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Birth defects are the leading cause of infant mortality in the United States, accounting for more than 20% of all infant deaths.[1] Birth defects also contribute substantially to morbidity and […]

Rates of neural tube defects have decreased since folic acid fortification of the food supply in the United States. The authors' objective was to evaluate the associations between neural tube defects and maternal folic acid intake among pregnancies conceived after fortification. This is a multicenter, case-control study that uses data from the National Birth Defects Prevention Study, 1998-2003. Logistic regression was used to compute crude and adjusted odds ratios between cases and controls assessing maternal periconceptional use of folic acid and intake of dietary folic acid. Among 180 anencephalic cases, 385 spina bifida cases, and 3, 963 controls, 21.1%, 25.2%, and 26.1%, respectively, reported periconceptional use of folic acid supplements. Periconceptional supplement use did not reduce the risk of having a pregnancy affected by a neural tube defect. Maternal intake of dietary folate was not significantly associated with neural tube defects. In this study conducted among pregnancies conceived after mandatory folic acid fortification, the authors found little evidence of an association between neural tube defects and maternal folic acid intake. A possible explanation is that folic acid fortification reduced the occurrence of folic acid-sensitive neural tube defects. Further investigation is warranted to possibly identify women who remain at increased risk of preventable neural tube defects. [ABSTRACT FROM AUTHOR]

OBJECTIVE: This study was undertaken to investigate the association between congenital heart defects (CHD), and maternal homocysteine, smoking, and the MTHFR 677 C>T polymorphism. STUDY DESIGN: Plasma homocysteine concentrations, smoking status, and MTFHR 677 genotypes were determined in 275 white women who had pregnancies affected by CHDs and 118 white women who had a normal pregnancy. RESULTS: Homocysteine concentrations were significantly higher among women who had affected pregnancies (P < .0001). The highest estimated risk for having a CHD-affected pregnancy was among women who were smokers, were in the highest quartile for homocysteine, and had the MTHFR 677 CC genotype (odds ratio [OR] 11.8; 95% CI 2.6-53.3). CONCLUSION: Many CHDs are due to a complex interaction between lifestyle factors and genetic susceptibilities. Our results suggest that the combined effect of elevations in maternal homocysteine, smoking, and the MTHFR 677 C>T polymorphism increase the risk of having a CHD-affected pregnancy. [ABSTRACT FROM AUTHOR]

BACKGROUND: Women who have had pregnancies that were affected by nonsyndromic congenital heart defects have alterations in the homocysteine-methionine pathway that may indicate increased exposure to oxidative stress or reduced antioxidant defense or both. OBJECTIVE: Our goal was to establish a maternal metabolic risk profile for nonsyndromic congenital heart defects that would enhance current preventive strategies. DESIGN: Using a case-control design, we measured biomarkers of the transsulfuration pathway in a population-based sample of women whose pregnancies were affected by congenital heart defects (331 cases) and in a control group of women (125 controls). Plasma concentrations of reduced and oxidized glutathione, vitamin B-6, homocysteine, cysteine, cysteinylglycine (CysGly), and glutamylcysteine (GluCys) were compared between cases and controls after adjustment for lifestyle and sociodemographic variables. RESULTS: After covariate adjustment, cases had significantly lower mean plasma concentrations of reduced glutathione (P < 0.0001), GluCys (P < 0.0001), and vitamin B-6 (P = 0.0023) and significantly higher mean concentrations of homocysteine (P < 0.0001) and oxidized glutathione (P < 0.0001) than did controls. CONCLUSIONS: Biomarkers of oxidative stress involved in the transsulfuration pathway were significantly higher in women with pregnancies affected by congenital heart defects than in women without such a history. Further analysis of relevant biomarkers of oxidative stress and genetic and environmental factors is required to define the basis for the observed alterations. Identifying the nature and extent of alterations in biomarkers of oxidative stress may suggest primary intervention strategies and provide clues to a greater understanding of the pathogenesis of congenital heart defects. Copyright © 2005 American Society for Clinical Nutrition [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to examine the risk of birth defects in relation to diabetes mellitus and the lack of use of periconceptional vitamins or supplements that contain folic acid.Study Design: The National Birth Defects Prevention Study (1997-2004) is a multicenter, population-based case-control study of birth defects (14,721 cases and 5437 control infants). Cases were categorized into 18 types of heart defects and 26 noncardiac birth defects. We estimated odds ratios for independent and joint effects of preexisting diabetes mellitus and a lack of periconceptional use of vitamins or supplements that contain folic acid.Results: The pattern of odds ratios suggested an increased risk of defects that are associated with diabetes mellitus in the absence vs the presence of the periconceptional use of vitamins or supplements that contain folic acid.Conclusion: The lack of periconceptional use of vitamins or supplements that contain folic acid may be associated with an excess risk for birth defects due to diabetes mellitus. [ABSTRACT FROM AUTHOR]

Oculogastrointestinal neurodevelopmental syndrome has been described in seven previously published individuals who harbor biallelic pathogenic variants in the CAPN15 gene. Biallelic missense variants have been reported to demonstrate a phenotype of eye abnormalities and developmental delay, while biallelic loss of function variants exhibit phenotypes including microcephaly and craniofacial abnormalities, cardiac and genitourinary malformations, and abnormal neurologic activity. We report six individuals from three unrelated families harboring biallelic deleterious variants in CAPN15 with phenotypes overlapping those previously described for this disorder. Of the individuals affected, four demonstrate radiographic evidence of the classical triad of Dandy-Walker malformation including hypoplastic vermis, fourth ventricle enlargement, and torcular elevation. Cerebellar anomalies have not been previously reported in association with CAPN15-related disease. Here, we present three unrelated families with findings consistent with oculogastrointestinal neurodevelopmental syndrome and cerebellar pathology including Dandy-Walker malformation. To corroborate these novel clinical findings, we present supporting data from the mouse model suggesting an important role for this protein in normal cerebellar development. Our findings add six molecularly confirmed cases to the literature and additionally establish a new association of Dandy-Walker malformation with biallelic CAPN15 variants, thereby expanding the neurologic spectrum among patients affected by CAPN15-related disease., (© 2023 Wiley Periodicals LLC.)

Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. The resulting background of less than one artifactual mutation per 10 7 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DS-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues⁠, a considerable advancement compared to currently used in vivo gene mutation assays., Competing Interests: Declaration of Competing Interest G.A.P., F.Y.L, J.E.H., E.K.S., L.N.W., D.N., C.C.V., and J.J.S. are employees (or former employees) and equity holders of TwinStrand Biosciences, Inc. F.Y.L, E.K.S., L.N.W., C.C.V., and J.J.S. are authors on one or more duplex sequencing-related patents., (Published by Elsevier B.V.)

The risk of congenital heart defects (CHDs) may be influenced by maternal genes, fetal genes, and their interactions. Existing methods commonly test the effects of maternal and fetal variants one-at-a-time and may have reduced statistical power to detect genetic variants with low minor allele frequencies. In this article, we propose a gene-based association test of interactions for maternal-fetal genotypes (GATI-MFG) using a case-mother and control-mother design. GATI-MFG can integrate the effects of multiple variants within a gene or genomic region and evaluate the joint effect of maternal and fetal genotypes while allowing for their interactions. In simulation studies, GATI-MFG had improved statistical power over alternative methods, such as the single-variant test and functional data analysis (FDA) under various disease scenarios. We further applied GATI-MFG to a two-phase genome-wide association study of CHDs for the testing of both common variants and rare variants using 947 CHD case mother-infant pairs and 1306 control mother-infant pairs from the National Birth Defects Prevention Study (NBDPS). After Bonferroni adjustment for 23,035 genes, two genes on chromosome 17, TMEM107 (p = 1.64e-06) and CTC1 (p = 2.0e-06), were identified for significant association with CHD in common variants analysis. Gene TMEM107 regulates ciliogenesis and ciliary protein composition and was found to be associated with heterotaxy. Gene CTC1 plays an essential role in protecting telomeres from degradation, which was suggested to be associated with cardiogenesis. Overall, GATI-MFG outperformed the single-variant test and FDA in the simulations, and the results of application to NBDPS samples are consistent with existing literature supporting the association of TMEM107 and CTC1 with CHDs., (© 2023 The Authors. Genetic Epidemiology published by Wiley Periodicals LLC.)

Conotruncal heart defects (CTDs) are heart malformations that affect the cardiac outflow tract and typically cause significant morbidity and mortality. Evidence from epidemiological studies suggests that maternal folate intake is associated with a reduced risk of heart defects, including CTD. However, it is unclear if folate-related gene variants and maternal folate intake have an interactive effect on the risk of CTDs. In this study, we performed targeted sequencing of folate-related genes on DNA from 436 case families with CTDs who are enrolled in the National Birth Defects Prevention Study and then tested for common and rare variants associated with CTD. We identified risk alleles in maternal MTHFS (OR meta = 1.34; 95% CI 1.07 to 1.67), maternal NOS2 (OR meta = 1.34; 95% CI 1.05 to 1.72), fetal MTHFS (OR meta = 1.35; 95% CI 1.09 to 1.66), and fetal TCN2 (OR meta = 1.38; 95% CI 1.12 to 1.70) that are associated with an increased risk of CTD among cases without folic acid supplementation. We detected putative de novo mutations in genes from the folate, homocysteine, and transsulfuration pathways and identified a significant association between rare variants in MGST1 and CTD risk. Results suggest that periconceptional folic acid supplementation is associated with decreased risk of CTD among individuals with susceptible genotypes.

Background: There is emerging evidence that children with complex congenital heart defects (CHDs) are at increased risk for childhood lymphoma, but the mechanisms underlying this association are unclear. Thus, we sought to evaluate the role of DNA methylation patterns on "CHD-lymphoma" associations., Methods: From >3 million live births (1988-2004) in California registry linkages, we obtained newborn dried bloodspots from eight children with CHD-lymphoma through the California BioBank. We performed case-control epigenome-wide association analyses (EWAS) using two comparison groups with reciprocal discovery and validation to identify differential methylation associated with CHD-lymphoma., Results: After correction for multiple testing at the discovery and validation stages, individuals with CHD-lymphoma had differential newborn methylation at six sites relative to two comparison groups. Our top finding was significant in both EWAS and indicates PPFIA1 cg25574765 was hypomethylated among individuals with CHD-lymphoma (mean beta = 0.04) relative to both unaffected individuals (mean beta = 0.93, p = 1.5 × 10 -12 ) and individuals with complex CHD (mean beta = 0.95, p = 3.8 × 10 -8 ). PPFIA1 encodes a ubiquitously expressed liprin protein in one of the most commonly amplified regions in many cancers (11q13). Further, cg25574765 is a proposed marker of pre-eclampsia, a maternal CHD risk factor that has not been fully evaluated for lymphoma risk in offspring, and the tumor microenvironment that may drive immune cell malignancies., Conclusions: We identified associations between molecular changes present in the genome at birth and risk of childhood lymphoma among those with CHD. Our findings also highlight novel perinatal exposures that may underlie methylation changes in CHD predisposing to lymphoma., (© 2022 Wiley Periodicals LLC.)

