A New Apolipoprotein Influencing Plasma Triglyceride Levels in Humans and Mice
Len Pennacchio, Lawrence Berkeley National Laboratory, Berkeley, CA; Michael Olivier, Medical College of Wisconsin, Milwaukee, WI; Jaroslav A Hubacek, Jonathan C Cohen, UT Southwestern Medical Center, Dallas, TX; David R Cox, Stanford Human Genome Center, Palo Alto, CA; Jamilla Fruchart, Pasteur Institute, Lille, France; Ronald M Krauss, Edward M Rubin, Lawrence Berkeley National Laboratory, Berkeley, CA
The apolipoprotein gene cluster on human chromosome 11q23 (apoAI/CIII/AIV) represents a well-studied region that influences a variety of plasma lipid parameters and atherosclerosis susceptibility in humans. To facilitate the identification of evolutionarily conserved sequences with potential function near this cluster, we determined the sequence of ∼200 kbp of orthologous mouse DNA and compared the mouse and human sequences. The presence of a stretch of inter-species sequence conservation approximately 30 kbp proximal to the apoAI/CIII/AIV gene cluster, led us to an interval that upon further analysis was shown to encode a new member (apoAV) of the chromosome 11 apolipoprotein gene cluster. ApoAV transcripts were identified predominantly in liver tissue from mice and humans. To assess the function of apoAV, we generated mice over-expressing a human apoAV transgene as well as apoAV knockout mice. We observed dramatic but opposite effects on plasma triglyceride in these two groups of animals; a 3-fold decrease in plasma triglycerides in the apoAV transgenics and a 4-fold increase in the knockout mice. To explore the relationship between apoAV and lipid parameters in humans, several single nucleotide polymorphisms (SNPs) across the apoAV locus were identified and genotyped in two independent patient populations of normo-lipidemics. A common haplotype was found to be significantly associated with increased plasma triglycerides in the two large independent studies each employing different study designs. These findings in humans and mice illustrate the utility of comparative sequence analysis as a means to prioritize regions of the genome for further analysis and suggest an important physiological role for apoAV in affecting plasma levels of triglyceride, a major risk factor for coronary artery disease in humans.
Mutations in the Protein Tyrosine Phosphatase Gene, PTPN11, Cause Noonan Syndrome
Marco Tartaglia, Ernest L. Mehler, Mount Sinai School of Medicine, New York, NY; Rosalie Goldberg, Albert Einstein College of Medicine, Bronx, NY; Giuseppe Zampino, Istituto Superiore di Sanita, Rome Italy; Andrew H. Crosby, Andra Ion, Steve Jeffery, Kamini Kalidas, St George’s Hospital Medical School, London, UK; Han G. Brunner, Hannie Kremer, Ineke van der Burgt, University Medical Centre, Nijmegen, Netherlands; Michael A. Patton, St George’s Hospital Medical School, London, UK; Raju S. Kucherlapati, Albert Einstein College of Medicine, Bronx, NY; Bruce D. Gelb, Mount Sinai School of Medicine, New York, NY
Noonan syndrome (NS) is an autosomal dominant trait with pulmonic stenosis and hypertrophic cardiomyopathy . The full spectrum of congenital heart defects in NS includes AV septal defects, PDA and aortic coarctation. Short stature, facial dysmorphism and skeletal anomalies are additional features. NS has been mapped to chromosome 12q24, but is genetically heterogeneous. Methods: To evaluate the positional candidate gene, PTPN11, which encodes a protein tyrosine phosphatase (PTP) with two scr-homology 2 (SH2) domains, the gene organization was determined from genomic sequences. PTPN11 exons and intron boundaries were amplified from 2 NS families whose trait linked to 12q24, and amplimers were sequenced. Identified changes were confirmed with restriction assays and excluded in >200 controls. Mutation analysis was performed with DNAs from 14 unrelated NS subjects. Energetics-based structural analysis was performed using the Monte Carlo with scaled collective variables technique. Results: Analysis of the 15 PTPN11 coding exons with two linked NS families revealed missense changes in exon 3 altering conserved N-SH2 residues (A72S and Q79R). Changes were present in all affecteds but not in the unaffecteds or controls. Mutation analysis of 14 NS samples revealed missense mutations in 7 (Q79R and 4 novel ones) altering residues in the N-SH2 and PTP domains. Mutated residues clustered at the site of N-SH2-PTP binding, an interaction that inactivates PTP activity. Structural analysis of two N-SH2 mutants in the active conformation revealed an increased population of low-energy states with small root mean square deviation compared to wild type, predicting reduced inactivation. Conclusions: PTPN11 mutations cause NS and account for about 50% of disease. All defects affected the N-SH2-PTP interaction, apparently with gain-of-function effects. These findings implicate PTPN11 in the control of cardiogenesis and cardiomyocyte growth.
