Contribution of Genetic Background, Traditional Risk Factors, and HIV-Related Factors to Coronary Artery Disease Events in HIV-Positive Persons

Background Persons infected with human immunodeficiency virus (HIV) have increased rates of coronary artery disease (CAD). The relative contribution of genetic background, HIV-related factors, antiretroviral medications, and traditional risk factors to CAD has not been fully evaluated in the setting of HIV infection. Methods In the general population, 23 common single-nucleotide polymorphisms (SNPs) were shown to be associated with CAD through genome-wide association analysis. Using the Metabochip, we genotyped 1875 HIV-positive, white individuals enrolled in 24 HIV observational studies, including 571 participants with a first CAD event during the 9-year study period and 1304 controls matched on sex and cohort. Results A genetic risk score built from 23 CAD-associated SNPs contributed significantly to CAD (P = 2.9×10−4). In the final multivariable model, participants with an unfavorable genetic background (top genetic score quartile) had a CAD odds ratio (OR) of 1.47 (95% confidence interval [CI], 1.05–2.04). This effect was similar to hypertension (OR = 1.36; 95% CI, 1.06–1.73), hypercholesterolemia (OR = 1.51; 95% CI, 1.16–1.96), diabetes (OR = 1.66; 95% CI, 1.10–2.49), ≥1 year lopinavir exposure (OR = 1.36; 95% CI, 1.06–1.73), and current abacavir treatment (OR = 1.56; 95% CI, 1.17–2.07). The effect of the genetic risk score was additive to the effect of nongenetic CAD risk factors, and did not change after adjustment for family history of CAD. Conclusions In the setting of HIV infection, the effect of an unfavorable genetic background was similar to traditional CAD risk factors and certain adverse antiretroviral exposures. Genetic testing may provide prognostic information complementary to family history of CAD.

Antipredator defences of young are independently determined by genetic inheritance, maternal effects and own early experience in mouthbrooding cichlids.

1. Predation is a prime force of natural selection. Vulnerability to predation is typically highest early in life, hence effective antipredator defences should work already shortly after birth. Such early defences may be innate, transmitted through non-genetic parental effects or acquired by own early experience. 2. To understand potential joint effects of these sources of antipredator defences on pheno- typic expression, they should be manipulated within the same experiment. We investigated innate, parental and individual experience effects within a single experiment. Females of the African cichlid Simochromis pleurospilus were exposed to the offspring predator Ctenochromis horei or a benign species until spawning. Eggs and larvae were hand-reared, and larvae were then exposed to odour cues signalling the presence or absence of predators in a split-brood design. 3. Shortly after independence of maternal care, S. pleurospilus undergo a habitat shift from a deeper, adult habitat to a shallow juvenile habitat, a phase where young are thought to be par- ticularly exposed to predation risk. Thus, maternal effects induced by offspring predators pres- ent in the adult habitat should take effect mainly shortly after independence, whereas own experience and innate antipredator responses should shape behaviour and life history of S. pleurospilus during the later juvenile period. 4. We found that the manipulated environmental components independently affected different offspring traits. (i) Offspring of predator-exposed mothers grew faster during the first month of life and were thus larger at termination of maternal care, when the young migrate from the adult to the juvenile habitat. (ii) The offspring’s own experience shortly after hatching exerted lasting effects on predator avoidance behaviour. (iii) Finally, our results suggest that S. pleuro- spilus possess a genetically inherited ability to distinguish dangerous from benign species. 5. In S. pleurospilus, maternal effects were limited to a short but critical time window, when young undergo a niche shift. Instead, own environmental sampling of predation risk combined with an innate predisposition to correctly identify predators appears to prepare the young best for the environment, in which they grow up as juveniles.

Genetic variations in complement factors in patients with congenital thrombotic thrombocytopenic purpura with renal insufficiency.

