Abstracts S14 Poster Group I – Immunogenetics

1 2 EFFECT OF NIGELLA SATIVA (BLACK SEED) OIL ON A NOVEL HLA-B*44 ALLELE IDENTIFIED IN THE CZECH CYTOKINE PRODUCTION OF SPLENIC MONONUCLEAR FAMILY AND CONFIRMED BY SEQUENCING-BASED TYPING CELLS IN ALLERGEN SENSITIZED MICE Frantisek Mrazek*, Ingrid Fae¶, Zuzana Ambruzova*, Edgar Faber+, Suna Büyüköztürka, Aslý Gelincika, Ferhan Özþekera, Sema Gençb, Karel Indrak+, Gottfried F. Fischer¶ & Martin Petrek* Fatma Oðuzc, Bayram Kýrand, Gaye Yýllard, Sacide Erdene, Filiz Aydýnc, *Tissue Typing Laboratory, Department of and +Department Mahmut Çarinc. Istanbul University, Istanbul Faculty of Medicine, of Haemato-oncology, Medical Faculty, Palacky University Olomouc, Department of Allergy and Clinical Immunology Czech Republic; e-mail: [email protected] Background: Nigella sativa is known to have benefi cial effects on a wide ¶Institute for Blood Group Serology, University of Vienna, Austria range of diseases including asthma. But its mechanism of action in asthma A novel HLA-B*44 allele was identifi ed in a patient with leukaemia and other allergic diseases is not clear. (Caucasoid, Czech ancestry) and his mother during an intrafamilial donor Objective: This study was planned to evaluate the effects of N. Sativa on search for haematopoietic stem cell transplantation. The presence of a novel cytokine production of splenic mononuclear cells in ova-sensitized mice. allele was initially proposed after the class I typing of the immediate family. Methods: Nineteen two-month-old BALB/c mice were given 0.3 ml of There was a discrepancy between HLA-B types obtained by serology and N.sativa oil by oro-eosophageal cannula once a day for a month. The control DNA techniques in the patient and his mother. PCR-SSP typing at “two group consisting of 10 mice took 0.3 ml 0.9 % saline solution by the same digits” level revealed B*44 allelic group whereas by serology it seemed route for the same period. On the third week of the study, all mice were more like B21. PCR-SSO typing resulted in an unique reaction pattern. sensitized by means of intraperitoneal injections of 20 µg of ovalbumin “Four digits” typing of B*44 by PCR-SSP was not matching to any known (OVA-Grade VI, Sigma). Ova injections were repeated three times with allele either. Sequencing based typing of the HLA-B locus revealed a novel 7 day intervals. One week later from the last injection, all mice were sacrifi ed by means of cervical dislocation. Then the splenic mononuclear allele identical with B*4405 except one base exchange located within the cells (MNCs) of mice were cultured with OVA or Concavalin A (Con-A). exon 3. Further serological testing using another panel confi rmed that the From the culture supernatants, IL-4, IL-10 and IFN-γ were assessed by novel B*44 allele shows unusual serological reactivity: positive reactions means of ELISA. were observed rather with “broad” B21 sera than with B44 sera. Results: The cytokine production of splenic MNCs of mice that were Here we report a novel HLA-B*44 allele identifi ed in the Czech family. given N.sativa oil for 30 days was not signifi cantly different than those that The sequence of the novel allele will be submitted to the EMBL took saline solution instead. database and to the WHO Nomenclature Committee. Conclusion: N.sativa oil seems not to have an immunomodulatory effect Supported by the Czech Govt. funding MSM6198959205. on Th1 and Th2 cell responsiveness to allergen stimulation. Certainly, the other probable ways of action of N.sativa on various diseases and asthma need to be clarifi ed.

3 4 CHARACTERIZATION OF A NOVEL ALLELE HLA-CW*160102 IDENTIFICATION OF A NOVEL HLA-DRB1*14 ALLELE BY Sonia Nesci, Ornella Buffi , Anca Iliescu, Michele Luchetti*, Teresa Sista, SEQUENCE-BASED TYPING Gabriele Rinaldi Thomas Karvunidis, Pavel Jindra, Alexandra Dorner, Gotfried Fischer, Laboratorio di Tipizzazione Tissutale, Azienda Ospedale San Salvatore, Vladimir Koza Via Lombroso, 61100 Pesaro, Italy Department of Hematology and Oncology, University Hospital Pilsen, *Istituto di Clinica Medica - Ematologia ed Immunologia Clinica, Polo Pilsen, Czech Republic Didattico, Ancona Forty-eight different amino acid variants of the HLA-DRB1*14 allelic A novel HLA-Cw allele was identifi ed during a routine HLA analysis group are known currently (10/2004). Here we report the identifi cation of of an Italian donor selected from Italian Bone Marrow Donor Registry a new HLA-DRB1*14 allele in 21-years-old healthy Caucasoid woman. for a Talassemic patient proposed for an unrelated bone marrow The allele was detected by sequence-base typing after receiving an atypical transplantation. PCR-SSP result pattern during the routine HLA class II typing of a new DNA was isolated from peripheral blood and the sequence of HLA-C healthy bone marrow donor from Czech National Marrow Donors Registry. exons 1, 2 and 3 was performed with primers located in 5’ UT region and When compared to all of known HLA-DRB1*14 alleles we have found two 3’ intron 3. The purifi ed PCR product was cloned into the pDrive Cloning nonsynonymous and one silent nucleotide substitutions within the exon 2 Vector (Qiagen). Nine individual clones were obtained and sequenced. of the novel allele. The fi rst nucleotide substitution (C→T) at position 181 Six out nine were assigned to Cw*160102 and all showed the presence of (cod 32) results in amino acid exchange of Tyrosine replacing Histidine. a single nucleotide substitution at codon 67 (CCG vs CCA) in the exon 2. The second substitution (T→A) is localized at position 197 (cod 37) where The difference at position 201 did not cause amino acid change. Tyrosine replaces Phenylalanine. Finally, the silent (G→A, CAG→CAA respectively, encoding Glutamine) is localized at position 189 (cod 34). The both nonsynonymous substitution are localized at - binding groove of β-chain of HLA-DRB1 peptide. Our sequencing data were confi rmed by SBT performed at the HLA typing laboratory in Vienna, Austria. The offi cial assignment of the novel HLA-DRB1*14 allele by the WHO Nomenclature Committee is in the progress now.

Genes and Abstracts S15 5 6 CCR5 GENE EXPRESSION AND EBV LOAD IN PATIENTS WITH DRB1*1211: A NOVEL HLA-DRB1*12 ALLELE HAEMATOLOGICAL DISORDERS: PRELIMINARY STUDY Peter A. Horn, David DeLuca, Murielle Verboom, Kerstin Müller, Emilia Jaskula, Katarzyna Bogunia-Kubik, Andrzej Lange Pavel Jindra, Vladimír Koza, Rainer Blasczyk Institute of Immunology and Experimental Therapy / Lower Silesian Department of Transfusion Medicine, Hannover Medical School, Center for Cellular Transplantation, Wroclaw, Poland Hannover, Germany Epstein-Barr (EBV) infection is a serious complication in the Currently 10 different amino acid variants of the HLA-DRB1*12 family recipients of allogeneic haematopoietic stem cell transplants (HSCT). The are known. We report here the identifi cation of a new HLA-DRB1*12 allele recent reports have suggested a potential role of chemokine receptor CCR5 in a healthy Caucasian. The new allele was detected by sequencing-based in perpetuation of viral infection. In the present study the level of CCR5 typing (PCR-SBT) during routine high resolution typing of a male potential gene expression was related with a number of EBV copies in 23 patients unrelated donor from the Czech National Marrow Donors Registry. suffering from haematological disorders. At the same time points, the viral Compared to DRB1*120101, to which it is closest, the new variant load and the numbers of mRNA CCR5 copies were assessed employing is characterized by a nonsynonymous nucleotide exchange (T→C) at real-time PCR technique. In addition CCR5delta32 polymorphism was nucleotide position 126 of exon 2, which was previously considered a analysed. constant position, indicating that it is likely to be caused by a single point The presence of the CCR5delta32 allele (associated with defective CCR5 mutation. It results in the amino acid exchange Phe→Leu at position 47 of expression) was found only in one patient. This patient lacked EBV infection the mature polypeptide, which is part of the peptide binding site at pocket E. although he presented with over 298 000 copies of CCR5 mRNA/100000 Until now, only phenylalanine and tyrosine have been observed at this cells of peripheral blood. The higher numbers of EBV copies were detected position across all of DRB. This substitution could directly affect position 7 in patients with enhanced CCR5 gene expression. Among 15 patients with of a 9-mer peptide. Furthermore, this residue appears to serve as a keystone, EBV infection, 14 had more than 50 000 copies of CCR5 mRNA/100 000 joining both alpha helices and the beta sheet. Thus, the effective shortening cells as compared to 3 out of 8 patients lacking EBV infection (having of phenylalanine to leucine at position 47 could have a general effect on the less than 10 of EBV DNA copies/100 000 cells) (0.93 vs 0.38, p=0.009). binding pocket. Accordingly, peptide presentation can be expected to be No association was observed between the polymorphism and expression substantially altered compared to DRB1*1201. of the CCR5 gene. It appeared that patients with increased CCR5 gene The name DRB1*1211 has been offi cially assigned by the WHO expression more frequently presented with elevation of EBV copies. These Nomenclature Committee in December 2004 and the nucleotide sequence is results might suggest that the expression of functional CCR5 plays a role available in the EMBL and GenBank Nucleotide Sequence Database under in initiation/perpetuation of EBV infection. This work was supported by the accession number AJ870921. ALLOSTEM grant.

