Integrated multi-omic characterization of congenital heart disease.

The heart, the first organ to develop in the embryo, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of patients with CHD survive into adulthood, but many suffer premature death from heart failure and non-cardiac causes1. Here, to gain insight into this disease progression, we performed single-nucleus RNA sequencing on 157,273 nuclei from control hearts and hearts from patients with CHD, including those with hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot, two common forms of cyanotic CHD lesions, as well as dilated and hypertrophic cardiomyopathies. We observed CHD-specific cell states in cardiomyocytes, which showed evidence of insulin resistance and increased expression of genes associated with FOXO signalling and CRIM1. Cardiac fibroblasts in HLHS were enriched in a low-Hippo and high-YAP cell state characteristic of activated cardiac fibroblasts. Imaging mass cytometry uncovered a spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD, in agreement with the predilection in CHD to infection and cancer2. Our comprehensive phenotyping of CHD provides a roadmap towards future personalized treatments for CHD.

Molecular Pathways and Animal Models of Tetralogy of Fallot and Double Outlet Right Ventricle.

Tetralogy of Fallot and double-outlet right ventricle are outflow tract (OFT) alignment defects situated on a continuous disease spectrum. A myriad of upstream causes can impact on ventriculoarterial alignment that can be summarized as defects in either i) OFT elongation during looping morphogenesis or ii) OFT remodeling during cardiac septation. Embryological processes underlying these two developmental steps include deployment of second heart field cardiac progenitor cells, establishment and transmission of embryonic left/right information driving OFT rotation and OFT cushion and valve morphogenesis. The formation and remodeling of pulmonary trunk infundibular myocardium is a critical component of both steps. Defects in myocardial, endocardial, or neural crest cell lineages can result in alignment defects, reflecting the complex intercellular signaling events that coordinate arterial pole development. Importantly, however, OFT alignment is mechanistically distinct from neural crest-driven OFT septation, although neural crest cells impact indirectly on alignment through their role in modulating signaling during SHF development. As yet poorly understood nongenetic causes of alignment defects that impact the above processes include hemodynamic changes, maternal exposure to environmental teratogens, and stochastic events. The heterogeneity of causes converging on alignment defects characterizes the OFT as a hotspot of congenital heart defects.

Brainstem and cerebellar volumes at magnetic resonance imaging are smaller in fetuses with congenital heart disease.

BACKGROUND: Congenital heart disease is associated with an increased risk of smaller brain volumes and structural brain damage, and impaired growth of supratentorial brain structures in utero has been linked to poor neurodevelopmental outcomes. However, little is known on brainstem and cerebellar volumes in fetuses with congenital heart disease. Moreover, it is not clear whether impaired infratentorial growth, if present, is associated with only certain types of fetal cardiac defects or with supratentorial brain growth, and whether altered biometry is already present before the third trimester. OBJECTIVE: This study aimed to investigate brainstem and cerebellar volumes in fetuses with congenital heart disease and to compare them to infratentorial brain volumes in fetuses with normal hearts. Secondarily, the study aimed to identify associations between infratentorial brain biometry and the type of cardiac defects, supratentorial brain volumes, and gestational age. STUDY DESIGN: In this retrospective case-control study, 141 magnetic resonance imaging studies of 135 fetuses with congenital heart disease and 141 magnetic resonance imaging studies of 125 controls with normal hearts at 20 to 37 gestational weeks (median, 25 weeks) were evaluated. All cases and controls had normal birthweight and no evidence of structural brain disease or genetic syndrome. Six types of congenital heart disease were included: tetralogy of Fallot (n=32); double-outlet right ventricle (n=22); transposition of the great arteries (n=27); aortic obstruction (n=24); hypoplastic left heart syndrome (n=22); and hypoplastic right heart syndrome (n=14). First, brainstem and cerebellar volumes of each fetus were segmented and compared between cases and controls. In addition, transverse cerebellar diameters, vermian areas, and supratentorial brain and cerebrospinal fluid volumes were quantified and differences assessed between cases and controls. Volumetric differences were further analyzed according to types of cardiac defects and supratentorial brain volumes. Finally, volume ratios were created for each brain structure ([volume in fetus with congenital heart disease/respective volume in control fetus] × 100) and correlated to gestational age. RESULTS: Brainstem (cases, 2.1 cm3 vs controls, 2.4 cm3; P<.001) and cerebellar (cases, 3.2 cm3 vs controls, 3.4 cm3; P<.001) volumes were smaller in fetuses with congenital heart disease than in controls, whereas transverse cerebellar diameters (P=.681) and vermian areas (P=.947) did not differ between groups. Brainstem and cerebellar volumes differed between types of cardiac defects. Overall, the volume ratio of cases to controls was 80.8% for the brainstem, 90.5% for the cerebellum, and 90.1% for the supratentorial brain. Fetuses with tetralogy of Fallot and transposition of the great arteries were most severely affected by total brain volume reduction. Gestational age had no effect on volume ratios. CONCLUSION: The volume of the infratentorial brain, which contains structures considered crucial to brain function, is significantly smaller in fetuses with congenital heart disease than in controls from midgestation onward. These findings suggest that impaired growth of both supra- and infratentorial brain structures in fetuses with congenital heart disease occurs in the second trimester. Further research is needed to elucidate associations between fetal brain volumes and neurodevelopmental outcomes in congenital heart disease.

