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Neural tube defects (NTDs) are common birth defects (~1 in 1000 pregnancies in the US and Europe) that have complex origins, including environmental and genetic factors. A low level of maternal folate is one well-established risk factor, with maternal periconceptional folic acid supplementation reducing the occurrence of NTD pregnancies by 50-70%. Gene variants in the folate metabolic pathway (e.g., MTHFR rs1801133 (677 C > T) and MTHFD1 rs2236225 (R653Q)) have been found to increase NTD risk. We hypothesized that variants in additional folate/B12 pathway genes contribute to NTD risk.
A tagSNP approach was used to screen common variation in 82 candidate genes selected from the folate/B12 pathway and NTD mouse models. We initially genotyped polymorphisms in 320 Irish triads (NTD cases and their parents), including 301 cases and 341 Irish controls to perform case–control and family based association tests. Significantly associated polymorphisms were genotyped in a secondary set of 250 families that included 229 cases and 658 controls. The combined results for 1441 SNPs were used in a joint analysis to test for case and maternal effects.
Nearly 70 SNPs in 30 genes were found to be associated with NTDs at the p < 0.01 level. The ten strongest association signals (p-value range: 0.0003–0.0023) were found in nine genes (MFTC, CDKN2A, ADA, PEMT, CUBN, GART, DNMT3A, MTHFD1 and T (Brachyury)) and included the known NTD risk factor MTHFD1 R653Q (rs2236225). The single strongest signal was observed in a new candidate, MFTC rs17803441 (OR = 1.61 [1.23-2.08], p = 0.0003 for the minor allele). Though nominally significant, these associations did not remain significant after correction for multiple hypothesis testing.
To our knowledge, with respect to sample size and scope of evaluation of candidate polymorphisms, this is the largest NTD genetic association study reported to date. The scale of the study and the stringency of correction are likely to have contributed to real associations failing to survive correction. We have produced a ranked list of variants with the strongest association signals. Variants in the highest rank of associations are likely to include true associations and should be high priority candidates for further study of NTD risk.
Keywords:Neural tube defects; Spina bifida; Folic acid; One-carbon metabolism; Candidate gene
Neural tube defects (NTDs) are one of the most common birth defects, with a historical prevalence of ~1 in 1000 in the US [1,2]. The NTD rate is now closer to ~5 in 10,000 in areas with folic acid fortification, such as the US [3] and many European countries [4]. Between 21 and 28 days after conception, the neural plate folds and closes to form the neural tube; this structure later develops into the brain and spinal cord. Failure of the neural tube to close most commonly leads to spina bifida or anencephaly, although encephalocele, craniorachischisis and iniencephaly can also occur [5].
It is known that both environmental and genetic factors contribute to the development of NTDs. The most established environmental factor is dietary folate; significantly lower levels of folate are observed in mothers with an NTD pregnancy [6], and periconceptional folate supplementation can reduce the risk of an NTD pregnancy by up to 75% [7][8][9]. There is also growing evidence of the importance of cobalamin (vitamin B12) in the etiology of NTDs. Like folate, lower vitamin B12 levels have been reported in mothers with an NTD pregnancy [6,10-17].
Genetic factors also contribute to NTDs. Compared to the general population, there is a 10–20 fold higher recurrence risk to siblings in families with an NTD child [18-20]. This, combined with the recognition of the importance of maternal folate, has led many groups to evaluate genetic polymorphisms related to the folate metabolic pathway as risk factors for NTDs. The best studied genetic risk factor is a single nucleotide polymorphism (SNP) in 5, 10-methylene-tetrahydrofolate reductase (MTHFR). The 677 C > T polymorphism results in the substitution of a valine for an alanine at codon 222 (A222V), leading to a thermolabile isoform of the protein [21]. A significantly higher frequency of the MTHFR 677 TT genotype has been observed in NTD cases in many populations (reviewed in [22]). Genetic variants associated with NTDs have been reported in other genes encoding folate- and vitamin B12-related proteins, such as methylenetetrahydrofolate dehydrogenase (NADP + dependent) 1, methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase (MTHFD1) [23-26], methylenetetrahydrofolate dehydrogenase (NADP + dependent) 1-like (MTHFD1L) [27], dihydrofolate reductase (DHFR) [28,29], methionine synthase reductase (MTRR) [16,26,30-33], and the transcobalamin II receptor (TCblR) [34].
As genotyping technology has advanced, the scale of studies attempting to identify genetic NTD risk factors has grown from single SNP analyses to simultaneous evaluation of dozens of variants. Several studies have evaluated specific candidate polymorphisms with evidence of functional changes and/or disease risk (87 variants in 45 genes, [35]; 48 SNPs in 11 genes [36]; 64 SNPs in 34 genes [37]). In contrast, other studies have examined all common variation in candidate genes via tagging SNPs (tagSNPs) in specific genes of interest (118 tagSNPs in 14 folate-related genes [26]; 37 tagSNPs in 6 transcriptional activator genes [38]). In the current study, we also took the tagSNP approach to evaluate 1441 SNPs in 82 candidate genes for NTD risk.
Common genetic variation in 82 candidate genes (Figure 1) was tested for association with NTDs. Results were generated in two stages. In the first stage, four broad tests of association were performed on all SNPs using a subset of samples. In the second stage, SNPs of interest identified in the initial analysis were then typed in the complete cohort to maximize the power to detect an effect, and a wider range of genetic models were applied to the combined dataset to evaluate the potential contribution of all SNPs to case or maternal risk of NTDs.
Figure 1 . Classification Distribution of 82 Selected Candidate Genes. Categories of selected candidate genes (legend) and their distribution (pie chart) are shown. Over half of the genes selected for study are known to encode a protein with enzymatic function related to the folate/vitamin B12 pathway.
Initial analyses
The primary sample set (320 NTD case families and 341 controls) was genotyped for 1517 tagging SNPs intended to capture common genetic variation in 82 candidate genes related to folate/vitamin B12 metabolism, transport of folate or vitamin B12, or transcriptional or developmental processes implicated in NTD mouse models (Table 1). Genotype data was successfully obtained for 1320 SNPs. Four tests of association were performed; two tests were to detect NTD case risk and two tests were to detect maternal risk for an NTD pregnancy. There were 203 SNPs in 54 genes that were significant (p < 0.05) by at least one test of association. A gene-based approach was used to select SNPs from fifteen genes to be genotyped in the secondary sample set. Five genes (mitochondrial folate transporter/carrier (MFTC), megalin (LRP2, low density lipoprotein receptor-related protein 2), DNA (cytosine-5-)-methyltransferase 3 beta (DNMT3B), phosphatidylethanolamine N-methyltransferase (PEMT), and euchromatic histone-lysine N-methyltransferase (EHMT1)) contained at least one SNP that was positive for both tests of a case effect or both tests of a maternal effect. An additional four genes (5-methyltetrahydrofolate-homocysteine methyltransferase 1 (MTR), cubilin (CUBN, intrinsic factor-cobalamin receptor), T (Brachyury homolog (mouse)), and AT rich interactive domain 1A (ARID1A)) contained more than five SNPs significant for any of the four tests of association. The remaining six genes (adenosine deaminase (ADA), ferritin, heavy polypeptide 1 (FTH1), cystathionase (CTH), peptidyl arginine deiminase, type IV (PADI4), low density lipoprotein receptor-related protein 6 (LRP6), and serine hydroxymethyltransferase 1 (SHMT1)) were selected based on a combination of factors, including the number of positive SNPs, their level of significance, and biological plausibility. Any SNP in these genes significant by any of the four tests of association (Table 2) was selected for genotyping in the secondary sample set (258 additional case families and 658 additional controls).
Table 1. Candidate Genes Selected for Evaluation of Common Genetic Variation as NTD Risk Factors
Table 2. SNPs Selected for Study in the Combined NTD Cohort
Combined analyses
In the combined analyses each SNP was evaluated for contributing to NTD risk by twelve association tests: case and maternal effects were each evaluated using three case–control (or mother-control) tests and three family-based tests (see Methods). There were 68 SNPs in 30 genes that showed an association at the p < 0.01 level by any of the twelve tests (Table 3). Of these, twelve genes contained a single associated SNP. The remaining 56 SNPs were found in 18 genes. Not all associations were independent. Areas of interest were covered by tagSNPs as well as additional SNPs for physical coverage; as a result there were seven SNP pairs in this set with a linkage disequilibrium (LD) relationship above the threshold (r2 ≥ 0.8) selected for tagging in this study (Figure 2, ARID1A_rs11247593 and ARID1A_rs11247594, CUBN_rs7070148 and CUBN_rs2273737, GART_rs2070388 and GART_rs4817580, MFTC_rs17803441 and MFTC_rs3134260, MTHFR_rs17367504 and MTHFR_rs17037425, MTR_rs10733117 and MTR_rs10925260, and PEMT_rs4646402 and PEMT rs1108579). Notably, for many genes, the associated SNPs occur in the same haplotype block (solid spine of LD based on D' relationships), implying a single association signal for that gene (Figure 2, GART, COMT, MFTC, MTHFR, ARID1A, MTR, FOLH1, MTHFD1, PEMT, RAI1, ENOSF1). A single signal probably also accounts for the significant SNP pairs seen in PDGFRA (D' = 0.65) and CDKN2A (D' = 0.79). Genes exhibiting more than one independent SNP association include FTCD (D' = 0.10), MTHFD1L (two separate haplotype blocks and two SNPs, D' ≤ 0.57), CUBN (one haplotype block and a SNP, D' ≤ 0.20), ALDH1A2 (D' = 0.13) and ADA (a weak haplotype block and a SNP, D' ≤ 0.19).
