Nucleotide alterations in the HLA-C class I gene can cause aberrant splicing and marked changes in RNA levels in a polymorphic context-dependent manner.

Polymorphisms of HLA genes, which play a crucial role in presenting peptides with diverse sequences in their peptide-binding pockets, are also thought to affect HLA gene expression, as many studies have reported associations between HLA gene polymorphisms and their expression levels. In this study, we devised an ectopic expression assay for the HLA class I genes in the context of the entire gene, and used the assay to show that the HLA-C*03:03:01 and C*04:01:01 polymorphic differences observed in association studies indeed cause different levels of RNA expression. Subsequently, we investigated the C*03:23N null allele, which was previously noted for its reduced expression, attributed to an alternate exon 3 3' splice site generated by G/A polymorphism at position 781 within the exon 3. We conducted a thorough analysis of the splicing patterns of C*03:23N, and revealed multiple aberrant splicing, including the exon 3 alternative splicing, which overshadowed its canonical counterpart. After confirming a significant reduction in RNA levels caused by the G781A alteration in our ectopic assay, we probed the function of the G-rich sequence preceding the canonical exon 3 3' splice site. Substituting the G-rich sequence with a typical pyrimidine-rich 3' splice site sequence on C*03:23N resulted in a marked elevation in RNA levels, likely due to the enhanced preference for the canonical exon 3 3' splice site over the alternate site. However, the same substitution led to a reduction in RNA levels for C*03:03:01. These findings suggested the dual roles of the G-rich sequence in RNA expression, and furthermore, underscore the importance of studying polymorphism effects within the framework of the entire gene, extending beyond conventional mini-gene reporter assays.

Genetic Association between Farrowing Rates and Swine Leukocyte Antigen Alleles or Haplotypes in Microminipigs.

We have previously reported specific swine leukocyte antigen (SLA) haplotype associations with significant effects on several reproduction performance traits in a highly inbred miniature pig population of Microminipigs (MMPs). In this study, to clarify the effects on farrowing rates of SLA similarity between mating partners in the MMP population, we compared the farrowing rates as a measure of reproductive success after 1063-cumulative matings among the following three groups of mating partners: (1) completely sharing SLA class I or class II haplotypes or alleles between partners (CS), (2) only one sharing the haplotypes or alleles (OS), and (3) non-sharing the haplotypes or alleles (NS). Average farrowing rates in CS groups consisting of completely sharing SLA class II haplotypes or DRBI and DQB1 alleles were lowest in the three groups. Moreover, lower farrowing rates were indicated in mating pairs with smaller amino acid pairwise genetic distances of SLA-1, SLA-3, DRB1 and DQB1 alleles between the pairs. These results suggested that the dissimilarity of SLA class I and class II alleles between mating partners markedly improved reproductive performance; therefore, SLA alleles or haplotypes are potentially useful genetic markers for the selection of mating pairs in breeding programs and epistatic studies of reproductive traits of MMPs.

Sequence Variations Within HLA-G and HLA-F Genomic Segments at the Human Leukocyte Antigen Telomeric End Associated With Acute Graft-Versus-Host Disease in Unrelated Bone Marrow Transplantation.

Acute graft-versus-host disease (aGVHD) is defined as a syndrome of an immunological response of graft to the host that occurs early after allogeneic hematopoietic stem cell transplantation (HCT). This disease is frequently observed even in HCT matched for human leukocyte antigen (HLA) alleles at multiple gene loci. Although the HLA region represents complex and diverse genomic characteristics, detailed association analysis is required for the identification of uncharacterized variants that are strongly associated with aGVHD. We genotyped three loci, OR2H2, HLA-F-AS1, and HLA-G, that are located in the 460 kb of HLA telomeric region and statistically analyzed the genotypes including HLA-DPB1 with clinical and transplantation outcomes using 338 unrelated bone marrow transplantation (UR-BMT) patient-donor pairs who were matched for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 (HLA-10/10). Multivariate analyses demonstrated that HLA-F-AS1 and HLA-DPB1 mismatches were associated with grade II-IV aGVHD (hazard ratio (HR), 1.76; 95% CI, 1.07-2.88; p = 0.026; and HR, 1.59; CI, 1.02-2.49; p = 0.042, respectively). There was no confounding between HLA-F-AS1 and HLA-DPB1 (p = 0.512), suggesting that the HLA-F-AS1 mismatch has a strong effect on aGVHD independently of HLA-DPB1. Moreover, a stratified analysis suggested possible associations of HLA-F-AS1, HLA-DPB1, and/or HLA-G mismatches with grade II-IV aGVHD and the more severe grade III-IV aGVHD. These findings provide new insights into understanding the molecular mechanism of aGVHD caused by HLA-matched UR-BMT.

