Jorge Martinez-Laso

HLA-G, -E, -F preworkshop: tools and protocols for analysis of non-classical class I genes transcription and protein expression.

Non-classical MHC class I HLA-E, -F, and -G molecules differ from classical class I histocompatibility antigens by specific patterns of transcription, protein expression, and immunological functions. Restriction of the expression pattern of these non-classical antigens may play a key role in modulation of immune responses during pregnancy and diseases but remains to be additionally defined. A specific component of the second International Conference on HLA-G and the 13th HLA-G Histocompatibility Workshop will be dedicated to the analysis of transcription and expression of non-classical class I genes in normal and pathological tissues. In a first step, referred to as the preworkshop, we here report the analysis and conclusions of a working group which was constituted to gather and validate optimal reagents and protocols allowing RT-PCR analysis of HLA-E, -F, -G transcript levels and flow cytometry and immunochemistry analysis of HLA-G expression in cells and tissues. As a result of this work, use of specific primers and probes detecting alternative transcripts of HLA-E, -F, and G have been validated in transfected cells expressing differential pattern of HLA class I antigens. Analysis of the specificity and affinity of collected antibodies has allowed definition of reagents to be proposed for immunochemistry and flow cytometry analysis of HLA-G expression in normal and pathological tissues during the workshop. This work has allowed constitution of an extended workshop group which is now initiating analysis of non-classical class I transcription and expression in various cells and tissues, a collective contribution that will additionally refine our view of the expression of these antigens in normal and pathological situations.

Relatedness among Basques, Portuguese, Spaniards, and Algerians studied by HLA allelic frequencies and haplotypes

Abstract HLA-A, -B, -DRB1, -DQA1, and DQB1 alleles were studied in Iberian and Algerian populations by serology and DNA sequence methodologies. The genetic and cultural relatedness among Basques, Spaniards, and paleo-North Africans (Berbers or Tamazights) was established. Portuguese people have also maintained a certain degree of cultural and ethnic-specific characteristics since ancient times. The results of the present HLA study in Portuguese populations show that they have features in common with Basques and Spaniards from Madrid: a high frequency of the HLA-haplotypes A29-B44-DR7 (ancient western Europeans), A2-B7-DR15 (ancient Europeans and paleo-North Africans), and A1-B8-DR3 (Europeans) are found as common characteristics. Portuguese and Basques do not show the Mediterranean A33-B14-DR1 haplotype, suggesting a lower admixture with Mediterraneans; Spaniards and Algerians do have this haplotype in a relatively high frequency, indicating a more extensive Mediterranean genetic influence. The paleo-North African haplotype A30-B18-DR3 present in Basques, Algerians, and Spaniards is not found in Portuguese either. The Portuguese have a characteristic unique among world populations: a high frequency of HLA-A25-B18-DR15 and A26-B38-DR13, which may reflect a still detectable founder effect coming from ancient Portuguese, i.e., oestrimnios and conios; Basques and Algerians also show specific haplotypes, A11-B27-DR1 and A2-B35-DR11, respectively, probably showing a relatively lower degree of admixture. A neighbor-joining dendrogram place Basques, Portuguese, Spaniards, and Algerians closer to each other and more separated from other populations. Genetic, cultural, geological, and linguistic evidence also supports the hypothesis that people coming from a fertile Saharan area emigrated towards the north (southern Europe, Mesopotamia, the Mediterranean Islands, and the North African coast) when the climate changed drastically to hotter and drier ca 10 000 years B.C.

HLA ALLELE AND HAPLOTYPE FREQUENCIES IN ALGERIANS: RELATEDNESS TO SPANIARDS AND BASQUES

The powerful genetic polymorphism of the HLA system has been used to identify individuals and populations. Ethnic groups may be characterized by specific HLA allele frequencies and particular extended HLA haplotypes; also, genetic relationships among these groups may be deduced. In the present study, serology and DNA typing were used to detect HLA-A, -B, -C, -DR, and -DQ alleles in each individual and to calculate characteristic haplotypes in Algerians. These results were compared to those previously obtained in other populations, particularly northern Mediterraneans; genetic distances and their respective dendrograms place Basques and Spaniards closer to Algerians than to other Europeans. Also, characteristic Basque and/or Spanish haplotypes are found in Algerians; i.e., A30-B18-Cw5-DR3-DQ2 and A1-B57-Cw7-DR7-DQ2. This supports the evidence that the Algerian population, mainly its paleo-North African component (Berbers), has a common descent with Basques and Spaniards, probably reflecting a preneolithic relationship between Iberians and paleo-North Africans.

