Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses was used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 × 10–15) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 × 10–8) and mtDNA replication (p = 1.2 × 10–7). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell-related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 × 10–4).

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Chronic idiopathic myelofibrosis (MF) is a clonal hematopoietic disorder that leads to progressive marrow fibrosis and peripheral cytopenias. Very little is known about the role of aberrant DNA methylation in the pathobiology of this disease. Whole genome methylation was analyzed by a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 9 patients with IMF were compared to 9 age-matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays using cDNA from the same samples. Methylation analysis showed that myelofibrosis samples clustered separately from normal and anemic controls when grouped by unsupervised clustering based on Pearson’s correlation coefficient. On the other hand, global gene expression demonstrated no clear cut differences between myelofibrosis and control samples suggesting that methylation profiling has greater discriminatory power when compared to conventional gene expression profiling. A high correlation (r=0.88–0.96) was observed between whole genome methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can act as a valid surrogates for epigenomic analysis. Further analysis showed that genes aberrantly methylated in all myelofibrosis samples included v-myc, histone 2A, TNF, TNF Receptor1, FGF14 and others. Functional pathway analysis by Ingenuity showed that pathways involved in Inflammation and Cell signaling were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes for chemokine CXCL13, APC, IL-3, STAT2 and others. The pathways most activated by hypomethylation were involved in hematopoiesis and cell growth and proliferation demonstrating the biological validity of our analysis. Thus, our data demonstrates that myelofibrosis is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and lead to translational studies with agents targeting DNA methylation.

In addition to genetic alterations, epigenetic events have been recently proposed to play a role in the pathogenesis and progression of various malignancies. We wanted to study the role of both global genetic and epigenetic changes during leukemic progression by combining Whole genome methylation, Gene expression and DNA copy number alteration analyses. We used a cell line model of LGL derived from the same patient at two different stages of disease (Daibata, Int J Cancer.2004;108(6):845) for comparison. The first cell line, MOTN1, is IL-2 dependant and was derived during indolent course of LGL, while PLT-2 typifies the aggressive phenotype and is IL-2 independent in vitro. Whole genome methylation was analyzed by the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, 2 color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Gene expression analysis was performed using 37K oligo maskless arrays and high density array comparative genomic hybridization (aCGH) was performed at 6Kb resolution (Nimblegen). aCGH showed significantly higher sensitivity when compared to conventional cytogenetic analysis and revealed that a total of 548 genes were deleted and 635 amplified during leukemic progression. Gene amplification led to significantly increased mean expression of important genes such as CDK6, Fyn kinase, DICER, MAP3K9 and deletion led to decreased gene expression of genes implicated in many cancer pathways by Ingenuity functional analysis. Methylation analysis showed that there was a trend towards hypomethylation with disease progression. The mean change in the HpaII/MspI (HELP) Log (2) ratio was 0.67, where a negative value indicates methylation and positive value indicates hypomethylation. Several genes implicated in hematologic diseases were found to be hypomethylated and overexpressed, including CDK8, IGF binding protein 5, HDAC4 and MAPK14. Methylation and suppression of various genes belonging to the suppressive TGF-β and TNF pathways was observed during leukemic evolution. A comparison of primary LGL samples with normal NK cells and CD4 lymphocytes demonstrated a consistent trend towards global hypomethylation. Significantly, many genes hypomethylated in the cell lines were found to be hypomethylated in 2 primary patient samples of NK and T LGL as well; these included members of RAS oncogene family, and many cell cycle regulators. These results show that interplay of both genetic and epigenetic events may be responsible for transformation of malignancies. For example, in our model we observed that pro-proliferative cyclin dependant protein kinases can be overactivated by both mechanisms, CDK6 by gene amplification and CDK8 by DNA hypomethylation. These findings strongly suggest that progression of LGL from the stable to the aggressive phase is associated with significant epigenetic changes, in addition to cytogenetic alterations.