7 8 UNEXPECTENCIES IN INHERITANCE OF HLA-A19 SPECIFICITY REFERENCE STRAND CONFORMATION ANALYSIS FOR Yulia Zaretskaya, Svetlana Aleshenko, Yury Lednev, Tamara Tehugreeva, DETERMINATION OF HLA POLYMORPHISMS Zemphira Mamilliaeva. Research Center for Hematology, 1Libor Kolesar, 1Peter Novota, 2Eva Ivaskova, 1Antonij Slavcev 4A Novozykovsky lane, Moscow, Russia 1Dept. of Immunology, 2Czech Bone Marrow Donor Registry Classic states that fusion of gametes into a zygote is a random event Institute for Clinical and Experimental Medicine (IKEM) where the chances of all gametes are equal. Following analyzing The methods used for analysis of HLA gene polymorphisms differ of the HLA-A19 splits (A29, 30, 31, 32, 33) it has been established that according to their resolution capacity. We compared the resolution of gametes carrying the A19 splits have an advantage in genetic transmission Reference Strand Conformation Analysis (RSCA) with that of three other compared to the alternative haplotype in the same HLA-phenotype. The 28 methods, i.e. Sequence Specifi c Oligonucleotide Probes (SSOP), Sequence families were typed serologically and by PCP-SSP on purpose of allogeneic Specifi c Primers (SSP) and Sequence Based Typing (SBT). BMT to the leukemia patients who were among siblings in the families Ninety-six healthy individuals were typed. Fifty-four individuals where one or both parents were carrying “A19”. Among 46 offsprings 34 were typed for HLA-A and -B loci at low resolution (LR) – by SSP and expressed “A19”, and 12 children were “A19”-negative (with regard to SSOP - and at high resolution level (HR) by SSP and RSCA. The other 44 leukemia persistence, 28 persons were sick and 18 were healthy). The ratio individuals were typed for DRB1 and DQB1 loci at LR level and as well at of alternative shares (“A19”-positive, 73.9% vs “A19”-negative, 26.1%) HR level by SSP and SBT. Polymorphisms in exon 2 of HLA class II genes can be considered as an example of Bernoulli’s binomial distribution. and in exons 2 and 3 of class I genes were defi ned. Calculation of standard deviations has shown that this ratio is signifi cant We found a 100% concordance between the SSP and SSOP typing results with 95% probability. Comparison of “A19” inheritance in leukemia at the LR level of all (A, B DRB1 and DQB1) loci. HR typing of class I by affected and healthy offsprings has revealed no signifi cant difference RSCA revealed 87% concordance with the SSP method for locus A and (χ2=0.81; p>0.05) suggesting that “A19” was preferably inherited by both 100% concordance for locus B. HR typing of class II by SBT reached the sick and healthy siblings. The second study comprised 22 families where 100% concordance with SSP results in all tested loci. only one haplotype of the four parental haplotypes carried A19. Among 37 Conclusion: To our needs the RSCA method was not successful in HR offsprings, 25 individuals inherited “A19” (67.6%), while 12 (32.4%) were typing, while by combining the SSP and the SBT system we are able to “A19”-negative. Difference in “A19” alternative shares was also signifi cant. distinguish a vast majority of HLA alleles. Third study included one family where 4 offsprings have acquired A31-B51, while just one offspring has received the alternative A26-B27 haplotype from the mother. Essence of the discovered phenomenon is in natural selection which exists at the level of the gametes fusion. Selection is supposedly controlled by genes encoding some HLA-specifi cities, most likely the A19 splits.

Genes and Immunity Abstracts S16 9 10 IDENTIFICATION OF A NOVEL HLA-B*07 ALLELE -DRB ANALYSIS OF DIFFERENT NON-HUMAN PRIMATE Peter A. Horn, Anja Hundt, Kerstin Müller, Rainer Blasczyk SPECIES BY DENATURING GRADIENT GEL ELECTROPHORESIS Department of Transfusion Medicine, Hannover Medical School, (DGGE) Hannover, Germany Annemiek JM Rouweler, Gaby GM Doxiadis, Nanine de Groot, We here report the identifi cation of a new HLA-B*07 allele in a healthy Ronald E Bontrop, Biomedical Primate Research Centre, Rijswijk, male Caucasian. The new allele was initially typed as B*0713 by PCR- The Netherlands SSP. Because of the infrequence of that allele, a sequencing-based typing In contrast to humans, -DRB loci of rhesus monkeys and other macaques (PCR-SBT) was performed to confi rm that result. This yielded the detection are known to be not only polymorphic but also polygenic. Thus, simple of the novel allele. Surprisingly, it is closest to B*070201, while it differs typing techniques as direct sequencing of the most polymorphic exon 2 from B*0713 in 12 positions in exon 2. Compared to B*070201, the are not applicable for -DRB typing of those species. Denaturing gradient new variant is characterized by a nonsynonymous nucleotide exchange gel electrophoresis (DGGE) has been proven to be a rapid and reliable (C→T) at nucleotide position 118 of exon 2. Previously this was considered technique for distinguishing -DRB patterns in macaques as well as in some a constant position, suggesting that it is likely to be caused by a single point New World monkey species. Therefore, the DGGE technique was tested for mutation. It results in the amino acid exchange Ala→Val at position 64 of its usefulness in pre-screening of -DRB exon 2 of 26 different non-human the mature polypeptide. Since this position is located in an outer loop of primate species and one Lemuroidae. From 75% of the species tested a the HLA molecule, it is highly unlikely to affect peptide binding or T-cell clear -DRB banding pattern could be distinguished. By direct sequencing receptor interaction. Thus, the newly found allele has a very low alloreactive of certain bands more than 50% could be assigned to a unique -DRB allele. potential in case of a mismatch to the most common HLA-B allele B*0702. Furthermore, most species show a complex banding pattern indicating the The nucleotide sequence has been submitted to the EMBL nucleotide presence of more than one -DRB locus per haplotype. Some species, of sequence database and is available in the EMBL, GenBank and DDBJ which several individuals have been tested, show also polymorphism of Nucleotide Sequence Database under the accession number AJ870971. -DRB loci. The data demonstrate that the technique used is applicable as pre-screening method for a variety of non-human primate species. The results allow intra-species comparisons concerning identity, diversity and/ or polymorphism, which are needed in transplantation research, analysis of immune related diseases, or colony management. Additionally, phylogenetic analyses of the resulting Mhc-DRB alleles permit inter- species comparisons that can be helpful in solving evolutionary issues.

