B21 Haplotype Research Articles

Revealing novel and conservative T-cell epitopes with MHC B2 restriction on H9N2 avian influenza virus (AIV)

B2 haplotype major histocompatibility complex (MHC) has been extensively reported to confer resistance to various avian diseases. But its peptide-binding motif is unknown, and the presenting peptide is rarely identified. Here, we identified its peptide-binding motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) invitro using Random Peptide Library-based MHC I LC-MS/MS analysis. To further clarify the structure basis of motif, we determined the crystal structure of the BF2∗02:01-PB2552-560 complex at 1.9Å resolution. We found that BF2∗02:01 had a relatively wide antigen-binding groove, and the structural characterization of pockets was consistent with the characterization of peptide-binding motif. The wider features of the peptide-binding motif and increased number of peptides bound by BF2∗02:01 than BF2∗04:01 might resolve the puzzles for the presence of potential H9N2 resistance in B2 chickens. Afterward, we explored the H9N2 avian influenza virus (AIV)-induced cellular immune response in B2 haplotype chickens invivo. We found that ratio of CD8+ T cell and kinetic expression of cytotoxicity genes including Granzyme K, interferon-γ, NK lysin, and poly-(ADP-ribose) polymerase in peripheral blood mononuclear cells were significantly increased in defending against H9N2 AIV infection. Especially, we selected 425 epitopes as candidate epitopes based on the peptide-binding motif and further identified four CD8+ T-cell epitopes on H9N2 AIV including NS198-106, PB2552-560, NP182-190, and NP455-463 via ELI-spot interferon-γ detections after stimulating memory lymphocytes with peptides. More importantly, these epitopes were found to be conserved in H7N9 AIV and H9N2 AIV. These findings provide direction for developing effective T cell epitope vaccines using well-conserved internal viral antigens in chickens.

Contribution of the TCRβ Repertoire to Marek’s Disease Genetic Resistance in the Chicken

Marek's disease (MD) is a lymphoproliferative disease of chickens induced by Marek's disease virus (MDV), an oncogenic α-herpesvirus. MDV has increased in virulence, prompting continued efforts in both improved vaccines and enhanced genetic resistance. Model pairs of genetically MD-resistant and MD-susceptible chickens that were either MHC-matched or MHC-congenic allowed characterization of T cell receptor (TCR) repertoires associated with MDV infection. MD-resistant chickens showed higher usage of Vβ-1 TCRs than susceptible chickens in both the CD8 and CD4 subsets in the MHC-matched model, and in the CD8 subset only in the MHC-congenic model, with a shift towards Vβ-1+ CD8 cells during MDV infection. Long and short read sequencing identified divergent TCRβ loci between MHC-matched MD-resistant and MD-susceptible chickens, with MD-resistant chickens having more TCR Vβ1 genes. TCR Vβ1 CDR1 haplotype usage in MD-resistant x MD-susceptible F1 birds by RNAseq indicated that the most commonly used CDR1 variant was unique to the MD-susceptible line, suggesting that selection for MD resistance in the MHC-matched model optimized the TCR repertoire away from dominant recognition of one or more B2 haplotype MHC molecules. Finally, TCR downregulation during MDV infection in the MHC-matched model was strongest in the MD-susceptible line, and MDV reactivation downregulated TCR expression in a tumor cell line.

Marek's disease virus-specific T cells proliferate, express antiviral cytokines but have impaired degranulation response.

The major histocompatibility complex (MHC) haplotype is one of the major determinants of genetic resistance and susceptibility of chickens to Marek’s disease (MD) which is caused by an oncogenic herpesvirus; Marek’s disease virus (MDV). To determine differential functional abilities of T cells associated with resistance and susceptibility to MD, we identified immunodominant CD4+TCRvβ1 T cell epitopes within the pp38 antigen of MDV in B19 and B21 MHC haplotype chickens using an ex vivo ELISPOT assay for chicken IFN-gamma. These novel pp38 peptides were used to characterize differential functional abilities of T cells as associated with resistance and susceptibility to MD. The results demonstrated an upregulation of cytokines (IL-2, IL-4, IL-10) and lymphocyte lysis-related genes (perforin and granzyme B) in an antigen specific manner using RT-PCR. In the MD-resistant chickens (B21 MHC haplotype), antigen-specific and non-specific response was highly skewed towards Th2 response as defined by higher levels of IL-4 expression as well as lymphocyte lysis-related genes compared to that in the MD-susceptible chicken line (B19 MHC haplotype). Using CD107a degranulation assay, the results showed that MDV infection impairs cytotoxic function of T cells regardless of their genetic background. Taken together, the data demonstrate an association between type of T cell response to pp38 and resistance to the disease and will shed light on our understanding of immune response to this oncogenic herpesvirus and failure to induce sterile immunity.

