immunogenetics, Institute for Dept. of Immunology-, "Aghia Sophia" Children's Clinical and Experimental Medicine, Prague, Czech Republic, 1H&I Hospital, Athens - Greece DNA 1DNA Reference Laboratory, National Blood Service, Bristol, UK; 2University Medical Centre, Utrecht, The Netherlands Cytokines are known to be important mediators of the immune response. Several cytokine genes bear polymorphisms which cause variations in cytokine HLA Class I antigens are still typed by serology for some Bone Marrow production. Certain genotypes have been associated with the susceptibility to registries and this is also the case for solid organ transplantation in some autoimmune and infectious diseases and the outcome of organ and hematopoetic histocompatibility laboratories. Problems may arise when HLA antigens are stem cell transplantation (HSCT). We evaluated the polymorphic variants of 5 typed by serological or DNA methods only. Serology can miss or cytokine genes in a randomly selected group of 78 healthy Greek individuals. incorrectly assign some antigens while DNA typing can incorrectly assign The allele and genotype frequencies have been determined by PCR-SSP an antigen which is not actually expressed by a null allele. Such errors in analysis (One Lambda), for the following cytokine genes: TNF- (-308 G/A), tissue typing may have disastrous implications for allogenic transplants. A IL6 (-174 G/C), IFN- (Intron 1-874 A/T), TGF 1 (codon 10 C/T and codon 25 combination of serological and DNA-based genotyping methods (including G/C) and IL10 (-1082 G/A, -819 C/T and -592 C/A). Percentages of genotype PCR-SSP, PCR-SSOP and SBT) helps to avoid such problems and frequencies are presented in the table: especially facilitates the identification of null and low-expression alleles, although allele specific typing is necessary for their final definition. TNFα IL-6 IFNγ TGFβ1 TGFβ1 IL-10 IL-10 IL-10 Serological typing has been carried out on 1800 donors from the Czech (-308) (-174) (+874) (codon (codon (-1082) (-819) (-592) 10) 25) Bone Marrow Donor Registry (CBMD), 77 patients and 200 healthy GG GG TT CC GG GG CC CC individuals from a local panel. These samples were retyped using the 83,3% 51,3% 25,6% 19,2% 87,2% 20,5% 55,1% 55,1% automated reverse PCR-SSOP and PCR-SSP technology (low resolution GA GC TA CT GC GA CT CA LiPA and Olerup SSP GenoVision kits). The presence of A*24 null or low 16,7% 34,6% 47,4% 46,1% 12,8% 42,3% 34,6% 34,6% expession allele was implicated in one donor. His phenotype was A2; B44, AA CC AA TT CC AA TT AA B55; Cw1, Cw7 (determined by serology), DRB1*1301, DRB1*16011, 0% 14,1% 26,9% 34,6% 0% 37,2% 10,3% 10,3% DQB1*0603, DQB1*0502. Allele specific PCR-SSP (Olerup SSP HLA-A9 kit) demostrated the presence of an A*24020102L allele. A*24020102L has Allele and genotype frequencies were correlated with other available population an identical sequence to that of A*24020101 but with a single nucleotide data in Caucasians and non Caucasians. The majority of the cytokine substitution (A instead G) at the 244th position in intron 2 (position 708 genotypes, in our study, did not present significant deviation from the according HLA-A Genomic Sequence Alignments given by IMGT database frequencies reported in other European populations (2 U.K., 1 Portugese). This (http://www.anthonynolan.org.uk/HIG/seq/nuc/text/agen_nt.txt). SBT was analysis may be useful for subsequent clinical studies on diseases and used to confirm our results. transplantation. This research was supplied by grant of Ministry of Health of the Czech Republic NM/6522-3

Genes and Immunity Abstracts S29

112 113 DIVERSITY OF NONCLASSIC HLA GENES AMONG RECOMBINATION BETWEEN THE HLA-DR AND HLA-DQ RUSSIANS LOCI WITHIN A FAMILY ?