The Case of a 44-Year-Old Survivor of Unrepaired Tetralogy of Fallot, Right Aortic Arch and Abdominal Aortopulmonary Collateral Vessels.

The most common congenital cyanotic heart disease is described in the literature as the Tetralogy of Fallot. This abnormality is characterized by the presence of ventricular septal defect (VSD), obstruction of the right ventricular (RV) outflow tract, right ventricular hypertrophy, and overriding aorta. In patients with pulmonary atresia with ventricular septal defect (PA/VSD), major aortopulmonary collateral arteries (MAPCA) are common; however, although some of them do not have PA/VSD, they do have other particular anatomical variants. The case we are presenting in this article is a rare mild symptomatic adult noncorrected TOF, with preserved RV function, right aortic arch, and MAPCAs ("classic" thoracic MAPCAs but also abdominal MAPCAs). The anatomy of a complex congenital defect is well illustrated by cardiac magnetic resonance (CMR) and computer tomography angiography (CTA), and these imaging techniques are mostly used to understand the relative clinical "silence" TOF. Imaging scans thus play a key role in the evaluation of these patients, being very important to know the indications and limitations of each method, but also to learn to combine them with each other depending on the clinical picture of the patient's presentation. Additionally, the close collaboration between clinicians and imagers is essential for a correct, complete and detailed preoperative evaluation, being subsequently essential for cardiovascular surgeons, the whole team thus deciding the best therapeutic management.

A rare mutant of OFD1 gene responsible for Joubert syndrome with significant phenotype variation.

Joubert syndrome (JBTS), a rare genetic disorder resulted from primary cilium defects or basal-body dysfunction, is characterized by agenesis of cerebellar vermis and abnormal brain stem. Both genotypes and phenotypes of JBTS are highly heterogeneous. The identification of pathogenic gene variation is essential for making a definite diagnosis on JBTS. Here, we found that hypoplasia of cerebellar vermis occurred in three male members in a Chinese family. Then, we performed whole exome sequencing to identify a novel missense mutation c.599T > C (p. L200P) in the OFD1 gene which is the candidate gene of X-linked JBTS (JBST10). The following analysis showed that the variant was absent in the 1000 Genomes, ExAC and the 200 female controls; the position 200 Leucine residue was highly conserved across species; the missense variant was predicted to be deleterious using PolyPhen-2, PROVEAN, SIFT and Mutation Taster. The OFD1 expression was heavily lower in the proband and an induced male fetus compared with a healthy male with a wild-type OFD1 gene. The in vitro expression analysis of transiently transfecting c.599T or c.599C plasmids into HEK-293T cells confirmed that the missense mutation caused OFD1 reduction at the protein level. And further the mutated OFD1 decreased the level of Gli1 protein, a read-out of Sonic hedgehog (SHH) signaling essential for development of central neural system. A known pathogenic variant c.515T > C (p. L172P) showed the similar results. All of these observations suggested that the missense mutation causes the loss function of OFD1, resulting in SHH signaling impairs and brain development abnormality. In addition, the three patients have Dandy-Walker malformation, macrogyria and tetralogy of Fallot, respectively, the latter two of which are firstly found in JBTS10 patients. In conclusion, our findings expand the context of genotype and phenotype in the JBTS10 patients.

