Redox-proteomics involves the large scale analysis of oxidative protein post-translational modifications. In particular, cysteine residues have become the subject of intensifying research interest because of their redox-reactive thiol side chain. Certain reactive cysteine residues can function as redox-switches, which sense changes in the local redox-environment by flipping between the reduced and oxidized state. Depending on the reactive oxygen or nitrogen species, cysteine residues can receive one of several oxidative modifications, each with the potential to confer a functional effect. Modification of these redox-switches has been found to play an important role in oxidative-signaling in the cardiovascular system and elsewhere. Due to the labile and dynamic nature of these modifications, several targeted approaches have been developed to enrich, identify and characterize the status of these critical residues. Here, we review the various proteomic strategies and limitations for the large scale analysis of the different oxidative cysteine modifications.
redox-proteomics; mass spectrometry; biotin switch assay; cysteine modification
Exome sequencing is a recently implemented method to discover rare mutations for Mendelian disorders. Less is known about its feasibility to identify genes for complex traits. We used exome sequencing to search for rare variants responsible for a complex trait, low levels of serum high-density lipoprotein cholesterol (HDL-C).
Methods and Results
We conducted exome sequencing in a large French Canadian family with 75 subjects available for study of which 27 had HDL-C values < the 5th age-sex specific population percentile. We captured ~50 Mb of exonic and transcribed sequences of three closely related family members with HDL-C levels <5th age-sex percentiles and sequenced the captured DNA. Approximately 82,000 variants were detected in each individual of which 41 rare non-synonymous variants were shared by the sequenced affected individuals after filtering steps. Two rare non-synonymous variants in the ATP-binding cassette, sub-family A (ABC1), member 1 (ABCA1) and lipoprotein lipase (LPL) genes predicted to be damaging were investigated for co-segregation with the low HDL-C trait in the entire extended family. The carriers of either variant had low HDL-C levels and the individuals carrying both variants had the lowest HDL-C values. Interestingly, the ABCA1 variant exhibited a sex effect which was first functionally identified, and subsequently, statistically demonstrated using additional French Canadian families with ABCA1 mutations.
This complex combination of two rare variants causing low HDL-C in the extended family would not have been identified using traditional linkage analysis, emphasizing the need for exome sequencing of complex lipid traits in unexplained familial cases.
genetics; HDL cholesterol; Exome sequencing; Rare variants
Hundreds of non-synonymous single nucleotide variants (nsSNVs) have been identified in the two most common LQTS-susceptibility genes (KCNQ1 and KCNH2). Unfortunately, a ~3% background rate of rare KCNQ1 and KCNH2 nsSNVs amongst healthy individuals complicates the ability to distinguish rare pathogenic mutations from similarly rare yet presumably innocuous variants.
Methods and Results
In this study, 4 tools [1) conservation across species, 2) Grantham values, 3) SIFT, and 4) PolyPhen] were used to predict “pathogenic” or “benign” status for nsSNVs identified across 388 clinically “definite” LQTS cases and 1344 ostensibly healthy controls. From these data, estimated predictive values (EPVs) were determined for each tool independently, in concert with previously published protein topology-derived EPVs, and synergistically when ≥ 3 tools were in agreement. Overall, all 4 tools displayed a statistically significant ability to distinguish between case-derived and control-derived nsSNVs in KCNQ1, whereas each tool, except Grantham values, displayed a similar ability to differentiate KCNH2 nsSNVs. Collectively, when at least 3 of the 4 tools agreed on the “pathogenic” status of C-terminal nsSNVs located outside the KCNH2/Kv11.1 cyclic nucleotide binding domain, the topology-specific EPV improved from 56% to 91%.
While in silico prediction tools should not be used to predict independently the pathogenicity of a novel, rare nSNV, our results support the potential clinical utility of the synergistic use of these tools to enhance the classification of nsSNVs, particularly for Kv11.1’s difficult to interpret C-terminal region.
genetic variation; genetics; ion channels; long-QT syndrome; pediatrics
Data regarding the familial aggregation of left ventricular (LV) geometry and its relations to parental heart failure (HF) are limited.
Methods and Results
We evaluated concordance of LV geometry within 1093 nuclear families in 5758 participants of the Original (parents; N=2351) and Offspring (N=3407) cohorts of the Framingham Heart Study undergoing routine echocardiography in mid-to-late adulthood. LV geometry was categorized based on cohort- and sex-specific 80th percentile cutoffs of LV mass and relative wall thickness (RWT) into normal (both <80th percentile), concentric remodeling (LV mass<80th percentile, RWT>80th percentile), concentric hypertrophy (both >80th percentile) and eccentric hypertrophy (LV mass>80th percentile, RWT<80th percentile). Within nuclear families, LV geometry was concordant among related pairs (parent-child, sibling-sibling) (P=0.0015), but not among unrelated spousal pairs (P=0.60), a finding that remained unchanged after adjusting for clinical covariates known to influence LV remodeling (age, systolic blood pressure, body mass index), excluding individuals with prevalent HF and myocardial infarction, and varying the thresholds for defining LV geometry. The prevalence of abnormal LV geometry was higher in family members of affected individuals, with recurrence risks of 1.4 for concentric remodeling (95%CI, 1.2–1.7) and eccentric hypertrophy (95%CI, 1.1–1.8), and 3.9 (95%CI, 3.2–4.6) for concentric hypertrophy. In a subset of 1497 offspring, we observed an association between parental HF (N=458) and eccentric hypertrophy in offspring (P<0.0001).
Our investigation of a two-generational community-based sample demonstrates familial aggregation of LV geometry, with the greatest recurrence risk for concentric LV geometry, and establishes an association of eccentric LV geometry with parental HF.
echocardiography; remodeling; risk factors
Thoracic aortic aneurysms leading to acute aortic dissections (TAAD) are the major diseases that affect the thoracic aorta. Approximately 20% of patients with TAAD have a family history of TAAD, and these patients present younger with more rapidly enlarging aneurysms than patients without a family history of aortic disease.
Methods and Results
A large family with multiple members with TAAD inherited in an autosomal dominant manner was identified. The ascending aortic aneurysms were associated with slow enlargement, a low risk of dissection, and decreased penetrance in women. Genome-wide linkage analysis was performed and a novel locus on chromosome 12 was identified for the mutant gene causing disease in this family. Of the 12 male members who carry the disease-linked microsatellite haplotype, nine had ascending aortic aneurysms with an average diameter of 4.7 cm and average age of 55 years (age range, 32-76) at the time of diagnosis; only one individual had progressed to acute aortic dissection and no other members with aortic dissections were identified. Women harboring the disease-linked haplotype did not have thoracic aortic disease, including an 84 year old woman. Sequencing of 9 genes within the critical interval at the chromosome 12 locus did not identify the mutant gene.
Mapping a locus for ascending thoracic aortic aneurysms associated with a low risk of aortic dissection supports our hypothesis that genes leading to familial disease can be associated with less aggressive thoracic aortic disease.
acute aortic dissection genes; aneurysm; genome-wide analysis; mapping; risk prediction
Fatty acids provide energy and structural substrates for the heart and brain and may influence resuscitation from sudden cardiac arrest (SCA). We investigated whether genetic variation in fatty acid metabolism pathways was associated with SCA survival.
Methods and Results
Subjects (mean age 67, 80% male, Caucasian) were out-of-hospital SCA patients found in ventricular fibrillation in King County, WA. We compared subjects who survived to hospital admission (n=664) with those who did not (n=689), and subjects who survived to hospital discharge (n=334) with those who did not (n=1019). Associations between survival and genetic variants were assessed using logistic regression adjusting for age, gender, location, time to arrival of paramedics, whether the event was witnessed, and receipt of bystander CPR. Within-gene permutation tests were used to correct for multiple comparisons. Variants in five genes were significantly associated with SCA survival. After correction for multiple comparisons, SNPs in ACSL1 and ACSL3 were significantly associated with survival to hospital admission. SNPs in ACSL3, AGPAT3, MLYCD, and SLC27A6 were significantly associated with survival to hospital discharge.
Our findings indicate that variants in genes important in fatty acid metabolism are associated with SCA survival in this population.
epidemiology; fatty acids; genetics; heart arrest
Genome-wide association studies have identified multiple variants associating with coronary artery disease (CAD) and myocardial infarction (MI). Whether a combined genetic risk score (GRS) is associated with prevalent and incident MI in high risk subjects remains to be established.
Methods and Results
In subjects undergoing cardiac catheterization (n=2597) we identified cases with a history of MI onset at age <70 years and controls ≥70 years old without prior MI, and followed them for incident MI and death. Genotyping was performed for 11 established CAD/MI variants and a GRS calculated based on average number of risk alleles carried at each locus weighted by effect size. Replication of association findings was sought in an independent angiographic cohort (n=2702). The GRS was significantly associated with prevalent MI, occurring before age 70 years, compared to older controls (≥70 years) with no history of MI (p<0.001). This association was successfully replicated in a second cohort yielding a pooled p value of <0.001. The GRS modestly improved the c-statistic in models of prevalent MI with traditional risk factors. However, the association was not statistically significant when elderly controls without MI but with stable angiographic CAD were examined (pooled p=0.11). Finally, during a median 2.5 year follow-up, only a non-significant trend was noted between the GRS and incident events, which was also not significant in the replication cohort.
A GRS of 11 CAD/MI variants is associated with prevalent MI but not near term incident adverse events in two independent angiographic cohorts. These findings have implications for understanding the clinical utility of genetic risk scores for secondary as opposed to primary risk prediction.
cardiovascular disease risk factors; coronary artery disease; myocardial infarction; Genetic Risk Score
Universal principles underlying network science, and their ever-increasing applications in biomedicine, underscore the unprecedented capacity of systems biology based strategies to synthesize and resolve massive high throughput generated datasets. Enabling previously unattainable comprehension of biological complexity, systems approaches have accelerated progress in elucidating disease prediction, progression, and outcome. Applied to the spectrum of states spanning health and disease, network proteomics establishes a collation, integration, and prioritization algorithm to guide mapping and decoding of proteome landscapes from large-scale raw data. Providing unparalleled deconvolution of protein lists into global interactomes, integrative systems proteomics enables objective, multi-modal interpretation at molecular, pathway, and network scales, merging individual molecular components, their plurality of interactions, and functional contributions for systems comprehension. As such, network systems approaches are increasingly exploited for objective interpretation of cardiovascular proteomics studies. Here, we highlight network systems proteomic analysis pipelines for integration and biological interpretation through protein cartography, ontological categorization, pathway and functional enrichment and complex network analysis.
ATP-sensitive K+ channel; bioinformatics; complex network analysis; KATP channel; Kir6.2; genetics; heart disease; metabolism; network biology; proteome; regenerative medicine; SUR2A; stem cells; systems biology
Methods and Results
We developed a novel, integrative method (combining animal models, transcriptomics, bioinformatics, molecular biology, and trait-extreme phenotypes) to identify candidate genes for essential hypertension and the metabolic syndrome. We first undertook transcriptome profiling on adrenal glands from blood pressure extreme mouse strains: the hypertensive BPH and hypotensive BPL. Microarray data clustering revealed a striking pattern of global underexpression of intermediary metabolism transcripts in BPH. The MITRA algorithm identified a conserved motif in the transcriptional regulatory regions of the underexpressed metabolic genes, and we then hypothesized that regulation through this motif contributed to the global underexpression. Luciferase reporter assays demonstrated transcriptional activity of the motif, via transcription factors HOXA3, SRY, and YY1. We finally hypothesized that genetic variation at HOXA3, SRY, and YY1 might predict blood pressure and other metabolic syndrome traits in humans. Tagging variants for each locus were associated with BP in a human population BP extreme sample, with the most extensive associations for YY1 tagging SNP rs11625658, on SBP, DBP, BMI, and fasting glucose. Meta-analysis extended the YY1 results into two additional large population samples, with significant effects preserved on DBP, BMI, and fasting glucose.
The results outline an innovative, systematic approach to the genetic pathogenesis of complex cardiovascular disease traits, and point to transcription factor YY1 as a potential candidate gene involved in essential hypertension and the cardio-metabolic syndrome.
BPH mouse strain; complex trait; essential (genetic) hypertension; human genetics; metabolic syndrome
Hypertrophic cardiomyopathy and dilated cardiomyopathy arise from mutations in genes encoding sarcomere proteins including MYH7, MYBPC3, and TTN. Genetic diagnosis of cardiomyopathy relies on complete sequencing of the gene coding regions, and most pathogenic variation is rare. The 1000 Genomes project is an ongoing consortium designed to deliver whole genome sequence information from an ethnically diverse population and therefore is a rich source to determine both common and rare genetic variants.
Methods and Results
We queried the 1000 Genomes database of 1,092 individuals for exonic variants within three sarcomere genes MHY7, MYBPC3, and TTN. We focused our analysis on protein-altering variation, including nonsynonymous single nucleotide polymorphisms, insertion/deletion polymorphisms, or splice site altering variants. We identified known and predicted pathogenic variation in MYBPC3 and MYH7 at a higher frequency than what would be expected based on the known prevalence of cardiomyopathy. We also found substantial variation, including protein-disrupting sequences, in TTN.
Cardiomyopathy is a genetically heterogeneous disorder caused by mutations in multiple genes. The frequency of predicted pathogenic protein altering variation in cardiomyopathy genes suggests that many of these variants may be insufficient to cause disease on their own but may modify phenotype in a genetically susceptible host. This is suggested by the high prevalence of TTN insertion/deletions in the 1000 Genomes cohort. Given the possibility of additional genetic variants that modify the phenotype of a primary driver mutation, broad-based genetic testing should be employed.
cardiomyopathy; hypertrophic cardiomyopathy; myosin heavy chain; myosin-binding subunit; titin
editorial; paraoxonase; clopidogrel; pharmacogenetics; percutaneous coronary intervention
High levels of cardiac troponin T measured by a highly sensitive assay (hs-cTnT) are strongly associated with incident coronary heart disease (CHD) and heart failure (HF). No large-scale genome-wide association study (GWAS) of hs-cTnT has been reported to date. We sought to identify novel genetic variants that are associated with hs-cTnT levels.
Methods and Results
We performed a GWAS in 9,491 European-Americans and 2,053 African-Americans free of CHD and HF from 2 prospective cohorts: the Atherosclerosis Risk in Communities Study (ARIC) and the Cardiovascular Health Study (CHS). GWASs were conducted in each study and race stratum. Fixed-effect meta-analyses combined the results of linear regression from 2 cohorts within each race stratum, and then across race strata to produce overall estimates and p-values. The meta-analysis identified a significant association at chromosome 8q13 (rs10091374, p = 9.06 × 10−9) near the nuclear receptor coactivator 2 (NCOA2) gene. Over-expression of NCOA2 can be detected in myoblasts An additional analysis using logistic regression and the clinically motivated 99th percentile cut-point detected a significant association at 1q32 (rs10091374, p = 9.06 × 10−8) in the gene TNNT2, which encodes the cardiac troponin T protein itself. The hs-cTnT-associated SNPs were not associated with CHD in a large case-control study, but rs12564445 was significantly associated with incident HF in ARIC European-Americans (HR = 1.16, p-value = 0.004).
We identified 2 loci, near NCOA2 and in the TNNT2 gene, at which variation was significantly associated with hs-cTnT levels. Further use of the new assay should enable replication of these results.
genetics; genome-wide association study; troponin
Current clinical diagnosis of long-QT syndrome (LQTS) includes genetic testing of family members of mutation positive patients. The present study was designed to assess the clinical course of individuals who are found negative for the LQTS-causing mutation in their families.
Methods and Results
Multivariate Cox proportional hazards model was used to assess the risk for cardiac events (comprising syncope, aborted cardiac arrest [ACA], or sudden cardiac death [SCD]) from birth through age 40 years among 1828 subjects from the LQTS Registry who were found negative for their family LQTS-causing mutation. The median QTc of study subjects was 423 msec (interquartile-range: 402–442 msec). The cumulative probability of a first syncope through age 40 years was 15%. However, only 2 patients (0.1%) experienced ACA and none died suddenly during follow-up. Independent risk factors for syncope in genotype negative subjects included female gender (HR 1.60, p = 0.002), prolonged QTc (HR = 1.63 per 100 msec increment, p = 0.02), family history of ACA or SCD (HR = 1.89, p = 0.002), and LQT2 vs. LQT1 family mutation (HR = 1.41, p = 0.03). Subgroup analysis showed that the presence of the K897T polymorphism in the LQT2 gene in an affected family was associated with an 11-fold (p = 0.001) increase in the risk of recurrent syncope in genotype negative subjects.
Our findings suggest that cardiac events among genotype-negative family members of LQTS patients are dominated by nonfatal syncopal episodes without occurrence of sudden cardiac death. The risk for nonfatal events in this population may be mediated by the presence of common polymorphisms in LQTS genes.
gene mutation; genetic polymorphisms; long-QT syndrome; sudden cardiac death arrhythmia; syncope
The primary role of natriuretic peptide receptor-3 (NPR3) or NPR-C is in the clearance of natriuretic peptides that play an important role in modulating intravascular volume and vascular tone. Genetic variation in NPR3 has been associated with variation in blood pressure and obesity. Despite the importance of NPR3, sequence variation in the gene has not been addressed using DNA from different ethnic populations. We set out to identify and functionally characterize genetic variation in NPR3 in 3 ethnic groups.
Methods and Results
DNA samples from 96 European American, 96 African American, and 96 Han Chinese American healthy subjects were used to resequence NPR3 exons, splice junctions, and flanking regions. We identified 105 polymorphisms, 50 of which were novel, including 8 nonsynonymous single-nucleotide polymorphisms, 7 were novel. Expression constructs were created for the nonsynonymous single-nucleotide polymorphisms. HEK293 cells were transfected with constructs for wild type and variant allozymes; and recombinant proteins were measured by quantitative Western blot analysis. The most significant change in NPR3 protein was observed for the Arg146 variant allozyme, with 20% of wild-type protein, primarily because of autophagy-dependent degradation. NPR3 structural modeling confirmed that the Arg146 variant protein was not compatible with wild-type conformation and could result in protein misfolding or instability.
Multiple novel NPR3 genetic polymorphisms were identified in 3 ethnic groups. The Arg146 allozyme displayed a significant decrease in protein quantity because of degradation mediated predominantly by autophagy. This genetic variation could have a significant effect on the metabolism of natriuretic peptides with potential clinical implications.
natriuretic peptide receptor-3; natriuretic peptides; pharmacogenetics; polymorphism
Degradation of extracellular matrix support in the large abdominal arteries contribute to abnormal dilation of aorta, leading to abdominal aortic aneurysms, and matrix metalloproteinase-9 (MMP-9) is the predominant enzyme targeting elastin and collagen present in the walls of the abdominal aorta. Previous studies have suggested a potential association between MMP-9 genotype and abdominal aortic aneurysm, but these studies have been limited only to the p-1562 and (CA) dinucleotide repeat microsatellite polymorphisms in the promoter region of the MMP-9 gene. We determined the functional alterations caused by 15 MMP-9 single-nucleotide polymorphisms (SNPs) reported to be relatively abundant in the human genome through Western blots, gelatinase, and promoter–reporter assays and incorporated this information to perform a logistic-regression analysis of MMP-9 SNPs in 336 human abdominal aortic aneurysm cases and controls.
Methods and Results
Significant functional alterations were observed for 6 exon SNPs and 4 promoter SNPs. Genotype analysis of frequency-matched (age, sex, history of hypertension, hypercholesterolemia, and smoking) cases and controls revealed significant genetic heterogeneity exceeding 20% observed for 6 SNPs in our population of mostly white subjects from Northern Wisconsin. A step-wise logistic-regression analysis with 6 functional SNPs, where weakly contributing confounds were eliminated using Akaike information criteria, gave a final 2 SNP (D165N and p-2502) model with an overall odds ratio of 2.45 (95% confidence interval, 1.06–5.70).
The combined approach of direct experimental confirmation of the functional alterations of MMP-9 SNPs and logistic-regression analysis revealed significant association between MMP-9 genotype and abdominal aortic aneurysm.
aneurysm; case-control study; genetic association; MMP; single nucleotide polymorphism genetics
editorial; monocytes; cardiovascular outcomes; inflammation
Heritable and idiopathic pulmonary arterial hypertension (PAH) are phenotypically identical and associated with mutations in several genes related to TGF beta signaling, including bone morphogenetic protein receptor type 2 (BMPR2), activin receptor-like kinase 1 (ALK1), endoglin (ENG), and mothers against decapentaplegic 9 (SMAD9). Approximately 25% of heritable cases lack identifiable mutations in any of these genes.
Methods and Results
We used whole exome sequencing to study a three generation family with multiple affected family members with PAH but no identifiable TGF beta mutation. We identified a frameshift mutation in Caveolin-1 (CAV1), which encodes a membrane protein of caveolae abundant in the endothelium and other cells of the lung. An independent de novo frameshift mutation was identified in a child with idiopathic PAH. Western blot analysis demonstrated a reduction in caveolin-1 protein, while lung tissue immunostaining studies demonstrated a reduction in normal caveolin-1 density within the endothelial cell layer of small arteries.
Our study represents successful elucidation of a dominant Mendelian disorder using whole exome sequencing. Mutations in CAV1 are associated in rare cases with PAH. This may have important implications for pulmonary vascular biology as well as PAH-directed therapeutic development.
bioinfomatics genes; genetics; BMPR2; caveolae; pulmonary hypertension
About half of people with Down syndrome (DS) exhibit some form of congenital heart disease (CHD). However, trisomy for human chromosome 21 (Hsa21) alone is insufficient to cause CHD as half of all people with DS have a normal heart, suggesting that genetic modifiers interact with dosage sensitive gene(s) on Hsa21 to result in CHD. We hypothesize that a threshold exists in both Down syndrome and euploid populations for the number of genetic perturbations that can be tolerated before CHD results.
Methods and Results
We ascertained a group of individuals with DS and complete atrioventricular septal defect (AVSD) and sequenced two candidate genes for CHD, CRELD1, which is associated with AVSD in people with or without DS, and HEY2, whose mouse ortholog produces septal defects when mutated. Several deleterious variants were identified but the frequency of these potential modifiers was low. We crossed mice with mutant forms of these potential modifiers to the Ts65Dn mouse model of Down syndrome. Crossing loss-of-function alleles of either Creld1 or Hey2 onto the trisomic background caused a significant increase in the frequency of CHD, demonstrating an interaction between the modifiers and trisomic genes. We showed further that although either of these mutant modifiers is benign by itself, they interact to affect heart development when inherited together.
Using mouse models of Down syndrome and of genes associated with congenital heart disease we demonstrate a biological basis for an interaction that supports a threshold hypothesis for additive effects of genetic modifiers in the sensitized trisomic population.
congenital heart disease; genetic modifier; Down syndrome
personalized medicine; coronary artery disease; genotyping; SNP; preventive cardiology
A functional polymorphism in the inhibitory IgG-Fc receptor FcγRIIB influences intravenous immunoglobulin (IVIG) response in Kawasaki Disease (KD) a vasculitis preferentially affecting the coronary arteries in children. We tested the hypothesis that the polymorphisms in the activating receptors (Fcγ RIIA, Fcγ RIIIA and Fcγ RIIIB) also influence susceptibility, IVIG treatment response, and coronary artery disease (CAD) in KD patients.
Methods and Results
We genotyped polymorphisms in the activating FcγRIIA, FcγRIIIA and FcγRIIIB genes using pyrosequencing in 443 KD patients, including 266 trios and 150 single parent-child pairs, in northwest US and genetically determined race with 155 ancestry information markers. We used the FBAT program to test for transmission disequilibrium and further generated pseudo-sibling controls for comparisons to the cases. The FcγRIIA-131H variant showed an association with KD (p = 0.001) with ORadditive = 1.51 [1.16–1.96], p = 0.002) for the primary combined population, which persisted in both Caucasian (p = .04) and Asian (p = .01) subgroups and is consistent with the recent genome-wide association study. We also identified over-transmission of FcγRIIIB-NA1 among IVIG non-responders (p = 0.0002), and specifically to Caucasian IVIG non-responders (p = 0.007). Odds ratios for overall and Caucasian non-responders were respectively 3.67 [1.75–7.66], p = 0.0006 and 3.60 [1.34–9.70], p = 0.01. Excess NA1 transmission also occurred to KD with CAD (ORadditive = 2.13 [1.11–4.0], p = 0.02).
A common variation in FcγRIIA is associated with increased KD susceptibility. The FcγRIIIB-NA1, which confers higher affinity for IgG compared to NA2, is a determining factor for treatment response. These activating FcγRs play an important role in KD pathogenesis and mechanism of IVIG anti-inflammatory.
coronary disease; pediatrics; Kawasaki disease; IVIG treatment response; FcγR
The occurrence of a congenital heart defect has long been thought to have a multifactorial basis, but the evidence is indirect. Complex trait analysis could provide a more nuanced understanding of congenital heart disease.
Methods and Results
We assessed the role of genetic and environmental factors on the incidence of ventricular septal defects caused by a heterozygous Nkx2-5 knockout mutation. We phenotyped >3100 hearts from a second generation intercross of the inbred mouse strains C57BL/6 and FVB/N. Genetic linkage analysis mapped loci with LOD scores of 5-7 on chromosomes 6, 8 and 10 that influence the susceptibility to membranous VSD in Nkx2-5+/- animals. The chromosome 6 locus overlaps one for muscular VSD susceptibility. Multiple logistic regression analysis for environmental variables revealed that maternal age is correlated with the risk of membranous and muscular VSD in Nkx2-5+/- but not wild-type animals. The maternal age effect is unrelated to aneuploidy or a genetic polymorphism in the affected individuals. The risk of a VSD is not only complex but dynamic. Whereas the effect of genetic modifiers on risk remains constant, the effect of maternal aging increases over time.
Enumerable factors contribute to the presentation of a congenital heart defect. The factors that modify rather than cause congenital heart disease substantially affect risk in predisposed individuals. Their characterization in a mouse model offers the potential to narrow the search space in human studies and to develop alternative strategies for prevention.
genetic variation; heart defects; congenital; Nkx2-5; genetic modifier; maternal age
A number of single gene defects have been identified in patients with isolated or nonsyndromic congenital heart defects (CHD). However, due to significant genetic heterogeneity candidate gene approaches have had limited success in finding high-risk alleles in most cases.
Use exome sequencing to identify high-risk gene variants in a family with highly penetrant pleiotropic CHD.
DNA samples from 2 members of a family with diverse CHD were analyzed by exome sequencing. Variants were filtered to eliminate common variants and sequencing artifacts and then prioritized based upon the predicted effect of the variant and on gene function. The remainder of the family was screened using PCR, high resolution melting analysis and DNA sequencing to evaluate variant segregation.
After filtering, more than 2000 rare variants (including single nucleotide substitutions and indels) were shared by the 2 individuals. Of these, 46 were non-synonymous, 3 were predicted to alter splicing, and 6 resulted in a frameshift. Prioritization reduced the number of variants potentially involved in CHD to 18. None of the variants completely segregated with CHD in the kindred. However, one variant, Myh6 Ala290Pro, was identified in all but one affected individual. This variant was previously identified in a patient with tricuspid atresia and large secundum ASD.
It is likely that next generation sequencing will become the method of choice for unraveling the complex genetics of CHD, but information gained by analysis of transmission through families will be crucial.
Atrial Septal Defects; Exome Sequencing; Myosin Heavy Chain 6; Mutations
Diabetic patients have an increased risk of developing atherosclerosis and related complications compared to non-diabetic individuals. The increased cardiovascular risk associated with diabetes is due in part to genetic variations that influence both glucose homeostasis and atherosclerotic lesion growth. Mouse strains C57BL/6J (B6) and BALB/cJ (BALB) exhibit distinct differences in fasting plasma glucose and atherosclerotic lesion size when deficient in apolipoprotein E (Apoe−/− . Quantitative trait locus (QTL) analysis was performed to determine genetic factors influencing the two phenotypes.
Methods and Results
266 female F2 mice were generated from an intercross between B6.Apoe−/− and BALB.Apoe−/− mice and fed a Western diet for 12 weeks. Atherosclerotic lesions in the aortic root, fasting plasma glucose, and body weight were measured. 130 microsatellite markers across the entire genome were genotyped. Four significant QTLs, Ath1 on chromosome (Chr) 1, Ath41 on Chr2, Ath42 on Chr5, and Ath29 on Chr9, and one suggestive QTL on Chr4, were identified for atherosclerotic lesion size. Four significant QTLs, Bglu3 and Bglu12 on Chr1, Bglu13 on Chr5, Bglu15 on Chr12, and two suggestive QTLs on Chr9 and Chr15 were identified for fasting glucose levels on the chow diet. Two significant QTLs, Bglu3 and Bglu13, and one suggestive locus on Chr8 were identified for fasting glucose on the Western diet. One significant locus on Chr1 and two suggestive loci on Chr9 and Chr19 were identified for body weight. Ath1 and Ath42 coincided with Bglu3 and Bglu13, respectively, in the confidence interval.
We have identified novel QTLs that have major influences on atherosclerotic lesion size and glucose homeostasis. The colocalization of QTLs for atherosclerosis and diabetes suggests possible genetic connections between the two diseases.
Atherosclerosis; type 2 diabetes; quantitative trait locus; hyperglycemia
Proteases hydrolyze peptide bonds, thereby controlling the function of proteins and peptides on the posttranslational level. In the cardiovascular system, proteases play pivotal roles in the regulation of blood pressure, coagulation and other essential physiological processes. Accordingly, proteases are prime targets for therapeutic interventions and diagnostics. Proteases are part of complex proteolytic networks comprised of enzymes, inhibitors, activators, substrates and cleavage products. Analyzing these networks on a system-wide level is essential to understanding cardiovascular function and how dysregulation can lead to pathological conditions. Mass spectrometry-based quantitative and dynamic proteomics approaches are leading the way to enhance our knowledge of proteolytic networks such as the renin-angiotensin-system. Here, we critically review proteomics tools utilized in protease biology and provide an overview on how these methods can be used to characterize and validate protease function.
peptides; biomarker; proteomics; cardiovascular diseases; mass spectrometry