Silvia Pulignani

Maternal and Paternal Environmental Risk Factors, Metabolizing GSTM1 and GSTT1 Polymorphisms, and Congenital Heart Disease

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 <0.0001) and paternal (OR 2.5, 95% CI 1.6 to 3.8, p <0.0001) occupational/environmental exposures increased the risk of CHD. Also, a significant additive risk (OR 4.5, 95% CI 2.5 to 8.3, p <0.0001) was found when both parents were exposed to toxicants. Both maternal (OR 3.6, 95% CI 1.1 to 11.2, p = 0.03) and paternal (OR 3.3, 95% CI 1.0 to 10.8, p = 0.03) exposure to toxicants increased the CHD risk in children who carried the combined null GST genotypes. The effect for the combined null GST genotypes was also stronger (OR 6.5, 95% CI 1.5 to 28.0) when both parents were exposed. In conclusion, paternal smoking and exposure to toxicants for both parents affect the risk of children with CHD. Polymorphisms in GST genes can modify a persons risk of toxicant exposure-induced disease.

Congenital Heart Disease: The Crossroads of Genetics, Epigenetics and Environment

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 with intermediate atrioventricular canal defect, and aortic coarctation: Functional characterization of a de novo TBX5 mutation

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.

Genetics of congenital heart defects: is it not all in the DNA?

Wereadwith great interest the paper byWu et al on the genetic analysis of the promoter region of the GATA4 gene in patients with ventricular septal defect (VSD). The authors provide new important ideas about the potential role of genetic variants within regulatory region rather than the codifying sequences that may contribute to the etiology of VSD, the most common type of congenital heart defect (CHD). In this study, 5 heterozygous sequence variants were found within the promoter region of GATA4 gene in VSD patients but in none of the healthy controls. Although these variants do not interrupt the regulatory promoter regions, they seem to significantly alter the transcriptional activity of GATA4 gene promoter, which may contribute to the VSD. In another study published by the same authors, functional analysis showed that sequence variants within promoter regions significantly enhance the transcriptional activities of the NKX2-5 gene, which may lead to upregulated NKX2-5 gene expression, contributing to the VSD etiology. We believe that these observations underlie that the causative factors and the molecular mechanisms involved in the CHD etiology still remain largely elusive. In recent years, several lines of evidence have highlighted the importance of GATA4, in association with a variety of binding partners like NKX2-5, in a specific transcriptional complex that confer tissue-specific gene expression during cardiogenesis and that can be altered during the development of CHD. Indeed, mutations leading to gene haploinsufficiency in key cardiac transcription factors (TFs) are responsible for inherited and sporadic CHDs. Nevertheless, the study of genetic basis of CHDs is complicated by the fact that a given structural defect can be caused by more than one gene because TFs

miRNome Profiling in Bicuspid Aortic Valve-Associated Aortopathy by Next-Generation Sequencing

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.

Genetic and Epigenetic Mechanisms Linking Air Pollution and Congenital Heart Disease

Epidemiological studies strongly suggest that parental air pollutants exposure during the periconceptional period may play a major role in causing fetal/newborn malformations, including a frequent heterogeneity in the methods applied and a difficulty in estimating the clear effect of environmental toxicants. Moreover, only some couples exposed to toxicants during the pre-conception period give birth to a child with congenital anomalies. The reasons for such phenomena remain elusive but they can be explained by the individual, innate ability to metabolize these contaminants that eventually defines the ultimate dose of a biological active toxicant. In this paper, we reviewed the major evidence regarding the role of parental air pollutant exposure on congenital heart disease (CHD) risk as well as the modulating effect on detoxification systems. Finally, major epigenetic alterations induced by adverse environment contaminants have been revised as possible mechanisms altering a correct heart morphogenesis.

[Genetic screening of Gata4 and Nkx2.5 mutations in hereditary congenital heart defects: 5 familial cases].

Single gene mutations in Gata4 and Nkx2.5 genes have been identified as a causative factor for various clinical forms of hereditary congenital heart diseases (CHDs), especially for cardiac septal defects. However, the role of Gata4 and Nkx2.5 mutations in familial CHD is not clear yet. We report 5 cases of familial CHD with a positive history of cardiac septal defects. Our data suggest that mutations of either the Gata4 or Nkx2.5 genes are very uncommonly found in familial cases of CHD, supporting the genetic heterogeneity of cardiac congenital defects and the limitation of genetic testing in clinical setting.

MicroRNAs and Congenital Heart Disease: Where Are We Now?

