Purpose:Genome sequencing (GS) presents a powerful approach to uncover disease-causing genetic variants. We used GS to examine single vs dual molecular causes in some of the most complicated pediatric cases-those with both a neoplasm and a birth defect. Methods:From our pediatric biobank, we selected 1463 children with a major congenital malformation, such as cleft lip/palate or internal organ defect, including 827 cases with a pediatric-onset cancer. The cohort includes nearly 40% non-White and/or multiracial individuals. We implemented GS as a first-tier diagnostic method and hypothesized that in most cases, a single disease-causing variant would explain their complex disease pictures. We developed a novel variant annotation and prioritization algorithm to provide a molecular diagnosis. Results:Our algorithm uncovered 361 disease-causing single-nucleotide variants/insertion/deletions in patients with compatible phenotypes (n = 324/1373; 23.6%), including 207 known and 120 novel variants in 167 genes. In addition, we identified aneuploidies (n = 41; 3%) and disease-causing copy-number variations, including haploinsufficient regions, de novo pathogenic variants and variable degrees of mosaicism (n = 65, 4.7%). Likely deleterious variants were identified in 2 candidate genes, GNG13 and RTKN2. Most cases had a single molecular cause for the cancer and the congenital anomaly, with notable exceptions of dual molecular causes. Conclusion:In children with severe and complex phenotypes, our findings demonstrate that GS revealed causative molecular underpinnings, including novel causes. A single genetic defect may underlie phenotypes of high complexity that appear unrelated, with double molecular findings identified in the same patient on rare occasions.

AIMS:Type 1 diabetes (T1D) exhibits sex differences in genetic risk, yet most genetic studies treat sex as a covariate rather than a modifier of risk. We hypothesized that sex-stratified genome-wide association studies (GWAS) would uncover sex-specific genetic architecture and improve risk prediction. METHODS:We performed GWAS in 6,599 T1D cases (3,483 males, 3,109 females, 7 undetermined) and 12,350 controls (6,665 males, 5,658 females, 27 undetermined) of European ancestry, testing additive models and sex-stratified analyses. For mechanistic insights, we performed scRNA-seq of PBMCs from nine matched male-female pediatric pairs. Finally, we tested male-, female-, and standard polygenic risk scores (PRS) in an independent cohort (471 T1D cases, 2,300 controls). RESULTS:Sex-stratified analyses identified 215 genome wide significant SNPs (P < 5x10-8) with heterogeneity: 119 male-specific and 94 female-specific. Integration of scRNA-seq data revealed 41 sex-specific T1D genes with cell type-specific differential expression. In the independent cohort, sex-specific PRS outperformed the combined PRS: in males, AUC = 0.668 versus 0.623 (p < 2.2x10-16); in females, AUC = 0.719 versus 0.635 (p < 2.2x10-16). CONCLUSIONS:Sex-stratified GWAS reveal novel T1D risk loci influenced by sex. Incorporating sex-specific effect sizes into PRS enhances risk discrimination, underscoring the value of sex-aware genetic analyses for precise prediction of T1D.

BackgroundExposure to fine particulate matter (PM2.5) increases asthma severity and reduces glucocorticoid responsiveness in children, yet the molecular mechanisms underlying PM2.5 sensitivity remain unclear. We previously identified a PM2.5-sensitive asthma phenotype and developed a PM2.5 sensitivity polygenic risk score (sPRS) correlated with asthma exacerbations and lung function decline.Research questionWe sought to determine whether genetic variants contributing to PM2.5 sensitivity converge on specific biological pathways or transcriptional regulators, and whether children with a high sPRS exhibit immune transcriptional signatures consistent with heightened PM2.5 susceptibility.MethodsGenes implicated by sPRS variants were mapped using regulatory annotation tools and evaluated for pathway and transcription factor target enrichment. Peripheral blood mononuclear cells (PBMCs) from high- and low-sPRS children matched on long-term ambient PM2.5 exposure were profiled using single-cell RNA sequencing. Donor-level pseudobulk differential expression was performed using a paired quasi-likelihood negative binomial framework, followed by exploratory pathway enrichment and perturbagen signature analyses.ResultssPRS-implicated genes were enriched for transcriptional regulators linked to SMAD2/3- and MAPK-associated signaling, suggesting TGF-β1-related pathway involvement. No genes reached false-discovery-rate-adjusted significance at the donor level in this small, matched cohort. However, secondary pathway-level analyses demonstrated concordant enrichment across multiple immune populations in inflammatory and stress-response signaling programs previously linked to PM2.5 exposure. Perturbagen signature analyses likewise highlighted small-molecule regulators of TGF-β1-associated pathways.InterpretationThese integrative genomic and transcriptomic analyses nominate TGF-β1-SMAD/MAPK signaling as a biologically plausible axis of genetic susceptibility to PM2.5 in pediatric asthma. Given the modest sample size and indirect nature of enrichment-based inference, these findings should be considered hypothesis-generating and motivate targeted functional validation.

