Claudia P. Cabrera

Genome-wide association analysis identifies novel blood pressure loci and offers biological insights into cardiovascular risk.

Elevated blood pressure is the leading heritable risk factor for cardiovascular disease worldwide. We report genetic association of blood pressure (systolic, diastolic, pulse pressure) among UK Biobank participants of European ancestry with independent replication in other cohorts, and robust validation of 107 independent loci. We also identify new independent variants at 11 previously reported blood pressure loci. In combination with results from a range of in silico functional analyses and wet bench experiments, our findings highlight new biological pathways for blood pressure regulation enriched for genes expressed in vascular tissues and identify potential therapeutic targets for hypertension. Results from genetic risk score models raise the possibility of a precision medicine approach through early lifestyle intervention to offset the impact of blood pressure–raising genetic variants on future cardiovascular disease risk.

Genome-wide analysis of epistasis in body mass index using multiple human populations

We surveyed gene–gene interactions (epistasis) in human body mass index (BMI) in four European populations (n<1200) via exhaustive pair-wise genome scans where interactions were computed as F ratios by testing a linear regression model fitting two single-nucleotide polymorphisms (SNPs) with interactions against the one without. Before the association tests, BMI was corrected for sex and age, normalised and adjusted for relatedness. Neither single SNPs nor SNP interactions were genome-wide significant in either cohort based on the consensus threshold (P=5.0E−08) and a Bonferroni corrected threshold (P=1.1E−12), respectively. Next we compared sub genome-wide significant SNP interactions (P<5.0E−08) across cohorts to identify common epistatic signals, where SNPs were annotated to genes to test for gene ontology (GO) enrichment. Among the epistatic genes contributing to the commonly enriched GO terms, 19 were shared across study cohorts of which 15 are previously published genome-wide association loci, including CDH13 (cadherin 13) associated with height and SORCS2 (sortilin-related VPS10 domain containing receptor 2) associated with circulating insulin-like growth factor 1 and binding protein 3. Interactions between the 19 shared epistatic genes and those involving BMI candidate loci (P<5.0E−08) were tested across cohorts and found eight replicated at the SNP level (P<0.05) in at least one cohort, which were further tested and showed limited replication in a separate European population (n>5000). We conclude that genome-wide analysis of epistasis in multiple populations is an effective approach to provide new insights into the genetic regulation of BMI but requires additional efforts to confirm the findings.

IGSF10 mutations dysregulate gonadotropin‐releasing hormone neuronal migration resulting in delayed puberty

Early or late pubertal onset affects up to 5% of adolescents and is associated with adverse health and psychosocial outcomes. Self‐limited delayed puberty (DP) segregates predominantly in an autosomal dominant pattern, but the underlying genetic background is unknown. Using exome and candidate gene sequencing, we have identified rare mutations in IGSF10 in 6 unrelated families, which resulted in intracellular retention with failure in the secretion of mutant proteins. IGSF10 mRNA was strongly expressed in embryonic nasal mesenchyme, during gonadotropin‐releasing hormone (GnRH) neuronal migration to the hypothalamus. IGSF10 knockdown caused a reduced migration of immature GnRH neurons in vitro, and perturbed migration and extension of GnRH neurons in a gnrh3:EGFP zebrafish model. Additionally, loss‐of‐function mutations in IGSF10 were identified in hypothalamic amenorrhea patients. Our evidence strongly suggests that mutations in IGSF10 cause DP in humans, and points to a common genetic basis for conditions of functional hypogonadotropic hypogonadism (HH). While dysregulation of GnRH neuronal migration is known to cause permanent HH, this is the first time that this has been demonstrated as a causal mechanism in DP.

Exploring hypertension genome-wide association studies findings and impact on pathophysiology, pathways, and pharmacogenetics.

Hypertension is a major risk factor for global mortality. Recent genome‐wide association studies (GWAS) have led to successful identification of many genetic loci influencing blood pressure, although these studies account for less than 5% of heritability. While genetic discovery efforts continue, it is timely to pause and reflect on what information has been gained to date from reported loci. Knowledge from GWAS findings inform our understanding of the pathways and pleiotropy underpinning hypertension and aid in the identification of potential druggable targets. By reviewing blood pressure loci we aim to determine how much potential the current observations have for future clinical utility. WIREs Syst Biol Med 2015, 7:73–90. doi: 10.1002/wsbm.1290

Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5 +/Tbx5 + population rapidly replaced the Tbx5 low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study.

Artesunate Protects Against the Organ Injury and Dysfunction Induced by Severe Hemorrhage and Resuscitation.

