Histocompatibility Complex Genes Research Articles

Pan-TRK Immunohistochemistry and NTRK Gene Fusions in Primary Carcinomas of the Liver.

Gene fusions involving NTRK are not common in solid tumors. The aim of this study was to investigate the TRK protein expression and molecular characteristics of gene fusions in primary liver carcinomas. A total of 110 hepatocellular carcinomas (HCC) and 69 intrahepatic cholangiocarcinomas were retrieved for tissue microarray (TMA) construction and clinicopathologic characterization. Immunohistochemistry (IHC) for pan-TRK was initially performed on TMA slides and evaluated for staining intensity. Twelve (10.9%) of 110 HCC showed weak cytoplasmic TRK expression by IHC on TMA, while all others, including 69 intrahepatic cholangiocarcinomas, were negative for TRK. The TRK expression did not correlate with patient's age, sex, tumor differentiation, or tumor stage. The 12 cases were then validated by IHC on whole sections but all turned out to be negative. Further, RNA sequencing analysis did not detect any NTRK fusions in all 12 HCC cases; however, it did identify many fusions frequently involving genes that encode mitochondrial and ribosomal proteins, microRNAs, and some transcription factors. A few fusions were recurrent, including MT-ATP6/MT-ATP8 fusion (n=9, 75%), Ig κ light chain gene IGKV/IGKJ fusion (n=5, 41.7%), and histocompatibility complex gene HLA-C/HLA-B fusion (n=4, 33.3%). In summary, NTRK fusion is very rare in primary liver carcinomas. IHC on TMA for TRK expression yields high false positive results, which should be validated on whole sections and confirmed by molecular genetic studies such as RNA sequencing. Many fusions involving genes other than NTRK are detected in HCC, the significance of which warrants further studies.

Disease Prevention, Genetic Selection, and Vaccination Based on BoLA-DRB3.2 Polymorphism: A Model for Immunogenetic Studies

Unearthing the immune defects associated genes and genetic variations may lead to locating novel targetable elements and introducing the underlying mechanisms and pathways of the desired condition. The major histocompatibility complex (MHC) genes are essential for protein antigens presentation and inducing immune response as well as are associated with production and phenotypic traits. The MHC class II genes of cattle and buffaloes, Bovine Leukocyte Antigen (BoLA) or Buffalo Leukocyte Antigen (BuLA), are located on the short arm of chromosome 23. It has been demonstrated that the second exon of BoLA-DRB3 (BoLA-DRB3.2) is highly polymorphic, having more than one hundred identified alleles, that each of them can form special binding pockets for corresponding antigenic peptides. Concerning the populations of cows, unique native and hybrid, and buffaloes are distinctly divided into different regions of Iran, analysis and interpretation of the polymorphisms’ expression status of this locus can be implemented to find better breeding strategies like selecting highly resistant animals to infection diseases, in herds. It also can help to develop effective and novel vaccines regarding allele frequencies in populations; different allelic variants of MHC class II binding to different peptides. Immunogenic evaluation of animals’ genome/genes characteristics has always been used as a model for the study of similar genes in humans.

Familial multiple sclerosis in patients with Von Hippel-Lindau disease

BackgroundMultiple sclerosis (MS) is a progressive autoimmune demyelinating disorder. Recent studies suggest that a combination of genetic susceptibility and environmental insult contributes to its pathogenesis. Many candidate genes have been discovered to modulate susceptibility for developing MS by genome wide association studies (GWAS); these include major histocompatibility complex (MHC) genes and non-MHC genes. MS cases in the context of genetic diseases may provide different approaches and clues towards identifying novel genes and pathways involved in MS pathogenesis. Here, we present a case series of two related patients with concomitant Von Hippel-Lindau disease (VHLD) and MS.Case presentationWe present two patients, a mother (case 1) and daughter (case 2), who developed superimposed relapsing-remitting multiple sclerosis in the background of the autosomal dominant genetic disorder VHLD. Several tumors characteristic of VHLD developed in both cases with pancreatic and renal neoplasms and cerebellar hemangioblastomas. In addition, both patients developed clinical symptoms consistent with multiple sclerosis, supported by radiologic lesions disseminating in time and space.ConclusionThough non-MHC susceptibility genes remain elusive in MS, we present the striking finding of superimposed multiple sclerosis in a mother and daughter with VHLD. The VHL gene is known to be the primary regulator of Nrf2, the well-established target of the FDA-approved therapeutic dimethyl fumarate. These cases provide support for further studies to determine whether VHLD pathway related genes represent a novel genetic link in multiple sclerosis.

