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Background and aims Mitochondrial DNA copy number (mtDNA-CN) depletion has been recently associated with an increased cardiovascular risk. However, the integrity of mtDNA is another key aspect of the energy metabolism and mitochondrial function. We investigated the prognostic role of peripheral blood common mitochondrial deletion (mtDNA4977) and mtDNA-CN on long-term major adverse cardiac events (MACEs) and all-cause mortality in a cohort of patients with coronary artery disease (CAD). Methods Within the Italian GENOCOR (Genetic Mapping for Assessment of Cardiovascular Risk) cohort, we studied 515 patients (450 males, 65 ± 8 years) with known or suspected stable CAD. mtDNA4977 deletion and mtDNA-CN were assessed in peripheral blood using qRT-PCR. Results During a mean follow-up of 4.5 ± 1.1 years, 78 (15%) patients had MACEs (15 cardiac deaths, 17 nonfatal myocardial infarction and 46 coronary revascularizations) and 28 patients died for non-cardiac causes. Patients with high levels of mtDNA4977 deletion (>75th) had increased risk of MACEs (log rank = 7.2, p=0.007) and all-cause mortality (log rank = 5.7, p=0.01) compared with patients with low mtDNA4977 deletion (≤75th). Multivariate Cox regression analysis showed that log mtDNA4977 was a significant predictor of MACEs (HR = 2.17; 95% CI, 1.31–3.59; p=0.003) and all-cause mortality (HR = 2.03; 95% CI: 1.13–3.65, p=0.02). Log mtDNA-CN was not significantly associated with MACEs or all-cause mortality. However, patients with high mtDNA4977 deletion (>75th) and low mtDNA-CN ( Conclusions Mitochondria DNA damage was associated with an increased risk of MACEs and all-cause mortality in patients with stable CAD, confirming the critical role of mitochondrial dysfunction in atherosclerosis.

Disturbed flow has been suggested to contribute to aneurysm susceptibility in bicuspid aortic valve (BAV) patients. Lately, flow has emerged as an important modulator of DNA methylation. Hear we combined global methylation analysis with in vitro studies of flow-sensitive methylation to identify biological processes associated with BAV-aortopathy and the potential contribution of flow. Biopsies from non-dilated and dilated ascending aortas were collected from BAV (n = 21) and tricuspid aortic valve (TAV) patients (n = 23). DNA methylation and gene expression was measured in aortic intima-media tissue samples, and in EA.hy926 and primary aortic endothelial cells (ECs) isolated from BAV and TAV exposed to oscillatory (±12 dynes/cm2) or laminar (12 dynes/cm2) flow. We show methylation changes related to epithelial-mesenchymal-transition (EMT) in the non-dilated BAV aorta, associated with oscillatory flow related to endocytosis. The results indicate that the flow-response in BAV ECs involves hypomethylation and increased expression of WNT/β-catenin genes, as opposed to an angiogenic profile in TAV ECs. The EMT-signature was exasperated in dilated BAV aortas. Aberrant EMT in BAV aortic walls could contribute to increased aneurysm susceptibility, and may be due to disturbed flow-exposure. Perturbations during the spatiotemporally related embryonic development of ascending aorta and semilunar valves can however not be excluded.

Aging is one of the main risk factors for cardiovascular disease, resulting in a progressive organ and cell decline. This study evaluated a possible joint impact of two emerging hallmarks of aging, leucocyte telomere length (LTL) and common mitochondrial DNA deletion (mtDNA4977), on major adverse cardiovascular events (MACEs) and all-cause mortality in patients with coronary artery disease (CAD). We studied 770 patients (673 males, 64.8 &plusmn, 8.3 years) with known or suspected stable CAD. LTL and mtDNA4977 deletion were assessed in peripheral blood using qRT-PCR. During a median follow-up of 5.4 &plusmn, 1.2 years, MACEs were 140 while 86 deaths were recorded. After adjustments for confounding risk factors, short LTLs and high mtDNA4977 deletion levels acted independently as predictors of MACEs (HR: 2.2, 95% CI: 1.2&ndash, 3.9, p = 0.01 and HR: 1.7, 95% CI: 1.1&ndash, 2.9, p = 0.04, respectively) and all-cause mortality events (HR: 2.1, 95% CI: 1.1&ndash, 4.6, p = 0.04 and HR: 2.3, 95% CI: 1.1&ndash, 4.9, p = 0.02, respectively). Patients with both short LTLs and high mtDNA4977 deletion levels had an increased risk for MACEs (HR: 4.3, 95% CI: 1.9&ndash, 9.6, p = 0.0006) and all-cause mortality (HR: 6.0, 95% CI: 2.0&ndash, 18.4, p = 0.001). The addition of mtDNA4977 deletion to a clinical reference model was associated with a significant net reclassification improvement (NRI = 0.18, p = 0.01). Short LTL and high mtDNA4977 deletion showed independent and joint predictive value on adverse cardiovascular outcomes and all-cause mortality in patients with CAD. These findings strongly support the importance of evaluating biomarkers of physiological/biological age, which can predict disease risk and mortality more accurately than chronological age.