11 12 CYTOKINE POLYMORPHISM IN PATIENTS WITH CHILDHOOD CYTOKINE GENE POLYMORPHISMS AND SUSCEPTIBILITY ACUTE LEUKEMIA TO REJECTION IN CORNEAL TRANSPLANTATION Fatma Oguz, Kursat Ozdilli, Yeliz Duvarcı, Sema Anak, Mahmut Carin, Sandra Balseiro*1,2, Paulo Santos1, Ramon Salvado3, Pedro Faria4, Gunduz Gedikoglu IU, Istanbul Medical Faculty, Department of Medical Maria Joao Quadrado4, Joaquim Murta4, Fernando Regateiro1 (1) Histocompatibility Center, Coimbra-Portugal; (2) Fac Science and Cytokines are necessary for normal hematopoiesis in the bone marrow Technology Univ. Coimbra; (3) Coimbra Hospital Center; (4) Coimbra and provide a means of fi ne-tuning bone marrow function in response to University Hospitals stimulation. Our aim is to analyze cytokine gene profi les in patients with The aim of this study was to analyse the association of cytokine gene leukemia in order to clarify the pathogenesis of acute leukemia. Childhood polymorphisms with corneal graft rejection. ALL and acute myeloid leukemia (AML) patients were diagnosed in a single In 41 individuals submitted to corneal transplantation, PCR-SSP institution. Patients meeting the standart criteria for high-risk were prepared genotyping was performed for IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, for a possible Hematopoietic Stem Cell Transplantation (HSCT). Genotyping IFN-γ, TGF-β, TNF-α, IL-1R, IL-1RA and IL-4RA using the 13th IHW was performed by PCR-SSP using in 45 childhood ALL patients (27 male, 18 tray kit (U. Heidelberg). female), 45 childhood AML patients (22 male, 23 female) and 60 (42 male, Pro-infl ammatory cytokine IFN-γ high producer genotype TT increased 18 female) unrelated healthy individuals. Genotype frequencies between the risk of acute corneal rejection (p<0.05). IL-2 genotype TG/TT, ALL,AML patients and controls were compared using Chi-Square Yates, intermediate producer, showed a signifi cant association with rejection Fisher’s Exact Tests and Stepwise Logistic Regression Analysis (SLRA). (p<0.05). High producer genotypes of IL-10 (GCC) and IL-6 (GG) seemed We found higher frequency in IL-10(GCC/ATA), TNF-a(GA) genotype to induce tolerance to the recipients, whereas the low producer genotypes polymorphism was signifi cantly higher in the patients with ALL and AML of this cytokines seemed to induce corneal rejection but lacking statistical so it may predispose to acute leukemias. TGF-b (TC/GG) genotype may signifi cance. The remaining genotypes presented similar frequencies in predispose to AML. On the other hand TGF-b(TT/GG) genotype and TNF- both groups. a(GG) genotypes might be the preventive factor for acute leukemias. Pro-infl ammatory cytokines seemed to have the most important role in GENOTYPE ALL ALL vs. AML AML vs. CONTROL n=45 CONTROL n=45 CONTROL n=60 corneal graft rejection. Cytokine genotyping could be useful to predict the n % p n % p n % risk of corneal transplantation candidates. TNF-a(GA) 14 31,1 0,003* 10 22,2 0,044* 5 8,3 TNF-a(GG) 31 68,1 0,014* 35 77,8 0,14 53 88,3 TGF-b(TC/GG) 16 25,5 0,43 22 49 0,03* 17 28,3 TGF-b(TT/GG) 14 31,1 0,81 7 15,6 0,039* 20 33,3 IL-10 (GCC/ATA) 17 37,8 0,004* 15 33 0,027* 8 13,3 We believe studies like this will eventually help to understand the pathogenesis of ALL and AML the role cytokines play in those, but larger groups of studies must be done in future.

Genes and Immunity Abstracts S17 13 14 HLA-B*8102, A NEW ALLELE FOUND IN AN EXTERNAL EXTENDED IMMUNOGENETIC PROFILES COULD BE PROFICIENCY TESTING SCHEME PREDICTIVE FOR LONGEVITY D. Kriks, I. Faé, M. Lau, C. Voorter, W.R. Mayr, G.F. Fischer, Milena Ivanova, Anastasia Myhailova, Snejina Mihailova, Penka Nikolova, Medical University of Vienna Elissaveta Naumova. Medical University, Sofi a, Bulgaria A new human leukocyte allele HLA-B*8102 has been identifi ed in a cell The aim of the present study was to determine the role of immunogenetic provided by the UCLA International Cell Exchange scheme. The nucleotide factors in successful aging and longevity. HLA and cytokine genes were sequences of exons 2 and 3 of the new allele are identical with HLA-B*8101. analyzed by PCR-SSP and PCR-SBT methods in 57 healthy elderly Four nucleotide differences, however, are observed in exon 1, three of them individuals, 12 families with longevity members and 56 unrelated young representing non-synonymous base changes. Although different from the controls from the Bulgarian population. Analysis of extended HLA HLA-B*8101 allele, the exon 1 sequence of HLA-B*8102 is found in haplotypes, defi ned by inheritance in the families, showed increased several other HLA-B alleles including HLA-B*4801. This patchwork of frequencies of protective DRB1*11 and DRB1*16 haplotypes in elderly sequence motifs suggests that the separation of B*4801 and B*8102 came individuals. No differences were observed for gene profi les of IL-6 and by a double recombination event, introducing a ‘discriminating’ exon 2 IFN-γ. Signifi cant differences were found for 2 IL-10 haplotypes with sequence in a common ancestor. In a further step, there could have been possible correlation to gene expression level. The haplotype 1082A, a recombination between exon 1 and 2 of HLA-B*8102 alleles giving rise -819C, -592C (low) was decreased, while -1082G, -819C, -592C (high) to the HLA-B*8101 allele Peptides derived from the leader sequence of was prevalent in elderly. This effect was more pronounced in homozygous certain HLA-B molecules modulate the interaction between HLA-E and the individuals. Similarly, 2 extended genotypes of TGF-B showed signifi cant inhibitory CD94/NKG2A receptor of NK cells (4). Although HLA-B*8101 differences. The genotype (c10)C/C, (c25)G/G, (-988)C/C, (-800)G/G (low) and HLA-B*8102 differ in their leader peptide sequences, it is unlikely that was decreased, while the genotype (c10)T/C,(c25)G/G,(-988)C/C,(-800) they differ in their interaction with HLA-E, because both alleles contain the G/A (high) was increased in elderly individuals. Two known polymorphisms nucleotide motif encoding the probable HLA-E binding peptide. of TNF-A: +489G/G and -1031T/T were decreased in elderly. Extended TNF-A genotypes, based on 7 SNPs, associated with longevity were identifi ed. Analysis of IL-12B genotypes, based on SNPs at positions 1188, 1217, and 2124 also showed signifi cant differences. In conclusion our results indicated the role of HLA and cytokine genes for successful aging. Extended immunogenetic profi les able to control infl ammatory status could contribute to maintaining healthy status in elderly and could be an informative predictive factor for longevity.