A novel swab storage gel is superior to dry swab DNA collection, and enables long-range high resolution next generation sequencing HLA typing from buccal cell samples.

HLA sequence-based DNA typing (SBT) by long-range PCR amplification (LR PCR) and next-generation sequencing (NGS) is a high-throughput DNA sequencing method (LR-NGS-SBT) for the efficient and sensitive detection of novel and null HLA alleles to the field-4 level of allelic resolution without phase ambiguity. However, the accuracy and reliability of the HLA typing results using buccal cells (BCs) and saliva as genetic source materials for the LR-NGS-SBT method are dependent largely on the quality of the extracted genomic DNA (gDNA) because a large degree of gDNA fragmentation can result in insufficient PCR amplification with the incorrect assignment of HLA alleles because of allele dropouts. In this study, we developed a new cost-efficient swab storage gel (SSG) for wet swab collection of BCs (BC-SSG) and evaluated its usefulness by performing different DNA analytical parameters including LR-NGS-SBT to compare the quality and quantity of gDNA extracted from BCs (in SSG or air dried), blood and saliva of 30 subjects. The BC-SSG samples after 5 days of storage revealed qualitative and quantitative DNA values equivalent to that of blood and/or saliva and better than swabs that were only air-dried (BC-nSSG). Moreover, all the gDNA extracted from blood, saliva and BC-SSG samples were HLA-typed successfully to an equivalent total of 408 alleles for each sample type. Therefore, the application of BC-SSG collection media for LR-NGS-SBT has benefits over BC dried samples (dry swabs) such as reducing retesting and the number of untestable BC samples because of insufficient DNA amplification.

Identification of three novel HLA-DRA alleles by next-generation sequencing.

Three novel HLA-DRA alleles, DRA*01:03, DRA*01:04, and DRA*01:05 alleles with unique amino acid sequences.

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs.

Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.

Capturing Differential Allele-Level Expression and Genotypes of All Classical HLA Loci and Haplotypes by a New Capture RNA-Seq Method.

The highly polymorphic human major histocompatibility complex (MHC) also known as the human leukocyte antigen (HLA) encodes class I and II genes that are the cornerstone of the adaptive immune system. Their unique diversity (>25,000 alleles) might affect the outcome of any transplant, infection, and susceptibility to autoimmune diseases. The recent rapid development of new next-generation sequencing (NGS) methods provides the opportunity to study the influence/correlation of this high level of HLA diversity on allele expression levels in health and disease. Here, we describe the NGS capture RNA-Seq method that we developed for genotyping all 12 classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5) and assessing their allelic imbalance by quantifying their allele RNA levels. This is a target enrichment method where total RNA is converted to a sequencing-ready complementary DNA (cDNA) library and hybridized to a complex pool of RNA-specific HLA biotinylated oligonucleotide capture probes, prior to NGS. This method was applied to 161 peripheral blood mononuclear cells and 48 umbilical cord blood cells of healthy donors. The differential allelic expression of 10 HLA loci (except for HLA-DRA and HLA-DPA1) showed strong significant differences (P < 2.1 × 10-15). The results were corroborated by independent methods. This newly developed NGS method could be applied to a wide range of biological and medical questions including graft rejections and HLA-related diseases.

Detection of 6pLOH in an aplastic anemia patient by in phase HLA genotyping.

Recent studies have reported loss of heterozygosity in the chromosome 6p arms (6pLOH) of acquired aplastic anemia (AA) patients, and in tumor cells trying to escape the autoimmune system. We thus sought to establish detection methods for LOH to investigate the mechanisms underlying AA and tumor immunity. Herein, we report our evaluation of 6pLOH in a patient with severe AA patient using super-high resolution, single-molecule, sequence-based typing (SS-SBT). The highest ratios of 6pLOH detection were observed during the patient's treatment with granulocyte colony stimulating factor (G-CSF). This result suggested that most of the neutrophil precursor cells stimulated by G-CSF already had LOH in the HLA lesion. The SS-SBT method is a simple NGS method that provides complete HLA allele coverage.

Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform.

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

Influence of swine leukocyte antigen haplotype on serum antibody titers against swine erysipelas vaccine and reproductive and meat production traits of SLA-defined selectively bred Duroc pigs.

