Our Research

Despite advances in sequencing technologies and variant interpretation frameworks, many variants identified in genetic testing, particularly missense variants, remain classified as variants of uncertain significance (VUS), posing ongoing challenges in diagnosis and clinical management. This review explores the ongoing challenges of VUS interpretation in clinical genomics and its impact on patients, clinicians, and healthcare systems. We summarize established strategies that support VUS resolution, including large population reference databases, data-sharing initiatives, computational prediction tools, consensus-based guidelines, deep phenotyping, and functional validation assays. Building on these foundations, we highlight emerging approaches that leverage multi-omics analyses, high-throughput experimental platforms (e.g., saturation genome editing and cell-based morphological assays), and artificial intelligence-driven tools to improve scalability and interpretive accuracy. Together, these complementary approaches aim to reduce uncertainty, increase diagnostic yield, and enhance the clinical utility of genomic testing in both research and precision medicine.

Children with critical illness are vulnerable to nutritional deficits, which may contribute to increased susceptibility to infection, impaired immune function and delayed recovery. Optimizing nutritional support during acute illness is therefore essential to improving clinical outcomes. The complex interplay between critical illness, nutrition and infection is nuanced and requires careful consideration to minimize morbidity and mortality. In this narrative review, we synthesize current evidence and considerations regarding optimizing nutritional management to minimize infectious complications across neonatal and pediatric intensive care settings.

BACKGROUND: Haploinsufficiency of the RNA splicing regulator RBFOX2 has been linked to congenital heart disease, including hypoplastic left heart syndrome, yet its pathogenic mechanisms remain unresolved.

METHODS: Human induced pluripotent stem cells heterozygous or null for RBFOX2 were created and their cardiomyocyte (CM) derivatives were characterized in monolayer culture and engineered heart tissues. In addition to cellular phenotyping, molecular profiling by RNA sequencing and enhanced crosslinking and immunoprecipitation sequencing was performed to detect changes in gene expression, exon usage, and RBFOX2 (RNA-binding fox-1 homolog 2) binding. These data sets were integrated to identify RBFOX2-bound RNAs with altered expression and splicing and to compare them with publicly available data sets from progressive CM differentiation time points. Rescue experiments were performed in induced pluripotent stem cell-derived CMs, engineered heart tissues, and zebrafish.

RESULTS: We demonstrate that RBFOX2 is essential for CM differentiation by driving transcriptome maturation through gene expression- and alternative splicing-based isoform switching in transcripts primarily encoding sarcomere, cytoskeletal, and focal adhesion components, including the critical Z-disc factor ACTN2 (alpha-actinin-2). We further find that threshold levels of RBFOX2 facilitate autoregulatory splicing of mutually exclusive exons, generating early and late RBFOX2 isoforms with distinct functions. In heterozygous CMs, this threshold is not reached, resulting in isoform imbalance and production of an aberrant transcript, which further reduces functional RBFOX2 levels to well below 50% of wild-type. Finally, we demonstrate that ACTN2 overexpression rescues heterozygous, but not null, phenotypes, suggesting that ACTN2 activates an RBFOX2-dependent feedback loop that promotes transcriptome maturation.

CONCLUSIONS: Our data demonstrate that reduced RBFOX2 dosage and impaired autoregulation stall CM differentiation by inhibiting transcriptome maturation, contributing to congenital heart disease pathogenesis and potentially increasing heart failure susceptibility following surgical intervention.

OBJECTIVE: To determine the incremental risks of mortality and major neonatal morbidities associated with congenital heart disease (CHD) beyond prematurity alone.

STUDY DESIGN: Secondary analysis of two North American neonatal registries (2010-2022) including infants <35 weeks' gestation: 11,221 with CHD from the Children's Hospitals Neonatal Consortium and 720,846 without CHD from the Vermont Oxford Network. The primary outcome was in-hospital mortality. Secondary outcomes were major neonatal morbidities.

RESULTS: Preterm infants with CHD had fourfold higher odds of in-hospital mortality (OR 4.0, 95% CI 3.76-4.21). Mortality was similar at ≤26 weeks' gestation, but the relative impact of CHD increased with advancing gestational age. CHD was associated with significantly increased risks of severe bronchopulmonary dysplasia, intraventricular hemorrhage, and necrotizing enterocolitis.

CONCLUSION: The impact of CHD on mortality is gestational age dependent, with prematurity predominating at the lowest gestational ages and CHD increasingly driving mortality as gestational maturity improves.

Folate deficiency is associated with increased risk of conotruncal heart defects (CTHD), but its interaction with genetic factors remains unclear. Our objective was to investigate genome-wide associations between common genetic variants and birth before versus after universal folic acid fortification among children with CTHD and other heart defects. Sequencing data were available through the Pediatric Cardiac Genomics Consortium, aligned to the human reference genome (GRCh38/hg38), and jointly processed to ensure uniform variant detection and minimize batch effects. Analyses were restricted to individuals with European-inferred genetic ancestry, limiting generalizability to other ancestral groups. Genome-wide association study (GWAS) models compared allele frequencies across fortification eras among all participants (n = 1285), the subset with CTHD (n = 534), and the remaining individuals with other heart defects (n = 751). Functional enrichment was assessed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Among the full analytic group, eight loci had at least two suggestive variants before compared to after fortification. Among the subset with CTHD, two variants located in DHRS3 (rs7551703, odds ratio [OR] 2.10, and rs6541043, OR 2.21) and four variants located in PPARGC1B showed suggestive association after fortification (OR 4.47-4.51). Enriched biological pathway terms were consistent with cardiac developmental processes. In summary, this study examined the association between folic acid fortification and genetic risk for CTHD and other heart defects and identified associations with fortification era that suggest some shift in risk following fortification. Identified pathways suggest gene-nutrient interactions may modulate cardiac developmental pathways, underscoring the relationship between maternal folate status and cardiovascular development.