15 16 HLA II CLASS ALLELE DISTRIBUTION IN ADOLESCENT EXTERNAL QUALITY ASSESSMENT OF ABO BLOOD PATIENTS WITH PULMONARY TUBERCULOSIS GROUPING BY DNA-BASED METHODS Ludmila N. Bubnova, Irina E. Pavlova, Tatiana V. Glazanova, Susan Corbin, Jonathan Downing, Chris Darke Ludmila I. Archakova, Ludmila A. Skvortsova Welsh Blood Service, Pontyclun, CF72 9WB, UK Russian Research Institute of hematology and transfusiology, ABO blood grouping is easily achieved by PCR using 12 sequence-specifi c St. Petersburg Reseasrch Institute of Phthisiopulmonology primers in 8 PCR mixtures under the same conditions as routine HLA To increase the effi cacy of diagnostics and therapy of pulmonary Class I typing (EJI 2003; 30: 295). This is a convenient method of tuberculosis (PTB) the identifi cation of genes, especially HLA II class, confi rming ABO groups of, e.g., solid organ donors and has the added which determine susceptibility or resistance to tuberculous infection seems security of identifying the two principal histocompatibility systems on the of actual. same DNA sample [Vox Sang 2004; 87 (Suppl. 3): 76]. In addition, it may be The purpose of our study was to evaluate the frequency of HLA-DRB1* valuable for A1, A2 typing when considering transplanting a group O patient and DQB1* alleles in adolescent patients with pulmonary tuberculosis. We with an A2 donor kidney. The UK National External Quality Assessment have examined 38 patients with PTB aged from 21 to 55 years (32 - with Schemes for Histocompatibility and Immunogenetics endeavour to keep infi ltrative tuberculosis and 6 – with fi brocavernous tuberculosis) all of abreast of evolving practices. Accordingly, it has operated a sub-scheme whom have undergone full clinic examination at the hospital. HLA-DRB*1 for ‘ABO Grouping by DNA-Based Methods’ since 2002. This uses the and –DQB1* genes were typed by PCR-SSP method; control group for 12 whole blood samples distributed for its ‘DNA HLA Typing’ scheme. HLA-DRB1* locus consisted of 346 healthy persons, and for DQB1* locus The sub-scheme is not currently scored or assessed but allows interested – of 78 healthy persons, residents of North-West region of Russia. laboratories to compare their ABO DNA typing fi ndings with those of We have established the following statistically signifi cant differences others under the conditions of an EQA exercise. in HLA II class gene frequency in PTB patients as compared to normal In 2004 there were just six participating laboratories. All typed the 12 values: 1. Increased HLA-DRB1*08 (16.2% vs. 6.1%, chi-square=5.25); 2. samples (one sample not tested by one laboratory) whose consensus ABO Increased HLA-DRB1*09 (7.9% vs. 2.3%, chi-square=3.85); 3. Decreased genotypes were: A1A1 (n=1), A1O1 (n=3), A2O1 (n=1), O1O1 (n=4), A1B DRB1*15 (13.2% vs. 28.3%, chi-square=4.01); 4. Decreased HLA- (n=2), A2B (n=1). There was a single non-consensus fi nding: one laboratory DQB1*03 (36.8% vs. 69.5%, chi-square=6.50); Increased HLA-DQB1*04 assigned a consensus A1B sample as B1. (10.5% vs. 2.4%, chi-square=4.89). Only a minority of laboratories currently appear to use DNA-based methods Thus, our study has demonstrated the defi nite HLA II class alleles for ABO grouping. However, since this method has several important which serve as predisposing (DRB1*08, DRB1*09, DQB1*04) or advantages it should be seriously considered by clinical histocompatibility protective (DRB1*15, DQB1*03) factors in the development of pulmonary testing laboratories. tuberculosis in adults.

Genes and Immunity Abstracts S18 17 18 IDENTIFICATION OF A NEW DRB1 ALLELE IN THREE FAMILY RESULTS OF THE 9TH TRIAL OF THE HLA CLASS I MEMBERS USING SEQUENCE BASED TYPING PROFICIENCY TESTING FOR CENTRAL-EAST EUROPE Wilfried Levering, Cindy van Assenbergh, Kees Sintnicolaas. Katarzyna Bogunia-Kubik, Andrzej Lange Laboratory for Histocompatibility and Immunogenetics, Sanquin Blood Institute of Immunology and Experimental Therapy, Polish Academy of Bank South West Region, Rotterdam, The Netherlands Sciences, Wroclaw, Poland The novel allele described in this report was found in three siblings of In 2004 we organized the IX round of the Profi ciency Testing of HLA a patient considered for haematopoietic stem cell transplantation. DNA class I Typing for Central-East Europe. Five new laboratories (from was typed by PCR-SSP using DRB1 “low resolution” kits and DRB1*01 Gdansk, Kaunas, Lodz, Prague and Riga) joined our workshop. Eight blood “high resolution” kits (Olerup-SSPTM), by PCR-SSOP using DRB1 LiPA and/or 10 DNA samples were sent to 30 institutions from 10 countries (InnoLiPATM), and by SBT (Protrans S4 HLA-ModulesTM). PCR-SSP and involved in HLA typing. There were 17 Polish laboratories participated, 4 PCR-SSOP resulted in the absence of interpretation due to inconclusive from the Czech Republic, 2 from Lithuania, one from Hungary, Bulgaria, reaction patterns in both techniques. SBT identifi ed a variant DRB1*01 Croatia, Estonia, Latvia, Russia and Turkey. All participants provided HLA allele with several mismatches in exon 2. This variant is most similar to typing data. The HLA-A and B serological typing results were obtained DRB1*0111 with mismatches (G to A) at position 299, (C to G) at position from 25 institutions and for HLA-C locus from 17 laboratories. Genetical 305, (G to C) at position 307, and (C to G) at position 308. These mismatches typing of HLA-A and B was performed by 18 institutions while the HLA-C lead to amino acid changes from arginine (AGG) to lysine (AAG) at codon specifi cities were analysed with the use of DNA typing by 15 laboratories. 71, from alanine (GCC) to glycine (GGC) at codon 73, and from alanine This time a signifi cant improvement was observed with respect to the (GCG) to arginine (CGG) at codon 74. The substitutions at codons 73 and HLA class I typing serology and DNA typing. There was no requisite 74 have not been reported for any other HLA-DRB1*01 allele so far. The 75% consensus for one HLA-A (A10 subtyping) and one HLA-C antigen HLA type of the involved haplotype in the three siblings is HLA-A*0201; (the presence of C17). The other serological typing results of HLA-A, B*3501; Cw*0401; DRB1*01 variant; DQB1*0501. The sequence has B and C locus were erroneous in 4.1%, 3.1 % and 5.9%, respectively. Some been submitted to the EMBL Nucleotide Sequence Database. discrepancies appeared also in genetical typing in 1.7%, 1.2% and 2.9% for HLA-A, B and C specifi cities, respectively. Taking together discrepancies constituted less than 4% of serological typing results and only 2% of DNA typings (7.2% and 5% in the 8th round, respectively). These data confi rm the usefulness and effectiveness of the profi ciency testing exercises and imply that still there is a need for the continuation of the workshop.

