Analysis of Archived Residual Newborn Screening Blood Spots After Whole Genome Amplification

Deidentified newborn screening bloodspot samples (NBS) represent a valuable potential resource for genomic research if impediments to whole exome sequencing of NBS deoxyribonucleic acid (DNA), including the small amount of genomic DNA in NBS material, can be overcome. For instance, genomic analysis of NBS could be used to define allele frequencies of disease-associated variants in local populations, or to conduct prospective or retrospective studies relating genomic variation to disease emergence in pediatric populations over time. In this study, we compared the recovery of variant calls from exome sequences of amplified NBS genomic DNA to variant calls from exome sequencing of non-amplified NBS DNA from the same individuals. Results: Using a standard alignment-based Genome Analysis Toolkit (GATK), we find 62,000-76,000 additional variants in amplified samples. After application of a unique kmer enumeration and variant detection method (RUFUS), only 38,000-47,000 additional variants are observed in amplified gDNA. This result suggests that roughly half of the amplification-introduced variants identified using GATK may be the result of mapping errors and read misalignment. Conclusions: Our results show that it is possible to obtain informative, high-quality data from exome analysis of whole genome amplified NBS with the important caveat that different data generation and analysis methods can affect variant detection accuracy, and the concordance of variant calls in whole-genome amplified and non-amplified exomes.National Institute of Health P01HD067244, NS076465, R01ES021006Nutritional Science

Association between CFL1 gene polymorphisms and spina bifida risk in a California population

BACKGROUND: CFL1 encodes human non-muscle cofilin (n-cofilin), which is an actin-depolymerizing factor and is essential in cytokinesis, endocytosis, and in the development of all embryonic tissues. Cfl1 knockout mice exhibit failure of neural tube closure at E10.5 and die in utero. We hypothesized that genetic variation within the human CFL1 gene may alter the protein's function and result in defective actin depolymerizing and cellular activity during neural tube closure. Such alterations may be associated with an increased risk for neural tube defects (NTDs). METHODS: Having re-sequenced the human CFL1 gene and identified five common single nucleotide polymorphisms (SNPs) in our target population, we investigated whether there existed a possible association between the genetic variations of the CFL1 gene and risk of spina bifida. Samples were obtained from a large population-based case-control study in California. Allele association, genotype association and haplotype association were evaluated in two different ethnicity groups, non-Hispanic white and Hispanic white. RESULTS: Homozygosity for the minor alleles of the SNPs studied (rs652021, rs665306, rs667555, rs4621 and rs11227332) appeared to produce an increased risk for spina bifida. Subjects with the haplotype composed of all minor alleles (CCGGT) appeared to have increased spina bifida risk (OR = 1.6, 95% CI: 0.9~2.9), however, this finding is not statistically significant likely due to limited sample size. CONCLUSION: The sequence variation of human CFL1 gene is a genetic modifier for spina bifida risk in this California population

Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex

<p>Abstract</p> <p>Background</p> <p>The reduced folate carrier (<it>RFC1</it>) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine <it>RFC1 </it>gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 <it>RFC1</it>^-/-embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation.</p> <p>Results</p> <p>Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 <it>RFC1</it>^-/-vs. <it>RFC1</it>^+/+embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuity™ Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of <it>RFC1</it>^-/-embryos, while cubilin protein was widely misexpressed.</p> <p>Conclusion</p> <p>Inactivation of <it>RFC1 </it>impacts the expression of several ligands and interacting proteins in the cubilin-amnionless-megalin complex that are involved in the maternal-fetal transport of folate and other nutrients, lipids and morphogens such as sonic hedgehog (Shh) and retinoids that play critical roles in normal embryogenesis.</p

Autism-Like Behavior and Epigenetic Changes Associated with Autism as Consequences of In Utero Exposure to Environmental Pollutants in a Mouse Model

We tested the hypothesis that in utero exposure to heavy metals increases autism-like behavioral phenotypes in adult animals and induces epigenetic changes in genes that have roles in the etiology of autism. Mouse dams were treated with cadmium, lead, arsenate, manganese, and mercury via drinking water from gestational days (E) 1–10. Valproic acid (VPA) injected intraperitoneally once on (E) 8.5 served as a positive control. Young male offspring were tested for behavioral deficits using four standardized behavioral assays. In this study, in utero exposure to heavy metals resulted in multiple behavioral abnormalities that persisted into adulthood. VPA and manganese induced changes in perseverative/impulsive behavior and social dominance behavior, arsenic caused changes only in perseverative/impulsive behavior, and lead induced abnormalities in social interaction in comparison to the control animals. Brain samples from Mn, Pb, and VPA treated and control animals were evaluated for changes in CpG island methylation in promoter regions and associated changes in gene expression. The Chd7 gene, essential for neural crest cell migration and patterning, was found to be hypomethylated in each experimental animal tested compared to water-treated controls. Furthermore, distinct patterns of CpG island methylation yielded novel candidate genes for further investigation