The relationship between MHC−peptide interaction and resistance to virus in chickens

IntroductionThe MHC‐peptide interaction has a subtle influence on host resistance to virus. This paper aims to study the relationship between MHC‐peptide interaction and MHC‐related virus‐resistance.MethodsBy 3D homology modeling, the structure of chicken BF2 molecule BF2*0201 (PDB code: 4d0d) was studied and compared with the known structures of BF2 molecule BF2*0401 (PDB code: 4e0r) to elucidate the characteristics of BF2*0201‐binding antigenic peptides.ResultsThe results show that due to the amino acid difference between the two binding groove of 4e0r and 4d0d, the size of the binding groove of the two are 1130 ų and1380 ų respectively, indicating the amino acid species that 4e0r binding peptide has lower selectivity than 4d0d; and because of large side chain conformation of Arg (especially Arg111) of 4e0r replaced by small side chain Tyr111 of 4d0d, the volume of central part of the binding groove of 4d0d is obviously larger than that of 4e0r, indicating that the restrictive of binding antigenic peptides for 4d0d is narrower than that of 4e0r; and on account of the chargeability of the binding groove of the two are different, namely the binding groove chargeability of 4e0r (strong positive polarity) and 4d0d (weak negative polarity).ConclusionThere are generally more peptides presented by the BF2 of B2 haplotype than by that of B4 haplotype, leading to more resistance of B2 than that of B4 to virus.

Differential Virus-Specific IFN-Gamma Producing T Cell Responses to Marek's Disease Virus in Chickens With B19 and B21 MHC Haplotypes.

Marek’s disease virus (MDV), the etiologic agent for Marek’s disease (MD), causes a deadly lymphoproliferative disease in chickens. Causes of the well-documented association between genetically defined lines of chicken and resistance to MD remain unknown. Here, the frequencies of IFN-gamma producing pp38 and MEQ-specific T cell responses were determined in line N (B21 haplotype; MD-resistant) and line P2a (B19 haplotype, MD-susceptible) chickens after infection with vaccine and/or virulent (RB1B) strains of MDV using both standard ex vivo and cultured chIFN-gamma ELISPOT assays. Notably, MDV infection of naïve and vaccinated MD-resistant chickens induced higher frequencies of IFN-gamma producing MDV-specific T cell responses using the cultured and ex vivo ELISPOT assay, respectively. Remarkably, vaccination did not induce or boost MEQ-specific effector T cells in the susceptible chickens, while it boosted both pp38-and MEQ-specific response in resistant line. Taken together, our results revealed that there is a direct association between the magnitude of T cell responses to pp38 and MEQ of MDV antigens and resistance to the disease.

Study on the contrast of the MHC-peptide interaction of B2/B21 haplotype and MHC-related virus resistance in chickens.

IntroductionThree‐dimensional (3D) structures of MHC class I exert some influence by the MHC–peptide interaction over host resistance to the virus. The thesis aims at studying the connection between MHC–peptide interaction of B2/B21 haplotype and MHC‐related resistance to the virus.MethodsThe structure of chicken MHC class I BF2*0201 from B2 haplotype was studied and contrasted with that of BF2*2101 from B21 haplotype by using DNAMAN and PyMol software.ResultsThe amino acid difference resulted in the difference in size and changeability of the binding groove of the two, resulting in different choices on the binding polypeptide. 3bew's (the crystal structure of BF2*2101 bound to peptide RV10) small side chain His111 replaces the short side chain Tyr111 of 4cvx (the crystal structure of BF2*0201 bound to peptide YL9), and the very small amino acid of Ser69 and Ser97 make the middle of the 3bew's binding groove become apparently broad and bound restrictive of amino acid smaller. Moreover, due to the specific amino acids—Arg9, Asp24, and Asp73 of 4cvx and Arg9, Asp24, and His111 of 3bew, the effect of the polypeptide and the binding groove differ between the two, and 3bew tends to bind polypeptides with negatively charged amino acids, but the large space in the middle can also accommodate other amino acids. Contrasted with the binding groove characteristic of 4cvx, it can be said that the selectivity of 3bew is higher than that of 4cvx in the amino acid type of the binding polypeptide, so the B21 haplotype has more host resistance to the virus than that of the B2 haplotype in chicken.ConclusionThere are usually various kinds of peptides presented by the BF2*2101 molecules of B21 haplotypes, resulting in resistance to pathogenic microorganisms, such as Rous sarcoma virus and/or Marek's disease virus. These findings may have an important theoretical foundation for screening of virus antigen, vaccine design, and genetic resistance breeding.

