Innovative approach to identify multigenomic and environmental interactions associated with birth defects in family-based hybrid designs.

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.

African Americans' Perceptions of Adherence to Medications and Lifestyle Changes Prescribed to Treat Hypertension.

More than 80 million Americans have hypertension (HTN), and African Americans (AAs) are disproportionately affected. AAs also have lower rates of adherence to HTN treatment. It is important to understand AAs' perceptions of adherence to develop effective interventions. The aim of this study is to examine AAs' perceptions of adherence to medications and lifestyle changes prescribed to treat HTN. In this qualitative study, we used purposive sampling to recruit Southern AAs with HTN aged 21 and older from a free, faith-based clinic. We recorded individual, in-person interviews about perceptions related to adherence to treatment of HTN and analyzed verbatim transcripts using content analysis and constant comparison. We also conducted medical record audits. Twenty-nine AAs participated (52% female, 38% were <50 years of age, 52% had taken anti-HTN medications for ≥5 years). Audits indicated that 65% had uncontrolled HTN during the previous year. Two main themes included causes of HTN and ways to improve blood pressure. Perceived causes of HTN included diet, stress, unhealthy actions, genes, and obesity. Ways to improve HTN included using cultural treatments "passed down," increasing exercise, reducing stress, and losing weight. Many reported using home remedies to control HTN, including drinking pickle juice. More than half of this sample had uncontrolled HTN. They identified influences of culture on perceptions of adherence including causes and treatment of HTN, and possibly detrimental home remedies. It is imperative that clinicians identify culturally appropriate interventions for this high-risk group.

Uterine responses to feeding soy protein isolate and treatment with 17ß-estradiol differ in ovariectomized female rats.

There are concerns regarding reproductive toxicity from consumption of soy foods, including an increased risk of endometriosis and endometrial cancer, as a result of phytoestrogen consumption. In this study, female rats were fed AIN-93G diets made with casein (CAS) or soy protein isolate (SPI) from postnatal day (PND) 30, ovariectomized on PND 50 and infused with 5 µg/kg/d 17ß-estradiol (E2) or vehicle. E2 increased uterine wet weight (P<0.05). RNAseq analysis revealed that E2 significantly altered expression of 1991 uterine genes (P<0.05). SPI feeding had no effect on uterine weight and altered expression of far fewer genes than E2 at 152 genes (P<0.05). Overlap between E2 and SPI genes was limited to 67 genes. Functional annotation analysis indicated significant differences in uterine biological processes affected by E2 and SPI and little evidence for recruitment of estrogen receptor (ER)α to the promoters of ER-responsive genes after SPI feeding. The major E2 up-regulated uterine pathways were carcinogenesis and extracellular matrix organization, whereas SPI feeding up-regulated uterine peroxisome proliferator activated receptor (PPAR) signaling and fatty acid metabolism. The combination of E2 and SPI resulted in significant regulation of 504 fewer genes relative to E2 alone. The ability of E2 to induce uterine proliferation in response to the carcinogen dimethybenz(a)anthracene (DMBA) as measured by expression of PCNA and Ki67 mRNA was suppressed by feeding SPI (P<0.05). These data suggest that SPI is a selective estrogen receptor modulator (SERM) interacting with a small sub-set of E2-regulated genes and is anti-estrogenic in the presence of endogenous estrogens.

Obstructive heart defects associated with candidate genes, maternal obesity, and folic acid supplementation.

Right-sided and left-sided obstructive heart defects (OHDs) are subtypes of congenital heart defects, in which the heart valves, arteries, or veins are abnormally narrow or blocked. Previous studies have suggested that the development of OHDs involved a complex interplay between genetic variants and maternal factors. Using the data from 569 OHD case families and 1,644 control families enrolled in the National Birth Defects Prevention Study (NBDPS) between 1997 and 2008, we conducted an analysis to investigate the genetic effects of 877 single nucleotide polymorphisms (SNPs) in 60 candidate genes for association with the risk of OHDs, and their interactions with maternal use of folic acid supplements, and pre-pregnancy obesity. Applying log-linear models based on the hybrid design, we identified a SNP in methylenetetrahydrofolate reductase (MTHFR) gene (C677T polymorphism) with a main genetic effect on the occurrence of OHDs. In addition, multiple SNPs in betaine-homocysteine methyltransferase (BHMT and BHMT2) were also identified to be associated with the occurrence of OHDs through significant main infant genetic effects and interaction effects with maternal use of folic acid supplements. We also identified multiple SNPs in glutamate-cysteine ligase, catalytic subunit (GCLC) and DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B) that were associated with elevated risk of OHDs among obese women. Our findings suggested that the risk of OHDs was closely related to a combined effect of variations in genes in the folate, homocysteine, or glutathione/transsulfuration pathways, maternal use of folic acid supplements and pre-pregnancy obesity.

Conotruncal heart defects and common variants in maternal and fetal genes in folate, homocysteine, and transsulfuration pathways.