Maternal–fetal metabolic gene–gene interactions and risk of neural tube defects

Single-gene analyses indicate that maternal genes associated with metabolic conditions (e.g., obesity) may influence the risk of neural tube defects (NTDs). However, to our knowledge, there have been no assessments of maternal-fetal metabolic gene-gene interactions and NTDs. We investigated 23 single nucleotide polymorphisms among 7 maternal metabolic genes (ADRB3, ENPP1, FTO, LEP, PPARG, PPARGC1A, and TCF7L2) and 2 fetal metabolic genes (SLC2A2 and UCP2). Samples were obtained from 737 NTD case-parent triads included in the National Birth Defects Prevention Study for birth years 1999–2007. We used a 2-step approach to evaluate maternal-fetal gene-gene interactions. First, a case-only approach was applied to screen all potential maternal and fetal interactions (n=76), as this design provides greater power in the assessment of gene-gene interactions compared to other approaches. Specifically, ordinal logistic regression was used to calculate the odds ratio (OR) and 95% confidence interval (CI) for each maternal-fetal gene-gene interaction, assuming a log-additive model of inheritance. Due to the number of comparisons, we calculated a corrected p-value (q-value) using the false discovery rate. Second, we confirmed all statistically significant interactions (q<0.05) using a log-linear approach among case-parent triads. In step 1, there were 5 maternal-fetal gene-gene interactions with q<0.05. The “top hit” was an interaction between maternal ENPP1 rs1044498 and fetal SLC2A2 rs6785233 (interaction OR=3.65, 95% CI: 2.32–5.74, p=2.09×10−8, q=0.001), which was confirmed in step 2 (p=0.00004). Our findings suggest that maternal metabolic genes associated with hyperglycemia and insulin resistance and fetal metabolic genes involved in glucose homeostasis may interact to increase the risk of NTDs

A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele

Orofacial clefts, including cleft lip and palate (CL/P), and neural tube defects (NTDs) are among the most common congenital anomalies but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesised that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) not only causes spinal NTDs in Axial defects (Axd) mice, but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate, abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion which disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesise that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans

Maternal Occupational Exposure to Polycyclic Aromatic Hydrocarbons: Effects on Gastroschisis among Offspring in the National Birth Defects Prevention Study

Background: Exposure to polycyclic aromatic hydrocarbons (PAHs) occurs in many occupational settings. There is evidence in animal models that maternal exposure to PAHs during pregnancy is associated with gastroschisis in offspring; however, to our knowledge, no human studies examining this association have been conducted

Diabetes and Obesity-Related Genes and the Risk of Neural Tube Defects in the National Birth Defects Prevention Study

Few studies have evaluated genetic susceptibility related to diabetes and obesity as a risk factor for neural tube defects (NTDs). The authors investigated 23 single nucleotide polymorphisms among 9 genes (ADRB3, ENPP1, FTO, LEP, PPARG, PPARGC1A, SLC2A2, TCF7L2, and UCP2) associated with type 2 diabetes or obesity. Samples were obtained from 737 NTD case-parent triads included in the National Birth Defects Prevention Study during 1999–2007. Log-linear models were used to evaluate maternal and offspring genetic effects. After application of the false discovery rate, there were 5 significant maternal genetic effects. The less common alleles at the 4 FTO single nucleotide polymorphisms showed a reduction of NTD risk (for rs1421085, relative risk (RR) = 0.73 (95% confidence interval (CI): 0.62, 0.87); for rs8050136, RR = 0.79 (95% CI: 0.67, 0.93); for rs9939609, RR = 0.79 (95% CI: 0.67, 0.94); and for rs17187449, RR = 0.80 (95% CI: 0.68, 0.95)). Additionally, maternal LEP rs2071045 (RR = 1.31, 95% CI: 1.08, 1.60) and offspring UCP2 rs660339 (RR = 1.32, 95% CI: 1.06, 1.64) were associated with NTD risk. Furthermore, the maternal genotype for TCF7L2 rs3814573 suggested an increased NTD risk among obese women. These findings indicate that maternal genetic variants associated with glucose homeostasis may modify the risk of having an NTD-affected pregnancy

Genes encoding critical transcriptional activators for murine neural tube development and human spina bifida: a case-control study

<p>Abstract</p> <p>Background</p> <p>Spina bifida is a malformation of the neural tube and is the most common of neural tube defects (NTDs). The etiology of spina bifida is largely unknown, although it is thought to be multi-factorial, involving multiple interacting genes and environmental factors. Mutations in transcriptional co-activator genes-<it>Cited2</it>, <it>p300</it>, <it>Cbp</it>, <it>Tfap2α</it>, <it>Carm1 </it>and <it>Cart1 </it>result in NTDs in murine models, thus prompt us to investigate whether homologues of these genes are associated with NTDs in humans.</p> <p>Methods</p> <p>Data and biological samples from 297 spina bifida cases and 300 controls were derived from a population-based case-control study conducted in California. 37 SNPs within <it>CITED2</it>, <it>EP300</it>, <it>CREBBP</it>, <it>TFAP2A</it>, <it>CARM1 </it>and <it>ALX1 </it>were genotyped using an ABI SNPlex assay. Odds ratios and 95% confidence intervals were calculated for alleles, genotypes and haplotypes to evaluate the risk for spina bifida.</p> <p>Results</p> <p>Several SNPs showed increased or decreased risk, including <it>CITED2 </it>rs1131431 (OR = 5.32, 1.04~27.30), <it>EP300 </it>rs4820428 (OR = 1.30, 1.01~1.67), <it>EP300 </it>rs4820429 (OR = 0.50, 0.26~0.50, in whites, OR = 0.7, 0.49~0.99 in all subjects), <it>EP300 </it>rs17002284 (OR = 0.43, 0.22~0.84), <it>TFAP2A </it>rs3798691 (OR = 1.78, 1.13~2.87 in Hispanics), <it>CREBBP </it>rs129986 (OR = 0.27, 0.11~0.69), <it>CARM1 </it>rs17616105 (OR = 0.41, 0.22~0.72 in whites). In addition, one haplotype block in <it>EP300 </it>and one in <it>TFAP2A </it>appeared to be associated with increased risk.</p> <p>Conclusions</p> <p>Modest associations were observed in <it>CITED2</it>, <it>EP300</it>, <it>CREBBP</it>, <it>TFAP2A </it>and <it>CARM1 </it>but not <it>ALX1</it>. However, these modest associations were not statistically significant after correction for multiple comparisons. Searching for potential functional variants and rare causal mutations is warranted in these genes.</p