A novel nonsense mutation in NPR2 gene causing Acromesomelic dysplasia, type Maroteaux in a consanguineous family in Southern Punjab (Pakistan).

BACKGROUND: Acromesomelic dysplasia, type Maroteaux (AMDM) is a rare skeletal dysplasia following autosomal recessive mode of inheritance and characterized by abnormal growth plates, short and abnormal bones in the extremities and spine. OBJECTIVE: Present study was designed to report the molecular basis of AMDM in enrolled consanguineous family from Pakistan. METHODS: A consanguineous family from Vehari District in Pakistan having multiple siblings suffering from AMDM was enrolled in present study. Whole exome sequencing (WES) approach was adopted to identify causative agent of AMDM. Human full length NPR2 gene and sequence with nonsense mutation was amplified by using Myc-tagged pXN vector and transformed in E. coli DH5α cells to confirm mutation. SDS-PAGE and Western blotting were done to confirm the production of truncated protein. Computational three dimensional structure generation through homology modeling approach was done to compare protein structure between patients and controls. RESULTS: WES reveled a nonsense mutation (c.613 C>T, p.R205X) in exon 1 of NPR2 gene leading to premature termination codon in mRNA of NPR2 gene resulting in a truncated protein with 204 amino acid residues that was confirmed by SDS-PAGE and Western blotting. Sanger sequencing confirmed that mutation in all subjects and mutation followed Mendalian pattern of inheritance. Multiple sequence alignment by ClustalW revealed that mutated domain of NPR2 is conserved region. Proetin structure comparison revealed a significant structural part of NPR2 was missing in truncated protein as compared to control. CONCLUSION: We are reporting that a novel nonsense mutation (c.613 C>T, p.R205X) in exon 1 of NPR2 gene is causing AMDM in a consanguineous Pakistani family.

LncRNA HIF1A-AS1 contributes to ventricular remodeling after myocardial ischemia/reperfusion injury by adsorption of microRNA-204 to regulating SOCS2 expression.

Objectives: Long non-coding RNAs (lncRNAs) serve pivotal roles in heart disease, while the role of lncRNA hypoxia-inducible factor 1α-antisense RNA 1 (HIF1A-AS1) is rarely mentioned. Therefore, the objective of this study was to investigate the mechanism of lncRNA HIF1A-AS1 regulating suppressor of cytokine signaling 2 (SOCS2) expression by adsorption of microRNA-204 (miR-204) on ventricular remodeling after myocardial ischemia-reperfusion (I/R) injury in mice. Methods: The mouse model of I/R was established by left coronary artery occlusion. The expression of HIF1A-AS1, miR-204 and SOCS2 was determined. The mice were injected with HIF1A-AS1-siRNA, miR-204 mimics or their controls to investigate their effects on cardiac function and ventricular remodeling of mice after I/R injury. The binding relationship between HIF1A-AS1 and miR-204 as well as between miR-204 and SOCS2 were verified. Results: HIF1A-AS1 and SOCS2 were upregulated and miR-204 was downregulated in myocardial tissues in mice after I/R injury. LVEDD, LVEDS, LVEDP, LVMI and RVMI expression reduced while LVEF, LVFS, +dp/dt max and - dp/dt max increased through knockdown HIF1A-AS1 and upregulated miR-204. The expression of BNP, cTnI, LDH, CK, TNF-α, IL-1ß, IL-6 and ß-MHC reduced, and the expression of α-MHC increased when HIF1A-AS1 was poorly expressed and miR-204 was highly expressed. Silencing HIF1A-AS1 and upregulating miR-204 inhibited apoptosis of cells. LncRNA HIF1A-AS1 could act as ceRNA to adsorb miR-204 to suppress miR-204 expression and elevate SOCS2 expression. Conclusion: Our study provides evidence that downregulation of HIF1A-AS1 and upregulation of miR-204 could alleviate ventricular remodeling and improve cardiac function in mice after myocardial I/R injury via regulating SOCS2.

ROC Anatomy-Getting the Most Out of Your Diagnostic Test.

Clinical decision-making relies heavily on making a correct diagnosis. Clinicians have a responsibility to understand the full spectrum of the diagnostic information conveyed by a physical exam finding, laboratory test result, or imaging. Many laboratory tests, such as troponin and B-type natriuretic peptide (BNP), are continuous tests with many possible results. Yet, there is a tendency to dichotomize tests into positive and negative, and use sensitivity and specificity to describe the test characteristics. This approach can lead to waste of important diagnostic information and substandard clinical decision-making. The aim of this paper is to demonstrate the role of ROC curves in developing a more comprehensive understanding of diagnostic information portrayed by continuous tests to augment clinical decision-making.

[Up-regulation of atrial natriuretic peptide and GATA3 levels in peripheral blood of children with respiratory syncytial virus bronchiolitis].

