Fetal Single Nucleotide Polymorphisms Research Articles

A capture-based method of prenatal cell-free DNA screening for autosomal recessive non-syndromic hearing loss.

This study aimed to develop and validate a prenatal cell-free DNA (cfDNA) screening method that uses capture-based enrichment to genotype fetal autosomal recessive disorders. This method was applied in pregnancies at high risk of autosomal recessive non-syndromic hearing loss (ARNSHL) to assess its accuracy and effectiveness. This assay measured the allele counts in both white blood cell DNA and cfDNA from the blood samples of pregnant women using a capture-based next-generation sequencing method. It then applied a binomial model to infer the fetal genotypes with the maximum likelihood. Ninety-four pregnant couples that were carriers of variants of ARNSHL in GJB2 or SLC26A4 were enrolled. The fetal genotypes deduced using this screening method were compared with the results of genetic diagnosis using amniocentesis. Of the 94 couples, 65 carried more than one variant, resulting in 170 single-nucleotide polymorphism (SNP) loci to be inferred in the fetuses. Of the 170 fetal SNP genotypes, 150 (88.2%) had high confidence calls and 139 (92.7%) of these matched the genotypes obtained by amniocentesis result. Out of the remaining 20 (11.8%) cases with low-confidence calls, only 14 (70.0%) were concordant with genetic diagnosis using amniocentesis. The concordance rate was 100% for sites where the maternal genotype was wild-type homozygous. The discordance was site-biased, with each locus showing a consistent direction of discordance. Genetic diagnosis identified a total of 19 wild-type homozygotes, 46 heterozygotes, 19 compound heterozygotes, and 10 pathogenic homozygotes. This screening method correctly genotyped 81.9% (77/94) of fetuses and demonstrated a sensitivity of 89.7% and a specificity of 89.2% for correctly identifying ARNSHL. This capture-based method of prenatal screening by cfDNA demonstrated strong potential for fetal genotyping of autosomal recessive disorders.

Lipid Metabolic Genes and Maternal Supraphysiological Hypercholesterolemia: An Analysis of Maternal-fetal Interaction.

The joint associations of maternal and fetal single nucleotide polymorphisms (SNPs) of lipid metabolic genes with the risk of maternal supraphysiological hypercholesterolemia (MSPH) are unclear. This study aims to investigate the associations of maternal/fetal SNPs of APOE, LPL, LDLR, PCSK9, and SCARB1 with the risk of MSPH and explore whether the maternal-fetal pairing pattern of the risk alleles can affect MSPH risk. A nested case-control study was conducted that included 182 pregnant women with MSPH and 182 with maternal physiological hypercholesterolemia. Maternal venous and umbilical venous blood were collected to detect the SNPs of genes. The primary outcome was MSPH. Logistic regression model was used to determine the associations of SNPs with risk of MSPH. The C-allele in maternal APOE rs429358 T > C (adjusted odds ratio [OR] = 1.72, P = 0.033), G-allele in fetal APOE rs440446 C > G (adjusted OR = 1.62, P = 0.012) and T-allele in fetal LPL rs263 C > T (adjusted OR = 1.53, P = 0.011) increased the risk of MSPH. The A-allele in maternal LDLR rs7258950 G > A decreased the risk of MSPH (adjusted OR = 0.67, P = 0.028). For maternal-fetal pairing analysis, the variant concordance of PCSK9 rs2149041, rs7523141, rs7523242, rs7525649, and LDLR rs7258950 were associated with the decreased risk of MSPH under the dominant model. The variant concordance of other SNPs of PCSK9, APOE, LDLR, LPL, and SCARB1 were associated with the increased risk of MSPH. This study supports the hypothesis that maternal and fetal genetic polymorphisms of lipid metabolic genes are associated with the risk of MSPH. The maternal-fetal variant concordance is also associated with this risk.

Do Genetic Variants Modify the Effect of Smoking on Risk of Preeclampsia in Pregnancy?

