BackgroundAlthough aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic background, drug resistance and relapse in ALL is poorly understood.ResultsWe surveyed the DNA methylation levels of 435,941 CpG sites in samples from 764 children at diagnosis of ALL and from 27 children at relapse. This survey uncovered four characteristic methylation signatures. First, compared with control blood cells, the methylomes of ALL cells shared 9,406 predominantly hypermethylated CpG sites, independent of cytogenetic background. Second, each cytogenetic subtype of ALL displayed a unique set of hyper- and hypomethylated CpG sites. The CpG sites that constituted these two signatures differed in their functional genomic enrichment to regions with marks of active or repressed chromatin. Third, we identified subtype-specific differential methylation in promoter and enhancer regions that were strongly correlated with gene expression. Fourth, a set of 6,612 CpG sites was predominantly hypermethylated in ALL cells at relapse, compared with matched samples at diagnosis. Analysis of relapse-free survival identified CpG sites with subtype-specific differential methylation that divided the patients into different risk groups, depending on their methylation status.ConclusionsOur results suggest an important biological role for DNA methylation in the differences between ALL subtypes and in their clinical outcome after treatment.

Common SNPs in the chromosome 17q12-q21 region alter the risk for asthma, type 1 diabetes, primary biliary cirrhosis, and Crohn disease. Previous reports by us and others have linked the disease-associated genetic variants with changes in expression of GSDMB and ORMDL3 transcripts in human lymphoblastoid cell lines (LCLs). The variants also alter regulation of other transcripts, and this domain-wide cis-regulatory effect suggests a mechanism involving long-range chromatin interactions. Here, we further dissect the disease-linked haplotype and identify putative causal DNA variants via a combination of genetic and functional analyses. First, high-throughput resequencing of the region and genotyping of potential candidate variants were performed. Next, additional mapping of allelic expression differences in Yoruba HapMap LCLs allowed us to fine-map the basis of the cis-regulatory differences to a handful of candidate functional variants. Functional assays identified allele-specific differences in nucleosome distribution, an allele-specific association with the insulator protein CTCF, as well as a weak promoter activity for rs12936231. Overall, this study shows a common disease allele linked to changes in CTCF binding and nucleosome occupancy leading to altered domain-wide cis-regulation. Finally, a strong association between asthma and cis-regulatory haplotypes was observed in three independent family-based cohorts (p = 1.78 x 10(-8)). This study demonstrates the requirement of multiple parallel allele-specific tools for the investigation of noncoding disease variants and functional fine-mapping of human disease-associated haplotypes.

Cis-acting variants altering gene expression are a source of phenotypic differences. The cis-acting components of expression variation can be identified through the mapping of differences in allelic expression (AE), which is the measure of relative expression between two allelic transcripts. We generated a map of AE associated SNPs using quantitative measurements of AE on Illumina Human1M BeadChips. In 53 lymphoblastoid cell lines derived from donors of European descent, we identified common cis variants affecting 30% (2935/9751) of the measured RefSeq transcripts at 0.001 permutation significance. The pervasive influence of cis-regulatory variants, which explain 50% of population variation in AE, extend to full-length transcripts and their isoforms as well as to unannotated transcripts. These strong effects facilitate fine mapping of cis-regulatory SNPs, as demonstrated by dissection of heritable control of transcripts in the systemic lupus erythematosus-associated C8orf13-BLK region in chromosome 8. The dense collection of associations will facilitate large-scale isolation of cis-regulatory SNPs.

Genome scans for asthma have identified suggestive or significant linkages on 17 different chromosomes, including chromosome 12, region q13-23, housing the vitamin D receptor (VDR) gene. Through interaction with VDR, 1,25-dihydroxyvitamin D3 mediates numerous biological activities, such as regulation of helper T-cell development and subsequent cytokine secretion profiles. Variants of the VDR have been found to be associated with immune-mediated diseases that are characterized by an imbalance in helper T-cell development, such as Crohn's disease and tuberculosis. The VDR, hence, is a good candidate to be investigated for association with asthma, which is characterized by enhanced helper T-cell type 2 activity. Here, we examined VDR genetic variants in an asthma family-based cohort from Quebec. We report six variants to be strongly associated with asthma and four with atopy (0.0005 < or = p < or = 0.05). Analysis of the linkage disequilibrium pattern and haplotypes also revealed significant association with both phenotypes (0.0004 < or = p < or = 0.01). The findings have been replicated by another research team in a second but not in a third cohort. These results identify VDR variants as genetic risk factors for asthma/atopy and implicate a non-human leukocyte antigen immunoregulatory molecule in the pathogenesis of asthma and atopy.

Using data from a genome-wide association study of 907 individuals with childhood acute lymphoblastic leukemia (cases) and 2,398 controls and with validation in samples totaling 2,386 cases and 2,419 controls, we have shown that common variation at 9p21.3 (rs3731217, intron 1 of CDKN2A) influences acute lymphoblastic leukemia risk (odds ratio = 0.71, P = 3.01 × 10−11), irrespective of cell lineage.

The identification of human sequence polymorphisms that regulate gene expression is key to understanding human genetic diseases. We report a survey of human genes that demonstrate allelic differences in gene expression, reflecting the presence of putative allele-specific cis-acting factors of either genetic or epigenetic nature. The expression of allelic transcripts in heterozygous samples is assessed directly by relative quantitation of intragenic marker alleles in messenger or heteronuclear RNA derived from cells or tissues. This survey used 193 single-nucleotide polymorphisms (SNPs) from 129 genes expressed in lymphoblastoid cell lines, to identify 23 genes (18%) with common allele-specific transcripts whose expression deviated from the expected equimolar ratio. A subset of these deviations, or "allelic imbalances," can be observed in multiple samples derived from reference CEPH ("Centre d'Etude du Polymorphisme Humain") pedigrees and demonstrate a spectrum of patterns of transmission, including cosegregation of allelic skewing across generations compatible with Mendelian inheritance as well as random monoallelic expression for three genes (IL1A, HTR2A, and FGB). Additional studies for BTN3A2 provide evidence of SNPs and haplotypes in complete linkage disequilibrium with high- and low-expressing transcripts. The pipeline described herein offers tools for efficient identification and characterization of allelic expression allowing identification of regulatory sequence variants as well as epigenetic variation affecting human gene expression.

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The recent development of whole genome association studies has lead to the robust identification of several loci involved in different common human diseases. Interestingly, some of the strongest signals of association observed in these studies arise from non-coding regions located in very large introns or far away from any annotated genes, raising the possibility that these regions are involved in the etiology of the disease through some unidentified regulatory mechanisms. These findings highlight the importance of better understanding the mechanisms leading to inter-individual differences in gene expression in humans. Most of the existing approaches developed to identify common regulatory polymorphisms are based on linkage/association mapping of gene expression to genotypes. However, these methods have some limitations, notably their cost and the requirement of extensive genotyping information from all the individuals studied which limits their applications to a specific cohort or tissue. Here we describe a robust and high-throughput method to directly measure differences in allelic expression for a large number of genes using the Illumina Allele-Specific Expression BeadArray platform and quantitative sequencing of RT-PCR products. We show that this approach allows reliable identification of differences in the relative expression of the two alleles larger than 1.5-fold (i.e., deviations of the allelic ratio larger than 60∶40) and offers several advantages over the mapping of total gene expression, particularly for studying humans or outbred populations. Our analysis of more than 80 individuals for 2,968 SNPs located in 1,380 genes confirms that differential allelic expression is a widespread phenomenon affecting the expression of 20% of human genes and shows that our method successfully captures expression differences resulting from both genetic and epigenetic cis-acting mechanisms.