Objective To investigate the relationship between changes in atrial natriuretic peptide (ANP) signal and GATA3 expression in peripheral blood of children with bronchiolitis, and to explore the possible mechanism of ANP signal in the pathogenesis of bronchiolitis.Methods 20 normal children, 16 children with mild bronchiolitis, and 14 medium-severe children were enrolled. ELISA was used to detect the level of ANP and interleukin-4(IL-4) in plasma. Real-time fluorescent quantitative PCR was performed to determine the mRNA expression of natriuretic peptide receptor A(NPRA)and GATA3 in peripheral blood mononuclear cells(PBMCs) .Western blot analysis was used to determine the protein expression of NPRA and GATA3. Results As the degree of inflammation of bronchiolitis increases, the level of ANP and IL-4 in plasma increased significantly, and the mRNA and protein expression of NPRA and GATA3 in PBMCs also increased. Conclusion The levels of ANP, NPRA, IL-4 and GATA3 increased in peripheral blood of children with bronchiolitis.

[Detection of guanylate cyclase C mRNA and cytokeratin 20 mRNA in peripheral blood and analysis of prognosis in early to moderate colorectal cancer patients].

OBJECTIVE: To investigate the associations of guanylate cyclase C (GC-C) mRNA and cytokeratin 20 (CK20) mRNA with metastasis and prognosis in early to moderate colorectal cancer patients. METHODS: GC-C mRNA and CK 20 mRNA in peripheral blood of 74 colorectal cancer patients without distant metastasis were detected by fluorescent quantitative PCR (FQ-PCR). Based on their clinicopathological and postoperative follow-up data, the relationship and clinical significance of these data with metastasis hazards and prognosis factors were analyzed. RESULTS: The positive rate of GC-C mRNA in 74 colorectal cancer patients was 33.8% (25/74), and CK20 mRNA was 31.1% (23/74). The 1-, 2-, 3- year disease-free survival rates of patients were 94.6%, 82.4% and 78.4% respectively. There were significant differences in positive rates of GC-C mRNA and CK20 mRNA, tumor differentiation, mesentery lymph node metastasis, tumor embolus in vessel and postoperative chemotherapy associated with 3-year disease free survival rate by Kaplan-Meier analysis (all P<0.05). While mesentery lymph node metastasis and tumor embolus in vessel were independent risk factors of 3-year disease-free survival (P<0.05). CK20 mRNA and tumor embolus in vessel were independent risk factors of 3-year disease-free survival by analysis stratified with clinical stage (P<0.05). CONCLUSIONS: Detection of CK20 mRNA and GC-C mRNA in peripheral blood may be important for early detection of early metastasis of colorectal cancer.

Natriuretic peptide system: a link between fat mass and cardiac hypertrophy and hypertension in fat-fed female rats.

The present study was designed to develop an animal model of hypertension and cardiac hypertrophy associated with obesity in female rats. Furthermore, we studied the involvement of the natriuretic peptide system in the mechanisms of these conditions. Obesity was induced in Wistar rats by a high fat diet and ovariectomy. The rats were divided into four groups: ovariectomized or sham-operated with high-fat diet and ovariectomized or sham-operated with control diet. After 24 weeks of diet, rats were killed, and their tissues were removed. Cardiac atrial natriuretic peptide (ANP), clearance receptor (NPr-C) gene expression was determined by PCR. ANP concentrations were measured in plasma. Ovariectomized fat-fed rats (OF) showed increased body weight, visceral fat depot and blood pressure and decreased sodium excretion compared to other groups. Also, these rats showed higher heart-to-body weight and cell diameters of ventricular cardiomyocytes and lower cardiac ANP mRNA and plasma ANP than the control group. The adipocyte and renal NPr-C mRNA of OF rats were higher than the control group. These data showed that combined ovariectomy and high fat diet elicited obesity, hypertension and cardiac hypertrophy. These results suggest that the impairment of the natriuretic peptide system may be one of the mechanisms involved not only in development of hypertension but also in cardiac hypertrophy associated with obesity in ovariectomized rats.

B-type natriuretic peptide infusions in acute myocardial infarction.

