Epigenetic-genetic chromosome dosage approach for fetal trisomy 21 detection using an autosomal genetic reference marker

Background

The 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.

Methodology

We 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 Findings

Using 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.

Conclusions

As 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.     

High Resolution Size Analysis of Fetal DNA in the Urine of Pregnant Women by Paired-End Massively Parallel Sequencing

Background

Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine.

Methodology

We have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine.

Principal Findings

Cell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length.

Conclusions

With the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded.     

Systematic identification of placental epigenetic signatures for the noninvasive prenatal detection of Edwards syndrome

Background

Noninvasive prenatal diagnosis of fetal aneuploidy by maternal plasma analysis is challenging owing to the low fractional and absolute concentrations of fetal DNA in maternal plasma. Previously, we demonstrated for the first time that fetal DNA in maternal plasma could be specifically targeted by epigenetic (DNA methylation) signatures in the placenta. By comparing one such methylated fetal epigenetic marker located on chromosome 21 with another fetal genetic marker located on a reference chromosome in maternal plasma, we could infer the relative dosage of fetal chromosome 21 and noninvasively detect fetal trisomy 21. Here we apply this epigenetic-genetic (EGG) chromosome dosage approach to detect Edwards syndrome (trisomy 18) in the fetus noninvasively.

Principal Findings

We have systematically identified methylated fetal epigenetic markers on chromosome 18 by methylated DNA immunoprecipitation (MeDIP) and tiling array analysis with confirmation using quantitative DNA methylation assays. Methylated DNA sequences from an intergenic region between the VAPA and APCDD1 genes (the VAPA-APCDD1 DNA) were detected in pre-delivery, but not post-delivery, maternal plasma samples. The concentrations correlated positively with those of an established fetal genetic marker, ZFY, in pre-delivery maternal plasma. The ratios of methylated VAPA-APCDD1(chr18) to ZFY(chrY) were higher in maternal plasma samples of 9 male trisomy 18 fetuses than those of 27 male euploid fetuses (Mann-Whitney test, P = 0.029). We defined the cutoff value for detecting trisomy 18 fetuses as mean+1.96 SD of the EGG ratios of the euploid cases. Eight of 9 trisomy 18 and 1 of 27 euploid cases showed EGG ratios higher than the cutoff value, giving a sensitivity of 88.9% and a specificity of 96.3%.

Conclusions

Our data have shown that the methylated VAPA-APCDD1 DNA in maternal plasma is predominantly derived from the fetus. We have demonstrated that this novel fetal epigenetic marker in maternal plasma is useful for the noninvasive detection of fetal trisomy 18.     

Non-Invasive Prenatal Detection of Trisomy 13 Using a Single Nucleotide Polymorphism- and Informatics-Based Approach

Purpose

To determine how a single nucleotide polymorphism (SNP)- and informatics-based non-invasive prenatal aneuploidy test performs in detecting trisomy 13.

Methods

Seventeen trisomy 13 and 51 age-matched euploid samples, randomly selected from a larger cohort, were analyzed. Cell-free DNA was isolated from maternal plasma, amplified in a single multiplex polymerase chain reaction assay that interrogated 19,488 SNPs covering chromosomes 13, 18, 21, X, and Y, and sequenced. Analysis and copy number identification involved a Bayesian-based maximum likelihood statistical method that generated chromosome- and sample-specific calculated accuracies.

Results

Of the samples that passed a stringent DNA quality threshold (94.1%), the algorithm correctly identified 15/15 trisomy 13 and 49/49 euploid samples, for 320/320 correct copy number calls.

Conclusions

This informatics- and SNP-based method accurately detects trisomy 13-affected fetuses non-invasively and with high calculated accuracy.     

Association of MDR1 gene SNPs and haplotypes with the tacrolimus dose requirements in Han Chinese liver transplant recipients

Background

This work seeks to evaluate the association between the C/D ratios (plasma concentration of tacrolimus divided by daily dose of tacrolimus per body weight) of tacrolimus and the haplotypes of MDR1 gene combined by C1236T (rs1128503), G2677A/T (rs2032582) and C3435T (rs1045642), and to further determine the functional significance of haplotypes in the clinical pharmacokinetics of oral tacrolimus in Han Chinese liver transplant recipients.