Resumen Introduccion y objetivos Los polimorfismos de nucleotido unico situados en un lugar de union de microacidos ribonucleicos (miARN) pueden tener diferentes efectos en la expresion genica, y ello puede influir en el riesgo de enfermedad. Este estudio tiene como objetivo evaluar la asociacion existente entre los polimorfismos de nucleotido unico y los haplotipos presentes en la region 3’UTR del gen GATA4 y el riesgo de cardiopatia congenita. Metodos Se utilizaron algoritmos de bioinformatica para analizar los polimorfismos de nucleotido unico en los presuntos lugares de union de miARN en la region 3’UTR del gen GATA4 y para calcular la diferencia de energia de hibridacion libre (ΔFE, kcal/mol) para cada alelo de tipo natural (wild-type) en comparacion con cada variante alelica. Resultados Formaron la poblacion de estudio 146 pacientes caucasicos (73 varones; edad, 6,68 ± 7,79 anos) y 265 recien nacidos sanos (147 varones). Se considero que la suma de todos los ΔFE predecia la importancia biologica de los polimorfismos de nucleotido unico al unirse a mas miARN. A continuacion se determino el genotipo de los 4 polimorfismos (+1158 C > T, + 1256 A > T, + 1355 G > A, +1521 C > G) que tenian el valor predicho de ΔFE total mas alto (9,91, 14,85, 11,03 y 21,66 kcal/mol respectivamente) en un estudio de casos y controles (146 pacientes y 250 controles). Al aplicar una correccion por multiplicidad de pruebas, tan solo el alelo +1158 T mostro una diferencia significativa entre los pacientes y los controles. El analisis de los haplotipos puso de manifiesto que el haplotipo T-T-G-C (mas infrecuente en los pacientes con cardiopatias congenitas que en los controles) se asociaba a una disminucion del riesgo significativa (p = 0,03), mientras que el haplotipo muy infrecuente C-A-A-C, que se daba de manera muy poco comun en los controles (0,3%) en comparacion con los pacientes con la enfermedad (2,4%), se asociaba a un aumento de 4 veces en el riesgo de enfermedad (p = 0,04). Conclusiones Las variantes frecuentes de la region 3’UTR del gen GATA4 interaccionan de manera conjunta y con ello afectan a la susceptibilidad a la cardiopatia congenita, probablemente mediante la alteracion de la regulacion postranscripcional de los miARN.

Compelling evidence has shown that microRNAs (miRs) are involved in the pathophysiology of BAV-associated aortopathy. The purpose of this study was to assess the biological role as well as the circulating expression of two miRs (miR-424-3p and miR-3688-3p) that have been previously identified as significantly dysregulated in thoracic aortic aneurysm specimens of BAV patients. Bioinformatic tools were used to predict miR gene targets followed by functional validation transfecting synthetic miR mimics and negative controls into human aortic smooth muscle cells (HASMCs). Levels of miRs and target genes were evaluated by qRT-PCR. The circulating miR expression profile analysis was assessed on plasma samples collected from a cohort of 72 patients with aortopathy including 39 BAV (33 males; 58 ± 13 years) and 33 TAV patients (26 males; 67 ± 9 years). Computational analysis revealed that SMAD7 and YAP1 were potential targets of miR-424-3p and miR-3688-3p, respectively. Transfection with mimics confirmed a significantly decreased gene expression of SMAD7 and YAP1 compared to mimic negative control (p = 0.04 and p = 0.0005, respectively) or blank control (p = 0.01 and p = 0.0007, respectively). Overexpression of miR-3688-3p also significantly upregulated pro-apoptotic caspase-3 gene expression compared to mimic negative control (p = 0.02) or blank control (p = 0.01). Furthermore, a significant down-regulation of the circulating miR-424-3p was observed in BAV compared to TAV patients (p = 0.001). In multiple linear regression analysis, the aortic valve morphology (β = − 0.29, p = 0.04) and the presence of aortic stenosis (β = − 0.28, p = 0.03) had a significant effect on the miR-424-3p expression. In conclusion, our study demonstrated that miR-424-3p and miR-3688-3p directly targeted SMAD7 and YAP1 in HASMCs, pivotal genes of the TGF-β and Hippo-signaling pathways. Circulating miR-424-3p was also found to be significantly decreased in BAV patients when compared to TAV patients, especially in patients with aortic stenosis. Further large studies of well-characterized BAV patient cohorts are needed to define the clinical significance of the miR-424-3p.