Natural Killer (NK) cells are integral components of the innate immune system, recognizing and eliminating virus-infected cells. They may play a crucial role in the immune response and contribute to the complications associated with Single Ventricle/Hypoplastic Left Heart Syndrome (SV/HLHS). Utilizing single-cell RNA sequencing (scRNA-seq), NK cells from peripheral blood mononuclear cells (PBMCs) were analyzed in three de-identified SV/HLHS cases and three healthy controls. This study identified two novel NK cell subpopulations that could not be detected by conventional scRNA-seq pipelines or traditional flow cytometry. These subpopulations exhibit distinct gene expression profiles linked to the heterogeneity of immune responsiveness and stress adaptation in NK cells. In SV/HLHS patients, one cluster showed a significant upregulation of androgen response and downregulation of heme metabolism compared to healthy controls. Our study offers new insights into the fine-tuning of immune modulation that could help mitigate complications in SV/HLHS. It suggests that while NK cells in SV/HLHS adapt to support survival in a challenging physiological environment, these adaptations may compromise their ability to effectively respond to additional stresses, such as infections and inflammation.

BACKGROUND:Single ventricle and hypoplastic left heart syndrome (SV/HLHS) has detrimental effects on multiple organ systems, including peripheral blood mononuclear cells (PBMCs), and can weaken the immune system, exacerbating the risk of infection and various cardiovascular complications. METHODS:Using single-cell RNA sequencing (scRNA-seq), we studied PBMCs from 33 pediatric patients (10 females and 23 males) with SV/HLHS. By a pair-wide study design, the SV/HLHS patients were compared to 33 controls without heart disease. RESULTS:Four cell types account for the top 62% cumulative importance of disease effects on gene expression in different cell types, that is, Th1/17 cells, TFH cells, NK cells, and Th2 cells. Significant sex differences were observed in TFH Cells, with less prominent effects in females. A total of 6659 genes in different cell types were significantly differentially expressed (DE). Hierarchical clustering by WGCNA analysis of the DE genes revealed that DE genes in NK cells are most closely related to those in SV/HLHS. A total of 822 genes showed cell-specific DE with opposite directions in different cell types, highlighting overrepresented MYC and IFN-γ activity in T cell and NK cell populations, as well as underrepresentation in monocytes and Treg cells. CONCLUSION:This study elucidates the complex transcriptome landscape in PBMCs in patients with SV/HLHS, emphasizing the differential impacts on various cell types. New insights are gained into the precise modulation of MYC and IFN-γ activity in SV/HLHS, which may help balance immune responses and reduce harmful inflammation, and promote effective tissue repair and infection control.

Craniofrontonasal syndrome (CFNS; MIM #304110) is a rare craniofacial disorder characterized by hypertelorism, a broad nasal root with a bifid nasal tip, orofacial clefting, and genital malformations caused by pathogenic variants in the X-linked gene EFNB1 (MIM *300035). CFNS exhibits sex-specific heterogeneity, with increased severity in females likely secondary to cellular interference related to random X-inactivation, resulting in mosaic EFNB1 expression. Previous studies have identified over 140 variants in EFNB1, but approximately 20% of CFNS have negative molecular testing, either due to a yet undiscovered causal gene or causal variants in regulatory regions not covered by traditional genetic testing methodologies. Here, we report a two-generation family with a clinical diagnosis of CFNS and negative clinical molecular testing. Research short-read genome testing identified a 2-Mb inversion together with two smaller deletions (13- and 7-bp), about 106-Kb downstream of EFNB1, which cosegregated with CFNS. Patient-derived fibroblasts reprogrammed into induced pluripotent stem cells (iPSCs) demonstrated two distinct iPSC populations in affected females, where one or other of the two X chromosomes was inactivated. In vitro assays further demonstrated that iPSCs with the active X chromosome bearing the inversion, exhibited a significant increase in EFNB1 expression, suggesting allelic imbalance contributes to mosaic EFNB1 expression. These findings nominate a novel causal variant type of CFNS, conclude a 43-year diagnostic odyssey for an affected family, and offer new hope for family planning for affected individuals.

