The Phosphatase CSW Controls Life Span by Insulin Signaling and Metabolism Throughout Adult Life in Drosophila

Noonan syndrome and related disorders are caused by mutations in genes encoding for proteins of the RAS-ERK1/2 signaling pathway, which affect development by enhanced ERK1/2 activity. However, the mutations’ effects throughout adult life are unclear. In this study, we identify that the protein most commonly affected in Noonan syndrome, the phosphatase SHP2, known in Drosophila as corkscrew (CSW), controls life span, triglyceride levels, and metabolism without affecting ERK signaling pathway. We found that CSW loss-of-function mutations extended life span by interacting with components of the insulin signaling pathway and impairing AKT activity in adult flies. By expressing csw-RNAi in different organs, we determined that CSW extended life span by acting in organs that regulate energy availability, including gut, fat body and neurons. In contrast to that in control animals, loss of CSW leads to reduced homeostasis in metabolic rate during activity. Clinically relevant gain-of-function csw allele reduced life span, when expressed in fat body, but not in other tissues. However, overexpression of a wild-type allele did not affect life span, showing a specific effect of the gain-of-function allele independently of a gene dosage effect. We concluded that CSW normally regulates life span and that mutations in SHP2 are expected to have critical effects throughout life by insulin-dependent mechanisms in addition to the well-known RAS-ERK1/2-dependent developmental alterations.Fil: Ruzzi, Leonardo Rubén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Schilman, Pablo Ernesto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: San Martín, Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Lew, Sergio Eduardo. Universidad de Buenos Aires. Facultad de Ingeniería. Instituto de Ingeniería Biomédica; ArgentinaFil: Gelb, Bruce D.. Icahn School of Medicine at Mount Sinai; Estados UnidosFil: Pagani, Mario Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentin

Cathepsin K Deficiency Reduces Elastase Perfusion-Induced Abdominal Aortic Aneurysms in Mice

Objective: Cathepsin K (CatK) is one of the most potent mammalian elastases. We have previously shown increased expression of CatK in human abdominal aortic aneurysm (AAA) lesions. Whether this protease participates directly in AAA formation, however, remains unknown. Methods and Results: Mouse experimental AAA was induced with aortic perfusion of a porcine pancreatic elastase. Using this experimental model, we demonstrated that absence of CatK prevented AAA formation in mice 14 days postperfusion. CatK deficiency significantly reduced lesion CD4 T-cell content, total lesion and medial cell proliferation and apoptosis, medial smooth muscle cell (SMC) loss, elastinolytic CatL and CatS expression, and elastin fragmentation, but it did not affect AAA lesion Mac-3 macrophage accumulation or CD31 microvessel numbers. In vitro studies revealed that CatK contributed importantly to CD4 T-cell proliferation, SMC apoptosis, and other cysteinyl cathepsin and matrix metalloproteinase expression and activities in SMCs and endothelial cells but played negligible roles in microvessel growth and monocyte migration. AAA lesions from CatK-deficient mice showed reduced elastinolytic cathepsin activities compared with those from wild-type control mice. Conclusion: This study demonstrates that CatK plays an essential role in AAA formation by promoting T-cell proliferation, vascular SMC apoptosis, and elastin degradation and by affecting vascular cell protease expression and activities.Other Research Uni

Systems Analysis Implicates WAVE2 Complex in the Pathogenesis of Developmental Left-Sided Obstructive Heart Defects.

Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to identify causative variants is limited. To identify causal CHD genes that are associated with specific molecular functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe CHD. This approach enabled the authors to identify an association between left ventricular outflow tract obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins brk1, nckap1, and wasf2 and the regulators of small GTPase signaling cul3a and racgap1 are critical to cardiac development

EM-mosaic detects mosaic point mutations that contribute to congenital heart disease.

BackgroundThe contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined.MethodsWe developed a new computational method, EM-mosaic (Expectation-Maximization-based detection of mosaicism), to analyze mosaicism in exome sequences derived primarily from blood DNA of 2530 CHD proband-parent trios. To optimize this method, we measured mosaic detection power as a function of sequencing depth. In parallel, we analyzed our cohort using MosaicHunter, a Bayesian genotyping algorithm-based mosaic detection tool, and compared the two methods. The accuracy of these mosaic variant detection algorithms was assessed using an independent resequencing method. We then applied both methods to detect mosaicism in cardiac tissue-derived exome sequences of 66 participants for which matched blood and heart tissue was available.ResultsEM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The estimated true frequency of mosaic variants above 10% mosaicism was 0.14/person in blood and 0.21/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction.ConclusionsWe estimate that ~ 1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants with relatively higher allele fraction. Although blood is a readily available DNA source, cardiac tissues analyzed contributed ~ 5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses

Transcription factor Ap-2alpha is necessary for development of embryonic melanophores, autonomic neurons and pharyngeal skeleton in zebrafish

