Reference Strand Conformation Analysis Research Articles

93-P: MIC AND NKG2D GENES POLYMORPHISM IN MEXICAN POPULATION

Aim Nowadays, different knowledge polymorphism in genes involved in the immune response helps to identify the susceptibility or risk in the development of different human diseases. Therefore, the objective was to determine the distribution of MIC gene polymorphisms and Single Nucleotide Polymorphisms (SNP) rs1049174, rs2255336 and rs2617160 of KLRK1 (NKG2D) gene in the Mexican population by their interaction, MICA/MICB-NKG2D. Methods The polymorphism of MICA and MICB genes and the SNPs of NKG2D were analyzed by Reference Strand Conformation Analysis (RSCA) and Sequence Specific Oligonucleotide Probes (SSOP)-Single Specific Primer (SSP) and allelic discrimination, respectively. We genotyped 336 samples of persons clinically healthy and MICA, MICB and NKG2D polymorphisms were identified by RSCA, SSOP-SSP and allelic discrimination, techniques, respectively. Results The most frequent allele were MICA ∗002:01 (33.28%), MICB ∗005:02 (63.09%), NKG2D rs1049174 ∗T (61.31%), NKG2D rs2255336 ∗C (61.31%) and NKG2D rs2617160 ∗G (93.30%). We also found that the most frequent combination of genotypes in the genes MICA/MICB was ∗002:01-∗002:01/∗005:02-∗005:02 with 7.44%, while the combination of genotypes at SNPs NKG2D in positions rs1049174/rs2255336/rs2617160 was ∗A-∗T/∗C-∗G/∗G-∗G with 45.53%. Conclusions The data obtained provide other genetic markers involved in the immune innate response that can be considered for the study of susceptibility or protection in different diseases of public health and participation in transplants in Mexican individuals. FOFOI 2005/1/I/016, CONACYT 14019.

MIC and NKG2D genes polymorphism in Mexican population

Event Abstract Back to Event MIC and NKG2D genes polymorphism in Mexican population Miriam Nieves-Ramirez1, Eyerahi Bravo-Flores1, Pedro Alegre-Crespo2, Maria Del Carmen Tapia-Lugo2 and Martha Perez-Rodriguez1* 1 Mexican Institute of Social Security, Pediatric Hospital, National Medical Center S-XXI, Mexico 2 Mexican Institute of Social Security, General Hospital 1A "Venados", Mexico Nowadays, different knowledge polymorphism in genes involved in the immune response helps to identify the susceptibility or risk in the development of different human diseases. Therefore, the objective was to determine the distribution of MIC gene polymorphisms and Single Nucleotide Polymorphisms (SNP) rs1049174, rs2255336 and rs2617160 of KLRK1 (NKG2D) gene in the Mexican population by their interaction, MICA/MICB-NKG2D. The polymorphism of MICA and MICB genes and the SNPs of NKG2D were analyzed by Reference Strand Conformation Analysis (RSCA) and Sequence Specific Oligonucleotide Probes (SSOP)-Single Specific Primer (SSP) and allelic discrimination, respectively. We genotyped 336 samples of persons clinically healthy and MICA, MICB and NKG2D polymorphisms were identified by RSCA, SSOP-SSP and allelic discrimination, techniques, respectively. The most frequent allele were MICA *002:01 (33.28%), MICB *005:02 (63.09%), NKG2D rs1049174 *T (61.31%), NKG2D rs2255336 *C (61.31%) and NKG2D rs2617160 *G (93.30%). We also found that the most frequent combination of genotypes in the genes MICA/MICB was *002:01-*002:01/*005:02-*005:02 with 7.44%, while the combination of genotypes at SNPs NKG2D in positions rs1049174/rs2255336/rs2617160 was *A-*T/*C-*G/*G-*G with 45.53%. The data obtained provide other genetic markers involved in the immune innate response that can be considered for the study of susceptibility or protection in different diseases of public health and participation in transplants in Mexican individuals. Acknowledgements Work performed with support FOFOI 2005/1/I/016, CONACYT 14019. Keywords: Polymorphism, Genetic, Polymorphism, Single Nucleotide, Polymorphism, Single-Stranded Conformational, Sequence Specific Oligonucleotide Probes, Single Specific Primer Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Nieves-Ramirez M, Bravo-Flores E, Alegre-Crespo P, Tapia-Lugo M and Perez-Rodriguez M (2013). MIC and NKG2D genes polymorphism in Mexican population. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00764 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Martha Perez-Rodriguez, Mexican Institute of Social Security, Pediatric Hospital, National Medical Center S-XXI, Mexico City, Mexico, meperezrodriguez@yahoo.com.mx Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Miriam Nieves-Ramirez Eyerahi Bravo-Flores Pedro Alegre-Crespo Maria Del Carmen Tapia-Lugo Martha Perez-Rodriguez Google Miriam Nieves-Ramirez Eyerahi Bravo-Flores Pedro Alegre-Crespo Maria Del Carmen Tapia-Lugo Martha Perez-Rodriguez Google Scholar Miriam Nieves-Ramirez Eyerahi Bravo-Flores Pedro Alegre-Crespo Maria Del Carmen Tapia-Lugo Martha Perez-Rodriguez PubMed Miriam Nieves-Ramirez Eyerahi Bravo-Flores Pedro Alegre-Crespo Maria Del Carmen Tapia-Lugo Martha Perez-Rodriguez Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Characterisation of MHC haplotypes in a breeding colony of Indonesian cynomolgus macaques reveals a high level of diversity