Role of Autoimmunity in Patients Transplanted for Acute Liver Failure of Unknown Origin: A Clinical and Graft Biopsy Analysis.

The etiology and prognosis of acute liver failure (ALF) remains unknown in a significant proportion of cases. Signs of autoimmunity may be present, but no consistent pattern has been observed. We aimed to analyze if pretransplant immunological findings, human leukocyte antigen (HLA) haplotypes, and clinical features among patients with an unknown etiology differ from those of autoimmune or other known etiologies. We also analyzed whether such signs impact posttransplant biopsy findings or complications. All adult ALF patients undergoing liver transplantation (LT) in Finland during 1987-2015 were followed to 2016. Data were collected from the LT registry, pathology database, and patient records. A total of 124 patients were included in the analysis. Study subgroups were acute autoimmune hepatitis (AIH; n=25), known non-AIH etiology (n=54), and unknown etiology (n=45). The unknown etiology group differed from the known non-AIH group with regard to the following pretransplant autoimmunity-associated features: positive perinuclear anti-neutrophil cytoplasmic antibodies (36% versus 8%; P=0.02) and higher mean immunoglobulin A (IgA; 3.2±1.7 versus 2.1±1.4, P=0.006) and immunoglobulin G (IgG; 12.7±4.3 versus 8.5±3.6, P=0.001). AIH-associated HLA haplotypes B8, DR3, and B8DR3 were more common in the AIH group (40%, 44%, and 36%, respectively) and in the unknown group (29%, 33%, and 29%, respectively) than in the known non-AIH group (11%, 17%, and 11%, respectively) or in the Finnish general population (17%, 18%, and 8%, respectively). However, these findings had no association with protocol biopsies, extrahepatic autoimmune diseases, or survival. Patients with≥1 rejection episode had higher pretransplant IgA (3.7±2.3 versus 2.6± 1.2; P=0.02) and IgG (16.4±10.2 versus 12.4±6.8; P=0.03) than those without rejections. Autoimmunity-associated pretransplant laboratory findings and HLA haplotypes were common in ALF of unknown etiology, but they showed minimal predictive value for posttransplant biopsy findings, clinical complications, or survival.

Resolving mitochondrial haplogroups B2 and B4 with next-generation mitogenome sequencing to distinguish Native American from Asian haplotypes

Mitochondrial haplogroup information can be useful in forensic contexts that rely primarily on mitochondrial DNA (mtDNA) testing, which often involve limited or degraded DNA. Due to the phylogeographic patterning of mtDNA in human populations, mitochondrial haplogroups are indicative of maternal ancestry (as mtDNA is a maternally inherited marker). In certain circumstances, maternal ancestry inferred from mitochondrial haplogrouping could be beneficial to forensic investigations. For example, ancestry information could assist in the identification of unknown service members from past conflicts, such as the World War II Battle of Tarawa involving American and Japanese forces. In this context, it could be useful to distinguish Native American mtDNA from Asian mtDNA to bolster the anthropological and circumstantial evidence leading to an identification or foreign national determination. Although most of the founding Native American haplogroups contain diagnostic variants in the mitochondrial control region (CR), haplogroup B2 does not, and this makes it more difficult to distinguish B2 from the parental B4 and closely related B4b haplogroups found in Asia. In this paper, the amount of mtDNA information required to distinguish Native American haplotypes from Asian haplotypes within haplogroup B was examined. Fifty-six samples belonging to subtypes of B2 and B4 were sequenced for the entire mitogenome. Haplogroups were estimated from three ranges of mitochondrial DNA (HV1 and 2, CR, and full mitogenome). Half of the samples could not be precisely haplogrouped without full mitogenome data, although enough variants were often provided to make an accurate B2 versus B4 distinction. Native American B2 haplotypes were distinguishable using CR data alone in 82% of samples, though the remaining samples required full mitogenome data for haplogroup B2 designation. The use of full mitogenome data consistently enables accurate haplogroup determination, and opens the possibility for gaining information on maternal ancestry.

Functional Alleles of Chicken BG Genes, Members of the Butyrophilin Gene Family, in Peripheral T Cells.