Table 3. p-values for Evaluated SNPs Showing Association (p < 0.01) with NTDs
Figure 2. Linkage Disequilibrium Relationships Between SNPs Significantly Associated with NTDs. Sixty-eight SNPs were associated with case or maternal effects (p < 0.01, uncorrected). Of these, 56 SNPs occur in the same gene as at least one other significantly associated SNP. Their D' (red scale) and r2 (gray scale) relationships are shown. These SNPs are plotted by relative position. Haplotype blocks defined by a Solid Spine of LD (D') are outlined in black. Each SNP is numbered and the gene and dbSNP accession number for each are: 1) MTHFR_rs17367504; 2) MTHFR_rs17037425; 3) ARID1A_rs11247593; 4) ARID1A_rs11247594; 5) ARID1A_rs11247596; 6) MTR_rs10733117; 7) MTR_rs10925260; 8) CUBN_rs11591606; 9) CUBN_rs1801222; 10) CUBN_rs11254375; 11) CUBN_rs7070148; 12) CUBN_rs2273737; 13) FOLH1_rs383028; 14) FOLH1_rs16906205; 15) MTHFD1_rs11627525; 16) MTHFD1_rs2236225; 17) ALDH1A2_rs7169289; 18) ALDH1A2_rs6493978; 19) PEMT_rs4646402; 20) PEMT_rs1108579; 21) PEMT_rs16961845; 22) PEMT_rs11656215; 23) RAI1_rs11654526; 24) RAI1_rs9914733; 25) RAI1_rs11658846; 26) ENOSF1_rs1059384; 27) ENOSF1_rs10502289; 28) ADA_rs6031682; 29) ADA_rs452159; 30) ADA_rs406383; 31) ADA_rs2299686; 32) ADA_rs427483; 33) ADA_rs6094017; 34) GART_rs2070388; 35) GART_rs4817580; 36) FTCD_rs7280485; 37) FTCD_rs2839127; 38) COMT_rs737865; 39) COMT_rs174675; 40) PDGFRA_rs2114039; 41) PDGFRA_rs9993187; 42) MTHFD1L_rs2295083; 43) MTHFD1L_rs17080476; 44) MTHFD1L_rs487637; 45) MTHFD1L_rs6923486; 46) MTHFD1L_rs12524884; 47) MTHFD1L_rs11155772; 48) MTHFD1L_rs12199063; 49) MTHFD1L_rs4869987; 50) MFTC_rs10112450; 51) MFTC_rs1865855; 52) MFTC_rs17803441; 53) MFTC_rs750606; 54) MFTC_rs3134260; 55) CDKN2A_rs7041637; 56) CDKN2A_rs3218009
We ranked SNPs by the lowest p-value for any test, accounting for relatedness to other highly significant SNPs. The nine genes exhibiting the 10 strongest association signals were: MFTC, CDKN2A, ADA, PEMT, CUBN, GART, ADA, DNMT3A, MTHFD1 and T (Brachyury) (Table 4). MFTC, ADA, PEMT and CUBN contained more than one significant SNP, and ADA showed evidence of two independent association signals.
Table 4. The Ten Independent*Association Signals Exhibiting the Lowest Uncorrected p-values for Any Test of NTD Association
SNPs in MFTC, PEMT and ADA account for seven of the top ten SNPs (Table 4). In MFTC, these two SNPs (rs17803441 and rs3134260) are essentially in perfect LD (D' = 1.0, r2 = 0.99). As expected, they yielded very similar evidence of NTD risk in a continuous model of logistic regression (rs17803441: OR = 1.61 [1.23-2.08], p = 0.0003, Risk Allele Frequency (RAF) = 0.07; rs3134260: OR = 1.56 [1.22-2.04], p = 0.0006, RAF = 0.07), as well as a recessive model of logistic regression (rs17803441: OR = 1.59 [1.19-2.08], p = 0.0013; rs3134260: OR = 1.54 [1.18-2.04], p = 0.0021). There are a total of five highly significant (p < 0.01) MFTC SNPs and they are all consistent with a case effect. MFTC rs10112450 is significant by the transmission disequilibrium test (TDT, GRR = 1.42, p = 0.0065, RAF = 0.79), and the remaining MFTC SNPs (rs1865855 and rs750606) show evidence of NTD risk to the case by logistic regression with a continuous or dominant model (p < 0.0091). Because all five of these SNPs fall in the same haplotype block (D' > 0.96), it is likely there is a single variant in this region responsible for the association signals.
Three SNPs in PEMT were in the top ten SNPs, and two of these are in high linkage disequilibrium (rs1108579 and rs4646402; D' = 1.0, r2 = 0.809). These SNPs also yield very similar evidence of NTD risk by TDT (rs1108579: GRR = 1.47, p = 0.0006, RAF = 0.52; rs4646402: GRR = 1.43, p = 0.0009, RAF = 0.57). PEMT rs11656215 is less strongly linked to these SNPs (r2 < 0.6), and shows association with NTD risk by TDT (GRR = 1.35, p = 0.0053, RAF = 0.49) and by log-linear analysis of a recessive model (GRR = 1.68 [1.24-2.28], p = 0.0008). A fourth highly significant SNP was found in the maternal analysis (rs16961845). It shows evidence of maternal risk in a recessive model of log-linear analysis (GRR = 1.92 [1.18-3.11], p = 0.0082), as well as recessive (p < 0.0048) and continuous (p < 0.0072) models of logistic regression. By r2 measures of LD, this SNP is the least related to the other three SNPs (r2 < 0.08), although all four of these SNPs fall in the same haplotype block (Figure 2).
Lastly, three SNPs in ADA were among the ten SNPs with the lowest p values. They show evidence of LD by D' (≥0.82) but less so by r2 (≤0.54). These three SNPs are all significantly associated with a maternal effect by logistic regression of a continuous model (rs2299686: OR = 1.30 [1.11-1.52], p = 0.0010, RAF = 0.45; rs427483: OR = 1.28 [1.10-1.52], p = 0.0018, RAF = 0.33; rs406383: OR = 1.33 [1.12-1.58], p = 0.0012, RAF = 0.25). Three other SNPs in ADA (rs6031682, rs452159 and rs6094017) also showed a highly significant association with a maternal effect in a continuous model of logistic regression (p < 0.0059). All six SNPs were highly significant (p < 0.01) for association with maternal risk in either a recessive model (rs406383, p = 0.0021) or dominant model (the other five SNPs, p ≤ 0.0063) of logistic regression. Additionally, ADA rs6031682 shows association with case risk in a continuous model of logistic regression (1.38 [1.10-1.73], p = 0.0052, RAF = 0.84). These six SNPs do not appear to be strongly linked; no haplotype blocks (solid spine of LD) larger than two SNPs were identified. It appears that rs6031682 is clearly outside of a degraded haplotype block consisting of the other five SNPs.
CUBN was the only other gene with more than one SNP found in the ten most strongly associated signals. CUBN rs7070148 and CUBN rs2273737 were both significant for association with maternal risk in a continuous model of logistic regression (OR = 1.64 [1.22-2.17], p = 0.0010, RAF = 0.90 and OR = 1.54 [1.18-2.04], p = 0.0021, RAF = 0.89, respectively). These SNPs are highly linked (D' = 0.935, r2 = 0.823). There are three other highly significant (p < 0.01) SNPs in CUBN. Two other SNPs in the same haplotype block with CUBN rs7070148 and CUBN rs2273737 (rs1801222 and rs11254375, D' < 0.76) also showed an association with maternal effects. A third SNP, CUBN rs11591606, is outside this block (D' < 0.20) and is associated with maternal risk in a dominant model of logistic regression (OR = 4.15 [1.51-11.39], p = 0.0058, RAF = 0.17).
Correction for multiple testing was performed for three of the twelve tests of association. No adjusted p-value was found to remain significant, and no further correction was performed.
This study represents a new scale of evaluation of genetic contribution to NTD risk. Common variants in 82 biologically plausible candidate genes were tested for association with NTDs in a large Irish population. Seventeen variants in nine genes account for the ten most significant associations observed. CDKN2A, GART, DNMT3A, MTHFD1 and T (Brachyury) contained a single SNP among the ten lowest p-values observed for all tests. In contrast, MFTC, ADA, PEMT, and CUBN each contained more than one such SNP. This seems to be due to strong LD relationships between the associated SNPs. The only exception is in ADA, which shows evidence of two strong, unrelated association signals.
ADA (adenosine deaminase) converts adenosine to inosine by removal of an amino group. Deficiency in this enzyme causes severe combined immunodeficiency disease (SCID), which is characterized by compromise of both T cells and B cells. Interestingly, ADA activity was significantly elevated in a study of 68 pregnant women carrying a fetus with a central nervous system malformation [39]; of these women, 17 had a spina bifida pregnancy. Consistent with this, six unrelated (r2 < 0.70), noncoding ADA SNPs were found in the current study to be associated with maternal risk of carrying an NTD pregnancy (p ≤ 0.006, uncorrected). Genetic variation in ADA may contribute to maternal risk of NTDs. In addition, this gene was the only one to exhibit two independent association signals among the top ten signals observed. This may indicate that there is more than one allele associated with risk or the same allele has recurred on more than one haplotype. ADA rs6031682 shows evidence of case effects (p = 0.0016) as well as maternal effects (p = 0.0019) in log-linear analyses of a dominant model, and it is clearly independent of the other significant ADA SNPs (D' < 0.19). It would be of interest to test the associated ADA SNPs in an independent study, especially since the scale of correction would be much smaller in a focused study.
