Increased prevalence of EPAS1 variant in cattle with high-altitude pulmonary hypertension.

High-altitude pulmonary hypertension (HAPH) has heritable features and is a major cause of death in cattle in the Rocky Mountains, USA. Although multiple genes are likely involved in the genesis of HAPH, to date no major gene variant has been identified. Using whole-exome sequencing, we report the high association of an EPAS1 (HIF2α) double variant in the oxygen degradation domain of EPAS1 in Angus cattle with HAPH, mean pulmonary artery pressure >50 mm Hg in two independent herds. Expression analysis shows upregulation of 26 of 27 HIF2α target genes in EPAS1 carriers with HAPH. Of interest, this variant appears to be prevalent in lowland cattle, in which 41% of a herd of 32 are carriers, but the variant may only have a phenotype when the animal is hypoxemic at altitude. The EPAS1 variant will be a tool to determine the cells and signalling pathways leading to HAPH.

Hepatitis C and HIV Coinfection for Social Workers in Public Health, Medical and Substance Use Treatment Settings.

Hepatitis C virus (HCV) infection is a leading cause of liver disease and liver-related deaths among those living with human immunodeficiency virus (HIV) infection. Due to recent pharmacologic advancements with direct acting antivirals, if detected and treated, HCV antiviral therapy can prevent liver disease progression and permanently cure coinfected patients of HCV. The objectives of this article are to inform public health social workers and social workers in medical and substance use treatment settings of the public health burden of HCV/HIV coinfection and to highlight state-of-the art pharmacologic advancements in HCV antiviral therapy.

Tgif1 regulates quiescence and self-renewal of hematopoietic stem cells.

TG-interacting factor 1 (TGIF1) is a transcriptional repressor that can modulate retinoic acid and transforming growth factor ß signaling pathways. It is required for myeloid progenitor cell differentiation and survival, and mutations in the TGIF1 gene cause holoprosencephaly. Furthermore, we have previously observed that acute myelogenous leukemia (AML) patients with low TGIF1 levels had worse prognoses. Here, we explored the role of Tgif1 in murine hematopoietic stem cell (HSC) function. CFU assays showed that Tgif1(-/-) bone marrow cells produced more total colonies and had higher serial CFU potential. These effects were also observed in vivo, where Tgif1(-/-) bone marrow cells had higher repopulation potential in short- and long-term competitive repopulation assays than wild-type cells. Serial transplantation and replating studies showed that Tgif1(-/-) HSCs exhibited greater self-renewal and were less proliferative and more quiescent than wild-type cells, suggesting that Tgif1 is required for stem cells to enter the cell cycle. Furthermore, HSCs from Tgif1(+/-) mice had a phenotype similar to that of HSCs from Tgif1(-/-) mice, while bone marrow cells with overexpressing Tgif1 showed increased proliferation and lower survival in long-term transplant studies. Taken together, our data suggest that Tgif1 suppresses stem cell self-renewal and provide clues as to how reduced expression of TGIF1 may contribute to poor long-term survival in patients with AML.

Role of BMPR2 alternative splicing in heritable pulmonary arterial hypertension penetrance.

BACKGROUND: Bone morphogenic protein receptor 2 (BMPR2) gene mutations are the most common cause of heritable pulmonary arterial hypertension. However, only 20% of mutation carriers get clinical disease. Here, we explored the hypothesis that this reduced penetrance is due in part to an alteration in BMPR2 alternative splicing. METHODS AND RESULTS: Our data showed that BMPR2 has multiple alternative spliced variants. Two of these, isoform-A (full length) and isoform-B (missing exon 12), were expressed in all tissues analyzed. Analysis of cultured lymphocytes of 47 BMPR2 mutation-positive heritable pulmonary arterial hypertension patients and 35 BMPR2 mutation-positive unaffected carriers showed that patients had higher levels of isoform-B compared with isoform-A (B/A ratio) than carriers (P=0.002). Furthermore, compared with cells with a low B/A ratio, cells with a high B/A ratio had lower levels of unphosphorylated cofilin after BMP stimulation. Analysis of exon 12 sequences identified an exonic splice enhancer that binds serine arginine splicing factor 2 (SRSF2). Because SRSF2 promotes exon inclusion, reduced SRSF2 expression would mean that exon 12 would not be included in final BMPR2 mRNA (thus promoting increased isoform-B formation). Western blot analysis showed that SRSF2 expression was lower in cells from patients compared with cells from carriers and that siRNA-mediated knockdown of SRSF2 in pulmonary microvascular endothelial cells resulted in elevated levels of isoform-B compared with isoform-A, ie, an elevated B/A ratio. CONCLUSIONS: Alterations in BMPR2 isoform ratios may provide an explanation of the reduced penetrance among BMPR2 mutation carriers. This ratio is controlled by an exonic splice enhancer in exon 12 and its associated splicing factor, SRSF2.