Maternal smoking is associated with as much as a 50% reduced risk of preeclampsia, despite increasing risk of other poor pregnancy outcomes that often co-occur with preeclampsia, such as preterm birth and fetal growth restriction. Researchers have long sought to understand whether this perplexing association is biologically based, or a result of noncausal mechanisms. We examined whether smoking-response genes modify the smoking-preeclampsia association to investigate potential biological explanations. We conducted a nested case-control study within the Norwegian Mother, Father and Child Birth Cohort (1999-2008) of 2,596 mother-child dyads. We used family-based log-linear Poisson regression to examine modification of the maternal smoking-preeclampsia relationship by maternal and fetal single nucleotide polymorphisms involved in cellular processes related to components of cigarette smoke (n = 1,915 with minor allele frequency ≥10%). We further investigated the influence of smoking cessation during pregnancy. Three polymorphisms showed overall (p < 0.001) multiplicative interaction between smoking and maternal genotype. For rs3765692 (TP73) and rs10770343 (PIK3C2G), protection associated with smoking was reduced with two maternal copies of the risk allele and was stronger in continuers than quitters (interaction p = 0.02 for both loci, based on testing 3-level smoking by 3-level genotype). For rs2278361 (APAF1) the inverse smoking-preeclampsia association was eliminated by the presence of a single risk allele, and again the trend was stronger in continuers than in quitters (interaction p = 0.01). Evidence for gene-smoking interaction was limited, but differences by smoking cessation warrant further investigation. We demonstrate the potential utility of expanded dyad methods and gene-environment interaction analyses for outcomes with complex relationships between maternal and fetal genotypes and exposures. · Maternal and fetal genotype may differentially influence preeclampsia.. · Smoking-related genes did not strongly modify smoking-preeclampsia association.. · Smoking cessation reduced strength of gene by smoking interactions..

Noninvasive prenatal paternity testing by means of SNP-based targeted sequencing.

ObjectiveTo develop a method for noninvasive prenatal paternity testing based on targeted sequencing of single nucleotide polymorphisms (SNPs).MethodSNPs were selected based on population genetics data. Target‐SNPs in cell‐free DNA extracted from maternal blood (maternal cfDNA) were analyzed by targeted sequencing wherein target enrichment was based on multiplex amplification using QIAseq Targeted DNA Panels with Unique Molecular Identifiers. Fetal SNP genotypes were called using a novel bioinformatics algorithm, and the combined paternity indices (CPIs) and resultant paternity probabilities were calculated.ResultsFetal SNP genotypes obtained from targeted sequencing of maternal cfDNA were 100% concordant with those from amniotic fluid‐derived fetal genomic DNA. From an initial panel of 356 target‐SNPs, an average of 148 were included in paternity calculations in 15 family trio cases, generating paternity probabilities of greater than 99.9999%. All paternity results were confirmed by short‐tandem‐repeat analysis. The high specificity of the methodology was validated by successful paternity discrimination between biological fathers and their siblings and by large separations between the CPIs calculated for the biological fathers and those for 60 unrelated men.ConclusionThe novel method is highly effective, with substantial improvements over similar approaches in terms of reduced number of target‐SNPs, increased accuracy, and reduced costs.

Development and comprehensive evaluation of a noninvasive prenatal paternity testing method through a scaled trial

PurposeTo eliminate the miscarriage risks caused by traditional invasive sampling methods, we develop a noninvasive prenatal paternity testing (NIPPT) method and evaluate its efficiency, reliability and sensitivity based on a scaled trial. MethodsWe use maternal cell-free DNA and massive parallel sequencing to obtain NIPPT genotypes for parents and fetuses based on quality-controlled genome-wide single nucleotide polymorphisms (SNPs). In a preliminary testing, data from 14 pregnant women and 7 negative controls are used for setting threshold of fetal genotyping in reference to postpartum children. After that, those from 349 cases with pregnancies of 6–35 gestational weeks (GW) and 9 negative controls from non-pregnant women who have fertility experience previously are in-depth evaluated. ResultsIn all cases, the biological fathers have been successfully identified from unrelated with a combined paternity index (CPI) of 3.58 × 1018 – 1.46 × 10165 for the cases versus 1.52 × 10−22 – 2.30 × 10-839 for the controls. For negative controls, fetal SNPs originating from previous pregnancies could not be detected. Our NIPPT results completely aligned with the invasive prenatal test results using PCR-CE STR methods. ConclusionNIPPT can be applied to determine paternity accurately from 6 weeks after conception until birth and may serve as an alternative prenatal paternity test advantageous to the currently-used methods.

Fetal and Maternal Genetic Variants Influencing Neonatal Vitamin D Status.

