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Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs).Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2.We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

Summary Objective Platelet-rich plasma (PRP) is an emerging therapeutic strategy for treatment of osteoarthritis (OA); however, there is a lack of preclinical and clinical evidence for its efficacy and its mechanism of action is unclear. In the current study, we utilized leukocyte poor-PRP (LP-PRP) and leukocyte rich-PRP (LR-PRP) to mimic clinical point of care formulations and assessed their potential to alter disease progression in a mouse model of post-traumatic OA. Method Three-month-old wild-type male FVB/N mice received destabilization of the medial meniscus (DMM) surgery to induce OA. To assess the efficacy of LP-PRP and LR-PRP, mice were given intraarticular injections at 2-, 7- and 28-days post-surgery. Mice were then assessed at 5-, 9-, and 13-weeks post-surgery for changes in chronic pain using the hot plate nociceptive assay. At 14-weeks, OA pathogenesis was evaluated using histology and phase-contrast μCT. Results Treatment with LP-PRP and to a lesser extent LR-PRP preserved cartilage volume and surface area compared to phosphate-buffered saline (PBS) as measured by phase-contrast μCT. However, both treatments had higher Osteoarthritis Research Society International (OARSI) and synovitis scores compared to sham, and neither substantially improved scores compared to PBS controls. With respect to thermal hyperalgesia, PBS-treated mice displayed reduced latency to response compared to sham, and LR-PRP but not LP-PRP improved latency to response at 5-, 9- and 13-weeks post-surgery compared to PBS. Conclusion The results of this study suggest that effects of PRP therapy on OA progression and disease-induced hyperalgesia may be leukocyte-dependent. And while LP-PRP and to a lesser extent LR-PRP protect from volume and surface loss, significant pathology is still seen within OA joints. Future work is needed to understand how the different components of PRP effect OA pathogenesis and pain, and how these could be modified to achieve greater therapeutic efficacy.

Mutations in the DNA methyltransferase 3A ( DNMT3A ) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a −/− bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a −/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a −/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

This JAMA Insights discusses RNA sequencing, which allows detection of qualitative and quantitative changes in RNA expression across the genome in clinical samples, and how it may improve molecular diagnostic rates achieved by diagnostic exome sequencing and whole-genome sequencing alone.

Mutations in the gene encoding DNA methyltransferase 3A (

The expression of microRNAs (miRNAs) is dysregulated in many types of cancers including osteosarcoma (OS) due to genetic and epigenetic alterations. Among these, miR-34c, an effector of tumor suppressor P53 and an upstream negative regulator of Notch signaling in osteoblast differentiation, is dysregulated in OS. Here, we demonstrated a tumor suppressive role of miR-34c in OS progression using in vitro assays and in vivo genetic mouse models. We found that miR-34c inhibits the proliferation and the invasion of metastatic OS cells, which resulted in reduction of the tumor burden and increased overall survival in an orthotopic xenograft model. Moreover, the osteoblast specific over expression of miR-34c increased survival in the osteoblast specific p53 mutant OS mouse model. We found that miR-34c regulates the transcription of several genes in Notch signaling (NOTCH1, JAG1 and HEY2) and in p53 mediated cell cycle and apoptosis (CCNE2, E2F5, E2F2 and HDAC1). More interestingly, we found that the metastatic free survival probability was increased among a patient cohort from TARGET OS which has lower expression of direct targets of miR-34c that was identified in our transcriptome analysis such as E2F5 and NOTCH1. In conclusion, we demonstrate that miR-34c is a tumor suppressive miRNA in OS progression in vivo. In addition, we highlight the therapeutic potential of targeting miR-34c in OS.

BACKGROUND: Transcriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq–guided method to diagnose individuals across a wide range of ages and clinical phenotypes. METHODS: One hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and skin fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network clinical site from 2014 to 2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis. RESULTS: The transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen–de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCK-associated encephalopathy, NSD2- and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Skin fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts. CONCLUSIONS: For our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA. TRIAL REGISTRATION: Not applicable. FUNDING: NIH Common Fund, BCM Intellectual and Developmental Disabilities Research Center, Eunice Kennedy Shriver National Institute of Child Health & Human Development.

Multiple myeloma (MM) is an almost always incurable malignancy of plasma cells. Despite the advent of new therapies, most patients eventually relapse or become treatment-refractory. Consequently, therapies with nonoverlapping mechanisms of action that are nontoxic and provide long-term benefit to patients with MM are greatly needed. To this end, we clinically tested an autologous multitumor-associated antigen (mTAA)-specific T cell product for the treatment of patients with high-risk, relapsed or refractory MM. In this study, we expanded polyclonal T cells from 23 patients with MM. T cells whose native T cell receptors were reactive toward five myeloma-expressed target TAAs (PRAME, SSX2, MAGEA4, Survivin, and NY-ESO-1) were enriched ex vivo. To date, we have administered escalating doses of these nonengineered mTAA-specific T cells (0.5 × 107 to 2 × 107 cells/m2) to 21 patients with MM, 9 of whom were at high risk of relapse after a median of 3 lines of prior therapy and 12 with active, relapsed or refractory disease after a median of 3.5 prior lines. The cells were well tolerated, with only two transient, grade III infusion-related adverse events. Furthermore, patients with active relapsed or refractory myeloma enjoyed a longer than expected progression-free survival and responders included three patients who achieved objective responses concomitant with detection of functional TAA-reactive T cell clonotypes derived from the infused mTAA product.

