Miguel Alvarez

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

Allelic diversity at the primateMhc-G locus: Exon 3 bears stop codons in allCercophitecinae sequences

Twenty-seven major histocompatibility complex (Mhc)-G exon 2, exon 3, and exon 2 and 3 allelic sequences were obtained together with 12 different intron 2 sequences.Homo sapiens, Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus, Macaca fascicularis, Macaca mulatta, andCercopithecus aethiops individuals were studied. Polymorphism does not follow the classical pattern of three hypervariable regions per domain and is found in all species studied; exon 3 (equivalent to the α2 protein domain) shows stop codons in theCercopithecinae group but not in thePongidae and human groups. Dendrograms show that cotton top tamarin (Saguinus oedipus) Mhc-G sequences are closer toHomo sapiens andPongidae than toCercopithecinae, probably due to the stop codons existing at exon 3 of the latter. There is a clear trans-species evolution of allelism inCercopithecinae and also in exon 2 of all the other apes studied, but a generation of allelism within each species may be present on exon 3 sequences. This discrepancy may be due to the preferential use of exon 2 over exon 3 at the mRNA splicing level within each species in order to obtain the appropriate functional G product.Mhc-G intron 2 shows conserved motifs in all species studied, particularly a 23 base pair deletion between positions 161 and 183 which is locus specific, and some of the invariant residues, important for peptide presentation, conserved in classical class 1 molecules from fish and reptiles to humans were not found inMhc-G alleles; the intron 2 Dendrogram also shows a particular pattern of allelism within each species. In summary,Mhc-G has substantial differences from other classical class I genes: polymorphism patterns, tissue distribution, gene structure, splicing variability, and probably an allelism variability within each species at exon 3. The G proteins may also be different. This indicates that theMhc-G function may not be peptide presentation to the clonotypic T-cell receptor.

DESCRIPTION OF A NEW HLA-E (E*01031) ALLELE AND ITS FREQUENCY IN THE SPANISH POPULATION

An HLA-E polymorphism study by oligotyping and DNA sequencing was carried out in the Spanish population. As a result, a new HLA-E allele (E*01031) initially assigned by polymerase chain reaction oligotyping as E*0104 was found. This allele presents a synonymous change at codon 77 (AAT-->AAC; Asn) when compared with the E*01032 allele. This position is located in the alpha-helix (alpha 1-domain) and is involved in the peptide binding region of the hypothetical HLA-E molecule. Among 60 Spanish individuals, HLA-E*0101 presents the highest phenotype frequency, followed in decreasing order by E*01032, E*01031 (new allele), and E*0102. Also, new partial intron 1 and complete intron 2 sequences from E*0101, E*01031, and E*01032 are described; the sequences are identical among the three forms. However, the intron 2 sequence of the E*0102 allele bears a two-base deletion not found in apes.

Description of a novel HLA-B35 (B∗3514) allele found in a mexican family of Nahua Aztec descent

A new allele, HLA-B*3514, has been found in a Mexican family from Nahua descent. Its exon 2 is identical to that of B*3501 allele, but exon 3 bears a 3-base difference at codons 152 and 156, which results in Val-->Glu and Leu-->Trp changes, respectively, in the corresponding HLA molecule at the peptide-binding site. These substitutions may have originated from a DNA stretch donation from an allele belonging to the B15 group, enabling HLA-B*3514 to cope with the presentation of a new set of antigenic peptides. The high frequency of serologic B35 in Amerindians, together with the variety of B35 alleles detected by DNA sequencing in these populations, suggest that a frequent B35 subtype was present in the founder population and that several B35 subtypes may have been recently generated, probably due to the abrupt arrival of new pathogens following European invasions.

A new HLA-B15 allele (B*1541) found in a Mexican of Nahua (Aztec) descent

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the following accession numbers: B*1541, a) exon 2, AF033501, b) exon 3, AF033502, c) intron 1, AF034961, d) intron 2, AF034962. The name B*1541 was officially assigned by the WHO Nomenclature Committee in January 1998. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al 1997), names will be assigned to new sequences as they are identified. A list of such new names will be published in the following WHO Nomenclature Report.

An evolutionary overview of the MHC-G polymorphism: clues to the unknown function(s)

The functions of the major histocompatibility complex-G (MHC-G) molecule are still unknown. The idea that this molecule may be involved in preventing the rejection of fetuses is suggested by only indirect evidence. In the present paper, we review the structure, in vitro function and tissue distribution of MHC-G genes and proteins in different primate species. From available data, we conclude the following. First, the nomenclature of MHC-G alleles needs to be revised. Rhesus monkey A/G gene cannot be orthologous to MHC-G because of a lack of structural similarity. Cotton-top tamarin G molecules (which are also structurally similar to E molecules) cannot be orthologous to classical class I molecules. Second, selective pressure to maintain a low degree of polymorphism appears to operate only at the peptide-binding region (PBR) of the MHC-G molecule. Thus, this observation contradicts the idea that the MHC-G leader peptide is the only functional part of the molecule, and suggests that G proteins may have an antigen presenting function to clonotypic T cell receptors besides the ability to interact with NK receptors. Third, MHC-G may also have a function in the thymus, because it is expressed in the thymic epithelium. MHC-G may be important for creating an appropriate T-cell repertoire. Fourth, the presence of HLA-G proteins in tumours and the specific absence of soluble HLA-G mRNA isoforms in the tissues taken from patients with autoimmune diseases suggest that MHC-G may have a role in the immune response and inflammation control.