Introduction Single-nucleotide polymorphisms (SNPs) in microRNA (miRNA) machinery genes may affect the regulatory capacity of miRNAs by impacting their biogenesis. The aim of the study was to analyze the association between SNPs in two key genes (DICER rs1057035T>C and XPO5 rs11077A>C) and coronary artery disease (CAD) risk as well as to examine their effects on circulating levels of vascular miRNAs. Materials and methods Within the Italian GENOCOR cohort, we studied a cohort of 557 patients (502 males, 57 ± 9 years) with angiographically documented CAD. A total of 443 healthy controls (262 males, 56 ± 12 years) was also enrolled. Genotyping was determined by using a TaqMan®SNP genotyping assay. Analysis of miR-132 and miR-140-3p was assessed in a subset of 70 CAD patients by using qRT-PCR. Results There were statistically significant differences between CAD patients and healthy controls in the distribution of both DICER and XPO5 genotypes (p = 0.03 and p = 0.02, respectively). Multivariate analysis showed a significantly decreased risk of CAD by 50% in patients with DICER rs105703CC genotype as compared to TC heterozygote and TT homozygote patients (ORadjusted = 0.50; CI: 0.30–0.83, p = 0.007). In a recessive model, the XPO5 rs11077CC genotype was associated with a 32% reduced risk of CAD (ORadjusted = 0.68; CI: 0.30–0.99 p = 0.047). XPO5 rs11077CC genotype was significantly associated with higher levels of both miRNA-132 (p = 0.04) and miRNA-140-3p (p = 0.03). Conclusions Genetic polymorphisms in DICER and XPO5 genes are associated with a decreased risk of CAD, probably by impacting expression levels of vascular and cardiac-specific miRNAs. Further studies are needed to better elucidate the biological relevance of both variants in CAD development.

Mitochondrial DNA copy number (mtDNA-CN) depletion has been recently associated with an increased cardiovascular risk. However, the integrity of mtDNA is another key aspect of the energy metabolism and mitochondrial function. We investigated the prognostic role of peripheral blood common mitochondrial deletion (mtDNAWithin the Italian GENOCOR (Genetic Mapping for Assessment of Cardiovascular Risk) cohort, we studied 515 patients (450 males, 65 ± 8 years) with known or suspected stable CAD. mtDNADuring a mean follow-up of 4.5 ± 1.1 years, 78 (15%) patients had MACEs (15 cardiac deaths, 17 nonfatal myocardial infarction and 46 coronary revascularizations) and 28 patients died for non-cardiac causes. Patients with high levels of mtDNAMitochondria DNA damage was associated with an increased risk of MACEs and all-cause mortality in patients with stable CAD, confirming the critical role of mitochondrial dysfunction in atherosclerosis.

Recent experimental studies showed that ablation of the aryl hydrocarbon receptor (AhR) as well as its activation by exogenous ligands disrupt the molecular networks involved in heart formation and function, leading to congenital heart disease (CHD). However, no evidence is available about the role of AhR in humans. We assessed the prevalence of a functional AhR genetic variant (p.Arg554Lys) in CHD patients as well as its joint effects with parental exposure. A total of 128 CHD patients (76 males; age 6.2 ± 6.7 years) and 274 controls (160 males; age at birth) were genotyped for the AhR polymorphism by using the TaqMan® Drug Metabolism Genotyping assay. Both case and control parents completed a structured questionnaire on demographic, lifestyle and preconception exposures. Genotype (p = 0.001) and allele (p