Second instance of co-occurring 22q11.2 deletion syndrome and Williams syndrome.

We present an 18-month-old male with Tetralogy of Fallot, retrognathia, short stature, global developmental delay, and dysmorphic features who was found to have dual diagnoses of both Williams syndrome and 22q11.2 deletion syndrome (22q11.2DS). To our knowledge, this is the second case of such a co-occurrence documented in the medical literature. Our patient presents with a blended physical phenotype of these two conditions and a behavioral phenotype that is distinct from what is typically observed in either disorder alone. We compare our patient's phenotype to the previously reported case and to the typical phenotypes for each individual condition. Additionally, we discuss why the occurrence of these two disorders together seems to be so rare, and the benefit of a genetics evaluation to an inpatient service team and the patient.

Congenital heart disease in school children in Lagos, Nigeria: Prevalence and the diagnostic gap.

Congenital heart disease (CHD) in low-and-middle income countries (LMIC) is often characterized by late presentation resulting from inadequate screening and healthcare access in these regions. Accurate estimates of the burden of CHD among school children are often lacking. The objective of this study was to determine the prevalence and distribution of CHD among school children in two communities (urban and semi-urban) in south western Nigeria. Using clinical assessment and portable echocardiography, 4107 school children aged 5 years to 16 years in Lagos, Nigeria, were selected using a multistage sampling procedure and screened for CHD. Diagnosis of CHD was made after echocardiography. Children identified with CHD were referred to a tertiary hospital for appropriate cardiac care. The 4,107 children screened had a mean age of 11.3 ± 2.7 years and 53.7% were females. Twenty seven children had echocardiography-confirmed CHD, representing a prevalence of CHD among school children in Lagos, Nigeria of 6.6 per 1000 children. Acyanotic CHD constituted 96.3% of detected cases. Two children diagnosed with CHD (Tetralogy of Fallot and severe pulmonary valve stenosis respectively) had successful intervention. The prevalence of previously undiagnosed CHD among school children in Lagos Nigeria is substantial and highlights gaps in the health care system and school health programs. Echocardiographic screening of school children provides an opportunity for missed early diagnosis and treatment of CHD and reduces the prevalence of first-diagnosed CHD in adulthood. Therefore, focused clinical examination of school children followed by echocardiography is a strategy that could bridge this diagnostic and treatment gap in CHD.

Ex vivo cardiovascular magnetic resonance diffusion weighted imaging in congenital heart disease, an insight into the microstructures of tetralogy of Fallot, biventricular and univentricular systemic right ventricle.