PEMT (phosphatidylethanolamine N-methyltransferase) plays a role in choline metabolism. It converts phosphatidylethanolamine to phosphatidylcholine in the liver; phosphatidylcholine is a major component of cell membranes. This role for choline can compete with its role as a methyl donor. Choline can be converted to betaine, which acts as a methyl donor in an alternate, folate-independent conversion of homocysteine to methionine. This link between folate and choline metabolism makes PEMT an interesting candidate gene, and interactions between PEMT SNPs have been reported to be associated with NTDs. In a case–control study, single SNP effects were not observed, although some compound genotypes for PEMT rs7946 and PEMT rs897453 were associated with decreased NTD risk [40]. The latter variant was not directly tested in the current study, and no association for PEMT rs7946 was observed in this Irish sample [37]. However, one related SNP pair (r2 = 0.80) and two other SNPs (r2 < 0.60) in PEMT falling in the same haplotype block (D' ≥ 0.69) showed NTD association (p < 0.0053, uncorrected) in the current study, suggesting a role for this gene in NTD risk. Unlike the other three SNPs that exhibited case effects, the least related SNP in this block (PEMT rs16961845, 0.69 ≤ D' ≤ 0.89) was positive for maternal effects by three tests of association. This intronic SNP is in strong r2 LD with 6 other intronic SNPs, making it difficult to speculate about its function. It is also difficult to discern whether the associated SNPs in this block represent independent signals for case risk and for maternal risk, or whether a single signal for a case effect is being detected. Therefore, further studies on the variation of this gene and NTDs are warranted.
MFTC (mitochondrial folate transporter/carrier, SLC23A32) transports folate from the cytoplasm into the mitochondria. Some folate metabolic reactions occur in both the cytoplasm and in mitochondria via compartment-specific enzymes. The mitochondrion produces the majority of the one carbon units used by the cell (reviewed in [41]). As the genes coding for these mitochondrial enzymes have been identified, they have been shown to be intriguing and relevant candidates for NTD studies. For example, we previously reported that the gene encoding 5, 10-methylene-tetrahydrofolate dehydrogenase 1-like (MTHFD1L) contains a polymorphism associated with NTDs [27]. Genetic variation affecting mitochondrial folate transport may also contribute to NTDs, as seen by our finding that 5 of 11 tested MFTC SNPs showed association (p < 0.01) with NTD risk in cases. This gene falls in a region of very high D' LD; the haplotype block containing these five SNPs extends ~92 kb and contains two other genes: DCAF13 (DDB1 and CUL4 associated factor 13) and CTHRC1 (collagen triple helix repeat containing 1). Any SNP in this large haplotype block could be the causative variant driving the observed associations. As the only coding SNP in MFTC, the best candidate is rs17803441 (R117H). However, as arginine and histidine are both polar, basic amino acids, this is a fairly conservative change. We observed a minor allele frequency of 0.07 in this study. Conservation of the more common arginine residue is observed in chimp, wolf, cow, mouse, rat and zebrafish, but not in chicken or invertebrates. All of the SNPs in high LD (r2 > 0.7) with MFTC rs17803441 (R117H) in this block are intronic or intergenic. This SNP also had the lowest p-value for any test of association of all SNPs tested in this study. It would be of great interest to determine in an independent population whether it contributes to NTD risk.
CUBN encodes the intestinal receptor responsible for the uptake of the vitamin B12-intrinsic factor complex. It is also expressed in the kidney, where it is involved in reabsorption of many proteins and vitamins, including vitamin B12. This gene spans more than 300 kb of DNA. The only reported SNP association in CUBN is for rs1907362, which was associated with case risk in a Dutch population [35]. In contrast, we observed two highly significant SNPs in CUBN (rs7070148 and rs2273737) associated with maternal NTD risk. Due to their high LD these SNPs represent a single association signal. There were three other highly associated (p < 0.01) SNPs in this gene. CUBN rs11591606 was associated with maternal risk, and is in a smaller haplotype block at the 3' end of the gene. Two other CUBN SNPs (rs1801222 [S253F] and rs11254375) were also highly associated (p < 0.01) with maternal risk and are in the same ~30 kb haplotype block with rs7070148 and rs2273737 at the 5' end of the gene. While there are many SNPs in this block that could be the causal risk SNP, rs1801222 is of interest since it is a coding SNP (S253F) that was significantly associated with lower serum vitamin B12 levels in a meta-analysis of three genome wide association studies of three Caucasian populations [42]. This does not prove that CUBN rs1801222 is the causal SNP in either study, but it is consistent with the hypothesis that this SNP or another CUBN polymorphism linked to it within this haplotype block lowers vitamin B12 levels and thereby increases risk of an NTD pregnancy.
Multiple highly significant SNPs in ADA, PEMT, MFTC and CUBN account for half of the ten strongest association signals observed. The remaining five association signals are equally compelling. MTHFD1 rs2236225 (R653Q) was previously reported as a maternal NTD risk factor in the current study population [23,25] and others [22,24], while the other four signals represent new associations. First, CDKN2A rs3218009 was associated with maternal risk for NTDs. CDKN2a is a tumor suppressor gene that codes for several isoforms, including ARF (alternate open reading frame), a protein that stabilizes p53. A subset of mice carrying p53 null alleles exhibit overgrowth of neural tissue, supporting the importance of this pathway in normal neural tube development. Second, the same highly significant p-value was obtained for GART rs2070388 by two tests for case effect: TDT and log-linear analysis of a dominant model of case effect. GART is a trifunctional enzyme (phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase) involved in de novo purine synthesis. For its phosphoribosylglycinamide activity, GART uses N10-formyl tetrahydrofolate as a one-carbon donor in the synthesis pathway of inosine monophosphate (IMP), a purine precursor. Interestingly, GART rs4817579 in intron 2 has been associated with cleft lip and/or palate plus dental anomalies [43]. This variant was not tested in the current study, and the absence of GART rs2070388 from the HapMap data prevents us from evaluating the relatedness of these markers.
Third, DNMT3A rs7560488 was associated with NTD risk in cases. This gene encodes a DNA methyltransferase involved in de novo methylation during development. The folate pathway generates S-adenosyl methionine, which is used by DNMT3A as a methyl donor. Fourth, T (Brachyury) rs10806845 was associated with maternal NTD risk. The T (Brachyury) gene encodes a transcription factor involved in mesoderm formation and differentiation, and mice null for T (Brachyury) do not survive to term due to a number of developmental abnormalities, including fusion of the neural tube to the gut. Although previous studies differ in whether genetic variation in T (Brachyury) contributes to NTD risk in cases [44-48], our observation may be the first indication of its contribution to maternal risk of carrying an affected fetus.
Although no associations remained significant after conservative adjustment for multiple tests, it remains very possible that some of the evaluated candidates do in fact contribute to NTDs. The scale of our study design (using twelve tests of association to evaluate 1441 candidate SNPs) could contribute to Type II errors. This possibility is supported by the fact that of three SNPs previously reported to be associated with NTDs in this cohort (MTHFR 677 C > T [49,50], MTHFD1 R653Q [23,25], TCblR G220R [34]) only one was observed to be associated in the current study design (Table 5). Only MTHFD1 R653Q was found to be significantly associated in the primary phase of the analysis, which was performed on approximately half the samples. MTHFR 677 C > T and TCblR G220R were only found to be associated (p < 0.05, uncorrected) when the full cohort of samples were used. This suggests the possibility that the stringency of correction may be too high. Additionally, it is important to note that MTHFD1 R653Q was ninth among the top ten association signals in this study (Table 4). This suggests that a number of the ten strongest association signals observed in this study play a role in NTD risk, and they should be high priority candidates for further study.
Table 5. Significant (Uncorrected) Analyses of SNPs Previously Reported as NTD Risk Factors in this Population
In summary, this study involves the largest evaluation of common genetic variation for NTD risk yet reported: 1441 SNPs in 82 candidate genes. While no SNP associations remained significant after correction for multiple tests, there is a strong possibility that the study design and/or stringency of correction has resulted in obscuring true associations. At least one established risk factor, MTHFD1 R653Q, was corrected away, suggesting our approach was extremely conservative. Therefore, variation in the top genes identified in this study should be examined in independent populations for NTD risk, especially since many of these genes (MFTC, CDKN2A, ADA, CUBN, DNMT3A, and T (Brachyury)) represent new avenues of investigation.