Functional genetics for studying the human immune system.

Although small numbers of immune-mediated diseases are inherited due to rare genetic mutations, most are multifactorial diseases caused by multiple elements including genetic and environmental factors. In the case of autoimmune diseases, many disease-susceptibility genes, including several in the major histocompatibility gene complex, have been reported, and over the past 10 years, genome-wide association studies (GWAS) have been used to analyze disease-susceptibility loci in representative diseases. Furthermore, many disease-susceptibility variants have been found to be related to gene expression levels. The expression of genes involved in disease pathogenesis is often cell-type-specific, and this is closely related to epigenome alterations. Genomic information is present even before the onset of a disease and has a clear causal relationship to the disease (i.e. the outcome). Therefore, it is important to establish functional genetics in human immunology to understand the pathogenesis of diseases using these pieces of information. We can then apply these results to drug discovery. Here, we will review these issues, especially focusing on autoimmune diseases, and discuss current and future directions of human immune system research.

Molecular Characterization of MHC Class I Genes in Four Species of the Turdidae Family to Assess Genetic Diversity and Selection.

In vertebrate animals, the molecules encoded by major histocompatibility complex (MHC) genes play an essential role in the adaptive immunity. MHC class I deals with intracellular pathogens (virus) in birds. MHC class I diversity depends on the consequence of local and global environment selective pressure and gene flow. Here, we evaluated the MHC class I gene in four species of the Turdidae family from a broad geographical area of northeast China. We isolated 77 MHC class I sequences, including 47 putatively functional sequences and 30 pseudosequences from 80 individuals. Using the method based on analysis of cloned amplicons (n = 25) for each species, we found two and seven MHC I sequences per individual indicating more than one MHC I locus identified in all sampled species. Results revealed an overall elevated genetic diversity at MHC class I, evidence of different selection patterns among the domains of PBR and non-PBR. Alleles are found to be divergent with overall polymorphic sites per species ranging between 58 and 70 (out of 291 sites). Moreover, transspecies alleles were evident due to convergent evolution or recent speciation for the genus. Phylogenetic relationships among MHC I show an intermingling of alleles clustering among the Turdidae family rather than between other passerines. Pronounced MHC I gene diversity is essential for the existence of species. Our study signifies a valuable tool for the characterization of evolutionary relevant difference across a population of birds with high conservational concerns.

MHC class I evolution; from Northern pike to salmonids

BackgroundSalmonids are of major importance both as farmed and wild animals. With the changing environment comes changes in pathogenic pressures so understanding the immune system of all salmonid species is of essence. Major histocompatibility complex (MHC) genes are key players in the adaptive immune system signalling infection to responding T-cells populations. Classical MHC class I (MHCI) genes, defined by high polymorphism, broad expression patterns and peptide binding ability, have a key role in inducing immunity. In salmonids, the fourth whole genome duplication that occurred 94 million years ago has provided salmonids with duplicate MHCI regions, while Northern Pike, a basal sister clade to salmonids, represent a species which has not experienced this whole genome duplication.ResultsComparing the gene organization and evolution of MHC class I gene sequences in Northern pike versus salmonids displays a complex picture of how many of these genes evolved. Regional salmonid Ia and Ib Z lineage gene duplicates are not orthologs to the Northern pike Z lineage sequences. Instead, salmonids have experienced unique gene duplications in both duplicate regions as well as in the Salmo and Oncorhynchus branch. Species-specific gene duplications are even more pronounced for some L lineage genes.ConclusionsAlthough both Northern pike as well as salmonids have expanded their U and Z lineage genes, these gene duplications occurred separately in pike and in salmonids. However, the similarity between these duplications suggest the transposable machinery was present in a common ancestor. The salmonid MHCIa and MHCIb regions were formed during the 94 MYA since the split from pike and before the Oncorhynchus and Salmo branch separated. As seen in tetrapods, the non-classical U lineage genes are diversified duplicates of their classical counterpart. One MHCI lineage, the L lineage, experienced massive species-specific gene duplications after Oncorhynchus and Salmo split approximately 25 MYA. Based on what we currently know about L lineage genes, this large variation in number of L lineage genes also signals a large functional diversity in salmonids.