PURPOSE: Common types of congenital heart disease exhibit a variety of structural and functional variations which may be accompanied by changes in the myocardial microstructure. We aimed to compare myocardial architecture from magnetic resonance diffusion tensor imaging (DTI) in preserved pathology specimens. MATERIALS AND METHODS: Pathology specimens (n = 24) formalin-fixed for 40.8 ± 7.9 years comprised tetralogy of Fallot (TOF, n = 10), dextro-transposition of great arteries (D-TGA, n = 8) five with ventricular septal defect (VSD), systemic right ventricle (n = 4), situs inversus totalis (SIT, n = 1) and levo-TGA (L-TGA, n = 1). Specimens were imaged using a custom spin-echo sequence and segmented automatically according to tissue volume fraction. In each specimen T1, T2, fractional anisotropy, mean diffusivity, helix angle (HA) and sheet angle (E2A) were quantified. Pathologies were compared according to their HA gradient, HA asymmetry and E2A mean value in each myocardial segment (anterior, posterior, septal and lateral walls). RESULTS: TOF and D-TGA with VSD had decreased helix angle gradient by - 0.34°/% and remained symmetric in the septum in comparison to D-TGA without VSD. Helix angle range was decreased by 45°. It was associated with a decreased HA gradient in the right ventricular (RV) wall, i.e. predominant circumferential myocytes. The sheet angle in the septum of TOF was opposing those of the left ventricular (LV) free wall. Univentricular systemic RV had the lowest HA gradient (- 0.43°/%) and the highest HA asymmetry (75%). HA in SIT was linear, asymmetric, and reversed with a sign change at about 70% of the depth at mid-ventricle. In L-TGA with VSD, HA was asymmetric (90%) and its gradients were decreased in the septum, anterior and lateral wall. CONCLUSION: The organization of the myocytes as determined by DTI differs between TOF, D-TGA, L-TGA, systemic RV and SIT specimens. These differences in cardiac structure may further enlighten our understanding of cardiac function in these diverse congenital heart diseases.

An unusual presentation of developmental anomalies of the cardiovascular system including tetralogy of fallot, double outlet right ventricle, patent foramen ovale and persistent right aortic arch in a Friesian calf.

BACKGROUND: Congenital heart diseases are occasionally encountered in the bovine species. Ventricular septal defects (VSD) and atrial septal defects (ASD) are reported to be the most common; however, a vast collection have been reported [1, 2]. Congenital heart diseases is thought to represent less than 3% of all congenital abnormalities in calves [3]. Various cardiac anomalies arise due to defective embryologic development such as defects of the septae or the cardiac chambers [2]. The exact aetiology of these congenial heart anomalies remains to be fully elucidated [4]. VSDs appear to be the most common congenital cardiac anomaly in calves. Other diseases can be subdivided into cyanotic (e.g. ASD or patent ductus arteriosus) and non-cyanotic (e.g. tetralogy of fallot or eisenmengers complex) [5, 6]. An exceptional presentation of an array of congenital anomalies was identified in a Friesian heifer calf. To the authors' knowledge this concurrent collection of congenital abnormalities has never been reported in this species. CASE PRESENTATION: A 3-day old Friesian heifer presented with a history since birth of regurgitation post feeding. The main finding on clinical examination was tachypnoea with a holosystolic murmur. Echocardiography identified a VSD, patent foramen ovale (PFO) (both with left to right blood flow) and tricuspid insufficiency. The calf was subsequently euthanised and underwent gross post-mortem examination. A persistent right aortic arch (PRAA) was identified. The cardiac anomalies identified on the echocardiogram were confirmed along with additional abnormalities; double outlet right ventricle (DORV), partial transposition of the great vessels, pulmonic stenosis, hypoplasia of the right branch of the pulmonary artery and right ventricular hypertrophy. The final diagnosis was Tetralogy of Fallot with DORV, PFO and PRAA. The lungs appeared oedematous and congested due to cardiac malfunction and cranioventral aspiration pneumonia. Free serous fluid was identified in the thoracic cavity. Unilateral renal agenesis of the left kidney was an incidental finding but is of note due to its coexistence with the cardiac abnormalities. CONCLUSIONS: This is an unusual case as it features numerous congenital abnormalities that appeared to negate each other allowing capability with life. To the authors' knowledge, this collection of concurrent cardiac anomalies has not been previously reported in bovines.

Separate arch origin of the left external carotid artery with common trunk giving rise to the left internal carotid artery and left subclavian artery.