19 20 CYTOKINE GENE EXPRESSION AND POLYMORPHISM IN POSITIVE ASSOCIATION OF FCGRIIA-131R AND FCGRIIIB-NA2 LIVER TRANSPLANTATION ALLELE WITH URINARY TRACT INFECTION IN CHILDREN Sandra Balseiro*1,2, Sofi a Vale-Pereira*1,2, Paulo Santos1, Rahmi Ozdemir, Afi g Berdeli, Yilmaz Tabel, Sevgi Mir, Alphan Cura Rosa Ballesteros3, Rui Perdigoto3, Fernando Regateiro1 Department of Pediatrics and Molecular Medicine Laboratory, Faculty of (1) Histocompatibility Center, Coimbra-Portugal; (2) Fac Science and Medicine University of Ege, Bornova, Izmir, Turkey Technology Univ. Coimbra; (3) Coimbra Univ. Hospitals. Backround: Receptors for the Fc fragments of immunoglobulin G In this study, we analysed the association of cytokine gene polymorphisms (IgG) (FcgRs) play a unique role in coupling the humoral and cellular and expression with human liver allograft rejection and infection. immune responses by targeting immune complexes (ICs) to effector PCR-SSP genotyping and real-time PCR relative quantifi cation of gene cells. Immunglobulin Fc Receptors (FcR) play an important role in the expression was performed in 111 orthotopic liver transplant recipients for host defense and the regulation of . These receptors have γ γ γ IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, TGF-β, TNF-α, IL-1R, three subclasses: Fc RI (CD64), Fc RII (CD32) and Fc RIII (CD16). γ γ γ IL-1RA and IL-4RA using the 13th IHW tray kit (U. Heidelberg). IgG-induced Fc RIIa (CD32a), Fc RIIIa (CD16a), Fc RIIIb (CD16b) IL-10 genotype ATA/ATA, IL-6 genotype CA/CG, low producers, IL-4 function displays heterogenity between individuals according to genetic structure and ethnicity. (-1098) intermediate producer genotype GT, and IL-2 high producer In this study we are investigated genotype distribution and allele frequency genotype GG/GG, showed a signifi cant association with acute rejection of three different FcγR genes and genotype and phenotype relation in (p<0.05). IL-10 genotype GCC/GCC, IL-4 (-1098) genotype TT, IL-6 urinary tract infections ( UTI ). GG/GG, high producers, showed a signifi cant association with tolerance Matherial and Method:154 children having a diagnosis of UTI (mean age (p<0.05). IL-10, IL-4, IL-6 and IL-2 expression were found upregulated ± SD : 7.21±4.19, girls/boys: 121/33) and 118 healthy adults as a control α β γ α in patients according to rejection. IL-1 , IL-1 (-511), IFN- , TNF- , and group were included in our study. Gram negative was the causative IL-1RA, high producer genotypes, were associated with infection (p<0.05), agent in %82 of the children with UTI. whereas IL-6 low producer genotype CG/CG was associated with protection The genetic genotyping of FcγR genes were performed by allel-specifi c from infection (p<0.001). IL-1α, IL-1β, IFN-γ, TNF-α and IL-6 expression PCR or PCR-RFLP based method from genomic DNA. ¨ were found upregulated in patients that developed infections. Results:The distribution of FcgRIIa- 131 R/R, FcgRIIa-131H/R genotype Cytokine genotyping and gene expression revealed signifi cant associations and FcγRIIa-131R allelle were found signifi cantly higher than control group with acute rejection and infection, suggesting their usefulness to predict and (p<0,05) . And also, FcγRIIIb NA2 allele were found statistically higher monitoring risk in these patients. when compared to control group (p<0,0001). There was no statistically signifi cant difference between two groups in regarding the FcgRIIIa * S.B and S.V.P. contributed equally to this work. polymorphism. Conclusion FcγRIIa and FcγRIIIb which belong to FcγR gene family, take part host’s adaptive immune response, against bacteria might be responsible of host’s sucseptibilty of UTI.

Genes and Immunity Abstracts S19 21 22 TAP1 AND TAP2 POLYMORPHISMS IN THE EASTERN A QUICK SBT METHOD FOR UNAMBIGUOUS HIGH ANDALUSIA REGION OF SOUTHERN SPAIN RESOLUTION HLA TYPING Carmen Mª Cabrera, Rocío Alvarado-Guerri, Isabel Maleno, Jason Stein, Damian Goodridge*, David Sayer*, Jan Capper, J. Ramón Vilchez, Federico Garrido, Miguel Ángel López-Nevot Malcolm McGinnis and Pete Krausa Servicio de Análisis Clínicos e Inmunologia, Hospital Universitario Virgen Atria Genetics, 884 Dubuque Ave., South San Francisco, CA, 94080USA de las Nieves. Granada. Spain *Conexio , Perth, Australia Transporter associated with antigen processing (TAP) genes are involved Continued improvements to DNA sequencing instrumentation, in the processing of endogenous peptides that bind to MHC class I commercial HLA reagents and the demand for allele level typing results, molecules. The possible functional signifi cance of TAP polymorphisms in have helped to defi ne DNA sequencing as the preferred method for HLA antigenic peptide transport is currently under debate. is typing. As this trend continues, the number of known HLA alleles continues one approach to investigating the evolutionary and functional signifi cance to rise as does the number of ambiguities among certain heterozygote allele of genetic polymorphisms. In our study, 105 unrelated individuals from combinations. Obtaining unambiguous allele level results often requires the Eastern Andalusia region of Southern Spain were analysed for TAP1 additional testing to separate the two alleles to determine the cis/trans and TAP2 polymorphisms and linkage disequilibrium between TAP1-TAP2 relationship. Two commonly used methods for accomplishing this are to and TAP1/TAP2-HLA DR, DP, and DQ genes. HLA-DR, DQ, DP, use a panel of Sequence Specifi c Primers (SSPs) or an additional group and TAP1 loci were genotyped by the PCR-SSO method, and TAP2 specifi c PCR followed by sequencing, both of which can be time consuming. alleles were typed using the ARMS-PCR technique. Comparing TAP A quicker and more convenient alternative to these methods is to use group polymorphisms of this population of Eastern Andalusia with the Spanish specifi c sequencing primers (GSSP) to generate hemizygous sequence from population, we detected the presence of alleles TAP1*D (3.3%), TAP2*D existing heterozygous PCR template. In this study we evaluated a series (2.4%), and TAP2*E (2.9%) that were not detected in the Spanish sample. of GSSP used to resolve common ambiguities at HLA-A, -B, -C, -DRB1 The genetic pool from Arab people of the Iberian Peninsula may have and -DPB1 genes. The test panel consisted of DNA samples obtained from contributed to these new TAP polymorphisms found in individuals in well characterized cells and were chosen based on known ambiguous typing Eastern Andalusia. No evidence was found in the study population of results. An initial generic PCR amplifi cation was performed followed by linkage disequilibrium between TAP1 and TAP2 or between the TAP genes generic and group specifi c sequencing. Results were analyzed with Assign and HLA-DR, DP and DQ. These results are consistent with the absence of SBT from Conexio Genomics which allows for convenient analysis of both co-evolution between TAP and MHC class II genes and with the hypothesis heterozygous and hemizygous sequences. Through the use of the software’s of selective neutrality. “resolution” function these results demonstrate the utility of GSSP and Assign SBT for resolving ambiguities.

23 24 cDRB ANALYSIS OF CALLITHRIX JACCHUS: HIGHLY ELUCIDATION OF EXON 1, 4 AND 5 SEQUENCES OF 39 CONDENSED DRB REGION CONFIGURATION POLYMOR- INFREQUENT HLA-B ALLELES PHISM Wendy TN Swelsen, Christina EM Voorter, Kang Yuen Chak and Marit v/d Wiel, Herbert P Brok, Nanine de Groot, Natasja G de Groot, Ella M van den Berg-Loonen Nel Otting, Gaby Doxiadis, Bert A ‘t Hart, Ronald E Bontrop Tissue Typing Laboratory, University Hospital Maastricht, Maastricht, BPRC, Rijswijk, The Netherlands The Netherlands The common marmoset (Callithrix jacchus), a New World monkey Currently more than 590 HLA-B alleles are identifi ed by sequence species, is often used as model for MS because of its high susceptibility analysis. Although the polymorphic exon 2 and 3 sequences of all HLA-B for experimental autoimmune encephalomyelitis. Genomic analysis of alleles are described, the sequences of the other exons of a number of Caja-DRB exon 2 revealed three loci with different degrees of infrequent B-alleles are unknown. polymorphism, which seem to be present on each haplotype. Caja-DRB1*03, In this study the exon 1, 4 and/or 5 sequences of 39 different HLA-B encoding the same EYSTS peptide binding motif as the human DRB1*03 alleles were elucidated by allele-specifi c sequencing. Overall these exon lineage, is as polymorphic as the DRB*W16 locus regarding its exon sequences showed identity with the majority of the known sequences from 2 variability. The third locus, DRB*W12, seems to be monomorphic. the corresponding allele groups, except for four alleles; B*4010, B*4415, Members of all three loci are also present on the transcript level. However, B*4416 and B*5606. The exon 1 sequence of B*4010 had nucleotide a reduced number of cDRB1*03 alleles is detected whereas the number of differences with all B*40 alleles, but was identical to the B*54, *55, *56 and cDRB*W16 alleles is comparable to the one of gDRB*W16. Furthermore, *59 allele groups. B*4416 differed from B*440201 at position 988, which 2 cDRB*W12 alleles seem to be transcribed. In contrast to the cDRB*W16 was previously considered a conserved position. B*4415 showed exon 1, alleles, most of the cDRB1*03 variants show a shorter polypeptide chain 4 and 5 sequences deviating from the other B*44 alleles, but identical to missing 8 amino acids in the cytoplasmatic domain. Therefore, we assume B*4501. The exon 1 and 4 sequences of B*5606 differed from other B*56 that DRB1*03 locus members are expressed at a low level indicating that alleles, but were in complete agreement with B*7801. The deviating exon some of these genes are pseudogenes or defunctional. From exon 2 analyses, sequences of B*4415 and B*5606 confi rmed the evolutionary origin of these the polymorphism of Caja-DRB was known to be mainly a result of alleles suggested by the sequences of exons 2 and 3. The polymorphism various exchangements of motifs, which holds true for the complete cDRB observed in exons 1, 4 and 5 merely refl ects the lineage-specifi city sequence, too. Such processes may be also the reason for the existence of of HLA-B. an additional Caja-cDRB allele with heterogeneous features, conserving the EYSTS motif of DRB1*03 in combination with characteristics of the DRB*W16 lineage. Thus, Caja-DRB haplotypes contain two to three loci, namely an oligomorphic DRB*W12, a variable DRB*W16 gene, and in some cases additionally a DRB1*03-like locus member.

