Publications

Inflammation during neonatal brain infections leads to significant secondary sequelae such as hydrocephalus, which often follows neonatal sepsis in the developing world. In 100 African hydrocephalic infants we identified the biological pathways that account for this response. The dominant bacterial pathogen was a Paenibacillus species, with frequent cytomegalovirus co-infection. A proteogenomic strategy was employed to confirm host immune response to Paenibacillus and to define the interplay within the host immune response network. Immune activation emphasized neuroinflammation, oxidative stress reaction, and extracellular matrix organization. The innate immune system response included neutrophil activity, signaling via IL-4, IL-12, IL-13, interferon, and Jak/STAT pathways. Platelet-activating factors and factors involved with microbe recognition such as Class I MHC antigen-presenting complex were also increased. Evidence suggests that dysregulated neuroinflammation propagates inflammatory hydrocephalus, and these pathways are potential targets for adjunctive treatments to reduce the hazards of neuroinflammation and risk of hydrocephalus following neonatal sepsis.

IMPORTANCE: Patients with congenital heart disease (CHD), the most common birth defect, have increased risks for cancer. Identification of the variables that contribute to cancer risk is essential for recognizing patients with CHD who warrant longitudinal surveillance and early interventions.

OBJECTIVE: To compare the frequency of damaging variants in cancer risk genes among patients with CHD and control participants and identify associated clinical variables in patients with CHD who have cancer risk variants.

DESIGN, SETTING, AND PARTICIPANTS: This multicenter case-control study included participants with CHD who had previously been recruited to the Pediatric Cardiac Genomics Consortium based on presence of structural cardiac anomaly without genetic diagnosis at the time of enrollment. Permission to use published sequencing data from unaffected adult participants was obtained from 2 parent studies. Data were collected for this study from December 2010 to April 2019.

EXPOSURES: Presence of rare (allele frequency, <1 × 10-5) loss-of-function (LoF) variants in cancer risk genes.

MAIN OUTCOMES AND MEASURES: Frequency of LoF variants in cancer risk genes (defined in the Catalogue of Somatic Mutations in Cancer-Cancer Gene Consensus database), were statistically assessed by binomial tests in patients with CHD and control participants.

RESULTS: A total of 4443 individuals with CHD (mean [range] age, 13.0 [0-84] years; 2225 of 3771 with reported sex [59.0%] male) and 9808 control participants (mean [range] age, 52.1 [1-92] years; 4967 of 9808 [50.6%] male) were included. The frequency of LoF variants in regulatory cancer risk genes was significantly higher in patients with CHD than control participants (143 of 4443 [3.2%] vs 166 of 9808 [1.7%]; odds ratio [OR], 1.93 [95% CI, 1.54-2.42]; P = 1.38 × 10-12), and among CHD genes previously associated with cancer risk (58 of 4443 [1.3%] vs 18 of 9808 [0.18%]; OR, 7.2 [95% CI, 4.2-12.2]; P < 2.2 × 10-16). The LoF variants were also nominally increased in 14 constrained cancer risk genes with high expression in the developing heart. Seven of these genes (ARHGEF12, CTNNB1, LPP, MLLT4, PTEN, TCF12, and TFRC) harbored LoF variants in multiple patients with unexplained CHD. The highest rates for LoF variants in cancer risk genes occurred in patients with CHD and extracardiac anomalies (248 of 1482 individuals [16.7%]; control: 1099 of 9808 individuals [11.2%]; OR, 1.59 [95% CI, 1.37-1.85]; P = 1.3 × 10-10) and/or neurodevelopmental delay (209 of 1393 individuals [15.0%]; control: 1099 of 9808 individuals [11.2%]; OR, 1.40 [95% CI, 1.19-1.64]; P = 9.6 × 10-6).

CONCLUSIONS AND RELEVANCE: Genotypes of CHD may account for increased cancer risks. In this cohort, damaging variants were prominent in the 216 genes that predominantly encode regulatory proteins. Consistent with their fundamental developmental functions, patients with CHD and damaging variants in these genes often had extracardiac manifestations. These data may also implicate cancer risk genes that are repeatedly varied in patients with unexplained CHD as CHD genes.