Study population
The recruitment of the Irish NTD families (cases and parents) and controls has been described [23,34,51,52]. Briefly, the cohort includes 586 families with an NTD case; 442 of these families are full family triads (DNA from case, mother and father). For this study, 570 of the NTD families had sufficient DNA and were divided into two sets, one for primary analysis and one for secondary (combined) analysis. The primary and secondary sample sets were matched as closely as possible for the following parameters: the number of complete NTD triads, the proportion of NTD cases with spina bifida vs. other NTDs, and NTD case gender (Table 6).
Table 6. Characteristics of the Irish NTD Sets Used in this Study
The control population (n = 999) is a random sample drawn from 56,049 blood samples donated by women at their first prenatal visit to the three major maternity hospitals in Dublin (1986–1990). A subset of controls (n = 341) was randomly selected for the primary screen, and the remaining controls were used in the secondary set.
Written, informed consent was obtained from study subjects, their parents or their guardians. Archived control samples were anonymized prior to analysis. The study was approved by the Ethics Research Committee of the Health Research Board (Dublin, Ireland) and the Institutional Review Board at the National Human Genome Research Institute (Bethesda, MD, USA). Extraction of genomic DNA from blood samples and buccal swabs was performed using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA).
SNP selection
Genes were chosen for study because they are involved in folate and vitamin B12 metabolism or other metabolic and signaling pathways implicated in the etiology of NTDs (Figure 1). Although previously published, MTHFR[52-54], MTHFD1[23,25], MTHFD1L[27], TCblR[34], and TP53[51] were reanalyzed in this study in order to compare the differing research strategy, which includes new genetic models of risk assessment. For each gene chosen, we evaluated the transcribed region of the gene and 10 kilobases (kb) upstream and downstream of the gene in an effort to include polymorphisms with potential proximal effects, such as promoter variants. In order to capture the common variation in each gene, SNPs genotyped by HapMap (Data Release 21, Phase II, Anon, 2003 [55]) were considered. A set of tagSNPs was identified using an algorithm based on optimizing for tagSNPs with maximal minor allele frequencies (MAFs) and an r2 threshold of 0.8, while maximizing the MAF of the selected tagSNP. In addition to this set of tagSNPs, validated variants from dbSNP [26] were also selected for physical coverage so that spacing between SNPs would be less than 20 kb within D' haplotype blocks and less than 5 kb between haplotype blocks. A total of 1517 SNPs were selected.
The selected 1517 SNPs were genotyped on the primary sample sets (320 NTD case families and 341 controls). Genotypes were generated by the Johns Hopkins University SNP Center (Baltimore, MD) using the Illumina GoldenGate assay (Illumina, San Diego, CA). Of the 1517 SNPs attempted, 1320 SNPs (87.2%) remained after filtering out low quality data (re-attempted on another platform, see below). The overall call rate for these 1320 SNPs was 98%. All but 150 of these SNPs had a call rate of >95%; the rest had an average call rate of 87.2% (±7.9%) and were re-genotyped on another platform (see below). Both the overall duplicate concordance rates and the Mendelian consistency rates were 99.99% for the 1320 accepted SNPs.
Based on analyses of the primary sample sets, 93 SNPs were genotyped in the secondary sample set (250 NTD case families and 658 controls). Genotypes were generated by KASPar chemistry at Kbiosciences (Herts, UK). Three SNPs failed on this platform: rs127317149, rs7367859 and rs7096079. For the 90 successfully genotyped SNPs, the overall duplicate concordance rate was 99.81% and the overall Mendelian consistency rate was 99.94%.
SNPs that could not be assayed (n = 197) or returned low call rates (<95%, n = 150) via Illumina GoldenGate chemistry were re-genotyped in the entire sample set (570 NTD families and 999 controls) by detection of allele-specific primer extension using matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF) mass spectrometry (Sequenom, San Diego, CA, USA). Genotyping data from SNPs with call rates above 95% were added to the final analyses (121/197 SNPs that failed and 106/150 SNPs that yielded low call rates). The overall duplicate concordance rate was 99.10% and the overall Mendelian consistency rate was 99.32% for these 227 SNPs.
To summarize, our final data set consisted of 1441 SNPs; 1320 high quality SNPs from the Illumina platform (93 of these SNPS were also typed in the secondary samples using the KASPar platform, and 106 of these SNPs were re-typed in the entire sample set using the Sequenom platform) plus 121 SNPs from the Sequenom platform.
Linkage disequilibrium
D' and r2 measures of LD for SNPs of interest were estimated based on control genotypes using Haploview [56]. Haplotype blocks were based on D' values using the Solid Spine of LD option in Haploview.
Statistical methods
The design for this study involved two stages of genotyping. Rather than use the secondary samples for a replication study, joint analysis of the combined dataset was performed since it generally provides greater power to detect a genetic effect [57]. In the initial analysis, all SNPs successfully typed with the Illumina GoldenGate assay (n = 1320, including the 150 high quality SNPs with call rates of <95%) were analyzed in the primary sample set by four tests of association. Two tests were performed to evaluate case effects: 1) Logistic regression, a 1-degree of freedom (DOF) test of association between the affected status and number of risk alleles; and 2) the Spielman transmission disequilibrium test (TDT, [58]). Similarly, two tests were performed to evaluate maternal effects: 1) Logistic regression, a 1-DOF test of association between the maternal status and number of risk alleles; and 2) log-linear modeling with 2-DOF to test for effect of the maternal genotype.
SNPs were selected to be genotyped on all samples (570 NTD case families and 999 controls) if they met the following criteria: 1) SNPs of interest reaching a significance level in the primary analysis (n = 93, Table 2); or 2) failed SNPs (n = 121) and SNPs with low call rates (<95%, n = 106). Final analyses were performed on the entire dataset and consisted of twelve association tests. NTD case–control and NTD mother-control comparisons were performed using continuous, recessive and dominant coded models of logistic regression to generate odds ratios (ORs) and 95% confidence intervals (CI). Six family-based tests of association were also applied using log-linear models. The NTD triads (case, mother and father) were analyzed for case effects using recessive, dominant and linear (TDT) coding while direct maternal effects were analyzed using recessive, dominant and 2-DOF models.
To correct for multiple comparison, the final analysis used the complete information on the 1441 SNPs. Correction was performed by multivariate permutation (N = 9,999 random permutations) for three of the tests used in the initial analysis: case–control logistic regression, mother-control logistic regression and the TDT. This method accounts for any linkage disequilibrium between SNPs. Multivariate permuting of triads for the TDT was performed by treating the test as a one-sample test and permuting the risk allele [59]. Independent permutations were performed for the cases and controls or mothers and controls to adjust for multiple comparisons in the tests of logistic regression. The results were combined by Bonferroni adjustment to account for all comparisons (including all SNPs because of our multivariate permutation approach) while controlling the probability of any false positives at 5%. Since a SNP could only be found significant based on this combined analysis, our method provides type I error control regardless of the SNP selection process or number of SNPs selected for genotyping on all samples.
DOF, Degree(s) of freedom; LD, Linkage disequilibrium; NTDs, Neural tube defects; RAF, Risk allele frequency; SNP, Single nucleotide polymorphism; TDT, Transmission disequilibrium test.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FP, AMM, JLM, PNK, BS, JMS and LCB formulated the study design and performed candidate gene selection. FP additionally performed SNP selection and drafted the manuscript. AMM additionally managed the DNA samples and their selection. AP-M, VBO'L and CS participated in candidate gene selection. JMS and KG-S prepared DNA. AM and JEV extracted DNA and performed genotyping. KMK, AS, JC-H and NS performed genotyping. PC developed the SNP selection algorithm. MC provided database management. JFT contributed to study design and performed the statistical analyses. All authors read, edited and approved the final manuscript.
These studies would not be possible without the participation of the affected families, and their recruitment by the Irish Association of Spina Bifida and Hydrocephalus and the Irish Public Health Nurses in Ireland. This study was supported by the Intramural Research Programs of the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Human Genome Research Institute.
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Pre-publication history
The pre-publication history for this paper can be accessed here:


