Genetic History of Aleuts of the Commander Islands as Revealed by the Analysis of the HLA Class II Gene Variability

Variability of the HLA class II genes (alleles of the DRB1, DQA1, and DQB1 loci) was investigated in a sample of Aleuts of the Commanders (n = 31), whose ancestors inhabited the Commander Islands for many thousand years. Among 19 haplotypes revealed in the Aleuts of the Commanders, at most eight were inherited from the native inhabitants of the Commander Islands. Five of these haplotypes (DRB1*0401-DQA1*0301-DQB1*0301, DRB1*1401-DQA1*0101-DQB1*0503, DRB1*0802-DQA1*0401-DQB1*0402, DRB1*1101-DQA1*0501-DQB1*0301, and DRB1*1201-DQA1*0501-DQB1*0301) were typical of Beringian Mongoloids, i.e., Coastal Chukchi and Koryaks, as well as Siberian and Alaskan Eskimos. Genetic contribution of the immigrants to the genetic pool of the proper Aleuts constituted about 52%. Phylogenetic analysis based on Transberingian distribution of the DRB1 allele frequencies favored the hypothesis on the common origin of the Paleo-Aleuts, Paleo-Eskimos, and the Indians from the northwestern North America, whose direct ancestors survived in Beringian/southwestern Alaskan coastal refugia during the late Ice Age.     

HLA genes in populations of the Aleutian islands

We typed a subset of the Aleut population for HLA loci (HLA-A, HLA-B, HLA-DRB1, HLA-DQB1) to obtain an HLA profile, which was compared to other Eurasian and Amerindian populations for studying Aleut origin and its significance on the peopling of the Americas. Allele frequencies at the four loci were identified in an Aleut sample using standard indirect DNA sequencing methods. Genetic distances with Amerindians and Eurasians were obtained by comparing Aleut allele frequencies with a worldwide population database (13,164 chromosomes). The most frequently extended HLA haplotypes were also calculated. We also generated Aleut relatedness dendrograms and calculated correspondence relatedness in a multidimensional scale. Both neighbor-joining dendrograms and correspondence analysis separated Aleuts from Eskimos and Amerindians. Aleuts are closer genetically to Europeans, including Scandinavians and English. Our results are concordant with those obtained by Y-chromosome analysis, suggesting that most male Aleut ancestors of our sample came mainly from Europe.     

Transcriptional regulation of HLA class II and invariant chain genes

Class II (Ia) antigens are coded for by a family of genes located in the human MHC (HLA). These genes are regulated in a complex manner, being constitutively expressed, inducibly expressed, or not expressed, depending on the cell type examined. 6.1.6 is a variant of a normal B lymphoblastoid line that has lost expression of all class II molecules and has previously been shown to have a defect in the regulation of class II genes. In this report, we have examined those genes by Southern and Northern blotting and have found that 6.1.6 is severely deficient in mRNA for all class II genes examined, although the genes are structurally intact. P30, a partial revertant of 6.1.6, re-expresses mRNA for a subset of class II genes. mRNA for the class II-associated invariant chain is substantially reduced but not absent in 6.1.6.     

Structure and polymorphism of the HLA class II SB light chain genes

The HLA Class II region contains at least three groups of loci, DR, DC and SB, which play an important role in the immune response. The antigens encoded at these loci are heterodimers composed of an alpha and a beta chain. The sequence of a complete Class II beta cDNA clone whose sequence agrees closely with the limited N-terminal protein sequence available for the SB beta chain is reported. In addition the structure and coding sequence of genomic SB beta clones of two different SB haplotypes has been obtained and allows definition of some polymorphic regions. The SB beta gene appears to undergo alternate splicing at its 3' end, resulting in expression of two different intracytoplasmic regions. Partial sequencing of a second non-allelic SB beta-like gene, SX beta, indicates that it is a pseudogene.     

Detection of polymorphism of HLA class II genes using synthetic oligonucleotides

The recent sequence determination of exons of various HLA class II genes has allowed us to study the polymorphism of coding sequences of these genes on a sample of HLA-DR typed unrelated individuals. From this sequence determination have emerged polymorphic hypervariable areas. A 24-mer oligonucleotide has been synthetized, which corresponds to the first hypervariable region of the beta 1 domain of a DR-beta molecule. This oligonucleotide hybridized only with the DNA from DR3 or DR5 individuals tested, even under stringent conditions of washing. However, the existence of strong linkage disequilibrium among the 3 or 4 DR genes of the D region does not allow us to conclude that this sequence is epitope-specific.     