Genes and Immunity Abstracts S20 25 26 ELUCIDATION OF DQA1 INTRON SEQUENCES AND CYTOKINE GENE POLYMORPHISMS CAN EFFECT HEPATIC EVOLUTIONARY IMPLICATIONS FAILURE IN PATIENTS INFECTED WITH HEPATITIS B VIRUS Christina EM Voorter and Ella M van den Berg-Loonen; Tissue Typing Bilkay Basturk1, Zeki Karasu2, Murat Kılıç3, Sezgin Ulukaya4 Sedat Laboratory, University Hospital Maastricht, Maastricht, the Netherlands Boyacioglu5 1 Baskent University Faculty of Medicine Immunology Unit From an evolutionary point of view HLA-DQA1 is one of the most intriguing genes from the MHC region, because it is thought to be the most Cytokines play a key role in the regulation of immune response. The aim of this study was to investigate the association of the cytokine gene polymorphisms with ancient class II gene. Although polymorphism of class II genes is mainly the development of cirrhosis due to hepatitis B virus infection. located in exon 2, DQA1 is an exception with extended polymorphism The study included 27 patients with cirrhosis (due to HBV), 23 patients with found in all exons. Of the 28 DQA1 alleles presently known, 19 can not be HBV carriers and 60 healthy controls. All genotyping (TNF-alpha, TGF-beta, identifi ed on basis of exon 2 alone, additional information from the other IL-10, IL-6, IFN-gamma) experiments were performed using sequence specifi c exons is necessary. For evolutionary comparison and to assess the degree of primers (PCR-SSP) by using commercial kit. The distribution of cytokine genes polymorphism outside the exons, the sequences of the 5′ UT region and the polymorphisms were compared between patients with cirrhosis, HBV carriers 5′ end of intron 1 were determined. and healthy controls by the χ2 test. Thirty different homozygous cell lines were sequenced, encompassing 15 When the results of the patient compared to those of the healthy controls, it was different DQA1 alleles. The alleles were evenly divided over the different found TNF-alpha -308 G/G polymorphism (p: 0.002) and TGF-beta codon 10 allele groups. DQA1 typing of all cell lines was obtained by sequencing codon 25 T/C-G/G polymorfi sm (p:0.007) were signifi cantly higher in the patient with HBs positive group than the control group. IL-10 GCC/GCC haplotype was exons 1-4. From the 5′ UT region 147 bp were elucidated and 277 bp from higher in patients with cirrhosis (% 29.6) than the control group (%10.0) but ′ the 5 end of intron 1. no statistically signifi ciant was found. No statistically signifi cant difference in The sequences revealed major nucleotide differences between the different cytokine genes was found between patients with cirrhosis and HBV carriers. lineages, whereas within each lineage few differences were present. Each Chronic Hepatitis B virus infection is a major global health problem. In endemic lineage showed unique nucleotide assignments both in the 5′ UT and the areas, chronic HBV is leading cause of death because of the development of liver intron 1 region. The lineage DQA1*04/06 was in the lead with 20 unique failure and liver cancer. Individuals with an inadequate primary immune response positions, part of them resulting from a remarkable nucleotide shift in to hepatitis B are at increased risk of developing chronic HBV. The balance among intron 1. In total 16/147 (10.9%) were polymorph in the 5′ UT the Th1 (pro-infl ammatory cytokines such as TNF-alpha and interleukin-1), region and 45/277 (16.2%) in the 5´ part of intron 1. In 2 cases (DQA1*0103 Th2 (anti-infl ammatory cytokines such as interleukin-10 and interleukin-4) and T reg (regulatory cytokines such as TGF-beta) cells extremely important for the and *0303) differences were found in intron 1 between two cell lines development of immune response. Cytokines participates induction and effector with identical exon sequences. The same phenomenon was encountered phases of immune and infl ammatory response and levels of their production are previously in the 3’ end of intron 1 and intron 2. This implicates conservative associated with polymorphisms of cytokine genes. selection of the exons within a given lineage. Our study shows that TNF-alpha -308 G/G polymorphism and TGF-beta codon 10, codon 25 T/T-G/G gene polymorphism can be risk factors for the development of cirrhosis due to HBV. These polymorphisms may be valuable predictor determinants for the development of cirrhosis due to HBV infection.

27 28 A SYNONYMOUS SUBSTITUTION IN EXON 3 RESULTS IN AN HLA HAPLOTYPE WITH A DELETION AT THE HLA-A ALTERNATIVE SPLICING AND EXPRESSION LOSS OF THE LOCUS: A CASE REPORT A*010101VAR ALLELE Luca Mascaretti*, Mariarosa Riva*, Francesca Crosti, Elena Sala, J. Reinders1, A. Houben1, A. van Dijk1, P. van der Weide1, Y. Arts-Hilkes1, Francesca Poli, Loretta Crespiatico, Sara Frison, Elena Longhi, Adriana E.H. Rozemuller1, A. Dormy3, A. Mulder2, E.J. Petersen1, H.G. Otten1 Balduzzi, Paola Corti, Attilio Rovelli. *HLA Laboratory, Blood and M.G.J. Tilanus1 Transfusion Center, San Gerardo Hospital, Monza (Italy) 1UMC Utrecht and 2LUMC Leiden, Netherlands and 3Histocompatibility We report an HLA haplotype with a deletion at the HLA-A locus in a Laboratory, EFS Alsace, Strasbourg, France 7 year old patient of Caucasian origin affected by Late Onset Globoid Cell A new variant of the HLA-A*010101 allele, designated as HLA- Leukodystrophy, a rare autosomic recessive disease due to in the A*010101var, was found in a patient requiring a stem cell transplantation. galactosylceramidase gene localized on 14. Allogeneic bone The new variant was identifi ed due to discrepant results between serological marrow transplantation (BMT) in selected cases has proved successful. typing (A2,-) and routine DNA typing (A*010101,0206). DNA typing Materials and methods: HLA typing was performed with lympho- revealed the presence of a substitution from G to T at position 597, codon cytotoxicity, low and high resolution PCR-SSP and SBT. Microsatellites 175, of exon 3 of this new HLA-A*010101var allele. This variation results close to the HLA-A locus (D6S2700, D6S2705, D6S265, D6S510, RF, in a synonymous amino acid substitution. Additional FACS analysis with D6S276) and the HFE gene were determined in order to assess the extent monoclonal specifi c for the A1 and A2 molecule confi rmed cell of deletion. membrane expression loss of the A*010101var allele. RNA sequenced- Results: the patient (HLA-A1) inherited HLA-A1 from her father based-typing (SBT) revealed that 24 nucleotides, corresponding to codons (HLA-A1, 24) and apparently no HLA-A specifi city from her mother 175 through 182 in exon 3, were alternatively spliced in the A*010101var (HLA-A30). High resolution typing could not demonstrate any known allele. One-dimensional isoelectric focusing (1D-IEF) on total cell lysate HLA-A null allele. SBT for the patient only identifi ed HLA-A*010101. from a cell line established from patient’s cells, showed no cell surface The most probable hypothesis seemed a deletion at the HLA-A locus. All or cytoplasmic A*010101var expression, confi rming serology and FACS microsatellites analysed showed no deletion suggesting that the genomic results. The silent mutation resulted in the introduction of a new splice site, DNA loss was closer to the HLA-A locus, between the D6S510 and the RF with a higher affi nity for splicing than the conserved exon 3 / intron 3 splice microsatellites. BMT was carried out with a donor bearing a single DQB1* site giving rise to this new HLA-A null allele. The alternative splicing of the mismatch; although engraftment was successful and no GvHD evidenced, A*010101var allele prevented the formation of a stable class I molecule on disease progression could not be halted. the cell surface, giving rise to this new HLA-A null allele. Conclusion: an inherited haplotype carrying a small deletion at the HLA-A locus was described. Further investigation will include a karyotype analysis.

