Deciphering the fine nucleotide diversity of full HLA class I and class II genes in a well-documented population from sub-Saharan Africa.

With the aim to understand how next-generation sequencing (NGS) improves both our assessment of genetic variation within populations and our knowledge on HLA molecular evolution, we sequenced and analysed 8 HLA loci in a well-documented population from sub-Saharan Africa (Mandenka). The results of full-gene NGS-MiSeq sequencing compared with those obtained by traditional typing techniques or limited sequencing strategies showed that segregating sites located outside exon 2 are crucial to describe not only class I but also class II population diversity. A comprehensive analysis of exons 2, 3, 4 and 5 nucleotide diversity at the 8 HLA loci revealed remarkable differences among these gene regions, notably a greater variation concentrated in the antigen recognition sites of class I exons 3 and some class II exons 2, likely associated with their peptide-presentation function, a lower diversity of HLA-C exon 3, possibly related to its role as a KIR ligand, and a peculiar molecular diversity of HLA-A exon 2, revealing demographic signals. Based on full-length HLA sequences, we also propose that the most frequent DRB1 allele in the studied population, DRB1*13:04, emerged from an allelic conversion involving 3 potential alleles as donors and DRB1*11:02:01 as recipient. Finally, our analysis revealed a high occurrence of the DRB1*13:04-DQA1*05:05:01-DQB1*03:19 haplotype, possibly resulting from a selective sweep due to protection to Onchorcerca volvulus, a prevalent pathogen in West Africa. This study unveils highly relevant information on the molecular evolution of HLA genes in relation to their immune function, calling for similar analyses in other populations living in contrasting environments.

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.

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.

Sequence of a novel HLA-A2 allele in a haematopoietic stem cell donor of the international registry.

We report here the sequence of a new human leucocyte antigen-A2 allele, A*9251, identified in a volunteer haematopoietic stem cell donor of the international registry. A*9251 differs from A*02010101 by two nucleotides at codons 113-114, resulting in a single His>Asp substitution at codon 114.

DNA typing by microbead arrays and PCR-SSP: apparent false-negative or -positive hybridization or amplification signals disclose new HLA-B and -DRB1 alleles.

Human leukocyte antigen (HLA) typing by polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) hybridization on solid phase (microbead assay) or polymerase chain reaction-sequence-specific primers (PCR-SSP) requires interpretation softwares to detect all possible allele combinations. These programs propose allele calls by taking into account false-positive or false-negative signal(s). The laboratory has the option to validate typing results in the presence of strongly cross-reacting or apparent false-negative signals. Alternatively, these seemingly aberrant signals may disclose novel variants. We report here four new HLA-B (B*5620 and B*5716) and HLA-DRB1 alleles (DRB1*110107 and DRB1*1474) that were detected by apparent false-negative or -positive hybridization or amplification patterns, and ultimately resolved by sequencing. To avoid allele misassignments, a comprehensive evaluation of acquired data as documented in a quality assurance system is therefore required to confirm unambiguous typing interpretation.

Additional diversity within the B70 serotype: identification of B*1580 in a Caucasoid blood donor.

Anew B70 variant, B*1580, has been identified in a Swiss Caucasoid blood donor. Sequencing of exons 2 and 3 revealed that the HLA-B*1580 differs from its closest matching allele B*1518 by two substitutions in exon 3, leading to two amino acid changes, threonine to isoleucine and leucine to isoleucine at codons 94 and 95, respectively. The complete human leukocyte antigen type of the donor is: A*2402, A*2601; B*5101, B*1580; Cw*0704, Cw*1402/05; DRB1*0801, DQB1*0402. The B*1580 is a new member of the B70 cluster, characterized by the SEE motif at positions 24, 45, and 46 in the alpha1-domain. Substitutions at codons 94 and 95, also found in some B62 and B75 alleles, do not appear to interfere with the B70 serological reactivity. Based on sequence similarity and linkage with Cw*0704, the rare alleles B*1509, B*1529, B*1564, and B*1580 are possibly derived from the B*1518 haplotype.

Sequence of HLA-DRB1*0431 allele.

A novel HLA-DR4 allele has been detected in a volunteer bone marrow donor using a reverse microtiter plate oligotyping assay. Exon 2 cloning and sequencing revealed the new HLA-DRB1*0431 allele which differs from DRB1*0408 by two nucleotide changes at codon 74 leading to an Ala/Leu substitution. Although typical of DR8 alleles, Leu74 polymorphism was not sufficient to confer any serological reactivity with DR8 alloantisera. This rare DR4 subtype was identified in an individual of East European origin. The HLA typing of this donor is: A2,A31; B18,B51; Cw*0701,Cw*15; DRB1*1602,DRB1*0431; DRB4*01,DRB5*02; DQB1*0301,DQB1*0502.

Sequence of a new HLA-DR allele, DRB5*0106.

We report here the exon 2 sequence of a new HLA-DRB5 allele, DRB5*0106, that was identified in two volunteer bone marrow donors from the Swiss national registry. This new allele differs from DRB5*0101 by five amino acids at positions 67, 70, 71, 85 and 86. It is associated with DRB1*1501 and DQB1*0602. This unusual DRB1*1501-DRB5*0106 association increases the complexity of the DR2 group, although it appears to be very rare, at least in our population. HLA-DRB5*0106 can be readily detected upon DR generic oligotyping, provided the two probes that mark the major DRB5 subtypes, DRB5*0101 and DRB5*02, respectively, are included in the assay.

Sequence of HLA-DRB1*0818 in a bone marrow donor.

Exon 2 of a new HLA-DR8 subtype, DRB1*0818, was cloned and sequenced in a volunteer bone marrow donor. This new allele differs from DRB1*0805 by one single nucleotide in codon 67 resulting in an amino acid substitution from phenylalanine to isoleucine. Codon 67 dimorphism appears to be more frequent in DR8 and DR11 haplotypes. The HLA typing of the donor was A*0201/33 B*51/4403 Cw*0202/14 DRB1*0408/0818 DQB1*0302/0402. This new HLA-DR8 variant is very rare, at least in Caucasoids.