Congenital heart disease and pulmonary arterial hypertension in South America (2013 Grover Conference series).

South America is a territory of 17,819,100 km(2), where ∼388 million people live in 13 countries. In the region, access to medical assistance (e.g., for treatment of cardiovascular disorders) is relatively easy in metropolitan areas but difficult in remote places such as the Andes and the Amazon. Altitudes up to ∼6,700 m influence the prevalence of congenital heart disease (CHD) and pulmonary arterial hypertension (PAH). In tertiary centers, CHD is now treated earlier in life but remains an important etiology of PAH. In adolescents and adults with PAH assisted at institutions devoted to treatment of cardiovascular disorders, the relative frequency of PAH-CHD (∼50%-60%) is even higher than that of idiopathic PAH. In one big tertiary center in São Paulo, Brazil, the prevalence of advanced PAH in children and adults with CHD is 1.2% and 4.2%, respectively. In young patients with cardiac septal defects (aged up to 2 years), pulmonary vascular abnormalities are a matter of concern in the decision about operability in 4.9% of cases. Access to specific PAH drugs is not uniform in South America, being unrealistic in remote places. In big cities, there are real possibilities for management of complex CHD, neonatal disorders, and even cardiac transplantation. Research activities have been implemented at clinical, translational, and basic levels. However, because of social and economic inequalities and political issues, access to best standards of medical care remains a problem in the region as a whole.

Genetic and environmental risk factors in congenital heart disease functionally converge in protein networks driving heart development.

Congenital heart disease (CHD) occurs in ∼1% of newborns. CHD arises from many distinct etiologies, ranging from genetic or genomic variation to exposure to teratogens, which elicit diverse cell and molecular responses during cardiac development. To systematically explore the relationships between CHD risk factors and responses, we compiled and integrated comprehensive datasets from studies of CHD in humans and model organisms. We examined two alternative models of potential functional relationships between genes in these datasets: direct convergence, in which CHD risk factors significantly and directly impact the same genes and molecules and functional convergence, in which risk factors significantly impact different molecules that participate in a discrete heart development network. We observed no evidence for direct convergence. In contrast, we show that CHD risk factors functionally converge in protein networks driving the development of specific anatomical structures (e.g., outflow tract, ventricular septum, and atrial septum) that are malformed by CHD. This integrative analysis of CHD risk factors and responses suggests a complex pattern of functional interactions between genomic variation and environmental exposures that modulate critical biological systems during heart development.

Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition. METHODS: Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients. RESULTS: In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r(2)=0.33 and r(2)=0.34 respectively, p<0.0015). In patients with a low platelet count (<150x10(9) platelets/L), both densities were increased relative to controls (82% and 33% respectively, p<0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r(2)=0.33 and r(2)=0.29, p<0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p<0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p<0.05). CONCLUSIONS: There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count.

NKX2.5 mutations in patients with non-syndromic congenital heart disease.

BACKGROUND: Cardiac development is a complex and multifactorial biological process. Heterozygous mutations in the transcription factor NKX2.5 are between the first evidence of a genetic cause for congenital heart defects in human beings. In this study, we evaluated the presence and frequency of mutations in the NKX2.5 gene on 159 unrelated patients with a diverse range of non-syndromic congenital heart defects (conotruncal anomalies, septal defects, left-sided lesions, right-sided lesions, patent ductus arteriosus and Ebstein's anomaly). METHODS: The coding region of the NKX2.5 locus was amplified by polymerase chain reaction and mutational analysis was performed using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. RESULTS: We identified two distinct mutations in the NKX2.5 coding region among the 159 (1.26%) individuals evaluated. An Arg25Cys mutation was identified in a patient with Tetralogy of Fallot. The second mutation found was an Ala42Pro in a patient with Ebstein's anomaly. CONCLUSIONS: The association of NKX2.5 mutations is present in a small percentage of patients with non-syndromic congenital heart defects and may explain only a few cases of the disease. Screening strategies considering the identification of germ-line molecular defects in congenital heart disease are still unwarranted and should consider other genes besides NKX2.5.

ALDH1A2 (RALDH2) genetic variation in human congenital heart disease.

BACKGROUND: Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus. METHODS: One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay. RESULTS: We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls. CONCLUSION: In summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.

De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot.