HLA allele and haplotype frequencies in Algerians

Abstract The powerful genetic polymorphism of the HLA system has been used to identify individuals and populations. Ethnic groups may be characterized by specific HLA allele frequencies and particular extended HLA haplotypes; also, genetic relationships among these groups may be deduced. In the present study, serology and DNA typing were used to detect HLA-A, -B, -C, -DR, and -DQ alleles in each individual and to calculae characteristic haplotypes in Algerians. These results were compared to those previously obtained in other populations, particularly northern Mediterraneans; genetic distances and their respective dendrograms place Basques and Spaniards closer to Algerians than to other Europeans. Also, characteristic Basque and/or Spanish haplotypes are found in Algerians; i.e., A30-B18-Cw3-DR3-DQ2 and Al-B57-Ctv7-DR7-DQ2. This supports the evidence that the Algerian population, mainly its paleo-North African component (Berbers), has a common descent with Basques and Spaniards, probably reflecting a preneolithic relationship between Iberians and paleo-North Africans.

A new HLA-G allele (HLA-G * 0105N) and its distribution in the Spanish population

Six different HLA-G subtypes ( HLA-G*01011, -G*01012, -G*01013, -G*0102, -G*0103, and -G*0104) have been defined by the WHO Nomenclature Committee for Factors of the HLA System.HLA-G is an interesting nonclassical class I gene with the following characteristics: 1) a low degree of polymorphism (Morales et al. 1993; Yamashita et al. 1996); 2)HLA-G expression is restricted to trophoblast cells where it is the only Mhc molecule expressed; low mRNA transcript levels have been detected in a wide variety of cells in fetal and adult tissues (Onno et al. 1994); 3) alternative splicing of HLA-G transcripts gives rise to four membrane-bound and two soluble isoforms (Fujii et al. 1994); 4) polymorphic changes do not follow the three hypervariable regions per domain model and do not affect either the antigen or T-cell receptor binding sites (Arnaiz-Villena et al. 1996); and 5) stop codons have been found in homozygousity at theMhc-G exon 3 in the Cercopithecinaefamily of primates. Altogether this suggests that alternative splicing forms without exon 3 could play a role in cellular physiology other than antigen presenting (Castro et al. 1996). In the present study a new HLA-G subtype is described in the Spanish population; this allele shows linkage disequilibrium with the HLA A30-B13 haplotype and bears a nucleotide deletion either in the third position of codon 129 or the first of codon 130. Genomic DNA from 114 unrelated healthy Spanish individuals was isolated from peripheral blood lymphocytes using standard methods. Amplification of exon 2, exon 3, or exon 2 + intron 2 + exon 3 was performed using HLA-Gspecific primers as previously described (Morales et al. 1993). A total of 20 samples were further studied by singlestrand conformational polymorphism because they showed blanks or typing anomalies. Four showed a rare mobility pattern; polymerase chain reaction (PCR) products from exon 2 + intron 2 + exon 3 were purified, inserted into the pGEM-T vector, and sequenced in an Applied Biosystems (Foster City, CA) DNA automated sequencer. Two new HLA-G sequences were obtained: one ( HLA-G*0104, present in two unrelated samples) has been recently described in a Japanese population study (Yamashita et al. 1996), and the other (present in two other unrelated samples and namedHLA-G*0105N) has not yet been described. HLA-G*0104 has a non-synonymous leucine to isoleucine substitution at the first base of codon 110 (exon 3) compared withG*01013 (Fig. 1); this change is the first non-synonymous variation found at this codon in humans. This position does not seem to be important in the binding site region for processed antigen or in T-cell receptor interactions. A study of this allele in 114 unrelated Spanish individuals using a PCR-restriction fragment length polymorphism (RFLP) method (Morales et al. 1993) indicates that it is present in 100% of HLA-A23(n = 7)and 70% of HLA-A24 (n = 17)-positive Spaniards and has a frequency of 21.05% (n = 114, see Figure 1 legend). It is also found in four HLA-A19and oneHLA-A2/-A11-positive samples; strong linkage disequilibrium has already been described betweenHLA-A and -G alleles (Morales et al. 1993). The newHLA-G*0105Nallele shows a cytosine deletion either at the third base of codon 129 or the first base of codon 130 (exon 3; Fig. 1). This deletion changes the reading frame from this position, yielding a different amino acid sequence of the protein, which most likely would finish with a stop codon at the beginning of the α3 domain (exon 4), ifHLA-G*0105Nexon 4 DNA sequence is identical toHLA-G*01011 exon 4 DNA sequence; the cytosine deletion at codon 129/130 is not a PCR artefact because it has been observed in two different samples and in different PCR-RFLP assays. Stop codons in homozygousity have been found at exon 3 of Mhc-G sequences from The nucleotide sequence data reported in this paper have been subitted to the GenBank the nucleotide sequence database and have been assigned the accession number L78073. The name G*0105N was offficially assigned by the WHO Nomenclature Committee in October 1996

