5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals1–5. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC)1,2,4–7. 5hmC and TET proteins have been implicated in stem cell biology and cancer1,4,5,8,9, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS5. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the ‘poised’ chromatin signature found at developmentally-regulated genes in ES cells.

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues1–3. Patient-specific induced pluripotent stem (iPS) cells4 represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of telomerase reverse transcriptase (TERT)5–7. We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that multiple telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to increase TERC expression may be therapeutically beneficial in DC patients.

Background Sideroblastic anemias are heterogeneous congenital and acquired bone marrow disorders characterized by pathologic iron deposits in mitochondria of erythroid precursors. Among the congenital sideroblastic anemias (CSAs), the most common form is X-linked sideroblastic anemia, due to mutations in 5-aminolevulinate synthase (ALAS2). A novel autosomal recessive CSA, caused by mutations in the erythroid specific mitochondrial transporter SLC25A38, was recently defined. Other known etiologies include mutations in genes encoding the thiamine transporter (SLC19A2), the RNA-modifying enzyme pseudouridine synthase 1 (PUS1), a mitochondrial ATP-binding cassette transporter (ABCB7), glutaredoxin 5 (GLRX5), as well as mitochondrial DNA deletions. Despite these known diverse causes, in a substantial portion of CSA cases a presumed genetic defect remains unknown. Procedure In the context of the recent discovery of SLC25A38 as a major novel cause, we systematically analyzed a large cohort of previously unreported CSA patients. Sixty CSA probands (28 females, 32 males) were examined for ALAS2, SLC25A38, PUS1, GLRX5, and ABCB7 mutations. SLC19A2 and mitochondrial DNA were only analyzed if characteristic syndromic features were apparent. Results Twelve probands had biallelic mutations in SLC25A38. Seven ALAS2 mutations were detected in eight sporadic CSA cases, two being novel. We also identified a novel homozygous null PUS1 mutation and novel mitochondrial DNA deletions in two patients with Pearson syndrome. No mutationswere encountered in GLRX5, ABCB7, or SLC19A2. Conclusions The remaining undefined probands (43%) can be grouped according to gender, family and clinical characteristics, suggesting novel X-linked and autosomal recessive forms of CSA.

In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases.

There are no national standards for time between patient arrival and the initiation of scheduled chemotherapy (time to chemotherapy [TTC]). Delays in this process have a negative impact on patient care and the use of health care resources. At the University of Virginia Cancer Center, mean TTC in 2015 was 12.1 hours and mean length of stay (LOS) was 5.45 days at baseline. We formed a multidisciplinary team that participated in ASCO’s Quality Training Program. We aimed to improve TTC by 10% over 6 months. We used Plan-Do-Study-Act (PDSA) cycles as quality improvement (QI) models and used XmR charts to evaluate the interventions. The first PDSA cycle involved amending the chemotherapy consent process; mean TTC and LOS improved to 9.3 hours and 4.65 days, respectively. The second PDSA cycle involved shifting pharmacist review of chemotherapy orders to before admission rather than after patient arrival. Mean TTC remained at 9.4 hours (net 22% improvement from baseline) and LOS improved to 4.33 days (net 21% improvement). Our team surpassed the 10% improvement goal for TTC. This QI project faced a few limitations. Our baseline data set was a retrospective cohort review. In addition, oncology patients have a wide range of individual clinical needs that may have an impact on TTC. Delays in TTC have an impact on oncologic care at many medical centers. Our project highlights the need for guidance on this issue. We recommend that other institutions form multidisciplinary teams and also use QI tools to assess delays and implement changes.

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Background Acute myeloid leukemia patients receive anthracycline-containing induction chemotherapy. Anthracyclines cause cardiotoxicity; however, there is a paucity of data reflecting the risk of cardiotoxicity in the acute myeloid leukemia population, and risk factors for development of reduced left ventricular ejection fraction are not well established in this population. Methods A retrospective cohort study of adult acute myeloid leukemia patients receiving anthracycline-containing induction chemotherapy between March 2011 and August 2017 was performed. Baseline and all additional cardiac monitoring within one year of induction were collected. Home medications and new medication initiation were determined via the electronic health record and new outpatient prescriptions. Results Of 97 evaluable patients, 25 (25.8%) developed reduced left ventricular ejection fraction and 18 (18.6%) experienced clinical heart failure within one year of induction. The median difference from baseline to lowest left ventricular ejection fraction was −5.0 percentage points, with a range of +10.0 to −52.5. The median time to onset of reduced left ventricular ejection fraction was 27 days, at a median cumulative anthracycline dose of 270 mg/m2. No patient-specific or medication-specific factors were significantly associated with the risk of developing reduced left ventricular ejection fraction. Of 14 patients started on medical management for reduced left ventricular ejection fraction, 10 (71%) responded to therapy. Conclusions In this retrospective analysis, we observed that acute myeloid leukemia patients experienced reduced left ventricular ejection fraction more quickly and at lower doses than previously reported in the solid tumor population. Reduced left ventricular ejection fraction was at least partially reversible in most patients started on medical management. Although no factors were significantly associated with decreased cardiomyopathy risk, future assessment of cardioprotective medications may be warranted.

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