Inactivation of the Osteopontin Gene Enhances Vascular Calcification of Matrix Gla Protein-Deficient Mice: Evidence for Osteopontin as an Inducible Inhibitor In Vivo
Mei Y. Speer, University of Washington, Department of Bioengineering, Seattle, WA; Marc D McKee, McGill University, Department of Anatomy and Cell Biology and Faculty of Dentistry, Montreal, Quebec, Canada; Lucy Liaw, Maine Medical Center Research Institute, Scarborough, Maine, OR; Thorsten Schinke, Gerard Karsenty, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; Elyse Tung, Cecilia M Giachelli, University of Washington, Department of Bioengineering, Seattle, WA
Vascular calcification is widespread in individuals with atherosclerosis and diabetes, yet little is known about the mechanisms regulating this pathological process. Osteopontin (OPN) is expressed during bone remodeling, and is abundant in human calcified arteries, but not normal vessels. To examine the role of OPN in vascular calcification, we crossed OPN mutant mice with matrix Gla protein (MGP) mutant mice that were previously shown to develop vascular calcification. We found that mice deficient in MGP alone (MGP-/-OPN+/+) showed calcification in their aortas as early as 2 weeks after birth (∼ 0.3 mmol/g dry weight), as measured by calcium content; and expression of OPN was greatly upregulated in the calcified arteries, and to a smaller degree in serum, compared to wild-type littermates. The OPN expressed in the calcified vessels was observed adjacent to the mineral and co-localized to cells of the calcified medial layer. Mice deficient in both MGP and OPN (MGP-/-OPN-/-) had twice as much arterial calcification than MGP-/-OPN+/+ (MGP-/-OPN-/- = 0.6 mmol/g dry weight, P<0.05), suggesting an inhibitory effect of OPN in vascular calcification. Moreover, these mice died significantly earlier (4.4 ± 0.5 weeks) than MGP-/-OPN+/+ littermates (6.6 ± 2.2 weeks, n=5–9, P<0.05). The cause of death in these animals was found to be their vascular ruptures followed by heamorrhage, most likely due to severe vascular calcification. Immunohistochemical staining of calcified arteries showed that smooth muscle specific genes, SM-a-actin and SM22, were greatly decreased in the calcified arteries. These studies are the first to demonstrate a role for OPN as an inducible inhibitor of ectopic calcification in arteries in vivo.
