Sex differences in metabolic adaptation in infants with cyanotic congenital heart disease.

BACKGROUND: Female infants with congenital heart disease (CHD) face significantly higher postoperative mortality rates after adjusting for cardiac complexity. Sex differences in metabolic adaptation to cardiac stressors may be an early contributor to cardiac dysfunction. In adult diseases, hypoxic/ischemic cardiomyocytes undergo a cardioprotective metabolic shift from oxidative phosphorylation to glycolysis which appears to be regulated in a sexually dimorphic manner. We hypothesize sex differences in cardiac metabolism are present in cyanotic CHD and detectable as early as the infant period. METHODS: RNA sequencing was performed on blood samples (cyanotic CHD cases, n = 11; controls, n = 11) and analyzed using gene set enrichment analysis (GSEA). Global plasma metabolite profiling (UPLC-MS/MS) was performed using a larger representative cohort (cyanotic CHD, n = 27; non-cyanotic CHD, n = 11; unaffected controls, n = 12). RESULTS: Hallmark gene sets in glycolysis, fatty acid metabolism, and oxidative phosphorylation were significantly enriched in cyanotic CHD females compared to male counterparts, which was consistent with metabolomic differences between sexes. Minimal sex differences in metabolic pathways were observed in normoxic patients (both controls and non-cyanotic CHD cases). CONCLUSION: These observations suggest underlying differences in metabolic adaptation to chronic hypoxia between males and females with cyanotic CHD. IMPACT: Children with cyanotic CHD exhibit sex differences in utilization of glycolysis vs. fatty acid oxidation pathways to meet the high-energy demands of the heart in the neonatal period. Transcriptomic and metabolomic results suggest that under hypoxic conditions, males and females undergo metabolic shifts that are sexually dimorphic. These sex differences were not observed in neonates in normoxic conditions (i.e., non-cyanotic CHD and unaffected controls). The involved metabolic pathways are similar to those observed in advanced heart failure, suggesting metabolic adaptations beginning in the neonatal period may contribute to sex differences in infant survival.

A practical gestational age-based algorithm for timely detection of hypothyroidism in premature infants.

OBJECTIVES: To assess utility and accuracy of a gestational age-based screening targeting premature infants to detect congenital hypothyroidism. STUDY DESIGN: A prospective cohort study was conducted in infants <35 weeks' gestational age with clinical outcomes at 2-3 years of age. Patients received newborn screenings at 24 hours and 10-14 days of life. Free T4 (FT4) and thyroid-stimulating hormone (TSH) levels were measured at one month of life and repeated based on algorithm by corrected gestational age. RESULTS: Among infants <35 weeks gestation (n = 938), the incidence of hypothyroidism requiring treatment was 1:58. TSH levels at one month of age was predictive of treatment (AUC 0.96, 95% CI 0.88-1). The optimal TSH threshold of 8 mIU/L (8 µU/ml) increased the specificity to 0.97 and sensitivity to 0.88. Following initiation of treatment for hypothyroidism during NICU hospitalization, 43.8% (n = 7) were diagnosed with permanent congenital hypothyroidism. CONCLUSIONS: Our study supports a gestational age-based screening algorithm for early detection of hypothyroidism in premature infants.

Genomic testing and molecular diagnosis among infants with congenital heart disease in the neonatal intensive care unit.

OBJECTIVE: To evaluate patterns of genetic testing among infants with CHD at a tertiary care center. STUDY DESIGN: We conducted a retrospective observational cohort study of infants in the NICU with suspicion of a genetic disorder. 1075 of 7112 infants admitted to BCH had genetic evaluation including 329 with CHD and 746 without CHD. 284 of 525 infants with CHD admitted to CMHH had genetic evaluation. Patterns of testing and diagnoses were compared. RESULTS: The rate of diagnosis after testing was similar for infants with or without CHD (38% [121/318] vs. 36% [246/676], p = 0.14). In a multiple logistic regression, atrioventricular septal defects were most high associated with genetic diagnosis (odds ratio 29.99, 95% confidence interval 2.69-334.12, p < 0.001). CONCLUSIONS: Infants with suspicion of a genetic disorder with CHD had similar rates of molecular diagnosis as those without CHD. These results support a role for genetic testing among NICU infants with CHD.

Challenges in the clinical understanding of genetic testing in birth defects and pediatric diseases.