Genes and Immunity Abstracts S21 29 30 UNILATERAL NECROTISING TOXOPLASMIC DISULFIDE BRIDGE DISRUPTION IN THE HLA CLASS I ALPHA RETINOCHOROIDITIS AS THE ONLY MANIFESTATION OF A 2 DOMAIN LEADS TO A LOW SURFACE EXPRESSION OF THE PEPTIDE TRANSPORTER (TAP) DEFICIENCY CORRESPONDING HLA MOLECULE Anne Parissiadis, Patrick Lenoble, Dominique Fricker, Jean-Pierre Kaimo Hirv, Ulrich Pannicke, Joannis Mytilineos and Klaus Schwarz. Cazenave, Daniel Hanau, Anne Dormoy, and Henri de la Salle Department of Transplantation Immunology, IKT Ulm gGmbH, Laboratoire d’Histocompatibilité and INSERM U.725, EFS-Alsace, Helmholtzstrasse 10, 89081 Ulm, Germany. 67065 Strasbourg cedex, France We have identifi ed a novel HLA-A*30 allele with a single nucleotide Up to now, all described peptide transporter (TAP) defi ciencies have substitution in exon 3 (G563C) which at the protein level results in the been observed in a context of familial consanguinity, where a homozygous disruption of the disulfi de bridge in the alpha 2 domain (C164S). This inactivating mutation in one of the two subunits of the TAP leads to a severe disulfi de bridge disruption is believed to affect the stable binding between reduction in cell surface expression of HLA class I molecules. Although the alpha 2 domain and both, the beta-2 microglobulin and the peptide, a couple of asymptomatic TAP-defi cient adults have been described, the clinical manifestations generally consist of recurrent bacterial infections which in turn ends up with the reduction or the loss of the MHC class I cell of the respiratory tract and/or granulomatous skin lesions. We report here surface expression due to protein instability. the case of a 14 year-old boy, whose parents are totally unrelated, and Serological typing of the individual carrying the new allele was negative who was referred to our ophthalmology department for right posterior for HLA-A30. To assess the cell surface expression more precisely, the uveitis with vitreous infl ammation and detachment of the retina associated binding of three different anti-HLA-A30 monoclonal antibodies (mab) with a severe retinal necrosis. Clinical examination and serology led to was measured by fl ow cytometry at different temperatures since it was the diagnosis of ocular toxoplasmosis. Since an ocular localization of hypothesized that stabilization of the molecule may be optimal at lower toxoplasmosis is encountered in immunodefi cient patients (suffering from temperatures. A very low but detectable HLA-A30 expression could be malignant haemopathy, idiopathic CD4 defi ciency or HIV infection, under observed with all three mab at 30°C and two of the three mab at 37°C. therapy to prevent organ transplant rejection or for iatrogenic causes), The measured mean fl uorescence intensity (mfi ) was signifi cantly higher the immunocompetence of the patient was evaluated. This allowed us to at 30°C with all antibodies, when compared to the mfi at 37°C. Real-time demonstrate (i) by serotyping, an absence of HLA class I molecules on quantifi cation of HLA-A*30 mRNA was carried out with the Quantitect the plasma membrane, which was confi rmed by fl ow cytometry (95% SYBR® Green PCR Kit and HLA-A*30 specifi c primers. The HLA-A*30 reduction), (ii) by PCR-SSP, an HLA-homozygous genotype, iii) by complementation, the presence of a mutation in TAP1 gene, and iv) by gene mRNA levels were not reduced. sequencing, a single mutation in the TAP1 gene (creating a stop codon in In conclusion, the disulfi de bridge disruption in the alpha 2 domain of the the C-terminal cytoplasmic domain, at the position of Q 522). HLA class I molecules leads to a signifi cant reduction but not to the loss of Thus, while TAP-defi cient patients do not seem to be particularly the cell surface expression of the protein, probably due to the conformational susceptible to viral infections, they would appear to be “defi cient” in the changes of the HLA class I heavy chain. face of an intracellular parasitic invasion like infection with Toxoplasma gondii. Finally, in addition to other known immunological disorders, TAP- defi ciency should now be considered in case of ocular toxoplasmosis.

31 32 INVESTIGATION OF INTERLEUKIN-10 GENE A HUMAN MELANOMA CELL LINE PROGRESSES FROM HLA POLYMORPHISMS IN THE DEVELOPMENT OF PHENOTYPE II TO PHENOTYPE I BY DOWNREGULATION OF ATHEROSCLEROSIS APM COMPONENTS AFTER PASSAGE IN NUDE MICE Jane Merrifi eld, W. Martin Howell, Philip R. Evans, Shu Ye. Departments Laura Paco, Eva Jiménez, Isabel Maleno, Carmen Cabrera, of Molecular Pathology and University Human Genetics, Southampton José R. Vilchez, Angel García Lora, Miguel A. López-Nevot, General Hospital, Southampton, SO16 6YD, UK. Pierre Coulie*, Federico Garrido. Servicio de Análisis Clínicos e The object of this investigation was to determine the effects of single Inmunologia Hospital Universitario Virgen de las Nieves. Granada. nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) gene Spain.*Ludwig Institute for Cancer Research, Belgium in the development of atherosclerosis. ANDO-2 is a human melanoma cell line that has lost one HLA haplotype The study group consisted of DNA samples from 1178 Caucasian patients (HLA phenotype II). The lost HLA haplotype is HLA-A*0201, B*1302, with angiographically documented coronary atherosclerosis. Seven IL-10 Cw*0602, DRB1*0101, DQB1*0501 and the retained one is HLA-A*3201, SNPs (–3575, –1349, –1082 and –592, +19, +177 and +953) were included B*4001, Cw*0304, DRB1*1501, DQB1*0602. It expresses only HLA class in this study. SNP genotyping was performed using the 5′ nuclease assay I alleles and expresses no HLA class II alleles from the retained haplotype, for allelic discrimination (TaqMan®). Statistical analysis was performed as determined by FACS using allele-specifi c mAbs. Balb/c nude mice using the Hardy-Weinberg and chi-square (χ2) tests between different were subcutaneously injected with ANDO-2 (5x 10 cells/ml). After four patient sub-groups whilst haplotype analysis was performed using the weeks, a 1-cm tumour was excised and the cells were disaggregated and Snphap programme. harvested in culture medium. This variant, designated ANDO-2-NUDE, No genotype was found to be associated with development of myocardial had no HLA Class I expression and was classifi ed as HLA phenotype I infarction. In contrast, when comparing one and two vessel disease versus but it was HLA class II positive. No structural lesion of β-2 microglobulin three-vessel disease in untreated patients, one genotype (IL-10 –1082 GG) gene (e.g., deletion) was detected using STR markers from 15q. After and one haplotype (TAGCCC) were shown to be signifi cantly associated treatment with 800 und/ml IFN-γ for 48 hr, ANDO-2-NUDE re-expressed with disease severity (P=0.007, P=0.045 respectively). HLA class I. When antigen processing machinery (APM) components This study shows that IL-10 genotype may mediate atherosclerosis were analyzed by RT-PCR, a coordinated transcriptional downregulation severity, although the association is likely to be complex and requires of TAP-1, TAP-2, Tapasin, LMP-2 and LMP-7 was detected. INF-γ further investigation. One hypothesis is that genotypes associated with treatment upregulates these APM components, thereby restoring HLA class high IL-10 expression are a risk factor for atherosclerosis severity acting I expression. ANDO-2 and its variant ANDO-2-NUDE offer a good model via IL-10 down-regulation of VEGF expression, which is protective in to determine the transcriptional factors that control normal mRNA levels of atherosclerosis. APM components.

