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1.  Sequencing and comparative analysis of the gorilla MHC genomic sequence 
Major histocompatibility complex (MHC) genes play a critical role in vertebrate immune response and because the MHC is linked to a significant number of auto-immune and other diseases it is of great medical interest. Here we describe the clone-based sequencing and subsequent annotation of the MHC region of the gorilla genome. Because the MHC is subject to extensive variation, both structural and sequence-wise, it is not readily amenable to study in whole genome shotgun sequence such as the recently published gorilla genome. The variation of the MHC also makes it of evolutionary interest and therefore we analyse the sequence in the context of human and chimpanzee. In our comparisons with human and re-annotated chimpanzee MHC sequence we find that gorilla has a trimodular RCCX cluster, versus the reference human bimodular cluster, and additional copies of Class I (pseudo)genes between Gogo-K and Gogo-A (the orthologues of HLA-K and -A). We also find that Gogo-H (and Patr-H) is coding versus the HLA-H pseudogene and, conversely, there is a Gogo-DQB2 pseudogene versus the HLA-DQB2 coding gene. Our analysis, which is freely available through the VEGA genome browser, provides the research community with a comprehensive dataset for comparative and evolutionary research of the MHC.
doi:10.1093/database/bat011
PMCID: PMC3626023  PMID: 23589541
2.  Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project 
Immunogenetics  2008;60(1):1-18.
The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine.
doi:10.1007/s00251-007-0262-2
PMCID: PMC2206249  PMID: 18193213
Major histocompatibility complex; Haplotype; Polymorphism; Retroelement; Genetic predisposition to disease; Population genetics
3.  Evolution and comparative analysis of the MHC Class III inflammatory region 
BMC Genomics  2006;7:281.
Background
The Major Histocompatibility Complex (MHC) is essential for immune function. Historically, it has been subdivided into three regions (Class I, II, and III), but a cluster of functionally related genes within the Class III region has also been referred to as the Class IV region or "inflammatory region". This group of genes is involved in the inflammatory response, and includes members of the tumour necrosis family. Here we report the sequencing, annotation and comparative analysis of a tammar wallaby BAC containing the inflammatory region. We also discuss the extent of sequence conservation across the entire region and identify elements conserved in evolution.
Results
Fourteen Class III genes from the tammar wallaby inflammatory region were characterised and compared to their orthologues in other vertebrates. The organisation and sequence of genes in the inflammatory region of both the wallaby and South American opossum are highly conserved compared to known genes from eutherian ("placental") mammals. Some minor differences separate the two marsupial species. Eight genes within the inflammatory region have remained tightly clustered for at least 360 million years, predating the divergence of the amphibian lineage. Analysis of sequence conservation identified 354 elements that are conserved. These range in size from 7 to 431 bases and cover 15.6% of the inflammatory region, representing approximately a 4-fold increase compared to the average for vertebrate genomes. About 5.5% of this conserved sequence is marsupial-specific, including three cases of marsupial-specific repeats. Highly Conserved Elements were also characterised.
Conclusion
Using comparative analysis, we show that a cluster of MHC genes involved in inflammation, including TNF, LTA (or its putative teleost homolog TNF-N), APOM, and BAT3 have remained together for over 450 million years, predating the divergence of mammals from fish. The observed enrichment in conserved sequences within the inflammatory region suggests conservation at the transcriptional regulatory level, in addition to the functional level.
doi:10.1186/1471-2164-7-281
PMCID: PMC1654159  PMID: 17081307
4.  The Leukocyte Receptor Complex in Chicken Is Characterized by Massive Expansion and Diversification of Immunoglobulin-Like Loci 
PLoS Genetics  2006;2(5):e73.
The innate and adaptive immune systems of vertebrates possess complementary, but intertwined functions within immune responses. Receptors of the mammalian innate immune system play an essential role in the detection of infected or transformed cells and are vital for the initiation and regulation of a full adaptive immune response. The genes for several of these receptors are clustered within the leukocyte receptor complex (LRC). The purpose of this study was to carry out a detailed analysis of the chicken (Gallus gallus domesticus) LRC. Bacterial artificial chromosomes containing genes related to mammalian leukocyte immunoglobulin-like receptors were identified in a chicken genomic library and shown to map to a single microchromosome. Sequencing revealed 103 chicken immunoglobulin-like receptor (CHIR) loci (22 inhibitory, 25 activating, 15 bifunctional, and 41 pseudogenes). A very complex splicing pattern was found using transcript analyses and seven hypervariable regions were detected in the external CHIR domains. Phylogenetic and genomic analysis showed that CHIR genes evolved mainly by block duplications from an ancestral inhibitory receptor locus, with transformation into activating receptors occurring more than once. Evolutionary selection pressure has led not only to an exceptional expansion of the CHIR cluster but also to a dramatic diversification of CHIR loci and haplotypes. This indicates that CHIRs have the potential to complement the adaptive immune system in fighting pathogens.
