Histone 3 Methyltransferases Alter Melanoma Initiation and Progression Through Discrete Mechanisms.

Perturbations to the epigenome are known drivers of tumorigenesis. In melanoma, alterations in histone methyltransferases that catalyze methylation at histone 3 lysine 9 and histone 3 lysine 27-two sites of critical post-translational modification-have been reported. To study the function of these methyltransferases in melanoma, we engineered melanocytes to express histone 3 lysine-to-methionine mutations at lysine 9 and lysine 27, which are known to inhibit the activity of histone methyltransferases, in a zebrafish melanoma model. Using this system, we found that loss of histone 3 lysine 9 methylation dramatically suppressed melanoma formation and that inhibition of histone 3 lysine 9 methyltransferases in human melanoma cells increased innate immune response signatures. In contrast, loss of histone 3 lysine 27 methylation significantly accelerated melanoma formation. We identified FOXD1 as a top target of PRC2 that is silenced in melanocytes and found that aberrant overexpression of FOXD1 accelerated melanoma onset. Collectively, these data demonstrate how histone 3 lysine-to-methionine mutations can be used to uncover critical roles for methyltransferases.

Risk Factors and Timing of Acute Myocardial Infarction Associated With Pregnancy: Insights From the National Inpatient Sample.

Background Pregnancy increases the risk of acute myocardial infarction (AMI). The purpose of this study was to examine timing and risk factors for AMI in pregnancy and poor outcome. Methods and Results National Inpatient Sample (2003-2015) was screened in pregnancy, labor and delivery, and postpartum. There were 11 297 849 records extracted with 913 instances of AMI (0.008%). One hundred eleven (12.2%) women experienced AMI during labor and delivery, 338 (37.0%) during pregnancy and most during the postpartum period (464; 50.8%). The prevalence of AMI in pregnancy has increased (P=0.0005). Most major adverse cardiovascular and cerebrovascular events occurred in the postpartum period (63.5%). Inpatient mortality was 4.5%. Predictors of AMI include known coronary artery disease (odds ratio [OR], 517.4; 95% CI, 420.8-636.2), heart failure (OR, 8.2; 95% CI, 1.9-35.2), prior valve replacement (OR, 6.4; 95% CI, 2.4-17.1), and atrial fibrillation (OR, 2.7; CI, 1.5-4.7; P<0.001). Risk factors of traditional atherosclerosis including hyperlipidemia, obesity, tobacco history, substance abuse, and thrombophilia were identified (P<0.001). Gestational hypertensive disorders (eclampsia OR, 6.0; 95% CI, 3.3-10.8; preeclampsia OR, 3.2; 95% CI, 2.5-4.2) were significant risk factors in predicting AMI. Risk factors associated with major adverse cardiovascular and cerebrovascular events included prior percutaneous coronary intervention (OR, 6.6; 95% CI, 1.4-31.2) and pre-eclampsia (OR, 2.3; 95% CI, 1.3-3.9). Conclusions AMI is associated with modifiable, nonmodifiable, and obstetric risk factors. These risk factors can lead to devastating adverse outcomes and highlight the need for risk factor modification and public health resource initiatives toward the goal of decreasing AMI in the pregnant population.

Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

In vivo severity ranking of Ras pathway mutations associated with developmental disorders.

Germ-line mutations in components of the Ras/MAPK pathway result in developmental disorders called RASopathies, affecting about 1/1,000 human births. Rapid advances in genome sequencing make it possible to identify multiple disease-related mutations, but there is currently no systematic framework for translating this information into patient-specific predictions of disease progression. As a first step toward addressing this issue, we developed a quantitative, inexpensive, and rapid framework that relies on the early zebrafish embryo to assess mutational effects on a common scale. Using this assay, we assessed 16 mutations reported in MEK1, a MAPK kinase, and provide a robust ranking of these mutations. We find that mutations found in cancer are more severe than those found in both RASopathies and cancer, which, in turn, are generally more severe than those found only in RASopathies. Moreover, this rank is conserved in other zebrafish embryonic assays and Drosophila-specific embryonic and adult assays, suggesting that our ranking reflects the intrinsic property of the mutant molecule. Furthermore, this rank is predictive of the drug dose needed to correct the defects. This assay can be readily used to test the strengths of existing and newly found mutations in MEK1 and other pathway components, providing the first step in the development of rational guidelines for patient-specific diagnostics and treatment of RASopathies.