text: Streeter Lecka, Getty Images [ Knicks Squeak Past Bobcats With J.R. Smith's Buzzer Beater [VIDEO] By Jeff Monaski December 6, 2012 J.R. Smith struggled to find his rhythm all night, but the Knicks shooting guard was able to roll his shoulders and shake his hips after hitting the game-winning bucket as time expired in New York's 100-98 win over the Bobcats in Charlotte on Wednesday. Read More|Comment Category: Sports, Utica Rome Sports Tags: carmelo anthony, j.r. smith, kemba walker, NBA, new york knicks Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!

subset: commoncrawl


text:
|Accession Number: G-17678 (MetaCyc)
Species: Spinacia oleracea
A chloroplastic enzyme with fructose-1,6-bisphosphate aldolase activity was isolated from the leaves of well developed spinach (Spinacia oleracea L. Monopa) plants [Schnarrenberger86]. This enzyme was separated from its cytosolic homolog isolated simultaneously (see cytosolic fructose-bisphosphate aldolase). In addition to fructose 1,6-bisphosphate, the enzyme was shown to catalyze the aldol cleavage of β-D-fructofuranose 1-phosphate. The chrloroplastic enzyme represented 92% of the total leaf aldolase activity. Additionally, this chloroplast aldolase was shown to have a Km (F1,6P) value 5 times higher than that of the cytosol aldolase. However, the Km (F1P) values for either enzymes are relatively similar [Schnarrenberger86].
Locations: chloroplast
Molecular Weight of Polypeptide: 42.468 kD (from nucleotide sequence)
Unification Links: Entrez-Nucleotide:X66814 , Pride:P16096 , Protein Model Portal:P16096 , UniProt:P16096
Relationship Links: InterPro:IN-FAMILY:IPR000741 , InterPro:IN-FAMILY:IPR013785 , Panther:IN-FAMILY:PTHR11627 , Pfam:IN-FAMILY:PF00274 , Prosite:IN-FAMILY:PS00158
GO Terms:
|Cellular Component:
|GO:0009507 - chloroplast [Schnarrenberger86]
Created 17-Jan-2007 by Tissier C , TAIR
Enzymatic reaction of: fructose-1-phosphate aldolase (chloroplastic fructose-bisphosphate aldolase)
EC Number: 22.214.171.124
β-D-fructofuranose 1-phosphate <=> glycerone phosphate + D-glyceraldehyde
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.
The reaction is favored in the direction shown.
Kinetic Parameters:
Enzymatic reaction of: fructose-1,6-bisphosphate aldolase (chloroplastic fructose-bisphosphate aldolase)
EC Number: 184.108.40.206
fructose 1,6-bisphosphate <=> glycerone phosphate + D-glyceraldehyde 3-phosphate
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.
This reaction is reversible.
In Pathways: superpathway of anaerobic sucrose degradation , sucrose biosynthesis I (from photosynthesis) , glycolysis I (from glucose 6-phosphate)
Kinetic Parameters:
Schnarrenberger86: Schnarrenberger C, Kruger I (1986). "Distinction between Cytosol and Chloroplast Fructose-Bisphosphate Aldolases from Pea, Wheat, and Corn Leaves." Plant Physiol 80(2);301-304. PMID: 16664617
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December 16, 2007 8:25 PM
A few things you may consider to do before you go:
For those of you who are already done with finals, congratulations! You have survived another (or your first one if you are a Freshman) rigorous semester at the nation's #1 public university! For those of you who still have papers/presentations/exams next week, keep the good work and you are almost there!
Here are a few things you may consider to do before you go:
1). Sell your textbooks
You can go to Cal Student Store or Neds to sell back your books. However, if you feel that the price at which they are buying back your stuff is not satisfying, just visit comegetused.com to register and list your item there!
(Disclaimer: comegetused.com is a textbook classifieds network owned and operated by a Berkeley alumnus and an UCLA student. Though there is no charge for the service, there is no guarantee that someone will buy your book)
2). Do some laundry
Personally, I have piled up some laundry the past two weeks because I just don't have the leisure to do them! But hey, you don't want your dirty clothing to sit there (in a basket at the corner in your closet) until you come back in January. Technically, you could pack your laundry into a suitcase and bring them home. But you are a college student who has the ability to take care of yourself. So let's finish them before you leave.
3). Finish spending your meal points if you have a meal plan
Wasting meal points = wasting your money!
4). Have fun!!!
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Adjust Your Mac's Volume More Precisely
Quick Geek Tip: Adjust Your Mac's Volume
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Learn more after the jump.
If you're listening to music with headphones, using this additional control isn't as noticeable (unless, of course, you're rocking a pair of DJ-quality headphones like those Beats I love so much), but after attaching external speakers to my computer, I noticed the difference immediately. It creates more of a seamless increase and decrease in volume, similar to how turning a volume dial sounds.


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A beautiful two-story living room with a sunny bay window graces the front of the home and gives you access to a private courtyard. The semi-formal dining area is filled with windows and showcases a curved staircase. The kitchen is airy with a large island and lots of counter space, and adjoins a relaxing family room. Upstairs, you'll find a wonderful master suite that features an open vanity with double sinks and a nice sized walk-in closet. Choose to have two more bedrooms upstairs, or one bedroom and an open loft—perfect as a study or play area.
Ivory Homes has been ranked #1 among home builders in Utah for 25 consecutive years! Ivory Homes builds beautiful custom Utah homes (estate homes, townhomes, condos and luxury homes) in over 55 Utah communities (including active adult), many of which include rich amenities like pools, parks, and trails. If you are looking for a Utah home builder in Salt Lake, Utah County, Wasatch, Davis, Weber, Tooele, or Washington County (St. George), Ivory Homes is the homebuilder for you.
© 2011 Ivory Homes Ltd. All Rights Reserved.


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- Musician's Friend Best Selection, Price & Service. Guaranteed.
Call our Gear Heads
at: 800-449-9128
Call our Gear Heads
at: 800-449-9128
Cleans, polishes and protects pianos, guitars, and drums.
The Trick Ultimate Instrument Care Kit contains 25 - 10" x 12" premoistened Ultimate Carnauba Wipes and a polishing cloth. The wipes are the easiest, fastest, and most convenient way to keep instruments looking like new. They work on any instrument finish and won't scratch, streak, or swirl as they remove fingerprints and smoke, dust, and sweat residues. The polishing cloth will not scratch even the most delicate finishes and its 100,000 microfiber strands per square inch grab, lift, and hold grime like a magnet.
Order today and keep everyone in the band's instrument ready for showtime.
Review Snapshot®by PowerReviews
Reviewed by 1 customer
Comments about Trick Drums Ultimate Instrument Care Kit:
I've used tons of polishes and "filth repelants" that did little more than remove finger prints. This stuff is great. Makes a guitar shine.One towel will shine several guitars nicely, so if you are only polishing one guitar pull half of the towel out and cut it off to save a little.
Our product catalog varies by country due to manufacturer restrictions. If you change the Ship-To country, some or all of the items in your cart may not ship to the new destination.


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COMPANY PRESS RELEASE: Schnuck Markets, Inc. will sell its seven stores in metro Kansas City to Hy-Vee, Inc. of West Des Moines, Iowa, effective Aug. 25. Terms of the asset purchase, pre-approved by the Federal Trade Commission, were not disclosed.
Virtually all of Schnucks' 900 associates working in its Kansas City stores will be offered comparable positions with Hy-Vee. "We appreciate the dedication of our Kansas City employees since we entered the market in 1991 and the opportunity to serve our Kansas City customers. While it's difficult to make the decision to leave the area, we realized we were at a crossroads with two options," said Craig D. Schnuck, chairman and chief executive officer. "One was to make a substantial investment in new stores and sales promotion to expand our Kansas City presence in order to reach a sufficient size and scale to be profitable. The other was to see if another supermarket operator might want to grow by buying our operation." Two other factors contributed to Schnucks' decision to sell. One was the over-stored condition of the Kansas City grocery market, and the tendency of local supermarket operators to build stores in growth areas before the surrounding population is large enough to support a profitable operation. The other was the high cost of transporting products from Schnucks' St. Louis warehouses to its Kansas City stores. Those costs are significantly higher on a per-item basis than in most of Schnucks' other markets. Schnucks received a strong response from potential buyers, and pursued discussions with the highest bidders this summer. "We decided on Hy-Vee's offer because it makes everybody a winner. Our outstanding employees will retain their jobs, our customers will continue to have great stores in which to shop, and we are able to recoup our investment," Schnuck said. "We'll use the proceeds to fund our ongoing expansion in other markets." Scott C. Schnuck, president and chief operating officer, added, "We're confident our employees will be joining a well-managed and progressive company with solid Midwestern values similar to our own. Hy-Vee offers a bright and secure future working in stores where customers come first."
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No. 02306113
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London W1T 3JH, UK
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Marni updates a pair of standard black brogues with a shot of grey flannel. The texture clash is a sharp look that will do well to complement your new Marni backpack. Fall into these at Mr. Porter.
Marni Flannel-Panelled Leather Brogues
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After the latest update, I went to use it and instead of a list of quick responses I got a white screen with "I'm here!" in a large font which then disappeared and the watch went back to the watch face.
I cleared all the data, same issue.
Then I uninstalled it and reinstalled it. Same again.
Has anyone got the same issue?
Can anyone confirm it is working for them?
If it is working for you, do you have the smartwatch xposed module too?