BACKGROUND: We investigated the association between conotruncal heart defects (CTDs) and maternal and fetal single nucleotide polymorphisms (SNPs) in 60 genes in the folate, homocysteine, and transsulfuration pathways. We also investigated whether periconceptional maternal folic acid supplementation modified associations between CTDs and SNPs METHODS: Participants were enrolled in the National Birth Defects Prevention Study between 1997 and 2008. DNA samples from 616 case-parental triads affected by CTDs and 1645 control-parental triads were genotyped using an Illumina® Golden Gate custom SNP panel. A hybrid design analysis, optimizing data from case and control trios, was used to identify maternal and fetal SNPs associated with CTDs RESULTS: Among 921 SNPs, 17 maternal and 17 fetal SNPs had a Bayesian false-discovery probability of <0.8. Ten of the 17 maternal SNPs and 2 of the 17 fetal SNPs were found within the glutamate-cysteine ligase, catalytic subunit (GCLC) gene. Fetal SNPs with the lowest Bayesian false-discovery probability (rs2612101, rs2847607, rs2847326, rs2847324) were found within the thymidylate synthetase (TYMS) gene. Additional analyses indicated that the risk of CTDs associated with candidate SNPs was modified by periconceptional folic acid supplementation. Nineteen maternal and nine fetal SNPs had a Bayesian false-discovery probability <0.8 for gene-by-environment (G × E) interactions with maternal folic acid supplementation. CONCLUSION: These results support previous studies suggesting that maternal and fetal SNPs within folate, homocysteine, and transsulfuration pathways are associated with CTD risk. Maternal use of supplements containing folic acid may modify the impact of SNPs on the developing heart.

Feeding soy protein isolate and treatment with estradiol have different effects on mammary gland morphology and gene expression in weanling male and female rats.

Isoflavones are phytochemical components of soy diets that bind weakly to estrogen receptors (ERs). To study potential estrogen-like actions of soy in the mammary gland during early development, we fed weanling male and female Sprague-Dawley rats a semipurified diet with casein as the sole protein source from postnatal day 21 to 33, the same diet substituting soy protein isolate (SPI) for casein, or the casein diet supplemented with estradiol (E2) at 10 µg/kg/day. In contrast to E2, the SPI diet induced no significant change in mammary morphology. In males, there were 34 genes for which expression was changed ≥2-fold in the SPI group vs. 509 changed significantly by E2, and 8 vs. 174 genes in females. Nearly half of SPI-responsive genes in males were also E2 responsive, including adipogenic genes. Serum insulin was found to be decreased by the SPI diet in males. SPI and E2 both downregulated the expression of ERα (Esr1) in males and females, and ERß (Esr2) only in males. Chromatin immunoprecipitation revealed an increased binding of ERα to the promoter of the progesterone receptor (Pgr) and Esr1 in both SPI- and E2-treated males compared with the casein group but differential recruitment of ERß. ER promoter binding did not correlate with differences in Pgr mRNA expression. This suggests that SPI fails to recruit appropriate co-activators at E2-inducible genes. Our results indicate that SPI behaves like a selective estrogen receptor modulator rather than a weak estrogen in the developing mammary gland.

Lack of periconceptional vitamins or supplements that contain folic acid and diabetes mellitus-associated birth defects.

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.

Association between selected folate pathway polymorphisms and nonsyndromic limb reduction defects: a case-parental analysis.

Inadequate folate status resulting from either genetic variation or nutritional deficiencies has been associated with an increased risk of congenital malformations including orofacial clefting, limb, cardiac and neural tube defects. Few epidemiological studies have examined the association between limb reduction defects (LRDs) and folate-related genetic polymorphisms other than MTHFR 677C→T. We conducted a case-parental analysis of 148 families who participated in the National Birth Defects Prevention Study to examine the association between nonsyndromic transverse and longitudinal LRDs with five single nucleotide polymorphisms (SNPs) in genes encoding enzymes in folate and methionine pathways. Log-linear Poisson regression, adapted for analysis of case-parental data assuming an additive genetic model, was used to estimate genetic relative risks and 95% confidence intervals for the association between LRDs and each SNP. Among women who did not take multivitamin supplements, the MTHFR 677T variant acts via the offspring's genome to increase the risk of LRDs. No association between LRDs and any fetal SNP was found among women who used multivitamin supplements. These results suggest the possibility that initiating folic acid supplementation prior to pregnancy may reduce the risk of having a LRD-affected pregnancy, especially in women whose offspring inherit one or two copies of the MTHFR 677T variant.

Neural tube defects and maternal folate intake among pregnancies conceived after folic acid fortification in the United States.

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.

Hospitalizations of newborns with folate-sensitive birth defects before and after fortification of foods with folic acid.

