Roger Horton

DNA methylation profiling of human chromosomes 6, 20 and 22

DNA methylation is the most stable type of epigenetic modification modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of six annotation categories showed that evolutionarily conserved regions are the predominant sites for differential DNA methylation and that a core region surrounding the transcriptional start site is an informative surrogate for promoter methylation. We find that 17% of the 873 analyzed genes are differentially methylated in their 5′ UTRs and that about one-third of the differentially methylated 5′ UTRs are inversely correlated with transcription. Despite the fact that our study controlled for factors reported to affect DNA methylation such as sex and age, we did not find any significant attributable effects. Our data suggest DNA methylation to be ontogenetically more stable than previously thought.

A second major histocompatibility complex susceptibility locus for multiple sclerosis

Variation in the major histocompatibility complex (MHC) on chromosome 6p21 is known to influence susceptibility to multiple sclerosis with the strongest effect originating from the HLA‐DRB1 gene in the class II region. The possibility that other genes in the MHC independently influence susceptibility to multiple sclerosis has been suggested but remains unconfirmed.

Genetic Analysis of Completely Sequenced Disease-Associated MHC Haplotypes Identifies Shuffling of Segments in Recent Human History

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.

Recruitment of heterogeneous nuclear ribonucleoprotein A1 in vivo to the LMP/TAP region of the major histocompatibility complex.

Sequences containing the matrix recognition signature were identified adjacent to theLMP/TAP gene cluster in the human and mouse major histocompatibility complex class II region. These sequences were shown to function as nuclear matrix attachment regions (MARs). Three of the five human MARs and the single mouse MAR recruit heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) in vivo during transcriptional up-regulation of the major histocompatibility complex class II genes. The timing of this recruitment correlates with a rise in mature TAP1 mRNA. Two of the human MARs bind hnRNP-A1 in vitro directly within a 35-bp sequence that shows over 90% similarity to certain Alu repeat sequences. This study shows that MARs recruit and bind hnRNP-A1 upon transcriptional up-regulation.

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.

Accessing HLA Sequencing Data Through the 6ace Database

The chromosome 6 database (6ace) is one of a suite of databases available at the Sanger Centre, which serve the human genome sequencing communities of several chromosomes (1, 6, 20, 22, and X). Data may be retrieved in graphical or textural form using interactive windows and menus or simple command texts. The database and its management system are based on ACEDB and can be accessed via three main routes, a graphics interface, a text interface, and a Web interface. Here, we describe 6ace with particular emphasis on how to access major histocompatibility complex (MHC) and human leukocyte antigen (HLA) associated data.