We investigated possible associations of SLA class II haplotypes with serum antibody titers against a swine erysipelas vaccine, reproductive and meat production traits using a population of selective breeding Duroc pigs. In the selective breeding Duroc pigs, four SLA class II-DRB1 and -DQB1 alleles were assigned by using PCR-sequence specific primer technique. Low-resolution haplotype (Lr)-0.30 and/or Lr-0.13 were deduced from the SLA class II alleles in the population of SLA-defined Duroc pigs. SLA-homozygous piglets with the Lr-0.30 haplotype had relatively lower serum antibody titers against the vaccine compared to those with Lr-0.13. In contrast, there were no statistically significant differences in reproductive performance between the SLA-defined pigs with two SLA class II haplotypes. Weaning and rearing rates until the body weight of 105 kg was reached in homozygous piglets with Lr-0.30 were significantly lower than those in homozygous piglets with Lr-0.13. The SLA-defined pigs had lower birth and weaning weights, body weights at 60 days of age, and daily weight gains than non-selective breeding Duroc pigs. Furthermore, the SLA-defined pigs had slightly lower back fat thickness compared to the non-selective breeding pigs. The rib eye areas of homozygous or heterozygous pigs with Lr-0.13 were larger than those of homozygous pigs with Lr-0.30 and non-selective breeding pigs. These data suggested that SLA haplotypes had the potential as useful genetic markers for selective breeding in the population of SLA-defined Duroc pigs.

Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs.

The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA)-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs) by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.

Association of swine leukocyte antigen class II haplotypes and immune-related traits in a swine line selected for resistance to mycoplasmal pneumonia.

By selective breeding for five generations, a Landrace line has been recently established to improve resistance to mycoplasmal pneumonia of swine (MPS), daily gain (DG), back fat thickness (BF), and plasma cortisol concentrations (COR). To clarify the involvement of swine leukocyte antigen (SLA) polymorphisms in the selection process, we investigated possible associations of 11 SLA-class II haplotypes with selected traits or immune parameters. Pigs with the low-resolution SLA haplotype Lr-0.23 or Lr-0.13, which increased in frequency with the passage of generations, had less severe pathological lesions of MPS, increased leukocyte phagocytic activity, and higher white blood cell counts. In contrast, Lr-0.12 and Lr-0.2, which decreased in subsequent generations, were weakly associated with more severe pathological lesions of MPS. Therefore, in the studied Landrace line, the Lr-0.23 and Lr-0.13 haplotypes are potentially useful genetic markers for selecting and breeding animals with less severe pathological lesions of MPS.

Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing.

BACKGROUND: HLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1. RESULTS: We developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template. CONCLUSIONS: In this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.

Genomic sequence analysis of the MHC class I G/F segment in common marmoset (Callithrix jacchus).

The common marmoset (Callithrix jacchus) is a New World monkey that is used frequently as a model for various human diseases. However, detailed knowledge about the MHC is still lacking. In this study, we sequenced and annotated a total of 854 kb of the common marmoset MHC region that corresponds to the HLA-A/G/F segment (Caja-G/F) between the Caja-G1 and RNF39 genes. The sequenced region contains 19 MHC class I genes, of which 14 are of the MHC-G (Caja-G) type, and 5 are of the MHC-F (Caja-F) type. Six putatively functional Caja-G and Caja-F genes (Caja-G1, Caja-G3, Caja-G7, Caja-G12, Caja-G13, and Caja-F4), 13 pseudogenes related either to Caja-G or Caja-F, three non-MHC genes (ZNRD1, PPPIR11, and RNF39), two miscRNA genes (ZNRD1-AS1 and HCG8), and one non-MHC pseudogene (ETF1P1) were identified. Phylogenetic analysis suggests segmental duplications of units consisting of basically five (four Caja-G and one Caja-F) MHC class I genes, with subsequent expansion/deletion of genes. A similar genomic organization of the Caja-G/F segment has not been observed in catarrhine primates, indicating that this genomic segment was formed in New World monkeys after the split of New World and Old World monkeys.

Differentiation ability of multipotent hematopoietic stem/progenitor cells detected by a porcine specific anti-CD117 monoclonal antibody.

CD117 is a cytokine receptor expressed on the surface of hematopoietic stem cells with a likely role in cell survival, proliferation and differentiation. In order to study the differentiation activity of porcine CD117 hematopoietic cells in vitro and in vivo we prepared an anti-swine CD117 Mab (2A1) with high specificity for flow-cytometrical analysis. The 2A1 Mab did not recognize mouse or human mast cells suggesting that 2A1 is species-specific. Swine bone marrow (BM) CD117+ cells differentiated in vitro mainly into erythroid and monocyte lineages in the methylcellulose-based colony assay. When the swine BM CD117+ cells were transplanted in vivo into immunodeficient NOG (NOD/SCID/IL-2gc-null) mice, a significant amount of swine CD45+ leukocytes, including CD3 positive T cells, were developed in the mice. These results revealed that the swine BM CD117+ cells possess hematopoietic stem/progenitor activity and when monitored in immunodeficient mice or in vitro they can develop into lymphoid, erythroid, and myeloid cells efficiently with the new monoclonal antibody.

Modified S/MAR episomal vectors for stably expressing fluorescent protein-tagged transgenes with small cell-to-cell fluctuations.

