Key Points Germ line variants in TERT, SH2B3, TET2, ATM, CHEK2, PINT, and GFI1B are associated with JAK2 V617F clonal hematopoiesis and MPNs. Age-related JAK2 V617F clonal hematopoiesis is found in ∼2 out of 1000 individuals in the general population.

Sildenafil causes pulmonary vasodilation, thus potentially reducing impairments of hypoxia-induced pulmonary hypertension on exercise performance at altitude. The purpose of this study was to determine the effects of sildenafil during normoxic and hypoxic exercise. We hypothesized that 1) sildenafil would have no significant effects on normoxic exercise, and 2) sildenafil would improve cardiac output, arterial oxygen saturation (SaO2), and performance during hypoxic exercise. Ten trained men performed one practice and three experimental trials at sea level (SL) and simulated high altitude (HA) of 3,874 m. Each cycling test consisted of a set-work-rate portion (55% work capacity: 1 h SL, 30 min HA) followed immediately by a time trial (10 km SL, 6 km HA). Double-blinded capsules (placebo, 50, or 100 mg) were taken 1 h before exercise in a randomly counterbalanced order. For HA, subjects also began breathing hypoxic gas (12.8% oxygen) 1 h before exercise. At SL, sildenafil had no effects on any cardiovascular or performance measures. At HA, sildenafil increased stroke volume (measured by impedance cardiography), cardiac output, and SaO2 during set-work-rate exercise. Sildenafil lowered 6-km time-trial time by 15% (P<0.05). SaO2 was also higher during the time trial (P<0.05) in response to sildenafil, despite higher work rates. Post hoc analyses revealed two subject groups, sildenafil responders and nonresponders, who improved time-trial performance by 39% (P<0.05) and 1.0%, respectively. No dose-response effects were observed. During cycling exercise in acute hypoxia, sildenafil can greatly improve cardiovascular function, SaO2, and performance for certain individuals.

High-altitude anorexia leads to a hormonal response pattern modulated by both hypoxia and caloric restriction (CR). The purpose of this study was to compare altitude-induced neuroendocrine changes with or without energy imbalance and to explore how energy sufficiency alters the endocrine acclimatization process. Twenty-six normal-weight, young men were studied for 3 wk. One group [hypocaloric group (HYPO), n = 9] stayed at sea level and consumed 40% fewer calories than required to maintain body weight. Two other groups were deployed to 4,300 meters (Pikes Peak, CO), where one group (ADQ, n = 7) was adequately fed to maintain body weight and the other [deficient group (DEF), n = 10] had calories restricted as above. HYPO experienced a typical CR-induced reduction in many hormones such as insulin, testosterone, and leptin. At altitude, fasting glucose, insulin, and epinephrine exhibited a muted rise in DEF compared with ADQ. Free thyroxine, thyroid-stimulating hormone, and norepinephrine showed similar patterns between the two altitude groups. Morning cortisol initially rose higher in DEF than ADQ at 4,300 meters, but the difference disappeared by day 5. Testosterone increased in both altitude groups acutely but declined over time in DEF only. Adiponectin and leptin did not change significantly from sea level baseline values in either altitude group regardless of energy intake. These data suggest that hypoxia tends to increase blood hormone concentrations, but anorexia suppresses elements of the endocrine response. Such suppression results in the preservation of energy stores but may sacrifice the facilitation of oxygen delivery and the use of oxygen-efficient fuels.

Macrophages are activated by IFN-γ, a proinflammatory and proatherogenic cytokine that mediates its downstream effects primarily through STAT1. IFN-γ signaling induces phosphorylation of two STAT1 residues: Tyr701 (Y701), which facilitates dimerization, nuclear translocation, and DNA binding; and Ser727 (S727), which enables maximal STAT1 transcription activity. Immunosuppressive molecules such as adenosine in the cellular microenvironment can reduce macrophage inflammatory and atherogenic functions through receptor-mediated signaling pathways. We hypothesized that adenosine achieves these protective effects by interrupting IFN-γ signaling in activated macrophages. This investigation demonstrates that adding adenosine to IFN-γ-stimulated murine RAW 264.7 and human THP-1 macrophages results in unique modulation of STAT1 serine and tyrosine phosphorylation events. We show that adenosine inhibits IFN-γ-induced STAT1 S727 phosphorylation by &gt;30% and phosphoserine-mediated transcriptional activity by 58% but has no effect on phosphorylation of Y701 or receptor-associated JAK tyrosine kinases. Inhibition of the adenosine A3 receptor with a subtype-specific antagonist (MRS 1191 in RAW 264.7 cells and MRS 1220 in THP-1 cells) reverses this adenosine suppressive effect on STAT1 phosphoserine status by 25–50%. Further, RAW 264.7 A3 receptor stimulation with Cl-IB-MECA reduces IFN-γ-induced STAT1 transcriptional activity by 45% and STAT1-dependent gene expression by up to 80%. These data suggest that A3 receptor signaling is key to adenosine-mediated STAT1 modulation and anti-inflammatory action in IFN-γ-activated mouse and human macrophages. Because STAT1 plays a key role in IFN-γ-induced inflammation and foam cell transformation, a better understanding of the mechanisms underlying STAT1 deactivation by adenosine may improve preventative and therapeutic approaches to vascular disease.