We present a case of a 3-year-old boy with tetralogy of Fallot having the direct origin of the left external carotid artery from the aorta with a common trunk giving rise to the left internal carotid artery and left subclavian artery, in a right-sided aortic arch. We also highlight the potential implications in management.

Telocytes in the Myocardium of Children with Congenital Heart Disease Tetralogy of Fallot.

Telocytes, a new type of interstitial stem cells with long thin processes that form a three-dimensional network around cardiomyocytes, vessels, and nerve fibers were described in the myocardium of children with tetralogy of Fallot. Two types of morphologically different telocytes, spindle-shaped and rounded, were identified. Contacts of telocytes with stem cells and interstitial macrophages were found. Telocytes were more common in the immature myocardium, where the assembly of myofibrils in cardiomyocytes was not completed and small Ki-67+ cardiomyocyte progenitor cells were present. Telocytes expressed immunohistochemical markers CD117, vimentin, CD34, and CD44. Localization and ultrastructural characteristics of telocytes suggested their participation in stem cell differentiation, coordination of neoangiogenesis, and paracrine regulation of all components of the interstitium.

Effect of Losartan on Right Ventricular Dysfunction: Results From the Double-Blind, Randomized REDEFINE Trial (Right Ventricular Dysfunction in Tetralogy of Fallot: Inhibition of the Renin-Angiotensin-Aldosterone System) in Adults With Repaired Tetralogy of Fallot.

BACKGROUND: The effect of angiotensin II receptor blockers on right ventricular (RV) function is still unknown. Angiotensin II receptor blockers are beneficial in patients with acquired left ventricular dysfunction, and recent findings have suggested a favorable effect in symptomatic patients with systemic RV dysfunction. The current study aimed to determine the effect of losartan, an angiotensin II receptor blocker, on subpulmonary RV dysfunction in adults after repaired tetralogy of Fallot. METHODS: The REDEFINE trial (Right Ventricular Dysfunction in Tetralogy of Fallot: Inhibition of the Renin-Angiotensin-Aldosterone System) is an investigator-initiated, multicenter, prospective, 1:1 randomized, double-blind, placebo-controlled study. Adults with repaired tetralogy of Fallot and RV dysfunction (RV ejection fraction [EF] <50%) but without severe valvular dysfunction were eligible. Patients were randomly assigned between losartan (150 mg daily) and placebo with target treatment duration between 18 and 24 months. The primary outcome was RV EF change, determined by cardiovascular MRI in intention-to-treat analysis. RESULTS: Of 95 included patients, 47 patients received 150 mg losartan daily (age, 38.0±12.4 years; 74% male), and 48 patients received placebo (age, 40.6±11.4 years; 63% male). Overall, RV EF did not change in patients allocated to losartan (n=42) (44.4±5.1% to 45.2±5.0%) and placebo (n=46) (43.2±6.3% to 43.6±6.9%). Losartan did not significantly improve RV EF in comparison with placebo (+0.51%; 95% confidence interval, -1.0 to +2.0; P=0.50). No significant treatment effects were found on secondary outcomes: left ventricular EF, peak aerobic exercise capacity, and N-terminal pro-brain natriuretic peptide (P>0.30 for all). In predefined subgroup analyses, losartan did not have a statistically significant impact on RV EF in subgroups with symptoms, restrictive RV, RV EF<40%, pulmonary valve replacement, or QRS fragmentation. However, in a post hoc analysis, losartan was associated with improved RV EF in a subgroup (n=30) with nonrestrictive RV and incomplete remodeling (QRS fragmentation and previous pulmonary valve replacement) (+2.7%; 95% confidence interval, +0.1 to +5.4; P=0.045). CONCLUSIONS: Losartan had no significant effect on RV dysfunction or secondary outcome parameters in repaired tetralogy of Fallot. Future larger studies may determine whether there might be a role for losartan in specific vulnerable subgroups. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02010905.

Haploinsufficiency of vascular endothelial growth factor related signaling genes is associated with tetralogy of Fallot.

