Heteroplasmic mitochondrial DNA variants in cardiovascular diseases

Mitochondria are implicated in the pathogenesis of cardiovascular diseases (CVDs) but the reasons for this are not well understood. Maternally-inherited population variants of mitochondrial DNA (mtDNA) which affect all mtDNA molecules (homoplasmic) are associated with cardiometabolic traits and the risk of developing cardiovascular disease. However, it is not known whether mtDNA mutations only affecting a proportion of mtDNA molecules (heteroplasmic) also play a role. To address this question, we performed a high-depth (~1000-fold) mtDNA sequencing of blood DNA in 1,399 individuals with hypertension (HTN), 1,946 with ischemic heart disease (IHD), 2,146 with ischemic stroke (IS), and 723 healthy controls. We show that the per individual burden of heteroplasmic single nucleotide variants (mtSNVs) increases with age. The age-effect was stronger for low-level heteroplasmies (heteroplasmic fraction, HF, 5–10%), likely reflecting acquired somatic events based on trinucleotide mutational signatures. After correcting for age and other confounders, intermediate heteroplasmies (HF 10–95%) were more common in hypertension, particularly involving non-synonymous variants altering the amino acid sequence of essential respiratory chain proteins. These findings raise the possibility that heteroplasmic mtSNVs play a role in the pathophysiology of hypertension.     

Decreased Levels of BAG3 in a Family With a Rare Variant and in Idiopathic Dilated Cardiomyopathy

The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole‐exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10‐nucleotide deletion in the BCL2‐associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C‐terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end‐stage HF undergoing cardiac transplantation when compared with non‐failing controls. Diminished levels of BAG3 protein may be associated with both familial and non‐familial forms of dilated cardiomyopathy. J. Cell. Physiol. 229: 1697–1702, 2014. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Neither cardiac mitochondrial DNA variation nor copy number contribute to congenital heart disease risk

The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p = 6.8E?21), which were lower than in ventricle tissue (1,340 ± 280, p = 1.4E?4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean = 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p = 2.6E?3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r = 0.975) and RNA VAF (r = 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.     

The use constructed wetlands with horizontal sub-surface flow for various types of wastewater

Constructed wetlands with horizontal sub-surface flow (HF CWs) have been used for wastewater treatment for more than 30 years. Most HF CWs have been designed to treat municipal or domestic wastewater. Nowadays, municipal HF CWs focus not only on common pollutants but also on special parameters such as pharmaceuticals, endocrine disruptive chemicals or linear alkylbenzensulfonates (LAS). At present, HF CWs are used to treat many other types of wastewater. Industrial applications include wastewaters from oil refineries, chemical factories, pulp and paper production, tannery and textile industries, abattoir, distillery and winery industries. In particular, the use of HF CWs is becoming very common for treatment of food-processing wastewaters (e.g., production and processing of milk, cheese, potatoes, sugar). HF constructed wetlands are also successfully used to treat wastewaters from agriculture (e.g., pig and dairy farms, fish farm effluents) and various runoff waters (agriculture, airports, highway, greenhouses, plant nurseries). HF CWs have also effectively been used to treat landfill leachate. Besides the use as a single unit, HF CWs are also used in combination with other types of constructed wetlands in hybrid systems.     

Association of bovine β‐casein protein variant I with milk production and milk protein composition

The aim of this study was to detect new polymorphisms in the bovine β‐casein (β‐CN) gene and to evaluate association of (new) β‐CN protein variants with milk production traits and milk protein composition. Screening of the β‐CN gene in genomic DNA from 72 Holstein Friesian (HF) bulls resulted in detection of 19 polymorphisms and revealed the presence of β‐CN protein variant I in the Dutch HF population. Studies of association of β‐CN protein variants with milk composition usually do not discriminate protein variant I from variant A2. Association of β‐CN protein variants with milk composition was studied in 1857 first‐lactation HF cows and showed that associations of protein variants A2 and I were quite different for several traits. β‐CN protein variant I was significantly associated with protein percentage and protein yield, and with α_s1‐casein (α_s1‐CN), α_s2‐casein (α_s2‐CN), κ‐casein (κ‐CN), α‐lactalbumin (α‐LA), β‐lactoglobulin (β‐LG), casein index and casein yield. Inferring β‐κ‐CN haplotypes showed that β‐CN protein variant I occurred only with κ‐CN variant B. Consequently, associations of β‐κ‐CN haplotype IB with protein percentage, κ‐CN, α‐LA, β‐LG and casein index are likely resulting from associations of κ‐CN protein variant B, while associations of β‐κ‐CN haplotype IB with α_s1‐CN and α_s2‐CN seem to be resulting from associations of β‐CN variant I.     