Significance Mutations that reduce the function of the de novo DNA methyltransferase, DNMT3A, are very common in patients with clonal hematopoiesis and hematopoietic malignancies. Restoring the function of DNMT3A in mouse hematopoietic cells deficient for this protein corrects the DNA methylation defect in an ordered fashion, and partially restores abnormalities in gene expression and myeloid population skewing. These data may be relevant for therapeutic strategies designed to restore DNMT3A activity in patients with diseases caused by mutations in this gene., Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a−/− bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a−/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a−/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

Introduction Somatic mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) are the most common cause of clonal hematopoiesis, and among the most common initiating events for acute myeloid leukemia (AML). Constitutional DNMT3A mutations cause DNMT3A Overgrowth Syndrome, associated with obesity, overgrowth, behavioral problems, and increased risk of hematopoietic malignancies after years of latency [1]. Studies of germline and somatic Dnmt3a knockout mice have identified focal, canonical hypomethylation phenotypes in hematopoietic cells. This hypomethylation phenotype is thought to contribute to the epigenetic "fitness" of hematopoietic cells to acquire cooperating mutations that cause clonal progression and AML. However, the kinetics of methylation loss following mutational inactivation of DNMT3A in hematopoietic cells is essentially unknown. Methods Bone marrow from C57Bl/6-CD45.2 Dnmt3a fl/fl -ERT2-cre mice was transplanted into lethally irradiated C57Bl/6-CD45.1 recipients. Engraftment frequencies of >80% were detected in virtually all mice. Inactivation of Dnmt3a was induced with 9 doses of Tamoxifen (tam) given by gavage on MWF for three consecutive weeks. Bone marrow samples were serially collected for whole-genome bisulfite sequencing (WGBS) and analysis as previously described [1-4]. We compared these data to WGBS data from previously published doxycycline inducible DNMT3A transgene addback data, described in [3]. Results Cre-mediated deletion of exon 19 (flanked by loxP sites) leads to out-of-frame splicing of the mRNAs encoding all active isoforms of Dnmt3a. To limit Dnmt3a inactivation to hematopoietic cells, we used a transplantation model, which allowed us to eliminate the effects Dnmt3a loss in non-hematopoietic tissues. Floxing efficiency in the bone marrow was >90% 1 week after the last dose of Tam, and these levels persisted until the last mice in the cohort were analyzed 35 weeks later. We defined differentially methylated regions (DMRs) using WGBS data derived from WT vs Dnmt3a knockout (KO) bone marrow samples, using previously described criteria [4]. We identified 33,519 focal, highly canonical DMRs in the KO samples; 33,504 (99.96%) of these were hypomethylated, compared to WT marrow. Passively plotting the methylation values of the Dnmt3afl/fl + tam ("takeaway") samples for these DMRs over time (Figure 1a) revealed a slow, progressive, ordered loss of methylation, although the degree of methylation loss did not reach KO levels by 36 weeks. In contrast, remethylation of most of these DMRs with transgene-driven DNMT3A "addback" in KOmice revealed methylation 'correction' that started rapidly (within 1-2 weeks), and that approached wild-type levels by 24 weeks (Figure 1b). Combined, these data show that methylation loss after Dnmt3a inactivation occurs much more slowly than remethylation of Dnmt3a-dependent sites following restoration of DNMT3A activity. This trend was true across all functionally annotated regions of the genome, including gene bodies, promoters, enhancers, transcriptional start sites (TSS) and CpG islands. Importantly, DMRs that lost methylation rapidly in the takeaway setting, also gained methylation rapidly with addback, suggesting that there is a canonical set of genomic regions where DNA methylation is actively "remodeled". These "Dynamic DMRs" are significantly smaller (and had fewer CpGs) than DMRs associated with slower rates of remodeling. Conclusions These data describe a very slow rate of methylation loss following Dnmt3a inactivation in hematopoietic cells. We also describe regions of the genome (Dynamic DMRs) where DNA methylation is actively remodeled in hematopoietic cells, presumably by the collaborative action of de novo DNA methylases (Dnmt3a and Dnmt3b) vs. the DNA demethylases (i.e. the TET family of enzymes). Since clonal hematopoiesis patients with DNMT3A mutations can have very long latent periods before developing hematopoietic malignancies, these data suggest that long latency may be (at least in part) related to the very slow loss of DNA methylation at relevant regions of the genome after a DNMT3A allele acquires an inactivating somatic mutation. Figure 1 Figure 1. Disclosures Leight: Amgen: Current Employment.

The gene that encodes DNA methyltransferase 3A (DNMT3A) is mutated in nearly 40% of normal karyotype acute myeloid leukemia (AML) patients. More importantly, DNMT3A mutations are almost invariably the initiating event for the disease; however, the mechanisms by which they contribute to AML initiation are not yet clear [1, 2]. Regardless, ancestral clones containing the DNMT3A mutation are hard to eradicate, and often persist in clinical remission [3, 4]. We have recently shown that the most common DNMT3A mutation in AML patients (R882H; R878H in mice) is a dominant negative mutation that causes a focal, canonical pattern of DNA hypomethylation that is present in pre-leukemic hematopoietic cells, and appears to evolve further in fully transformed AML cells [5]. However, it is not yet clear how the DNMT3AR882H mutation can cause the clonal expansion of HSPCs, or how it makes HSPCs more susceptible to transformation by secondary mutations. We have utilized the conditional Dnmt3aR878H knock-in model described by Guryanova et. al. [6] and established an hematopoietic-specific model of clonal hematopoiesis driven by DNMT3AR882 mutations. With this model, we set out to investigate the epigenetic and functional consequences of Dnmt3aR878H expression in murine hematopoietic cells. The bone marrow cells of 6-week-old Dnmt3aR878H x Vav1-CREmice are >95% floxed; although these mice have a distinct focal hypomethylation phenotype, it is not as striking as that of Dnmt3a deficient bone marrow cells (this is expected, since the dominant negative R882H mutation reduces DNMT3A activity by 80%, not 100%). Importantly, Dnmt3aR878H and WT RNA are expressed at a precise 50:50 ratio, mimicking the ratio found in human patients with this mutation [7]. To establish the "baseline" methylation status of Dnmt3a+/+ vs. Dnmt3aR878H/+ bone marrow cells, we harvested marrow from 6-week-old littermates and performed whole genome bisulfite sequencing on 7 independent mice from each genotype. The R878H bone marrow cells have focal, canonical regions of DNA hypomethylation that strongly resemble those seen in human patients with DNMT3AR882 mutations. 2,621 differentially methylated regions (DMRs) exist in the Dnmt3aR878H/+ marrow samples, and >99% are hypomethylated. Dnmt3a-/- bone marrow cells have > 8,000 DMRs compared to Dnmt3a+/+ bone marrow cells, highlighting the intermediate methylation phenotype in Dnmt3aR878H marrow (Figure 1A). Further, the hypomethylation phenotype is stable with aging, and does not progress between weeks 2 and 52 of life (data not shown). Previous studies utilizing bulk RNA sequencing demonstrated very few differentially expressed genes (DEGs) in primary human AML samples with DNMT3AR882H. Therefore, we utilized single cell RNA sequencing to identify DEGs in the progenitor populations of Dnmt3aR878H hematopoietic cells. In addition to the methylation phenotype outlined above, we have identified a set of 117 DEGs (30 genes up and 87 genes down) in lineage negative, c-KIT positive cells from Dnmt3aR878H bone marrow, including Hspa1a and Hspa1b, Cxcl2 and Cxcl12, Fosb and C1q complement genes, with pathway analyses suggesting dysregulation of the proteasome, chemokine and inflammatory signaling, and apoptotic and proliferative pathways (Figure 1B). These pathways have all been implicated in cancer, and may provide a rationale for the Dnmt3aR878H -driven pre-leukemic phenotype. Extensive hematopoietic immunophenotyping has revealed subtle population changes in progenitor and stem populations of 6-week old Dnmt3aR878H mice; we identified significantly lower granulocyte-monocyte progenitors (GMP) and multipotent progenitors (MPP), and a significant increase in SLAM cells. Although these differences are significant, they do not result in any gross lineage population skewing in the bone marrow or peripheral blood. In sum, these data define some of the epigenetic consequences of the Dnmt3aR878H mutation in hematopoietic cells, and establish this mouse model as a valid one for the study of this mutation. This is the first report of the pre-leukemic state mediated by Dnmt3aR878H and these data have important implications in the design of future pharmacological agents targeting this mutation in patients with AML. Disclosures No relevant conflicts of interest to declare.