PURPOSE: To determine disease-associated single-gene variants in conotruncal defects, particularly tetralogy of Fallot (TOF). METHODS: We analyzed for rare loss-of-function and deleterious variants in FLT4 (VEGFR3) and other genes in the vascular endothelial growth factor (VEGF) pathway, as part of a genome sequencing study involving 175 adults with TOF from a single site. RESULTS: We identified nine (5.1%) probands with novel FLT4 variants: seven loss-of-function, including an 8-kb deletion, and two predicted damaging. In ten other probands we found likely disruptive variants in VEGF-related genes: KDR (VEGFR2; two stopgain and two nonsynonymous variants), VEGFA, FGD5, BCAR1, IQGAP1, FOXO1, and PRDM1. Detection of VEGF-related variants (19/175, 10.9%) was associated with an increased prevalence of absent pulmonary valve (26.3% vs. 3.4%, p < 0.0001) and right aortic arch (52.6% vs. 29.1%, p = 0.029). Extracardiac anomalies were rare. In an attempt to replicate findings, we identified three loss-of-function or damaging variants in FLT4, KDR, and IQGAP1 in ten independent families with TOF. CONCLUSION: Loss-of-function variants in FLT4 and KDR contribute substantially to the genetic basis of TOF. The findings support dysregulated VEGF signaling as a novel mechanism contributing to the pathogenesis of TOF.

Two-stage approach for surgical treatment of tetralogy of Fallot in underweight children: Clinical and morphological outcomes.

BACKGROUND: Two-stage surgery including right ventricular outflow tract (RVOT) stenting with subsequent total surgical repair (TSG) has been suggested as a promising curative option in infants with tetralogy of Fallot (ToF) having comorbidities such as low body weight. However, data on clinical outcomes of such approach and tissue response to RVOT stenting in underweight infants are scarce. METHODS: We recruited 16 underweight (<3 kg; average weight, 2.2 ± 0.4 and 4.7 ± 0.9 kg at the time of RVOT stenting and TSG, respectively) infants (1-3 months of age, average 28.2 ± 4.3 and 100.2 ± 22.3 days at the time of RVOT stenting and TSG, respectively) with ToF and performed RVOT stenting with the subsequent TSG. Excised stents were embedded into epoxy resin and stained by toluidine blue and basic fuchsin. RESULTS: Fifteen infants had a favorable clinical outcome, probably due to the rapid increase in the body weight, blood oxygen saturation, and left ventricular end-diastolic volume to body surface area ratio indicative of improved pulmonary perfusion. Histological analysis revealed an endothelial cell monolayer at the stent surface with notable neovascularization of stented tissues, which could potentially explain the abovementioned clinical and echocardiography improvements. The only death occurred immediately after RVOT stenting and was caused by a massive subdural hematoma, possibly provoked by grade 2 intraventricular hemorrhage 12 days before the stenting. CONCLUSIONS: We confirm RVOT stenting with the subsequent TSG as a safe and efficient surgical approach for the treatment of underweight children with ToF.

Clinical Predictors of Right Ventricular Myocardial Fibrosis in Patients With Repaired Tetralogy of Fallot.

BACKGROUND: This study aimed to identify the clinical predictors of the degree of right ventricular (RV) myocardial fibrosis in patients with repaired tetralogy of Fallot (TOF) with special focus on the RV pressure load.Methods and Results:From April 2004 to March 2017, 30 patients with repaired TOF underwent pulmonary valve replacement and concomitant RV myocardial biopsy. The stroke volume ratio (RV stroke volume/left ventricular stroke volume), RV end-diastolic volume index, and right-to-left ventricular systolic pressure ratio were evaluated with respect to their prognostic value for the degree of RV myocardial fibrosis. Significant positive linear correlations were detected between the stroke volume ratio and the degree of RV myocardial fibrosis (P=0.003, r=0.52). Patients with a right-to-left ventricular systolic pressure ratio >0.45 showed a significantly greater degree of RV myocardial fibrosis under an equivalent stroke volume ratio. CONCLUSIONS: Under conditions of RV volume overload, a right-to-left ventricular systolic pressure ratio >0.45 was a predisposing factor for progression of RV myocardial fibrosis in patients with repaired TOF.