γδ T cells recognize a wide variety of ligands in mammals, among them members of the butyrophilin (BTN) family. Nothing is known about γδ T cell ligands in chickens, despite there being many such cells in blood and lymphoid tissues, as well as in mucosal surfaces. The major histocompatibility complex (MHC) of chickens was discovered because of polymorphic BG genes, part of the BTN family. All but two BG genes are located in the BG region, oriented head-to-tail so that unequal crossing-over has led to copy number variation (CNV) as well as hybrid (chimeric) genes, making it difficult to identify true alleles. One approach is to examine BG genes expressed in particular cell types, which likely have the same functions in different BG haplotypes and thus can be considered “functional alleles.” We cloned nearly full-length BG transcripts from peripheral T cells of four haplotypes (B2, B15, B19, and B21), and compared them to the BG genes of the B12 haplotype that previously were studied in detail. A dominant BG gene was found in each haplotype, but with significant levels of subdominant transcripts in three haplotypes (B2, B15, and B19). For three haplotypes (B15, B19, and B21), most sequences are closely-related to BG8, BG9, and BG12 from the B12 haplotype. We found that variation in the extracellular immunoglobulin-variable-like (Ig-V) domain is mostly localized to the membrane distal loops but without evidence for selection. However, variation in the cytoplasmic tail composed of many amino acid heptad repeats does appear to be selected (although not obviously localized), consistent with an intriguing clustering of charged and polar residues in an apparent α-helical coiled-coil. By contrast, the dominantly-expressed BG gene in the B2 haplotype is identical to BG13 from the B12 haplotype, and most of the subdominant sequences are from the BG5-BG7-BG11 clade. Moreover, alternative splicing leading to intron read-through results in dramatically truncated cytoplasmic tails, particularly for the dominantly-expressed BG gene of the B2 haplotype. The approach of examining “functional alleles” has yielded interesting data for closely-related genes, but also thrown up unexpected findings for at least one haplotype.

MHC-B haplotypes impact susceptibility and resistance to RSV-A infection.

We investigated the impact ofhaplotypeofmajor histocompatibility complex(MHC)-B on the outcome of infection of Synthetic Dam Line (SDL) broiler strain withRous Sarcoma Virus(RSV).Genomicanalysis ofMHC-Bhaplotypes, revealed a total of 12 known standardhaplotypesthat constituted to twenty-five differentgenotypesand one newhaplotypeof 217 bp size, designated BX. The inoculation ofRSV-A in SDL chicks resulted in the development oftumorsof progressive or regressivephenotypeswith varyingtumor profile index(TPI).HaplotypesB2, B21and B22had lowTPIscores (1 or 2) with lessmortalityand were resistant toRSV-Atumor. ThehaplotypesB13, B13.1., B15, B15.1.and B15.2.had significantly higherTPIscores (5 or 6), indicating a susceptibility toRSV-A. Thegenotype, Bx/Bx, had a meanTPIscore of 3.67 ± 1.33, which was closer to the resistanthaplotype.Sequence analysisof the newhaplotype(BX) revealed 99.5% similarity with B2haplotype.Metastaseswas observed in 44% of chicks and comprised of mixed fibrosarcoma and myxosarcoma.

RNA sequencing demonstrates large-scale temporal dysregulation of gene expression in stimulated macrophages derived from MHC-defined chicken haplotypes.

Discovering genetic biomarkers associated with disease resistance and enhanced immunity is critical to developing advanced strategies for controlling viral and bacterial infections in different species. Macrophages, important cells of innate immunity, are directly involved in cellular interactions with pathogens, the release of cytokines activating other immune cells and antigen presentation to cells of the adaptive immune response. IFNγ is a potent activator of macrophages and increased production has been associated with disease resistance in several species. This study characterizes the molecular basis for dramatically different nitric oxide production and immune function between the B2 and the B19 haplotype chicken macrophages.A large-scale RNA sequencing approach was employed to sequence the RNA of purified macrophages from each haplotype group (B2 vs. B19) during differentiation and after stimulation. Our results demonstrate that a large number of genes exhibit divergent expression between B2 and B19 haplotype cells both prior and after stimulation. These differences in gene expression appear to be regulated by complex epigenetic mechanisms that need further investigation.