Black cohosh (BC; Actaea racemosa L.), a top-selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose-related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross-sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC-naïve women. Twenty-three women were enrolled in the BC-exposed group and 28 in the BC-naïve group. Use of any brand of BC-only supplement for at least 3 months was required for inclusion in the BC-exposed group. Supplements were analyzed for chemical composition to allow cross-product comparisons. All participants were healthy, with no known exposures (e.g., x-rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings., (© 2022 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagen Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Rapid genome sequencing in critically ill infants is increasingly identified as a crucial test for providing targeted and informed patient care. We report the outcomes of a pilot study wherein eight critically ill neonates received rapid whole genome sequencing with parental samples in an effort to establish a prompt diagnosis. Our pilot study resulted in a 37.5% diagnostic rate by whole genome sequencing alone and an overall 50% diagnostic rate for the cohort. We describe how the diagnoses led to identification of additional affected relatives and a change in management, the limitations of rapid genome sequencing, and some of the challenges with sample collection. Alongside this pilot study, our site simultaneously established a research protocol pipeline that will allow us to conduct research-based genomic testing in the cases for which a diagnosis was not reached by rapid genome sequencing or other available clinical testing. Here we describe the benefits, limitations, challenges, and potential for rapid whole genome sequencing to be incorporated into routine clinical evaluation in the neonatal period.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases 1-3 . SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing 4,5 . Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission., (© 2022. The Author(s).)

Newborn screening (NBS) dramatically improves outcomes in severe childhood disorders by treatment before symptom onset. In many genetic diseases, however, outcomes remain poor because NBS has lagged behind drug development. Rapid whole-genome sequencing (rWGS) is attractive for comprehensive NBS because it concomitantly examines almost all genetic diseases and is gaining acceptance for genetic disease diagnosis in ill newborns. We describe prototypic methods for scalable, parentally consented, feedback-informed NBS and diagnosis of genetic diseases by rWGS and virtual, acute management guidance (NBS-rWGS). Using established criteria and the Delphi method, we reviewed 457 genetic diseases for NBS-rWGS, retaining 388 (85%) with effective treatments. Simulated NBS-rWGS in 454,707 UK Biobank subjects with 29,865 pathogenic or likely pathogenic variants associated with 388 disorders had a true negative rate (specificity) of 99.7% following root cause analysis. In 2,208 critically ill children with suspected genetic disorders and 2,168 of their parents, simulated NBS-rWGS for 388 disorders identified 104 (87%) of 119 diagnoses previously made by rWGS and 15 findings not previously reported (NBS-rWGS negative predictive value 99.6%, true positive rate [sensitivity] 88.8%). Retrospective NBS-rWGS diagnosed 15 children with disorders that had been undetected by conventional NBS. In 43 of the 104 children, had NBS-rWGS-based interventions been started on day of life 5, the Delphi consensus was that symptoms could have been avoided completely in seven critically ill children, mostly in 21, and partially in 13. We invite groups worldwide to refine these NBS-rWGS conditions and join us to prospectively examine clinical utility and cost effectiveness., Competing Interests: Declaration of interests K.P.H., C.M.K., S.S.M., and D.T. are employees and shareholders of Illumina, Inc. G.D,A., B.M., S.L., and T.D. are employees and shareholders of Alexion Pharmaceuticals. E.F. and M.G.R. are employees and shareholders of Fabric Genomics, Inc. M.K. and S.S. are employees and shareholders of Genomenon, Inc. C.K., G.P., S.S., S.P., and A.R.W. are employees and shareholders of TileDB, Inc. S.K. is an employee and shareholder of Luna PBC, Inc. S.K. has filed a patent related to this work., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Motivation: CpG sites within the same genomic region often share similar methylation patterns and tend to be co-regulated by multiple genetic variants that may interact with one another., Results: We propose a multi-trait methylation random field (multi-MRF) method to evaluate the joint association between a set of CpG sites and a set of genetic variants. The proposed method has several advantages. First, it is a multi-trait method that allows flexible correlation structures between neighboring CpG sites (e.g. distance-based correlation). Second, it is also a multi-locus method that integrates the effect of multiple common and rare genetic variants. Third, it models the methylation traits with a beta distribution to characterize their bimodal and interval properties. Through simulations, we demonstrated that the proposed method had improved power over some existing methods under various disease scenarios. We further illustrated the proposed method via an application to a study of congenital heart defects (CHDs) with 83 cardiac tissue samples. Our results suggested that gene BACE2, a methylation quantitative trait locus (QTL) candidate, colocalized with expression QTLs in artery tibial and harbored genetic variants with nominal significant associations in two genome-wide association studies of CHD., Availability and Implementation: https://github.com/chenlyu2656/Multi-MRF., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Obstructive heart defects (OHDs) share common structural lesions in arteries and cardiac valves, accounting for ~25% of all congenital heart defects. OHDs are highly heritable, resulting from interplay among maternal exposures, genetic susceptibilities, and epigenetic phenomena. A genome-wide association study was conducted in National Birth Defects Prevention Study participants (N discovery  = 3978; N replication  = 2507), investigating the genetic architecture of OHDs using transmission/disequilibrium tests (TDT) in complete case-parental trios (N discovery&#95;TDT  = 440; N replication&#95;TDT  = 275) and case-control analyses separately in infants (N discovery&#95;CCI  = 1635; N replication&#95;CCI  = 990) and mothers (case status defined by infant; N discovery&#95;CCM  = 1703; N replication&#95;CCM  = 1078). In the TDT analysis, the SLC44A2 single nucleotide polymorphism (SNP) rs2360743 was significantly associated with OHD (p discovery  = 4.08 × 10 -9 ; p replication  = 2.44 × 10 -4 ). A CAPN11 SNP (rs55877192) was suggestively associated with OHD (p discovery  = 1.61 × 10 -7 ; p replication  = 0.0016). Two other SNPs were suggestively associated (p < 1 × 10 -6 ) with OHD in only the discovery sample. In the case-control analyses, no SNPs were genome-wide significant, and, even with relaxed thresholds ( ×  discovery  < 1 × 10 -5 and p replication  < 0.05), only one SNP (rs188255766) in the infant analysis was associated with OHDs (p discovery  = 1.42 × 10 -6 ; p replication  = 0.04). Additional SNPs with p discovery  < 1 × 10 -5 were in loci supporting previous findings but did not replicate. Overall, there was modest evidence of an association between rs2360743 and rs55877192 and OHD and some evidence validating previously published findings., (© 2022 Wiley Periodicals LLC.)