The molecular mechanisms underlying thoracic aortic aneurysm (TAA) in patients with bicuspid aortic valve (BAV) are incompletely characterized. MicroRNAs (miRNAs) may play a major role in the different pathogenesis of aortopathy. We sought to employ next-generation sequencing to analyze the entire miRNome in TAA tissue from patients with BAV and tricuspid aortic valve (TAV). In the discovery stage, small RNA sequencing was performed using the Illumina MiSeq platform in 13 TAA tissue samples (seven patients with BAV and six with TAV). Gene ontology (GO) and KEGG pathway analysis were used to identify key pathways and biological functions. Validation analysis was performed by qRT-PCR in an independent cohort of 30 patients with BAV (26 males; 59.5 ± 12 years) and 30 patients with TAV (16 males; 68.5 ± 9.5 years). Bioinformatic analysis identified a total of 489 known mature miRNAs and five novel miRNAs. Compared to TAV samples, 12 known miRNAs were found to be differentially expressed in BAV, including two up-regulated and 10 down-regulated (FDR-adjusted p-value ≤ 0.05 and fold change ≥ 1.5). GO and KEGG pathway enrichment analysis (FDR-adjusted p-value < 0.05) identified different target genes and pathways linked to BAV and aneurysm formation, including Hippo signaling pathway, ErbB signaling, TGF-beta signaling and focal adhesion. Validation analysis of selected miRNAs confirmed the significant down-regulation of miR-424-3p (p = 0.01) and miR-3688-3p (p = 0.03) in BAV patients as compared to TAV patients. Our study provided the first in-depth screening of the whole miRNome in TAA specimens and identified specific dysregulated miRNAs in BAV patients.

Congenital heart disease (CHD) is a genetically heterogeneous disease. Targeted next-generation sequencing (NGS) offers a unique opportunity to sequence multiple genes at lower cost and effort compared to Sanger sequencing. We tested a targeted NGS of a specific gene panel in a relatively large population of non-syndromic CHD patients. The patient cohort comprised 68 CHD patients (45 males; 8.3 ± 1.7 years). Amplicon libraries for 16 CHD-strictly related genes were generated using a TruSeq® Custom Amplicon kit (Illumina, CA) and sequenced using the Illumina MiSeq platform. Sequence data were processed through the MiSeq Reporter and wANNOVAR softwares. After applying stringent filtering criteria, 20 missense variants in 9 genes were predicted to be damaging and were validated by Sanger sequencing with 100% concordance. Fourteen variants were present in public databases with very rare allele frequency, of which four variants (p.Arg25Cys in NKX2-5, p.Val763Ile in ZFPM2, p.Arg1398Gln and Gly1826Asp in MYH6) have been previously linked to CHD or cardiomyopathy. The remaining six variants in four genes (GATA4, NKX2-5, NOTCH1, TBX1) were novel mutations, currently not found in public databases, and absent in 200 control alleles of healthy subjects. Four patients (5.8%) carried two missense variants (1 compound heterozygote in the same gene and 3 double heterozygotes in different genes), with possibly synergistic deleterious effects. Targeted NGS is a powerful and efficient tool to detect DNA sequence variants in multiple genes, providing the opportunity for discovery of the co-occurrence of two or more missense rare variants.

Congenital heart diseases (CHDs) are recognized as the most common type of birth malformations. Although recent advances in pre- and neonatal diagnosis as well as in surgical procedures have reduced the morbidity and mortality for many CHD, the etiology for CHD remains undefined. In non-syndromic and isolated (without a familial history or a Mendelian inheritance) forms of CHDs, a multifactorial pathogenesis with interplay between inherited and non-inherited causes is recognized. In this paper, we discuss the current knowledge of the potential molecular mechanisms, mediating abnormal cardiac development in non-syndromic and isolated CHD, including mutations in cardiac transcription factors, the role of somatic mutations and epigenetic alterations as well as the influence of gene-environment interactions. In the near future, the advent of high-throughput genomic technologies with the integration of system biology will expand our understanding of isolated, non-syndromic CHDs for their prevention, early diagnosis and therapy.