The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/- mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/- mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/- mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.

DNMT3A mutations occur in ~25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3AR882H, has dominant negative activity that reduces DNA methylation activity by ~80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3AR882 mutations. Non-leukemic hematopoietic cells with DNMT3AR882H displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing, and was essentially absent in AMLs with DNMT3AR882 mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3AR882, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.

We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of the patients with de novo myelodysplastic syndrome (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity-binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis.

Genome-wide association studies (GWAS) have identified 36 loci associated with body mass index (BMI), predominantly in populations of European ancestry. We conducted a meta-analysis to examine the association of3.2 million SNPs with BMI in 39,144 men and women of African ancestry and followed up the most significant associations in an additional 32,268 individuals of African ancestry. We identified one new locus at 5q33 (GALNT10, rs7708584, P = 3.4 × 10(-11)) and another at 7p15 when we included data from the GIANT consortium (MIR148A-NFE2L3, rs10261878, P = 1.2 × 10(-10)). We also found suggestive evidence of an association at a third locus at 6q16 in the African-ancestry sample (KLHL32, rs974417, P = 6.9 × 10(-8)). Thirty-two of the 36 previously established BMI variants showed directionally consistent effect estimates in our GWAS (binomial P = 9.7 × 10(-7)), five of which reached genome-wide significance. These findings provide strong support for shared BMI loci across populations, as well as for the utility of studying ancestrally diverse populations.

In conducting genome-wide association studies (GWAS), analytical approaches leveraging biological information may further understanding of the pathophysiology of clinical traits. To discover novel associations with estimated glomerular filtration rate (eGFR), a measure of kidney function, we developed a strategy for integrating prior biological knowledge into the existing GWAS data for eGFR from the CKDGen Consortium. Our strategy focuses on single nucleotide polymorphism (SNPs) in genes that are connected by functional evidence, determined by literature mining and gene ontology (GO) hierarchies, to genes near previously validated eGFR associations. It then requires association thresholds consistent with multiple testing, and finally evaluates novel candidates by independent replication. Among the samples of European ancestry, we identified a genome-wide significant SNP in FBXL20 (P = 5.6 × 10(-9)) in meta-analysis of all available data, and additional SNPs at the INHBC, LRP2, PLEKHA1, SLC3A2 and SLC7A6 genes meeting multiple-testing corrected significance for replication and overall P-values of 4.5 × 10(-4)-2.2 × 10(-7). Neither the novel PLEKHA1 nor FBXL20 associations, both further supported by association with eGFR among African Americans and with transcript abundance, would have been implicated by eGFR candidate gene approaches. LRP2, encoding the megalin receptor, was identified through connection with the previously known eGFR gene DAB2 and extends understanding of the megalin system in kidney function. These findings highlight integration of existing genome-wide association data with independent biological knowledge to uncover novel candidate eGFR associations, including candidates lacking known connections to kidney-specific pathways. The strategy may also be applicable to other clinical phenotypes, although more testing will be needed to assess its potential for discovery in general.

The DNA methyltransferases DNMT3A and DNMT3B are primarily responsible for de novo methylation of specific cytosine residues in CpG dinucleotides during mammalian development. While loss-of-function mutations in DNMT3A are highly recurrent in acute myeloid leukemia (AML), DNMT3A mutations are almost never found in AML patients with translocations that create oncogenic fusion genes such as PML-RARA, RUNX1-RUNX1T1, and MLL-AF9. Here, we explored how DNMT3A is involved in the function of these fusion genes. We used retroviral vectors to express PML-RARA, RUNX1-RUNX1T1, or MLL-AF9 in bone marrow cells derived from WT or DNMT3A-deficient mice. Additionally, we examined the phenotypes of hematopoietic cells from Ctsg-PML-RARA mice, which express PML-RARA in early hematopoietic progenitors and myeloid precursors, with or without DNMT3A. We determined that the methyltransferase activity of DNMT3A, but not DNMT3B, is required for aberrant PML-RARA–driven self-renewal ex vivo and that DNMT3A is dispensable for RUNX1-RUNX1T1– and MLL-AF9–driven self-renewal. Furthermore, both the PML-RARA–driven competitive transplantation advantage and development of acute promyelocytic leukemia (APL) required DNMT3A. Together, these findings suggest that PML-RARA requires DNMT3A to initiate APL in mice.