While many genetic diseases have effective treatments, they frequently progress rapidly to severe morbidity or mortality if those treatments are not implemented immediately. Since front-line physicians frequently lack familiarity with these diseases, timely molecular diagnosis may not improve outcomes. Herein we describe Genome-to-Treatment, an automated, virtual system for genetic disease diagnosis and acute management guidance. Diagnosis is achieved in 13.5 h by expedited whole genome sequencing, with superior analytic performance for structural and copy number variants. An expert panel adjudicated the indications, contraindications, efficacy, and evidence-of-efficacy of 9911 drug, device, dietary, and surgical interventions for 563 severe, childhood, genetic diseases. The 421 (75%) diseases and 1527 (15%) effective interventions retained are integrated with 13 genetic disease information resources and appended to diagnostic reports ( https://gtrx.radygenomiclab.com ). This system provided correct diagnoses in four retrospectively and two prospectively tested infants. The Genome-to-Treatment system facilitates optimal outcomes in children with rapidly progressive genetic diseases., (© 2022. The Author(s).)

Background: Discharge from the NICU is a highly complex process. Multidisciplinary survey results and chart audits identified gaps in the timeliness and efficiency of discharge in our NICU. Using the define-measure-analyze-improve-control quality improvement framework, we aimed to increase the percentage of patients discharged before 11:00 am from a baseline mean of 9.4% to 50% without adversely impacting caregiver readiness to discharge., Methods: We used a fishbone diagram to identify causes of late and inefficient NICU discharge. A Pareto chart and Impact-Effort matrix were used to select targets for improvement efforts. Plan-do-study-act (PDSA) cycles established a goal unit discharge time, created a discharge checklist, prioritized rounding on discharging patients, set expectations for caregiver education completion, and increased nurse knowledge and comfort with providing caregiver education., Results: The mean percent of patients discharged before 11:00 am increased from 9.4% to 52.4%, exceeding our aim. Median discharge time improved from 13:30 pm to 11:15 am (P < .001). Discharge was more efficient as demonstrated by significantly earlier completion of many discharge tasks. These improvements did not adversely impact reported caregiver readiness to discharge (75% vs 77%, P = .76)., Conclusions: Quality improvement methods can significantly improve the timeliness and efficiency of NICU discharge. Improvement in this complex process may be facilitated by a multidisciplinary team that offers diverse perspectives, unique process and methodologic knowledge, and the ability to appeal to all unit stakeholders. Lessons learned from this project may benefit other teams working to improve their ICU discharge process., (Copyright © 2022 by the American Academy of Pediatrics.)

Objectives: To assess associations between maternal smoking and congenital heart defects (CHDs) in offspring., Study Design: We performed a retrospective case-control study using data for cases of CHD (n = 8339) and nonmalformed controls (n = 11 020) from all years (1997-2011) of the National Birth Defects Prevention Study. Maternal self-reported smoking 1 month before through 3 months after conception was evaluated as a binary (none, any) and categorical (light, medium, heavy) exposure. Multivariable logistic regression was used to estimate aOR and 95% CIs. Stratified analyses were performed for septal defects according to maternal age, prepregnancy body mass index, and maternal race/ethnicity., Results: Multiple CHDs displayed modest associations with any level of maternal periconceptional smoking independent of potential confounders; the strongest associations were for aggregated septal defects (OR, 1.5; 95% CI, 1.3-1.7), tricuspid atresia (OR, 1.7; 95% CI, 1.0-2.7), and double outlet right ventricle (DORV) (OR, 1.5; 95% CI, 1.1-2.1). Tricuspid atresia and DORV also displayed dose-response relationships. Among heavy smokers, the highest odds were again observed for tricuspid atresia (aOR 3.0; 95% CI, 1.5-6.1) and DORV (aOR 1.5; 95% CI, 1.1-2.2). Heavy smokers ≥35 years old more frequently had a child with a septal defect when compared with similarly aged nonsmokers (aOR 2.3; 95% CI, 1.4-3.9)., Conclusions: Maternal periconceptional smoking is most strongly associated with septal defects, tricuspid atresia, and DORV; the risk for septal defects is modified by maternal age., (Copyright © 2021 Elsevier Inc. All rights reserved.)