Sergeev I.V., Trofimov D.Yu., Groudakova E.G., Alexeev L.P. Thomas M.C. Binder1, Julia M. Krizak1, Cornelia Brendel2, Ulrich Institute of Immunology, Moscow Finckh3, Peter Kühnl1, Thomas H. Eiermann1 1)Institute of Transfusion Medicine and Transplantation Immunology Both structure and polymorphism of nonclassic HLA genes currently attracts & 3)Institute of , University Hospital Hamburg- attention of investigators. LMP2, TAP1, DMA and MIC-A genes belong to Eppendorf, 2) Hospital for Haematology, Oncology and Immunology, nonclassic HLA genes, they encode immune system proteins and are involved in University Marburg; Germany transport, processing and formation of antigenic peptides/HLA molecule complexes. The fresh data on their construction associated to susceptibility with Although unusual associations of HLA-DR and HLA-DQ alleles have indicated different pathologies contributes to increasing interest to study these genes’ that recombination between these genes have occurred in the past, a most recent polymorphism. For example the results of recent researches show HLA MIC-A crossover event has been reported in the literature only once before. associations to type 1 diabetes [Gambelunghe et al.]. On the occasion of HLA-typing of a family of potential stem cell donors, we The purpose of our research was both to investigate nonclassic HLA genes have identified a male individual, who seems to be homozygous carrying the polymorphism in Russian population and to determine possible associations HLA haplotype A24, B7, DRB1*1501, DQA1*05, DQB1*0602 twice. between MIC-A gene polymorphism and type 1 diabetes among Russian On the first point of view, the young man can’t be the son of his parents, subjects. because only his mother, but not his father are carrying the equivalent Methods. In our research we used the following methods: PCR-RFLP haplotype. His fathers haplotypes are the common A1, B7, DRB1*1501, (restriction fragments length polymorphism), PCR-ACRS (amplification created DQA1*05, DQB1*0602 and the unusual A24, B7, DRB1*1501, DQA1*05, restriction sites), and microsatellite analysis for MIC-A gene. We determined DQB1*0201. (The second haplotype were also found in several members of this the following alleles frequencies among the population under study: DMA family.) (0101-83%, 0102-10%, 0103-4%, 0104-3%), LMP2 (H-74%, R-26%), TAP1 The single tandem repeats (STR) analysis confirmed the parenthood of his (01-83%, 02011-10,5%, 02012-4%, 03-2%, 04-0,5%), MIC-A (A4-18%, A5- father and his mother. The micro satellite marker analysis also excluded 12%, A5.1-38%, A6-14%, A9-18%). uniparental (maternal) disomy or a major chromosomal deletion on chromosome We genotyped as well Russian type 1 diabetic patients (n=65). The 6. The ‘homozygous haplotype’ could result either from a HLA-A1 deletion or comparison between MIC-A alleles’ frequencies determined among diabetic from a crossing over event during his father's spermatogenesis. Further patients (A4-13%, A5-16%, A5.1-39%, A6-10%, and A9-22%) and healthy investigations will help to answer this intriguing question. control did not reveal associations between MIC-A alleles and type 1 diabetes.