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits1, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait2,3. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways ( P=0.016) and that underlie skeletal growth defects ( P

Importance Tests that predict outcomes for patients with acute myeloid leukemia (AML) are imprecise, especially for those with intermediate risk AML. Objectives To determine whether genomic approaches can provide novel prognostic information for adult patients with de novo AML. Design, Setting, and Participants Whole-genome or exome sequencing was performed on samples obtained at disease presentation from 71 patients with AML (mean age, 50.8 years) treated with standard induction chemotherapy at a single site starting in March 2002, with follow-up through January 2015. In addition, deep digital sequencing was performed on paired diagnosis and remission samples from 50 patients (including 32 with intermediate-risk AML), approximately 30 days after successful induction therapy. Twenty-five of the 50 were from the cohort of 71 patients, and 25 were new, additional cases. Exposures Whole-genome or exome sequencing and targeted deep sequencing. Risk of identification based on genetic data. Main Outcomes and Measures Mutation patterns (including clearance of leukemia-associated variants after chemotherapy) and their association with event-free survival and overall survival. Results Analysis of comprehensive genomic data from the 71 patients did not improve outcome assessment over current standard-of-care metrics. In an analysis of 50 patients with both presentation and documented remission samples, 24 (48%) had persistent leukemia-associated mutations in at least 5% of bone marrow cells at remission. The 24 with persistent mutations had significantly reduced event-free survival vs the 26 who cleared all mutations (median [95% CI]: 6.0 months [95% CI, 3.7-9.6] for persistent mutations vs 17.9 months [95% CI, 11.3-40.4] for cleared mutations, log-rank P P P = .003; HR, 2.86 [95% CI, 1.39-5.88], P = .004). Among the 32 patients with intermediate cytogenetic risk, the 14 patients with persistent mutations had reduced event-free survival compared with the 18 patients who cleared all mutations (median [95% CI]: 8.8 months [95% CI, 3.7-14.6] for persistent mutations vs 25.6 months [95% CI, 11.4-not estimable] for cleared mutations, log-rank P = .003; HR, 3.32 [95% CI, 1.44-7.67], P = .005) and reduced overall survival (median [95% CI]: 19.3 months [95% CI, 7.5-42.3] for persistent mutations vs 46.8 months [95% CI, 22.6-not estimable] for cleared mutations, log-rank P = .02; HR, 2.88 [95% CI, 1.11-7.45], P = .03). Conclusions and Relevance The detection of persistent leukemia-associated mutations in at least 5% of bone marrow cells in day 30 remission samples was associated with a significantly increased risk of relapse, and reduced overall survival. These data suggest that this genomic approach may improve risk stratification for patients with AML.

In addition to measuring gene expression levels, next-generation sequencing (NGS)-based RNA-sequencing methods (RNA-seq) can also reveal unknown transcripts and splicing isoforms, and provide quantitative measurements of alternatively spliced isoforms. The total RNA complement of a cell or a population of cells, also known as transcriptome, can be analyzed by this technology. RNA-seq greatly extends the possibilities of transcriptome studies to the analysis of gene isoforms, translocation events, nucleotide variations, and posttranscriptional base modifications. Due to its flexibility and the declining cost of massively parallel sequencing, NGS-based RNA-seq is becoming an increasingly attractive tool to investigate the full range of transcripts and to reveal the complex landscape and dynamics of the transcriptome. However, significant challenges remain for RNA-seq to migrate into clinical practice for diagnostic and predictive applications. This chapter discusses the concepts and considerations of RNA-seq and methylome sequencing, and describes the basic approaches and challenges associated with these applications.