Several genetic polymorphisms determine vitamin D status. We aimed to estimate the strength of association of established 25-hydroyxvitamin D (25OHD)-associated variants in the mother and in the fetus, with 25OHD concentration in newborn umbilical cord plasma. We randomly selected 578 mother and child dyads from the prospective Norwegian Mother and Child Cohort study. 25OHD was assayed in maternal samples taken shortly after delivery and in cord samples. We genotyped the mother and child for single nucleotide polymorphisms in or near GC, DHCR7, CYP2R1, and CYP24A1, previously confirmed to be associated with 25OHD, and computed genetic risk score (GRS). The genetic associations were replicated in an independent sample of 594 subjects. Although both fetal and maternal GRS were associated with cord 25OHD, only fetal GRS remained significantly associated with cord 25OHD in a regression model with maternal and fetal GRS simultaneously (1.6 nmol/L per fetal 25OHD-increasing allele; 95% confidence interval, 0.6 to 2.5, P = 0.0001). Two fetal single nucleotide polymorphisms in the GC gene (rs2282679 and rs222040) were the strongest genetic predictors of cord 25OHD [4.0 (2.1 to 5.9) and 3.0 (1.3 to 4.8) nmol/L per fetal allele, P < 0.001], followed by rs12785878 in DHCR7 [2.0 (0.1 to 3.8) nmol/L, P = 0.037]. The independent replication sample gave similar results. With fetal genotype included in a regression model with environmental factors, R2 for cord 25OHD was 0.28. We show that fetal 25OHD-modifying genotype was a stronger predictor of cord 25OHD than corresponding maternal genotype. This raises interesting questions about fetal acquisition and placental transfer of 25OHD.

A Population-Based Study of Effects of Genetic Loci on Orofacial Clefts

Prior genome-wide association studies for oral clefts have focused on clinic-based samples with unclear generalizability. Prior samples were also small for investigating effects by cleft type and exclusively studied isolated clefts (those occurring without other birth defects). We estimated the effects of 17 top loci on cleft types in both isolated and nonisolated cases in the largest consortium to date of European-descent population-based studies. Our analytic approach focused on a mother-child dyad case-control design, but it also allowed analyzing mother-only or child-only genotypes to maximize power. Our total sample included 1,875 cases with isolated clefts, 459 cases with nonisolated clefts, and 3,749 controls. After correcting for multiple testing, we observed significant associations between fetal single-nucleotide polymorphisms (SNPs) at IRF6, PAX7, 8q21.3, 8q24, KIAA1598-VAX1, and MAFB and isolated cleft lip only (CLO) and cleft lip and palate (CLP). Significant associations were observed between isolated CLO and fetal SNPs near TPM1 and NOG1 and between CLP and fetal SNPs at ABCA4-ARHGAP29, THADA, FOXE1, and SPRY2. Overall, effects were similar for isolated CLO and CLP, except for ABCA4-ARHGAP29. A protective effect was observed for the fetal NOG1 SNP on cleft palate only, opposite in direction to the effect on CLO. For most fetal SNPs, a dose-response allelic effect was observed. No evidence of parent-of-origin or maternal genome effects was observed. Overall, effect direction and magnitude were similar between isolated and nonisolated clefts, suggesting that several loci are modifiers of cleft risk in both isolated and nonisolated forms. Our results provide reliable estimates of the effects of top loci on risks of oral clefts in a population of European descent.

Maternal obesity and tobacco use modify the impact of genetic variants on the occurrence of conotruncal heart defects.

Conotruncal heart defects (CTDs) are among the most severe birth defects worldwide. Studies of CTDs indicate both lifestyle behaviors and genetic variation contribute to the risk of CTDs. Based on a hybrid design using data from 616 case-parental and 1645 control-parental triads recruited for the National Birth Defects Prevention Study between 1997 and 2008, we investigated whether the occurrence of CTDs is associated with interactions between 921 maternal and/or fetal single nucleotide polymorphisms (SNPs) and maternal obesity and tobacco use. The maternal genotypes of the variants in the glutamate-cysteine ligase, catalytic subunit (GCLC) gene and the fetal genotypes of the variants in the glutathione S-transferase alpha 3 (GSTA3) gene were associated with an elevated risk of CTDs among obese mothers. The risk of delivering infants with CTDs among obese mothers carrying AC genotype for a variant in the GCLC gene (rs6458939) was 2.00 times the risk among those carrying CC genotype (95% confidence interval: 1.41, 2.38). The maternal genotypes of several variants in the glutathione-S-transferase (GST) family of genes and the fetal genotypes of the variants in the GCLC gene interacted with tobacco exposures to increase the risk of CTDs. Our study suggests that the genetic basis underlying susceptibility of the developing heart to the adverse effects of maternal obesity and tobacco use involve both maternal and embryonic genetic variants. These results may provide insights into the underlying pathophysiology of CTDs, and ultimately lead to novel prevention strategies.