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"The more laws that are written, the more criminals are produced." - "The more laws and order are made prominent, the more thieves and robbers there will be." - Lao Tze
"Good people do not need laws to tell them to act responsibly, while bad people will find a way around the laws." -Plato
Ephesians 6:10-17
Hammock Forums is my social network.


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The term "marketable" is defined as being in good demand or suitable for sale. It is a word used to describe someone used by major corporations to sell their product. It deals with an image or brand.
Beautiful women have always been marketable. You could invent the world's worse product, but if you attach a sexy model on the cover, you just sold a million cases.
I have found the 25 hottest marketable female athletes in the world and compiled them in a list for your enjoyment. So put aside the cell phone and hold all calls while you enjoy the beautiful ladies.


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Coupon Round-Up (7/21/2013)
This Hip2Save.com Deal was hand-posted on Sunday, July 21st, 2013 at 4:33 pm.
Buy 3 get 1 Free SoyJoy single bars
*Go here and change your zip code to 12345 and then click on the link above!
$1/1 any SoyJoy 5-Pack
*Go here and change your zip code to 12345 and then click on the link above!
$2/1 DiGiorno Pizzeria (Facebook) – still available!
$1/2 Bottles of Welch's Summer Sparkling
$1/1 Starbucks Iced Coffee
*Be sure to print this coupon now as it will be a $1 money-maker at CVS starting 7/28!
$0.40/2 Pillsbury Crescent Dinner Rolls
Buy One Get One Free Harvest Snaps (3.3 oz Snapea Crisps or 3.0 oz Lentil Snaps) – new link!
$1/1 any 16 oz Jar of Peanut Butter & Co. Peanut Butter (Facebook)
$0.75/1 any Welch's 100% Juice or 100% Juice Blend
$1/2 any Ken's Products
New Ibotta Offers:
Buy any On the Border Tortilla Chips AND any Ortega Salsa in the same shopping trip, earn $1.50 cash
Buy any 16-32oz box of Krusteaz Pancake mix AND Driscoll's Blueberries 18 or 32 oz. container in the same shopping trip, earn $1.50 cash
Buy fTGI Fridays Snack Chips Any variety 2 oz. bag or larger, earn $0.50 cash
Buy Brawny Paper Towels Any variety 2-pack or larger, earn $1.00 cash
Buy any Edge Shave Gel 5 oz. or larger, earn $0.50 cash
Buy any GOYA Beans canned variety, earn $0.50 cash
Buy any Fresh Gourmet Premium Croutons 5 oz. bag, earn $0.50 cash
Buy any Newman's Own Salad Dressing 16 oz only, earn $0.50 cash
Buy any Daisy Regular or Light varieties, earn $0.50 cash
Buy any size Pebbles Cereal Fruity or Cocoa varieties, earn $0.50 cash
Buy any Emeril's Cooking Spray, earn $0.50 cash
Buy any size FLA-VOR-ICE, earn $0.50 cash
Buy any Jergens Natural Glow Moisturizer, earn $0.75 cash
Buy any Johnsonville Bratwurst, earn $0.75 cash
Personal Care:
$3/1 any Pearls Branded Product
FREE Tena samples
$1/1 GoodNites Bed Mats
$2/1 GoodNites Underwear
$0.50/1 any Carefree Product
$1/1 any Excedrin Extra Strength 24 Count or Larger
$1/1 any Excedrin Migraine 24 Count or Larger
$0.50/2 any Glad Odor Shield Trash Bags 13 Gallon, 45 Count or Higher
$1.25/2 any Glad Odor Shield Trash Bags 13 Gallon, 45 Count or Higher
$0.50/1 any Glad Designer Series Medium Rectangle Containers 4 Count
$2/1 any Final Touch Fresh Expressions In Wash Scent Booster or Fabric Softener (Facebook)
Store Coupons/Restaurant Deals:
(Photo Credit: Wheel 'n Deal Mama!)
Target: Keep your eyes peeled in your mailbox for a new Save Smart Target coupon booklet with over $30 in coupon savings for Back-to-School inside! A few coupons include a FREE $5 gift card with any $50 purchase, $2 off $10+ Crayola Purchase, $1 off two BIC school supplies (Writing instruments, Correction tools, markers/highlighters), a FREE 24 count up & up crayons with purchase of two up & up school supplies listed: 8-10 count broad tip markers, 12 or 24 count colored pencils, 24 count crayons, and more!
BonTon: $10 off regular or sale price purchase of $30 or more AND $20 off regular or sale price purchase of $60 or more, valid 7/21-7/23.
Olive Garden: Buy one adult entrée and receive a second of equal or lesser value for 1/2 off (excludes kids entrées, soup, Garden Salad, pizza, tax, and gratuity) – fill out form.
Whole Foods: Check out the email I received from reader, Jesse –
Not sure if this is worth mentioning, but our daughter uses Earth's Best Organic Infant Formula, and for us it is $29.99 at Whole Foods vs. $33.99 at QFC/Kroger Affiliate. Anyhow, at WF the tubs of Earth's Best have $5 coupon peelies good until 2014. That's a pretty sweet deal in my book :) Cheaper than Amazon's price currently.
Old Orchard: Old Orchard has released a new survey for fan club members to take to earn 500 points. Also there is a new game hub where you can start your own tournaments and still earn points for playing games and for your achievements. If you're not a member, you can sign up now to start taking advantage of these point earning opportunities (plus, you'll get 1,000 Rewards Points just for signing up!).
(Thanks, Brittany, Brandice, The Savvy Bump, Growing Up Natural, Wheel 'n Deal Mama, Jennifer, Thriving on Thrifty, Cincy Shopper, Stephanie, and Printable Coupons and Deals!)
23 Hip Readers Commented
Olive Garden is B1G1 half off (not free)
Hey Collin how is the starbucks drink coupon going to make a 1.00 money maker? I printed them.
They are on sale next week at CVS for $1.50, but then you get a $1.50 Extra Care Buck, so it will be free, but if you have the Coupon, then it will be a $1.00 money maker.
Thanks. So they are just giving them away?
Ya cuz it's a relatively new product that taste completely awful lol. This is coming from someone who lives on Starbucks.
Totally agree with Adria , LOve Starbucks, LOve coffee but this one is awful.
My Target flyer had a coupon for $5 GC when you spend $75, not $50. Some other people got the same thing. Clearly some of us spend way too much at Target!
As for how to get the Target flyers, there's no sign up. I didn't start getting them until I got the Target Red Card (just the debit one, not the credit one). You can try that if you don't already have one. Other people started getting them after signing up for a registry.
Mine came with that same deal – $5 for $75 purchase and I'm in Atlanta. I wonder if the amount is a regional thing.
I got the Target mailer but there wasn't a coupon for a $5 gift card for spending anything. I'm in Milwaukee.
same here no coupon, I'm in appleton WI
I can no longer print coupons. It tells me to be sure I don't have 'save document' selected and I don't. I have tried selecting it and then unselecting it and still no-go. Any ideas?
Starbucks coupon is saying–
Sorry, this coupon has already been
printed the number of times allowed or
this coupon link is no longer valid.
That's weird. I was just able to print them. (8:30 CST)
Same message here. I guess the coupon didn't reset from 2 months ago?
I am getting the same message and never printed it before, so it's probably just out of prints. :(
Got the Target mailer w a $5gc w a $50 purchase :) I'm in CT.
Hmm wish I knew how to get the target mailer, and I have a red card :(
Ibotta is a fun and exciting way to earn cash every time you shop – and it's free! It works in over 40 grocery chains nationwide, and Ibotta users have already earned over $1 million. If you sign up today, there's a special $10 Share The Love Bonus waiting for you! http://ibotta.com/r/tsoZyQ
Want to get a app that makes you money for scanning things in the store or just going to the store like you do anyways. Download this app from this link you will get 50 kicks on shopkick, a fun app that lets you earn rewards for shopping. http://get.shopkick.com/oregano9099
Can anyone tell me how to sign up to get the Target mailers? I have a Target.com account, but I'm not getting them. Thanks!
Somebody help me! I'm 1 person short for the final fun in the sun bonus! PLEASE sign up using my link! =) https://ibotta.com/register?friend=BfHRw