We previously identified recurrent mutations in the de novo DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). The most common DNMT3A mutation in AML (R882H) creates a dominant negative protein that reduces DNA methylation activity by ~80% in AML cells, and causes canonical patterns of DNA hypomethylation in the AML genome (Russler-Germain et al, Cancer Cell 2014). Approaches to restore DNMT3A activity in these AML genomes may be therapeutically relevant, but only if remethylation can return these genomes to their native methylation state. To begin to address whether DNA methylation can be restored in hematopoietic cells that are deficient for Dnmt3a, we performed an Òadd-backÓ experiment using a transgenic mouse model system. First, to characterize the genome-wide effect of Dnmt3a loss on DNA methylation in hematopoietic cells, we carried out whole-genome bisulfite sequencing (WGBS) on the total bone marrow cells of wild type (WT) mice, vs. marrow derived from littermates homozygous for a germline Dnmt3a null mutation (Dnmt3a-/-, Okano et al Cell, 1999), which have overtly normal hematopoiesis. Total bone marrow cells from Dnmt3a-/- mice have a canonical pattern of DNA hypomethylation at specific CpG residues and regions in the genome; many of the CpGs are virtually unmethylated in specific regions, suggesting that the normal Dnmt3a-dependent methylation ÒmarkÓ was added in stem/progenitor cells, and then maintained in all lineages. To define the timing and specificity of remethylation in Dnmt3a-/- mice, we crossed heterozygotes from this line with transgenic mice containing a tetracycline-inducible human wild type DNMT3A cDNA (DNMT3A Tg mice), and a second transgenic mouse containing the rtTA coactivator, expressed from the Rosa26 locus. When DNMT3A Tg+, rtTA+ mice are fed Doxycycline (Dox) chow for one week, WT human DNMT3A is expressed in the marrow at a level ~4 times higher that of endogenous murine Dnmt3a. Since Dnmt3a-/- mice die of severe runting at about three weeks of age, we harvested the marrow cells from Dnmt3a-/-, DNMT3A Tg+, rtTA+mice at 2 weeks of age, and transplanted them into lethally irradiated C57Bl/6 recipients. The marrow was allowed to engraft for four weeks. Half of the mice were then given Dox chow, and half were untreated. Whole bone marrow was isolated from pairs of mice (+ vs. -Dox), DNA was purified, and then subjected to whole genome bisulfite sequencing (WGBS). WGBS produced methylation data on >93% of the CpGs in the mouse reference sequence with a median coverage of 10-12x per sample. Differential methylation analysis was performed on 2 kb tiled windows across the whole genome, revealing 108, 797 differentially methylated regions (DMRs) that were hypomethylated (Table 1). Dnmt3a-/-, DNMT3A Tg+, rtTA+bone marrow from mice without Dox (i.e. no DNMT3A was expressed) demonstrated no evidence for remethylation at any time after transplant. However, if mice were treated with Dox for only 2 weeks, 59% of these DMRs were remethylated, increasing to 70% at 4 weeks, and 83% by week 9. Data from weeks 12 and 24 are pending. Patterns of remethylation for a subset of 560 differentially methylated CpGs (DMCpGs) are shown in Figure 1. These CpGs all had methylation values of >= 90% in WT mouse bone marrow cells, and 66% methylated at a time point, yellow = 33%-66%, and green = < 33%). Most CpGs are remethylated after only 2 weeks of DNMT3A expression, but some are delayed, and become remethylated later (yellow at week 2, changing to red at 4 or 9 weeks). A small subset of these CpGs remethylate very slowly, if at all (green). Without the induction of DNMT3A (-Dox, lower panel), little or no remethylation is detected. Inspection of specific regions of the genome showed that regions that are normally unmethylated in the bone marrow cells of WT mice are rarely methylated by adding back human DNMT3A in this system (data not shown), showing that remethylation is highly specific. Although the mechanisms involved in specifying remethylation patterns are not yet clear, these data may have important implications for therapeutically restoring DNMT3A activity in AML patients with DNMT3A mutations that reduce its activity. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia is heterogeneous with respect to clinical outcome and molecular pathogenesis. Approximately 20% of AML cases are refractory to induction chemotherapy, and about 50% of patients ultimately relapse within a time interval that ranges from months to years. At the molecular level, diverse chromosomal abnormalities and genetic mutations have been observed across patients1. Although several clinical factors (age, white blood cell count), cytogenetic aberrations (t[15;17] translocation, loss of chromosome 5) 2-4, and genetic mutations (DNMT3A, FLT3) have been associated with differences in survival 5,6, these factors are of limited prognostic utility. Moreover, few studies have integrated sequence data with clinical and cytogentic factors to build predictive models of patient outcome. Here, we sought to identify genomic predictors of refractory disease or early relapse. We used whole genome and exome sequencing to analyze the genomes of 71 adult de novo AML patients treated with anthracycline and cytarabine-based induction chemotherapy. Of these, 34 had refractory disease or relapsed within 6 months, 12 relapsed in 6-12 months, and 25 had a long first remission (>12 months). We also developed an enhanced exome sequencing (EES) approach to identify and follow leukemia-associated variants over time. In 12 additional patients that achieved morphologic remission after induction chemotherapy, we used EES to identify and track variants at time of diagnosis, time of morphologic remission (roughly 30 days later), and a final time point corresponding to eventual relapse (n=8) or extended remission (n=4). No novel coding or non-coding variants present at the time of diagnosis were found to be predictive of refractory disease or early relapse. Using EES, however, we were able to detect leukemia-associated variants in the initial remission bone marrow in all eight patients who eventually relapsed. One persistent leukemia-associated variant was also detected in one patient still in remission, but all other variants in that patient were eliminated. We also detected 64 somatic variants that became enriched following chemotherapy, but were not detected in the original leukemic cells. These may represent relapse-specific variants or oligoclonal hematopoiesis after bone marrow recovery. Overall, our data suggest that the persistence of leukemia-associated variants after bone marrow recovery from cytotoxic therapy is strongly correlated with relapse, and may be used to complement more traditional, morphologic measures of leukemic cell clearance. 1. Cancer Genome Atlas Research N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England Journal of Medicine 2013;368:2059-74. 2. Byrd JC, Mrozek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100:4325-36. 3. Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010;116:354-65. 4. Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. The New England Journal of Medicine 2008;358:1909-18. 5. Kihara R, Nagata Y, Kiyoi H, et al. Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients. Leukemia 2014;28:1586-95. 6. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. The New England Journal of Medicine 2010;363:2424-33. Citation Format: Jeffery M. Klco, Christopher A. Miller, Malachi Griffith, Allegra Petti, David H. Spencer, Shamika Ketkar-Kulkarni, Lukas D. Wartman, Matthew Christopher, Tamara L. Lamprecht, Jacqueline E. Payton, Jack Baty, Sharon E. Heath, Obi L. Griffith, Dong Shen, Jasreet Hundal, Gue Su Chang, Robert S. Fulton, Michelle O'laughlin, Catrina Fronick, Vincent Magrini, Ryan Demeter, David E. Larson, Shashikant Kulkarni, Bradley A. Ozenberger, John S. Welch, Matthew J. Walker, Timothy A. Graubert, Peter Westervelt, Jerald P. Radich, Daniel C. Link, Elaine R. Mardis, John F. DiPersio, Richard K. Wilson. Genomic approaches for risk assessment in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr PR11.