Northern fowl mite (Ornithonyssus sylviarum) effects on metabolism, body temperatures, skin condition, and egg production as a function of hen MHC haplotype

The northern fowl mite, Ornithonyssus sylviarum, is the most damaging ectoparasite on egg-laying hens in the United States. One potential strategy for management is breeding for mite resistance. Genes of white leghorn chickens linked to the major histocompatibility complex (MHC) were previously identified as conferring more (B21 haplotype) or less (B15 haplotype) mite resistance. However, immune responses can be energetically costly to the host and affect the economic damage incurred from mite infestations. We tested energy costs (resting metabolic rate) of mite infestations on egg-laying birds of both MHC B-haplotypes. Resting metabolic rates were documented before (pre-) mite infestation, during (mid-) infestation, and after peak (late) mite infestation. Mite scores, economic parameters (egg production, feed consumption), and physiological aspects such as skin inflammation and skin temperature were recorded weekly. Across experiments and different infestation time points, resting metabolic rates generally were not affected by mite infestation or haplotype, although there were instances of lower metabolic rates in infested versus control hens. Skin temperatures were recorded both at the site of mite feeding damage (vent) and under the wing (no mites), which possibly would reflect a systemic fever response. Ambient temperatures modified skin surface temperature, which generally was not affected by mites or haplotype. Feed conversion efficiency was significantly worse (4.9 to 17.0% depending on trial) in birds infested with mites. Overall egg production and average egg weight were not affected significantly, although there was a trend toward reduced egg production (2 to 8%) by infested hens. The MHC haplotype significantly affected vent skin inflammation. Birds with the mite-resistant B21 haplotype showed earlier onset of inflammation, but a reduced overall area of inflammation compared to mite-susceptible B15 birds. No significant differences in resting energy expenditure related to mite infestation or immune responses were detected. Potential breeding for resistance to mite infestation using these two haplotypes appears to be neutral in terms of impact on hen energy costs or production efficiency, and may be an attractive option for future mite control.

Macrophages from disease resistant B2 haplotype chickens activate T lymphocytes more effectively than macrophages from disease susceptible B19 birds

Resistance to respiratory pathogens, including coronavirus-induced infection and clinical illness in chickens has been correlated with the B (MHC) complex and differential ex vivo macrophage responses. In the current study, in vitro T lymphocyte activation measured by IFNγ release was significantly higher in B2 versus B19 haplotypes. AIV infection of macrophages was required to activate T lymphocytes and prior in vivo exposure of chickens to NP AIV plasmid enhanced responses to infected macrophages. This study suggests that the demonstrated T lymphocyte activation is in part due to antigen presentation by the macrophages as well as cytokine release by the infected macrophages, with B2 haplotypes showing stronger activation. These responses were present both in CD4 and CD8 T lymphocytes. In contrast, T lymphocytes stimulated by ConA showed greater IFNγ release of B19 haplotype cells, further indicating the greater responses in B2 haplotypes to infection is due to macrophages, but not T cells. In summary, resistance of B2 haplotype chickens appears to be directly linked to a more vigorous innate immune response and the role macrophages play in activating adaptive immunity.

Strains of the Group I Lineage of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch of Cucurbitaceous Crops, are Predominant in Brazil.

Bacterial fruit blotch (BFB), caused by the seedborne bacterium Acidovorax citrulli, is an economically important threat to cucurbitaceous crops worldwide. Since the first report of BFB in Brazil in 1990, outbreaks have occurred sporadically on watermelon and, more frequently, on melon, resulting in significant yield losses. At present, the genetic diversity and the population structure of A. citrulli strains in Brazil remain unclear. A collection of 74 A. citrulli strains isolated from naturally infected tissues of different cucurbit hosts in Brazil between 2000 and 2014 and 18 A. citrulli reference strains from other countries were compared by pulsed-field gel electrophoresis (PFGE), multilocus sequence analysis (MLSA) of housekeeping and virulence-associated genes, and pathogenicity tests on seedlings of different cucurbit species. The Brazilian population comprised predominantly group I strains (98%), regardless of the year of isolation, geographical region, or host. Whole-genome restriction digestion and PFGE analysis revealed that three unique and previously unreported A. citrulli haplotypes (assigned as haplotypes B22, B23, and B24) occurred in Brazil. The greatest diversity of A. citrulli (four haplotypes) was found among strains collected from the northeastern region of Brazil, which accounts for more than 90% of the country's melon production. MLSA clearly distinguished A. citrulli strains into two well-supported clades, in agreement with observations based on PFGE analysis. Five Brazilian A. citrulli strains, representing different group I haplotypes, were moderately aggressive on watermelon seedlings compared with four group II strains that were highly aggressive. In contrast, no significant differences in BFB severity were observed between group I and II A. citrulli strains on melon and squash seedlings. Finally, we observed a differential effect of temperature on in vitro growth of representative group I and II A. citrulli haplotypes. Specifically, of 18 group II strains tested, all grew at 40 and 41°C, whereas only 3 of 15 group I strains (haplotypes B8[P], B3[K], and B15) grew at 40°C. Three strains representing haplotype B8(P) were the only group I strains that grew at 41°C. These results contribute to a better understanding of the genetic diversity of A. citrulli associated with BFB outbreaks in Brazil, and reinforce the efficiency of MLSA and PFGE analysis for assessing population structure. This study also provides the first evidence to suggest that temperature might be a driver in the ecological adaptation of A. citrulli populations.