Epigenome-wide DNA methylation assay reveals placental epigenetic markers for noninvasive fetal single-nucleotide polymorphism genotyping in maternal plasma.

The use of DNA methylation difference between maternal blood cell and fetal (placental) DNA is one of the main areas of interest for the development of fetal epigenetics markers in maternal plasma. We employed a methylation array (HumanMethylation450 array, Illumina, Inc.) to identify novel biomarkers that are specially hypermethylated in placental DNA versus maternal blood cells in a genome-wide basis. Validation by bisulfite genomic sequencing was performed and the priority was given to potential targets that harbor differential methylated CpG sites overlapped with at least two methylation-sensitive restriction enzyme (MSRE) recognizing sites, as well as one polymorphic single-nucleotide polymorphism (SNP), within a short DNA stretch. Three candidate regions of PSMB8, SKI, and CHST11 gene were selected for developing a preliminary polymerase chain reaction assay with MSRE digestion of maternal plasma DNA. SNP genotypes were confirmed by direct sequencing. We identified 2944 and 5218 fetal-specific hypermethylated CpG sites in the first- and third-trimester placenta, respectively, of which 2613 were overlapped, suggesting a consistency of differential methylation during the whole pregnancy. The array results were confirmed by bisulfite genomic sequencing. The preliminary tests in maternal plasma showed that postdigestion hypermathylated versions of these candidate molecules were detectable only in pregnant women. We further revealed that methylated targets in maternal plasma possessed the fetal SNP genotypes. The present studies systematically identified hypermethylated sites in fetal tissues and preliminarily demonstrated that some of the fetal epigenetic markers that contain informative SNPs have great potential for noninvasive fetal genetic diagnosis.

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

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

Single-nucleotide polymorphism associations with preterm delivery: a case–control replication study and meta-analysis

The aim of this study was to replicate single-nucleotide polymorphism (SNP) associations with preterm birth (PTB; birth at <37 completed weeks of gestation) and synthesize currently available evidence using meta-analysis. Spontaneous PTB cases and controls were selected from an existing cohort. Candidate SNPs were taken from an existing genotype panel. A systematic review was conducted for each SNP in the panel to determine suitability as a PTB candidate. Those with significant associations previously reported in Caucasians were selected for replication. Candidate SNPs were already genotyped in cases and controls and clinical data were accessed from state perinatal and cerebral palsy databases. Association analysis was conducted between each SNP and PTB, and meta-analysis was conducted if there were ≥ 3 studies in the literature. Maternal and fetal SNPs were considered as separate candidates. A cohort of 170 cases and 583 controls was formed. Eight SNPs from the original panel of genotyped SNPs were selected as PTB candidates and for replication on the basis of systematic literature review results. In our cohort, fetal factor V Leiden (FVL) was significantly associated with PTB (odds ratio (OR): 2.6, 95% confidence interval (CI): 1.31-5.17), and meta-analysis confirmed this association (OR: 2.71, 95% CI: 1.15-6.4). Replication and meta-analysis support an increased risk of PTB in Caucasians with the fetal FVL mutation.

X-Chromosomal Maternal and Fetal SNPs and the Risk of Spontaneous Preterm Delivery in a Danish/Norwegian Genome-Wide Association Study