Recent reports have revealed that cynomolgus macaques obtained from different geographic origins may be more or less suitable for particular studies depending on the specific question(s) being addressed, e.g. Mauritian cynomolgus macaques are particularly suitable for detailed immunological studies against a limited genetic background while less conserved populations may be more appropriate to predict breadth of vaccine coverage in the genetically diverse human population. We have characterised MHC haplotypes in 90 Indonesian cynomolgus macaques using microsatellite and reference strand conformational analysis. Thirty unique haplotypes were defined in the cohort, emphasising the high degree of diversity in this population of cynomolgus macaques. The majority of haplotypes were present at a frequency of ≤ 6%. Transcription profiles indicated that each haplotype was associated with two to eight transcribed class I alleles. The results corroborate previous reports of the extensive MHC diversity of Indonesian cynomolgus macaques and provide additional data to inform colony management decisions. Further, definition of the MHC diversity of the population satisfies one of the prerequisites to MHC association studies and detailed immunological investigations in this outbred non-human primate species.

Methods for MHC genotyping in non‐model vertebrates

Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems.

MHC haplotype frequencies in a UK breeding colony of Mauritian cynomolgus macaques mirror those found in a distinct population from the same geographic origin

Mauritian cynomolgus macaques have greatly restricted genetic diversity in the MHC region compared to other non-human primates; however, the frequency of common MHC haplotypes among captive-bred populations has not been reported. Microsatellite PCR was used to determine MHC haplotype frequencies among captive macaques at a UK breeding facility. Allele-specific PCR and reference strand conformational analysis were used to determine the allele expression profile of a subset of animals. Haplotypes H3 (21%) and H1 (19%) were most common in the captive population of Mauritian cynomolgus macaques. Predicted alleles were detected by allele-specific PCR-SSP in 98% of animals. Allele expression profiles were similar in animals with identical haplotypes. Mauritian cynomolgus macaques in the UK breeding facility have restricted MHC diversity comparable to a previously described population. Microsatellite-derived haplotypes are highly predictive of allele expression. A selective breeding program has been established to produce MHC-identical animals for biomedical research.

Host HLA B*allele-associated multi-clade Gag T-cell recognition correlates with slow HIV-1 disease progression in antiretroviral therapy-naïve Ugandans.