Phylogeny and rapid Northern and Southern Hemisphere speciation of goldfinches during the Miocene and Pliocene Epochs

Abstract. Mitochondrial cytochrome b (cyt b) from 25 out of 31 extant goldfinches, siskins, greenfinches and redpolls (genus Carduelis) has been sequenced from living samples taken around the world, specimens have also been photographed. Phylogenetic analysis consistently gave the same groups of birds, and this grouping was generally related to geographical proximity. It has been supposed that Pleistocene glaciations played a crucial role in the origin of extant diversity and distribution of Northern Hemisphere vertebrates. Molecular comparison of most extant songbird species belonging to the genus Carduelis does not support this assertion. The fossil record of chicken and pheasant divergence time has been used to calibrate the molecular clock; cyt b DNA dendrograms suggest that speciation in Carduelinae birds occurred during the Miocene and Pliocene Epochs (9 – 2 million years ago) in both the Northern and Southern Hemispheres. Only about 4% average amount of nucleotide substitution per lineage is found between the most distant Carduelis species; this suggests a remarkably rapid radiation when compared with the radiation of other passerine songbird genera. In addition, a continuum of small songbird speciation may be found during the Miocene Epoch in parallel with speciation of other orders (i.e. Galliformes, chicken/pheasant). Pleistocene glaciations may have been important in subspeciation (i.e. Eastern European grey-headed goldfinches/Western European black-headed goldfinches) and also in ice-induced vicariance (isolation) (i.e. siskin in Western Europe vs. siskin in Far East Asia) around the world. European isolated Serinus citrinella (citril finch) is not a canary, but a true goldfinch. South American siskins have quickly radiated in the last 4 million years coinciding with the emergence of the Isthmus of Panama; probably, a North American siskin related to C. notata invaded a suitable and varied biotope (the South American island) for Carduelis birds. North American goldfinches may be renamed as siskins, because they have a distant genetic relationship with European goldfinches. Genus Acanthis could be dropped, and thus redpolls should be separated from twite and linnet, the latter (Europeans) probably being related to American goldfinches. Also, reproductive barriers are observed between closely related species and not between other more distant ones. Finally, a tentative classification for genus Carduelis species is suggested.