AbstractBackgroundSingle ventricle and hypoplastic left heart syndrome (SV/HLHS) patients require lifelong medical monitoring and management to address potential complications and optimize their health. The consequence of SV/HLHS had detrimental effects on multiple organ systems, including on peripheral blood mononuclear cells (PBMCs) and can weaken the immune system, exacerbating the risk of infection and various cardiovascular complications.MethodsUsing single-cell RNA sequencing (scRNA-seq), we studied PBMCs from 33 pediatric patients (10 females and 23 males) with SV/HLHS. By a pair-wide study design, the SV/HLHS patients were compared to 33 controls without heart diseases.ResultsFour cell types account for the top 62% cumulative importance of disease effects on gene expression in different cell types, i.e., [T cells, CD4+, Th1/17], [T cells, CD4+, TFH], [NK cells], and [T cells, CD4+, Th2]. Significant sex differences were observed in [T cells, CD4+, TFH], with less prominent effects in female patients. A total of 6659 genes in different cell types were significantly differentially expressed (DE). Hierarchical clustering by WGCNA analysis of the DE genes revealed that DE genes in NK cells are most closely related to those in SV/HLHS. A total of 822 genes showed cell specific DE with opposite directions in different cell types, highlighting overrepresented MYC and IFN-γ activity in T cell and NK cell populations, as well as underrepresentation in monocytes and Treg cells.ConclusionThis study elucidates the complex transcriptome landscape in PBMCs in patients with SV/HLHS, emphasizing the differential impacts on various cell types. New insights are gained into the precise modulation of MYC and IFN-γ activity in SV/HLHS, which may help balance immune responses and reduce harmful inflammation, and promote effective tissue repair and infection control.

We utilized single-cell RNA sequencing (scRNA-seq) to examine peripheral blood mononuclear cells (PBMCs) from patients with Single Ventricle/Hypoplastic Left Heart Syndrome (SV/HLHS), and demonstrated a more pronounced correlation between gene expression in Natural Killer (NK) cells and SV/HLHS compared to other PBMC cell types. Our scRNA-seq analysis of NK cells in this study identified two distinct clusters with gene expression patterns linked to immune responsiveness and adaptation to stress. While this finding underscores the heterogeneity of NK cells, it provides new insights into fine-tuning of immune modulation that could prevent complications in SV/HLHS. Specifically, our study suggests that while NK cells in SV/HLHS are adapting to support survival in a challenging physiological environment, these adaptations may compromise their ability to manage additional stresses such as infections and inflammation.

Pseudohypoparathyroidism type 1B (PHP1B) is associated with epigenetic changes in the maternal allele of the imprinted GNAS gene that inhibit expression of the α subunit of Gs (Gsα), thereby leading to parathyroid hormone resistance in renal proximal tubule cells where expression of Gsα from the paternal GNAS allele is normally silent. Although all patients with PHP1B show loss of methylation for the exon A/B differentially methylated region (DMR), some patients with autosomal dominant PHP1B (AD-PHP1B) and most patients with sporadic PHP1B have additional methylation defects that affect the DMRs corresponding to exons XL, AS1, and NESP. Because the genetic defect is unknown in most of these patients, we sought to identify the underlying genetic basis for AD-PHP1B in 2 multigenerational families with broad GNAS methylation defects and negative clinical exomes. Genome sequencing identified small GNAS variants in each family that were also present in unrelated individuals with PHP1B in a replication cohort. Maternal transmission of one GNAS microdeletion showed reduced penetrance in some unaffected patients. Expression of AS transcripts was increased, and NESP was decreased, in cells from affected patients. These results suggest that the small deletion activated AS transcription, leading to methylation of the NESP DMR with consequent inhibition of NESP transcription, and thereby provide a potential mechanism for PHP1B.