114 115 RELATIONSHIPS BETWEEN MEDITERRANEANS AND SUB- HLA-GENES DIVERSITY AMONG 5 RUSSIAN GROUPS FROM SAHARAN AFRICANS ACCORDING TO HLA, OTHER GENES DIFFERENT REGIONS OF RUSSIAN EUROPEAN PART. AND HISTORY. Boldyreva M.N., Trofimov D.Y., Jankevich T.E., Bogatova O.V., Antonio Arnaiz-Villena, Eduardo Gómez-Casado, Juan Moscoso, Jorge Zamora, Gouskova I.A., Philippova E.V., Sergeev I.V., Alexeev L.P. Institute Ernesto Lowy, José Manuel Martín-Villa, Mercedes Pérez-Blas, and Jorge of Immunology Russia Ministry of Health Martínez-Laso. Department of Immunology and Molecular Biology, Hosp. 12 de We previously examined the distribution of HLA-alleles and haplotypes among Octubre, Universidad Complutense, Madrid, Spain. E-mail: 2 Russian population groups: 1. Pomors (n=81) from the North of Russia, the [email protected]. White sea coast; 2. Moscovites (n=146) from Moscow, Central Russia. We determined certain differences between HLA-profiles of the examined HLA genes allele distribution has been studied in Mediterranean and sub- population groups. The present study adds for the comparative analysis three Saharan populations. Their relatedness has been tested by genetic distances, more Russian population groups. They are as follows: 3. Russians (n=126) neighbour-joining dendrograms and correspondence analyses. The population from Kostroma region, the Volga middle basin; 4. Russians (n=46) from genetic relationships have been compared with the history of the classical Astrakhan, the Volga lower basin; 5. Russians (n=102) from Udmurtia, Middle populations living in the area. A revision of the historic postulates would have Ural. to be undertaken, particularly in the cases when genetics and history are overtly We performed low resolution typing of HLA-DRB1 genes and high resolution discordant. HLA genomics shows that: 1) Greeks share an important part of typing of HLA-DQA1, and DQB1 genes using SSP method. “Arlequin” their genetic pool with sub-Saharan Africans (Ethiopians and west Africans) software was applied to estimate haplotypes and FST haplotypic frequencies. also supported by Chr 7 Markers. The gene flow from Black Africa to Greece We revealed insignificant differences between Moscovites and both Russians may have occurred in Pharaonic times or when Saharan people emigrated after from Astrakhan and from Udmurtia, but significant differences between the present hyperarid conditions were established (5000 years B.C.). 2) Turks Moscovites and Russians from Kostroma region as well as from Pomors, in (Anatolians) do not significantly differ from other Mediterraneans, indicating particular. Pomors most differ from Astrakhan Russians. The differences that while the Asians Turks carried out an invasion with cultural significance between Pomors and both Russians from Udmurtia and from Kostroma region (language), it is not genetically detectable. 3) Kurds and Armenians are were equal. Both Russians from Udmurtia and from Kostroma region genetically very close to Turks and other Middle East populations. 4) There is significantly differ from Astrakhan Russian but did not significantly differ no HLA genetic trace of the so called Aryan invasion, which has only been between themselves. defined on doubtful linguistic bases. 5) , including , are related The obtained data suggest that Russians from different regions of the country to north-African Berbers. 6) Present-day Algerian and Moroccan urban and as well as other population groups could bear genetic features of neighboring country people show an indistinguishable Berber HLA profile. populations (Tatar, Udmurt, Mari, Bashkir, etc.). In our further studies we intend to perform HLA-genotyping of other nationalities neighboring Russians.