Acute myeloid leukemia is heterogeneous with respect to clinical outcome and molecular pathogenesis. Approximately 20% of AML cases are refractory to induction chemotherapy, and about 50% of patients ultimately relapse within a time interval that ranges from months to years. At the molecular level, diverse chromosomal abnormalities and genetic mutations have been observed across patients1. Although several clinical factors (age, white blood cell count), cytogenetic aberrations (t[15;17] translocation, loss of chromosome 5) 2-4, and genetic mutations (DNMT3A, FLT3) have been associated with differences in survival 5,6, these factors are of limited prognostic utility. Moreover, few studies have integrated sequence data with clinical and cytogentic factors to build predictive models of patient outcome. Here, we sought to identify genomic predictors of refractory disease or early relapse. We used whole genome and exome sequencing to analyze the genomes of 71 adult de novo AML patients treated with anthracycline and cytarabine-based induction chemotherapy. Of these, 34 had refractory disease or relapsed within 6 months, 12 relapsed in 6-12 months, and 25 had a long first remission (>12 months). We also developed an enhanced exome sequencing (EES) approach to identify and follow leukemia-associated variants over time. In 12 additional patients that achieved morphologic remission after induction chemotherapy, we used EES to identify and track variants at time of diagnosis, time of morphologic remission (roughly 30 days later), and a final time point corresponding to eventual relapse (n=8) or extended remission (n=4). No novel coding or non-coding variants present at the time of diagnosis were found to be predictive of refractory disease or early relapse. Using EES, however, we were able to detect leukemia-associated variants in the initial remission bone marrow in all eight patients who eventually relapsed. One persistent leukemia-associated variant was also detected in one patient still in remission, but all other variants in that patient were eliminated. We also detected 64 somatic variants that became enriched following chemotherapy, but were not detected in the original leukemic cells. These may represent relapse-specific variants or oligoclonal hematopoiesis after bone marrow recovery. Overall, our data suggest that the persistence of leukemia-associated variants after bone marrow recovery from cytotoxic therapy is strongly correlated with relapse, and may be used to complement more traditional, morphologic measures of leukemic cell clearance. 1. Cancer Genome Atlas Research N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England Journal of Medicine 2013;368:2059-74. 2. Byrd JC, Mrozek K, Dodge RK, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100:4325-36. 3. Grimwade D, Hills RK, Moorman AV, et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010;116:354-65. 4. Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. The New England Journal of Medicine 2008;358:1909-18. 5. Kihara R, Nagata Y, Kiyoi H, et al. Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients. Leukemia 2014;28:1586-95. 6. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. The New England Journal of Medicine 2010;363:2424-33. This abstract is also presented as a poster at the Translation of the Cancer Genome conference. Citation Format: Jeffery M. Klco, Christopher A. Miller, Malachi Griffith, Allegra Petti, David H. Spencer, Shamika Ketkar-Kulkarni, Lukas D. Wartman, Matthew Christopher, Tamara L. Lamprecht, Jacqueline E. Payton, Jack Baty, Sharon E. Heath, Obi L. Griffith, Dong Shen, Jasreet Hundal, Gue Su Chang, Robert S. Fulton, Michelle O'laughlin, Catrina Fronick, Vincent Magrini, Ryan Demeter, David E. Larson, Shashikant Kulkarni, Bradley A. Ozenberger, John S. Welch, Matthew J. Walker, Timothy A. Graubert, Peter Westervelt, Jerald P. Radich, Daniel C. Link, Elaine R. Mardis, John F. DiPersio, Richard K. Wilson. Genomic approaches for risk assessment in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR03.

Obesity is globaLy prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined aSociations betwEn body maS index and ĝ̂1/42.8 miLion SNPs in up to 123,865 individuals with targeted foLow up of 42 SNPs in up to 125,931 aDitional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci aSociated with body maS index (P < 5-10-8), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly aSociated loci may provide new insights into human body weight regulation. © 2010 Nature America, Inc. All rights reserved.

Fenofibrate therapy reduces serum triglycerides (TG) and increases high-density lipoprotein-cholesterol (HDL-C) and thus addresses the atherogenic dyslipidemia associated with metabolic syndrome (MetS). Our hypothesis is that genetic factors contribute to the variability of lipid response to fenofibrate differently in subjects with MetS and without MetS.We investigated the association in 25 candidate genes with lipid responses to a 3-weeks trial on fenofibrate in subjects with and without MetS. We employed growth curve mixed models to generate the response phenotypes to fenofibrate in TG, HDL-C, and low-density lipoprotein-cholesterol (LDL-C) and examined the genetic associations accounting for family dependencies.After correcting for multiple testing (p0.05) and accounting for significant differences in the association effect sizes between subjects with and without MetS (p0.05), variants of APOA5 (rs662799) and APOE (rs429358) were associated with HDL-C and LDL-C responses in MetS subjects, while APOA4 (rs675) was associated with TG response in non-MetS subjects. There was also suggestive evidence that MetS may interact with APOA4 (p=0.017), APOA5 (p=0.06), and APOE (p=0.09) to the variation to lipid responses.Genetic effects that contributed to the variability of lipid responses to fenofibrate may differ in subjects with and without MetS. This research may provide guidance for more personalized and effective therapies.

Chronic kidney disease (CKD) is a significant public health problem, and recent genetic studies have identified common CKD susceptibility variants. The CKDGen consortium performed a meta-analysis of genome-wide association data in 67,093 individuals of European ancestry from 20 predominantly population-based studies in order to identify new susceptibility loci for reduced renal function as estimated by serum creatinine (eGFRcrea), serum cystatin c (eGFRcys) and CKD (eGFRcrea 60 ml/min/1.73 m 2; n = 5,807 individuals with CKD (cases)). Follow-up of the 23 new genome-wide-significant loci (P 5 × 10 8) in 22,982 replication samples identified 13 new loci affecting renal function and CKD (in or near LASS2, GCKR, ALMS1, TFDP2, DAB2, SLC34A1, VEGFA, PRKAG2, PIP5K1B, ATXN2, DACH1, UBE2Q2 and SLC7A9) and 7 loci suspected to affect creatinine production and secretion (CPS1, SLC22A2, TMEM60, WDR37, SLC6A13, WDR72 and BCAS3). These results further our understanding of the biologic mechanisms of kidney function by identifying loci that potentially influence nephrogenesis, podocyte function, angiogenesis, solute transport and metabolic functions of the kidney. © 2010 Nature America, Inc. All rights reserved.

Fifteen known type 2 diabetes (T2D) gene variants were assessed for their associations with T2D status in 228 T2D families from the Framingham Heart Study (FHS) Original, Offspring, and Children Cohorts. Bayesian approach was used to test single-single-nucleotide polymorphism (SNP) association followed by logistic regression. Bayesian and logic regression approaches were used to test multiple SNP association searching for the best combinations of variants followed by logistic regression reconfirmation. The significant variants for T2D risk were also tested for their main and interacting effects on triglyceride (TG)/high-density lipoprotein (HDL) ratio change derived from four point measures across time. This slope phenotype was made available using mixed model growth curve approach from 155 T2D families in the FHS Offspring Cohort. Results CDKN2B rs10811661 (p = 0.042), TCF7L2 rs4506565 (p = 0.004), and JAZF1 rs864745 (p = 0.04) were individually associated with risk of T2D (OR = 1.0-2.0; effect size