Maternal nutrition is an important factor for infant neurodevelopment. However, prior magnetic resonance imaging (MRI) studies on maternal nutrients and infant brain have focused mostly on preterm infants or on few specific nutrients and few specific brain regions. We present a first study in term-born infants, comprehensively correlating 73 maternal nutrients with infant brain morphometry at the regional (61 regions) and voxel (over 300 000 voxel) levels. Both maternal nutrition intake diaries and infant MRI were collected at 1 month of life (0.9 ± 0.5 months) for 92 term-born infants (among them, 54 infants were purely breastfed and 19 were breastfed most of the time). Intake of nutrients was assessed via standardized food frequency questionnaire. No nutrient was significantly correlated with any of the volumes of the 61 autosegmented brain regions. However, increased volumes within subregions of the frontal cortex and corpus callosum at the voxel level were positively correlated with maternal intake of omega-3 fatty acids, retinol (vitamin A) and vitamin B12, both with and without correction for postmenstrual age and sex (P < 0.05, q < 0.05 after false discovery rate correction). Omega-3 fatty acids remained significantly correlated with infant brain volumes after subsetting to the 54 infants who were exclusively breastfed, but retinol and vitamin B12 did not. This provides an impetus for future larger studies to better characterize the effect size of dietary variation and correlation with neurodevelopmental outcomes, which can lead to improved nutritional guidance during pregnancy and lactation.

Neurodevelopmental abnormalities are the most common noncardiac complications in patients with congenital heart disease (CHD). Prenatal brain abnormalities may be due to reduced oxygenation, genetic factors, or less commonly, teratogens. Understanding the contribution of these factors is essential to improve outcomes. Because primary sulcal patterns are prenatally determined and under strong genetic control, we hypothesized that they are influenced by genetic variants in CHD. In this study, we reveal significant alterations in sulcal patterns among subjects with single ventricle CHD (n = 115, 14.7 ± 2.9 years [mean ± standard deviation]) compared with controls (n = 45, 15.5 ± 2.4 years) using a graph-based pattern-analysis technique. Among patients with CHD, the left hemisphere demonstrated decreased sulcal pattern similarity to controls in the left temporal and parietal lobes, as well as the bilateral frontal lobes. Temporal and parietal lobes demonstrated an abnormally asymmetric left-right pattern of sulcal basin area in CHD subjects. Sulcal pattern similarity to control was positively correlated with working memory, processing speed, and executive function. Exome analysis identified damaging de novo variants only in CHD subjects with more atypical sulcal patterns. Together, these findings suggest that sulcal pattern analysis may be useful in characterizing genetically influenced, atypical early brain development and neurodevelopmental risk in subjects with CHD.

BACKGROUND: Nucleated red blood cells (nRBCs) are associated with adverse outcomes for pediatric and adult intensive care patients.

METHODS: The association between nRBC count and mortality was examined in an observational cohort of patients admitted to the neonatal intensive care unit from December 2015-December 2018.

RESULTS: Among the 1059 patients with at least one nRBC count obtained, 45 infants (4.2%) experienced in-hospital mortality prior to NICU discharge, the primary outcome measured in this study. Infants with any nRBC count >0 had a significantly higher risk of mortality (5.3% [45/849] vs. 0% [0/351], p < 0.001 by Fisher exact), and time to mortality decreased with higher nRBC counts (Spearman correlation -0.59, p < 0.001). The association between nRBC count and mortality remained significant even when restricting only to infants who were older than 7 days at time of nRBC count.

CONCLUSION: Among neonatal intensive care unit patients, including those >7 days old, nRBCs are associated with significantly elevated mortality risk. A prospective study to better characterize clinical co-variants is necessary to better establish the use of nRBCs as a predictor of mortality.

Although DNA methylation is the best characterized epigenetic mark, the mechanism by which it is targeted to specific regions in the genome remains unclear. Recent studies have revealed that local DNA methylation profiles might be dictated by cis-regulatory DNA sequences that mainly operate via DNA-binding factors. Consistent with this finding, we have recently shown that disruption of CTCF-binding sites by rare single nucleotide variants (SNVs) can underlie cis-linked DNA methylation changes in patients with congenital anomalies. These data raise the hypothesis that rare genetic variation at transcription factor binding sites (TFBSs) might contribute to local DNA methylation patterning. In this work, by combining blood genome-wide DNA methylation profiles, whole genome sequencing-derived SNVs from 247 unrelated individuals along with 133 predicted TFBS motifs derived from ENCODE ChIP-Seq data, we observed an association between the disruption of binding sites for multiple TFs by rare SNVs and extreme DNA methylation values at both local and, to a lesser extent, distant CpGs. While the majority of these changes affected only single CpGs, 24% were associated with multiple outlier CpGs within ±1kb of the disrupted TFBS. Interestingly, disruption of functionally constrained sites within TF motifs lead to larger DNA methylation changes at nearby CpG sites. Altogether, these findings suggest that rare SNVs at TFBS negatively influence TF-DNA binding, which can lead to an altered local DNA methylation profile. Furthermore, subsequent integration of DNA methylation and RNA-Seq profiles from cardiac tissues enabled us to observe an association between rare SNV-directed DNA methylation and outlier expression of nearby genes. In conclusion, our findings not only provide insights into the effect of rare genetic variation at TFBS on shaping local DNA methylation and its consequences on genome regulation, but also provide a rationale to incorporate DNA methylation data to interpret the functional role of rare variants.

