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
and
polymorphic differences observed in association studies indeed cause different levels of RNA expression. Subsequently, we investigated the
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
, 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
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
. 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.
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.
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 (
, and
) 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
and
) showed strong significant differences (
< 2.1 × 10
). 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.
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.
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.
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.
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.
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.
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.
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.
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.
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