Genes and Immunity Abstracts S30

116 117 MICROSATELLITE POLYMORHISM IN THE HEME EVIDENCE FOR AN ANCESTRAL RECOMBINATION OXYGENASE-1 GENE PROMOTER IN A GERMAN BETWEEN THE HLA-DR AND HLA-DQ LOCI CAUCASIAN POPULATION. Thomas M.C. Binder1, Julia M. Krizak1, Cornelia Brendel2, Ulrich Lemonia Skoura1, Asimina Fylaktou1, Daniel Czachurski2, Pascal Finckh3, Peter Kühnl1, Thomas H. Eiermann1 1)Institute of Transfusion Medicine and Transplantation Immunology Berberat2, Zafiris Polymenidis1, Gehard Opelz2, Joannis & 3)Institute of Human Genetics, University Hospital Hamburg- Mytilineos2. Tranplant Immunology Laboratories of the Eppendorf, 2)Hospital for Haematology, Oncology and Immunology, 1Hippokration Hospital, Thessaloniki, Greece and the 2University University Marburg; Germany of Heidelberg, Heidelberg, Germany. The limited diversity of haplotypes encompassing HLA-DR and HLA-DQ Heme oxygenase-1 (HO-1), a key enzyme in heme catabolism, functions as a indicates an extremely low recombination rate between these genes. A most stress-rensponsive protein providing cellular protection against heme- and non- recent recombination event has been reported in the literature only once before. heme-mediated oxidant injury. A (GT)n dinucleotide repeat has been identified On the occasion of HLA-typing of potential stem cell donors, we have in the 5' -flanking region of the HO-1 gene. This repeat shows a length identified segregation of the unusual HLA haplotype A24, B7, DRB1*1501, polymorphism that may modulate the level of gene transcription and which was DQA1*05, DQB1*0201 in an East Frisian family. This haplotype has not been described to be associated with disorders connected to the development of observed previously and most likely reflects a rare ancestral recombinational oxidative stress, such as cardiovascular diseases. event between the HLA-DR and the HLA-DQ loci. In order to define the allele and genotype distribution in a German Caucasian To verify this unusual haplotype, about 30 members of the 4 generation family population, genomic DNA of 200 healthy, unrelated individuals was amplified were typed for HLA class I by the lymphocyte microcytotoxicity test (LCT). by PCR with a fluorescently labeled sense primer and a non labeled antisense Polymerase chain reaction (PCR) with sequence-specific primers (SSP), primer. PCR products were subsequently analyzed with a laser-based automated sequence-specific oligonucleotide probes (SSO), and sequence based typing DNA sequencer (ABI 3100). The number of repeats for each individual was (SBT) were used for HLA class II typing. calculated by the GeneScan software. The results confirmed the unusual haplotype in several of the family members. Following reports in the literature, HO-1 (GT)n alleles were grouped into three subclasses: M alleles (= intermediate HO-1 induction - 25-29 repeats) were found to be the most common subclass with a frequency of 54,2 % (n=217), followed by the high HO-1 inducing S alleles (<25 repeats - 34,8 %, n=139), and the low HO-1 inducing L alleles ( 30 repeats - 11,0 %, n=44). Our results may provide a useful reference for association studies between HO-1 microsatellite polymorphisms and diseases.