Holt-Oram syndrome (HOS) is a rare autosomal dominant disorder characterized by upper limb defects and congenital heart defects (CHD), which are often simple septal and conduction defects, less frequently complex CHDs. We report on a 9 year-old boy with clinical and radiologic features of HOS consisting of bilateral asymmetric hypoplastic thumbs, generalized brachydactyly, limited supination due to radioulnar synostosis, and sloping shoulders, and intermediate atrioventricular canal defect (AVCD) with aortic coarctation. A de novo, previously described mutation, (Arg279ter) was identified in the TBX5 gene. Molecular characterization of this mutation was carried out due to the atypical CHD. In order to investigate whether the mutated transcript of TBX5 was able to escape the post-transcriptional surveillance mechanism and to produce a truncated TBX5 protein, we analyzed the TBX5 transcript, and protein pattern in HOS, and WT cardiac tissues. Our results demonstrate that the mutant TBX5 transcript is cleared by the cellular mechanism of surveillance. This data provides some support for the hypothesis that a dominant negative mutation, which strongly impairs the WT allele, might be too hazardous to be maintained. The literature suggests that HOS is relatively common among syndromes associated with AVCD.

Somatic mutations and dysregulation by microRNAs (miRNAs) may have a pivotal role in the Congenital Heart Defects (CHDs). The purpose of the study was to assess both somatic and germline mutations in the GATA4 and NKX2.5 genes as well as to identify 3'UTR single nucleotide polymorphisms (SNPs) in the miRNA target sites. We enrolled 30 patients (13 males; 13.4±8.3 years) with non-syndromic CHD. GATA4 and NKX2.5 genes were screened in cardiac tissue of sporadic and in blood samples of familial cases. Computational methods were used to detect putative miRNAs in the 3'UTR region and to assess the Minimum Free Energy of hybridization (MFE, kcal/mol). Difference of MFEs (ΔMFE) ≥4 kcal/mol between alleles was considered biologically relevant on miRNA binding. The sum of all ΔMFEs (|ΔMFEtot|=∑|ΔMFE|) was calculated in order to predict the biological importance of SNPs binding more miRNAs. No evidence of novel GATA4 and NKX2.5 mutations was found both in sporadic and familial patients. Bioinformatic analysis revealed 27 putative miRNAs binding to identified SNPs in the 3'UTR of GATA4. ΔMFE ≥4 kcal/mol between alleles was obtained for the +354A>C (miR-4299), +587A>G (miR-604), +1355G>A (miR-548v, miR-139-5p) and +1521C>G (miR-583, miR-3125, miR-3928) SNPs. The +1521C>G SNP showed the highest ΔMFEtot (21.66 kcal/mol). Luciferase reporter assays indicated that miR-583 was dose-dependently effective in regulating +1521 C allele compared with +1521 G allele. Based on the analysis of 100 CHD cases and 204 healthy newborns, the +1521 G allele was also associated with a lower risk of CHD (OR=0.5, 95% CI 0.3-0.9, p=0.03), likely due to the relatively low binding of the miRNA and high levels of protein. These results suggest that common SNPs in the 3'UTR of GATA4 alter miRNA gene regulation contributing to the pathogenesis of CHDs.

Introduction: Individuals with bicuspid aortic valves (BAV) are at increased risk of ascending aortic aneurysm than individuals with tricuspid aortic valves (TAV), but the underlying mechanism is not fully understood. Aberrant DNA methylation has been described in various human diseases, and we have shown that key enzymes in the methylation machinery are differentially expressed in the aortic intima-media of BAV and TAV patients. In the present study, we assessed the hypothesis that DNA methylation may play an important role during aneurysm formation in BAV. We undertook a global methylation approach to delineate biological processes associated with BAV aortopathy, using TAV as a reference. Methods: Ascending aortic biopsies were collected from 21 BAV and 24 TAV patients, with either a non-dilated or a dilated aorta, at the time of surgery. Global DNA methylation was measured in the intima-media layer using Illumina 450k Array. Gene expression was analyzed in the same samples using Affymetrix Exon Array. Results: Compared with TAV, the BAV dilated aorta was hypomethylated (P=0.031), correlating with an up-regulation of global gene expression. A total of 4913 differentially methylated regions (DMRs) were identified and Hallmark analysis of the DMR-associated genes with a fold change of 10% (n=3147) showed a gene signature of Epithelial Mesenchymal Transition (EMT) (FDR q=2.91e-29). This was further confirmed by functional annotation analysis of hypomethylated DMRs using the Genomic Regions Enrichment of Annotations Tool (Stanford University), showing association to actin filament bundle (P=7.09e-12), stress fibers (P=1.72e-11) and adherence junctions (P=2.97e-8). Interestingly, analysis of non-dilated BAV and TAV aorta revealed that genes involved in EMT were the most differentially methylated genes prior to dilatation (FDR q=1.18e-6). We further confirmed the EMT-related molecular signature by immunostaining of some key players of EMT. In conclusion, epigenetic profiling clearly revealed differential methylation between BAV and TAV aorta, particularly in EMT-related genes. Aberrant EMT in the ascending aorta prior to dilatation may constitute the basis for the increased aneurysm susceptibility in BAV patients.