Genes and Immunity Abstracts S22 Population Genetics and Disease Predisposing Genes

33 34 HLA CLASS I AND CHEMOKINE GENE EXPRESSION A NEW HLA-DRB1 ALLELE: DRB1* 1152 IN RENAL CELL CARCINOMAS Giuseppina Ozzella, Palmina I. Monaco, Angela Iacona, Elide Calcagni, José María Romero, José Manuel Cózar, Julia Cantón, Antonio Garrido, Claudio Cortini, Daniela Piancatelli, Anna Aureli, Giuseppe Tufano, Teresa Cabrera, Pilar Jiménez, Susana Pedrinaci, Miguel Tallada, Antonina Piazza and Domenico Adorno. Federico Garrido, Francisco Ruiz-Cabello. Servicio Análisis Clínicos e C.N.R. Institute for Organ Transplantation, Rome - Italy Inmunologia, Hospital Universitario Virgen de las Nieves. Granada. Spain Searching for a potential related bone marrow donor, a new HLA- DRB1*11 allele, DRB1*1152, was identifi ed in three members of a Moroccan Berber Immune mechanisms have been suggested to play a role in the natural family. The doubtful presence of a new allele raised from some discrepancies disease course of RCC. The objective of this study was to examine factors among family members’ DRB1 low resolution typing. So, all members were that may be involved in the signifi cant survival benefi t of immunotherapy studied performing PCR SSP high resolution typing. Father’s DRB1 typing for RCC patients. A low frequency of total or HLA haplotype loss was surely permitted to assign DRB1*1104. It was not possible to defi ne the second found and, in parallel, the tumour tissue expressed more HLA classI/ allele because of an ambiguity between DRB1*1117 and some DRB1*14 alleles, B2m mRNA. These data signifi cantly differ from those reported in other due to a false positive reaction. HLA class II serological typing of father assigned epithelial tumours. Furthermore, chemokines and their receptors play key DR11, DR14. Instead, it seemed possible to assign DRB1*1117 allele to the roles in leukocyte traffi cking and are also implicated in cancer metastasis. patient and his matched brother, without any mismatch, just because father’s false positive reaction was now used to assign DRB1*1401 allele, inherited from We identifi ed a number of chemokines that were highly expressed in RCC mother. Sister’s high resolution typing permitted to assign, without any ambiguity, versus normal tissue. RT-PCR analysis showed expression of antiangiogenic DRB1*1104 inherited from father and DRB1*1401 from mother. All these agents I-TAC and IP-10, and proinfl ammatory chemokines MIP-1a and results supported the presence of a new DRB1*11 allele. Molecular cloning and RANTES in RCC tissues and very weak or undetectable expression in direct sequencing confi rmed that the new allele differs from DRB1* 1117 by one most of the normal kidney tissues. In contrast, the angiogenic SDF-1 was nucleotide substitution at position 265 (T to C) of exon 2, encoding an amino acid more strongly detected in the normal tissue. This chemokine production substitution of Tyrosine (TAC) to Histidine (CAC) at codon 60. The nucleotide pattern was correlated with a preferential recruitment of CD4+ Th1- sequence reported was submitted to GenBank database in March 2004 obtaining polarized effector memory cells that express CXCR3/CCR5, monocytes the accession number AY574194. The name DRB1* 1152 (HWS10002397) has been offi cially assigned by the WHO Nomenclature Committee for Factors of and NK cells. The composition of the tumour infi ltrate was different in HLA System in May 2004. more advanced tumours. In conclusion, upregulation of HLA class I gene Comparison of DRB1*1152 with all DRB1*14 allelic sequences on the IMGT expression in addition to chemokine expression in the tumour environment HLA database showed that the nucleotide substitution at codon 60 (CAC) was may contribute to the successful response of RCC to immunotherapy. shared with some DRB1*14 alleles. This may explain the class II serological typing that assigned in the father an erroneous DR14.

35 36 HLA ASSOCIATIONS WITH HIV-1 INFECTION IN BULGARIAN DISTRIBUTION OF HPA-GENES AND GENOTYPES IN THE PATIENTS POPULATION OF RUSSIAN UNRELATED DONORS Fani Martinova, Roumiana Markova, Ivailo Elenkov, Vera Popova, Larisa.L.Golovkina, Tatyana Makarik, Andrey B. Sudarikov Velislava Terzieva, Maria Yankova Hematologic Scientifi c Centre of Medical Science Russian Academy, Hospital “Pirogov”, NCIPD, Infect. Dis. Hospital, Sofi a, Bulgaria Moscow, Russia Due to the major role of the HLA locus in controlling the immune Race and populational distinctions in prevalence of HPA-genes (human response, associations between HLA genes and progression to AIDS have platelet ) are known. Defi nition of frequency allele variants and been extensively studied. The aim of the our study was to determine which genotypes HPA matters for immunogenetic and transfusion. The research HLA alleles from class I (HLA-A, -B) and class II (HLA-DRB1) were problem - studying of distribution of HPA-genes among Russians. A method presented in HIV-1 infected patients. of research - polymerase chain reaction with allele-specifi c primers of fi rm HLA-A, B and DRB1 alleles were tested in 17 patients with chronic HIV- “Protrans” for identifi cation of twelve allele genes “á” and “b” of loci 1 infection. 200 control healthy individual were tested too. Fifteen of the HPA-1,-2,-3,-4,-5 and -6. A material - DNA leukocytes of peripheral blood patients were under highly active antiretroviral therapy (HAART) (2NRTIs of 229 unrelated donors. Allele genes HPA- HPA-4A and HPA-6a met at and 1PI). all surveyed persons, HPA-1a - at 94,32 %, HPA-2a –at 98,25%, HPA-3a The HLA alleles were identifi ed using polymerase chain reaction (PCR)- - at 78,60 %, HPA-3b - at 70,74 % and HPA-5a – at 99,13 of Russians. amplifi ed DNA hybridized with sequence-specifi c probe (SSP). Allele genes HPA-1b,-2b,-5b met approximately identical frequency - at The frequencies of HLA class I antigens B*47 (p=0.0258, OR=13.665), 27,95 %, 21,83 % and 20,09 % of individuals. Frequencies of genotypes B*58 (p=0.0258, OR=13.665) and B*65 (p=0.0003) were increased in all are established: HPA-1a/a - 0,72, -1a/b - 0,22, -1b/b - 0,06; HPA-2a/a-0,78,- HIV-1 positive patients in comparison with the controls. The frequency of 2a/b-0,20; -2b/b-0,017; HPA-3a/a-0,297,-3a/b-0,49,-3b/b-0,214; HPA-5a/ the antigen B*35 was weakly and non-signifi cantly increased. HLA-B*7 a-0,8,-5a/b-0,19, 5b/b-0,009. Genotypes HPA-4b/b, HPA-6b/b have not and B*57 alleles were missing in all patients. been revealed. Gene frequencies have made 0,83 and 0,17 for HPA-1a and The frequencies of HLA class II antigens DRB1*01 and DRB1*13(*06) HPA-1b; 0,88 and 0,12 for HPA-2a and HPA-2b; 0,54 and 0,46 HPA-3a and have been found increased in patients. The frequency of DRB1*11(*05) has HPA-3b; 1,0 and 0 for HPA-4a, 6a and-4b,-6b; 0,895 and 0,105 for HPA-5a been established decreased in all patients in comparison with controls. and HPA-5b. The allele and genotypes frequencies of HPA at inhabitants Our fi ndings, although preliminary, suggest that HLA alleles infl uence of Russia are comparable to frequencies of genes in the East European resistance or susceptibility in a small cohort of HIV-1 infected Bulgarians, population. investigated up to now. Further studies are in progress to evaluate the correlation between host genetic profi les, progression to AIDS and the clinical outcome of the disease.

Genes and Immunity