Global dysregulation of gene expression in macrophages from different chicken haplotypes correlates with disease resistance

Abstract Disease resistance and susceptibility has been associated with the major histocompatibility complex (MHC) in several species. This study aimed to better characterize the molecular basis for dramatically different nitric oxide production and associated immune function between the B2 and the B19 haplotype chicken macrophages in response to IFNγ stimulation, previously shown by our laboratory. A next generation RNA sequencing approach was employed to analyze gene expression from purified macrophages from each haplotype group (B2 vs. B19) across 9 time points spanning seven days. Ultimately, our data shows global dysregulation of gene expression in B19 haplotypes correlating with increased disease susceptibility of this haplotype. Our results demonstrate that transcription factors (TF) exhibit divergent expression between B2 and B19 haplotype cells both prior to and after stimulation. Our results also reveal that miRNAs exhibit divergent expression between B2 and B19 haplotype cells both, prior to, and after stimulation with IFNγ. In contrast to TFs, miRNAs can modulate gene product levels post-transcriptionally. We also identify ubiquitin-related enzymes and factors that exhibit divergent expression between B2 and B19 haplotype cells. Some of our results indicate that snoRNAs exhibit divergent expression between B2 and B19 haplotype cells, which is noteworthy because snoRNAs can modulate genes epigenetically. Taken together our results suggest that the global dysregulation of gene expression observed in B19 haplotypes can at least in part be attributed to differential epigenetic regulation.

A high-density SNP panel reveals extensive diversity, frequent recombination and multiple recombination hotspots within the chicken major histocompatibility complex B region between BG2 and CD1A1.

BackgroundThe major histocompatibility complex (MHC) is present within the genomes of all jawed vertebrates. MHC genes are especially important in regulating immune responses, but even after over 80 years of research on the MHC, much remains to be learned about how it influences adaptive and innate immune responses. In most species, the MHC is highly polymorphic and polygenic. Strong and highly reproducible associations are established for chicken MHC-B haplotypes in a number of infectious diseases. Here, we report (1) the development of a high-density SNP (single nucleotide polymorphism) panel for MHC-B typing that encompasses a 209,296 bp region in which 45 MHC-B genes are located, (2) how this panel was used to define chicken MHC-B haplotypes within a large number of lines/breeds and (3) the detection of recombinants which contributes to the observed diversity.MethodsA SNP panel was developed for the MHC-B region between the BG2 and CD1A1 genes. To construct this panel, each SNP was tested in end-point read assays on more than 7500 DNA samples obtained from inbred and commercially used egg-layer lines that carry known and novel MHC-B haplotypes. One hundred and one SNPs were selected for the panel. Additional breeds and experimentally-derived lines, including lines that carry MHC-B recombinant haplotypes, were then genotyped.ResultsMHC-B haplotypes based on SNP genotyping were consistent with the MHC-B haplotypes that were assigned previously in experimental lines that carry B2, B5, B12, B13, B15, B19, B21, and B24 haplotypes. SNP genotyping resulted in the identification of 122 MHC-B haplotypes including a number of recombinant haplotypes, which indicate that crossing-over events at multiple locations within the region lead to the production of new MHC-B haplotypes. Furthermore, evidence of gene duplication and deletion was found.ConclusionsThe chicken MHC-B region is highly polymorphic across the surveyed 209-kb region that contains 45 genes. Our results expand the number of identified haplotypes and provide insights into the contribution of recombination events to MHC-B diversity including the identification of recombination hotspots and an estimation of recombination frequency.Electronic supplementary materialThe online version of this article (doi:10.1186/s12711-015-0181-x) contains supplementary material, which is available to authorized users.