Allelic diversity at the primate major histocompatibility complex DRB6 locus

The HLA-DRB6 gene (also called DRBσ/V1) has been found only in about 26% of human HLA haplotypes, i.e.; DR1, DRw10, and DR2-bearing ones (Corell et al. 1991). In contrast, exon-2 DRB6 sequences have been obtained from all tested primates: nine chimpanzees (Pan troglodytes), three gorillas (Gorilla gorilla) and three orangutans (Pongo pygmaeus); other apes which had already been sequenced (one gorilla and one chimpanzee) also had the DRB6 gene. Thus, all apes tested from three different species, some of them evolutionary separated by at least 14–16 million years, bear the DRB6 gene. In addition, more than one gene copy per haplotype has been found in one chimpanzee; this, together with the apparent loss of this gene in some of the human DR haplotypes, may indicate that the DR genome has undergone evolutionary changes more recently and more actively than class I or III genes. In addition, ten different and presumably allelic DRB6 exon-2 sequences have been obtained, and some of them coming from different species are more similar to each other than the one from the same species; this finding goes in favor of the trans-species theory of major histocompatibility complex polymorphism generation. Also, data are presented supporting that DRB6 may be one of the eldest genes of the DRB family, thus one of the first to diverge from the ancestral DRB gene.

The Old World sparrows (genus Passer) phylogeography and their relative abundance of nuclear mtDNA pseudogenes.

The phylogenetic relationships of genus Passer (Old World sparrows) have been studied with species covering their complete world living range. Mitochondrial (mt) cyt b genes and pseudogenes have been analyzed, the latter being strikingly abundant in genus Passer compared with other studied songbirds. The significance of these Passer pseudogenes is presently unclear. The mechanisms by which mt cyt b genes become pseudogenes after nuclear translocation are discussed together with their mode of evolution, i.e., transition/transversion mitochondrial ratio is decreased in the nucleus, as is the constraint for variability at the three codon positions. However, the skewed base composition according to codon position (in 1st position the percentage is very similar for the four bases, in 2nd position there are fewer percentage of A and G and more percentage of T, and in 3rd codon position fewer percentage of G and T and is very rich in A and C) is maintained in the translocated nuclear pseudogenes. Different nuclear internal mechanisms and/or selective pressures must exist for explaining this nuclear/mitochondrial differential DNA base evolutive variability. Also, the phylogenetic usefulness of pseudogenes for defining relationships between closely related lineages is stressed. The analyses suggest that the primitive genus Passer species comes from Africa, the Cape sparrow being the oldest: P. hispaniolensis italiae is more likely conspecific to P. domesticus than to P. hispaniolensis. Also, Passer species are not included within weavers or Estrildinae or Emberizinae, as previously suggested. European and American Emberizinae sparrows are closely related to each other and seem to be the earliest species that radiated among the studied songbirds (all in the Miocene Epoch).

HLA genes in the Chuvashian population from European Russia: admixture of Central European and Mediterranean populations.

HLA alleles have been determined for the first time in individuals from the Chuvashian population by DNA typing and sequencing. HLA-A, -B, -DR, and -DQ allele frequencies and extended haplotypes have also been determined, and the results compared to those for Central Europeans, Siberians and other Asians, Caucasians, Middle Easterners, and Mediterranean peoples. Genetic distances, neighbor-joining dendrograms, and correspondence analysis have been performed. Present-day Chuvash speak an Altaic-Turkic language and are genetically related to Caucasians (Georgians), Mediterraneans, and Middle Easterners, and not only to Central or Northern Europeans; Chuvash contain little indications of Central Asian-Altaic gene flow. Thus, present-day Chuvash who speak an Altaic-Turkic language are probably more closely related to ancient Mesopotamian-Hittites and northern European populations than to central Asia-Altaic people.

HLA molecular markers in Tuvinians: a population with both Oriental and Caucasoid characteristics

HLA class I and class II alleles have been studied for the first time in the Turkish‐speaking Tuvinian population, which lives in Russia, North of Mongolia and close to the Altai mountains. Comparisons have been done with about 11000 chromosomes from other worldwide populations, and extended haplotypes, genetic distances, neighbor joining dendrograms and correspondence analyses have been calculated. Tuvinians show an admixture of Mongoloid and Caucasoid characters, the latter probably coming from the ancient Kyrgyz background or, less feasibly, more recent Russian Caucasoid admixture. However, Siberian population traits are not found and thus Tuvinians are closer to Central Asian populations. Siberians are more related to Na‐Dene and Eskimo American Indians; Amerindians (from nowadays Iberian–America) are not related to any other group, including Pacific Islanders, Siberians or other American Indians. The ‘more than one wave’ model for the peopling of the Americas is supported.