Background The National Birth Defects Prevention Study (NBDPS) is a multisite, population-based, case-control study of genetic and nongenetic risk factors for major structural birth defects. Eligible women had a pregnancy affected by a birth defect or a liveborn child without a birth defect between 1997 and 2011. They were invited to complete a telephone interview to collect pregnancy exposure data and were mailed buccal cell collection kits to collect specimens from themselves, their child (if living), and their child's father. Over 23,000 families representing more than 30 major structural birth defects provided DNA specimens. Methods To evaluate their utility for exome sequencing (ES), specimens from 20 children with colonic atresia were studied. Evaluations were conducted on specimens collected using cytobrushes stored and transported in open versus closed packaging, on native genomic DNA (gDNA) versus whole genome amplified (WGA) products and on a library preparation protocol adapted to low amounts of DNA. Results The DNA extracted from brushes in open packaging yielded higher quality sequence data than DNA from brushes in closed packaging. Quality metrics of sequenced gDNA were consistently higher than metrics from corresponding WGA products and were consistently high when using a low input protocol. Conclusions This proof-of-principle study established conditions under which ES can be applied to NBDPS specimens. Successful sequencing of exomes from well-characterized NBDPS families indicated that this unique collection can be used to investigate the roles of genetic variation and gene-environment interaction effects in birth defect etiologies, providing a valuable resource for birth defect researchers.

Results from association studies are traditionally corroborated by replicating the findings in an independent data set. Although replication studies may be comparable for the main trait or phenotype of interest, it is unlikely that secondary phenotypes will be comparable across studies, making replication problematic. Alternatively, there may simply not be a replication sample available because of the nature or frequency of the phenotype. In these situations, an approach based on complementary pairs stability selection for genome-wide association study (ComPaSS-GWAS), is proposed as an ad-hoc alternative to replication. In this method, the sample is randomly split into two conditionally independent halves multiple times (resamples) and a GWAS is performed on each half in each resample. Similar in spirit to testing for association with independent discovery and replication samples, a marker is corroborated if its p-value is significant in both halves of the resample. Simulation experiments were performed for both nongenetic and genetic models. The type I error rate and power of ComPaSS-GWAS were determined and compared to the statistical properties of a traditional GWAS. Simulation results show that the type I error rate decreased as the number of resamples increased with only a small reduction in power and that these results were comparable with those from a traditional GWAS. Blood levels of vitamin pyridoxal 5 '-phosphate from the Trinity Student Study (TSS) were used to validate this approach. The results from the validation study were compared to, and were consistent with, those obtained from previously published independent replication data and functional studies.

The epilepsies affect around 65 million people worldwide and have a substantial missing heritability component. We report a genome-wide mega-analysis involving 15,212 individuals with epilepsy and 29,677 controls, which reveals 16 genome-wide significant loci, of which 11 are novel. Using various prioritization criteria, we pinpoint the 21 most likely epilepsy genes at these loci, with the majority in genetic generalized epilepsies. These genes have diverse biological functions, including coding for ion-channel subunits, transcription factors and a vitamin-B6 metabolism enzyme. Converging evidence shows that the common variants associated with epilepsy play a role in epigenetic regulation of gene expression in the brain. The results show an enrichment for monogenic epilepsy genes as well as known targets of antiepileptic drugs. Using SNP-based heritability analyses we disentangle both the unique and overlapping genetic basis to seven different epilepsy subtypes. Together, these findings provide leads for epilepsy therapies based on underlying pathophysiology.

The genetic variation in Northern Asian populations is currently undersampled. To address this, we generated a new genetic variation reference panel by whole-genome sequencing of 175 ethnic Mongolians, representing six tribes. The cataloged variation in the panel shows strong population stratification among these tribes, which correlates with the diverse demographic histories in the region. Incorporating our results with the 1000 Genomes Project panel identifies derived alleles shared between Finns and Mongolians/Siberians, suggesting that substantial gene flow between northern Eurasian populations has occurred in the past. Furthermore, we highlight that North, East, and Southeast Asian populations are more aligned with each other than these groups are with South Asian and Oceanian populations.