One-Year Follow-up After Tetralogy of Fallot Total Repair Preserving Pulmonary Valve and Avoiding Right Ventriculotomy.

BACKGROUND: We reviewed our revised surgical strategy for tetralogy of Fallot (TOF) total correction to minimize early exposure to significant pulmonary regurgitation (PR) and to avoid right ventriculotomy (RV-tomy). Methods and Results: Since February 2016, we have tried to preserve, first, pulmonary valve (PV) function to minimize PR by extensive commissurotomy with annulus saving; and second, RV infundibular function by avoiding RV-tomy. With this strategy, we performed total correction for 50 consecutive patients with TOF until May 2018. We reviewed the early outcomes of 27 of 50 patients who received follow-up for ≥3 months. Mean patient age at operation was 10.2±5.0 months, and mean body weight was 8.8±1.2 kg. The preoperative pressure gradient at the RV outflow tract and the PV z-score were improved at most recent echocardiography from 82.0±7.1 to 26.8±6.4 mmHg, and from -2.35±0.49 to -0.55±0.54, respectively, during 11.1±1.6 months of follow-up after operation. One patient required re-intervention for residual pulmonary stenosis. Twenty-two patients had less than moderate PR (none, 1; trivial, 8; mild, 13), and 5 patients had moderate PR. There was no free or severe PR. CONCLUSIONS: At 1-year follow-up, the patients who underwent total TOF correction with our revised surgical strategy had acceptable results in terms of PV function. The preserved PV had a tendency to grow on short-term follow-up.

Silencing mutations in JAG1 gene may play crucial roles in the pathogenesis of Tetralogy of Fallot.

JAG1 gene through Notch signaling is implicated in cell fate decisions in early cardiac development, and mutations in several proteins in the pathway have been involved in various disorders. Tetralogy of Fallot (TOF) is the most frequent form of complicated congenital heart disease. The abnormality of TOF begins through the first eight weeks of fetal growth and is confused with ventricular septal defects, obstruction to right ventricular outflow tract, aortic dextroposition, and right ventricular hypertrophy. Hence the existence of mutations in JAG1 gene in Iranian patients with TOF is evaluated. The clinical data and peripheral blood samples were collected from 44 sporadic nonsyndromic patients with TOF and compared to 44 healthy individuals. DNA was extracted, and the exon 6 of the JAG1 gene was amplified by PCR then the PCR products were purified and sequenced. The age range in patients and the control group was 2-36 years, and the mean and standard deviation (SD) of the age in patients was (11.69 ± 7.85 years) and in control group (11.63 ± 7.99 years).  Finally, the samples were successfully sequenced, then analyzed and one synonymous variant (c.765C>T; p.Y255Y) was observed in 38 patients with frequency (86.4%) and three controls with frequency (6.8%). The c.765C>T variant is significantly associated with the pathogenesis of TOF in Iranian population.

[Ultrastructural features of cardiomyocytes in infants with tetralogy of Fallot in the first year of life]. / Osobennosti ul'trastruktury kardiomiotsitov u detei pervykh let zhizni s tetradoi Fallo.