BackgroundSome HIV infected individuals remain asymptomatic for protracted periods of time in the absence of antiretroviral therapy (ART). Virological control, CD4 T cell loss and HIV-specific responses are some of the key interrelated determinants of HIV-1 disease progression. In this study, possible interactions between viral load, CD4 T cell slopes, host genetics and HIV-specific IFN-γ responses were evaluated in chronically HIV-1-infected adults.Methodolology/Principal FindingsMultilevel regression modeling was used to stratify clade A or D HIV-infected individuals into disease progression groups based on CD4 T cell slopes. ELISpot assays were used to quantify the frequency and magnitude of HIV-induced IFN-γ responses in 7 defined rapid progressors (RPs) and 14 defined slow progressors (SPs) at a single time point. HLA typing was performed using reference strand conformational analysis (RSCA). Although neither the breadth nor the magnitude of the proteome-wide HIV-specific IFN-γ response correlated with viral load, slow disease progression was associated with over-representation of host immunogenetic protective HLA B* alleles (10 of 14 SPs compared to 0 of 7; p = 0.004, Fisher's Exact) especially B*57 and B*5801, multiclade Gag T-cell targeting (71%, 10 of 14 SPs compared to 14%, 1 of 7 RPs); p = 0.029, Fisher's Exact test and evident virological control (3.65 compared to 5.46 log10 copies/mL in SPs and RPs respectively); p<0.001, unpaired student's t-testConclusionsThese data are consistent with others that associated protection from HIV disease with inherent host HLA B allele-mediated ability to induce broader Gag T-cell targeting coupled with apparent virological control. These immunogenetic features of Gag-specific immune response which could influence disease progression may provide useful insight in future HIV vaccine design.

Relevance of Polymerase Chain Reaction technique in immunogenetics and histocompatibility

During the last decade of the previous century, among many of developing sciences, molecular biology had the most important position. The greatest influence on the development of scientific knowledge had been brought by discovery of PCR technique, that showed numerous advantages in comparison to the existing techniques. The capability of PCR technique to amplify specific DNA sequences, in a simple, automatic procedure, accelerated the development of many fields in molecular biology researches such as cloning, identification of new genes and pathogenes, determination and quantification of nucleotide sequences, thus opening fields of experimental investigations and its clinical application. PCR technique is widely used in various investigations from anthropological researches to those in molecular biology, from basic genetic investigations to clinical diagnostics. In the field of immunogenetics and histocompatibility, there are several HLA genotyping techniques: PCR-SSOP, detection by sequence specific oligonucleotide probes. PCR-SSP, detection by sequence specific primers, RSCA, detection by Reference-Strand Conformation Analysis, SBT-Sequence Based Typing. The development of these techniques has enabled the testing of the HLA alleles as well as detection of shared nucleotide sequences between alleles on the same or/and different loci and the existence of some locus-specific nucleotide sequences in coding (exon) and noncoding (intron) regions. The application of PCR technique in immunogenetics made it possible to define genetic polymorphism in HLA system, which contributed to a significant progress in the field of tissue and organ transplantation, population and genetic studies as well as in studies of disease association and HLA alleles.

PCR-Based Methodology for Molecular Microchimerism Detection and Quantification

Peripheral blood microchimerism after pregnancy or solid organ transplantation has been widely studied, but a consensus on its detection has not yet been adopted. The objective of this study was to establish a panel of reproducible molecular polymerase chain reaction (PCR)-based methods for detection and quantification of foreign cells in an individual. We analyzed length polymorphisms generated by short tandem repeat (STR) and variable number tandem repeat (VNTR) markers. Human leukocyte antigen (HLA)-A and -B polymorphisms were detected by reference strand conformation analysis (RSCA). Class II polymorphisms on HLA-DRB1 locus were analyzed both by classical PCR-sequence-specific primers (SSP) and by quantitative PCR (Q-PCR). Also, sex-determining region-y gene (SRY) gene allowed specific male donor discrimination and quantification by Q-PCR in female recipients. Binomial statistical distribution analysis was used for each molecular technique to determine the number of PCR replicates of each sample. This analysis allowed the detection of the lowest detectable microchimerism level, when present. We could detect microchimerism in more than 96% and more than 86% of cases at levels as low as 1:10(5) and 1:10(6) donor per recipient cells (DPRC), respectively, using Q-PCR for SRY or for nonshared HLA-DRB1 alleles. These techniques allowed as low as 1 genome-equivalent cell detection. Lower levels (nanochimerism) could be detected but not quantified because of technique limitations. However, classical PCR methods allowed detection down to 1:10(4) DPRC for HLA-DRB1 PCR-SSP. The clinical application of these techniques in solid organ transplanted recipients showed microchimerism levels ranging from 1:10(4) to 1:10(6) DPRC after kidney or heart transplantation, and 1 log higher (1:10(3) to 1:10(6) DPRC) after liver transplantation. In conclusion, the standardization of molecular microchimerism detection techniques will allow for comparable interpretation of results in microchimerism detection for diagnostic or research studies.