Similar patterns of genetic diversity and linkage disequilibrium in Western chimpanzees (Pan troglodytes verus) and humans indicate highly conserved mechanisms of MHC molecular evolution

BackgroundMany species are threatened with extinction as their population sizes decrease with changing environments or face novel pathogenic threats. A reduction of genetic diversity at major histocompatibility complex (MHC) genes may have dramatic effects on populations’ survival, as these genes play a key role in adaptive immunity. This might be the case for chimpanzees, the MHC genes of which reveal signatures of an ancient selective sweep likely due to a viral epidemic that reduced their population size a few million years ago. To better assess how this past event affected MHC variation in chimpanzees compared to humans, we analysed several indexes of genetic diversity and linkage disequilibrium across seven MHC genes on four cohorts of chimpanzees and we compared them to those estimated at orthologous HLA genes in a large set of human populations.ResultsInterestingly, the analyses uncovered similar patterns of both molecular diversity and linkage disequilibrium across the seven MHC genes in chimpanzees and humans. Indeed, in both species the greatest allelic richness and heterozygosity were found at loci A, B, C and DRB1, the greatest nucleotide diversity at loci DRB1, DQA1 and DQB1, and both significant global linkage disequilibrium and the greatest proportions of haplotypes in linkage disequilibrium were observed at pairs DQA1 ~ DQB1, DQA1 ~ DRB1, DQB1 ~ DRB1 and B ~ C. Our results also showed that, despite some differences among loci, the levels of genetic diversity and linkage disequilibrium observed in contemporary chimpanzees were globally similar to those estimated in small isolated human populations, in contrast to significant differences compared to large populations.ConclusionsWe conclude, first, that highly conserved mechanisms shaped the diversity of orthologous MHC genes in chimpanzees and humans. Furthermore, our findings support the hypothesis that an ancient demographic decline affecting the chimpanzee populations – like that ascribed to a viral epidemic – exerted a substantial effect on the molecular diversity of their MHC genes, albeit not more pronounced than that experienced by HLA genes in human populations that underwent rapid genetic drift during humans’ peopling history. We thus propose a model where chimpanzees’ MHC genes regenerated molecular variation through recombination/gene conversion and/or balancing selection after the selective sweep.

Allelic diversity and patterns of selection at the major histocompatibility complex class I and II loci in a threatened shorebird, the Snowy Plover (Charadrius nivosus)

BackgroundUnderstanding the structure and variability of adaptive loci such as the major histocompatibility complex (MHC) genes is a primary research goal for evolutionary and conservation genetics. Typically, classical MHC genes show high polymorphism and are under strong balancing selection, as their products trigger the adaptive immune response in vertebrates. Here, we assess the allelic diversity and patterns of selection for MHC class I and class II loci in a threatened shorebird with highly flexible mating and parental care behaviour, the Snowy Plover (Charadrius nivosus) across its broad geographic range.ResultsWe determined the allelic and nucleotide diversity for MHC class I and class II genes using samples of 250 individuals from eight breeding population of Snowy Plovers. We found 40 alleles at MHC class I and six alleles at MHC class II, with individuals carrying two to seven different alleles (mean 3.70) at MHC class I and up to two alleles (mean 1.45) at MHC class II. Diversity was higher in the peptide-binding region, which suggests balancing selection. The MHC class I locus showed stronger signatures of both positive and negative selection than the MHC class II locus. Most alleles were present in more than one population. If present, private alleles generally occurred at very low frequencies in each population, except for the private alleles of MHC class I in one island population (Puerto Rico, lineage tenuirostris).ConclusionSnowy Plovers exhibited an intermediate level of diversity at the MHC, similar to that reported in other Charadriiformes. The differences found in the patterns of selection between the class I and II loci are consistent with the hypothesis that different mechanisms shape the sequence evolution of MHC class I and class II genes. The rarity of private alleles across populations is consistent with high natal and breeding dispersal and the low genetic structure previously observed at neutral genetic markers in this species.