Advances in prenatal/neonatal genetic screening practices and next generation sequencing (NGS) technologies have made the detection of molecular causes of pediatric diseases increasingly more affordable, accessible, and rapid in return of results. In the past, families searching for answers often required diagnostic journeys leading to delays in targeted care and missed diagnoses. Non-invasive prenatal NGS is now used routinely in pregnancy, significantly altering the obstetric approach to early screening and evaluation of fetal anomalies. Similarly, exome sequencing (ES) and genome sequencing (GS) were once only available for research but are now used in patient care, impacting neonatal care and the field of neonatology as a whole. In this review we will summarize the growing body of literature on the role of ES/GS in prenatal/neonatal care, specifically in neonatal intensive care units (NICU), and the molecular diagnostic yield. Furthermore, we will discuss the impact of advances in genetic testing in prenatal/neonatal care and discuss challenges faced by clinicians and families. Clinical application of NGS has come with many challenges in counseling families on interpretation of diagnostic results and incidental findings, as well as re-interpretation of prior genetic test results. How genetic results may influence medical decision-making is highly nuanced and needs further study. The ethics of parental consent and disclosure of genetic conditions with limited therapeutic options continue to be debated in the medical genetics community. While these questions remain unanswered, the benefits of a standardized approach to genetic testing in the NICU will be highlighted by two case vignettes.

Narrative review of single ventricle: where are we after 40 years?

Background and Objective: Key medical and surgical advances have been made in the longitudinal management of patients with "functionally" single ventricle physiology, with the principles of Fontan circulation applied to other complex congenital heart defects. The purpose of this article is to review all of the innovations, starting from fetal life, that led to a change of strategy for single ventricle. Methods: Our literature review included all full articles published in English language on the Cochrane, MedLine, and Embase with references to "single ventricle" and "univentricular hearts", including the initial history of the treatments for this congenital heart defects as well as the innovations reported within the last decades. Key Content and Findings: All innovations introduced have been analyzed, including: (I) fetal diagnosis and interventions, in particular to prevent or reduce brain damages; (II) neonatal care; (III) post-natal diagnosis; (IV) interventional cardiology procedures; (V) surgical procedures, including neonatal palliations, hybrid procedures, bidirectional Glenn and variations, Fontan completion, biventricular repair; (VI) peri-operative management; (VII) Fontan failure, with Fontan take-down and conversion, and mechanical circulatory support; (VIII) transplantation, including heart, heart and lung, heart and liver; (IX) exercise; (X) pregnancy; (XI) adolescents and adults without Fontan completion; (XII) future studies, including experimental studies on animals, computational studies, genetics, stem cells and bioengineering. Conclusions: These last 40 years have certainly changed the course of natural history for children born with any form of "functionally" single ventricle, thanks to the improvement in diagnostic and treatment techniques, and particularly to the increased knowledge of the morphology and function of these complex hearts, from fetal to adult life. There is still much left unexplored and room for improvement, and all efforts should be concentrated in collaborations among different institutions and specialties, focused on the same matter.

Congenital heart defects and copy number variants associated with neurodevelopmental impairment.

A genetic etiology is identifiable in 20%-30% of patients with congenital heart defects (CHD). Chromosomal microarray analysis (CMA) can detect copy number variants (CNV) associated with CHD. In previous studies, the diagnostic yield of postnatal CMA testing ranged from 4% to 28% in CHD patients. However, incidental pathogenic CNV and variants of unknown significance are often discovered without any known association with CHD. The study objective was to describe the rate of pathogenic CNV associated with neurodevelopmental impairment (NDI) and compare clinical findings in CHD neonates with genetic results. A single-center retrospective review was performed on all consecutive newborns with CHD admitted to a tertiary neonatal intensive care unit from January 2013 to March 2019 (n = 525). CHD phenotypes were classified as per the National Birth Defect Prevention Study. CMA detected pathogenic CNV in 21.3% (61/287) of neonates, and karyotype or fluorescence in situ hybridization detected aneuploidies in an additional 11% of the overall cohort (58/525). Atrioventricular septal defects and conotruncal defects showed the highest diagnostic yield by CMA (28.6% and 27.2%, respectively). Among neonates with pathogenic CNV on CMA, 78.7% (48/61) were associated with NDI. Neonates with pathogenic CNV were smaller in length at birth compared to those with benign CNV or variants of unknown significance (p = 0.005) and were more likely to be discharged with an enteral feeding tube (p = 0.027). CMA can discover genetic variants associated with NDI and are common in neonates with CHD. Genetic testing in the neonatal period can heighten awareness of genetic risk for NDI.

Can Erythropoietin Reduce Hypoxemic Neurological Damages in Neonates With Congenital Heart Defects?