DNA polymorphism of HLA class II genes in primary biliary cirrhosis

We investigated the DNA restriction fragment length polymorphism of the major histocompatibility complex class II genes: HLA-DRB,-DQA,-DQB, DPA,-DPB, the serologically defined HLA-A, B, C, DR antigens, and the primed lymphocyte typing defined HLA-DP antigens in 23 Danish patients with primary biliary cirrhosis (PBC) and in healthy Danes. The following genetic markers were found with increased frequencies in PBC: HLA-B8 (relative risk, RR=2.4, P<0.05, corrected P>0.05), HLA-DR3 (RR=3.4, P<0.01, corrected P<0.05), the DRB3 ^* 01/02/03 (DRw52) associated DRB Bgl II 9.1 kilobase (kb) fragment (RR= 2.9; P<0.05, corrected P>0.05), the DQA1 ^* 0501 associated DQA Taq I4.8 kb fragment (RR=3.1; P<0.05, corrected P>0.05), the DQB1 ^* 0201 (DQw2) associated DQB Hin dIII 11.5 kb fragment (RR=3.1; P<0.05, corrected P>0.05). No DNA fragments specific for DRB1 ^* 0301 (DR3) could be identified. The frequencies in PBC of other genetic markers including DRw8, DRB1 ^* 08, HLA-DP antigens, DPA, and DPB genes did not differ significantly from those in controls. The associations between PBC and B8, DR3, DQA1 ^* 0501, and DQB ^* 0201, which are frequently found together on the same haplotype, are at variance with recent reports on associations between PBC and Drw8. The discrepancy suggests that PBC is genetically heterogenous.     

Detection of novel sequence heterogeneity and haplotypic diversity of HLA class II genes

Nucleic acid sequences of the second exons of HLA-DRB1, –DRB3/4/5, –DQB1, and –DQA1 genes were determined from 43 homozygous cell lines, representing each of the known class II haplotypes, and from 30 unrelated Caucasian subjects, comprising 60 haplotypes. This systematic sequence analysis was undertaken in order to a) determine the existence of sequence microheterogeneity among cell lines which type as identical by methods other than sequencing; b) determine whether direct sequencing of class II genes will identify the presence of more extensive sequence polymorphism at the population level than that identified with other typing methods; c) accurately determine the molecular composition of the known class II haplotypes; and d) study their evolutionary relatedness by maximum parsimony analysis. The identification of seven previously unidentified haplotypes carrying five new allelic amino acid sequences suggests that sequence microheterogeneity at the population level may be more frequent than previously thought. Maximum parsimony analysis of these haplotypes allowed their evolutionary classification and indicates that the higher mutation rate at DRB1 compared to DQB1 loci in most haplotypic groups is inversed in specific haplotype lineages. Furthermore, the extent and localization of gene conversions and point mutations at class II loci in the evolution of these haplotypes is significantly different at each locus. Identification of additional HLA class II molecular microheterogeneity suggests that direct sequence analysis of class II HLA genes can uncover new allelic sequences in the population and may represent a useful alternative to current typing methodologies to study the effects of sequence allelism in organ transplantation.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M35890 through M35953.     

Evolution of HLA class II molecules: Allelic and amino acid site variability across populations

Analysis of the highly polymorphic beta1 domains of the HLA class II molecules encoded by the DRB1, DQB1, and DPB1 loci reveals contrasting levels of diversity at the allele and amino acid site levels. Statistics of allele frequency distributions, based on Watterson's homozygosity statistic F, reveal distinct evolutionary patterns for these loci in ethnically diverse samples (26 populations for DQB1 and DRB1 and 14 for DPB1). When examined over all populations, the DQB1 locus allelic variation exhibits striking balanced polymorphism (P < 10(-4)), DRB1 shows some evidence of balancing selection (P < 0.06), and while there is overall very little evidence for selection of DPB1 allele frequencies, there is a trend in the direction of balancing selection (P < 0.08). In contrast, at the amino acid level all three loci show strong evidence of balancing selection at some sites. Averaged over polymorphic amino acid sites, DQB1 and DPB1 show similar deviation from neutrality expectations, and both exhibit more balanced polymorphic amino acid sites than DRB1. Across ethnic groups, polymorphisms at many codons show evidence for balancing selection, yet data consistent with directional selection were observed at other codons. Both antigen-binding pocket- and non-pocket-forming amino acid sites show overall deviation from neutrality for all three loci. Only in the case of DRB1 was there a significant difference between pocket- and non-pocket-forming amino acid sites. Our findings indicate that balancing selection at the MHC occurs at the level of polymorphic amino acid residues, and that in many cases this selection is consistent across populations.     