BackgroundRecent epidemiological studies suggest that the maternal genome is an important contributor to spontaneous preterm delivery (PTD). There is also a significant excess of males among preterm born infants, which may imply an X-linked mode of inheritance for a subset of cases. To explore this, we examined the effect of maternal and fetal X-chromosomal single nucleotide polymorphisms (SNPs) on the risk of PTD in two independent genome-wide association studies and one replication study.MethodsParticipants were recruited from the Danish National Birth Cohort and the Norwegian Mother and Child cohort studies. Data from these two populations were first analyzed independently, and then combined in a meta-analysis. Overall, we evaluated 12,211 SNPs in 1,535 case-mother dyads and 1,487 control-mother dyads. Analyses were done using a hybrid design that combines case-mother dyads and control-mother dyads, as implemented in the Haplin statistical software package. A sex-stratified analysis was performed for the fetal SNPs. In the replication study, 10 maternal and 16 fetal SNPs were analyzed using case-parent triads from independent studies of PTD in the United States, Argentina and Denmark.ResultsIn the meta-analysis, the G allele at the maternal SNP rs2747022 in the FERM domain containing 7 gene (FRMD7) increased the risk of spontaneous PTD by 1.2 (95% confidence interval (CI): 1.1, 1.4). Although an association with this SNP was confirmed in the replication study, it was no longer statistically significant after a Bonferroni correction for multiple testing.ConclusionWe did not find strong evidence in our data to implicate X-chromosomal SNPs in the etiology of spontaneous PTD. Although non-significant after correction for multiple testing, the mother’s G allele at rs2747022 in FRMD7 increased the risk of spontaneous PTD across all populations in this study, thus warranting further investigation in other populations.

The impact of glucocorticoid polymorphisms on markers of neonatal respiratory disease after antenatal betamethasone administration

We previously demonstrated that maternal and fetal genotypes are associated independently with neonatal respiratory distress syndrome. The objective of the current study was to determine the impact of maternal and fetal single-nucleotide polymorphisms (SNPs) in key betamethasone pathways on respiratory outcomes that serve as markers for severity of disease. DNA was obtained from women who were given betamethasone and from their infants. Samples were genotyped for 73 exploratory drug metabolism and glucocorticoid pathway SNPs. Clinical variables and neonatal outcomes were obtained. Logistic regression analysis that controlled for relevant clinical variables to determine SNP impact on bronchopulmonary dysplasia (BPD), the need for respiratory support, and surfactant therapy use was performed. Data from 109 women who delivered 117 infants were analyzed: 14.5% of the infants experienced BPD; 70.8% of the infants needed some respiratory support after birth, and 27.5% of the infants needed surfactant therapy. In a multivariable regression analysis, gestational age at delivery was associated with most neonatal respiratory outcomes (P ≤ .01), and chorioamnionitis was associated with BPD (P < .03). The following genotypes were associated with respiratory severity outcomes: BPD-fetal Importin 13 gene (IPO13; rs4448553; odds ratio [OR], 0.01; 95% confidence interval [CI], 0.00-0.92); surfactant use-maternal IPO13 (rs2428953 and 2486014; OR, 13.8; 95% CI, 1.80-105.5; and OR, 35.5; 95% CI, 1.71-736.6, respectively). Several discrete maternal and fetal SNPs in the IPO13 family may be associated with neonatal respiratory outcomes after maternal antenatal corticosteroid treatment for anticipated preterm birth.

Evaluation of single‐nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups

Determination of fetal blood groups in maternal plasma samples critically depends on adequate amplification of fetal DNA. We evaluated the routine inclusion of 52 single-nucleotide polymorphisms (SNPs) as internal reference in our polymerase chain reaction (PCR) settings to obtain a positive internal control for fetal DNA. DNA from 223 plasma samples of pregnant women was screened for RHD Exons 3, 4, 5, and 7 in a multiplex PCR including 52 SNPs divided into four primer pools. Amplicons were analyzed by single-base extension and the GeneScan method in a genetic analyzer. Results of D screening were compared to standard RHD genotyping of amniotic fluid or real-time PCR of fetal DNA from maternal plasma. The vast majority of all samples (97.8%) demonstrated differences in maternal and fetal SNP patterns when tested with four primer pools. These differences were not observed in less than 2.2% of the samples most probably due to an extraction failure for adequate amounts of fetal DNA. Comparison of the fetal genotypes with independent results did not reveal a single false-negative case among samples (n = 42) with positive internal control and negative fetal RHD typing. Coamplification of 52 SNPs with RHD-specific sequences for fetal blood group determination introduces a valid positive control for the amplification of fetal DNA to avoid false-negative results. This new approach does not require a paternal blood sample. It may also be applicable to other assays for fetal genotyping in maternal blood samples.