Congenital heart defects (CHD), the most common cause of birth defects with increasing birth prevalence, affect nearly 1% of live births worldwide. Cyanotic CHD are characterized by hypoxemia, with subsequent reduced oxygen delivery to the brain, especially critical during brain development, beginning in the fetus and continuing through the neonatal period. Therefore, neonates with CHD carry a high risk for neurological comorbidities, even more frequently when there are associated underlying genetic disorders. We review the currently available knowledge on potential prevention strategies to reduce brain damage induced by hypoxemia during fetal development and immediately after birth, and the role of erythropoietin (EPO) as a potential adjunctive treatment. Maternal hyper-oxygenation had been studied as a potential therapeutic to improve fetal oxygenation. Despite demonstrating some effectiveness, maternal hyper-oxygenation has proven to be impractical for extensive clinical application, thus prompting the investigation of specific pathways for pharmacological intervention. Among those, the role of antioxidant pathways and Hypoxia Inducible Factors (HIF) have been studied for their involvement in the protective response to hypoxic injury. One of the proteins induced by HIF, EPO, has properties of being anti-apoptotic, antioxidant, and protective for neurons, astrocytes, and oligodendrocytes. In human trials, EPO administration in neonates with hypoxic ischemic encephalopathy (HIE) significantly reduced the neurological hypoxemic damages in several reported studies. Currently, it is unknown if the mechanisms of pathophysiology of cyanotic CHD are like HIE. Neonates with cyanotic CHD are exposed to both chronic hypoxemia and episodes of acute ischemia-reperfusion injury when undergo cardiopulmonary bypass surgery requiring aortic cross-clamp and general anesthesia. Our review supports future trials to evaluate the potential efficiency of EPO in reducing the hypoxemic neurologic damages in neonates with CHD. Furthermore, it suggests the need to identify early biomarkers of hypoxia-induced neurological damage, which must be sensitive to the neuroprotective effects of EPO.

The current state of prenatal detection of genetic conditions in congenital heart defects.

The incidence of congenital heart defect (CHD) has increased over the past fifty years, partly attributed to routine fetal anatomical examination by sonography during obstetric care and improvements in ultrasound technology and technique. Fetal findings on ultrasound in addition to maternal biomarkers are the backbone of first- and second-trimester screening for common genetic conditions, namely aneuploidy. Since the introduction of non-invasive prenatal testing (NIPT) using next-generation sequencing to sequence cell-free fetal DNA, the detection rate of common trisomies as well as sex chromosomal aneuploidies have markedly increased. As the use of NIPT continues to broaden, the best means of incorporating NIPT into prenatal care is less clear and complicated by misunderstanding of the limitations and non-diagnostic role of NIPT by clinicians and families. In other advancements in prenatal genetic testing, recommendations on the role of chromosomal microarray (CMA) for prenatal diagnosis has led to its increasing use to identify genetic conditions in fetuses diagnosed with CHD. Lastly, as whole exome sequencing (WES) becomes more available and affordable, the next clinical application of next-generation sequencing in prenatal diagnostic testing is on the horizon. While newer genetic tests may provide answers in terms of genetic diagnosis, even more questions will likely ensue for clinicians, researchers, and parents. The objective of this review is to provide the perspective of the evolution of maternal and fetal obstetric care against the backdrop of advancing genetic technology and its impact on families and clinicians.

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs. Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

The Cardiac Neural Crest Cells in Heart Development and Congenital Heart Defects.

The neural crest (NC) is a multipotent and temporarily migratory cell population stemming from the dorsal neural tube during vertebrate embryogenesis. Cardiac neural crest cells (NCCs), a specified subpopulation of the NC, are vital for normal cardiovascular development, as they significantly contribute to the pharyngeal arch arteries, the developing cardiac outflow tract (OFT), cardiac valves, and interventricular septum. Various signaling pathways are shown to orchestrate the proper migration, compaction, and differentiation of cardiac NCCs during cardiovascular development. Any loss or dysregulation of signaling pathways in cardiac NCCs can lead to abnormal cardiovascular development during embryogenesis, resulting in abnormalities categorized as congenital heart defects (CHDs). This review focuses on the contributions of cardiac NCCs to cardiovascular formation, discusses cardiac defects caused by a disruption of various regulatory factors, and summarizes the role of multiple signaling pathways during embryonic development. A better understanding of the cardiac NC and its vast regulatory network will provide a deeper insight into the mechanisms of the associated abnormalities, leading to potential therapeutic advancements.

Hippo-Yap Pathway Orchestrates Neural Crest Ontogenesis.