Reference strand conformational analysis (RSCA) is a valuable tool in identifying MHC-DRB sequences in three species of Aotus monkeys

The Aotus monkey has been of great value in the pre-clinical study of malaria vaccine candidates. Several components of this primate's immune system have been studied and they display great similarity to their human counterparts. Cloning and sequencing studies have revealed extensive sequence polymorphisms in Aotus MHC-DRB with very high similarities to several human allelic lineages, grouping at least nine distinct MHC-DRB lineages. As the efficacy of peptide vaccines in this animal model may be strongly influenced by exon 2 MHC-DRB polymorphism, the availability of a reliable and rapid MHC-DRB typing method for three species of Aotus (Aotus nancymaae, Aotus vociferans and Aotus nigriceps) is necessary. Reference strand conformational analysis (RSCA) was used here for differentiating the distinctive Aotus MHC-DRB sequences' mobility using five fluorescently labelled references proved to be very useful for resolving closely related sequences, establishing the number of sequences transcribed in a particular monkey and their identity. The RSCA method's reliability in terms of identifying Aotus MHC-DRB sequences will facilitate evaluating individual responsiveness to vaccines and prompt studies associating susceptibility/resistance to infectious agents or auto-immune disease, for which Aotus monkeys may be considered to be an appropriate animal model.

28 EXPRESSION OF MHC CLASS I IN THE PLACENTA AND THE LYMPHOCYTES OF BOVINE SOMATIC CLONES

A major limitation for the development of somatic cloning in cattle is the low efficiency for producing live offspring because only 5 to 25% of the reconstructed embryos reach full-term development, with variation between the genotypes. Hill et al. (2002 Biol. Reprod. 67, 55–63) reported that early fetal losses were due to inappropriate expression of trophoblast major histocompatibility complex Class 1 (MHC1) antigens on the placenta of bovine somatic clones. This abnormal expression was present in all clones, with more cells expressing MHC1 in those with growth retardation. These clones, however, originated from only one genotype, and only one antibody recognizing a monomorphic determinant of bovine MHCI was used. In the present study, MHC1 expression was examined throughout gestation in bovine clone placentas originating from 3 different genotypes. Placentas were collected at Days 32, 62, 180, 260, and 280 (term) in clones (n = 8) and in controls obtained by artificial insemination (n = 6). Four different monoclonal antibodies that recognize different determinants of MHC1 molecules (ILA-A88, IL-A19, H58A, and the β2-microglobulin, i.e. the MHC1 light chain) were used. Results showed MHC1 expression in the maternal tissue but not on the fetal side, regardless of group, gestational age, or fetal growth, although patchy expression of β2-microglobulin could be found in both clones and controls in the fetal mesenchyme in some but not all samples. In conclusion, MHC1 protein expression appears normally down-regulated in the placenta of clones, but the antibodies used do not discriminate between classical and non-classical expression. The mRNA expression of classical and non-classical MHC1 genes is currently being explored. These results cast doubt on the hypothesis that the observed fetal losses would be the result of an immunologic rejection due to inappropriate expression of some placental MHC1 genes. Although early observations suggest that immune functions appear normal (Lanza et al. 2001 Science 294, 1893–1894; Chavatte-Palmer et al. 2006 Reprod. Fert. Dev. 18, 121 abst), immunological deficiencies have been reported in some bovine somatic clones after birth (Renard et al. 1999 The Lancet 353, 1489–1491), and they may be related to an abnormal expression of MHC1 genes. We therefore determined the MHC1 haplotype of cell donors from 4 genotypes and 3 of their healthy clones using the Reference Strand Conformation Analysis method with DNA extracted from lymphocytes. Results showed that the MHC1 type was identical, as could be expected in clones. Animals that died of immune deficiencies, however, may have had abnormal MHC1 expression. Further work is therefore being performed on tissues obtained postmortem from cloned animals having been diagnosed with thymic atrophy.