OBJECTIVE: To analyze cardiomyocyte (CMC) ultrastructural changes in the right ventricle outflow tract (RVOT) of infants with tetralogy of Fallot (TF) in the first years of life and to compare the findings with clinical parameters in these patients. MATERIAL AND METHODS: Intraoperative RVOT myocardial biopsy specimens obtained from 51 patients aged 3-33 months with TF during radical correction of defect were investigated. CMC diameter and length were measured using the semithin myocardial sections stained with periodic acid-Schiff. The ultrathin sections were examined in the electron microscope. RESULTS: The diameter of CMCs in the RVOT of infants with TF varied significantly (7.3-17.0 µm) and averaged 10.8±2.2 µm; a large number of multinucleated CMCs were observed. There were ultrastructural signs of incomplete differentiation of CMCs: active myofibril assembly in the free sarcoplasmic region; gap junctions on the lateral surfaces of CMCs; and centrioli in their sarcoplasm. Myofibrillogenesis in babies under 6 months increased in response to hemodynamic overload and hypoxemia. In addition, organelles suggestive of the synthetic activity of CMCs, such as cisterns and vesicles of the Golgi complex and granular endoplasmic reticulum, were detected in the sarcoplasm of a number of CMCs. TF infants' myocardium also displayed focal disorders of CMC interposition; the change in the shape of myocytes was accompanied by the appearance of additional lateral insert discs. Some CMCs showed the abnormal localization of the nucleus beneath the sarcolemma, sarcoplasmic bulging areas, and dystrophic changes. CONCLUSION: There were ultrastructural features characteristic for the myocardium that was at the state of active growth and differentiation (increases in the diameter and length of CMCs and in the number of nuclei; myofibrillogenesis; signs of synthetic and proliferative activity along with insignificant dystrophic changes) in the CMCs of myocardial RVOT in infants with TF in the first years of life.

Congenital heart diseases and their association with the variant distribution features on susceptibility genes.

Congenital heart disease (CHD), one of the causes of childhood morbidity and mortality, is mainly triggered by a combination of environmental and genetic factors. Several susceptible genes, such as NKX2-5, GATA4 and TBX5, have been reported as closely related to heart and vessel development. CHD subtypes are classified into diverse clinical phenotypes, such as atrial septal defects (ASD), ventricular septal defects (VSD), tetralogy of Fallot (TOF), and Holt-Oram syndrome (HOS). Here, we summarize the associations of the genetic variants in these three genes with CHD subtypes. CHD-associated variants of NKX2-5 locate mainly in the tinman domain and the homeodomain. Mutations in the homeodomain are correlated with ASD and atrioventricular (AV) block subtypes. VSD-associated variants of GATA4 are mainly at its terminal ends. Variants of TBX5 gene are primarily in exons 3, 4, 5 and 7 and highly associated with HOS subtype. Hence, the variant distribution of NKX2-5, GATA4 and TBX5 are tightly associated with particular CHD subtypes. Further structure-modelling analysis revealed that these mutated amino acid residuals maintain their DNA-binding ability and structural stability. Therefore structural features of these genes may be used to predict the high risk of CHD subtypes in infants.

Histologic alterations in tetralogy of Fallot.

BACKGROUND: This study was performed to investigate the pre-existing histologic alterations at the time of complete repair in patients with tetralogy of Fallot (TOF) and evaluate their effects on the early postoperative outcomes. METHODS: Fourteen patients, seven with acyanotic TOF (SO2 > 90, group I) and seven with cyanotic TOF (SO2 < 90, group II), undergoing complete repair, were enrolled. Right ventricular biopsies were examined for cardiomyocyte injury and fibrosis by light microscopy and mitochondrial injury by electron microscopy. The association of the severity of histologic alterations and postoperative inotrope use, intensive care unit, and in-hospital stays were evaluated. RESULTS: Compared with group I, patients in group II had a higher inotrope score (p = 0.03) and longer intensive care unit (p = 0.01) and in-hospital stays (p = 0.04). Cardiomyocyte injury and mitochondrial damage scores were higher in group II (p = 0.01 and p = 0.02, respectively). Fibrosis was detected in all specimens but was more severe in group II (p < 0.001). However, we could not demonstrate any correlation between histologic alterations and early surgical outcomes. The history of spell was significantly associated with worse early surgical outcomes (p < 0.05). CONCLUSIONS: Pre-existing cardiomyocyte injury accompanied by mitochondrial damage and fibrosis were more pronounced in cyanotic TOF patients. Early repair may prevent the development of histopathologic alterations in these patients.