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1.  A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis 
Nature biotechnology  2008;26(7):779-785.
DNA methylation is an indispensible epigenetic modification of mammalian genomes. Consequently there is great interest in strategies for genome-wide/whole-genome DNA methylation analysis, and immunoprecipitation-based methods have proven to be a powerful option. Such methods are rapidly shifting the bottleneck from data generation to data analysis, necessitating the development of better analytical tools. Until now, a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling has been the inability to estimate absolute methylation levels. Here we report the development of a novel cross-platform algorithm – Bayesian Tool for Methylation Analysis (Batman) – for analyzing Methylated DNA Immunoprecipitation (MeDIP) profiles generated using arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). The latter is an approach we have developed to elucidate the first high-resolution whole-genome DNA methylation profile (DNA methylome) of any mammalian genome. MeDIP-seq/MeDIP-chip combined with Batman represent robust, quantitative, and cost-effective functional genomic strategies for elucidating the function of DNA methylation.
doi:10.1038/nbt1414
PMCID: PMC2644410  PMID: 18612301
2.  Association of Smoking Behavior with an Odorant Receptor Allele Telomeric to the Human Major Histocompatibility Complex 
Genetic testing  2008;12(4):481-486.
Smoking behavior has been associated in two independent European cohorts with the most common Caucasian human leukocyte antigen (HLA) haplotype (A1-B8-DR3). We aimed to test whether polymorphic members of the two odorant receptor (OR) clusters within the extended HLA complex might be responsible for the observed association, by genotyping a cohort of Hungarian women in which the mentioned association had been found. One hundred and eighty HLA haplotypes from Centre d’Etude du Polymorphisme Humain families were analyzed in silico to identify single-nucleotide polymorphisms (SNPs) within OR genes that are in linkage disequilibrium with the A1-B8-DR3 haplotype, as well as with two other haplotypes indirectly linked to smoking behavior. A nonsynonymous SNP within the OR12D3 gene (rs3749971T) was found to be linked to the A1-B8-DR3 haplotype. This polymorphism leads to a 97Thr → Ile exchange that affects a putative ligand binding region of the OR12D3 protein. Smoking was found to be associated in the Hungarian cohort with the rs3749971T allele (p = 1.05×10−2), with higher significance than with A1-B8-DR3 (p = 2.38×10−2). Our results link smoking to a distinct OR allele, and demonstrate that the rs3749971T polymorphism is associated with the HLA haplotype-dependent differential recognition of cigarette smoke components, at least among Caucasian women.
doi:10.1089/gte.2008.0029
PMCID: PMC2635552  PMID: 18939942
3.  Somatically acquired hypomethylation of IGF2 in breast and colorectal cancer 
Human Molecular Genetics  2008;17(17):2633-2643.
The imprinted insulin-like growth factor 2 (IGF2) gene is expressed predominantly from the paternal allele. Loss of imprinting (LOI) associated with hypomethylation at the promoter proximal sequence (DMR0) of the IGF2 gene was proposed as a predisposing constitutive risk biomarker for colorectal cancer. We used pyrosequencing to assess whether IGF2 DMR0 methylation is either present constitutively prior to cancer or whether it is acquired tissue-specifically after the onset of cancer. DNA samples from tumour tissues and matched non-tumour tissues from 22 breast and 42 colorectal cancer patients as well as peripheral blood samples obtained from colorectal cancer patients [SEARCH (n=case 192, controls 96)], breast cancer patients [ABC (n=case 364, controls 96)] and the European Prospective Investigation of Cancer [EPIC-Norfolk (n=breast 228, colorectal 225, controls 895)] were analysed. The EPIC samples were collected 2–5 years prior to diagnosis of breast or colorectal cancer. IGF2 DMR0 methylation levels in tumours were lower than matched non-tumour tissue. Hypomethylation of DMR0 was detected in breast (33%) and colorectal (80%) tumour tissues with a higher frequency than LOI indicating that methylation levels are a better indicator of cancer than LOI. In the EPIC population, the prevalence of IGF2 DMR0 hypomethylation was 9.5% and this correlated with increased age not cancer risk. Thus, IGF2 DMR0 hypomethylation occurs as an acquired tissue-specific somatic event rather than a constitutive innate epimutation. These results indicate that IGF2 DMR0 hypomethylation has diagnostic potential for colon cancer rather than value as a surrogate biomarker for constitutive LOI.
doi:10.1093/hmg/ddn163
PMCID: PMC2515372  PMID: 18541649
4.  Generation of a genomic tiling array of the human Major Histocompatibility Complex (MHC) and its application for DNA methylation analysis 
BMC Medical Genomics  2008;1:19.
Background
The major histocompatibility complex (MHC) is essential for human immunity and is highly associated with common diseases, including cancer. While the genetics of the MHC has been studied intensively for many decades, very little is known about the epigenetics of this most polymorphic and disease-associated region of the genome.
Methods
To facilitate comprehensive epigenetic analyses of this region, we have generated a genomic tiling array of 2 Kb resolution covering the entire 4 Mb MHC region. The array has been designed to be compatible with chromatin immunoprecipitation (ChIP), methylated DNA immunoprecipitation (MeDIP), array comparative genomic hybridization (aCGH) and expression profiling, including of non-coding RNAs. The array comprises 7832 features, consisting of two replicates of both forward and reverse strands of MHC amplicons and appropriate controls.
Results
Using MeDIP, we demonstrate the application of the MHC array for DNA methylation profiling and the identification of tissue-specific differentially methylated regions (tDMRs). Based on the analysis of two tissues and two cell types, we identified 90 tDMRs within the MHC and describe their characterisation.
Conclusion
A tiling array covering the MHC region was developed and validated. Its successful application for DNA methylation profiling indicates that this array represents a useful tool for molecular analyses of the MHC in the context of medical genomics.
doi:10.1186/1755-8794-1-19
PMCID: PMC2430202  PMID: 18513384
5.  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

Results 1-5 (5)