Genetic polymorphism of human factor H (beta 1H globulin)

Polyacrylamide gel isoelectric focusing (PAGIEF) of EDTA plasma and neuraminidase-treated plasma samples at pH 3.5-9.5 containing 8.0 M urea followed by an electroblotting with enzyme immunoassay was applied for the detection of factor H (HF) phenotypes in 536 unrelated Japanese blood donors living in Tokyo. In the major cathodal components, phenotypes of HF were classified into three common and five rare patterns, and these were considered to be controlled by two common and two rare alleles. The data suggest that the HF*Q0 allele also exists in the Japanese population. Family studies confirm the hypothesis that the HF polymorphism is controlled by autosomal codominant Mendelian inheritance.     

Genetics of heart failure

Heart failure (HF) occurs when the cardiac output, no longer compensated by endogenous mechanisms, fails to meet the metabolic demands of the body. In most populations, the prevalence of heart failure continues to rise, constituting a major public health burden, especially in developed countries. There is some evidence that the risk of HF in the general population depends on genetic predisposition, necessarily characterised by a very complex architecture. In a small, but probably underestimated proportion, HF is caused by Mendelian inherited forms of myocardial disease. The genetic background of these genetic conditions is a matter of intensive research that is already shedding light onto the genetics of common sporadic forms of HF. In this review, we briefly review the insights provided by candidate gene and genome-wide association approaches in common HF and then describe the main genetic causes of inherited heart muscle disease. Finally we present the current challenges and future research needs for both forms of HF. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.     

Cross-reacting and specific antigenic components in cystic fluid from metacestodes of Echinococcus granulosus and Taenia solium

Sera from confirmed patients of 5 hydatidosis, 67 neurocysticercosis and 89 other parasitic diseases were tested for specific antibody (IgG) levels by ELISA to cystic fluid antigens from metacestodes of Echinococcus granulosus (HF) and Taenia solium (CF). All hydatidosis sera reacted positively to both HF and CF while neurocysticercosis sera did in 49.3% to HF and 85.1% to CF. The frequencies of cross-reactions were lower in other parasitic diseases to both antigens. By SDS-PAGE, protein bands of 64, 35, 22 and 7 kilodaltons (kDa) were found common in HF and CF. SDS-PAGE/immunoblot exhibited that hydatidosis sera reacted crossly to CF at 135, 110, 100, 86, 64, 45, 39, 35 and 24 kDa bands while neurocysticercosis sera did to HF at 135, 100, 86, 64, 52, 39, 35, 29 and 24 kDa bands. These results indicated that protein bands of 135, 100, 86, 64, 39, 35 and 24 kDa were major common components in HF and CF. Protein bands of 7 kDa in HF and 15, 10 and 7 kDa in CF did not react crossly and were specific components in respective antigens.     

Systematic identification of common functional modules related to heart failure with different etiologies

The development of heart failure (HF) is a complex process that can be initiated by multiple etiologies. Identifying common functional modules associated with HF is a challenging task. Here, we developed a systems method to identify these common functional modules by integrating multiple expression profiles, protein interactions from four species, gene function annotations, and text information. We identified 1439 consistently differentially expressed genes (CDEGs) across HF with different etiologies by applying three meta-analysis methods to multiple HF-related expression profiles. Using a weighted human interaction network constructed by combining interaction data from multiple species, we extracted 60 candidate CDEG modules. We further evaluated the functional relevance of each module by using expression, interaction network, functional annotations, and text information together. Finally, five functional modules with significant biological relevance were identified. We found that almost half of the genes in these modules are hubs in the weighted network, and that these modules can accurately classify HF patients from healthy subjects. We also identified many significantly enriched biological processes that contribute to the pathophysiology of HF, including two new ones, RNA splicing and vesicle-mediated protein transport. In summary, we proposed a novel framework to analyze common functional modules related to HF with different etiologies. Our findings provide important insights into the complex mechanism of HF. Further biological experimentations should be required to validate these novel biological processes.     