118 119 ANALYSIS OF HLA HAPLOTYPE FREQUENCIES IN 906 BONE HLA GENES IN PALESTINIANS. MARROW DONORS FROM NORTHERN GREECE Antonio Arnaiz-Villena, Nagah Elaiwa#, Carlos Silvera, Ahmed Panagiota Lazidou, Eleni Papakyriazi, Euthimia Parapanissiou, Rostom#, Juan Moscoso, Eduardo Gómez-Casado, Luis Allende, Lemonia Skoura, Soultana Lalanga, Zafiris Polymenidis Pilar Varela, Jorge Zamora, Ernesto Lowy, and Jorge Martínez- Tissue typing Lab & Immunology Dpt.Hippokration Hosp. Laso Thessaloniki, Greece Department of Immunology and Molecular Biology, Hosp. 12 de We estimated HLA allele (AF) and haplotype frequencies (HF) in 906 healthy Octubre, Universidad Complutense, Madrid, Spain. E-mail: individuals from Bone Marrow Donors Registry GR-2. These individuals were [email protected]. selected out of a total of 2500 donors, based on their HLA –A,B,DRB1 #Laboratories & Blood Bank,El-Shifa Hospital, Gaza, Palestine. phenotype, assigned by molecular typing (PCR-SSP). The analysis was performed using the HFES program ver.3.00. Some modifications were made to The genetic profile of Palestinians has, for the first time, been studied by using the information file used for the allele and haplotype frequency estimation. (HLA) gene variability and haplotypes. The The most frequent HLA alleles are A*02, B*35 and DRB1*11 with AF comparison with other Mediterranean populations by using neighbor-joining 25.5%, 20.1% and 27.8% respectively. When two locus HF and Linkage dendrograms and correspondence analyses reveal that Palestinians are Disequilibrium (∆) values were estimated, significant differences were observed genetically very close to Jews and other Middle East populations, including between the observed and expected frequencies in the following haplotypes: Turks (Anatolians), Lebanese, Egyptians, Armenians and Iranians. Archaeologic A*02-B*51 HF 6.1% ∆=2.388, A*01-B*08 HF 2.03% ∆=1.5, A*02-B*18 HF and genetic data support that both Jews and Palestinians came from the ancient 4.07 ∆ =1.41, A*24-B*35 HF 4.09% ∆=1.32. Considering HLA A-DRB1 Canaanites, who extensively mixed with Egyptians, Mesopotamian and haplotypes, A*01-DRB1*0301 (HF 2.03%) had the highest ∆ value (1.27) while Anatolian peoples in ancient times. The relatively close relatedness of both Jews B*08-DRB1*0301 (HF 3.7 ∆=3.4), B*18-DRB1*11 (HF 5.3 ∆=2.41) and and Palestinians to western Mediterranean populations reflects the continuous B*35-DRB1*11(HF 7.10 ∆=1.50) found in strong linkage disequilibrium in circum-Mediterranean cultural and gene flow that have occurred in prehistoric HLA B-DRB1 haplotype study. The most frequent HLA A-B-DRB1 haplotypes and historic times. This flow overtly contradicts the demic diffusion model of are A*02-B*18-DRB1*11 (HF 3.367% ∆=2.55), A*24-B*35-DRB1*11 (HF western Mediterranean populations substitution by agriculturalists coming from 2.75 ∆ =1.88), A*02-B*51-DRB1*11 (HF 2.45 ∆ =1.54) and A*01-B*08- the Middle East in the Mesolithic-Neolithic transition. DRB1*0301 (HF 2.06% ∆=2.02). The study of HLA diversity as well as the linkage disequilibrium between HLA alleles on chromosome 6, have a variety of applications and will serve as reference for donor/recipient matching in organ and bone marrow transplantation.

Genes and Immunity Abstracts S31

120 121 MHC-G GENE IN HUMANS AND OTHER PRIMATES: THE HLA GENES IN THE CHUVASHIAN REGION POPULATION COMPLETE PROTEIN IS NOT NECESSARY FOR SURVIVAL. FROM EUROPEAN RUSSIA: ADMIXTURE OF CENTRAL EUROPEAN AND MEDITERRANEAN POPULATIONS. Antonio Arnaiz-Villena, Mª José Castro, Jorge Martínez-Laso, Pablo Morales, Juan Moscoso, Pilar Varela, Eduardo Gómez-Casado, Jorge Zamora, Ernesto Antonio Arnaiz-Villena1, Jorge Martínez-Laso1, Juan Moscoso1, Gregory Lowy, and Luis Allende. Livshits2, Jorge Zamora1, Eduardo Gómez-Casado1, Ernesto Lowy1, Carlos Department of Immunology and Molecular Biology, Hosp. 12 de Silvera-Redondo1, Kristin Melvin3, José Manuel Martín-Villa1, and Michael Octubre, Universidad Complutense, Madrid, Spain. E-mail: Crawford3 [email protected]. 1Department of Immunology and Molecular Biology, Hosp. 12 de Octubre, Universidad Complutense, Madrid, Spain. E-mail: [email protected]. The study of the MCH-G gene evolution during nearly 50 million years does not 2Department of Anatomy, Sackler Faculty of Medicine, Tel-Aviv, Israel support a role for the full molecule. New World monkeys MHC-G-like proteins 3Laboratory of Biological Anthropology, Department of are most probably classical presenting molecules, Old World Cercopithecinae Anthropology, University of Kansas, Lawrence, Kansas 66045. monkeys do not have a full MHC-G molecule and human individual homozygous for the HLA-G null allele are healthy and do not show birth HLA alleles have been determined for the first time in individuals pathologies. This is in contrast with “in vitro” studies which show a role for the from the Chuvashian population by DNA typing and sequencing. HLA-G protein. Some of the possible isoforms may suffice for functionality. HLA-A, -B, -DR, -DQ allele frequencies and extended haplotypes have been also determined, and the results compared to Central Europeans, Siberians and other Asians, Caucasians, Middle East people and Mediterraneans. Genetic distances, neighbor-joining dendrograms and correspondence analysis have been performed. Present day Chuvash speak an Altaic-Turkic language and are genetically related to Caucasians (Georgians), Mediterraneans and Middle East populations and not only to Central or Northern Europeans; Chuvash contain little indications of Central Asian- Altaic gene flow. Thus, present-day Chuvashes who speak an Altaic-Turkic language are probably more related to ancient Mesopotamian-Hittites and northern European populations than to central Asia-Altaic people.