Congenital heart defects (CHDs) are the most prevalent of all birth defects and the leading cause of death in the first year of life. The molecular causes of most CHDs remain largely unknown. The LIM homeodomain transcriptor factor ISL1 is a marker for undifferentiated cardiac progenitor cells that give rise to both the right ventricle and the inflow and outflow tracts, which are affected by several cardiovascular malformations. Contradictory findings about the role of the ISL1 rs1017 single-nucleotide polymorphism in increasing the risk of CHD have been reported. In this study, we aimed to investigate whether the ISL1 rs1017 genetic polymorphism conferred susceptibility to CHD in the white population. In a case-control study design, 309 patients with CHD (197 men [age 21.3 ± 25.2]) and 500 healthy controls (272 men [age 15.7 ± 21.3]) were genotyped for the ISL1 rs1017 polymorphism. No significant difference in the genotype and variant allele distributions was found between patients and controls. In addition, the ISL1 rs1017 polymorphism was not associated with the risk of CHD neither overall (p = 0.7) nor stratifying the population by sex and CHD classification. In conclusion, ISL1 common variant rs1017 is not associated with increased genetic risk of CHD in the white population.

The GSTP1 gene, highly expressed early in fetal life, is the most abundant phase 2 xenobiotic metabolism enzyme in a human placenta. Fetal inherited GSTP1 Ile105Val polymorphism may modify the metabolism and excretion of xenobiotics from fetal tissue and increase the risk of congenital heart disease (CHD). This study aimed to analyze the joint effects of GSTP1 genetic polymorphism (Ile105Val) and maternal environmental exposure on CHD risk. Within a case-control design, a total of 190 children with CHD (104 boys age 4 ± 5.6 years) and 190 healthy children (114 newborn boys) were genotyped for the GSTP1 Ile105Val polymorphism. Mothers completed a structured questionnaire on the demographics as well as the preconceptional and lifestyle exposures. A higher frequency of mothers of children with CHD (38 %) reported a positive history of exposure to toxicants (occupational and environmental) than mothers of healthy children (23 %) (p = 0.0013). Logistic regression analysis showed that maternal occupational and environmental exposures increased the risk of CHD (odds ratio, 2.6; 95 % confidence interval, 1.6-4.2; p < 0.0001). No significant differences in Ile105Val genotype frequencies were observed between the children with CHD and the healthy children (p = 0.9). Furthermore, case-control analysis showed no evidence of significant interaction between the maternal exposures and GSTP1 polymorphism. Maternal exposure to toxicants increased the risk of children with CHD. However, fetal GSTP1 Ile105Val polymorphism did not increase the risk of CHD.

Laser-driven electron accelerators are capable of producing high-energy electron bunches in shorter distances than conventional radiofrequency accelerators. To date, our knowledge of the radiobiological effects in cells exposed to electrons using a laser-plasma accelerator is still very limited. In this study, we compared the dose-response curves for micronucleus (MN) frequency and telomere length in peripheral blood lymphocytes exposed to laser-driven electron pulse and X-ray radiations. Additionally, we evaluated the effects on cell survival of in vitro tumor cells after exposure to laser-driven electron pulse compared to electron beams produced by a conventional radiofrequency accelerator used for intraoperative radiation therapy. Blood samples from two different donors were exposed to six radiation doses ranging from 0 to 2 Gy. Relative biological effectiveness (RBE) for micronucleus induction was calculated from the alpha coefficients for electrons compared to X rays (RBE = alpha laser/alpha X rays). Cell viability was monitored in the OVCAR-3 ovarian cancer cell line using trypan blue exclusion assay at day 3, 5 and 7 postirradiation (2, 4, 6, 8 and 10 Gy). The RBE values obtained by comparing the alpha values were 1.3 and 1.2 for the two donors. Mean telomere length was also found to be reduced in a significant dose-dependent manner after irradiation with both electrons and X rays in both donors studied. Our findings showed a radiobiological response as mirrored by the induction of micronuclei and shortening of telomere as well as by the reduction of cell survival in blood samples and cancer cells exposed in vitro to laser-generated electron bunches. Additional studies are needed to improve preclinical validation of the radiobiological characteristics and efficacy of laser-driven electron accelerators in the future. (c) 2016 by Radiation Research Society