Major Histocompatibility Gene Complex Polymorphism in Gerze Chicken and Some Pure Line Genotypes

Bu çalışmada Gerze tavuğu popülasyonuna ait 43 örnek ile ticari saf hatlardan oluşturulan 50 örneklik Saf hat popülasyonunun majör doku uyumluluğu gen kompleksi (MHC) bakımından moleküler incelenmesi amaçlanmıştır. Allelik çeşitlilik, MHC içerisinde yer alan MCW0371 ve LEI0258 mikrosatellit lokuslarının PCR yöntemi yardımıyla çoğaltılarak elde edilen DNA fragmentlerinin agaroz jel elektroforezi ile ayrımlanması ile hesaplanmıştır. Gerze popülasyonunda LEI0258 lokusunda elde edilen allel sayısı, etkili allel sayısı, gözlenen heterozigotluk ve beklenen heterozigotluk değerleri sırasıyla; 16, 10.82, 0.67, 0.91, Saf hat popülasyonunda ise 19, 11.84, 0.60, 0.92 olarak bulunmuştur. MCW0371 lokusu için sırasıyla; Gerze popülasyonunda 3, 2.68, 0, 0.63 ve Saf hat popülasyonunda 3, 1.96, 0, 0.49 olarak tespit edilmiştir. Popülasyonlar arasında Nei genetik uzaklık ve genetik benzerlik değerleri sırasıyla 0.419 ve 0.658 olarak hesaplanmıştır. Popülasyonlar arasında FST değeri 0.068 olarak bulunmuştur. Elde edilen bulgular, içerdiği allel deseni bakımından Gerze tavuğunun özgün bir genetik kaynak olarak korunmasının ve ıslah programlarına dahil edilmesinin önemli olduğu sonucuna varılmıştır.

Genetic diversity and evolution of the MHC class II DRB gene in the Japanese marten, Martes melampus (Carnivora: Mustelidae)

High polymorphism in major histocompatibility complex (MHC) genes plays an essential role in the adaptive immune system of vertebrates for defense against various pathogens. To further understand the evolution of MHC in wildlife, partial sequences of exon 2 in MHC class II DRB genes were determined and analyzed for the Japanese marten (Martes melampus), which is endemic to Japan. From 26 individuals, 17 DRB alleles (Mame-DRBs) and four presumed pseudogenes were identified, some of them geographically widespread and others of limited distribution. Mixed-effect model evolution (MEME) and single breakpoint recombination (SBP) analyses indicated six amino acid positions under positive selection and one recombination site, respectively, that are implicated in maintaining the diversity of Mame-DRBs. In a Bayesian phylogenetic tree, Mame-DRBs did not group monophyletically, but were scattered among seven sub-clades, each containing DRBs from one or more other mustelid species. This trans-species polymorphism of Mame-DRBs has resulted from long-term balancing selection.

MHC Class II variability in bare-nosed wombats (Vombatus ursinus)

Studies of gene diversity are used to investigate population dynamics, including immunological fitness. Aside from the selection of an appropriate gene target, an important factor that underpins these studies is the ability to recover viable DNA samples from native animals that are protected, threatened or difficult to sample or locate such as the bare-nosed wombat (Vombatus ursinus). In this study, we used genomic DNA extracted from muscle tissue samples and also identified the optimal method to extract DNA from fresh wombat scat samples to enable further analyses to be performed using non-invasive techniques. The DNA was probed via the polymerase chain reaction using previously targeted marsupial Major Histocompatibility Complex (MHC) gene primers. These genes are highly variable and associated with binding and presentation of pathogens in the immune system. Twenty-three unique MHC Class II DAB V. ursinus gene sequences were translated to 21 unique predicted peptide sequences from 34 individual tissue or scat samples. Vombatus ursinus MHC Class II DAB gene and peptide sequences were most similar to other marsupial DNA and peptide sequences. Further analysis also indicated the likelihood of MHC Class II DAB family membership through motif identification. Additional sampling is required to assess the full level of diversity of MHC Class II DAB genes among V. ursinus populations; however, this study is the first to identify MHC genes in a wombat and will advance immunological and disease studies of the species.