Feline leucocyte antigen class II polymorphism and susceptibility to feline infectious peritonitis

There are four outcomes to feline coronavirus (FCoV) infection: the development of feline infectious peritonitis (FIP, which is immune-mediated), subclinical infection, development of healthy lifelong carriers and a small minority of cats who resist infection (Addie and Jarrett, Veterinary Record 148 (2001) 649). Examination of the FCoV genome has shown that the same strain of virus can produce different clinical manifestations, suggesting that host genetic factors may also play a role in the outcome of infection. FIP is most prevalent amongst pedigree cats, although how much of this is due to them living in large groups (leading to higher virus challenge and stress which predisposes to FIP) and how much is due to genetic susceptibility is not known. If host genetics could be shown to play a role in disease, it may allow the detection of cats with a susceptibility to FIP and the development of increased population resistance through selective breeding. The feline leucocyte antigen (FLA) complex contains many genes that are central to the control of the immune response. In this preliminary study, we used clonal sequence analysis or reference strand conformational analysis (RSCA) to analyse the class II FLA–DRB of 25 cats for which the outcome of FCoV exposure was known. Individual cats were shown to have between two and six FLA–DRB alleles. There was no statistically significant association between the number of alleles and the outcome of FCoV infection. No particular allele appeared to be associated with either the development of FIP, resistance to FCoV, or the carrier status. However, the analysis was complicated by apparent breed variation in FLA–DRB and the small number of individuals in this study.

Mitochondrial DNA damage in lymphocytes: a role in immunosenescence?

An age-related increase of DNA damage/mutation has been previously reported in human lymphocytes. The high copy number and mutation rate make the mtDNA genome an ideal candidate for assessing damage and to act as a potential biomarker of ageing. In the present study, two assays were developed to evaluate the level of mtDNA 4977 and the accumulation of point mutations with age. A competitive polymerase chain reaction (PCR) methodology incorporating three primers was used to detect and quantify the levels of mtDNA 4977 and a novel heteroduplex reference strand conformational analysis (RSCA) technique was used to analyse the accumulation of point mutations. The assays were applied to an in vitro model of T cell ageing and ex vivo DNA samples from an elderly cohort of subjects and a younger control group. The mtDNA 4977 was detected in all the DNA samples examined but only a very low concentration was observed and no age-related increase or accumulation was observed. No accumulation of point mutations was identified using RSCA within the T cell clones as they were aged or the ex vivo lymphocytes from the elderly cohort. A higher level of variation was observed within the ex vivo DNA samples, verifying the high resolution of RSCA and its ability to identify different mtDNA species, although no correlation with age was observed. The low level of mtDNA damage observed with respect to the ex vivo lymphocyte DNA samples within this study may be due in part to the high turnover of blood cells/mtDNA, which may inhibit the accumulation of genetically abnormal mtDNA that may play a role in immunosenescence. A similar explanation may also apply to the in vitro model of T cell ageing if the vast majority of the cells are replicating rather than entering senescence.

HLA-B typing by reference strand mediated conformation analysis using a capillary-Based semiautomated genetic analyzer

The application of reference strand conformation analysis (RSCA) to HLA-A typing using the ABI PRISM 310 capillary based genetic analyzer has recently been described. This study outlines the development and validation of capillary RSCA for HLA-B typing. Mobility values for 93 HLA-B alleles were defined following electrophoresis of known controls through the system. Three fluorescently labelled references, labelled with three different dyes can be electrophoresed simultaneously. The technique was validated by comparing results from 296 cord blood donors with those obtained using reverse SSO. Following capillary RSCA 14.5% of samples required confirmatory typing, compared with a repeat rate of 5.1% following reverse SSO. In samples where no other typing was necessary there was 100% correlation between the two methods. Capillary RSCA for HLA-B typing is quick, easy to implement, and with the introduction of new FLRs and gel matrices has the potential to evolve into a high resolution typing method.