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

Background: Single ventricle (SV) and hypoplastic left heart syndrome (HLHS) present significant sex differences in terms of surgical outcomes and long-term prognosis between male and female patients. Peripheral blood mononuclear cells (PBMCs) drive tissue damage and cardiac dysfunction, contributing to the sex-specific clinical outcomes. Aims: We used single-cell RNA sequencing (scRNA-seq) to assess gene expression profiles in PBMCs, aiming to gain insights into sex effects and identify new biomarkers for assessing disease prognosis. Methods: We studied PBMCs from 32 cases with SV/HLHS. The experiments were conducted in 3 independent batches (9 females/11 males; 2 females/6 males; 2 females/2 males), using 10X Chromium Single Cell Gene Expression assay (10x Genomics, Single Cell 3' v3). Sequencing was performed using the Illumina NovaSeq6000. The data analysis was conducted using the Seurat R package, employing SCTransform for data normalizing and scaling (Satija, Farrell et al. 2015, Butler, Hoffman et al. 2018). To mitigate batch effects, the 3 batches were analyzed separately. Results: 42 genes were consistently identified of differential expression (DE) (|log 2 FC|≥0.25, P adj<0.05) in each of the 3 batches, including 38 upregulated (6 on ChrX) and 4 downregulated genes in females. Notably, 3 autosome genes were also identified of DE in SV/HLHS cases compared to controls: PPP1R15A upregulated in cases has lower expression in females; EVL and CLEC2D downregulated in cases have higher expression in females. The expression of these 3 genes is more significant in naïve CD4 + T cells, CD14 + Monocytes, NK cells, and naïve B cells. Conclusion: PPP1R15A mediates cellular stress responses and apoptosis (Blais, Filipenko et al. 2004). EVL negatively regulates cell migration (Lambrechts, Kwiatkowski et al. 2000). CLEC2D mediates immune sensing of cell death through the recognition of histone sequences (Lai, Cruz et al. 2020). The expression changes in females may mitigate inflammation and immune dysregulation in SV/HLHS patients. The significance of these genes as prognostic markers and potential targets for therapeutic intervention in SV/HLHS management warrants further research.

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2‑dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

Background: Peripheral blood mononuclear cells (PBMCs) are widely used as a model in the study of different human diseases. There is often a time delay from blood collection to PBMC isolation during the sampling process, which can result in an experimental bias, particularly when performing single cell RNA-seq (scRNAseq) studies. Methods: This study examined the impact of different time periods from blood draw to PBMC isolation on the subsequent transcriptome profiling of different cell types in PBMCs by scRNAseq using the 10X Chromium Single Cell Gene Expression assay. Results: Examining the five major cell types constituting the PBMC cell population, i.e., CD4+ T cells, CD8+ T cells, NK cells, monocytes, and B cells, both common changes and cell-type-specific changes were observed in the single cell transcriptome profiling over time. In particular, the upregulation of genes regulated by NF-kB in response to TNF was observed in all five cell types. Significant changes in key genes involved in AP-1 signaling were also observed. RBC contamination was a major issue in stored blood, whereas RBC adherence had no direct impact on the cell transcriptome. Conclusions: Significant transcriptome changes were observed across different PBMC cell types as a factor of time from blood draw to PBMC isolation and as a consequence of blood storage. This should be kept in mind when interpreting experimental results.

Vascular anomalies are malformations or tumors of the blood or lymphatic vasculature and can be life-threatening. Although molecularly targeted therapies can be life-saving, identification of the molecular etiology is often impeded by lack of accessibility to affected tissue samples, mosaicism or insufficient sequencing depth. In a cohort of 356 participants with vascular anomalies, including 104 with primary complex lymphatic anomalies (pCLAs), DNA from CD31+ cells isolated from lymphatic fluid or cell-free DNA from lymphatic fluid or plasma underwent ultra-deep sequencing thereby uncovering pathogenic somatic variants down to a variant allele fraction of 0.15%. A molecular diagnosis, including previously undescribed genetic causes, was obtained in 41% of participants with pCLAs and 72% of participants with other vascular malformations, leading to a new medical therapy for 63% (43/69) of participants and resulting in improvement in 63% (35/55) of participants on therapy. Taken together, these data support the development of liquid biopsy-based diagnostic techniques to identify previously undescribed genotype–phenotype associations and guide medical therapy in individuals with vascular anomalies. Genomic and cell-free DNA sequencing clarify the clinical diagnosis and inform treatment initiation in a cohort of 356 patients with vascular anomalies.

Complex chromosomal rearrangements involve the restructuring of genetic material within a single chromosome or across multiple chromosomes. These events can cause serious human disease by disrupting coding DNA and gene regulatory elements via deletions, duplications, and structural rearrangements. Here we describe a 5-year-old female with severe developmental delay, dysmorphic features, multi-suture craniosynostosis, and growth failure found to have a complex series of balanced intra- and inter-chromosomal rearrangements involving chromosomes 4, 11, 13, and X. Initial clinical studies were performed by karyotype, chromosomal microarray, and FISH with research-based short-read genome sequencing coupled with sanger sequencing to precisely map her breakpoints to the base pair resolution to understand the molecular basis of her phenotype. Genome analysis revealed two pathogenic deletions at 4p16.1-p15.32 and 4q31.1, accounting for her developmental delay and dysmorphism. We identified over 60 breakpoints, many with blunt ends and limited homology, supporting a role for non-homologous end joining in restructuring and resolution of the seminal chromoplexy event. We propose that the complexity of our patient's genomic rearrangements with a high number of breakpoints causes dysregulation of gene expression by three-dimensional chromatin interactions or topologically associating domains leading to growth failure and craniosynostosis. Our work supports an important role for genome sequencing in understanding the molecular basis of complex chromosomal rearrangements in human disease.

