16(th) IHIW: analysis of HLA population data, with updated results for 1996 to 2012 workshop data (AHPD project report).

We present here the results of the Analysis of HLA Population Data (AHPD) project of the 16th International HLA and Immunogenetics Workshop (16IHIW) held in Liverpool in May-June 2012. Thanks to the collaboration of 25 laboratories from 18 different countries, HLA genotypic data for 59 new population samples (either well-defined populations or donor registry samples) were gathered and 55 were analysed statistically following HLA-NET recommendations. The new data included, among others, large sets of well-defined populations from north-east Europe and West Asia, as well as many donor registry data from European countries. The Gene[rate] computer tools were combined to create a Gene[rate] computer pipeline to automatically (i) estimate allele frequencies by an expectation-maximization algorithm accommodating ambiguities, (ii) estimate heterozygosity, (iii) test for Hardy-Weinberg equilibrium (HWE), (iv) test for selective neutrality, (v) generate frequency graphs and summary statistics for each sample at each locus and (vi) plot multidimensional scaling (MDS) analyses comparing the new samples with previous IHIW data. Intrapopulation analyses show that HWE is rarely rejected, while neutrality tests often indicate a significant excess of heterozygotes compared with neutral expectations. The comparison of the 16IHIW AHPD data with data collected during previous workshops (12th-15th) shows that geography is an excellent predictor of HLA genetic differentiations for HLA-A, -B and -DRB1 loci but not for HLA-DQ, whose patterns are probably more influenced by natural selection. In Europe, HLA genetic variation clearly follows a north to south-east axis despite a low level of differentiation between European, North African and West Asian populations. Pacific populations are genetically close to Austronesian-speaking South-East Asian and Taiwanese populations, in agreement with current theories on the peopling of Oceania. Thanks to this project, HLA genetic variation is more clearly defined worldwide and better interpreted in relation to human peopling history and HLA molecular evolution.

Human leukocyte antigen-A, -B, -C, -DRB1 and -DQB1 allele and haplotype frequencies in an Albanian population from Kosovo.

HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 genotyping was performed in a sample of Albanian population from Kosovo. The comparison of the respective allele frequencies through Fst analysis resulted in a close relationship with the Albanians from Albania, the Bulgarians, FYROM Macedonians and Greeks, while the other neighbouring populations are slightly more distant.

Strategies to work with HLA data in human populations for histocompatibility, clinical transplantation, epidemiology and population genetics: HLA-NET methodological recommendations.

HLA-NET (a European COST Action) aims at networking researchers working in bone marrow transplantation, epidemiology and population genetics to improve the molecular characterization of the HLA genetic diversity of human populations, with an expected strong impact on both public health and fundamental research. Such improvements involve finding consensual strategies to characterize human populations and samples and report HLA molecular typings and ambiguities; proposing user-friendly access to databases and computer tools and defining minimal requirements related to ethical aspects. The overall outcome is the provision of population genetic characterizations and comparisons in a standard way by all interested laboratories. This article reports the recommendations of four working groups (WG1-4) of the HLA-NET network at the mid-term of its activities. WG1 (Population definitions and sampling strategies for population genetics' analyses) recommends avoiding outdated racial classifications and population names (e.g. 'Caucasian') and using instead geographic and/or cultural (e.g. linguistic) criteria to describe human populations (e.g. 'pan-European'). A standard 'HLA-NET POPULATION DATA QUESTIONNAIRE' has been finalized and is available for the whole HLA community. WG2 (HLA typing standards for population genetics analyses) recommends retaining maximal information when reporting HLA typing results. Rather than using the National Marrow Donor Program coding system, all ambiguities should be provided by listing all allele pairs required to explain each genotype, according to the formats proposed in 'HLA-NET GUIDELINES FOR REPORTING HLA TYPINGS'. The group also suggests taking into account a preliminary list of alleles defined by polymorphisms outside the peptide-binding sites that may affect population genetic statistics because of significant frequencies. WG3 (Bioinformatic strategies for HLA population data storage and analysis) recommends the use of programs capable of dealing with ambiguous data, such as the 'gene[rate]' computer tools to estimate frequencies, test for Hardy-Weinberg equilibrium and selective neutrality on data containing any number and kind of ambiguities. WG4 (Ethical issues) proposes to adopt thorough general principles for any HLA population study to ensure that it conforms to (inter)national legislation or recommendations/guidelines. All HLA-NET guidelines and tools are available through its website http://hla-net.eu.