Immunogenetic response of the bananaquit in the face of malarial parasites

BackgroundIn the arms race between hosts and parasites, genes involved in the immune response are targets for natural selection. Toll-Like Receptor (TLR) genes play a role in parasite detection as part of the innate immune system whereas Major Histocompatibility Complex (MHC) genes encode proteins that display antigens as part of the vertebrate adaptive immune system. Thus, both gene families are under selection pressure from pathogens. The bananaquit (Coereba flaveola) is a passerine bird that is a common host of avian malarial parasites (Plasmodium sp. and Haemoproteus sp.). We assessed molecular variation of TLR and MHC genes in a wild population of bananaquits and identified allelic associations with resistance/susceptibility to parasitic infection to address hypotheses of avian immune response to haemosporidian parasites.ResultsWe found that allele frequencies are associated with infection status at the immune loci studied. A consistent general trend showed the infected groups possessed more alleles at lower frequencies, and exhibited unique alleles, compared to the uninfected group.ConclusionsOur results support the theory of natural selection favoring particular alleles for resistance while maintaining overall genetic diversity in the population, a mechanism which has been demonstrated in some systems in MHC previously but understudied in TLRs.

Kapa Biosystems Library Amplification Kit (KK 2611, KK2612): Developmentof a method for typing the major histocompatibility gene complex in humans (HLA) using a next-generation sequencer (NGS)

Kapa Biosystems Library Amplification Kit (KK 2611, KK2612): Developmentof a method for typing the major histocompatibility gene complex in humans (HLA) using a next-generation sequencer (NGS)

Extensive shared polymorphism at non-MHC immune genes in recently diverged North American prairie grouse

Gene polymorphisms shared between recently diverged species are thought to be widespread and most commonly reflect introgression from hybridization or retention of ancestral polymorphism through incomplete lineage sorting. Shared genetic diversity resulting from incomplete lineage sorting is usually maintained for a relatively short period of time, but under strong balancing selection it may persist for millions of years beyond species divergence (balanced trans-species polymorphism), as in the case of the major histocompatibility complex (MHC) genes. However, balancing selection is much less likely to act on non-MHC immune genes. The aim of this study was to investigate the patterns of shared polymorphism and selection at non-MHC immune genes in five grouse species from Centrocercus and Tympanuchus genera. For this purpose, we genotyped five non-MHC immune genes that do not interact directly with pathogens, but are involved in signaling and regulate immune cell growth. In contrast to previous studies with MHC, we found no evidence for balancing selection or balanced trans-species polymorphism among the non-MHC immune genes. No haplotypes were shared between genera and in most cases more similar allelic variants sorted by genus. Between species within genera, however, we found extensive shared polymorphism, which was most likely attributable to introgression or incomplete lineage sorting following recent divergence and large ancestral effective population size (i.e., weak genetic drift). Our study suggests that North American prairie grouse may have attained relatively low degree of reciprocal monophyly at nuclear loci and reinforces the rarity of balancing selection in non-MHC immune genes.

Abstract 637: The class I HDAC inhibitor mocetinostat augments checkpoint inhibitor therapy via direct up regulation of antigen presentation transcriptional programs in tumor cells and increased T-cell clonality in tumors

Abstract Mocetinostat is a spectrum-selective class I/IV histone deacetylase (HDAC) inhibitor that augments checkpoint inhibitor therapy through enhanced antigen presentation capacity and a pro-immunogenic shift in the tumor microenvironment (TME). Mocetinostat up-regulated tumor antigen presentation machinery (i.e. major histocompatibility complex (MHC) genes and co-presentation molecules) and programmed cell death-ligand 1 (PD-L1) expression in a panel of non-small cell lung cancer (NSCLC) cell lines. Surprisingly, some of the most highly up regulated genes following mocetinostat treatment were MHC class II genes (~20 fold), which are normally expressed by antigen-presenting cells, but are silenced in most epithelial tissues and solid tumors. To elucidate the molecular mechanisms whereby mocetinostat regulates MHC class I and class II transcriptional programs in tumor cells, we performed chromatin immunoprecipitation DNA sequencing (ChIP-Seq). HDAC2 was detected at the promoters of many mocetinostat-regulated immune pathway genes. Further, mocetinostat treatment increased histone 3 lysine 27 acetylation and histone 3 lysine 4 trimethylation indicating an induction of active transcription at these loci. Class II transactivator (CIITA) is an interferon gamma (IFNγ)-sensitive regulator of MHC class II gene expression and was one of the top HDAC2/mocetinostat target genes based on ChIP-Seq data. In addition, CIITA and a representative MHC class II gene, HLA-DRA, were synergistically up regulated following mocetinostat and IFNγ treatment. To investigate the impact of Class I HDAC inhibition on the TME, syngeneic mouse tumor models were utilized. Mocetinostat treatment decreased intratumoral immune suppressive T regulatory cells (Tregs) and increased intratumoral CD8+ T cells. To gain additional insight into the functional effects of mocetinostat in the TME, we performed deep sequencing of the T-cell receptor in vehicle, mocetinostat, PD-L1 or combination-treated tumors. Mocetinostat-treated tumor cohorts exhibited increased T-cell clonality which likely reflects expansion of a subset of activated, antigen-specific T cells. Together, these data provide molecular insight into the mechanism whereby mocetinostat augments checkpoint inhibitor therapy by directly regulating tumor antigen presentation and through functional effects on the T cell repertoire. Citation Format: David M. Briere, Niranjan Sudhakar, Peter Olson, Jamie Christensen. The class I HDAC inhibitor mocetinostat augments checkpoint inhibitor therapy via direct up regulation of antigen presentation transcriptional programs in tumor cells and increased T-cell clonality in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 637. doi:10.1158/1538-7445.AM2017-637