Background: Genetic polymorphisms can explain some of the population- and individual-based variations in nutritional status biomarkers. Objective: We sought to screen the entire human genome for common genetic polymorphisms that influence folate-status biomarkers in healthy individuals. Design: We carried out candidate gene analyses and genome-wide association scans in 2232 young, healthy Irish subjects to evaluate which common genetic polymorphisms influence red blood cell folate, serum folate, and plasma total homocysteine. Results: The 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C -> T (rs1801133) variant was the major genetic modifier of all 3 folate-related biomarkers in this Irish population and reached genome-wide significance for red blood cell folate (P = 1.37 x 10(-17)), serum folate (P = 2.82 x 10(-11)), and plasma total homocysteine (P = 1.26 x 10(-19)) concentrations. A second polymorphism in the MTHFR gene (rs3753584, P = 1.09 x 10(-11)) was the only additional MTHFR variant to exhibit any significant independent effect on red blood cell folate. Other MTHFR variants, including the 1298A -> C variant (rs1801131), appeared to reach genome-wide significance, but these variants shared linkage disequilibrium with MTHFR 677C -> T and were not significant when analyzed in MTHFR 677CC homozygotes. No additional non-MTHFR modifiers of red blood cell or plasma folate were detected. Two additional genome-wide significant modifiers of plasma homocysteine were found in the region of the dipeptidase 1 (DPEP1) gene on chromosome 16 and the Twist neighbor B (TWISTNB) gene on chromosome 7. Conclusions: The MTHFR 677C -> T variant is the predominant genetic modifier of folate status biomarkers in this healthy Irish population. It is not necessary to determine MTHFR 677C -> T genotype to evaluate folate status because its effect is reflected in concentrations of standard folate biomarkers. The MTHFR 1298A -> C variant had no independent effect on folate status biomarkers. To our knowledge, this is the first genome-wide association study report on red blood cell folate and the first report of an association between homocysteine and TWISTNB.

In humans, transport of food-derived cobalamin (vitamin B-12) from the digestive system into the bloodstream involves three paralogous proteins: transcobalamin (TC), haptocorrin (HC), and intrinsic factor (IF). Each of these proteins contains two domains, an -domain and a -domain, which together form a cleft in which cobalamin binds. Zebrafish (Danio rerio) are thought to possess only a single cobalamin transport protein, referred to as Tcn2, which is a transcobalamin homolog. Here, we used CRISPR/Cas9 mutagenesis to create null alleles of tcn2 in zebrafish. Fish homozygous for tcn2-null alleles were viable and exhibited no obvious developmentally or behaviorally abnormal phenotypes. For this reason, we hypothesized that previously unidentified cobalamin-carrier proteins encoded in the zebrafish genome may provide an additional pathway for cobalamin transport. We identified genes predicted to code for two such proteins, Tcn-beta-a (Tcnba) and Tcn-beta-b (Tcnbb), which differ from all previously characterized cobalamin transport proteins as they lack the -domain. These -domain-only proteins are representative of an undescribed class of cobalamin-carrier proteins that are highly conserved throughout the ray-finned fishes. We observed that the genes encoding the three cobalamin transport homologs, tcn2, tcnba, and tcnbb, are expressed in unique spatial and temporal patterns in the developing zebrafish. Moreover, exogenously expressed recombinant Tcnba and Tcnbb bound cobalamin with high affinity, comparable with binding by full-length Tcn2. Taken together, our results suggest that this noncanonical protein structure has evolved to fully function as a cobalamin-carrier protein, thereby allowing for a compensatory cobalamin transport mechanism in the tcn2(-/-) zebrafish.

In humans, poor nutrition, malabsorption and variation in cobalamin (vitamin B12) metabolic genes are associated with hematological, neurological and developmental pathologies. Cobalamin is transported from blood into tissues via the transcobalamin (TC) receptor encoded by the CD320 gene. We created mice carrying a targeted deletion of the mouse ortholog, Cd320. Knockout (KO) mice lacking this TC receptor have elevated levels of plasma methylmalonic acid and homocysteine but are otherwise healthy, viable, fertile and not anemic. To challenge the Cd320 KO mice we maintained them on a vitamin B12-deficient diet. After 5 weeks on this diet, reproductive failure develops in Cd320 KO females but not males. In vitro, homozygous Cd320 KO embryos from cobalamin-deficient Cd320 KO dams develop normally to embryonic day (E) 3.5, while in vivo, few uterine decidual implantation sites are observed at E7.5, suggesting that embryos perish around the time of implantation. Dietary restriction of vitamin B12 induces a severe macrocytic anemia in Cd320 KO mice after 10-12 months while control mice on this diet are anemia-free up to 2 years. Despite the severe anemia, cobalamin-deficient KO mice do not exhibit obvious neurological symptoms. Our results with Cd320 KO mice suggest that an alternative mechanism exists for mice to transport cobalamin independent of the Cd320 encoded receptor. Our findings with deficient diet are consistent with historical and epidemiological data suggesting that low vitamin B12 levels in humans are associated with infertility and developmental abnormalities. Our Cd320 KO mouse model is an ideal model system for studying vitamin B12 deficiency.

The glycine cleavage system (GCS) is a complex of four enzymes enabling glycine to serve as a source of one-carbon units to the cell. We asked whether concentrations of glycine, dimethylglycine, formate, and serine in blood are influenced by variation within GCS genes in a sample of young, healthy individuals. Fifty-two variants tagging (r(2) < 0.9) the four GCS genes were tested; one variant, GLDC rs2297442-G, was significantly associated (p = .0007) with decreased glycine concentrations in serum.