Analysis of the HLA population data (AHPD) submitted to the 15th International Histocompatibility/Immunogenetics Workshop by using the Gene[rate] computer tools accommodating ambiguous data (AHPD project report).

During the 15th International Histocompatibility and Immunogenetics Workshop (IHIWS), 14 human leukocyte antigen (HLA) laboratories participated in the Analysis of HLA Population Data (AHPD) project where 18 new population samples were analyzed statistically and compared with data available from previous workshops. To that aim, an original methodology was developed and used (i) to estimate frequencies by taking into account ambiguous genotypic data, (ii) to test for Hardy-Weinberg equilibrium (HWE) by using a nested likelihood ratio test involving a parameter accounting for HWE deviations, (iii) to test for selective neutrality by using a resampling algorithm, and (iv) to provide explicit graphical representations including allele frequencies and basic statistics for each series of data. A total of 66 data series (1-7 loci per population) were analyzed with this standard approach. Frequency estimates were compliant with HWE in all but one population of mixed stem cell donors. Neutrality testing confirmed the observation of heterozygote excess at all HLA loci, although a significant deviation was established in only a few cases. Population comparisons showed that HLA genetic patterns were mostly shaped by geographic and/or linguistic differentiations in Africa and Europe, but not in America where both genetic drift in isolated populations and gene flow in admixed populations led to a more complex genetic structure. Overall, a fruitful collaboration between HLA typing laboratories and population geneticists allowed finding useful solutions to the problem of estimating gene frequencies and testing basic population diversity statistics on highly complex HLA data (high numbers of alleles and ambiguities), with promising applications in either anthropological, epidemiological, or transplantation studies.

HLA allele and haplotype frequencies in the Albanian population and their relationship with the other European populations.

Human leucocyte antigen (HLA) alleles are very interesting markers in identifying population relationships. Moreover, their frequency distribution data are important in the implementation of donor-recipient registry programs for transplantation purposes and also in determining the genetic predisposition for many diseases. For these reasons, we studied the HLA class I and II allele and haplotype frequencies in 160 healthy, unrelated Albanian individuals originating from all regions of the country. The HLA genotyping was performed through a 2-digit resolution SSOP method. The data were analysed with Arlequin and Phylip programs. No deviation was found from the Hardy-Weinberg equilibrium. A total of 17 A*, 30 B*, 12 Cw*, 13 DRB1* and 5 DQB1* alleles were identified. The six most frequent HLA-A-B-DRB1 haplotypes were A*02-B*18-DRB1*11 (5.60%), A*02-B*51-DRB1*16 (4.74%), A*01-B*08-DRB1*03 (3.48%), A*24-B*35-DRB1*11 (2.77%), A*02-B*51-DRB1*13 (2.21%), A*24-B*35-DRB1*14 (1.89%). Interestingly, 12 HLA-A-B-Cw-DRB1-DQB1 haplotypes occurred at a frequency >1%. When compared with the other populations, a close relationship was found with North Greek, Bulgarian, Macedonian, Romanian, Turkish, Cretan, Serbian, Croatian and Italian populations. A higher differentiation in allele frequency level was found with Western Europe populations. These data are the first report of HLA allele and haplotype distribution in an Albanian population inside this country. When compared with other populations, their distribution frequencies show close similarities with neighbouring populations of the entire Balkan area.

Diagnostic and prognostic significance of different antinuclear antibodies in more than 1000 consecutive Albanian patients with rheumatic diseases.