OBJECTIVE To evaluate the effects of artesunate on organ injury and dysfunction associated with hemorrhagic shock (HS) in the rat. BACKGROUND HS is still a common cause of death in severely injured patients and is characterized by impairment of organ perfusion, systemic inflammatory response, and multiple organ failure. There is no specific therapy that reduces organ injury/dysfunction. Artesunate exhibits pharmacological actions beyond its antimalarial activity, such as anticancer, antiviral, and anti-inflammatory effects. METHODS Rats were submitted to HS. Mean arterial pressure was reduced to 30 mm Hg for 90 minutes, followed by resuscitation. Rats were randomly treated with artesunate (2.4 or 4.8 mg/kg i.v.) or vehicle upon resuscitation. Four hours later, parameters of organ injury and dysfunction were assessed. RESULTS Artesunate attenuated the multiple organ injury and dysfunction caused by HS. Pathway analysis of RNA sequencing provided good evidence to support an effect of artesunate on the Akt-survival pathway, leading to downregulation of interleukin-1 receptor-associated kinase 1. Using Western blot analysis, we confirmed that treatment of HS rats with artesunate enhanced the phosphorylation (activation) of Protein kinase B (Akt) and endothelial nitric oxide synthase and the phosphorylation (inhibition) of glycogen synthase kinase-3β (GSK-3β). Moreover, artesunate attenuated the HS-induced activation of nuclear factor kappa B and reduced the expression of proinflammatory proteins (inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin 6). CONCLUSIONS Artesunate attenuated the organ injury/dysfunction associated with HS by a mechanism that involves the activation of the Akt-endothelial nitric oxide synthase survival pathway, and the inhibition of glycogen synthase kinase-3β and nuclear factor kappa B. A phase II clinical trial evaluating the effects of good manufacturing practice-artesunate in patients with trauma and severe hemorrhage is planned.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives 4.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially. http://creativecommons.org/licenses/by-nc-nd/4.0.Objective: To evaluate the effects of artesunate on organ injury and dysfunction associated with hemorrhagic shock (HS) in the rat. Background: HS is still a common cause of death in severely injured patients and is characterized by impairment of organ perfusion, systemic inflammatory response, and multiple organ failure. There is no specific therapy that reduces organ injury/dysfunction. Artesunate exhibits pharmacological actions beyond its antimalarial activity, such as anticancer, antiviral, and anti-inflammatory effects. Methods: Rats were submitted to HS. Mean arterial pressure was reduced to 30 mm Hg for 90 minutes, followed by resuscitation. Rats were randomly treated with artesunate (2.4 or 4.8 mg/kg i.v.) or vehicle upon resuscitation. Four hours later, parameters of organ injury and dysfunction were assessed. Results: Artesunate attenuated the multiple organ injury and dysfunction caused by HS. Pathway analysis of RNA sequencing provided good evidence to support an effect of artesunate on the Akt-survival pathway, leading to downregulation of interleukin-1 receptor-associated kinase 1. Using Western blot analysis, we confirmed that treatment of HS rats with artesunate enhanced the phosphorylation (activation) of Protein kinase B (Akt) and endothelial nitric oxide synthase and the phosphorylation (inhibition) of glycogen synthase kinase-3&bgr; (GSK-3&bgr;). Moreover, artesunate attenuated the HS-induced activation of nuclear factor kappa B and reduced the expression of proinflammatory proteins (inducible nitric oxide synthase, tumor necrosis factor-&agr;, and interleukin 6). Conclusions: Artesunate attenuated the organ injury/dysfunction associated with HS by a mechanism that involves the activation of the Akt-endothelial nitric oxide synthase survival pathway, and the inhibition of glycogen synthase kinase-3&bgr; and nuclear factor kappa B. A phase II clinical trial evaluating the effects of good manufacturing practice-artesunate in patients with trauma and severe hemorrhage is planned.

The biological impact of blood pressure associated genetic variants in the natriuretic peptide receptor C gene on human vascular smooth muscle.

Abstract Elevated blood pressure (BP) is a major global risk factor for cardiovascular disease. Genome-wide association studies have identified several genetic variants at the NPR3 locus associated with BP, but the functional impact of these variants remains to be determined. Here we confirmed, by a genome-wide association study within UK Biobank, the existence of two independent BP-related signals within NPR3 locus. Using human primary vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) from different individuals, we found that the BP-elevating alleles within one linkage disequilibrium block identified by the sentinel variant rs1173771 was associated with lower endogenous NPR3 mRNA and protein levels in VSMCs, together with reduced levels in open chromatin and nuclear protein binding. The BP-elevating alleles also increased VSMC proliferation, angiotensin II-induced calcium flux and cell contraction. However, an analogous genotype-dependent association was not observed in vascular ECs. Our study identifies novel, putative mechanisms for BP-associated variants at the NPR3 locus to elevate BP, further strengthening the case for targeting NPR-C as a therapeutic approach for hypertension and cardiovascular disease prevention.