The congenital form of thrombotic thrombocytopenic purpura (TTP) is caused by genetic mutations in ADAMTS13. Some, but not all, congenital TTP patients manifest renal insufficiency in addition to microangiopathic hemolysis and thrombocytopenia. We included 32 congenital TTP patients in the present study, which was designed to assess whether congenital TTP patients with renal insufficiency have predisposing mutations in complement regulatory genes, as found in many patients with atypical hemolytic uremic syndrome (aHUS). In 13 patients with severe renal insufficiency, six candidate complement or complement regulatory genes were sequenced and 11 missense mutations were identified. One of these missense mutations, C3:p.K155Q mutation, is a rare mutation located in the macroglobulin-like 2 domain of C3, where other mutations predisposing for aHUS cluster. Several of the common missense mutations identified in our study have been reported to increase disease-risk for aHUS, but were not more common in patients with as compared to those without renal insufficiency. Taken together, our results show that the majority of the congenital TTP patients with renal insufficiency studied do not carry rare genetic mutations in complement or complement regulatory genes.

A multifactorial analysis of obesity as CVD risk factor: use of neural network based methods in a nutrigenetics context

Obesity is a multifactorial trait, which comprises an independent risk factor for cardiovascular disease (CVD). The aim of the current work is to study the complex etiology beneath obesity and identify genetic variations and/or factors related to nutrition that contribute to its variability. To this end, a set of more than 2300 white subjects who participated in a nutrigenetics study was used. For each subject a total of 63 factors describing genetic variants related to CVD (24 in total), gender, and nutrition (38 in total), e.g. average daily intake in calories and cholesterol, were measured. Each subject was categorized according to body mass index (BMI) as normal (BMI ≤ 25) or overweight (BMI > 25). Two artificial neural network (ANN) based methods were designed and used towards the analysis of the available data. These corresponded to i) a multi-layer feed-forward ANN combined with a parameter decreasing method (PDM-ANN), and ii) a multi-layer feed-forward ANN trained by a hybrid method (GA-ANN) which combines genetic algorithms and the popular back-propagation training algorithm.

The genetic basis of craniofacial and dental abnormalities

The embryonic head development, including the formation of dental structures, is a complex and delicate process guided by specific genetic programs. Genetic changes and environmental factors can disturb the execution of these programs and result in abnormalities in orofacial and dental structures. Orofacial clefts and hypodontia/ oligodontia are examples of such abnormalities frequently seen in dental clinics. An insight into the mechanisms and genes involved in the formation of orofacial and dental structures has been gradually gained by genetic analysis of families and by the use of experimental vertebrate models such as the mouse and chick models. The development of novel clinical therapies for orofacial and dental pathological conditions depends very much on a detailed knowledge of the molecular and cellular processes that are involved in head formation.

Cardiac channelopathies: genetic and molecular mechanisms

Channelopathies are diseases caused by dysfunctional ion channels, due to either genetic or acquired pathological factors. Inherited cardiac arrhythmic syndromes are among the most studied human disorders involving ion channels. Since seminal observations made in 1995, thousands of mutations have been found in many of the different genes that code for cardiac ion channel subunits and proteins that regulate the cardiac ion channels. The main phenotypes observed in patients carrying these mutations are congenital long QT syndrome (LQTS), Brugada syndrome (BrS), catecholaminergic polymorphic ventricular tachycardia (CPVT), short QT syndrome (SQTS) and variable types of conduction defects (CD). The goal of this review is to present an update of the main genetic and molecular mechanisms, as well as the associated phenotypes of cardiac channelopathies as of 2012.