In an unselected population of 1390 consecutive Albanian patients with rheumatic diseases (RD) and other miscellaneous non-rheumatic diseases (MNRD), for whom antinuclear antibody (ANA) testing was requested, we calculated the diagnostic sensitivity, specificity and positive predictive value (PPV) of ANA positive results, ANA titres over 1:100, anti-native DNA (nDNA), anti-Sm, anti-U1 RNP, anti-SSA (Ro) anti-SSB (La) and anti-non-identified extractable nuclear antigen (NIENA) antibodies. The PPVs of these ANA types were found to be appreciable only for systemic lupus erythematosus (SLE); only the positive predictive value of ANA for SLE (26.4%) was lower than that for RA (34.3%). The anti-snRNP (Sm/U1RNP) positive SLE patients were more likely to have over 4 of the ARA criteria for SLE, ANA titres over 1:100, and anti-nDNA antibodies, in contrast with the anti-snRNP negative subgroup. On the other hand, the anti-ENA positive and anti-nDNA positive SLE patients generally showed higher frequencies of renal disease, over 4 of the criteria for SLE and ANA titres over 1:100, compared to anti-ENA positive and anti-nDNA negative patients. Our data suggest that the association of anti-snRNP antibodies with a more severe form of SLE is not to be attributed to these antibodies themselves, but rather to their close association with the concomitant presence of anti-nDNA antibodies.

Anti-phenolic glycolipid 1 IgM antibodies in leprosy patients and in their household contacts.

Though they have no apparent protective action, the specific antibodies are important markers of the infection with Mycobacterium leprae. For their detection we employed an ELISA method using as substrate a synthetic immunodominant disaccharide of phenolic glycolipid 1 antigen of M. leprae, conjugated with bovine serum albumin (D-BSA). Increased levels of anti-D-BSA antibodies of the IgM class were detected in 61.5% of the 13 leprosy patients and in 13.3% of their 53 household contacts, whereas they were not found in any of the 37 normal blood donors. A strong correlation (r = -0.846) was found between the antibody levels and the duration of the disease among the 12 patients with lepromatous leprosy. These preliminary data demonstrate the usefulness of this method for epidemiological studies and for the detection of cases with subclinical infection.

Impaired anti-pneumococcal antibody response in patients with AIDS-related persistent generalized lymphadenopathy.

Pre- and post-immunization serum antibodies to pneumococcal polysaccharides (PPS) and tetanus toxoid (TT) were measured in 25 patients with persistent generalized lymphadenopathy and serum antibodies to the human immunodeficiency virus (HIV). The increase in post-immunization anti-PPS antibodies was lower than 40% in 16/25 patients. Isotype analysis indicated that the IgM, IgA, IgG2, but not the IgG1 antibody responses were lower in patients that in healthy controls, whereas pre-immunization values were similar. For TT, no difference was found between the patients and the healthy group in total and IgG1 antibody response whereas IgG4 response was lower in patients. No significant association was found between the defect in anti-PPS antibody response and associated thrush or constitutional symptoms or other immunological parameters. These findings suggest that defective response to a thymo-independent polysaccharide antigen is a distinctive consequence of HIV infection.

Sequential determination of IgG subclasses and IgA specific antibodies in primary and reactivating toxoplasmosis.

During the course of T. gondii infection, we have analysed serum IgG and IgA antibodies responses in 50 immunocompetent with acquired infection and 19 immunocompromised patients with evidence of reactivated toxoplasmosis. Using an ELISA, IgG1, IgG2, IgG3 and IgA antibodies were found in sera of all patients, whereas IgG4 antibodies were usually not detectable. In immunocompetent patients, the predominant antibody isotype was IgG1 at the different stages of infection, presumably in relation with a T-cell control of humoral response during toxoplasmosis. In immunocompromised hosts (kidney or bone marrow transplanted and HIV infected patients), a sequential study was performed on serum samples taken before and after reactivation had occurred. The isotypic distribution of antibodies was similar to that observed in immunocompetent patients, but differences between groups of immunocompromised patients were detected when the kinetics of the antibody response was considered. The IgG and IgA antibody rise was lower in HIV1 infected patients with clinical toxoplasmosis; whatever was the peak antibody value, clinical symptoms appeared earlier in patients with a slower antibody response. This presumably reveals a functional T-cell abnormality, which may rely to the defective containment of the parasite in these patients.