The impact of drug metabolizing enzyme polymorphisms on outcomes after antenatal corticosteroid use

To determine the impact of maternal and fetal single nucleotide polymorphisms in key betamethasone pathways on neonatal outcomes. DNA was obtained from women given betamethasone and their infants. Samples were genotyped for 73 exploratory drug metabolism and glucocorticoid pathway single nucleotide polymorphisms. Clinical variables and neonatal outcomes were obtained. Logistic regression analysis using relevant clinical variables and genotypes to model for associations with neonatal respiratory distress syndrome was performed. One hundred nine women delivering 117 infants were analyzed. Sixty-four infants (49%) developed respiratory distress syndrome. Multivariable analysis revealed that respiratory distress syndrome was associated with maternal single nucleotide polymorphisms in CYP3A5 (odds ratio [OR], 1.63; 95% confidence interval [CI], 1.16-2.30) and the glucocorticoid resistance (OR, 0.28; 95% CI, 0.08-0.95) and fetal single nucleotide polymorphisms in ADCY9 (OR, 0.17; 95% CI, 0.03-0.80) and CYP3A7*1E (rs28451617; OR, 23.68; 95% CI, 1.33-420.6). Maternal and fetal genotypes are independently associated with neonatal respiratory distress syndrome after treatment with betamethasone for preterm labor.

Fetal and Maternal Candidate Single Nucleotide Polymorphism Associations With Cerebral Palsy: A Case-Control Study

Previous studies have suggested associations between certain genetic variants and susceptibility to cerebral palsy (CP). This study was designed to assess established and novel maternal and child genetic and epidemiologic risk factors for CP along with their interactions. DNA from 587 case and 1154 control mother-child pairs was analyzed. A panel of 35 candidate single nucleotide polymorphisms (SNPs) were examined and included SNPs in genes associated with (1) thrombophilia, (2) inflammation, and (3) risk factors for CP (eg, preterm birth). Comparisons were specified a priori and made by using a χ(2) test. There were 40 fetal and 28 maternal associations with CP when analyzed by CP subtype, gestational age, genotypes of apolipoprotein E, and haplotypes of mannose-binding-lectin. After Bonferroni correction for multiple testing, no fetal or maternal candidate SNP was associated with CP or its subtypes. Only fetal carriage of prothrombin gene mutation remained marginally associated with hemiplegia in term infants born to mothers with a reported infection during pregnancy. Odds ratio directions of fetal SNP associations were compared with previously reported studies and confirmed no trend toward association. Except for the prothrombin gene mutation, individual maternal and fetal SNPs in our candidate panel were not found to be associated with CP outcome. Past reported SNP associations with CP were not confirmed, possibly reflecting type I error from small numbers and multiple testing in the original reports.

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

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

Epigenetic-Genetic Chromosome Dosage Approach for Fetal Trisomy 21 Detection Using an Autosomal Genetic Reference Marker

BackgroundThe putative promoter of the holocarboxylase synthetase (HLCS) gene on chromosome 21 is hypermethylated in placental tissues and could be detected as a fetal-specific DNA marker in maternal plasma. Detection of fetal trisomy 21 (T21) has been demonstrated by an epigenetic-genetic chromosome dosage approach where the amount of hypermethylated HLCS in maternal plasma is normalized using a fetal genetic marker on the Y chromosome as a chromosome dosage reference marker. We explore if this method can be applied on both male and female fetuses with the use of a paternally-inherited fetal single nucleotide polymorphism (SNP) allele on a reference chromosome for chromosome dosage normalization.MethodologyWe quantified hypermethylated HLCS molecules using methylation-sensitive restriction endonuclease digestion followed by real-time or digital PCR analyses. For chromosome dosage analysis, we compared the amount of digestion-resistant HLCS to that of a SNP allele (rs6636, a C/G SNP) that the fetus has inherited from the father but absent in the pregnant mother.Principal FindingsUsing a fetal-specific SNP allele on a reference chromosome, we analyzed 20 euploid and nine T21 placental tissue samples. All samples with the fetal-specific C allele were correctly classified. One sample from each of the euploid and T21 groups were misclassified when the fetal-specific G allele was used as the reference marker. We then analyzed 33 euploid and 14 T21 maternal plasma samples. All but one sample from each of the euploid and T21 groups were correctly classified using the fetal-specific C allele, while correct classification was achieved for all samples using the fetal-specific G allele as the reference marker.ConclusionsAs a proof-of-concept study, we have demonstrated that the epigenetic-genetic chromosome dosage approach can be applied to the prenatal diagnosis of trisomy 21 for both male and female fetuses.