With more than 20% of infant deaths and about 40% of prenatal deaths each year, congenital heart diseases (CHD) are the most common congenital disorders in newborns, thus representing a growing burden for health care systems. Furthermore, an increasing number of children affected by even the most complex forms of CHD are surviving to adulthood, exceeding the pediatric CHD cohort and thus requiring lifelong specialized cardiac care.As a consequence, there is an urgent need to better understand the genetic and molecular mechanisms associated with the disease in order to establish new preventive strategies and treatments. Several decades of intensive research have revealed the highly complicated regulatory networks governing cardiovascular development, underscoring the importance of genetic factors as well as environmental cues in the pathogenesis of CHD. Recently, a novel mechanism involving posttranscriptional regulation by microRNAs (miRNAs) has emerged as a central regulator of embryonic heart development. MicroRNAs are a class of small and evolutionarily preserved regulatory RNAs, approximately 20 to 26 nucleotides in length at a mature stage. These molecules act at a posttranscriptional level primarily by an imperfect base pairing with the mRNA target in a sequence-specific manner, resulting in translational repression and gene silencing (Figure 1). From reports published to date, various forms of CHDs have been associated with altered expression of specific miRNAs and associated target genes, thus suggesting that a proper regulation of miRNA expression levels during cardiogenesis might be crucial for CHD prevention. During the last few decades, deciphering the significance of miRNA-mediated posttranscriptional regulation in CHD pathology has benefited from extensive model organism studies that have provided new insights into heart development. Specifically, earlier studies have examined the tissue-specific deletion of enzymes essential for miRNA biogenesis (eg, Dicer, Drosha, Ago2, DGCR8) in mice and zebrafish models. Indeed, knock-out models resulted in early embryonic lethality due to general growth arrest and defects in heart development, demonstrating the importance of miRNAs in the cardiovascular system. Such roles have been further confirmed by gainor lossof-function experiments, indicating that a fine balance in miRNA abundance is fundamental to maintaining the cardiac homeostasis and that a deviation from such a balance may play a substantial role in cardiovascular disease. Cardiac muscle is enriched with numerous miRNAs, such as miR-1, miR-133, miR-499 and miR-208, which are abundantly, but not exclusively, expressed in the cardiac tissue. Studies on the developing murine and Xenopus laevis heart demonstrated that misexpression of such muscle-specific microRNAs altered cardiac tissue formation, leading to a thin ventricular wall and early embryonic lethality due to severe cardiac malformations. Recent high-throughput technologies combining bioinformatic and molecular analysis have definitively established the power of such miRNAs to modulate, and even control, fundamental cardiac transcription factors, such as Gata4, MEF2C, Tbx1, cardiac myosin heavy chain genes and SRF, whose deregulation have been previously associated with the development of CHD. Additional ubiquitously expressed miRNAs have been functionally analyzed, including miR-17-92, miR-195, miR-196a and miR-363, and have been demonstrated to play a role in controlling myocardial differentiation during mammalian embryogenesis. Indeed, additional in vivo evidence has demonstrated that altered expression of such miRNAs may induce conotruncal heart malformations and/or septal defects in distinct animal models, probably by repressing key cardiac progenitor genes, such as Isl1, Tbx1, Hoxb8, and Hand1. In this regard, it is intriguing that miRNAs may act through feedback loops to regulate their own expression and that of their target mRNAs. This is the case of miR-1 in muscle cells, which can modify the expression of histone deacetylase 4 (HDAC4), a repressor of MEF2 transcription factor, which, in turn, activates miR-1 and other target mRNAs. This fine regulation feedback loop might play a key role in cellular proliferation and differentiation, probably contributing to structural and/or functional cardiacrelated disorders. Altogether, the findings of in vivo and in vitro studies have definitively created the optimal background for human studies, characterizing a wide range of miRNAs that might play a role in the development of CHD in humans when dysregulated. Notably, deregulated miRNA expression patterns in humans have been predominantly documented in cyanotic diseases and ventricular septal defects, highlighting the presence of an altered miRNA profile, mostly in the myocardial human tissue. Specifically, multiple differentially-expressed miRNAs, including miR-1, miR206, miR-421, miR-940, miR-181c and miR-138, have been reported to be associated with tetralogy of Fallot and septal Rev Esp Cardiol. 2019;72(1):7–9

Prognostic value of mitochondrial DNA4977 deletion and mitochondrial DNA copy number in patients with stable coronary artery disease

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 (<25th) had significantly increased risk for MACEs (HR: 3.73; 95% CI: 1.79-7.79; p=0.0005). 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.

Radiobiological Effectiveness of Ultrashort Laser-Driven Electron Bunches: Micronucleus Frequency, Telomere Shortening and Cell Viability.

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.