Soluble HLA class I and HLA class II antigens in the blood of HIV infected patients

The levels of HLA, class I, antigen and HLA-DR antigen in the blood sera of HIV-infected persons were determined by the enzyme immunoassay. The levels of soluble antigens HLR-DR and of HLA, class I, in serum samples containing only antibodies to HIV were elevated, respectively, 1.8- and 1.3-fold in comparison with the norm. The level of soluble HLA-DR antigen in samples containing the markers of other infections (HBsAg, antibodies to hepatitis C virus, anti-IgM antibodies to cytomegalovirus) was significantly higher in comparison with samples containing only antibodies to HIV and serum samples from healthy donors (p < 0.05). The concentration of soluble HLA antigen, class I, in the samples containing antibodies to HIV and markers of other infections was also elevated, but the statistically authentic increase in comparison with the normal level was observed only in the presence of the markers of HIV infection, hepatitis C and cytomegalovirus infection.     

Sequence analysis of HLA class II domains: characterization of the DQw3 family of DQB genes

HLA class II allelic variants within the DQw3-related family of genes carry distinct allo-specificities and have been implicated in specific HLA-disease associations, such as insulin-dependent diabetes mellitus. To investigate the nucleotide variations which characterize DQw3 genes, we applied a novel cDNA cloning strategy that uses a single-stranded vector/primer system to facilitate DNA sequencing of allelically variable gene families. Using a DQB-specific primer sequence and M13 bacteriophage as the cloning vector, direct cloning and sequencing of multiple DQB genes was performed without the need for second strand synthesis or for subcloning. Sequence analysis from eight lymphoblastoid cell lines selected to represent different ethnic backgrounds revealed three DQw3-related DQB genes, DQB3.1, 3.2, and 3.3, corresponding to the newly designated HLA-DQw7, w8, and w9 specificities, respectively. An unusual Pro-Pro couplet at codons 55–56 is characteristic of all DQw3-positive sequences and may be contributing to the broad DQw3 allospecificity. Comparisons among ethnically disparate DQw3-related sequences showed no additional expressed or silent nucleotide substitutions among these DQB alleles. Thus, polymorphism within the DQw3 family of genes appears to be extremely limited, with a paucity of nucleotide variations accumulated by evolutionary distance.     

Molecular studies of a rare DR2/LD-5a/DQw3 HLA class II haplotype. Multiple genetic mechanisms in the generation of polymorphic HLA class II genes

A cDNA library was constructed from a homozygous B lymphoblastoid cell line (REM) obtained from an individual of a long isolated American Indian tribe, the Warao. The REM cell line expresses serologically defined determinants, DR2 and DQw3, and the T lymphocyte-defined (Dw/LD) specificity, LD-5a. T cells can recognize differences between FJO (a DR2/DQw1 cell line that expresses the Dw specificity MN2) and REM for both DR and DQ molecules. cNDA clones encoding the polymorphic DR beta 1-, DR beta 2-, DQ beta-, and DQ alpha-chains were sequenced and compared with other DR and DQ gene sequences. The DR beta 1-sequence of REM is identical to the DR beta 1-sequence of FJO; the DR beta 2-sequence is also identical to that of FJO except for one amino acid difference at position 67 in the polymorphic first domain (Leu in REM, Phe in FJO) due to a single point mutation. The DQ beta-sequence is identical to that of DR4/DQw3 haplotype; the DQ alpha is different from the DQ alpha of DR4/DQw3 haplotype and identical to the DQ alpha of both the DR3/DQw2 haplotype of a Raji cell line and the DR5/DQw3 haplotype in deduced amino acid sequence. Taken together, these findings suggest that: 1) a single amino acid difference (position 67) in the third hypervariable region of the first domain of the DR beta 2-chain in the DR2 haplotype is apparently sufficient for stimulating T cell responses; 2) the DQw3 serologic specificity may be defined mainly by the DQ beta-rather than DQ alpha-chain; and 3) multiple genetic events have probably occurred to generate the rarely found REM (DR2/LD-5a/DQw3) haplotype.     

The chromosome region containing the highly polymorphic HLA class I genes displays limited large scale variability in the human population

The large-scale organization and polymorphism of the HLA class I region was investigated by pulsed field gel (PFG) fractionation of DNA from various HLA-typed cell lines cleaved by different 'rare cutter' restriction enzymes, followed by hybridization with 'general' and locus-specific HLA probes. Results indicate that (i) most HLA class I sequences are contained in a 340 kb MluI DNA fragment which also carries the HLA-A gene; (ii) HLA-A, -B and -C genes are present on different fragments bounded by 'HTF islands' (CpG-rich, unmethylated DNA regions containing multiple sites for 'rare cutter' enzymes) which generally coincide with the 5' regions of expressed genes; and (iii) very little fragment size polymorphism is seen, implying that expansion/contraction events in the HLA class I region due to unequal crossing over (as documented in the mouse class I system) are infrequently found in the human population.