Anne Parle-McDermott

The ATP-binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3.

Pleiotropic drug resistance (PDR) in the yeast Saccharomyces cerevisiae can arise from overexpression of ATP‐binding cassette (ABC) efflux pumps such as Pdr5 and Snq2. Mutations in the transcription factor genes PDR1 and PDR3 are also associated with PDR. We show here that a pdr1–3 mutant exhibits a PDR phenotype, including elevated resistance to the mutagen 4‐nitroquinoline‐N‐oxide, a known substrate for Snq2 but not for Pdr5. Northern analysis and immunoblotting demonstrated that the SNQ2 gene is 10‐fold overexpressed in a pdr1–3 gain‐of‐function mutant strain, whereas Snq2 expression is severely reduced in a Δpdr1 deletion strain, and almost abolished in a Δpdr1Δpdr3 double disruptant when compared to the PDR1 strain. However, expression of the Ste6 a‐factor pheromone transporter, another yeast ABC transporter not associated with PDR, is unaffected in pdr1–3 mutant cells and in strains carrying Δpdr1, Δpdr3, or Δpdr1Δpdr3 deletions. Finally, DNA footprint analysis revealed that the SNQ2 promoter contains three binding sites for Pdr3. Our results identify SNQ2 as a novel target for both Pdr1 and Pdr3, and demonstrate that the PDR phenotype of a pdr1–3 mutant strain results from overexpression of more than one ABC drug‐efflux pump.

Confirmation of the R653Q polymorphism of the trifunctional C1-synthase enzyme as a maternal risk for neural tube defects in the Irish population

The risk of neural tube defects (NTDs) is known to have a significant genetic component that could act through either the NTD patient and/or maternal genotype. The success of folic acid supplementation in NTD prevention has focused attention on polymorphisms within folate-related genes. We previously identified the 1958G>A (R653Q) polymorphism of the trifunctional enzyme MTHFD1 (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolate-cyclohydrolase, formyltetrahydrofolate synthetase; often referred to as ‘C1 synthase’) as a maternal risk for NTDs, but this association remains to be verified in a separate study to rule out a chance finding. To exclude this possibility, we genotyped an independent sample of mothers with a history of an NTD-affected pregnancy derived from the same Irish population. In this sample there was a significant excess of 1958AA homozygote mothers of NTD cases (n=245) compared to controls (n=770). The direction and magnitude of risk (odds ratio 1.49 (1.07–2.09), P=0.019) is consistent with our earlier finding. Sequencing of the MTHFD1 gene revealed that this association is not being driven by another common variant within the coding region. We have established that the MTHFD1 1958G>A polymorphism has a significant role in influencing a mothers risk of having an NTD-affected pregnancy in the Irish population.

The yeast ATP binding cassette (ABC) protein genes PDR10 and PDR15 are novel targets for the Pdr1 and Pdr3 transcriptional regulators.

The yeast transcription factors Pdr1 and Pdr3 control pleiotropic drug resistance (PDR) development, since they regulate expression of ATP‐binding cassette (ABC) drug efflux pumps through binding to cis‐acting sites known as PDREs ( R esponsive lements). In this report, we show by Northern blotting, gel shift mobility assays and DNase I footprinting that transcription of the ABC genes PDR10 and PDR15 is also controlled by Pdr1 and Pdr3. In addition, in vitro band shift assays demonstrate that a GST‐Pdr1 fusion protein can bind to the PDREs of PDR10 and PDR15. DNase I footprinting allowed the identification of the precise PDRE binding motifs, indicating the presence of a novel slightly degenerate PDRE motif in the PDR15 promoter. Finally, PDR10 and PDR15 mRNA levels vary dramatically in abundance in isogenic yeast strains carrying either Δpdr1, Δpdr3 and Δpdr1 Δpdr3 deletions or pdr1‐3 and pdr3‐2 gain‐of‐function mutations, demonstrating that both PDR10 and PDR15 are new members of the yeast PDR network.