122 123 CYTOKINE GENE POLYMORPHISM IN GREEK POPULATION HLA-DPB1-GENE POLYMORPHISM IN RUSSIA POPULATION

Panagiota Lazidou, Heleni Papakyriazi, Euthimia Parapanisiou, Lemonia Elena Groudakova, Ilya S, Dmitry T., Leonid V., Aligeydar R., Skoura, Soultana Lalanga, Zafiris Polymenidis Leonid A. Moscow Tissue typing &Immunology Dpt, Hippokration Hosp. Thessaloniki, Greece The purpose of our research was to study the HLA-DPB1 gene polymorphism in Russian population. We conducted analysis of the HLA-DPB1 alleles Cumulative evidence indicates the functional importance of several determined among healthy Russian subjects from Moscow. For “high polymorphisms of cytokine and their receptor genes. SNPs in regulatory regions resolution” typing method we used cycle sequencing of previously amplified or in coding regions can influence cytokine production levels while their likely DNA. The results’ detection was performed on automatic sequencing system involvement in human autoimmune diseases is suggested. The aim of this study “OpenGene” (“Visible Genetics, Ink.”, Canada). α was to determine the distribution of the SNPs of the following cytokines: IL-1 , The analysis on the DPB1 locus diversity revealed the following features of β β γ β α IL-1 , IL-1R, IL-1RA, IL-4, IL4-RA, IL-12 , -IFN, TGF- , TNF- , IL-2, I, DPB1 alleles’ distribution among the given Russian population group: IL-6 and IL-10. DNA samples from 104 healthy Greek individuals were the most frequent alleles were determined as follows: *0401 (39%), genotyped by PCR-SSP with kits from Heidelberg University. The most *02012(17,8%), *0402(12,3%), *0301(7,5%), *01011(4,8%). Such allelic frequent genotypes are shown in table below. distribution of DPB1 locus is actually characteristic for all studied Caucasian population groups. However the determined distribution of DPB1 specificities IL- 1A -889 IL- 1B -511 IL-1B +3962 IL-1R 1970 IL-1RA 11100 in Russian population is more alike to the data obtained among South Europeans C/C 53.3% C/C 47.7% C/C 57.7% C/C 47.1% TT 58.5% (Bulgarian, French). The results of our study do not yet show the likeness C/T 42.26% C/T 45.42% C/T 39.5% C/T 45.77% C/T 39% between Russian population and North Europeans whereas the previously IL- 12 -1188γ -IFN 5644 TGF- βCdn10 TNF IL-2 conducted analysis on DRB1 locus and DRB1-DQA1-DQB1 haplotypic A/A 21.78% A/A 36.72% Cdn25 -308,-238 -330,+160 combinations in Russian population revealed the distribution of DRB1 A/C 12.44 A/T 49.56% CG/TG 45% AG/GG 21.51% GG/GG 16.8% specificities and three-loci haplotipic combinations to correspond on whole to T/T 16.72% TG/TG 25.5% GG/GG 77% GG/TG 34.1% the data obtained both on Middle European and South European populations (Pole, French) as well as to North Europeans (Orcadian, German, English) IL-4 IL-6 -174, 565 IL-10 IL-10 IL 4RA+1902 populations of the Caucasian race. -1098,-590,-330 CA/GG 32.6% -1082,-819.-590 A/A 76.9% The obtained data could be further implied in establishing data base for bone GCC/TCC 22.1% GG/GG 59.8% ACC/ACC 13.9% ACC/GCC 26.7% A/G 24.1% marrow donors. TCC/TCC 36.8% ACC/ATA 20.4% ATA/GCC 19.6%