OBJECTIVE:Juvenile idiopathic arthritis (JIA) is the most common chronic immune-mediated joint disease among children and encompasses a heterogeneous group of immune-mediated joint disorders classified into 7 subtypes according to clinical presentation. However, phenotype overlap and biologic evidence suggest a shared mechanistic basis between subtypes. This study was undertaken to systematically investigate shared genetic underpinnings of JIA subtypes. METHODS:We performed a heterogeneity-sensitive genome-wide association study encompassing a total of 1,245 JIA cases (classified into 7 subtypes) and 9,250 controls, followed by fine-mapping of candidate causal variants at each genome-wide significant locus, functional annotation, and pathway and network analysis. We further identified candidate drug targets and drug repurposing opportunities by in silico analyses. RESULTS:In addition to the major histocompatibility complex locus, we identified 15 genome-wide significant loci shared between at least 2 JIA subtypes, including 10 novel loci. Functional annotation indicated that candidate genes at these loci were expressed in diverse immune cell types. CONCLUSION:This study identified novel genetic loci shared by JIA subtypes. Our findings identified candidate mechanisms underlying JIA subtypes and candidate targets with drug repurposing opportunities for JIA treatment.

Neuroblastoma is a childhood cancer that originates in the developing sympathetic nervous system. We previously reported a crucial role of mitochondrial DNA haplogroups in the pathology of neuroblastoma. To pinpoint mitochondrial DNA variants associated with neuroblastoma risk, we applied a mitochondrial genome imputation pipeline to the single nucleotide polymorphisms array data of 2 pediatric cohorts containing a total of 2404 neuroblastoma patients and 9310 cancer-free controls. All statistical tests were 2-sided. The single nucleotide variant, rs2853493, was statistically significantly associated with neuroblastoma risk in the discovery cohort (odds ratio = 0.62, 95% confidence interval = 0.53 to 0.72, P < .001) and further confirmed in the replication cohort (odds ratio = 0.75, 95% confidence interval = 0.62 to 0.90, P = .002). Further, expression quantitative trait loci analysis indicated genotypes of rs2853493 were associated with expression levels of MT-CYB gene expression in neuroblastoma cells, suggesting rs2853493 may confer risk to neuroblastoma via regulating the expression level of its nearby genes.

Ring-finger protein 213 (RNF213) encodes a protein of unknown function believed to play a role in cellular metabolism and angiogenesis. Gene variants are associated with susceptibility to moyamoya disease. Here, we describe two children with moyamoya disease who also demonstrated kidney disease, elevated aminotransferases, and recurrent skin lesions found by exome sequencing to have de novo missense variants in RNF213. These cases highlight the ability of RNF213 to cause Mendelian moyamoya disease in addition to acting as a genetic susceptibility locus. The cases also suggest a new, multi-organ RNF213-spectrum disease characterized by liver, skin, and kidney pathology in addition to severe moyamoya disease caused by heterozygous, de novo C-terminal RNF213 missense variants.

Ichthyosis follicularis, atrichia, and photophobia syndrome (IFAP syndrome) is a rare, X-linked disorder caused by pathogenic variants in membrane-bound transcription factor protease, site 2 (MBTPS2). Pathogenic MBTPS2 variants also cause BRESHECK syndrome, characterized by the IFAP triad plus intellectual disability and multiple congenital anomalies. Here we present a patient with ichthyosis, sparse hair, pulmonic stenosis, kidney dysplasia, hypospadias, growth failure, thrombocytopenia, anemia, bone marrow fibrosis, and chronic diarrhea found by research-based exome sequencing to harbor a novel, maternally inherited MBTPS2 missense variant (c.766 G>A; (p.Val256Leu)). In vitro modeling supports variant pathogenicity, with impaired cell growth in cholesterol-depleted media, attenuated activation of the sterol regulatory element-binding protein pathway, and failure to activate the endoplasmic reticulum stress response pathway. Our case expands both the genetic and phenotypic spectrum of BRESHECK syndrome to include a novel MBTPS2 variant and cytopenias, bone marrow fibrosis, and chronic diarrhea.