The 19-bp deletion polymorphism in intron-1 of dihydrofolate reductase (DHFR) may decrease rather than increase risk for spina bifida in the irish population

Periconceptional maternal folic acid supplementation can prevent up to 70% of pregnancies affected with neural tube defects (NTDs), including spina bifida. This has focused attention on folate‐related genes such as dihydrofolate reductase (DHFR) in a bid to identify the genetic factors that influence NTD risk through either the fetal or maternal genotype. We considered a novel intronic 19‐bp deletion polymorphism and two polymorphisms within the 3′ untranslated region (721A > T and 829C > T) of the DHFR gene as candidates for NTD risk. We studied NTD cases (n = 283), mothers of cases (n = 280), fathers of cases (n = 279), and controls (n = 256). We did not find the DHFR 829C > T polymorphism to be variable within the Irish population. The 19‐bp intron deletion and the 721A > T polymorphisms were found to be in linkage disequilibrium. In contrast to a previous study, the 19‐bp intron deletion allele did show a significant protective effect in mothers of NTD cases when present in one (relative risk 0.59 [95%CI: 0.39–0.89], P = 0.01) or two copies (relative risk 0.52 [95%CI: 0.32–0.86], P = 0.01). Analysis of mRNA levels revealed a small increase in expression (∼1.5‐fold) associated with the 19‐bp intron deletion polymorphism, but this was not significant. In conclusion, the DHFR intron 19‐bp deletion allele may be a protective NTD genetic factor by increasing DHFR mRNA levels in pregnant women.

Evaluation of common genetic variants in 82 candidate genes as risk factors for neural tube defects

BackgroundNeural 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.MethodsA 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.ResultsNearly 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.ConclusionsTo 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.

MTHFD1 R653Q Polymorphism Is a Maternal Genetic Risk Factor for Severe Abruptio Placentae

This study examined the relationship between folate/homocysteine‐related genetic polymorphisms: MTHFD1 1958G → A (R653Q), MTHFR 677C → T (A222V), MTHFR 1298A → C (E429A), and risk of severe abruptio placentae. We genotyped 62 women with a pregnancy history complicated by severe abruptio placentae and 184 control pregnancies. Analysis of the MTHFD1 1958G → A (R653Q) polymorphism showed increased frequency of the ‘QQ’ homozygote genotype in pregnancies affected by severe abruptio placentae compared to control pregnancies (odds ratio 2.85 (1.47–5.53), P = 0.002). In contrast to previous reports, the MTHFR polymorphisms 677C → T (A222V) and 1298A → C (E429A) were not associated with abruptio placentae risk in our cohort, when analyzed either independently or in combination. We conclude that women who are ‘QQ’ homozygote for the MTHFD1 1258G → A (R653Q) polymorphism are almost three times more likely to develop severe abruptio placentae during their pregnancy than women who are ‘RQ’ or ‘RR.’ Published 2005 Wiley‐Liss, Inc.

Folate-related gene polymorphisms as risk factors for cleft lip and cleft palate.