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Conference calling can provide vital connectivity in the event of absences. UIT has arranged for a "pay as you use it" conferencing capability. This service provides divisions and schools with an 800 number and a moderator PIN to conference up to 96 people who can call from anywhere in the world. You pay for the service only when you use it. To use Tufts' conference calling service, you must arrange for access in advance of your first intended call. To establish conference calling services, please send requests to email@example.com or call the Voice Services Helpdesk at (617) 627-4357.
For more information regarding Conferencing calling, please visit the Conference Calling Services page at UIT


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Change isn't always bad: trout in headwater streams show increased growth following wildfire.
Heck, Michael*,1, Gresswell, Robert2, 1 Oregon State University, Corvallis, OR2 USGS Northern Rocky Mountain Science Center, Bozeman, MT
ABSTRACT- Wildfire, a largely terrestrial perturbation, is broadly recognized as an agent of disturbance and ecological change in forested biomes. Effects of postfire conditions on biotic components of aquatic systems have been less well-documented, although hypothetically the two are strongly connected. In fact, the influence of wildfire may be most profound in headwater streams because of the tight linkage between aquatic and terrestrial ecosystems. By observing growth of a tertiary consumer (coastal cutthroat trout, Oncorhynchus clarkii clarkia), we sought to investigate how postfire conditions influence fish growth in headwater streams. During the summer of 2002, wildfire burned portions of three headwater catchments in western Oregon. Burn severities ranged from moderate to severe. An unburned catchment was selected as a control. For three years following the wildfire, all fish-bearing portions of the stream network in the four catchments were surveyed, and scale samples were collected for age and growth analysis. Despite considerable alterations in the physical stream environment following the wildfire, growth of coastal cutthroat trout appeared to be positively related to wildfire severity. It appears that increased solar radiation and water temperatures triggered a bottom-up ecosystem response resulting in greater growth of fish in the burned catchments. Contrary to the commonly held perception that wildfire is detrimental, these results suggest that over short time scales, the effects of wildfire on stream ecosystems may benefit some members of the aquatic community.
Key words: wildfire, growth, coastal cutthroat trout
All materials copyright The Ecological Society of America (ESA), and may not be used without written permission.


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Administration Opens Door to Wolf Hunting in Wyoming National Park Unit
|February 7, 2012
|Sharon Mader, Grand Teton Program Manager, National Parks Conservation Association, (O) 307-733-4680, (C) 307-690-5245, firstname.lastname@example.com
Jeff Billington, Senior Media Relations Manager, National Parks Conservation Association, (O) 202-419-3717, (C) 202-384-8894, firstname.lastname@example.com
Administration Opens Door to Wolf Hunting in Wyoming National Park Unit
JACKSON HOLE, WY — A plan brokered by the Department of the Interior (DOI) and the state of Wyoming could allow for an unprecedented wolf hunt in the John D. Rockefeller (JDR) Parkway, a 24,000 acre unit of the national park system that serves as a vital link for wildlife between Yellowstone and Grand Teton National Park. The National Parks Conservation Association (NPCA) is calling for DOI to secure changes in Wyoming's wolf plan to prohibit wolf hunting and state wolf control inside all national park units in Wyoming before wolves are removed from Endangered Species Act protections.
"National parks are supposed to provide sanctuary and refuge for wildlife," said NPCA Grand Teton Program Manager Sharon Mader. "The Administration is opening the door to wolf hunting in a national park unit immediately after this iconic species is removed from the 'endangered' list. These are the same wolves that Grand Teton and Yellowstone National Park visitors have enjoyed viewing for years and have become a mainstay attraction in our national parks."
The recovery of the gray wolf in the northern Rockies is one of the America's great conservation success stories. The DOI has proposed to remove the wolf in Wyoming from the Endangered Species list, but has given preliminary approval of a state plan that permits the hunting of wolves in the JDR Parkway, which connects Yellowstone and Grand Teton National Parks.
"Allowing a hunt in a national park immediately following the delisting of the wolf would be a black mark in what has otherwise been a positive conservation record for the Administration," said Tim Stevens NPCA's Northern Rockies Regional Director. "Fortunately, there is still time to correct this major problem by excluding all NPS lands in Wyoming from the possibility of a wolf hunt."
Both Grand Teton and the JDR Parkway are identified as inside Wyoming's 'Trophy Game Management Area' boundaries that would be subject to a hunt in the state's wolf management plan. While the NPS and state have clarified they do not anticipate hunting wolves within Grand Teton, both have made it clear that a wolf hunt within the Parkway could be considered. In addition, recent statements by the state of Wyoming indicate they have not given up on killing wolves on 1,400 acres of state-owned land within the boundaries of Grand Teton.
"The JDR Parkway is a critical corridor connecting the Yellowstone and Grand Teton wolves," noted Mader. "The state plan already excludes Yellowstone from wolf hunting and we are calling for them to do the same for the JDR Parkway and Grand Teton."
"Our national parks are supposed to be a safe haven for our iconic wildlife, not a free fire zone," said Stevens. "If this decision stands, the Administration is creating unnecessary problems for many years to come and this problem can and must be fixed before the delisting rule is finalized."


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The pirate-themed exhibits will feature artwork from literature and film to movie posters, books prints and other forms.
This is a family oriented event, so submissions must be appropriate. Artists are asked to provide easels or pedestals to display their pieces.
Submissions must be made for acceptance no later than Friday, Aug. 5, to Loo by e-mail at firstname.lastname@example.com.
Hung Sau "Assassin" Loo Ting hails from the province of Canton, China. During his travels and studies with the ancient mystics, Loo said he developed an obsession for ancient weaponry and a keen eye for all arts. Inspired by the beauty that surrounded him, Loo started painting and using odd metals and distressed substances for his artwork.
For more information, visit www.michiganpiratefestival.com; or call 842-5560, ext. 222.


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Ruby Rd Utility Chic Collection Earring
$9.60 Original Price $16.00
Savings: $6.40 (40%)
With a beautiful brass oxide finish, these drop earrings feature a hammered disc post and disc drop to create a feminine and chic accessory. Pair with your favorite outfit to complete a sophisticated look.
- 1.75-in. L
- Pierced earring
- Oxidized finish
- Metal; Post: surgical steel
- Belk Exclusive
- Imported
Shipping & Returns
Shipping your purchase is easy on belk.com. Depending on the destination of your package, you can expect it to arrive within 4-7 business days. Shipping Information
Returns are easy on belk.com. Easy returns
Is a mix of sensational color, fun fashion, fabulous details and unique styling which draws it's inpiration from an eclectic mix of sources.
This item is also part of the collection:
Ruby Rd Utility Chic II Jewelry Collection
Ruby Rd Utility Chic II Jewelry Collection