Postinfectious hydrocephalus (PIH), which often follows neonatal sepsis, is the most common cause of pediatric hydrocephalus worldwide, yet the microbial pathogens underlying this disease remain to be elucidated. Characterization of the microbial agents causing PIH would enable a shift from surgical palliation of cerebrospinal fluid (CSF) accumulation to prevention of the disease. Here, we examined blood and CSF samples collected from 100 consecutive infant cases of PIH and control cases comprising infants with non-postinfectious hydrocephalus in Uganda. Genomic sequencing of samples was undertaken to test for bacterial, fungal, and parasitic DNA; DNA and RNA sequencing was used to identify viruses; and bacterial culture recovery was used to identify potential causative organisms. We found that infection with the bacterium Paenibacillus, together with frequent cytomegalovirus (CMV) coinfection, was associated with PIH in our infant cohort. Assembly of the genome of a facultative anaerobic bacterial isolate recovered from cultures of CSF samples from PIH cases identified a strain of Paenibacillus thiaminolyticus This strain, designated Mbale, was lethal when injected into mice in contrast to the benign reference Paenibacillus strain. These findings show that an unbiased pan-microbial approach enabled characterization of Paenibacillus in CSF samples from PIH cases, and point toward a pathway of more optimal treatment and prevention for PIH and other proximate neonatal infections.

BACKGROUND: The contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined.

METHODS: We developed a new computational method, EM-mosaic (Expectation-Maximization-based detection of mosaicism), to analyze mosaicism in exome sequences derived primarily from blood DNA of 2530 CHD proband-parent trios. To optimize this method, we measured mosaic detection power as a function of sequencing depth. In parallel, we analyzed our cohort using MosaicHunter, a Bayesian genotyping algorithm-based mosaic detection tool, and compared the two methods. The accuracy of these mosaic variant detection algorithms was assessed using an independent resequencing method. We then applied both methods to detect mosaicism in cardiac tissue-derived exome sequences of 66 participants for which matched blood and heart tissue was available.

RESULTS: EM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The estimated true frequency of mosaic variants above 10% mosaicism was 0.14/person in blood and 0.21/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction.

CONCLUSIONS: We estimate that   1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants with relatively higher allele fraction. Although blood is a readily available DNA source, cardiac tissues analyzed contributed   5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses.

INTRODUCTION: To increase the rate of iron sufficiency among neonatal intensive care unit (NICU) patients from 16% to >35% within 12 months of implementing standardized assessment of reticulocyte hemoglobin (retHE).

METHODS: We implemented a quality improvement (QI) study to improve iron sufficiency in our out-born level III/IV NICU. We screened 2,062 admissions, of which 622 were eligible based on feeding status at discharge. QI interventions included educational efforts and guideline implementation. Our primary outcome measure was the percentage of patients with their discharge retHE measure within the normal range. We also tracked the process measure of the number of retHE tests performed and a balancing measure of the incidence of elevated retHE among patients receiving iron supplementation. Statistical process control (SPC) charts assessed for special cause variation.

RESULTS: The percentage of patients with a retHe within the normal range was significantly increased from a mean of 20% to 39% on SPC chart analysis. We measured significantly more retHE values after guideline implementation (11/mo to 24/mo) and found no cases of elevated retHE among patients receiving iron supplementation.

CONCLUSIONS: After the implementation of a standardized guideline, a higher rate of iron sufficiency was found in NICU patients at discharge. This work is generalizable to neonatal populations with the potential for a significant impact on clinical practice.

Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2, and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.