Surface expression, peptide repertoire, and thermostability of chicken class I molecules correlate with peptide transporter specificity

The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface expression level of MHC class I molecules varies as much as 10-fold between chicken haplotypes and is inversely correlated with diversity of peptide repertoire and with resistance to Marek's disease caused by an oncogenic herpesvirus. Here we show that the average thermostability of class I molecules isolated from cells also varies, being higher for high-expressing MHC haplotypes. However, we find roughly the same amount of class I protein synthesized by high- and low-expressing MHC haplotypes, with movement to the cell surface responsible for the difference in expression. Previous data show that chicken TAP genes have high allelic polymorphism, with peptide translocation specific for each MHC haplotype. Here we use assembly assays with peptide libraries to show that high-expressing B15 class I molecules can bind a much wider variety of peptides than are found on the cell surface, with the B15 TAPs restricting the peptides available. In contrast, the translocation specificity of TAPs from the low-expressing B21 haplotype is even more permissive than the promiscuous binding shown by the dominantly expressed class I molecule. B15/B21 heterozygote cells show much greater expression of B15 class I molecules than B15/B15 homozygote cells, presumably as a result of receiving additional peptides from the B21 TAPs. Thus, chicken MHC haplotypes vary in several correlated attributes, with the most obvious candidate linking all these properties being molecular interactions within the peptide-loading complex (PLC).

Genetic Determinants of Busulfan Clearance and Outcomes in Pediatric Patients Undergoing Hematopoietic Stem Cell Transplantation- Result of a Multicentric Prospective Study on Behalf of the Pediatric Disease Working Party of the European Blood and Marrow Transplantation Group

▪Busulfan (BU) dose adjustment following therapeutic drug concentration monitoring improves outcomes of hematopoietic stem cell transplantation (HSCT). Further improvement could be achieved through genotype-based BU dose adjustments. Studies with large and homogeneous samples are essential to establish the utility of genetic factors as a predictor of BU dose and outcomes of HSCT. In this multicentric study, we included 139 children aged between 0.1 to 19.9 years (65 females) receiving BU based myeloablative conditioning regimen prior to allogeneic HSCT. Genetic predictors of BU plasma levels, dose adjustment, and clinical outcomes post HSCT were prospectively evaluated. Patients received BU in a conventional 6 h dosing schedule as a two hours infusion for four days in combination with cyclophosphamide (CY; n=106), or CY and etoposide (15) or melphalan and CY (15).The remaining patients received BU with melphalan (2) or fludarabine (1). BU first dose pharmacokinetic parameters (PKs) in patients were estimated and were adjusted to steady state concentrations (Css) of 600-900 ng/mL. All the patients were genotyped for defining GSTA1*A1,*A2, *B1 and *B2 haplotypes, GSTM1, GSTT1 null alleles, CYP2C9*2,*3, CYP2C19*2,*17, CYP2B6*5, *9 and GSTP1 1578A>G, GSTP1 2293 C>T polymorphisms. Standard criteria were followed for defining clinical outcomes of HSCT such as neutrophil recovery, platelet recovery, acute graft versus host disease (aGvHD), sinusoidal obstruction syndrome (SOS), event-free survival (EFS), non-relapse mortality (NRM), hemorrhagic cystitis (HC), and graft failure (Ther Drug Monit. 2014;36:93-9; Bone Marrow Transplant. 2013; 48:939-46; Pharmacogenomics J. 2014; 14:263-71). PKs such as BU clearance (CL), area under the curve concentration (AUC), and Css were compared among genotype groups using non-parametric tests. The PKs and genetic variants or haplotypes were also associated with the clinical outcomes individually or in combinations in both univariate and multivariate analysis. Statistical significance was set at p<0.05. Higher BU CL, and lower AUC and Css levels were seen in GSTA1*A2 carriers compared to the other patients (p<0.05). The effect was more apparent in patients with malignancies (n=86; p<0.02) and in females (n=65; p<0.01). Patients with two GSTA1*A2 copies needed a higher BU dose adjustment than the other patients (p=0.01). Higher incidences of NRM (26.1 vs 5.7 %, p<0.0001) and lower EFS in patients with BU Css above median 631.0 ng/mL (21.4 vs 55.1%, p<0.0001, 70 patients had first dose Css above 631 ng/mL) has also been documented. Higher frequency of SOS was seen in individuals with two copies of GSTA1*B haplotype (28.6 Vs 8.6%; p=0.02). Higher incidences of aGVHD grade I-IV was seen in homozygous carriers of GSTA1*B haplotype (61.5 vs 30.4 %; p=0.008) and seems to be further increased in patients having also GSTP1 1578 GG genotype (75% Vs 32.2 %; p=0.006). Patients with malignancies carrying both GSTM1 and T1 null genotype had lower EFS compared to the remaining patients (n=86; 11.1 vs 59.7 %, p<0.0001). Higher incidences of HC before day 30 was seen in individuals with GSTM1 functional alleles compared to null allele carriers (n=138; 23.4 Vs 8.2 %; p=0.01) and was further potentiated through interaction with CYP2C9 (n=104; 25.7 Vs 5.8 %; p=0.004). In pediatric patients receiving conditioning regimen based on two alkylating agents with BU as one of the components, dosing algorithms for BU based on demographics and GSTA1 haplotype may improve the outcomes of HSCT. These observations also indicate that therapeutic window of BU in children might not be similar to that of adult patients especially in GSTM1 and T1 null allele carriers. This data also highlight that presence of both GSTM1 and T1 null genotypes are the independent risk factors for the occurrence of events in malignant patients receiving HSCT. DisclosuresNo relevant conflicts of interest to declare.