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Royal Corinthian Yacht Club, Cowes
The world of the private club is, to Cat and Matt, epitomised by the Drones Club - Bertie Wooster's London gentlemen's club. A fictional place where chinless hoorays lob bread rolls at each other, drink cocktails and hide from indomitable aunts.
An alternative view of the private club is offered by Dorothy L Sayers; in The Unpleasantness at the Bellona Club deep-buttoned chairs upholstered in blood-red leather are occupied by gin-sodden colonels whose whiskers twitch in time to their snoring.
Having managed to procure a ticket the Royal Corinthian, Cowes' 'Not New Year's Eve' party, M&C were unsure what to expect. They were advised to dress 'black tie', so knew it would be fancy. But should they wear wipe-clean attire in anticipation of a food fight, or perhaps Cat should bring her new Kindle to while away the hours to the rhythm of a ticking clock and the whisperings of sailing types?
Arriving promptly for once, Matt and Cat installed themselves at the bar. Matt rubbed his finger around the inside of his collar and Cat fiddled with the clasp on her satin clutch bag. Those of you who know the reviewing duo in person will gasp at the unlikely scenario of Matt in a tie and Cat in a party frock with a dainty clutch, but they had scrubbed up quite well. They certainly didn't draw any disparaging looks from their fellow guests so they figured that they'd made a good simulation of respectable party goers. Their spirits rose as jolly people entered, the place filled up with lively chatter and their glamorous acquaintances arrived. Drinks in hand the throng moved to the dining room at the sound of the gong.
Follow up:
A seating plan of sorts had been on display at the entrance to the dining room but, at all of the tables, there was much light-hearted jostling and place-reconfiguring. This seemed to be a traditional part of the evening. Cat, unaccustomed to being on the receiving end of such direction, was moved out of her first designated seat, directed to another and sat down with relief, as her five inch heels were starting to make their presence felt.
M and C's dining companions were all extremely pleasant and, although some were strangers to each other, the ice was soon broken by a team task. On each table there was a vast and robustly constructed toy wooden yacht, complete with rigging. Alongside was a pile of coloured and glittery papers, sellotape, scissors, string and pens. The object was to decorate and name the boat between courses. This was a fab way to get to know the others as the table's team beavered away to make the best-decorated ship in the room.
The waiting staff - faced with the chaos of all this paper, unfurled napkins being employed as sails, and the debris left by the obligatory party poppers - did an excellent job of serving the food. What's more, the food itself turned out to be impressively good. The starter of smoked haddock and scallop gratin was delightful, if slightly salty. However, this maritime dish was very in-keeping with the nautical theme of the table task and Cat was inspired to suggest that little paper sailors be made to decorate the rigging like cadets. Her dining companion Fran made a most excellent job of cutting out the paper matelots, even ensuring that each had a face from the adeptly-used snowman-themed paper. It became apparent that the purpose of some of the discreet pre-dinner shuffling had been to ensure that those known to be competent in the scissors and designing department had been positioned close to the toy boat, whilst those (like Matt) known to be unapologetically cack-handed incompetents had been kept away from it.
Four course meal plus coffee & half a bottle of wine
£45 per person
The main course was one of Cat's favourite dishes - fillet steak. Again, how the kitchen and waiting staff managed to cook and deliver about 150 plates of this spectacular meat simultaneously was nothing short of miraculous. The Rioja and thyme marinated tender steaks had all been cooked medium to satisfy the majority and were accompanied by delicious Dauphinoise potatoes, olivette carrots, green beans and tossed spinach. Not a hint of anything kept hot under a lamp, or oversteamed. Matt remarked that he'd struggle to serve a single steak in as good a state as this one, let alone more than a gross of the things. There was a pause in the hubbub while everyone chowed down.
Once the main course had been cleared away the atmosphere of the Corinthian Yacht Club - already a far cry from the Bellona Club - entered entertainingly into sociable Drones territory. No bread rolls had actually been thrown at this point but, the freely-flowing wine seemed to help the creative juices. The boat adjacent to M and C had been rigged with some impressive sails made of toilet paper and gaffer tape, billowing with the aid of a hairdryer (where did that come from?), employed to add an authentic gust. Teetering around the tables to see what her competitors were up to, Cat observed a variety of merrily creative solutions to the boat-decorating competition including one with a fiver sellotaped audaciously to the rigging; a most blatant bribe!
The next course was a twice-baked Westcombe cheddar soufflé with pear and prune chutney. This delicate dish is hard to get right in laboratory conditions so its success under such trying circumstances was to be applauded. The little flans were piping hot; the almost acid cheese complimented perfectly by the fruity chutney.
By now, the boat-decorating contest was coming to an end. Matt had employed his expertise to create a functional cannon out of cardboard and one of the remaining unexploded party poppers. The little sailors were standing to attention on the rigging and Cat had woven a crow's nest which was occupied by a paper sailor who for some reason had no legs - such inclusiveness was worthy of the Jubilee Sailing Trust. The boat was named 'POSH1' after the table's sophisticated hostess. As the judges approached, the confident team popped the poppers, including the cannon, and gabbled furiously pointing out the vessel's exquisite details. However, the the judges seemed more interested in figureheads - taking particular note of the ladies' cleavages. Un-PC it may be but plenty was on display, which may account for the table coming second in the contest! Hoorah!
Pudding was next to be delivered and Matt and Cat's table cleared some room from the mountains of snipped paper and spent plastic cartridges of party poppers. The dark chocolate and crème de menthe torte with warm spiced cherries was absolutely delightful; the bitterness of the torte soothed with the sweet cherries. Each of the four courses was beautifully presented, delicious and plentiful.
Coffee and saucy games with chocolate coins followed before the diners upped-sticks and made their way to the dance hall. Here they jiggled about to the retro sounds of the Accelerators. By this stage Cat's stupidly high shoes and Matt's tie had been discarded but no-one seemed to mind about this lapse in decorum - after the cleavage-ogling and chocolate coin larks, it seemed that anything goes.
Whilst the Royal Corinthian is not directly open to the public for casual dining, it is apparently available to all for events and is well-used in this way. If the club can gracefully produce such lovely food in such great quantities it must surely lend itself to events such as weddings and private parties where their art of mass catering would be essential. They really know how to throw a good party, with brilliant food delivered in a chaotic but very friendly environment. Bertie Wooster may have the edge on food fights but, unlike the mythical Drones, the Royal Corinthian is a very welcoming and accommodating place, that even allows women in. Recommended, what-ho!
Visit the website: http://www.rcyc.co.uk
19890 views
Categories: We love!, Cowes & East Cowes, Special events
6 comments (newest first)
Hi there Matt & Cat,
I've been following your site for a number of years. Well done!
I'm just setting up a website for people to crew on boats (sailing) in Cowes & the Solent. Whilst looking around the internet for a RCYC flag logo, the "POSH 1″ picture keeps popping up. I couldn't help myself in the end & clicked on the "Posh cleavage" to see what it was all about & well, here I am on your site again. Funny!
Would you like me to put a link on the site to you?
Matt and Cat respond: feel free Reg, and best of luck with the new site.
Shouldn't that be POSH 2?
We had our wedding reception at RCYC and the food was faultless, the service outstanding, we have sunday lunch there and you get a whole joint for however many are in your party, it is a great place.
I hear the caterers are called All About Catering. Fantastic food always.
Was lucky enough to go to the Isle of Wight 'Law Society' dinner dance here in November last year.
Food was good, service was good, but the band were very, very good. 'Off the Wall' a 70's/Soul band from the island.
Wow…what a good time! Happy New Year to my favorite reviewers!
Matt and Cat reply: Hi Mike, happy new year to you too. Thanks for your support over the years. We wondered if you got your own personalised steak branding irons from Father Christmas?


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Monthly Archives: May 2012
Update on Principal Place Exemption Tax
See below update regarding the reintroduction of the Principal Place of Residence concession. The rate will be 1% on the first $350,000 and then the normal amount of duty on top of the $350,000.00. Principal Place of Residence Exemption Reinstatement … Continue reading
Maintain a garden even if you're living in an apartment
When comparing the pros and cons of apartment living, a drawback that usually tends to arise is that apartments generally don't allow for occupants to have a garden. While many won't see this as an issue at all, others might … Continue reading
Port Douglas to host 3 Major Sporting Events This Weekend.
This weekend marks the start of the Cairns Airport Adventure Festival in Port Douglas, with 3 fantastic events to kick it off – the Coral Coast Triathlon, RRR Mountain Bike Race and Hekili Great Barrier Reef Ocean Challenge. It is … Continue reading
Tourist Arrivals & Departures
Tourist Arrivals and Departures Tourist arrivals hit record highs Overseas arrivals and departures Tourist arrivals hit record highs: The number of tourists arriving in Australia in March rose by 1.9 per cent to record highs. And the number of Australians … Continue reading
Port Douglas – Young Achiever Award 2012
FOR THE SECOND YEAR THE PORT DOUGLAS CHAMBER OF COMMERCE IS PLEASED TO SPONSOR THE YOUNG ACHIEVER AWARD 2012 NOMINATE YOURSELF OR SOMEONE YOU KNOW! THE TERMS AND CONDITIONS, INCLUDING THE NOMINATION FORM, ARE ATTACHED AND ARE ALSO … Continue reading
Reintroduction of the Principal Place of Residence Concession
See the below information update from the QLS regarding the reintroduction of the Principal Place of Residence Concession. The LNP Government has announced that it will be honouring its election commitment and reviving the principal place of residence (PPR) duty … Continue reading
Double Rate Cut Has Been Confirmed!
DOUBLE RATE CUT HAS BEEN CONFIRMED! The Reserve Bank of Australia today decided on a double rate cut (50 basis points) lowering the RBA's cash rate target to 3.75 percent from 4.25 percent which will equal a substantial saving in … Continue reading


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Nic + Zoe 'Spring Skies' Dress
|Item # 662346
Details & Care
Sinuous stripes with hazy patterning sweep around a soft dress in a feminine fit-and-flare silhouette accentuated with a wide, ruched waistband.
- Approx. length from shoulder to hem: 40".
- Measurements taken from size Medium and may vary slightly by size.
- Slips on over head.
- Surplice bodice.
- Unlined.
- Rayon/polyester/linen; hand wash.
- By Nic + Zoe; imported.
- Narrative.
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Diane Warren - Completely: An Anthology Of MusicItem #: WB.VF2147
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As performed by Diane Warren. Composed by Diane Warren.
Personality Songbooks. Piano/Vocal/Guitar Songbook. Arrangements for piano and voice with guitar chords. 9x12 inches. 164 pages. Published by Warner Brothers. (WB.VF2147)
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Completely * Don't Take Away My Heaven * When the Night Comes * Saving Forever for You * Set the Night to Music * Live for Loving You * Love Will Lead You Back * By the Time This Night is Over * Time, Love & Tenderness * Everything Changes * Look Away and many more.
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I could show you pictures of my mantel for the last four years, and you'd probably think it was exactly the same every year. I do usually make a little change or two, but I always use my Fontanini nativity on the mantel, so it is basically the same.
I've told the story before about finding this nativity set at an estate sale. If you would like to read it, please click here.
Christmas has begun at my house. If you've visited my blog during Christmas before, you know there is more Christmas to come. I'm linking this post to Seasonal Sunday at The Tablescaper. I really appreciate your visit. laurie


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