DNA methylation may be regulated by genetic variants within a genomic region, referred to as methylation quantitative trait loci (mQTLs). The changes of methylation levels can further lead to alterations of gene expression, and influence the risk of various complex human diseases. Detecting mQTLs may provide insights into the underlying mechanism of how genotypic variations may influence the disease risk. In this article, we propose a methylation random field (MRF) method to detect mQTLs by testing the association between the methylation level of a CpG site and a set of genetic variants within a genomic region. The proposed MRF has two major advantages over existing approaches. First, it uses a beta distribution to characterize the bimodal and interval properties of the methylation trait at a CpG site. Second, it considers multiple common and rare genetic variants within a genomic region to identify mQTLs. Through simulations, we demonstrated that the MRF had improved power over other existing methods in detecting rare variants of relatively large effect, especially when the sample size is small. We further applied our method to a study of congenital heart defects with 83 cardiac tissue samples and identified two mQTL regions, MRPS10 and PSORS1C1, which were colocalized with expression QTL in cardiac tissue. In conclusion, the proposed MRF is a useful tool to identify novel mQTLs, especially for studies with limited sample sizes., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Bladder exstrophy (BE) is a rare, lower ventral midline defect with the bladder and part of the urethra exposed. The etiology of BE is unknown but thought to be influenced by genetic variation with more recent studies suggesting a role for rare variants. As such, we conducted paired-end exome sequencing in 26 child/mother/father trios. Three children had rare (allele frequency ≤ 0.0001 in several public databases) inherited variants in TSPAN4, one with a loss-of-function variant and two with missense variants. Two children had loss-of-function variants in TUBE1. Four children had rare missense or nonsense variants (one per child) in WNT3, CRKL, MYH9, or LZTR1, genes previously associated with BE. We detected 17 de novo missense variants in 13 children and three de novo loss-of-function variants (AKR1C2, PRRX1, PPM1D) in three children (one per child). We also detected rare compound heterozygous loss-of-function variants in PLCH2 and CLEC4M and rare inherited missense or loss-of-function variants in additional genes applying autosomal recessive (three genes) and X-linked recessive inheritance models (13 genes). Variants in two genes identified may implicate disruption in cell migration (TUBE1) and adhesion (TSPAN4) processes, mechanisms proposed for BE, and provide additional evidence for rare variants in the development of this defect., (© 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

The emergence of the COVID-19 epidemic in the United States (U.S.) went largely undetected due to inadequate testing. New Orleans experienced one of the earliest and fastest accelerating outbreaks, coinciding with Mardi Gras. To gain insight into the emergence of SARS-CoV-2 in the U.S. and how large-scale events accelerate transmission, we sequenced SARS-CoV-2 genomes during the first wave of the COVID-19 epidemic in Louisiana. We show that SARS-CoV-2 in Louisiana had limited diversity compared to other U.S. states and that one introduction of SARS-CoV-2 led to almost all of the early transmission in Louisiana. By analyzing mobility and genomic data, we show that SARS-CoV-2 was already present in New Orleans before Mardi Gras, and the festival dramatically accelerated transmission. Our study provides an understanding of how superspreading during large-scale events played a key role during the early outbreak in the U.S. and can greatly accelerate epidemics., Competing Interests: Declaration of interests M.A.S. reports grants from the National Institutes of Health, European Research Council, and Wellcome Trust during the conduct of this research and grants and contracts from the Bill & Melinda Gates Foundation, Janssen Research and Development, Private Health Management, IQVIA, and the U.S. Department of Veterans Affairs outside the submitted work. S.L.L., R.R., and D.J.N. are employed by BioInfoexperts LLC. R.F.G. reports grants from the National Institutes of Health, the Coalition for Epidemic Preparedness Innovations, the Burroughs Wellcome Fund, the Wellcome Trust, the Center for Disease Prevention and Control, and the European & Developing Countries Clinical Trials Partnership. He is the co-founder and Chief Scientific Advisor of Zalgen Labs, a biotechnology company developing countermeasures to emerging viruses, including SARS-CoV-2. K.G.A. has received consulting fees and compensated expert testimony on SARS-CoV-2 and the COVID-19 pandemic., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Most congenital heart defects (CHDs) result from complex interactions among genetic susceptibilities, epigenetic modifications, and maternal environmental exposures. Characterizing the complex relationship between genetic, epigenetic, and transcriptomic variation will enhance our understanding of pathogenesis in this important type of congenital disorder. We investigated cis-acting effects of genetic single nucleotide polymorphisms (SNPs) on local DNA methylation patterns within 83 cardiac tissue samples and prioritized their contributions to CHD risk by leveraging results of CHD genome-wide association studies (GWAS) and their effects on cardiac gene expression., Results: We identified 13,901 potential methylation quantitative trait loci (mQTLs) with a false discovery threshold of 5%. Further co-localization analyses and Mendelian randomization indicated that genetic variants near the HLA-DRB6 gene on chromosome 6 may contribute to CHD risk by regulating the methylation status of nearby CpG sites. Additional SNPs in genomic regions on chromosome 10 (TNKS2-AS1 gene) and chromosome 14 (LINC01629 gene) may simultaneously influence epigenetic and transcriptomic variations within cardiac tissues., Conclusions: Our results support the hypothesis that genetic variants may influence the risk of CHDs through regulating the changes of DNA methylation and gene expression. Our results can serve as an important source of information that can be integrated with other genetic studies of heart diseases, especially CHDs.

Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant's mode of action. To address the limited approaches available for identifying epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration-response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4 h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (RGL1, NEU1, GPR183) or cellular stress-response genes (ATF3, CDKN1A, GADD45A) analyzed by TaqMan qPCR assays after 4 h of exposure. Whole transcriptomes were profiled after 4 h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.

Bisphenol A (BPA) is a chemical used in the manufacturing of plastics to which human exposure is ubiquitous. Numerous studies have linked BPA exposure to many adverse health outcomes prompting the replacement of BPA with various analogues including bisphenol-F (BPF) and bisphenol S (BPS). Other bisphenols are used in various consumer applications, such as 3,3',5,5'-Tetrabromobisphenol A (TBBPA), which is used as a flame retardant. Few studies to date have examined the effects of BPA and its analogues in stem cells to explore potential developmental impacts. Here we used transcriptomics to investigate similarities and differences of BPA and three of its analogues in the estrogen receptor negative, human embryonic stem cell line H9 (WA09). H9 cells were exposed to increasing concentrations of the bisphenols and analyzed using RNA-sequencing. Our data indicate that BPA, BPF, and BPS have similar potencies in inducing transcriptional changes and perturb many of the same pathways. TBBPA, the least structurally similar bisphenol of the group, exhibited much lower potency. All bisphenols robustly impacted gene expression in these cells, albeit at concentrations well above those observed in estrogen-positive cells. Overall, we provide a foundational data set against which to explore the transcriptional similarities of other bisphenols in embryonic stem cells, which may be used to assess the suitability of chemical grouping for read-across and for preliminary potency evaluation., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Previous research on risk factors for obstructive heart defects (OHDs) focused on maternal and infant genetic variants, prenatal environmental exposures, and their potential interaction effects. Less is known about the role of paternal genetic variants or environmental exposures and risk of OHDs. We examined parent-of-origin effects in transmission of alleles in the folate, homocysteine, or transsulfuration pathway genes on OHD occurrence in offspring. We used data on 569 families of liveborn infants with OHDs born between October 1997 and August 2008 from the National Birth Defects Prevention Study to conduct a family-based case-only study. Maternal, paternal, and infant DNA were genotyped using an Illumina Golden Gate custom single nucleotide polymorphism (SNP) panel. Relative risks (RR), 95% confidence interval (CI), and likelihood ratio tests from log-linear models were used to estimate the parent-of-origin effect of 877 SNPs in 60 candidate genes in the folate, homocysteine, and transsulfuration pathways on the risk of OHDs. Bonferroni correction was applied for multiple testing. We identified 3 SNPs in the transsulfuration pathway and 1 SNP in the folate pathway that were statistically significant after Bonferroni correction. Among infants who inherited paternally-derived copies of the G allele for rs6812588 in the RFC1 gene, the G allele for rs1762430 in the MGMT gene, and the A allele for rs9296695 and rs4712023 in the GSTA3 gene, RRs for OHD were 0.11 (95% CI: 0.04, 0.29, P = 9.16x10-7), 0.30 (95% CI: 0.17, 0.53, P = 9.80x10-6), 0.34 (95% CI: 0.20, 0.57, P = 2.28x10-5), and 0.34 (95% CI: 0.20, 0.58, P = 3.77x10-5), respectively, compared to infants who inherited maternally-derived copies of the same alleles. We observed statistically significant decreased risk of OHDs among infants who inherited paternal gene variants involved in folate and transsulfuration pathways., Competing Interests: The authors have declared that no competing interests exist.

Genes, including those with transgenerational effects, work in concert with behavioral, environmental, and social factors via complex biological networks to determine human health. Understanding complex relationships between causal factors underlying human health is an essential step towards deciphering biological mechanisms. We propose a new analytical framework to investigate the interactions between maternal and offspring genetic variants or their surrogate single nucleotide polymorphisms (SNPs) and environmental factors using family-based hybrid study design. The proposed approach can analyze diverse genetic and environmental factors and accommodate samples from a variety of family units, including case/control-parental triads, and case/control-parental dyads, while minimizing potential bias introduced by population admixture. Comprehensive simulations demonstrated that our innovative approach outperformed the log-linear approach, the best available method for case-control family data. The proposed approach had greater statistical power and was capable to unbiasedly estimate the maternal and child genetic effects and the effects of environmental factors, while controlling the Type I error rate against population stratification. Using our newly developed approach, we analyzed the associations between maternal and fetal SNPs and obstructive and conotruncal heart defects, with adjustment for demographic and lifestyle factors and dietary supplements. Fourteen and 11 fetal SNPs were associated with obstructive and conotruncal heart defects, respectively. Twenty-seven and 17 maternal SNPs were associated with obstructive and conotruncal heart defects, respectively. In addition, maternal body mass index was a significant risk factor for obstructive defects. The proposed approach is a powerful tool for interrogating the etiological mechanism underlying complex traits., (© 2020 Wiley Periodicals LLC.)