New DNA sequences for the human complement gene C4

In order to increase the knowledge on the human Cl genes polymorphism, sequencing of the most variable region (CM) has been carried out in four Tenth International Histocompatiiility Workshop iymphoblastoid cell lines: PLH, LKT3, YAR and DBB. Barn HI 926 bp fragments were amplifled (Belt et ai., 1985) using specific flanking primers (L3 and LA, Braun et al., 1990), by the PCR method. Purified products were cloned in a plasmid vector and recombinants were identified by differential migration in an 1% agarose gel electrophoresis. Double stranded DNA templates were sequenced using the Sanger’s dideoxy chain terminator method, with dye- iabelied dideoxy terminators and specific C4 oligonucieotidesL3, L5 and L6 (see Figure 1). Each sequence was coniirmed by analysis of 3 or 4 clones to avoid artifact aiieles due to the polymerase reaction. Four 883 bp sequences were obtained, which were assigned to the alleles tXX& C&Vi, C4A91 and CIIBNEW. Isotypic assignation was made based on the residues found in positions 1101, 1102, 1105 and 1106: proline, cysteine, leucine and aspartic acid for C4A and leucine, serine, isoleucine and histidine for c4B (Yu et ai., 1986). The obtained sequences have been compared with the already available ones. C4A2 sequence is identical in each polymorphic residue to C4A3, except codon 1054,

Description of HLA-A*6803 and A*68N in Mazatecan Indians from Mexico.

HLA-A2 and A28 are two of the most frequent HLA-A serologically defined antigens in the Mestizo and Indian Mexican populations (Weckmann et al. 1997, and unpublished data). In the present study, two new HLA-A*68 subtypes present in two unrelated individuals from the Mazatecan ethnic group were characterized and seen to belong to the A2/A28 family of antigens. They were also compared with other A*68 subtypes in order to define the possible mechanisms involved in the generation of these alleles. The Mazatecan Indians who inhabit Northern Oaxaca State in Mexico and some areas of Veracruz State are linguistically classified within the Macro-Mixtecan family, and they came originally from Eastern Mexico, as was recorded in 890 AD. Genomic DNA was isolated from peripheral blood lymphocytes using standard methods. A 5 9 HLA-A-specific primer (59AE1c: CAGACGCCGAGGATGGC) and a 3 9 HLA-A-specific primer (39Ai3c: GATCAGGGAGGCGCCCCG) were used in a polymerase chain reaction in order to obtain intron 1, exon 2, intron 2, and exon 3 sequences. DNA sequences were obtained as detailed by ArnaizVillena and co-workers (1992). The primers used for the sequencing process were ASEQ3 (Blasczyk et al. 1996) and ABSEQ5: CACAGTCTCCGGGTCCGA (local designation). Complete sequences of intron 1, exon 2, intron 2, and exon 3 from A*6803 and A*68N are shown in Fig. 1 together with those of HLAA*0204 and HLA-A*68012. HLA-A*0204 and HLA-A*68012 from Mazatecan individuals lymphocytes were also sequenced in our laboratory. TheHLA-A*68012allele has a one-base difference at codon 70 (CAG?CAC) when compared withHLA-A*6803(Fig. 1); this change results in an amino acid substitution (Gln ?His) with the corresponding neutral to positive change in polarity . Position 70 is situated at the α-helix of the first domain in the HLA molecule peptide binding site (Bjorkman et al. 1987). The residue interacts with the P2-binding pocket (B pocket) forming a salt bridge with Asp-74 (Madden et al. 1992). Analysis of non-coding regions (introns 1 and 2) of A*68012 andA*6803shows they are identical (Fig. 1). This suggests that one of them could have been generated from the other by a point mutation or by a gene conversion event that involved alleles like A2 or A24 (histidine at codon 70) (Arnett et al. 1995), which are highly frequent in Amerindians. On the other hand, theHLA-A*68N allele presents an identical intron 1, exon 2, intron 2, and exon 3 to those of HLA-A*6803except for a DNA stretch that includes the 3 9 end of intron 1 and the 5 9 end of exon 2 (from base 124 at intron 1 to codon 2 at exon 2) (see Fig. 1). A C?T change at position 124 is observed, whereas at exon 2-codon 2 a TCC?TCT synonymous change (Ser) is shown (Fig. 1). Serine at position 2 is placed on the β-sheet at the bottom of the peptide binding site. This residue is invariant in class I molecules and is involved in the α1 (position 2) andα2 (position 104) domain interactions (Bjorkman et al. 1987). Interestingly, the fragment inserted in A*68N is present in certainHLA-A2 alleles (A*0201, Cereb et al. 1996; A*0204, present work; A*0205 and A*0206, unpublished results). This fact suggests that A*68N may have been originated from A*6803which could have received a donation from this segment belonging to an HLA-A2 allele (perhapsA*0204 which is also detected in Mazatecans). Pathogen-driven polymorphism may explain the evolutive force generatingA*6803, because the change that is indicated with respect to A*68012 is productive and placed in the peptide binding region; the new AmerindianHLA-B proteins most probably diversified in the century after 1492, when 60–80 000 000 Amerindians were killed by measles, small pox, and influenza viruses introduced by European invaders (Dobbins 1993). However, A*68N andA*6803, which present a synonymous change at codon 2-exon 2, may have similar peptide presenting repertoires and the generation of one from the other may be better explained by a recombination or conversion event that involved intron 1 and exon 2 segments; this would be the first recorded example amongHLA class I or II genes not involving intron 2 and exon 3 segments (Martinez-Laso et al. 1995; Vargas-Alarco ́n et al. 1997).