Background: Estrogen receptor β (ESR2) may play a role in modulating prostate carcinogenesis through the regulation of genes related to cell proliferation and apoptosis. Methods: We conducted nested case-control studies in the Breast and Prostate Cancer Cohort Consortium (BPC3) that pooled 8,323 prostate cancer cases and 9,412 controls from seven cohorts. Whites were the predominant ethnic group. We characterized genetic variation in ESR2 by resequencing exons in 190 breast and prostate cancer cases and genotyping a dense set of single nucleotide polymorphisms (SNP) spanning the locus in a multiethnic panel of 349 cancer-free subjects. We selected four haplotype-tagging SNPs (htSNP) to capture common ESR2 variation in Whites; these htSNPs were then genotyped in all cohorts. Conditional logistic regression models were used to assess the association between sequence variants of ESR2 and the risk of prostate cancer. We also investigated the effect modification by age, body mass index, and family history, as well as the association between sequence variants of ESR2 and advanced-stage (≥T3b, N1, or M1) and high-grade (Gleason sum ≥8) prostate cancer, respectively. Results: The four tag SNPs in ESR2 were not significantly associated with prostate cancer risk, individually. The global test for the influence of any haplotype on the risk of prostate cancer was not significant (P = 0.31). However, we observed that men carrying two copies of one of the variant haplotypes (TACC) had a 1.46-fold increased risk of prostate cancer (99% confidence interval, 1.06-2.01) compared with men carrying zero copies of this variant haplotype. No SNPs or haplotypes were associated with advanced stage or high grade of prostate cancer. Conclusion: In our analysis focused on genetic variation common in Whites, we observed little evidence for any substantial association of inherited variation in ESR2 with risk of prostate cancer. A nominally significant (P < 0.01) association between the TACC haplotype and prostate cancer risk under the recessive model could be a chance finding and, in any event, would seem to contribute only slightly to the overall burden of prostate cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(10):1973–81)

The question of tagging single nucleotide polymorphism (tagSNP) transferability is an important one because many ongoing and upcoming Genome-Wide Association studies rely critically upon the validity, and practical feasibility of using a universal core set of tagSNPs. A series of recent studies analyzed performance of tagSNPs selected based on the HapMap. While these studies showed largely satisfactory transferability of the tagSNPs, they also reported that the level of transferability varies, substantively sometimes, especially when tagSNPs selected in one population were used in another distant population. We present a review of the literature about where and why tagSNP transferability may become a problem and suggest research directions that may help the resolution. Genet. Epidemiol. 2008. © 2007 Wiley-Liss, Inc.

Acute promyelocytic leukemia (APL) is an AML subtype that is characterized by aberrant expansion of immature myeloid progenitors and precursors that are arrested at the promyelocyte stage. Almost all APL cases are characterized by the t(15;17)(q22;q11.2) translocation that creates the PML-RARA fusion oncogene. Human APL cells are known to have a canonical expression signature and a specific methylation phenotype that is unique to this form of AML. Our laboratory previously created a mouse model of APL by expressing a human PML-RARA cDNA from the mouse Cathepsin G (Ctsg) locus (Ctsg-PML-RARA), which activates human PML- RARA expression in early myeloid progenitor cells, with peak expression in promyelocytes. After a long latent period (6-12 months), ~60% of these mice develop a clonal, APL-like myeloid malignancy. The long latent period is probably due to the requirement for cooperating mutations that synergize with PML-RARA to accelerate the disease. Human APL samples have a unique gene expression signature that distinguishes them from all other subtypes of AML. We evaluated RNA-Seq data derived from Poly A+ enriched cDNAs obtained from purified promyelocytes derived from 3 young (6 week old) WT and 3 Ctsg-PML-RARA mice. We identified 779 annotated genes that are significantly dysregulated in murine promyelocytes expressing PML-RARA with a log2 fold change >= 2 and P= 2. Differential expression analysis yielded 56 dysregulated lncRNA regions in PML-RARA expressing promyelocytes. To explore the association between gene dysregulation and DNA methylation in promyelocytes, we carried out whole-genome bisulfite sequencing using DNA derived from the purified promyelocytes of a 6 week old Ctsg-PML-RARA mouse, and a WT littermate. We generated a total of approximately 800 million sequencing reads, of which 78% mapped uniquely to the reference genome (mm9); we were able to map ~19 million CpGs with at least 10x coverage. Differential methylation analysis performed on ~4.5 million 1 Kb windows spanning the entire genome identified 17,633 differentially methylated regions with a mean difference of >= 25% and a q-value of < 0.01, the vast majority of which (17,264, 98%) were hypomethylated in the Ctsg-PML-RARA promyelocytes. These windows overlap several known genes, including Runx1, Jak2, Dnmt3a, Gata2, and the Hoxa and Hoxb gene clusters. Using more strict criteria (> 50% mean methylation difference), we identified 87 differentially methylated regions of at least 2 Kb in size. Of these 87 distinct regions, 74 (85%) were hypomethylated in PML-RARA promyelocytes, and 13 were hypermethylated; examples of both as shown in Figure 1. These data strongly suggest that PML-RARA has at least two distinct mechanisms by which it can modify DNA methylation. In regions where CpGs are hypomethylated, PML-RARA may be blocking the normal methylation of CpGs by the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b. In contrast, PML-RARA may be directing de novo methyltransferases to act on the hypermethylated regions. Regardless, these data, when coupled with comprehensive chromatin accessibility mapping and complete RNA sequencing data, should provide new insights into the mechanisms used by PML-RARA to alter gene expression and initiate APL. Figure1. Examples of differentially methylated regions. Black=WT cells. Red=PML-RARA expressing cells. Each CpG in the region is represented as a dot. Scale is 0-100% methylated at each position. Top panel: a region on chromosome 8 that is hypomethylated in PML-RARA expressing promyelocytes. Bottom panel: a region on chromosome 4 that is hypermethylated in PML-RARA expressing promyelocytes. Figure1. Examples of differentially methylated regions. Black=WT cells. Red=PML-RARA expressing cells. Each CpG in the region is represented as a dot. Scale is 0-100% methylated at each position. Top panel: a region on chromosome 8 that is hypomethylated in PML-RARA expressing promyelocytes. Bottom panel: a region on chromosome 4 that is hypermethylated in PML-RARA expressing promyelocytes. Disclosures No relevant conflicts of interest to declare.