M9.3 Association of maternal, paternal and fetal single nucleotide polymorphisms in vascular endothelial growth factor family genes with preeclampsia

To assess the ability of first trimester maternal serum (FTMS) soluble Fms-like tyrosine kinase (sFlt-1)/placental growth factor (PlGF) ratio in the first trimester threatened miscarriage (FTTM) to predict fetal loss in early pregnancy and adverse pregnancy outcomes when pregnancy continued to ⩾30 weeks to start suitable antenatal care as early as possible.A case control prospective cross-sectional study.320 cases of TM, 7–12 weeks’ gestation and 320 normal controls 7–12 weeks’ gestation without vaginal bleeding and other manifestations of FTTM and delivered at ⩾30 weeks’ gestation were enrolled in the study. All were primigravidae. Estimation of FTMS sFlt-1 and PlGF in the first trimester of TM and controls by enzyme-like immunosorbent assay (ELISA) and follow up of them till delivery were done. Adverse pregnancy outcomes encountered were gestational hypertension (GH), preeclampsia (PE), intrauterine growth restriction (IUGR), abruptio placentae, spontaneous preterm labor (PTL), placenta previa, preterm premature rupture of membranes (PPROM) and retained placenta.All adverse pregnancy outcomes were significantly (P < 0.05) higher in FTTM than in controls (44.52% vs. 17.5%). In FTTM MS concentrations of sFlt-1 and PlGF and sFlt-1/PlGF ratio were markedly reduced if fetal loss was encountered. sFlt-1/PlGF ratio <51.71 (cut-off point) predicted fetal loss with sensitivity 90.32%, specificity 98.47%, positive predicted value 93.33% and negative predictive value 97.60%. Patients that developed adverse pregnancy outcomes related to impaired placentation as GH, PE, IUGR, PTL <34 weeks’ gestation and abruptio placentae had reduced FTMS PlGF and increased sFlt-1/PlGF ratio, in both FTTM and controls. Soluble Flt-1/PlGF ratio >74.6 (cut-off point) predicted these adverse pregnancy outcomes with sensitivity 88.70%, specificity 95.40, positive predictive value 90.16% and negative predictive value 94.68% – adverse pregnancy outcomes not related to placental impairment as PLT >34 weeks’ gestation, PPROM, placenta previa and retained placenta were not predicted by sFlt-1/PlGF ratio because in these conditions FTMS concentration of these factors were normal.1-Adverse outcomes late in pregnancy (⩾30 weeks’ gestation) were more common in FTTM. 2-Diminished FTMS concentration of sFlt-1 and PlGF as well as sFlt-1/PlGF ratio <51.71 may predict fetal loss in FTTM. 3-In FTTM and normal controls FTMS PlGF was reduced and sFlt-1/PlGF ratio >74.6 may predict development of adverse outcomes characterized by impaired placentation. 4-Adverse pregnancy outcomes not related to impaired placentation were associated with normal FTMS concentration of sFlt-1 and PlGF and were not predicted by sFlt-1/PlGF ratio.

Association of fetal inflammation and coagulation pathway gene polymorphisms with neurodevelopmental delay at age 2 years

The purpose of this study was to evaluate the association between fetal inflammation and coagulation gene single-nucleotide polymorphisms (SNPs) and neurodevelopmental delay at age 2 years. We conducted a case-controlled secondary analysis of a randomized trial of single- vs multiple-course corticosteroids. Multiplex assay assessed 46 SNPs. Cases had mental developmental and/or psychomotor delay at age 2 years. Control subjects had normal neurodevelopment. One hundred twenty-five cases and 147 control subjects were analyzed. Allele frequencies were different between cases and control subjects for interleukin (IL)1beta-511 (P = .009), IL4R-148 (P = .03), IL6-174 (P = .02), and IL6-176 (P = .007). Genotype frequencies were different for IL1beta-511 (P = .03) and IL6-174 (P = .04). Results for IL1beta-511, IL4R-148, and IL6-176 remained significant after logistic regression analysis. IL1beta-511 and IL6-176 minor alleles were associated with increased risk of neurodevelopmental delay (odds ratio, 3.1; 95% confidence interval [CI], 1.2-8.2 and 2.2; 95% CI, 1.2-3.9, respectively). IL4R-148 minor allele was protective (odds ratio, 0.6; 95% CI, 0.4-0.9). Fetal SNPs in IL1beta, IL-4R, and IL-6 may be associated with neurodevelopmental delay at age 2 years.