Introduction and objectives Single-nucleotide polymorphisms within a microRNA binding site can have different effects on gene expression, influencing the risk of disease. This study aimed to evaluate the association between single-nucleotide polymorphisms and haplotypes in the 3’UTR of the GATA4 gene and congenital heart disease risk. Methods Bioinformatics algorithms were used to analyze single-nucleotide polymorphisms in putative microRNA-binding sites of GATA4 3’UTR and to calculate the difference in free energy of hybridization (ΔFE, kcal/mol) for each wild-type vs the variant allele. Results The study population comprised 146 Caucasian patients (73 males; 6.68 ± 7.79 years) and a 265 healthy newborn participants (147 males). The sum of all |ΔFE| was considered to predict the biological importance of single-nucleotide polymorphisms binding more microRNAs. Next, the 4 polymorphisms (+1158 C > T, +1256 A > T, +1355 G > A, +1521 C > G) with the highest predicted |ΔFE tot | (9.91, 14.85, 11.03, 21.66 kcal/mol, respectively) were genotyped in a case-control study (146 patients and 250 controls). Applying a correction for multiple testing only the +1158 T allele was found to be associated with a reduced risk showing significant difference between patients and controls. Haplotype analysis showed that the T-T-G-C haplotype (more uncommon in congenital heart diseases than in controls) was associated with a significantly decreased risk ( P = .03), while the rare C-A-A-C haplotype, which was very uncommon in controls (0.3%) compared with the disease (2.4%), was associated with a 4-fold increased risk of disease ( P = .04). Conclusions Common variants in 3’UTR of the GATA4 gene jointly interact, affecting the congenital heart disease susceptibility, probably by altering microRNA posttranscriptional regulation.

We present a laser-driven source of electron bunches with average energy 260 keV and picosecond duration, which has been setup for radiobiological tests covering the previously untested sub-MeV energy range. Each bunch combines high charge with short duration and sub-millimeter range into a record instantaneous dose rate, as high as 10(9) Gy s(-1). The source can be operated at 10 Hz and its average dose rate is 35 mGy s(-1). Both the high instantaneous dose rate and high level of relative biological effectiveness, attached to sub-MeV electrons, make this source very attractive for studies of ultrafast radiobiology on thin cell samples. The source reliability, in terms of shot-to-shot stability of features such as mean energy, bunch charge and transverse beam profile, is discussed, along with a dosimetric characterization. Finally, a few preliminary biological tests performed with this source are presented. .

Congenital heart defects (CHDs) are the most prevalent of all birth malformations arising from the complex interplay of environmental exposures and genes. Modifiable environmental risk factors are still largely unknown, especially for paternal exposure. The aim of the present study was to examine the association between the environmental exposures of both parents and CHD risk and to explore the modification effect of metabolizing gene polymorphisms in children who lack the genetic capacity to produce the glutathione S-transferase (GST) GSTM1 and GSTT1 enzymes. A total of 330 parents of a child with CHD and 330 parents of a child without any congenital malformations were compared in terms of lifestyle habits and toxicant exposure. GST gene polymorphisms were investigated in 180 patients with CHD (104 males, age 4.9 ± 5.8 years). Paternal smoking (≥15 cigarettes/day) was significantly associated with CHD risk (odds ratio [OR] 2.1, 95% confidence interval [CI] 1.3 to 3.5, p = 0.002). Both maternal (OR 2.6, 95% CI 1.6 to 4.2, p

Diseases related to lamin A/C mutations (laminopathies) are extremely heterogeneous. The common cardiac phenotype is idiopathic dilated cardiomyopathy with atrioventricular block and/or arrhythmias. Moreover, patients with lamin A/C gene mutations are at increased risk for sudden cardiac death. Here we present a family with a strong positive history of sudden cardiac death in presence of idiopathic dilated cardiomyopathy and cardiac conduction abnormalities, related to a novel lamin A/C mutation in exon 3.