Background: Maternal hypertension has been associated with congenital heart defect occurrence in several studies. We assessed whether maternal genotypes associated with this condition were also associated with congenital heart defect occurrence., Methods: We used data from the National Birth Defects Prevention Study to identify non-Hispanic white (NHW) and Hispanic women with (cases) and without (controls) a pregnancy in which a select simple, isolated heart defect was present between 1999 and 2011. We genotyped 29 hypertension-related single nucleotide polymorphisms (SNPs). We conducted logistic regression analyses separately by race/ethnicity to assess the relationship between the presence of any congenital heart defect and each SNP and an overall blood pressure genetic risk score (GRS). All analyses were then repeated to assess 4 separate congenital heart defect subtypes., Results: Four hypertension-related variants were associated with congenital heart defects among NHW women (N = 1,568 with affected pregnancies). For example, 1 intronic variant in ARHGAP2, rs633185, was associated with conotruncal defects (odds ratio [OR]: 1.3, 95% confidence interval [CI]: 1.1-1.6). Additionally, 2 variants were associated with congenital heart defects among Hispanic women (N = 489 with affected pregnancies). The GRS had a significant association with septal defects (OR: 2.1, 95% CI: 1.2-3.5) among NHW women., Conclusions: We replicated a previously reported association between rs633185 and conotruncal defects. Although additional hypertension-related SNPs were also associated with congenital heart defects, more work is needed to better understand the relationship between genetic risk for maternal hypertension and congenital heart defects occurrence., (© American Journal of Hypertension, Ltd 2020. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Hypospadias is a common birth defect where the urethral opening forms on the ventral side of the penis. We performed integrative methylomic, genomic, and transcriptomic analyses to characterize sites of DNA methylation that influence genital development. In case-control and case-only epigenome-wide association studies (EWAS) of preputial tissue we identified 25 CpGs associated with hypospadias characteristics and used one-sample two stage least squares Mendelian randomization (2SLS MR) to show a causal relationship for 21 of the CpGs. The largest difference was 15.7% lower beta-value at cg14436889 among hypospadias cases than controls (EWAS P = 5.4e-7) and is likely causal (2SLS MR P = 9.8e-15). Integrative annotation using two-sample Mendelian randomization of these methylation regions highlight potentially causal roles of genes involved in germ layer differentiation (WDHD1, DNM1L, TULP3), beta-catenin signaling (PKP2, UBE2R2, TNKS), androgens (CYP4A11, CYP4A22, CYP4B1, CYP4X1, CYP4Z2P, EPHX1, CD33/SIGLEC3, SIGLEC5, SIGLEC7, KLK5, KLK7, KLK10, KLK13, KLK14), and reproductive traits (ACAA1, PLCD1, EFCAB4B, GMCL1, MKRN2, DNM1L, TEAD4, TSPAN9, KLK family). This study identified CpGs that remained differentially methylated after urogenital development and used the most relevant tissue sample available to study hypospadias. We identified multiple methylation sites and candidate genes that can be further evaluated for their roles in regulating urogenital development.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

The National Toxicology Program tested two common radiofrequency radiation (RFR) modulations emitted by cellular telephones in a 2-year rodent cancer bioassay that included interim assessments of additional animals for genotoxicity endpoints. Male and female Hsd:Sprague Dawley SD rats and B6C3F1/N mice were exposed from Gestation day 5 or Postnatal day 35, respectively, to code division multiple access (CDMA) or global system for mobile modulations over 18 hr/day, at 10-min intervals, in reverberation chambers at specific absorption rates of 1.5, 3, or 6 W/kg (rats, 900 MHz) or 2.5, 5, or 10 W/kg (mice, 1,900 MHz). After 19 (rats) or 14 (mice) weeks of exposure, animals were examined for evidence of RFR-associated genotoxicity using two different measures. Using the alkaline (pH > 13) comet assay, DNA damage was assessed in cells from three brain regions, liver cells, and peripheral blood leukocytes; using the micronucleus assay, chromosomal damage was assessed in immature and mature peripheral blood erythrocytes. Results of the comet assay showed significant increases in DNA damage in the frontal cortex of male mice (both modulations), leukocytes of female mice (CDMA only), and hippocampus of male rats (CDMA only). Increases in DNA damage judged to be equivocal were observed in several other tissues of rats and mice. No significant increases in micronucleated red blood cells were observed in rats or mice. In conclusion, these results suggest that exposure to RFR is associated with an increase in DNA damage. Environ. Mol. Mutagen. 61:276-290, 2020. © 2019 Wiley Periodicals, Inc., (© 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.)