Somatic mutations in the DNA methyltransferase, DNMT3A, have been identified in >30% of de novo AML cases with a normal karyotype, and in >10% of patients with MDS and T-ALL. To understand whether mutations in DNMT3A alter hematopoietic development, we generated a transgenic mouse model capable of overexpressing either wild type human DNMT3A or the most common DNMT3A mutation found in AML cases (R882H, a hypomorphic variant that acts as a potent dominant negative inhibitor of WT DNMT3A, D. Germain et al., Cancer Cell 2014). Full-length human cDNAs encoding WT or R882H DNMT3A were cloned into a mammalian expression vector directly downstream from a tetracycline responsive element. This allows for the inducible expression of DNMT3A upon the expression of the rtTA coactivator, and the presence of Doxycycline (Dox). A single founder line for the WT DNMT3A allele, and two founder lines for the R882H DNMT3A allele, were established in the C57Bl6/J background. The WT DNMT3A transgene overexpressed 3.5x more human DNMT3A than endogenous murine DNMT3A in bone marrow cells; R882H DNMT3A transgenic line 1 expressed at a 4.5 fold excess, and R882H line 2 at a 16 fold excess. To determine whether overexpression of the R882H allele was associated with focal DNA hypomethylation in the bone marrow cells of mice (similar to that observed in human AML samples), we used a novel CpG capture approach with bisulfite sequencing to assess 200,000 genomic regions containing ~3 million CpGs in the bone marrow cells of 3 WT C57Bl6/J mice, 3 Dnmt3a null mice, and healthy transgenic mice noted above that had been on Dox chow for either 6 months or 1 year (transgenic mice do not develop hematopoietic malignancies even after one year of transgene induction). We were able to assess 1.6 million CpGs with 10X or greater coverage in all 14 samples. The Dnmt3a null marrow samples contained 188,367 differentially methylated CpGs (average of >25% difference compared to WT bone marrow, q value=99%); the hypomethylated CpGs were nearly identical in all three samples. Marrow cells from the two mice overexpressing the WT DNMT3A gene had only 338 differentially methylated CpGs compared to two matched rtTA control mice; of these, 337 were hypermethylated (>99%). For the two mice overexpressing the R882H allele in line 2 (16x overexpression), bone marrow cells had 2,356 differentially methylated CpGs, of which 2,316 were hypomethylated (98%). Of these CpGs, 1,745 (73%) overlapped with hypomethylated CpGs in the Dnmt3a null marrow samples, indicating that R882H overexpression causes hypomethylation in a subset of CpGs whose methylation in bone marrow cells is Dnmt3a dependent. Because none of our mice developed hematologic malignancies even after one year, but had shown significant hypomethylation in the bone marrow, we hypothesized that cooperating mutations were necessary to produce malignancy. We transduced whole bone marrow cells from four transgenic mice: WT DNMT3A Tg x rtTA; R882H-1 Tg x rtTA; R882H-2 Tg x rtTA; and rtTA only (the same samples analyzed for methylation changes in the previous paragraph) with an MSCV-derived virus containing a human FLT3-ITD cDNA, and transplanted the transduced cells into 8-10 lethally irradiated recipients. Mice of all genotypes succumbed to myeloproliferative disease, T-cell lymphoma, T-lymphoma/ALL, or T-ALL. Overall median latencies were: rtTA=155 days, WT DNMT3A Tg x rtTA=164 days, R882H Tg-1 x rtTA=108.5 days, R882H-2 Tg x rtTA=135.5 days. The average latency for T cell malignancies demonstrated even greater differences among the four genotypes: rtTA n=4, 160.8 +/- 12.49 days (SEM), WT DNMT3A Tg x rtTA n=5, 167.3 +/- 4.854, R882H Tg-1 x rtTA n=3, 124.7 +/- 17.7, R882H-2 Tg x rtTA n=4 124.5 days +/- 22.14. T malignancies derived from R882H expressing cells were especially homogeneous compared to other groups; these tumors were CD4/CD8 double positive in all hematopoietic compartments. Despite the small sample size, these results demonstrate a trend towards a decreased latency for T malignancies in R882H expressing marrow cells, using a FLT3-ITD viral transduction model. We are confirming these data with additional mice. Taken together, our results demonstrate a clear focal hypomethyation phenotype in the bone marrow cells of DNMT3A R882H overexpressing mice, which may lead to increased susceptibility to neoplastic transformation. Disclosures No relevant conflicts of interest to declare.

Chronic inflammation has been hypothesized to be a risk factor for prostate cancer. The Toll-like receptor 4 (TLR4) presents the bacterial lipopolysaccharide (LPS), which interacts with ligand-binding protein and CD14 (LPS receptor) and activates expression of inflammatory genes through nuclear factor-κB and mitogen-activated protein kinase signaling. A previous case-control study found a modest association of a polymorphism in the TLR4 gene [11381G/C, GG versus GC/CC: odds ratio (OR), 1.26] with risk of prostate cancer. We assessed if sequence variants of TLR4 were associated with the risk of prostate cancer. In a nested case-control design within the Health Professionals Follow-up Study, we identified 700 participants with prostate cancer diagnosed after they had provided a blood specimen in 1993 and before January 2000. Controls were 700 age-matched men without prostate cancer who had had a prostate-specific antigen test after providing a blood specimen. We genotyped 16 common (>5%) single nucleotide polymorphisms (SNP) discovered in a resequencing study spanning TLR4 to test for association between sequence variation in TLR4 and prostate cancer. Homozygosity for the variant alleles of eight SNPs was associated with a statistically significantly lower risk of prostate cancer (TLR4_1893, TLR4_2032, TLR4_2437, TLR4_7764, TLR4_11912, TLR4_16649, TLR4_17050, and TLR4_17923), but the TLR4_15844 polymorphism corresponding to 11381G/C was not associated with prostate cancer (GG versus CG/CC: OR, 1.01; 95% confidence interval, 0.79-1.29). Six common haplotypes (cumulative frequency, 81%) were observed; the global test for association between haplotypes and prostate cancer was statistically significant (χ2 = 14.8 on 6 degrees of freedom; P = 0.02). Two common haplotypes were statistically significantly associated with altered risk of prostate cancer. Inherited polymorphisms of the innate immune gene TLR4 are associated with risk of prostate cancer. (Cancer Res 2005; 65(24): 11771-8)