1737 Background: The somatic JAK2 V617F (V617F) mutation is found in approximately 95% of polycythemia vera (PV) and 50–60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. Detection of this mutation occurs primarily through genomic analysis of peripheral blood samples of myeloproliferative neoplasm (MPN) patients. This study demonstrates a novel broad screening mechanism for detection of V617F from myeloid cells in saliva samples from an online cohort of MPN patients and from a broader database of genotyped individuals. Methods: MPN patients were recruited primarily through social media and patient-mediated outreach efforts. Patients gave IRB-approved consent and completed surveys through an online 23andMe account. Participation in the MPN research initiative was free. In collaboration with an uncompensated panel of academic experts, online surveys were developed to collect patient-reported data on diagnosis and results of genetic testing for the V617F mutation. Participants were genotyped on SNP arrays based on the Illumina Omni Express, with additional custom content, including four probes for the V617F mutation. We estimated V617F mutation burden as the median across the four probes of B/(A+B) where A and B were normalized intensities for the reference and mutated alleles, respectively. We estimated sensitivity and specificity of our estimated V617F allele ratio, using the self-reported V617F-positive individuals to approximate the distribution of true positives, and 23andMe members from the broad database (outside of the MPN research cohort) and younger than 40 as likely true negatives. Results: Web-based recruitment efforts led to the accrual of over 900 MPN patients of different subtypes over 12 months. Of these participants, 745 patients have been genotyped and provided MPN-related health history and outcomes through online surveys. The cohort is disproportionately female (71%), with an average age of 53.1 ± 14.9 years. More than 57% of patients indicated a classic MPN diagnosis (PV, ET, PMF, or post PV/ET MF), 19.2% reported an atypical MPN diagnosis (systemic mastocytosis or hypereosinophilic syndrome), 14.5% indicated chronic myelogenous leukemia, and 9.3% of the cohort reported other diagnoses. From this MPN cohort, a total of 345 participants reported mutation test results. Consistent with expectations, the V617F variant was reported by patients in nearly all PV cases (105 of 110 patients with a self-reported diagnosis of PV), and at a lower rate in ET and MF cases (59 of 95 patients and 48 of 61 patients, respectively). In addition to self-reported data, we were specifically able to detect the V617F mutation using SNP array data. We determined a detection threshold for the V617F variant at which 86% of self-reported V617F carriers were scored as positive (i.e. test sensitivity), and 99.975% of young 23andMe customers were scored as negative (i.e. test specificity). Participants with a classic diagnosis in the MPN cohort who carry the V617F variant tended to be older (mean age=59.5 versus 54.6 years) and were less likely to be female (62.5% versus 72.0%) than non-carriers. At this threshold, we detected 133 individuals who likely carry the V617F mutation among a broader set of 23andMe participants (n= 99,700) who were not specifically recruited for the MPN study, corresponding to a prevalence of 0.14%. We estimated the positive predictive value of the test in this cohort, or the expected proportion of positive test results that are true, as 81%. Within the general 23andMe cohort, the frequency of the V617F variant increases with participant age, from a rate of 0.017% for age 20 to 30, to 1.1% for age 80 to 90. We also observed that patients with a high allele burden of V617F frequently had acquired uniparental disomy of chromosome 9p, as determined from calculated heterozygosity scores and no-call rates for this chromosomal arm. Conclusion: We can detect V617F with high specificity and sensitivity from myeloid cells in saliva. This non-invasive source for DNA extraction offers new possibilities for detecting genomic alterations and for finding novel genetic associations in MPN patients as well as the general population. This study demonstrates the feasibility of online technology to significantly accelerate recruitment and research progress in MPNs and other rare diseases. Disclosures: Barnholt: 23andMe, Inc.: Employment. Hinds:23andMe: Employment, Equity Ownership, Patents & Royalties. Kiefer:23andMe, Inc.: Employment. Do:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Eriksson:23andMe, Inc.: Employment, Equity Ownership, Patents & Royalties. Mountain:23andMe, Inc.: Consultancy, Employment, Honoraria, Patents & Royalties. Francke:23andMe, Inc.: Employment, Honoraria, Research Funding; Locus Development: Consultancy, Membership on an entity's Board of Directors or advisory committees. Tung:23andMe, Inc.: Employment. Mesa:23andMe, Inc.: Unpaid advisor and collaborator Other. Gotlib:23andMe, Inc.: Unpaid advisor and collaborator Other. Zehnder:23andMe, Inc.: Unpaid advisor and collaborator Other.