The genes that control development of embryonic melanocytes are poorly defined. Although transcription factor Ap-2a is expressed in neural crest (NC) cells, its role in development of embryonic melanocytes and other neural crest derivatives is unclear because mouse Ap-2a mutants die before melanogenesis. We show that zebrafish embryos injected with morpholino antisense oligonucleotides complementary to ap-2a (ap-2a MO) complete early morphogenesis normally and have neural crest cells. Expression of c-kit, which encodes the receptor for the Steel ligand, is reduced in these embryos, and, similar to zebrafish c-kit mutant embryos, embryonic melanophores are reduced in number and migration. The effects of ap-2a MO injected into heterozygous and homozygous c-kit mutants support the notion that Ap-2a works through C-kit and additional target genes to mediate melanophore cell number and migration. In contrast to c-kit mutant embryos, in ap-2a MO-injected embryos, melanophores are small and under-pigmented, and unexpectedly, analysis of mosaic embryos suggests Ap-2a regulates melanophore differentiation through cell non-autonomous targets. In addition to melanophore phenotypes, we document reduction of other neural crest derivatives in ap-2a MO-injected embryos, including jaw cartilage, enteric neurons, and sympathetic neurons. These results reveal that Ap-2a regulates multiple steps of melanophore development, and is required for development of other neuronal and nonneuronal neural crest derivatives.This work was supported by NIH grant HD22486 to J.S.E. and a Carver Foundation seed grant to R.A.C. C. d’., and M.A. were supported by grants ICM P99-137-f and Fondecyt 1031003. E.K.O. was supported by Grant T32 DC00040 (Bruce Gantz, PI)

The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Toward a Therapeutic Approach

The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach was held at the Renaissance Orlando at SeaWorld Hotel (August 2-4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion

Predictors of rapid aortic root dilation and referral for aortic surgery in Marfan syndrome

Few data exist regarding predictors of rapid aortic root dilation and referral for aortic surgery in Marfan syndrome (MFS). To identify independent predictors of the rate of aortic root (AoR) dilation and referral for aortic surgery, we investigated the data from the Pediatric Heart Network randomized trial of atenolol versus losartan in young patients with MFS. Data were analyzed from the echocardiograms at 0, 12, 24, and 36months read in the core laboratory of 608 trial subjects, aged 6months to 25 years, who met original Ghent criteria and had an AoR z-score (AoRz)>3. Repeated measures linear and logistic regressions were used to determine multivariable predictors of AoR dilation. Receiver operator characteristic curves were used to determine cut-points in AoR dilation predicting referral for aortic surgery. Multivariable analysis showed rapid AoR dilation as defined by change in AoRz/year>90th percentile was associated with older age, higher sinotubular junction z-score, and atenolol use (R-2=0.01) or by change in AoR diameter (AoRd)/year>90th percentile with higher sinotubular junction z-score and non-white race (R-2=0.02). Referral for aortic root surgery was associated with higher AoRd, higher ascending aorta z-score, and higher sinotubular junction diameter:ascending aorta diameter ratio (R-2=0.17). Change in AoRz of 0.72 SD units/year had 42% sensitivity and 92% specificity and change in AoRd of 0.34cm/year had 38% sensitivity and 95% specificity for predicting referral for aortic surgery. In this cohort of young patients with MFS, no new robust predictors of rapid AoR dilation or referral for aortic root surgery were identified. Further investigation may determine whether generalized proximal aortic dilation and effacement of the sinotubular junction will allow for better risk stratification. Rate of AoR dilation cut-points had high specificity, but low sensitivity for predicting referral for aortic surgery, limiting their clinical use. Clinical Trial Number ClinicalTrials.gov number, NCT00429364

Fgfr3 Is a Transcriptional Target of Ap2δ and Ash2l-Containing Histone Methyltransferase Complexes

Polycomb (PcG) and trithorax (trxG) proteins play important roles in establishing lineage-specific genetic programs through induction of chromatin modifications that lead to gene silencing or activation. Previously, we described an association between the MLL/SET1 complexes and a highly restricted, gene-specific DNA-binding protein Ap2δ that is required for recruitment of the MLL/SET1 complex to target Hoxc8 specifically. Here, we reduced levels of Ap2δ and Ash2l in the neuroblastoma cell line, Neuro2A, and analyzed their gene expression profiles using whole-genome mouse cDNA microarrays. This analysis yielded 42 genes that are potentially co-regulated by Ap2δ and Ash2l, and we have identified evolutionarily conserved Ap2-binding sites in 20 of them. To determine whether some of these were direct targets of the Ap2δ-Ash2l complex, we analyzed several promoters for the presence of Ap2δ and Ash2l by chromatin immunoprecipitation (ChIP). Among the targets we screened, we identified Fgfr3 as a direct transcriptional target of the Ap2δ-Ash2l complex. Additionally, we found that Ap2δ is necessary for the recruitment of Ash2l-containing complexes to this promoter and that this recruitment leads to trimethylation of lysine 4 of histone H3 (H3K4me3). Thus, we have identified several candidate targets of complexes containing Ap2δ and Ash2l that can be used to further elucidate their roles during development and showed that Fgfr3 is a novel direct target of these complexes

Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling

SummaryMany signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming

<i>De Novo</i> and Rare Variants at Multiple Loci Support the Oligogenic Origins of Atrioventricular Septal Heart Defects

Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk