Genetic variation in the non-canonical Wnt signalling pathway and predisposition to tetralogy of Fallot

Introduction Congenital cardiovascular malformation (CCM) is a prevalent group of conditions in humans. Tetralogy of Fallot (TOF) is the commonest cyanotic heart defect affecting 0.24/1000 newborns worldwide. Human CCM etiology has a genetic component with 17% of CCM attributable to particular genetic syndromes. The remaining 80% of "sporadic" cases nevertheless show a high heritability, suggesting complex polygenic inheritance modulated by environmental factors. Multiple previous studies have explored the involvement of the non-canonical Wnt signaling pathway, also known as the planar cell polarity pathway, in cardiogenesis but none has explored the relationship between genetic variation within the pathway and predisposition to TOF in humans. Methods Exonic sequences of 7 key genes within the pathway (ROCK1, ROCK2, Wnt11, Wnt5a, Dvl3, Dvl1l1 and ANKRD6) were obtained in 93 TOF probands as an initial mutational screen. All novel variants were genotyped in at least 465 TOF probands and 1465 controls and analyzed using in silico methods to determine impact in structure and splicing. Three variants were functionally assessed. Genotypes for tag SNPs in ROCK1 were also obtained. Results Twelve uncommon previously unreported variants were found within the seven genes: four non synonymous, 3 synonymous, 3 intronic (near exon/intron boundaries) and 2 located at untranslated regions. Two novel common variants were also found: one synonymous and one intronic. None of the variants was proven to be de novo. None of the probands carried more than one novel variant. Statistically significant differences in allele frequencies were found for ROCK1 C807T and WNT11 207 +47 G>T. WNT5A Asp119Ser was found as a singleton in one proband and not in controls. Functional experiments did not show splicing changes associated with ROCK1 C807T and WNT11 207 +47 G>T. Rs288979, an intronic common variant (mAF=0.051) was significantly associated with TOF (p=0.000015 OR: 0.61). Conclusions Results of this work suggest that genetic variation in the non canonical Wnt signaling genes is involved in the etiology of TOF in humans. The genetic predisposition to TOF seems to be given by a combination of rare variants (ROCK1 C807T, WNT11 207 +47 G>T and WNT5A Asp119Ser) associated with high ORs and common variants (rs288979) associated with low ORs. Further deep sequencing work in larger samples should unravel the remaining rare variants associated with TOF, while genome-wide association study (GWAS) may reveal the role for additional common variants.EThOS - Electronic Theses Online ServiceNewcastle University : Faculty of Medicine, Universidad de los Andes, ColombiaGBUnited Kingdo

Clinical Heterogeneity of Pulmonary Arterial Hypertension Associated With Variants in TBX4

Background: The knowledge of hereditary predisposition has changed our understanding of Pulmonary Arterial Hypertension. Genetic testing has been widely extended and the application of Pulmonary Arterial Hypertension specific gene panels has allowed its inclusion in the diagnostic workup and increase the diagnostic ratio compared to the traditional sequencing techniques. This is particularly important in the differential diagnosis between Pulmonary Arterial Hypertension and Pulmonary Venoocclusive Disease. Methods: Since November 2011, genetic testing is offered to all patients with idiopathic, hereditable and associated forms of Pulmonary Arterial Hypertension or Pulmonary Venoocclusive Disease included in the Spanish Registry of Pulmonary Arterial Hypertension. Herein, we present the clinical phenotype and prognosis of all Pulmonary Arterial Hypertension patients with disease-associated variants in TBX4. Results: Out of 579 adults and 45 children, we found in eight patients from seven families, disease-causing associated variants in TBX4. All adult patients had a moderate-severe reduction in diffusion capacity. However, we observed a wide spectrum of clinical presentations, including Pulmonary Venoocclusive Disease suspicion, interstitial lung disease, pulmonary vascular abnormalities and congenital heart disease. Conclusions: Genetic testing is now essential for a correct diagnosis work-up in Pulmonary Arterial Hypertension. TBX4-associated Pulmonary Arterial Hypertension has marked clinical heterogeneity. In this regard, a genetic study is extremely useful to obtain an accurate diagnosis and provide appropriate management.This project was founded by Project "Bases Gene´tico Moleculares de la Medicina de Precisio´n en la Hipertensio´n Arterial Pulmonar". Funder: Instituto Carlos III. Ministerio de Economı´a y Competitividad. https://www.isciii.es/Paginas/Inicio.aspx Award number: PI 18/01233 Grant Recipient: P E-

Clinical phenotypes and prognosis of dilated cardiomyopathy caused by truncating variants in the TTN Gene.

Background: Truncating variants in the TTN gene (TTNtv) are the commonest cause of heritable dilated cardiomyopathy. This study aimed to study the phenotypes and outcomes of TTNtv carriers. Methods: Five hundred thirty-seven individuals (61% men; 317 probands) with TTNtv were recruited in 14 centers (372 [69%] with baseline left ventricular systolic dysfunction [LVSD]). Baseline and longitudinal clinical data were obtained. The primary end point was a composite of malignant ventricular arrhythmia and end-stage heart failure. The secondary end point was left ventricular reverse remodeling (left ventricular ejection fraction increase by ≥10% or normalization to ≥50%). Results: Median follow-up was 49 (18–105) months. Men developed LVSD more frequently and earlier than women (45±14 versus 49±16 years, respectively; P=0.04). By final evaluation, 31%, 45%, and 56% had atrial fibrillation, frequent ventricular ectopy, and nonsustained ventricular tachycardia, respectively. Seventy-six (14.2%) individuals reached the primary end point (52 [68%] end-stage heart failure events, 24 [32%] malignant ventricular arrhythmia events). Malignant ventricular arrhythmia end points most commonly occurred in patients with severe LVSD. Male sex (hazard ratio, 1.89 [95% CI, 1.04–3.44]; P=0.04) and left ventricular ejection fraction (per 10% decrement from left ventricular ejection fraction, 50%; hazard ratio, 1.63 [95% CI, 1.30–2.04]; P<0.001) were independent predictors of the primary end point. Two hundred seven of 300 (69%) patients with LVSD had evidence of left ventricular reverse remodeling. In a subgroup of 29 of 74 (39%) patients with initial left ventricular reverse remodeling, there was a subsequent left ventricular ejection fraction decrement. TTNtv location was not associated with statistically significant differences in baseline clinical characteristics, left ventricular reverse remodeling, or outcomes on multivariable analysis (P=0.07). Conclusions: TTNtv is characterized by frequent arrhythmia, but malignant ventricular arrhythmias are most commonly associated with severe LVSD. Male sex and LVSD are independent predictors of outcomes. Mutation location does not impact clinical phenotype or outcomes.pre-print1,66 M

Novel genetic and molecular pathways in pulmonary arterial hypertension associated with connective tissue disease

Pulmonary Arterial Hypertension (PAH) is a severe complication of Connective Tissue Disease (CTD), with remarkable morbidity and mortality. However, the molecular and genetic basis of CTD-PAH remains incompletely understood. This study aimed to screen for genetic defects in a cohort of patients with CTD-PAH, using a PAH-specific panel of 35 genes. During recruitment, 79 patients were studied, including 59 Systemic Sclerosis patients (SSc) and 69 females. Disease-associated variants were observed in nine patients: 4 pathogenic/likely pathogenic variants in 4 different genes (TBX4, ABCC8, KCNA5 and GDF2/BMP9) and 5 Variants of Unknown Significance (VUS) in 4 genes (ABCC8, NOTCH3, TOPBP1 and CTCFL). One patient with mixed CTD had a frameshift pathogenic variant in TBX4. Two patients with SSc-PAH carried variants in ABCC8. A patient diagnosed with Systemic Lupus Erythematous (SLE) presented a pathogenic nonsense variant in GDF2/BMP9. Another patient with SSc-PAH presented a pathogenic variant in KCNA5. Four patients with SSc-PAH carried a VUS in NOTCH1, CTCFL, CTCFL and TOPBP1, respectively. These findings suggest that genetic factors may contribute to Pulmonary Vascular Disease (PVD) in CTD patients.Instituto de Salud Carlos III | Ref. PI 18/0123

The Free Radical Nature of Compound I From Horseradish Peroxidase and Chloroperoxidase

161 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1982.Compound Is were prepared from Horseradish Peroxidase (HRP), Chloroperoxidase (CPO), mesohemin substituted Horseradish Peroxidase (M-HRP) and deuterohemin substituted Horseradish Peroxidase. The Compound Is were analyzed by visible absorption, electron paramagnetic resonance (EPR), Mossbauer and electron nuclear double resonance (ENDOR) spectra. These spectral techniques were used to prove that one of the two oxidation equivalents associated with Compound I consists of a porphyrin centered pi-cation radical.Titration of the EPR and visible absorption spectra associated with Compound I formation and ENDOR spectra of deuterium substituted hemin demonstrated unequivocally that HRP Compound I has a pi-cation porphyrin centered radical associated with it.A g = 1.73 EPR signal was found to be associated with CPO Compound I. This EPR signal accounted for the theoretical spins which should be associated with Compound I assuming one unpaired spin per heme group. CPO Compound I Mossbauer spectra showed a strong magnetic broadening which could be accounted for by a strong interaction of a radical with the proven low spin ferryl iron center.EPR and Mossbauer spectra of M-HRP Compound I showed that it is very similar to HRP Compound I in conflict with the predictions of others. The EPR spectrum of D-HRP Compound I did not detect a major radical signal.It was proven that HRP, CPO and M-HRP Compound Is utilize a free radical, which is porphyrin centered, to store one of the two oxidation equivalents associated with Compound I formation.Oxygen 17 ENDOR spectroscopy was utilized to show that one of the oxygens from the peroxide used to form HRP and CPO Compound Is remains covalently attached to the intermediate. This peroxide derived oxygen associated with Compound I was shown to be non-exchangeable with the solvent water.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Customized massive parallel sequencing panel for diagnosis of pulmonary arterial hypertension

Pulmonary arterial hypertension is a very infrequent disease, with a variable etiology and clinical expressivity, making sometimes the clinical diagnosis a challenge. Current classification based on clinical features does not reflect the underlying molecular profiling of these groups. The advance in massive parallel sequencing in PAH has allowed for the describing of several new causative and susceptibility genes related to PAH, improving overall patient diagnosis. In order to address the molecular diagnosis of patients with PAH we designed, validated, and routinely applied a custom panel including 21 genes. Three hundred patients from the National Spanish PAH Registry (REHAP) were included in the analysis. A custom script was developed to annotate and filter the variants. Variant classification was performed according to the ACMG guidelines. Pathogenic and likely pathogenic variants have been found in 15% of the patients with 12% of variants of unknown significance (VUS). We have found variants in patients with connective tissue disease (CTD) and congenital heart disease (CHD). In addition, in a small proportion of patients (1.75%), we observed a possible digenic mode of inheritance. These results stand out the importance of the genetic testing of patients with associated forms of PAH (i.e., CHD and CTD) additionally to the classical IPAH and HPAH forms. Molecular confirmation of the clinical presumptive diagnosis is required in cases with a high clinical overlapping to carry out proper management and follow up of the individuals with the disease.Instituto de Salud Carlos III (España) | Ref. FISPI18/01233Xunta de Galicia | Ref. ED481A-2018/30

Combination of late gadolinium enhancement and genotype improves prediction of prognosis in non-ischaemic dilated cardiomyopathy