Tetralogy of Fallot (TOF), the most common severe congenital heart malformation, occurs sporadically, without other anomaly, and from unknown cause in 70% of cases. Through a genome-wide survey of 114 subjects with TOF and their unaffected parents, we identified 11 de novo copy number variants (CNVs) that were absent or extremely rare (<0.1%) in 2,265 controls. We then examined a second, independent TOF cohort (n = 398) for additional CNVs at these loci. We identified CNVs at chromosome 1q21.1 in 1% (5/512, P = 0.0002, OR = 22.3) of nonsyndromic sporadic TOF cases. We also identified recurrent CNVs at 3p25.1, 7p21.3 and 22q11.2. CNVs in a single subject with TOF occurred at six loci, two that encode known (NOTCH1, JAG1) disease-associated genes. Our findings predict that at least 10% (4.5-15.5%, 95% confidence interval) of sporadic nonsyndromic TOF cases result from de novo CNVs and suggest that mutations within these loci might be etiologic in other cases of TOF.

Lack of evidence of association between MTHFR C677T polymorphism and congenital heart disease in a TDT study design.

INTRODUCTION: Hyperhomocysteinemia is frequently associated with congenital defects of the heart and neural tube. A common missense mutation in the MTHFR gene (C to T substitution at position 677 changing valine to alanine) produces a variant with reduced enzymatic action, resulting in higher plasma levels of homocysteine. The aim of this study is to investigate whether MTHFR C677T functional genetic variant is associated with an increased risk of congenital heart disease (CHD) development using a family-based case-control design and the Transmission Disequilibrium Test (TDT) approach. METHODS: We selected 91 consecutive patients with congenital heart disease for the study. From these patients we were able to obtain samples on 147 parents. The C677T polymorphism at the MTHFR gene was determined from each participant. RESULTS: A statistically significant association was disclosed in univariate analysis using a family-based case-control design (p<0.0001 assuming an additive genetic model, p<0.0001 assuming a dominant genetic model, and p=0.01 assuming a recessive genetic model). This association was explained by an increased frequency of the T allele in patients as compared to their fathers. However, by using a TDT approach a highly non-significant result was obtained and no association could be defined between this locus and congenital heart disease. CONCLUSIONS: We did not find sufficient evidence for an association between MTHFR C677T genotype and congenital heart disease in our study group. Previous reports on such association may be due to population genetic structure.

Likelihood of left main coronary artery compression based on pulmonary trunk diameter in patients with pulmonary hypertension.

PURPOSE: In patients with pulmonary hypertension, extrinsic compression of the left main coronary artery by a dilated pulmonary trunk may cause angina, left ventricular ischemia, and sudden death. We assessed coronary artery compression in relation to pulmonary trunk diameter and other demographic, echocardiographic, hemodynamic, and scintigraphic variables. METHODS: Thirty-six patients (aged 15 to 86 years) with pulmonary hypertension, either idiopathic or associated with congenital heart disease, were enrolled. Left main coronary artery compression was defined angiographically as > or =50% obstruction associated with downward displacement of the vessel. Pulmonary trunk and aortic diameters were measured by transthoracic echocardiography. RESULTS: Twenty-six patients had angina, of whom 7 had left coronary artery compression. Compression was related to pulmonary trunk diameter (P = 0.002) and to the ratio of pulmonary trunk diameter to aortic diameter (P = 0.02). Compression was not seen at pulmonary artery diameters <40 mm; among 19 patients with values > or =40 mm, the rate was 37%. Similarly, compression did not occur at pulmonary trunk to aortic diameter ratios <1.21; among 27 patients with ratios > or =1.21, the rate was 26%. CONCLUSION: In pulmonary hypertension, noninvasive measurement of pulmonary trunk diameter may be helpful in determining the likelihood of left coronary artery compression and in selecting patients for diagnostic coronary angiography.

Hipertensão arterial por coarctação de aorta em adultos / Arterial hypertension due to coarctation of the aorta in adults

A hipertensão arterial secundária à coarctação da aorta constitui-se em uma situação preocupante pela sua alta prevalência, até mesmo entre os pacientes operados com sucesso. Ainda não existe um consenso que estabeleça a razão pela qual esse evento ocorre. Tanto os pacientes que seguem a história natural quanto aqueles que representam a evolução pós-operatória tardia da malformação estão expostos a riscos de morbidade e mortalidade cardiovascular decorrentes das complicações da hipertensão arterial. Neste artigo, são abordados os fatores relacionados à hipertensão arterial em portadores de coarctação de aorta, com ênfase nos aspectos anatômicos da malformação, da história natural e da evolução pós-operatória tardia. Também são discutidos os resultados da cirurgia sobre o comportamento da pressão arterial e sobre a morbidade e mortalidade cardiovascular em pacientes adultos, e as principais complicações da coarctação de aorta não tratada.