Background: The development of conotruncal heart defects (CTDs) involves a complex relationship among genetic variants and maternal lifestyle factors. In this article, we focused on the interactions between 13 candidate genes within folate, homocysteine, and transsulfuration pathways for potential association with CTD risk., Methods: Targeted sequencing was used for 328 case-parental triads enrolled in the National Birth Defects Prevention Study (NBDPS). To evaluate the interaction of two genes, we applied a conditional logistic regression model for all possible SNP pairs within two respective genes by contrasting the affected infants with their pseudo-controls. The findings were replicated in an independent sample of 86 NBDPS case-parental triads genotyped by DNA microarrays. The results of two studies were further integrated by a fixed-effect meta-analysis., Results: One SNP pair (i.e., rs4764267 and rs6556883) located in gene MGST1 and GLRX, respectively, was found to be associated with CTD risk after multiple testing adjustment using simpleM, a modified Bonferroni correction approach (nominal p-value of 4.62e-06; adjusted p-value of .04). Another SNP pair (i.e., rs11892646 and rs56219526) located in gene DNMT3A and MTRR, respectively, achieved marginal significance after multiple testing adjustment (adjusted p-value of .06)., Conclusion: Further studies with larger sample sizes are needed to confirm and elucidate these potential interactions., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Background: Hypospadias is a common male birth defect that has shown widespread variation in reported prevalence estimates. Many countries have reported increasing trends over recent decades., Objective: To analyze the prevalence and trends of hypospadias for 27 international programs over a 31-yr period., Design, Setting, and Participants: The study population included live births, stillbirths, and elective terminations of pregnancy diagnosed with hypospadias during 1980-2010 from 27 surveillance programs around the world., Outcome Measurements and Statistical Analysis: We used joinpoint regression to analyze changes over time in international total prevalence of hypospadias across programs, prevalence for each specific program, and prevalence across different degrees of severity of hypospadias., Results and Limitations: The international total prevalence of hypospadias for all years was 20.9 (95% confidence interval: 19.2-22.6) per 10000 births. The prevalence for each program ranged from 2.1 to 39.1 per 10000 births. The international total prevalence increased 1.6 times during the study period, by 0.25 cases per 10000 births per year (p<0.05). When analyzed separately, there were increasing trends for first-, second-, and third-degree hypospadias during the early 1990s to mid-2000s. The majority of programs (61.9%) had a significantly increasing trend during many of the years evaluated. Limitations include known differences in data collection methods across programs., Conclusions: Although there have been changes in clinical practice and registry ascertainment over time in some countries, the consistency in the observed increasing trends across many programs and by degrees of severity suggests that the total prevalence of hypospadias may be increasing in many countries. This observation is contrary to some previous reports that suggested that the total prevalence of hypospadias was no longer increasing in recent decades., Patient Summary: We report on the prevalence and trends of hypospadias among 27 birth defect surveillance systems, which indicate that the prevalence of hypospadias continues to increase internationally., (Copyright © 2019 European Association of Urology. All rights reserved.)

Background: Recent genome-wide association studies of hypospadias have implicated the role of genetic variants in or near the diacylglycerol kinase kappa (DGKK) gene. However, these variants are largely identified among samples of mild and moderate hypospadias cases. Therefore, we evaluated previously identified DGKK variants among second- and third-degree hypospadias cases and controls recruited in Arkansas, a state characterized by a high birth prevalence of hypospadias., Methods: Second- and third-degree hypospadias non-Hispanic white cases (n = 36 and n = 9, respectively) and controls (n = 45) were recruited at Arkansas Children's Hospital. Preputial tissue was collected on cases and controls between 2013 and 2017. Cases and controls were genotyped using the Illumina Infinium Global Screening Array. We used logistic regression models to assess the association of genotyped and imputed genetic variants mapped to the DGKK region with second- and third-degree hypospadias., Results: All families self-reported as non-Hispanic white and genetic principal component analyses did not demonstrate evidence of population stratification. Five DGKK variants previously reported as associated with hypospadias were identified in the genotype data. None of the variants were associated with second- or third-degree hypospadias (range of odds ratios = 0.7-0.9, all p > .05)., Conclusions: In our analyses, genetic variation in DGKK does not play a role in the development of moderate and severe hypospadias. Our findings provide support to the etiologic heterogeneity of hypospadias by all classifications of severity., (© 2019 Wiley Periodicals, Inc.)

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Obstructive heart defects (OHDs) are a major health concern worldwide. The platelet-derived growth factor (PDGF) genes are known to have regulatory functions that are essential for proper heart development. In a zebrafish model, Pdgfra was further demonstrated to interact with ethanol during craniofacial development. In this article, we investigated interactions between variants in PDGF genes and periconceptional alcohol exposure on the risk of OHDs by applying log-linear models to 806 OHD case and 995 control families enrolled in the National Birth Defects Prevention Study. The interactions between four variants in PDGFA and maternal binge drinking reached a nominal significance level. The maternal T allele of rs869978 was estimated to increase OHD risk among women who binge drink, while infant genotypes of rs2291591, rs2228230, rs1547904, and rs869978 may reduce the risk. Although none of these associations remain statistically significant after multiple testing adjustment and the estimated maternal effect may be influenced by unknown confounding factors, such as maternal smoking, these findings are consistent with previous animal studies supporting potential interactions between the PDGFRA gene and maternal alcohol exposure. Replication studies with larger sample sizes are needed to further elucidate this potential interplay and its influence on OHD risks.

When a comet assay, an increasingly popular in vivo genotoxicity test, shows a positive test result, interpretation of that response requires ruling out any confounding tissue site toxicity. Since the comet assay typically uses only two or three daily doses of test agent, precursor tissue changes indicative of toxicity may be easily overlooked. Using case examples for two flavoring agents, perillaldehyde and 4,5-epoxydec-2( trans )-enal, we highlight the role of pathology peer review in verifying precursor tissue changes indicative of tissue site toxicity, thereby increasing confidence in final interpretation of comet assay results. Given global deliberation regarding safety assessment of compounds entering the marketplace, we recommend consideration of pathology peer review for equivocal and positive comet assays so that interpretations are universally consistent.