Origins of marronage: Mitochondrial lineages of Jamaica's Accompong Town Maroons.

The Accompong Town Maroons are descendants of enslaved Africans who successfully waged war against British colonial rule and established an independent community in western Jamaica. There are discrepancies regarding Accompong Town Maroon ancestry with some scholars noting ancestry from both Africans and Taínos, Jamaica's indigenous population, while other scholars only acknowledge African ancestry. We considered the mitochondrial lineages of contemporary Accompong Town Maroons to address the question of ancestral origins. We sequenced a section of the mitochondrial DNA control region (np 16,024-16,569) and genotyped a panel of hierarchically selected haplogroup diagnostic SNPs for 50 individuals with genealogical ties to Accompong Town. Mitochondrial haplotypes were also compared with publically available Jamaican mitochondrial haplotypes using an exact test as well as haplotypes within the EMPOP public database to further access biogeographic origins. L-type mitochondrial haplogroups were observed in 96% of samples, and the remaining 4% belonged to haplogroup B2. Haplotype diversity was 0.922 (SD = 0.024) and not significantly different than the comparable Jamaican population. Of the two B2 haplotypes, one matched haplotypes throughout the Americas and East Asia and the other matched only in East Asia. These results suggest both African and indigenous American maternal ancestries within Accompong Town. Our data suggested that the maternal ancestry of contemporary Accompong Town Maroons is predominantly African and, despite claims to suggest otherwise, also indigenous American. Our study complemented Maroon oral histories, archeological data, and illuminated how colonization shaped human genetic variation within the Caribbean.

Establishment of six homozygous MHC-B haplotype populations associated with susceptibility to Marek’s disease in Chinese specific pathogen-free BWEL chickens

The highly polymorphic chicken major histocompatibility complex (MHC) is associated with different levels of immunologic responses to certain avian pathogens. MHC-B haplotype chickens are an important genetic resource for studying the genetic determination of pathogen resistance and susceptibility. The BWEL chicken population is the only specific pathogen-free (SPF) chickens bred and developed by the State Center of Poultry Genetic Resources of Laboratory Animals in China. In this study, we successfully established six homozygous MHC-B haplotype populations from the BWEL chickens using microsatellite marker technology, named as BW/G(1, 2, 3, 5, 6, 7) lines, and their molecular genotypes were matched to six serologically defined MHC-B haplotypes, B13, B15, B2, B5, B21 and B19, respectively. The sequences of BF genes exons 2 and 3 from four successive generations (F1–F4) of the BW/G(n) lines were completely consistent with those of serologically defined MHC-B haplotypes. Subsequently, six BW/G(n) line specific allo-antisera were prepared by immunization with red blood cells (RBCs) and hemagglutination tests results showed the BW/G(n) SPF chickens could be serologically differentiated. Additionally, susceptibility to Marek’s disease (MD) in the BW/G3 (B2 haplotype) and BW/G7 (B19 haplotype) lines were determined by comparing mortality, macroscopic and histopathological lesions, and viral loads in feather pulp. The BW/G7 line showed greater genetic susceptibility to the very virulent MD virus (MDV) strain than the BW/G3 line. The establishment of MHC-B haplotype chicken populations associated with susceptibility to MD will be helpful for studying host immune responses and further developing the more effective vaccines in the context of MHC specificities, and they are also very useful for an understanding of MHC genes architecture and function.