Further diversity at HLA-A and -B loci identified in Afro-Caribbean potential bone marrow donors.

Two novel HLA-A and three novel HLA-B alleles were identified within a group of Afro-Caribbean individuals who were recruited as potential donors for the Anthony Nolan Bone Marrow Trust Register. HLA typing was performed on DNA extracted from peripheral blood mononuclear cells using sequence-specific oligonucleotide (SSO) probes for HLA-A and -B loci. Eight individuals analysed exhibited hybridisation patterns for which a type could not be assigned. DNA from these individuals was further typed by two methodologies: direct sequencing of PCR products and reference strand conformation analysis (RSCA). The direct sequencing results allowed the identification of new alleles but did not allow confirmation of the cis/trans orientation of the new sequence motifs identified. RSCA analysis confirmed the results obtained by SSO and direct sequencing and in addition confirmed the cis/trans orientation of the new sequences. One individual possesses a new A*30 allele--A*3008 and two individuals possess an identical new A*74 allele--A*7404. The three novel HLA-B alleles were identified in three individuals: B*0812, B*1554 and B*4503 respectively. For the remaining two samples, A*2612 was identified. At present Caucasoid individuals, and therefore Caucasoid phenotypes, are predominantly represented on the various different volunteer bone marrow donor registries. The examples presented here highlight the potential for identification of further polymorphisms within the HLA system as more individuals from the much-needed ethnic minorities are recruited onto bone marrow donor registers.

HLA-B*4413 identified in a UK Caucasoid potential bone marrow donor.

We describe a novel allele belonging to the B*44 allele group. HLA-B*4413 was identified in a Caucasoid potential bone marrow donor from the Anthony Nolan Bone Marrow Trust register. Initial HLA typing of this donor revealed unusual serological reactivity. Further investigation was carried out by reference strand conformation analysis (RSCA) and sequence-based typing (SBT) using DNA extracted from an Epstein-Barr virus (EBV)-transformed B-cell line made from this donor (AMI005AN). In this individual, two B*44 alleles were revealed upon initial investigation: B*4409 and a previously unseen allele which has been named B*4413. B*4413 is identical to B*44031 except for a unique nucleotide substitution resulting in an amino acid difference at residue 61 in the alpha1 helix.

Sequence of HLA-A*6808.

A novel HLA-A*68 allele was identified by reference strand conformation analysis of a DNA sample studied for the International Cell Exchange program. Direct sequencing of HLA-A locus PCR products confirmed the presence of A*6602 and an A*68 allele which differed from A*68011 by two nucleotides at positions 538 and 539. The presence of this sequence motif in this allele which has been named A*6808 was verified by DNA sequencing of full-length A*6808 clones. No other coding differences between A*68011 and A*6808 were identified. The two nucleotide substitutions found in A*6808 effect an amino acid difference in the encoded protein at residue 156 within the peptide binding site which may evoke alloreactive immune response after HLA-A68 mismatched transplants.

Characterization of the HLA class I genotypes of a Venezuelan Amerindian group by molecular methods.

We have characterized the HLA class I genotypes of the Yucpa, a tribe of Venezuelan Amerindians, using molecular methods. The study was carried out on DNA extracted from unrelated individuals using low resolution ARMS-SSP typing with sequence-specific primers, high resolution typing using reference strand conformation analysis (RSCA), and for some samples sequence-based typing (SBT). The following class I alleles were found to be present in this tribe: for the HLA-A locus A*0204, A*0212, A*0213, A*2402, A*3101 and A*6801; for the B locus B*1522, B*3512, B*3905, B*3909, B*4004 and B*52012, and for C locus Cw*0102, Cw*0302/ 4, Cw*0401, Cw*0702 and Cw*1503. This is the first time these alleles have been described in this group, although all of them have previously been reported as being present in other Amerindian tribes. The study confirmed the high frequency of HLA-B39 which was previously observed in serological analysis of this tribe, and indicated that two different B*39 alleles were present in this population. The identification of the class I alleles by molecular methods for this ethnic group confirms the restricted polymorphism of the MHC molecules previously obtained by serology and has allowed a more accurate definition of the different alleles present in this population.