Neural crest (NC) cells are a migratory stem cell population in vertebrate embryogenesis that can give rise to multiple cell types, including osteoblasts, chondrocytes, smooth muscle cells, neurons, glia, and melanocytes, greatly contributing to the development of different tissues and organs. Defects in NC development are implicated in many human diseases, such as numerous syndromes, craniofacial aberration and congenital heart defects. Research on NC development has gained intense interest and made significant progress. Recent studies showed that the Hippo-Yap pathway, a conserved fundamental pathway with key roles in regulation of cell proliferation, survival, and differentiation, is indispensable for normal NC development. However, the roles and mechanisms of the Hippo-Yap pathway in NC development remain largely unknown. In this review, we summarize the key functions of the Hippo-Yap pathway indicated in NC induction, migration, proliferation, survival, and differentiation, as well as the diseases caused by its dysfunction in NC cells. We also discuss emerging current and future studies in the investigation of the Hippo-Yap pathway in NC development.

The value of serial newborn screening for congenital hypothyroidism using thyroxine (T4) in the neonatal intensive care unit.

OBJECTIVE: To evaluate the role of serial newborn screening of congenital hypothyroidism using thyroxine (T4) in the neonatal intensive care unit (NICU). SUBJECTS: Newborn screen results were reviewed from a single academic NICU during 2007-2016 (n = 6100). Thyroid function levels were reviewed in patients treated for hypothyroidism during that period. Duration of treatment was followed after discharge. RESULTS: Overall incidence of treated hypothyroidism was 1:103 with increasing incidence inversely related to birth weight. Among treated infants (n = 59), initial newborn screen demonstrated sensitivity and specificity of 74.1% and 84.9%, respectively; second screen demonstrated rates of 85.7% and 76.1%, respectively. Based on follow-up data, prevalence of permanent congenital hypothyroidism in our NICU population was 1:870 (n = 7); two patients would have been missed with a single screen. CONCLUSION: Abnormal T4 on newborn screening is common for preterm neonates. Higher rates of permanent congenital hypothyroidism highlight the need for screening beyond the newborn screen.

Mutations in folate transporter genes and risk for human myelomeningocele.

The molecular mechanisms linking folate deficiency and neural tube defect (NTD) risk in offspring remain unclear. Folate transporters (SLC19A1, SLC46A1, SLC25A32, and FOLH1) and folate receptors (FOLR1, FOLR2, and FOLR3) are suggested to play essential roles in transporting folate from maternal intestinal lumen to the developing embryo. Loss of function variants in these genes may affect folate availability and contribute to NTD risk. This study examines whether variants within the folate transporter and receptor genes are associated with an increased risk for myelomeningocele (MM). Exons and their flanking intron sequences of 348 MM subjects were sequenced using the Sanger sequencing method and/or next generation sequencing to identify variants. Frequencies of alleles of single nucleotide polymorphisms (SNPs) in MM subjects were compared to those from ethnically matched reference populations to evaluate alleles' associated risk for MM. We identified eight novel variants in SLC19A1 and twelve novel variants in FOLR1, FOLR2, and FOLR3. Pathogenic variants include c.1265delG in SLC19A1 resulting in an early stop codon, four large insertion deletion variants in FOLR3, and a stop_gain variant in FOLR3. No new variants were identified in SLC46A1, SLC25A32, or FOLH1. In SLC19A1, c.80A>G (rs1051266) was not associated with our MM cohort; we did observe a variant allele G frequency of 61.7%, higher than previously reported in other NTD populations. In conclusion, we discovered novel loss of function variants in genes involved in folate transport in MM subjects. Our results support the growing evidence of associations between genes involved in folate transport and susceptibility to NTDs.

Finding the genetic mechanisms of folate deficiency and neural tube defects-Leaving no stone unturned.

Neural tube defects (NTDs) occur secondary to failed closure of the neural tube between the third and fourth weeks of gestation. The worldwide incidence ranges from 0.3 to 200 per 10,000 births with the United States of American NTD incidence at around 3-6.3 per 10,000 dependent on race and socioeconomic background. Human NTD incidence has fallen by 35-50% in North America due to mandatory folic acid fortification of enriched cereal grain products since 1998. The US Food and Drug Administration has approved the folic acid fortification of corn masa flour with the goal to further reduce the incidence of NTDs, especially among individuals who are Hispanic. However, the genetic mechanisms determining who will benefit most from folate enrichment of the diet remains unclear despite volumes of literature published on studies of association of genes with functions related to folate metabolism and risk of human NTDs. The advances in omics technologies provides hypothesis-free tools to interrogate every single gene within the genome of NTD affected individuals to discover pathogenic variants and methylation targets throughout the affected genome. By identifying genes with expression regulated by presence of folate through transcriptome profiling studies, the genetic mechanisms leading to human NTDs due to folate deficiency may begin to be more efficiently revealed.