BACKGROUND: Natriuretic peptides have actions likely to ameliorate cardiac dysfunction. B-type natriuretic peptide (BNP) is indicated as treatment for decompensated cardiac failure. OBJECTIVE: To determine the utility of BNP in acute myocardial infarction (MI). DESIGN: Double-blind randomised placebo-controlled trial. SETTING: Tertiary hospital coronary care unit. PATIENTS: 28 patients with acute MI with delayed or failed reperfusion and moderate left ventricular dysfunction. INTERVENTIONS: Infusion of BNP or placebo for 60 hours after MI. MAIN OUTCOME MEASURES: Neurohormonal activation and renal function in response to BNP infusion, secondary end points of echocardiographic measures of left ventricular function and dimension. RESULTS: BNP infusion resulted in a significant rise in BNP (276 pg/l vs 86 pg/l, p = 0.001). NT-proBNP levels were suppressed by BNP infusion (p = 0.002). Atrial natriuretic peptide (ANP) and NT-proANP levels fell with a significant difference in the pattern between BNP infusion and placebo during the first 5 days (p<0.005). C-type natriuretic peptide (CNP) and NT-proCNP levels rose during the infusion with higher levels than placebo at all measurements during the first 3 days (p<0.01). Cyclic guanosine monophosphate (cGMP) was raised during the infusion period showing a peak of 23 pmol/l on day 2 (placebo 8.9 pmol/l, p = 0.002), with a correlation between BNP and cGMP levels (p<0.001). Glomerular filtration rate (GFR) fell with BNP infusion but was not significantly lower than with placebo (71.0 (5.6) vs 75.8 (5.4) ml/min/1.73 m2, p = 0.62). Patients receiving nesiritide exhibited favourable trends in left ventricular remodelling. CONCLUSIONS: Nesiritide, given soon after MI, induced increments in plasma cGMP and CNP and decrements in other endogenous cardiac peptides with a neutral effect on renal function and a trend towards favourable ventricular remodelling.

Left but not right cardiac hypertrophy in atrial natriuretic peptide receptor-deficient mice is prevented by angiotensin type 1 receptor antagonist losartan.

Mice with a genetic deletion of the atrial natriuretic peptide (ANP) receptor, guanylyl cyclase A (GC-A -/-), have chronic arterial hypertension and cardiac hypertrophy from the first day of life. To characterize the role of the angiotensin II and endothelin systems in the development of this cardiovascular phenotype, the effects of chronic treatment with either the angiotensin type I (AT1) receptor antagonist losartan or the endothelin A receptor antagonist BSF208075 were tested. Losartan almost completely reversed systemic arterial hypertension and left ventricular hypertrophy of GC-A -/- mice. This was accompanied by a marked regression of the left ventricular mRNA expression of cardiac hypertrophy markers such as ANP and brain natriuretic peptide and a significant reduction of left ventricular and pulmonary interstitial collagen accumulation. BSF208075 had no effect on any of these cardiovascular parameters. Intriguingly, GC-A -/- mice also showed a very marked right ventricular hypertrophy, which was not reversed by losartan or BSF208075 treatment. Analyses of components of the renin-angiotensin system (RAS) revealed an inhibition of renal and systemic RAS contrasting with increased local left ventricular angiotensin II levels in GC-A -/- mice. Collectively, the results suggest that RAS plays a more important role than the endothelin system in the pathogenesis of arterial hypertension as well as left ventricular hypertrophy and fibrosis in GC-A gene-disrupted mice.

Altered expression of vascular natriuretic peptide receptors in experimental hypertensive rats.

1. The aim of the present study was to determine whether the regulation of vascular natriuretic peptide receptors (NPR) is related to the local renin-angiotensin system (RAS). 2. Male Sprague-Dawley rats were made two-kidney, one-clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt hypertensive to activate and inhibit the RAS, respectively. Another model of hypertension was induced by treatment with an inhibitor of nitric oxide synthesis, namely NG-nitro-L-arginine methyl ester (L-NAME). 3. The mRNA expression of NPR-A, NPR-C, angiotensin- converting enzyme (ACE) and angiotensin AT1 receptors was determined in the thoracic aorta by semiquantitative reverse transcription-polymerase chain reaction. The particulate guanylyl cyclase activity stimulated by atrial natriuretic peptide (ANP) was also determined in the membrane fraction of the thoracic aorta. 4. The plasma concentrations of ANP were increased significantly in the three models of hypertension. Plasma renin activity was increased in 2K1C hypertension, decreased in DOCA-salt hypertension and not significantly altered in L-NAME hypertension. 5. The mRNA expression of NPR-A and NPR-C was decreased, whereas that of ACE and AT1 receptors was increased in 2K1C and L-NAME hypertension. The mRNA expression of NPR-A and NPR-C was increased, whereas that of ACE and AT1 receptors was decreased in DOCA-salt hypertension. 6. The particulate guanylyl cyclase activity was decreased in 2K1C and L-NAME hypertension and increased in DOCA-salt hypertension. 7. The vascular expression of NPR may be reciprocally regulated by local RAS activity.

[The examination of clinical prognostic importance of atrial and brain natriuretic peptide in patients with acute myocardial infarction]. / Badania nad klinicznym i prognostycznym znaczeniem przedsionkowego i mózgowego peptydu natriuretycznego u chorych na swiezy zawal miesnia sercowego.