CONTEXT: The prevalence of neural tube defects is reduced in populations of women who receive folic acid supplementation. Since 1998, grain products in the United States have been fortified with folic acid. Fortification may have additional benefits by reducing the national prevalence of newborn hospitalizations for other folate-sensitive birth defects. OBJECTIVE: Our purpose with this work was to compare rates of hospitalizations of newborns with folate-sensitive birth defects before and after implementation of fortification of grains. METHOD: National hospital discharge data from the Healthcare Cost and Utilization Project were used to compute rates of newborn hospitalizations for selected birth defects per 10,000 live births in the United States. Newborn hospitalization rates involving congenital anomalies recognizable at birth were analyzed for 5 years before fortification of grains and 5 years after fortification. Additional analyses compared changes in newborn hospitalization rates for birth defects by race/ethnicity, income, insurance status, and region of the country. RESULTS: Newborn hospitalization rates for spina bifida decreased 21% from 1993-1997 to 1998-2002. Newborn hospitalization rates also decreased for anencephaly (20%) and limb-reduction defects (4%). Decline in hospitalizations for spina bifida occurred more often among Hispanic newborns (33%) than among white (13%) or black (21%) newborns. Decline in limb-reduction defects was seen primarily among blacks (11%). Findings using hospitalization data were similar to recent reports using birth defect surveillance systems with the exception of findings for orofacial clefts and conotruncal heart defects. No reductions were noted in newborn hospitalizations for these anomalies. CONCLUSIONS: Results from this ecological study fail to demonstrate substantial declines in newborn hospitalizations beyond those anticipated from a reduction in neural tube defects. The society-wide impact of the fortification program on birth defects and other health conditions should continue to be monitored.

Awareness and use of folic acid among women in the lower Mississippi Delta.

CONTEXT: National and state efforts to increase folic acid awareness and use may not be reaching large segments of the population. PURPOSE: This study examines folic acid awareness and use among women of childbearing age in a representative, economically at-risk rural sample and identifies factors that influence awareness and use. METHODS: A cross-sectional random digit dialing telephone survey was completed with a representative sample of 646 women aged 14-45 years in 36 counties of the lower Mississippi Delta. Folic acid awareness and supplement use were estimated by percentages weighted to reflect the 36-county population. Pregnancy intentions and the ability to become pregnant were used to predict awareness and use among a subsample of sexually active women. FINDINGS: Compared to national samples, Delta women were less likely to have heard of folic acid (75% vs 64%) or to take a regular (5-7 days/wk) folic acid supplement (34% vs 22%). The proportion of women who took regular folic acid supplements was very low among some subgroups: African Americans (14%), those 14-19 years of age (12%), and those with low incomes (13%) and low educational levels (14%). Of the women who reported being sexually active, the ability to become pregnant more than doubled their likelihood of regular supplement use. CONCLUSIONS: The national folic acid campaign has not reached many women in the rural Mississippi Delta. A new mode of folic acid education is needed that is focused on low-income and young women and women not planning pregnancies. In the Delta and similar geographic regions, health care providers, black church leaders, and youth group leaders could be valuable advocates for folic acid.

Randomized trial of a physician-based intervention to increase the use of folic acid supplements among women.

OBJECTIVE: Fewer than one third of American women take folic acid daily, although many women report that they would take folic acid if their physicians advised them to do so. This study determined the impact of a physician intervention during routine gynecologic visits on folic acid supplementation. STUDY DESIGN: Patients were assigned randomly to receive brief folic acid counseling, a reminder phone call, and 30 folic acid tablets (n = 162 women; intervention group) or to receive counseling about other preventive health behaviors and a folic acid informational pamphlet (n = 160 women; control group). Self-reported folic acid use was compared at baseline and at 2 months. RESULTS: Of the 279 patients who completed the study, weekly folic acid intake increased in the intervention group by 68%, compared with 20% in the control group ( P =.008). No significant differences were found in daily intake. The women who were most influenced by the intervention were black and lower income and not planning pregnancies. CONCLUSION: With little effort expended to encourage folic acid use, gynecologists could potentially reduce the risk of folate-preventable birth defects among their patients by as much as 11%.

Folic acid use by women receiving routine gynecologic care.

OBJECTIVE: Many health professional groups recommend folic acid supplementation for all women able to become pregnant. In this study, we document folic acid supplement use among a sample of women receiving routine gynecologic care. METHODS: A short questionnaire was administered to 322 women aged 18-45 years who were seeking routine gynecologic care at participating clinics in Little Rock, Arkansas. Questions covered knowledge and use of folic acid supplements, pregnancy intention, and demographic and socioeconomic characteristics. Primary study outcomes were self-reported folic acid awareness, daily or weekly use of folic acid supplements, and intention to begin taking folic acid. Factors affecting study outcomes were examined individually by computing crude odd ratios and adjusted for other covariates using unconditional logistic regression. RESULTS: Although 61.8% of women reported awareness of the association between folic acid and birth defects prevention, only 27.1% of these women, and 22.7% of all study participants, reported daily use of a folic acid supplement. Substantially more women (39.8%) were taking a folic acid supplement at least once per week. Age, race, educational level, folic acid awareness, marital status, pregnancy intent, and other preventive health behaviors were the most important predictors of compliance. CONCLUSION: The results indicate a need for targeted interventions directed toward minority women, young women, and those of lower socioeconomic and educational status. The routine gynecologic visit is an ideal opportunity to counsel women of reproductive age to take folic acid daily. LEVEL OF EVIDENCE: III