Synopsis
The immune system developed to cope with a diverse array of pathogens, including infectious organisms. The detection of these pathogens by cells of the immune system is mediated by a large set of specific receptor proteins. Here the authors seek to understand how a particular subset of cell surface receptors of the domestic chicken, the chicken Ig-like receptors (CHIR), has evolved. They demonstrate that at least 103 such receptor loci are clustered on a single microchromosome and provide the first detailed analysis of this region. The sequences of the CHIR genes suggest the presence of inhibitory, activating, and bifunctional receptors, as well as numerous incomplete loci (pseudogenes) that appear to have evolved by duplications of an ancestral inhibitory receptor gene. Multiple regions of very high sequence variability were also identified within CHIR loci which, together with considerable expansion of the number of these genes, suggest that CHIR polypeptides are involved in critical functions in the immune system of the chicken.
doi:10.1371/journal.pgen.0020073
PMCID: PMC1458963  PMID: 16699593
5.  The Leukocyte Receptor Complex in Chicken Is Characterized by Massive Expansion and Diversification of Immunoglobulin-Like Loci 
PLoS Genetics  2006;2(5):e73.
The innate and adaptive immune systems of vertebrates possess complementary, but intertwined functions within immune responses. Receptors of the mammalian innate immune system play an essential role in the detection of infected or transformed cells and are vital for the initiation and regulation of a full adaptive immune response. The genes for several of these receptors are clustered within the leukocyte receptor complex (LRC). The purpose of this study was to carry out a detailed analysis of the chicken (Gallus gallus domesticus) LRC. Bacterial artificial chromosomes containing genes related to mammalian leukocyte immunoglobulin-like receptors were identified in a chicken genomic library and shown to map to a single microchromosome. Sequencing revealed 103 chicken immunoglobulin-like receptor (CHIR) loci (22 inhibitory, 25 activating, 15 bifunctional, and 41 pseudogenes). A very complex splicing pattern was found using transcript analyses and seven hypervariable regions were detected in the external CHIR domains. Phylogenetic and genomic analysis showed that CHIR genes evolved mainly by block duplications from an ancestral inhibitory receptor locus, with transformation into activating receptors occurring more than once. Evolutionary selection pressure has led not only to an exceptional expansion of the CHIR cluster but also to a dramatic diversification of CHIR loci and haplotypes. This indicates that CHIRs have the potential to complement the adaptive immune system in fighting pathogens.
Synopsis
The immune system developed to cope with a diverse array of pathogens, including infectious organisms. The detection of these pathogens by cells of the immune system is mediated by a large set of specific receptor proteins. Here the authors seek to understand how a particular subset of cell surface receptors of the domestic chicken, the chicken Ig-like receptors (CHIR), has evolved. They demonstrate that at least 103 such receptor loci are clustered on a single microchromosome and provide the first detailed analysis of this region. The sequences of the CHIR genes suggest the presence of inhibitory, activating, and bifunctional receptors, as well as numerous incomplete loci (pseudogenes) that appear to have evolved by duplications of an ancestral inhibitory receptor gene. Multiple regions of very high sequence variability were also identified within CHIR loci which, together with considerable expansion of the number of these genes, suggest that CHIR polypeptides are involved in critical functions in the immune system of the chicken.
doi:10.1371/journal.pgen.0020073
PMCID: PMC1458963  PMID: 16699593
6.  Genetic Analysis of Completely Sequenced Disease-Associated MHC Haplotypes Identifies Shuffling of Segments in Recent Human History 
PLoS Genetics  2006;2(1):e9.
The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II–related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR–DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.
Synopsis
A group of genes involved in the human immune system are contained within a surprisingly short section of Chromosome 6 that has long been recognised as the most important genomic region in relation to disease susceptibility. Discerning the actual genes playing a role in disease has proved difficult mainly because the region contains numerous genes and is also the most genetically variable in the genome. Within this jungle of variation, the research reported here has identified and characterised a discrete segment shared by two individuals that is virtually devoid of variation—a polymorphism desert. The conservation of this segment amongst a background of extreme variation suggests both an ancient origin and genetic exchange in early human history. These observations are important in evolutionary terms as they reveal a potential mechanism whereby certain genetic segments associated with favourable immune functions have spread across human populations. Within medical terms this may also explain contrasting disease risks in people from different ethnic backgrounds. Public access to these data will help researchers find specific variants conferring disease susceptibility or resistance and, as in this report, rule out regions for conveying specificity to certain diseases.
doi:10.1371/journal.pgen.0020009
PMCID: PMC1331980  PMID: 16440057
7.  Genetic Analysis of Completely Sequenced Disease-Associated MHC Haplotypes Identifies Shuffling of Segments in Recent Human History 
PLoS Genetics  2006;2(1):e9.
The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II–related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR–DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.
Synopsis
A group of genes involved in the human immune system are contained within a surprisingly short section of Chromosome 6 that has long been recognised as the most important genomic region in relation to disease susceptibility. Discerning the actual genes playing a role in disease has proved difficult mainly because the region contains numerous genes and is also the most genetically variable in the genome. Within this jungle of variation, the research reported here has identified and characterised a discrete segment shared by two individuals that is virtually devoid of variation—a polymorphism desert. The conservation of this segment amongst a background of extreme variation suggests both an ancient origin and genetic exchange in early human history. These observations are important in evolutionary terms as they reveal a potential mechanism whereby certain genetic segments associated with favourable immune functions have spread across human populations. Within medical terms this may also explain contrasting disease risks in people from different ethnic backgrounds. Public access to these data will help researchers find specific variants conferring disease susceptibility or resistance and, as in this report, rule out regions for conveying specificity to certain diseases.
doi:10.1371/journal.pgen.0020009
PMCID: PMC1331980  PMID: 16440057

Results 1-7 (7)