Natriuretic peptides ANP, BNP and CNP with their receptors A, B, and C play an important role in maintaining the homeostasis. They have vasodilating, diuretic and natriuretic actions and regulate the systemic resistance. These peptides have been proved to contribute in pathogenesis of many diseases, for example heart failure, hypertension, acute coronary events or hepatic and renal insufficiency. The aim of our study was the estimation whether ANP or BNP could be markers of postinfarct heart failure in patients after thrombolytic therapy. The survey was made in 96 patients with acute myocardial infarction (AMI), who were treated in Department of Cardiology of University School of Medicine in Wroclaw. Patients were divided into 2 groups. The first group consisted of 56 patients with reperfusion after thrombolytic therapy and the second group created 40 patients without reperfusion. All patients were administered acethylsalicylic acid, 100 mg r-tPA (recombined tissue plasminogen activator) i.v. within 90 min and heparin in typical doses for the first 3-5 days. Venous blood samples for ANP and BNP estimation were taken before thrombolysis and then in 1st, 3rd, 5th and 30th day after treatment. We also measured the ejection fraction and activity of CPK and CK-MB in all the patients. Patients stayed under clinical observation for 12 months. Our study showed that the levels of ANP and BNP increase in 1st day after treatment in all patients with AMI. The normalization of ANP and BNP occurs in 3rd day after treatment in patients with reperfusion and in patients without reperfusion there are increased levels of these peptides even in 30th day. The increased level of BNP (> 160 pg/ml) is a significant risk factor of left ventricle systolic dysfunction, renewed AMI and sudden death in all patients with AMI after thrombolytic therapy whereas the increased level of ANP (> 100 pg/ml)--only in patients without reperfusion. Taking BNP as a marker of postinfarct heart failure it is possible to asses the risk of this complication in first day after thrombolytic therapy and choose the most proper treatment.

Possibility of age regulation of the natriuretic peptide C-receptor in human platelets.

Natriuretic peptide binding sites on platelets have been hypothesized to act as clearance receptors; however, there is no clear definition of the function of this receptor. The aim of the study was: 1) to characterize natriuretic peptide receptors in human platelets by original competition study; 2) to evaluate a possible age modulation of these binding sites, since a delayed clearance of ANP in the elderly has been observed. The binding of 125I-ANP to intact platelets was completely inhibited by h-ANP, h-BNP, h-CNP and c-ANP, the selective ligand of the clearance receptor. IC50 values were 0.089+/-0.029, 0.703+/-0.104 and 1.19+/-0.13, 3.84+/-0.04 nmol/l, mean+/-SE, respectively (p<0.001 for IC50 value of h-ANP compared to the other natriuretic peptides). This observation on the receptor selectivity of natriuretic peptides in human platelets provides new evidence for the presence of the clearance receptor on platelets. In control subjects the Kd was 34.6+/-4.0 pmol/l and Bmax 13.6+/-0.92 fmol/10(9) platelets (mean+/-SE), (no.=46, mean age 41.7+/-2.1 years). Bmax was significantly reduced in older subjects (no.=25, mean age 53.2+/-1.5 years) with respect to the younger group (no.=21, mean age 28.0+/-0.87 years): 11.4+/-1.1 vs 16.1+/-1.4 fmol/10(9) cells, p=0.0096, respectively; moreover, a significant inverse relationship between Bmax and the subject's age was observed. This observation suggests a possible reduction of the natriuretic peptide clearance with aging, associated to a significant increase of plasma levels of natriuretic peptides.

Beta-adrenoceptor antagonist propranolol potentiates hypotensive action of natriuretic peptides.

Beta-adrenoceptor antagonists are known to increase plasma atrial natriuretic peptide (ANP) levels despite their hypotensive action. The aim of the present study was to examine the role of the ANP system in the antihypertensive effects of a beta-adrenoceptor antagonist. We investigated the effects of propranolol (75 mg kg(-1) day(-1), p.o., 4 weeks) on the ANP system in stroke-prone spontaneously hypertensive rats. Plasma ANP levels were significantly higher in the propranolol group than in the control group. Both receptor densities and mRNA levels of ANP(C) receptor were significantly decreased in the lung as the major site of ANP clearance from the circulation. In contrast, both central venous pressure and ANP mRNA levels in the heart were not significantly different between the two groups. Under both basal and ANP-stimulated conditions, the cGMP content in the aorta was significantly greater in the propranolol group than in the control group, whereas the basal and stimulated cGMP content of the kidney was similar in the two groups. Inhibition of endogenous ANP action by a specific ANP receptor antagonist, HS-142-1, produced a greater increase of blood pressure in the propranolol group than in the control group. These results suggest potentiation of natriuretic peptide activity as a new antihypertensive mechanism of the beta-adrenoceptor antagonist propranolol.