Slowed Conduction and Ventricular Arrhythmias Following Targeted Disruption of the Cardiac Sodium Channel
Alex Papadatos, Polly Wallerstein, Rosemary Ratcliff, Catherine Head, Peter Brady, University of Cambridge, Cambridge, UK; Klaus Benndorf, Universit[auml]t Jena, Jena, Germany; Richard Saumarez, Christopher Huang, Jamie Vandenberg, William Colledge, Andrew Grace, University of Cambridge, Cambridge, UK
Loss-of-function mutations of the cardiac sodium channel are associated with bradycardia, atrio-ventricular (AV) conduction disease and ventricular fibrillation. The mechanistic basis of these conditions is unresolved. Using established techniques we have generated mice with targeted deletion of the entire coding region of Scn5a providing an experimental system to examine both the pathophysiology of these conditions and the contribution of sodium channels to cardiac impulse initiation and propagation. Homozygous deletion resulted in intrauterine lethality between E10.5 and E11.5 whilst Scn5a+/- mice had normal survival. Whole-cell patch clamp analysis of adult ventricular myocytes showed significant reducitons in peak current densities for Scn5a+/- (21±2 pA/pF, n=5, mean±s.e.m.) compared to wild-type (37±4 pA/pF, n=6, P < 0.01). Half-activation voltages and slope factors for both steady-state activation and inactivation were indistinguishable. ECGs from Scn5a+/- mice showed bradycardia, increased P wave duration and prolonged PR interval (55.7±2.1 ms, n=9, vs. 41.0±1.2 ms in wild type, n=9, P<0.001) with some Scn5a+/- mice also having AV block or right axis deviation. Langendorff-perfused Scn5a+/- hearts lost all spontaneous electrical activity. During incremental atrial pacing impaired AV conduction was confirmed in Scn5a+/- hearts (anterograde Wenckebach conduction block occurred at 68.8±1.6ms, n=4, in Scn5a+/- vs. 41.8±1.4ms, n=4, in wild-type, P< 0.001). During ventricular pacing Scn5a+/- mice had increased stimulus-to-response latency (18.3±0.3ms, n=6, vs. 8.8±0.1ms, n=9), electrogram duration (64.2±0.4ms vs. 32.3±0.8ms) and ventricular effective refractory period (VERP, 52.2±2.5ms vs. 29.3 ± 0.4ms, all P<0.001 comparing Scn5a+/- to wild-type). Monomorphic ventricular tachycardia, of cycle-length 50–60ms with features indicative of re-entrant excitation, was observed consistently in Scn5a+/- but not wild-type mice. These data provide compelling evidence that a single mechanism, decreased sodium channel conductance, can account for the clinical manifestations of patients with loss-of-function SCN5A mutations in conditions such as Brugada syndrome and progressive cardiac conduction defect (PCCD).
Transgenic Mouse Model and Molecular Mechanism for Cardiac Arrhythmia
Qing Wang, Xiaol-Li Tian, The Cleveland Clinic Foundation, Cleveland, OH; Xiaoping Wan, Case Western Reserve University Metrohealth Campus, Cleveland, OH; Ling Wu, Mina K Chung, Patrick J Tchou, The Cleveland Clinic Foundation, Cleveland, OH; Glenn E Kirsch, Case Western Reserve University Metrohealth Campus, Cleveland, OH
Ventricular tachycardia (VT) degenerating into ventricular fibrillation (VF) kills 300,000–400,000 people annually in the US alone. Mutations in the cardiac sodium channel SCN5A are associated with several arrhythmias including long QT syndrome (LQT), cardiac conduction disease and idiopathic VF. To explore the molecular mechanisms underlying the pathogenesis of VT and VF, we generated transgenic mice with selective cardiac over-expression of an LQT mutation in SCN5A (N1325S). We found that the mutant mice demonstrated significantly prolonged QT interval as well as monomorphic VT, which frequently degenerated into polymorphic VT. Further degeneration of polymorphic VT to VF caused sudden death in the mutant mice. Cardiac cells from the mutant mice exhibited increased late persistent inward sodium currents and prolonged action potential duration. They also showed frequent early afterdepolarizations (EAD), delayed afterdepolarizations (DAD), or both. This report presents the establishment of the first mouse model for VT, VF, and sudden death by targeting a currently known human arrhythmogenic gene. Our work directly demonstrates a causal linkage between prolonged cardiac action potential, afterdepolarizations, VT/VF, and sudden death.