Prognostic importance of human leukocyte antigen DRβ1 gene and protein tyrosine phosphatase nonreceptor Type 22 gene polymorphism in rheumatoid arthritis

Background: Major histocompatibility complex (MHC) or human leukocyte antigen (HLA) gene and some of the non-MHC genes are associated with genetic predisposition in rheumatoid arthritis (RA). The aim of the present study was to find the prevalence of HLA DRβ1 alleles and protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene single-nucleotide polymorphism (SNP) in RA patients and also assessed their correlation with laboratory and clinical parameters. Materials and Methods: One hundred and fifty cases of RA and 100 healthy controls were studied. relation to RA patients. HLA DRβ1 typing was done by low-resolution sequence-specific primer polymerase chain reaction and auto-antibodies (Abs) (rheumatoid factor [RF], anti-cyclic citrullinated peptide 2 (CCP2) Ab, anti-nuclear Ab, double-stranded deoxyribose nucleic acid, anticardiolipin Ab) were done by enzyme-linked immunosorbent assay. Results: In patients with RA, the most common HLA DRβ1 allele was DRβ1*04 (23.3%), followed by DRβ1*10 (20.7%), DRβ1*03 (9.3%), and DRβ1*01 (8.7%). Contrary to this, some alleles of DRβ1 were significantly less expressed in RA such as DRβ1*07 (P = 0.000) and DRβ1*14 (P = 0.010). DRβ1*04 positive patients had significantly more RF positivity while DRβ1*10 positive patients had increased RF and anti-CCP2 Ab. DRβ1*04 positive cases had positive correlation with disease activity score. DRβ1*10 positive patients had negative correlation with the age of the RA patients. These patients have shown early onset of disease. PTPN22 C1858T SNP was found in only 4% cases of RA. No correlation was established with PTPN22 SNP positivity and clinical parameters. Conclusion: In our study, RA patients have less DRβ1*04 positivity as compared to the Western literature. In our area, DRβ1*10 is more common than DRβ1*01. HLA DRβ1*07 and *14 are found to be protective for RA. PTPN22 SNP has not shown any diagnostic or prognostic significance while a follow-up study may be useful for prognostic importance of HLA DRβ1 typing in RA.

The genome sequence and insights into the immunogenetics of the bananaquit (Passeriformes: Coereba flaveola).

Avian genomics, especially of non-model species, is in its infancy relative to mammalian genomics. Here, we describe the sequencing, assembly, and annotation of a new avian genome, that of the bananaquit Coereba flaveola (Passeriformes: Thraupidae). We produced ∼30-fold coverage of the genome with an assembly size of ca. 1.2Gb, including approximately 16,500 annotated genes. Passerine birds, such as the bananaquit, are commonly infected by avian malarial parasites (Haemosporida), which presumably drive adaptive evolution of immunogenetic loci within the host genome. In the context of our research on the distribution of avian Haemosporida, we specifically characterized immune loci, including toll-like receptor (TLR) and major histocompatibility complex (MHC) genes. Additionally, we identified novel molecular markers in the form of single nucleotide polymorphisms (SNPs), both genome-wide and within identified immune loci. We discovered nine TLR genes and four MHC genes and identified five other TLR- or MHC- associated genes. Genome-wide, over 6 million high-quality SNPs were annotated, including 568 within TLR genes and 102 in MHC genes. This newly described genome and immune characterization expands the knowledge base for avian genomics and phylogenetics and allows for immune genotyping in the bananaquit, providing tools for the investigation of host-parasite coevolution.