This analysis will be important for subsequent studies of association between cytokine genotypes and immune response in bone marrow or solid organ transplantation as well as some disease susceptibility and severity.

Genes and Immunity Abstracts S32

124 125 HLA ALLELES IN ISOLATED POPULATIONS FROM NORTH HLA-DPB1* POLYMORPHISMS IN GREEK POPULATION SPAIN: ORIGIN OF THE BASQUES AND THE ANCIENT IBERIANS. Katerina E. Tarassi, Diamanto I. Kouniaki, Theophilos I. Athanassiades, Chryssa A. Papasteriades. Pablo Sánchez-Velasco2, Eduardo Gómez-Casado1, Jorge Martínez-Laso1, Immunology - Histocompatibility Dept, Evangelismos Hospital, Juan Moscoso1, Jorge Zamora1, Ernesto Lowy1, Carlos Silvera-Redondo1, Athens, Greece. Alberto Cemborain2, Francisco Leyva-Cobián2, and Antonio Arnaiz-Villena1. 1Department of Immunology and Molecular Biology, Hosp. 12 de Octubre, Since the number of HLA-DPB1 identified alleles is continuously increasing Universidad Complutense, Madrid, Spain. E-mail: [email protected]. and their clinical relevance, particularly in Bone Marrow Transplantation, is 2Department of Immunology. Hospital Universitario Marqués de established, the aim of this work is to present the HLA-DPB1 polymorphism in Valdecilla. 39008 Santander, Spain. Greeks. The population studied consisted of 140 unrelated, unselected healthy individuals of Greek origin. Sixty-eight (68) of them were typed for HLA-A, -B, -DRB1, -DQA1 and -DQB1 alleles have been studied in three DPB1*0101-1901 using oligonucleotide typing, whereas 72 were typed for all relatively isolated populations of northern Spain from Cantabria (Pas Valleys known DPB1 alleles by PCR-SSOP and/or PCR-SSP. inhabitants or Pasiegos and Cabuernigos) and from the Basque Country (Arratia The DPB1* alleles detected are presented in the following table: Valley inhabitants). These populations have been compared to other neighbouring ones and other Mediterraneans by using neighbor-joining DPB1 Freq % DPB1 Freq % DPB1 Freq % dendrograms, plane genetic distances, correspondence and HLA haplotype (N=140) (N=140) (N=72) analyses. These populations are more related each other than to Spaniards and 0101 7.1 1101 5.7 2001 1.4 also are genetically close to North Africans. Pasiegos show a slight degree of 0201 40.0 1301 5.0 3101 1.4 admixture with central and western Europeans. The most feasible origin of these 0301 7.1 1401 1.4 3301 1.4 populations is regarded as being a remnant of the primitive Spanish population 0401 58.6 1601 2.1 3501 2.8 (ancient Iberians) or otherwise of North African refugees that could have 0402 33.6 1701 3.6 3701 1.4 occupied the area before their expulsion by the Catholic Kings after 1492 AD, 0501 1.4 3901 1.4 when North Africans definitively were expelled. The first theory is more favoured since there are no records of a permanent Muslim invasion in these 0801 0.7 5001 1.4 areas during Middle Ages. 0901 0.7 5101 1.4 1001 4.3 6001 1.4 Conclusions: In comparison to the data from the 11th and 12th IHW 1) DPB1*0201, 0401, 0402 are the most frequent alleles in Greeks, as in other Caucasians, but DPB1*0201 shows the highest frequency among all populations studied, 2) DPB1*1301, characterizing Blacks and Orientals, is presented in Greeks with the highest frequency among Caucasians (except Gypsy Spanish). So, it seems that DPB1* alleles distribution in Greeks show certain peculiarities, as we have also found in other HLA loci studied.