BACKGROUND Cleft lip with or without cleft palate (CLP) and cleft palate only (CPO) have an inherited component and, many studies suggest, a relationship with folate. Attempts to find folate-related genes associated with clefts have, however, often been inconclusive. This study examined four SNPs related to folate metabolism (MTHFR 677 C-->T, MTHFR 1298 A-->C, MTHFD1 1958 G-->A, and TC II 776 C-->G) in a large Irish population to clarify their relationship with clefts. METHODS Cases and their parents were recruited from major surgical centers performing cleft repairs in Ireland and a support organization. Data on risk factors, medical history, and DNA were collected. Controls were pregnant women from the greater Dublin area (n = 1,599). RESULTS CLP cases numbered 536 and CPO cases 426 after exclusions. CPO mothers were significantly more likely than controls to be MTHFR 677 TT, OR 1.50 (95% CI: 1.05-2.16; p = .03). Log-linear analysis showed a borderline association (p = .07). Isolated CPO case mothers were significantly more likely than controls to be homozygous for the MTHFD1 1958 G-->A variant, OR 1.50 (95%CI: 1.08-2.09; p = .02). When multiple cases were added, both CPO cases and case mothers were significantly more likely to be AA (p = .02 and p = .007, respectively). The CLP case-control and mother-control analyses also showed significant effects, ORs 1.38 (95% CI: 1.05-1.82; p = .03) and 1.39 (95% CI: 1.04-1.85; p = .03), respectively. CONCLUSIONS Associations were found for both CPO and CLP and MTHFD1 1958 G-->A in cases and case mothers. MTHFR 677 C-->T could be a maternal risk factor for clefts but the association was not strong. Because multiple comparisons were made, these findings require additional investigation. Given the known association between MTHFD1 1958 G-->A and NTDs, these findings should be explored in more detail.

Analysis of the MTHFR 1298A→C and 677C→T polymorphisms as risk factors for neural tube defects

AbstractThe thermolabile variant (677TT) of methylenetetrahydrofolate reductase (MTHFR) is a known risk factor for neural tube defects (NTDs). The relationship between a second MTHFR polymorphism (1298A→C) and NTD risk has been inconsistent between studies. We genotyped 276 complete NTD triads (mother, father and child affected with an NTD) and 256 controls for MTHFR 1298A→C. Our findings do not support a role for the 1298A→C polymorphism in NTDs (OR 0.85 (95% CI 0.49-1.47), p= 0.55), nor do we observe a combined effect with the 677C→T polymorphism.

A common variant in MTHFD1L is associated with neural tube defects and mRNA splicing efficiency

Polymorphisms in folate‐related genes have emerged as important risk factors in a range of diseases including neural tube defects (NTDs), cancer, and coronary artery disease (CAD). Having previously identified a polymorphism within the cytoplasmic folate enzyme, MTHFD1, as a maternal risk factor for NTDs, we considered the more recently identified mitochondrial paralogue, MTHFD1L, as a candidate gene for NTD association. We identified a common deletion/insertion polymorphism, rs3832406, c.781‐6823ATT(7–9), which influences splicing efficiency and is strongly associated with NTD risk. Three alleles of rs3832406 were detected in the Irish population with varying numbers of ATT repeats: Allele 1 consists of ATT7, whereas Alleles 2 and 3 consist of ATT8 and ATT9, respectively. Allele 2 of this triallelic polymorphism showed a decreased case risk as demonstrated by case–control logistic regression (P=0.002) and by transmission disequilibrium test (TDT) (P=0.001), whereas Allele 1 showed an increased case risk. Allele 3 showed no influence on NTD risk and represents the lowest frequency allele (0.15). Additional single nucleotide polymorphism (SNP) genotyping in the same genomic region provides additional supportive evidence of an association. We demonstrate that two of the three alleles of rs3832406 are functionally different and influence the splicing efficiency of the alternate MTHFD1L mRNA transcripts. Hum Mutat 30:1–7, 2009.

The impact of nutrition on differential methylated regions of the genome.

Nutrition has always played an important role in health and disease, ranging from common diseases to its likely contribution to the fetal origins of adult disease. However, deciphering the molecular details of this role is much more challenging. The impact of nutrition on the methylome, i.e., DNA methylation, has received particular attention in more recent years. Our understanding of the complexity of the methylome is evolving as efforts to catalog the DNA methylation differences that exist between different tissues and individuals continue. We review selected examples of animal and human studies that provide evidence that, in fact, specific genes and DNA methylation sites are subject to change during development and during a lifetime as a direct response to nutrition. Investigation of the methyl donors folate, choline, and methionine provide the most compelling evidence of a role in mediating DNA methylation changes. Although a number of candidate regions/genes have been identified to date, we are just at the beginning in terms of cataloging so-called nutrient-sensitive methylation variable positions in humans.