Polymorphism of HF (beta 1H-globulin) in three Asian populations (Bangladeshis,Tibetans and Indonesians)

The polymorphism of HF (beta 1H-globulin) was investigated in three Asian populations (Bangladeshis, Tibetans and Indonesians) by means of isoelectric focusing and immunoblotting. Phenotypes associated with three common alleles (HF*A, HF*B and HF*Q0) and a rare allele HF*A1 were identified. The observed numbers of phenotypes were in accordance with the numbers expected under the Hardy-Weinberg equilibrium. HF*A1 seems to be a unique allele of the East-Asian Mongoloids including Tibetans and Indonesians.     

Role of ion channels in heart failure and channelopathies

Heart failure (HF) is a complication of multiple cardiac diseases and is characterized by impaired contractile and electric function. Patients with HF are not only limited by reduced contractile function but are also prone to life-threatening ventricular arrhythmias. HF itself leads to remodeling of ion channels, gap junctions, and intracellular calcium handling abnormalities that in combination with structural remodeling, e.g., fibrosis, produce a substrate for an arrhythmogenic disorders. Not only ventricular life-threatening arrhythmias contribute to increased morbidity and mortality but also atrial arrhythmias, especially atrial fibrillation (AF), are common in HF patients and contribute to morbidity and mortality. The distinct ion channel remodeling processes in HF and in channelopathies associated with HF will be discussed. Further basic research and clinical studies are needed to identify underlying molecular pathways of HF pathophysiology to provide the basis for improved patient care and individualized therapy based on individualized ion channel composition and remodeling.     

Family-Based Association Test Using Both Common and Rare Variants and Accounting for Directions of Effects for Sequencing Data

Current family-based association tests for sequencing data were mainly developed for identifying rare variants associated with a complex disease. As the disease can be influenced by the joint effects of common and rare variants, common variants with modest effects may not be identified by the methods focusing on rare variants. Moreover, variants can have risk, neutral, or protective effects. Association tests that can effectively select groups of common and rare variants that are likely to be causal and consider the directions of effects have become important. We developed the Ordered Subset - Variable Threshold - Pedigree Disequilibrium Test (OVPDT), a combination of three algorithms, for association analysis in family sequencing data. The ordered subset algorithm is used to select a subset of common variants based on their relative risks, calculated using only parental mating types. The variable threshold algorithm is used to search for an optimal allele frequency threshold such that rare variants below the threshold are more likely to be causal. The PDT statistics from both rare and common variants selected by the two algorithms are combined as the OVPDT statistic. A permutation procedure is used in OVPDT to calculate the p-value. We used simulations to demonstrate that OVPDT has the correct type I error rates under different scenarios and compared the power of OVPDT with two other family-based association tests. The results suggested that OVPDT can have more power than the other tests if both common and rare variants have effects on the disease in a region.     

Human heart failure is accompanied by altered protein kinase A subunit expression and post-translational state

β-Adrenergic receptor blockade reduces total mortality and all-cause hospitalizations in patients with heart failure (HF). Nonetheless, β-blockade does not halt disease progression, suggesting that cAMP-dependent protein kinase (PKA) signaling downstream of β-adrenergic receptor activation may persist through unique post-translational states. In this study, human myocardial tissue was used to examine the state of PKA subunits. As expected, total myosin binding protein-C phosphorylation and Ser23/24 troponin I phosphorylation significantly decreased in HF. Examination of PKA subunits demonstrated no change in type II regulatory (RIIα) or catalytic (Cα) subunit expression, although site specific RIIα (Ser96) and Cα (Thr197) phosphorylation were increased in HF. Further, the expression of type I regulatory subunit (RI) was increased in HF. Isoelectric focusing of RIα demonstrated up to three variants, consistent with reports that Ser77 and Ser83 are in vivo phosphorylation sites. Western blots with site-specific monoclonal antibodies showed increased Ser83 phosphorylation in HF. 8-fluo-cAMP binding by wild type and phosphomimic Ser77 and Ser83 mutant RIα proteins demonstrated reduced Kd for the double mutant as compared to WT RIα. Therefore, failing myocardium displays altered expression and post-translational modification of PKA subunits that may impact downstream signaling.     

Sequence Kernel Association Tests for the Combined Effect of Rare and Common Variants

Recent developments in sequencing technologies have made it possible to uncover both rare and common genetic variants. Genome-wide association studies (GWASs) can test for the effect of common variants, whereas sequence-based association studies can evaluate the cumulative effect of both rare and common variants on disease risk. Many groupwise association tests, including burden tests and variance-component tests, have been proposed for this purpose. Although such tests do not exclude common variants from their evaluation, they focus mostly on testing the effect of rare variants by upweighting rare-variant effects and downweighting common-variant effects and can therefore lose substantial power when both rare and common genetic variants in a region influence trait susceptibility. There is increasing evidence that the allelic spectrum of risk variants at a given locus might include novel, rare, low-frequency, and common genetic variants. Here, we introduce several sequence kernel association tests to evaluate the cumulative effect of rare and common variants. The proposed tests are computationally efficient and are applicable to both binary and continuous traits. Furthermore, they can readily combine GWAS and whole-exome-sequencing data on the same individuals, when available, and are also applicable to deep-resequencing data of GWAS loci. We evaluate these tests on data simulated under comprehensive scenarios and show that compared with the most commonly used tests, including the burden and variance-component tests, they can achieve substantial increases in power. We next show applications to sequencing studies for Crohn disease and autism spectrum disorders. The proposed tests have been incorporated into the software package SKAT.     

Cas9HF1 enhanced specificity in Ustilago maydis

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.     

Methods for detecting associations with rare variants for common diseases: application to analysis of sequence data

Although whole-genome association studies using tagSNPs are a powerful approach for detecting common variants, they are underpowered for detecting associations with rare variants. Recent studies have demonstrated that common diseases can be due to functional variants with a wide spectrum of allele frequencies, ranging from rare to common. An effective way to identify rare variants is through direct sequencing. The development of cost-effective sequencing technologies enables association studies to use sequence data from candidate genes and, in the future, from the entire genome. Although methods used for analysis of common variants are applicable to sequence data, their performance might not be optimal. In this study, it is shown that the collapsing method, which involves collapsing genotypes across variants and applying a univariate test, is powerful for analyzing rare variants, whereas multivariate analysis is robust against inclusion of noncausal variants. Both methods are superior to analyzing each variant individually with univariate tests. In order to unify the advantages of both collapsing and multiple-marker tests, we developed the Combined Multivariate and Collapsing (CMC) method and demonstrated that the CMC method is both powerful and robust. The CMC method can be applied to either candidate-gene or whole-genome sequence data.     

Mapping rare and common causal alleles for complex human diseases

Advances in genotyping and sequencing technologies have revolutionized the genetics of complex disease by locating rare and common variants that influence an individual's risk for diseases, such as diabetes, cancers, and psychiatric disorders. However, to capitalize on these data for prevention and therapies requires the identification of causal alleles and a mechanistic understanding for how these variants contribute to the disease. After discussing the strategies currently used to map variants for complex diseases, this Primer explores how variants may be prioritized for follow-up functional studies and the challenges and approaches for assessing the contributions of rare and common variants to disease phenotypes.     

Pathophysiology of heart failure and frailty: a common inflammatory origin?

Frailty, a clinical syndrome that typically occurs in older adults, implies a reduced ability to tolerate biological stressors. Frailty accompanies many age‐related diseases but can also occur without overt evidence of end‐organ disease. The condition is associated with circulating inflammatory cytokines and sarcopenia, features that are shared with heart failure (HF). However, the biological underpinnings of frailty remain unclear and the interaction with HF is complex. Here, we describe the inflammatory pathophysiology that is associated with frailty and speculate that the inflammation that occurs with frailty shares common origins with HF. We discuss the limitations in investigating the pathophysiology of frailty due to few relevant experimental models. Leveraging current therapies for advanced HF and current known therapies to address frailty in humans may enable translational studies to better understand the inflammatory interactions between frailty and HF.