Poster Group 9 - Immunogenetics of Disease 126 127 THE ORIGIN OF SOUTHERN SIBERIA TUVINIANS: A COORDINATED DOWNREGULATION OF APM COMPONENTS POPULATION WITH BOTH ORIENTAL AND CAUCASOID IN MHC CLASS I-DEFICIENT METASTATIC TUMOUR CHARACTERISTICS. VARIANTS IMMUNOSELECTED BY T LYMPHOCYTES

Jorge Martínez-Laso2, Marina Sartakova1, Luis Allende2, Marisol Martinez, Angel M. Garcia-Lora, Ignacio Algarra*, Eva Jimenez and Vladimir Konenkov1, Juan Moscoso2, Carlos Silvera-Redondo2, Federico Garrido Serv. Analisis Clinicos, Hospital Univ. Virgen de las Nieves, Arancha Pacho2, Jorge Zamora2, Ernesto Lowy2, Eduardo Granada, Spain. *Dep. Ciencias de la Salud, Univ. de Jaén, Spain Gómez-Casado2, and Antonio Arnaiz-Villena2 1 Laboratory of Immunogenetics, Institute of Clinical Immunology, 630091 Our previous studies with the GR9 tumour model had shown that the MHC class Novosibirsk, Russia I phenotype of the metastatic tumour variants differs depending on the immune 2 Department of Immunology and Molecular Biology, H. 12 de status of the host: MHC class I-negative in immunocompetent BALB/c mice Octubre, Universidad Complutense, Madrid, Spain. E-mail: and MHC class I-positive in immunodeficient (nu/nu) mice. The results showed that in this tumour model the major factor contributing to the appearance of [email protected]. MHC class I-negative tumour variants is T cell immunoselection. HLA class I and class II alleles have been studied for the first time in the To explore the molecular mechanisms involved in the origin of these MHC Turkish-speaking Tuvinian population, which lives in Russia, North of class I-deficient tumour variants in our fibrosarcoma model, we analyzed the Mongolia and close to the Altai mountains. Comparisons have been done with expression of different components of the MHC class I antigen-processing about 11000 chromosomes from other worldwide populations, and extended machinery (APM). haplotypes, genetic distances, neighbour joining dendrograms and The MHC class I stability assays at 26ºC in the presence of exogenous 2- correspondence analyses have been calculated. Tuvinians show an admixture of microglobulin showed a partially recovered the cell surface expression of H-2 Mongoloid and Caucasoid characters, the latter probably coming from the class I molecules. Semiquantitative RT-PCR of different APM components ancient Kyrgyz background or, less feasibly, more recent Russian Caucasoid showed the constitutive expression of TAP-1, TAP-2, LMP-2, LMP-7, LMP-10, admixture. However, Siberian population traits are not found and thus Tuvinians tapasin and calnexin was downregulated in the B9 tumour clone as well as in all are closer to Central Asian populations. Siberians are more related to Na-Dene metastatic nodes obtained from immunocompetent BALB/c mice. Interestingly, and Eskimo American Indians; Amerindians (from nowadays Iberian-America) this was not the case for the nu/nu BALB/c-derived metastases. In conclusion, a are not related to any other group, including Pacific Islanders, Siberians or other coordinate transcriptional downregulation of multiple components of the APM American Indians. The `more than one wave' model for the peopling of the is responsible for the absence of H-2 class I antigen surface expression in Americas is supported. metastatic nodes generated in immunocompetent BALB/c mice.

Genes and Immunity