Recommendations for Genetic Testing to Reduce the Incidence of Anthracycline-induced Cardiotoxicity

AIM Anthracycline-induced cardiotoxicity (ACT) occurs in 57% of treated patients and remains an important limitation of anthracycline-based chemotherapy. In various genetic association studies, potential genetic risk markers for ACT have been identified. Therefore, we developed evidence-based clinical practice recommendations for pharmacogenomic testing to further individualize therapy based on ACT risk. METHODS We followed a standard guideline development process; including a systematic literature search, evidence synthesis and critical appraisal, and the development of clinical practice recommendations with an international expert group. RESULTS RARG rs2229774, SLC28A3 rs7853758 and UGT1A6 rs17863783 variants currently have the strongest and the most consistent evidence for association with ACT. Genetic variants in ABCC1, ABCC2, ABCC5, ABCB1, ABCB4, CBR3, RAC2, NCF4, CYBA, GSTP1, CAT, SULT2B1, POR, HAS3, SLC22A7, SCL22A17, HFE and NOS3 have also been associated with ACT, but require additional validation. We recommend pharmacogenomic testing for the RARG rs2229774 (S427L), SLC28A3 rs7853758 (L461L) and UGT1A6*4 rs17863783 (V209V) variants in childhood cancer patients with an indication for doxorubicin or daunorubicin therapy (Level B - moderate). Based on an overall risk stratification, taking into account genetic and clinical risk factors, we recommend a number of management options including increased frequency of echocardiogram monitoring, follow-up, as well as therapeutic options within the current standard of clinical practice. CONCLUSIONS Existing evidence demonstrates that genetic factors have the potential to improve the discrimination between individuals at higher and lower risk of ACT. Genetic testing may therefore support both patient care decisions and evidence development for an improved prevention of ACT.

Risk of Cerebral Venous Thrombosis in Obese Women.

IMPORTANCE Obesity is a risk factor for deep vein thrombosis of the leg and pulmonary embolism. To date, however, whether obesity is associated with adult cerebral venous thrombosis (CVT) has not been assessed. OBJECTIVE To assess whether obesity is a risk factor for CVT. DESIGN, SETTING, AND PARTICIPANTS A case-control study was performed in consecutive adult patients with CVT admitted from July 1, 2006 (Amsterdam), and October 1, 2009 (Berne), through December 31, 2014, to the Academic Medical Center in Amsterdam, the Netherlands, or Inselspital University Hospital in Berne, Switzerland. The control group was composed of individuals from the control population of the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis study, which was a large Dutch case-control study performed from March 1, 1999, to September 31, 2004, and in which risk factors for deep vein thrombosis and pulmonary embolism were assessed. Data analysis was performed from January 2 to July 12, 2015. MAIN OUTCOMES AND MEASURES Obesity was determined by body mass index (BMI). A BMI of 30 or greater was considered to indicate obesity, and a BMI of 25 to 29.99 was considered to indicate overweight. A multiple imputation procedure was used for missing data. We adjusted for sex, age, history of cancer, ethnicity, smoking status, and oral contraceptive use. Individuals with normal weight (BMI <25) were the reference category. RESULTS The study included 186 cases and 6134 controls. Cases were younger (median age, 40 vs 48 years), more often female (133 [71.5%] vs 3220 [52.5%]), more often used oral contraceptives (97 [72.9%] vs 758 [23.5%] of women), and more frequently had a history of cancer (17 [9.1%] vs 235 [3.8%]) compared with controls. Obesity (BMI ≥30) was associated with an increased risk of CVT (adjusted odds ratio [OR], 2.63; 95% CI, 1.53-4.54). Stratification by sex revealed a strong association between CVT and obesity in women (adjusted OR, 3.50; 95% CI, 2.00-6.14) but not in men (adjusted OR, 1.16; 95% CI, 0.25-5.30). Further stratification revealed that, in women who used oral contraceptives, overweight and obesity were associated with an increased risk of CVT in a dose-dependent manner (BMI 25.0-29.9: adjusted OR, 11.87; 95% CI, 5.94-23.74; BMI ≥30: adjusted OR, 29.26; 95% CI, 13.47-63.60). No association was found in women who did not use oral contraceptives. CONCLUSIONS AND RELEVANCE Obesity is a strong risk factor for CVT in women who use oral contraceptives.