Diversity in the Toll-Like Receptor Genes of the African Penguin (Spheniscus demersus).

The African penguin, Spheniscus demersus, is listed as Endangered by the IUCN Red List of Threatened Species due to the drastic reduction in population numbers over the last 20 years. To date, the only studies on immunogenetic variation in penguins have been conducted on the major histocompatibility complex (MHC) genes. It was shown in humans that up to half of the genetic variability in immune responses to pathogens are located in non-MHC genes. Toll-like receptors (TLRs) are now increasingly being studied in a variety of taxa as a broader approach to determine functional genetic diversity. In this study, we confirm low genetic diversity in the innate immune region of African penguins similar to that observed in New Zealand robin that has undergone several severe population bottlenecks. Single nucleotide polymorphism (SNP) diversity across TLRs varied between ex situ and in situ penguins with the number of non-synonymous alterations in ex situ populations (n = 14) being reduced in comparison to in situ populations (n = 16). Maintaining adaptive diversity is of vital importance in the assurance populations as these animals may potentially be used in the future for re-introductions. Therefore, this study provides essential data on immune gene diversity in penguins and will assist in providing an additional monitoring tool for African penguin in the wild, as well as to monitor diversity in ex situ populations and to ensure that diversity found in the in situ populations are captured in the assurance populations.

Population genetic diversity and geographical differentiation of MHC class II DAB genes in the vulnerable Chinese egret (Egretta eulophotes)

Major histocompatibility complex (MHC) genes are excellent markers for the study of adaptive genetic variation occurring over different geographical scales. The Chinese egret (Egretta eulophotes) is a vulnerable ardeid species with an estimated global population of 2600–3400 individuals. In this study, we sampled 172 individuals of this egret (approximately 6 % of the global population) from five natural populations that span the entire distribution range of this species in China. We examined their population genetic diversity and geographical differentiation at three MHC class II DAB genes by identifying eight exon 2 alleles at Egeu-DAB1, eight at Egeu-DAB2 and four at Egeu-DAB3. Allelic distributions at each of these three Egeu-DAB loci varied substantially within the five populations, while levels of genetic diversity varied slightly among the populations. Analysis of molecular variance showed low but significant genetic differentiation among five populations at all three Egeu-DAB loci (haplotype-based ϕST: 0.029, 0.020 and 0.042; and distance-based ϕST: 0.036, 0.027 and 0.043, respectively; all P < 0.01). The Mantel test suggested that this significant population genetic differentiation was likely due to an isolation-by-distance pattern of MHC evolution. However, the phylogenetic analyses and the Bayesian clustering analysis based on the three Egeu-DAB loci indicated that there was little geographical structuring of the genetic differentiation among five populations. These results provide fundamental population information for the conservation genetics of the vulnerable Chinese egret.

Major Histocompatibility Complex Class II in the red-tailed phascogale (Phascogale calura)

Diversity in major histocompatibility complex (MHC) genes can be correlated with the level of immunological fitness of an individual or group of individuals. This study tested published primer sets designed to amplify fragments of the MHC Class II DAB and DBB genes to amplify the equivalent gene fragments in red-tailed phascogales (Phascogale calura). Seventeen genomic DNA samples extracted from phascogale muscle tissue were used to amplify the initial DAB and DBB fragments; however, only DAB PCR proved successful. The fragments were 172 bp in length between the primers and had a high level of identity to other known marsupial MHC Class II DAB gene sequences (89–98%), including those of the koala (Phascolarctos cinereus), Tasmanian devil (Sarcophilus harrisii), common brushtail possum (Trichosurus vulpecula) and several wallaby species. Multiple sequence alignment revealed limited variability of MHC Class II genes between the individuals, but eight individual sequences in total. Genomic DNA was subsequently extracted from three fresh red-tailed phascogale scat samples and DAB fragments successfully amplified. The technique will allow for red-tailed phascogales to be sampled non-invasively in the wild and to determine the level of MHC diversity among individuals in the population.