We modified and tested scaffold/matrix attachment region (S/MAR) episomal vectors. The new vectors would be useful in obtaining cells stably expressing fluorescent protein-tagged transgenes with small, mostly within 10-fold cell-to-cell fluctuations. In the vectors, the same transcript directs episomal replication and expression of transgene/antibiotic marker, and only antibiotic selection without any other extra steps was sufficient to obtain desired stable cells, including those expressing two different proteins simultaneously. Furthermore, the two test cases (expression of human growth hormone in AtT20 and four protein kinase C isoforms in HEK293) would prove to be useful in visualizing and analyzing regulatory processes involving these proteins.

Polymorphic SVA retrotransposons at four loci and their association with classical HLA class I alleles in Japanese, Caucasians and African Americans.

Polymorphic insertion frequencies of the retrotransposons known as the "SVA" elements were investigated at four loci in the MHC class I genomic region to determine their allele and haplotype frequencies and associations with the HLA-A, -B or -C genes for 100 Japanese, 100 African Americans, 174 Australian Caucasians and 66 reference cell lines obtained from different ethnic groups. The SVA insertions representing different subfamily members varied in frequency between none for SVA-HF in Japanese and 65% for SVA-HB in Caucasians or African Americans with significant differences in frequencies between the three populations at least at three loci. The SVA loci were in Hardy-Weinberg equilibrium except for the SVA-HA locus which deviated significantly in African Americans and Caucasians possibly because of a genomic deletion of this locus in individuals with the HLA-A*24 allele. Strong linkage disequilibria and high percentage associations between the human leucocyte antigen (HLA) class I gene alleles and some of the SVA insertions were detected in all three populations in spite of significant frequency differences for the SVA and HLA class I alleles between the three populations. The highest percentage associations (>86%) were between SVA-HB and HLA-B*08, -B*27, -B*37 to -B*41, -B*52 and -B*53; SVA-HC and HLA-B*07; SVA-HA and HLA-A*03, -A*11 and -A*30; and SVA-HF and HLA-A*03 and HLA-B*47. From pairwise associations in the three populations and the homozygous cell line results, it was possible to deduce the SVA and HLA class I allelic combinations (haplotypes), population differences and the identity by descent of several common HLA-A allelic lineages.

HLA-A allele associations with viral MER9-LTR nucleotide sequences at two distinct loci within the MHC alpha block.

The study of the association of the Human Leukocyte Antigen (HLA) alleles and polymorphic retrotransposons such as Alu, HERV, and LTR at various loci within the Major Histocompatibility Complex allows for a better identification and stratification of disease associations and the origins of HLA haplotypes in different populations. This paper provides sequence and association data on two structurally polymorphic MER9-LTR retrotransposons that are located 54 kb apart and in close proximity to the multiallelic HLA-A gene involved in the regulation of the human immune system. Direct DNA sequencing and analysis of the PCR products identified DNA nucleotide variations between the MER9-LTR sequences at the two loci and their associations with HLA-A alleles as potential haplotype and evolutionary markers. All MER9-LTR sequences were haplotypic when associated with common HLA-A alleles. The number of SNP loci was 2.5 times greater for the solo LTR at the AK locus, which is located closer to the HLA-A gene than the solo or 3' LTR at the HG locus. Our study shows that the nucleotide variations of the MER9-LTR DNA sequences are additional informative markers in fine mapping HLA-A genomic haplotypes for future population, evolutionary, and disease studies.

Human endogenous retrovirus (HERVK9) structural polymorphism with haplotypic HLA-A allelic associations.

The frequency and HLA-A allelic associations of a HERVK9 DNA structural polymorphism located in close proximity to the highly polymorphic HLA-A gene within the major histocompatibility complex (MHC) genomic region were determined in Japanese, African Americans, and Australian Caucasians to better understand its human population evolutionary history. The HERVK9 insertion or deletion was detected as a 3' LTR or a solo LTR, respectively, by separate PCR assays. The average insertion frequency of the HERVK9.HG was significantly different (P < 1.083e(-6)) between the Japanese (0.59) and the African Americans (0.34) or Australian Caucasians (0.37). LD analysis predicted a highly significant (P < 1.0e(-5)) linkage between the HLA-A and HERVK9 alleles, probably as a result of hitchhiking (linkage). Evolutionary time estimates of the solo, 5' and 3' LTR nucleotide sequence divergences suggest that the HERVK9 was inserted 17.3 MYA with the first structural deletion occurring 15.1 MYA. The LTR/HLA-A haplotypes appear to have been formed mostly during the past 3.9 MY. The HERVK9 insertion and deletion, detected by a simple and economical PCR method, is an informative genetic and evolutionary marker for the study of HLA-A haplotype variations, human migration, the origins of contemporary populations, and the possibility of disease associations.