Connectivity map analysis of nonsense-mediated decay-positive BMPR2-related hereditary pulmonary arterial hypertension provides insights into disease penetrance.

The molecular mechanisms underlying the reduced penetrance seen in the nonsense-mediated decay-positive (NMD+) BMPR2 mutation-associated hereditary pulmonary arterial hypertension (HPAH) remain unknown. We reasoned that the cellular and genetic mechanisms behind this phenomenon could be uncovered by combining expression profiling with Connectivity Map (cMap) analysis. Cultured lymphocytes from 10 patients with HPAH and 10 matched familial control subjects, all with NMD+ BMPR2 mutations, were subjected to expression analysis. For each group, the expression data were combined before analysis. This generated a signature of 23 up-regulated and 12 down-regulated genes in patients with HPAH compared with control subjects (the "PAH penetrance signature"). Although gene set enrichment analysis of this signature was not uniquely informative, cMap analysis identified drugs with expression signatures similar to the PAH penetrance signature. Several of these drugs were predicted to influence reactive oxygen species (ROS) formation. This hypothesis was tested and confirmed in the same cells initially subjected to the expression analysis using quantitative biochemical detection of ROS concentration. We conclude that expression of the PAH penetrance signature represents an increased risk of developing clinical HPAH and that ROS formation may play a role in pathogenesis of HPAH. These results provide the first molecular insights into NMD+ BMPR2 related HPAH penetrance and highlight the potential utility of cMap analyses in pulmonary research.

High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression profiling of peripheral blood mononuclear cells.

High-altitude pulmonary hypertension (HAPH) is a consequence of chronic alveolar hypoxia, leading to hypoxic vasoconstriction and remodeling of the pulmonary circulation. Brisket disease in cattle is a naturally occurring animal model of hypoxic pulmonary hypertension. Genetically susceptible cattle develop severe pulmonary hypertension and right heart failure at altitudes >7,000 ft. No information currently exists regarding the identity of the pathways and gene(s) responsible for HAPH or influencing severity. We hypothesized that initial insights into the pathogenesis of the disease could be discovered by a strategy of (1) sequencing of functional candidates revealed by single nucleotide polymorphism (SNP) analysis and (2) gene expression profiling of affected cattle compared with altitude-matched normal controls, with gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA). We isolated blood from a single herd of Black Angus cattle of both genders, aged 12-18 months, by jugular vein puncture. Mean pulmonary arterial pressures were 85.6±13 mmHg STD in the 10 affected and 35.3±1.2 mmHg STD in the 10 resistant cattle, P<0.001. From peripheral blood mononuclear cells, DNA was hybridized to an Affymetrix 10K Gene Chip SNP, and RNA was used to probe an Affymetrix Bovine genome array. SNP loci were remapped using the Btau 4.0 bovine genome assembly. mRNA data was analyzed by the Partek software package to identify sets of genes with an expression that was statistically different between the two groups. GSEA and IPA were conducted on the refined expression data to identify key cellular pathways and to generate networks and conduct functional analyses of the pathways and networks. Ten SNPs were identified by allelelic association and four candidate genes were sequenced in the cohort. Neither endothelial nitric oxide synthetase, NADH dehydrogenase, TG-interacting factor-2 nor BMPR2 were different among affected and resistant cattle. A 60-gene mRNA signature was identified that differentiated affected from unaffected cattle. Forty-six genes were overexpressed in the affected and 14 genes were downregulated in the affected cattle by at least 20%. GSEA and Ingenuity analysis identified respiratory diseases, inflammatory diseases and pathways as the top diseases and disorders (P<5.14×10(-14)), cell development and cell signaling as the top cellular functions (P<1.20×10(-08)), and IL6, TREM, PPAR, NFkB cell signaling (P<8.69×10(-09)) as the top canonical pathways associated with this gene signature. This study provides insights into differences in RNA expression in HAPH at a molecular level, and eliminates four functional gene candidates. Further studies are needed to validate and refine these preliminary findings and to determine the role of transcribed genes in the development of HAPH.