Respiratory symptoms in preterm infants: burden of disease in the first year of life

Objective While respiratory symptoms in the first year of life are relatively well described for term infants, data for preterm infants are scarce. We aimed to describe the burden of respiratory disease in a group of preterm infants with and without bronchopulmonary dysplasia (BPD) and to assess the association of respiratory symptoms with perinatal, genetic and environmental risk factors. Methods Single centre birth cohort study: prospective recording of perinatal risk factors and retrospective assessment of respiratory symptoms during the first year of life by standardised questionnaires. Main outcome measures: Cough and wheeze (common symptoms), re-hospitalisation and need for inhalation therapy (severe outcomes). Patients: 126 preterms (median gestational age 28.7 weeks; 78 with, 48 without BPD) hospitalised at the University Children's Hospital of Bern, Switzerland 1999-2006. Results Cough occurred in 80%, wheeze in 44%, rehospitalisation in 25% and long term inhalation therapy in wheezers in 13% of the preterm infants. Using logistic regression, the main risk factor for common symptoms was frequent contact with other children. Severe outcomes were associated with maximal peak inspiratory pressure, arterial cord blood pH, APGAR and CRIB-Score. Conclusions Cough in preterm infants is as common as in term infants, whereas wheeze, inhalation therapy and re-hospitalisations occur more often. Severe outcomes are associated with perinatal risk factors. Preterm infants who did not qualify for BPD according to latest guidelines also showed a significant burden of respiratory disease in the first year of life.

Increased lipopolysaccharide-induced tumour necrosis factor-alpha, interferon-gamma and interleukin-10 production in atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is based on a genetic predisposition, but environmental factors may trigger skin inflammation. According to the hygiene hypothesis, decreased exposure to microbial products in early childhood does not allow sufficient maturation of the immune system that is associated with an increased risk of atopic sensitization. OBJECTIVES: The effect of lipopolysaccharide (LPS) on the cytokine production of peripheral blood mononuclear cells (PBMC) of AD patients and nonatopic controls was studied. PATIENTS AND METHODS: PBMC were isolated from heparinized blood of 10 patients with AD and 10 nonatopic individuals, suspended in culture medium and stimulated with LPS. Cytokine levels in the supernatants were measured by immunoassays. Results Upon stimulation with LPS, PBMC from AD patients produced significantly higher amounts of tumour necrosis factor-alpha, interferon-gamma and interleukin (IL)-10 compared with control PBMC. LPS stimulation blocked the increased spontaneous production of IL-4 and IL-5 by PBMC from AD patients, but had no effect on IL-13 production. CONCLUSIONS: These results demonstrate that the effects of LPS stimulation depend on both the type of cytokine and the origin of PBMC. Endotoxin exposure is suggested to modulate the disease course of AD.

Nitric oxide induces cell death in canine cruciate ligament cells by activation of tyrosine kinase and reactive oxygen species

BACKGROUND: There is increasing evidence suggesting that development of progressive canine cranial cruciate ligament (CCL) rupture involves a gradual degeneration of the CCL itself, initiated by a combination of factors, ranging from mechanical to biochemical. To date, knowledge is lacking to what extent cruciate disease results from abnormal biomechanics on a normal ligament or contrary how far preliminary alterations of the ligament due to biochemical factors provoke abnormal biomechanics. This study is focused on nitric oxide (NO), one of the potential biochemical factors. The NO-donor sodium nitroprusside (SNP) has been used to study NO-dependent cell death in canine cranial and caudal cruciate ligament cells and to characterize signaling mechanisms during NO-stimulation. RESULTS: Sodium nitroprusside increased apoptotic cell death dose- and time-dependently in cruciate ligamentocytes. Cells from the CCL were more susceptible to apoptosis than CaCL cells. Caspase-3 processing in response to SNP was not detected. Testing major upstream and signal transducing pathways, NO-induced cruciate ligament cell death seemed to be mediated on different levels. Specific inhibition of tyrosine kinase significantly decreased SNP-induced cell death. Mitogen activated protein kinase ERK1 and 2 are activated upon NO and provide anti-apoptotic signals whereas p38 kinase and protein kinase C are not involved. Moreover, data showed that the inhibition reactive oxygen species (ROS) significantly reduced the level of cruciate ligament cell death. CONCLUSIONS: Our data support the hypothesis that canine cruciate ligamentocytes, independently from their origin (CCL or CaCL) follow crucial signaling pathways involved in NO-induced cell death. However, the difference on susceptibility upon NO-mediated apoptosis seems to be dependent on other pathways than on these tested in the present study. In both, CCL and CaCL, the activation of the tyrosine kinase and the generation of ROS reveal important signaling pathways. In perspective, new efforts to prevent the development and progression of cruciate disease may include strategies aimed at reducing ROS.

Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.

BACKGROUND & AIMS Subtle inter-patient genetic variation and environmental factors combine to determine disease progression in non-alcoholic fatty liver disease (NAFLD). Carriage of the PNPLA3 rs738409 c.444C >G minor allele (encoding the I148M variant) has been robustly associated with advanced NAFLD. Although most hepatocellular carcinoma (HCC) is related to chronic viral hepatitis or alcoholic liver disease, the incidence of NAFLD-related HCC is increasing. We examined whether rs738409 C >G was associated with HCC-risk in patients with NAFLD. METHODS PNPLA3 rs738409 genotype was determined by allelic discrimination in 100 European Caucasians with NAFLD-related HCC and 275 controls with histologically characterised NAFLD. RESULTS Genotype frequencies were significantly different between NAFLD-HCC cases (CC=28, CG=43, GG=29) and NAFLD-controls (CC=125, CG=117, GG=33) (p=0.0001). In multivariate analysis adjusted for age, gender, diabetes, BMI, and presence of cirrhosis, carriage of each copy of the rs738409 minor (G) allele conferred an additive risk for HCC (adjusted OR 2.26 [95% CI 1.23-4.14], p=0.0082), with GG homozygotes exhibiting a 5-fold [1.47-17.29], p=0.01 increased risk over CC. When compared to the UK general population (1958 British Birth Cohort, n=1476), the risk-effect was more pronounced (GC vs. CC: unadjusted OR 2.52 [1.55-4.10], p=0.0002; GG vs. CC: OR 12.19 [6.89-21.58], p<0.0001). CONCLUSIONS Carriage of the PNPLA3 rs738409 C >G polymorphism is not only associated with greater risk of progressive steatohepatitis and fibrosis but also of HCC. If validated, these findings suggest that PNPLA3 genotyping has the potential to contribute to multi-factorial patient-risk stratification, identifying those to whom HCC surveillance may be targeted.

TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition, strongly associated with the metabolic syndrome, that can lead to progressive hepatic fibrosis, cirrhosis and hepatic failure. Subtle inter-patient genetic variation and environmental factors combine to determine variation in disease progression. A common non-synonymous polymorphism in TM6SF2 (rs58542926 c.449 C>T, p.Glu167Lys) was recently associated with increased hepatic triglyceride content, but whether this variant promotes clinically relevant hepatic fibrosis is unknown. Here we confirm that TM6SF2 minor allele carriage is associated with NAFLD and is causally related to a previously reported chromosome 19 GWAS signal that was ascribed to the gene NCAN. Furthermore, using two histologically characterized cohorts encompassing steatosis, steatohepatitis, fibrosis and cirrhosis (combined n=1,074), we demonstrate a new association, independent of potential confounding factors (age, BMI, type 2 diabetes mellitus and PNPLA3 rs738409 genotype), with advanced hepatic fibrosis/cirrhosis. These findings establish new and important clinical relevance to TM6SF2 in NAFLD.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.