Contributions of Function-Altering Variants in Genes Implicated in Pubertal Timing and Body Mass for Self-Limited Delayed Puberty

Context: Self-limited delayed puberty (DP) is often associated with a delay in physical maturation, but although highly heritable the causal genetic factors remain elusive. Genome-wide association studies of the timing of puberty have identified multiple loci for age at menarche in females and voice break in males, particularly in pathways controlling energy balance. Objective/Main Outcome Measures: We sought to assess the contribution of rare variants in such genes to the phenotype of familial DP. Design/Patients: We performed whole-exome sequencing in 67 pedigrees (125 individuals with DP and 35 unaffected controls) from our unique cohort of familial self-limited DP. Using a whole-exome sequencing filtering pipeline one candidate gene [fat mass and obesity–associated gene (FTO)] was identified. In silico, in vitro, and mouse model studies were performed to investigate the pathogenicity of FTO variants and timing of puberty in FTO+/− mice. Results: We identified potentially pathogenic, rare variants in genes in linkage disequilibrium with genome-wide association studies of age at menarche loci in 283 genes. Of these, five genes were implicated in the control of body mass. After filtering for segregation with trait, one candidate, FTO, was retained. Two FTO variants, found in 14 affected individuals from three families, were also associated with leanness in these patients with DP. One variant (p.Leu44Val) demonstrated altered demethylation activity of the mutant protein in vitro. Fto+/− mice displayed a significantly delayed timing of pubertal onset (P < 0.05). Conclusions: Mutations in genes implicated in body mass and timing of puberty in the general population may contribute to the pathogenesis of self-limited DP.

Enhanced energetic state and protection from oxidative stress in human myoblasts overexpressing BMI1.

Summary The Polycomb group gene BMI1 is essential for efficient muscle regeneration in a mouse model of Duchenne muscular dystrophy, and its enhanced expression in adult skeletal muscle satellite cells ameliorates the muscle strength in this model. Here, we show that the impact of mild BMI1 overexpression observed in mouse models is translatable to human cells. In human myoblasts, BMI1 overexpression increases mitochondrial activity, leading to an enhanced energetic state with increased ATP production and concomitant protection against DNA damage both in vitro and upon xenografting in a severe dystrophic mouse model. These preclinical data in mouse models and human cells provide a strong rationale for the development of pharmacological approaches to target BMI1-mediated mitochondrial regulation and protection from DNA damage to sustain the regenerative potential of the skeletal muscle in conditions of chronic muscle wasting.

Signatures of inflammation and impending multiple organ dysfunction in the hyperacute phase of trauma: A prospective cohort study.

Background Severe trauma induces a widespread response of the immune system. This “genomic storm” can lead to poor outcomes, including Multiple Organ Dysfunction Syndrome (MODS). MODS carries a high mortality and morbidity rate and adversely affects long-term health outcomes. Contemporary management of MODS is entirely supportive, and no specific therapeutics have been shown to be effective in reducing incidence or severity. The pathogenesis of MODS remains unclear, and several models are proposed, such as excessive inflammation, a second-hit insult, or an imbalance between pro- and anti-inflammatory pathways. We postulated that the hyperacute window after trauma may hold the key to understanding how the genomic storm is initiated and may lead to a new understanding of the pathogenesis of MODS. Methods and findings We performed whole blood transcriptome and flow cytometry analyses on a total of 70 critically injured patients (Injury Severity Score [ISS] ≥ 25) at The Royal London Hospital in the hyperacute time period within 2 hours of injury. We compared transcriptome findings in 36 critically injured patients with those of 6 patients with minor injuries (ISS ≤ 4). We then performed flow cytometry analyses in 34 critically injured patients and compared findings with those of 9 healthy volunteers. Immediately after injury, only 1,239 gene transcripts (4%) were differentially expressed in critically injured patients. By 24 hours after injury, 6,294 transcripts (21%) were differentially expressed compared to the hyperacute window. Only 202 (16%) genes differentially expressed in the hyperacute window were still expressed in the same direction at 24 hours postinjury. Pathway analysis showed principally up-regulation of pattern recognition and innate inflammatory pathways, with down-regulation of adaptive responses. Immune deconvolution, flow cytometry, and modular analysis suggested a central role for neutrophils and Natural Killer (NK) cells, with underexpression of T- and B cell responses. In the transcriptome cohort, 20 critically injured patients later developed MODS. Compared with the 16 patients who did not develop MODS (NoMODS), maximal differential expression was seen within the hyperacute window. In MODS versus NoMODS, 363 genes were differentially expressed on admission, compared to only 33 at 24 hours postinjury. MODS transcripts differentially expressed in the hyperacute window showed enrichment among diseases and biological functions associated with cell survival and organismal death rather than inflammatory pathways. There was differential up-regulation of NK cell signalling pathways and markers in patients who would later develop MODS, with down-regulation of neutrophil deconvolution markers. This study is limited by its sample size, precluding more detailed analyses of drivers of the hyperacute response and different MODS phenotypes, and requires validation in other critically injured cohorts. Conclusions In this study, we showed how the hyperacute postinjury time window contained a focused, specific signature of the response to critical injury that led to widespread genomic activation. A transcriptomic signature for later development of MODS was present in this hyperacute window; it showed a strong signal for cell death and survival pathways and implicated NK cells and neutrophil populations in this differential response.