Aims: Genotype and left ventricular scar on cardiac magnetic resonance (CMR) are increasingly recognized as risk markers for adverse outcomes in non-ischaemic dilated cardiomyopathy (DCM). We investigated the combined influence of genotype and late gadolinium enhancement (LGE) in assessing prognosis in a large cohort of patients with DCM. Methods and results: Outcomes of 600 patients with DCM (53.3 ± 14.1 years, 66% male) who underwent clinical CMR and genetic testing were retrospectively analysed. The primary endpoints were end-stage heart failure (ESHF) and malignant ventricular arrhythmias (MVA). During a median follow-up of 2.7 years (interquartile range 1.3–4.9), 24 (4.00%) and 48 (8.00%) patients had ESHF and MVA, respectively. In total, 242 (40.3%) patients had pathogenic/likely pathogenic variants (positive genotype) and 151 (25.2%) had LGE. In survival analysis, positive LGE was associated with MVA and ESHF (both, p < 0.001) while positive genotype was associated with ESHF (p = 0.034) but not with MVA (p = 0.102). Classification of patients according to genotype (G+/G−) and LGE presence (L+/L−) revealed progressively increasing events across L−/G−, L−/G+, L+/G− and L+/G+ groups and resulted in optimized MVA and ESHF prediction (p < 0.001 and p = 0.001, respectively). Hazard ratios for MVA and ESHF in patients with either L+ or G+ compared with those with L−/G− were 4.71 (95% confidence interval: 2.11–10.50, p < 0.001) and 7.92 (95% confidence interval: 1.86–33.78, p < 0.001), respectively. Conclusion: Classification of patients with DCM according to genotype and LGE improves MVA and ESHF prediction. Scar assessment with CMR and genotyping should be considered to select patients for primary prevention implantable cardioverter-defibrillator placementThis work was supported by grants from the Instituto de Salud Carlos III (ISCIII) (PI18/0004, PI19/01283, PI20/0320). (Co-funded by European Regional Development Fund/European Social Fund ‘A way to make Europe’/‘Investing in your future’). The Hospital Universitario Puerta de Hierro Majadahonda, the Hospital Clinic, the Hospital Vall d’Hebron, the Hospital General Universitario Gregorio Marañón and the Hospital Universitario Virgen de la Arrixaca are members of the European Reference Network for rare, low-prevalence, and complex diseases of the heart (ERN GUARD-Heart). The CNIC is supported by the ISCIII, MCIN, the Pro-CNIC Foundation, and the Severo Ochoa Centers of Excellence program (CEX2020-001041-S). Conflict of interest: none declare

Association between C677T polymorphism of methylene tetrahydrofolate reductase and congenital heart disease: meta-analysis of 7697 cases and 13,125 controls

BACKGROUND: Association between the C677T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene and congenital heart disease (CHD) is contentious. METHODS AND RESULTS: We compared genotypes between CHD cases and controls and between mothers of CHD cases and controls. We placed our results in context by conducting meta-analyses of previously published studies. Among 5814 cases with primary genotype data and 10 056 controls, there was no evidence of association between MTHFR C677T genotype and CHD risk (odds ratio [OR], 0.96 [95% confidence interval, 0.87-1.07]). A random-effects meta-analysis of all studies (involving 7697 cases and 13 125 controls) suggested the presence of association (OR, 1.25 [95% confidence interval, 1.03-1.51]; P=0.022) but with substantial heterogeneity among contributing studies (I(2)=64.4%) and evidence of publication bias. Meta-analysis of large studies only (defined by a variance of the log OR <0.05), which together contributed 83% of all cases, yielded no evidence of association (OR, 0.97 [95% confidence interval, 0.91-1.03]) without significant heterogeneity (I(2)=0). Moreover, meta-analysis of 1781 mothers of CHD cases (829 of whom were genotyped in this study) and 19 861 controls revealed no evidence of association between maternal C677T genotype and risk of CHD in offspring (OR, 1.13 [95% confidence interval, 0.87-1.47]). There was no significant association between MTHFR genotype and CHD risk in large studies from regions with different levels of dietary folate. CONCLUSIONS: The MTHFR C677T polymorphism, which directly influences plasma folate levels, is not associated with CHD risk. Publication biases appear to substantially contaminate the literature with regard to this genetic association