Methodology/Principal Findings

The tacrolimus blood concentrations were continuously recorded for one month after initial administration, and the peripheral blood DNA from a total of 62 liver transplant recipients was extracted. Genotyping of C1236T, G2677A/T and C3435T was performed, and SNP frequency, Hardy-Weinberg equilibrium, linkage disequilibrium, haplotypes analysis and multiple testing were achieved by software PLINK. C/D ratios of different SNP groups or haplotype groups were compared, with a p value<0.05 considered statistically significant. Linkage studies revealed that C1236T, G2677A/T and C3435T are genetically associated with each other. Patients carrying T-T haplotype combined by C1236T and G2677A/T, and an additional T/T homozygote at either position would require higher dose of tacrolimus. Tacrolimus C/D ratios of liver transplant recipients varied significantly among different haplotype groups of MDR1 gene.

Conclusions

Our studies suggest that the genetic polymorphism could be used as a valuable molecular marker for the prediction of tacrolimus C/D ratios of liver transplant recipients.     

Evaluation of a Novel Assay for Detection of the Fetal Marker RASSF1A: Facilitating Improved Diagnostic Reliability of Noninvasive Prenatal Diagnosis

Background

Analysis of cell free fetal (cff) DNA in maternal plasma is used routinely for non invasive prenatal diagnosis (NIPD) of fetal sex determination, fetal rhesus D status and some single gene disorders. True positive results rely on detection of the fetal target being analysed. No amplification of the target may be interpreted either as a true negative result or a false negative result due to the absence or very low levels of cffDNA. The hypermethylated RASSF1A promoter has been reported as a universal fetal marker to confirm the presence of cffDNA. Using methylation-sensitive restriction enzymes hypomethylated maternal sequences are digested leaving hypermethylated fetal sequences detectable. Complete digestion of maternal sequences is required to eliminate false positive results.

Methods

cfDNA was extracted from maternal plasma (n = 90) and digested with methylation-sensitive and insensitive restriction enzymes. Analysis of RASSF1A, SRY and DYS14 was performed by real-time PCR.

Results

Hypermethylated RASSF1A was amplified for 79 samples (88%) indicating the presence of cffDNA. SRY real time PCR results and fetal sex at delivery were 100% accurate. Eleven samples (12%) had no detectable hypermethylated RASSF1A and 10 of these (91%) had gestational ages less than 7 weeks 2 days. Six of these samples were male at delivery, five had inconclusive results for SRY analysis and one sample had no amplifiable SRY.

Conclusion

Use of this assay for the detection of hypermethylated RASSF1A as a universal fetal marker has the potential to improve the diagnostic reliability of NIPD for fetal sex determination and single gene disorders.     

Mrassf1a-Pap,a Novel Methylation-Based Assay for the Detection of Cell-Free Fetal DNA in Maternal Plasma

Objectives

RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma.

Methods

Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated.

Results

Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ∼3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%–100%).

Conclusions

Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.     

Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield

Objective

Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used.

Methods

Microfluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples.

Results

Total cell-free DNA quantity increased significantly with time in samples stored in K₃EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4°C did not prevent these changes.

Conclusion

When samples can be processed within eight hours of blood draw, K₃EDTA tubes can be used. Prolonged transfer times in K₃EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.     

Genetic variants in MARCO are associated with the susceptibility to pulmonary tuberculosis in Chinese Han population

Background

Susceptibility to tuberculosis is not only determined by Mycobacterium tuberculosis infection, but also by the genetic component of the host. Macrophage receptor with a collagenous structure (MARCO) is essential components required for toll like receptor-signaling in macrophage response to Mycobacterium tuberculosis, which may contribute to tuberculosis risk.

Principal Findings

To specifically investigated whether single nucleotide polymorphisms (SNPs) in MARCO gene are associated with pulmonary tuberculosis in Chinese Han population. By selecting tagging SNPs in MARCO gene, 17 tag SNPs were identified and genotyped in 923 pulmonary tuberculosis patients and 1033 healthy control subjects using a hospital based case-control association study. Single-point and haplotype analysis revealed an association in intron and exon region of MARCO gene. One SNP (rs17009726) was associated with susceptibility to pulmonary tuberculosis, where the carriers of the G allele had a 1.65 fold (95% CI = 1.32–2.05, p _corrected = 9.27E–5) increased risk of pulmonary tuberculosis. Haplotype analysis revealed that haplotype GC containing G allele of 17009726 and haplotype TGCC (rs17795618T/A, rs1371562G/T, rs6761637T/C, rs2011839C/T) were also associated with susceptibility to pulmonary tuberculosis (p _corrected = 0.0001 and 0.029, respectively).

Conclusions

Our study suggested that genetic variants in MARCO gene were associated with pulmonary tuberculosis susceptibility in Chinese Han population, and the findings emphasize the importance of MARCO mediated immune responses in the pathogenesis of tuberculosis.     

Selection of haplotype variables from a high-density marker map for genomic prediction

Background

Using haplotype blocks as predictors rather than individual single nucleotide polymorphisms (SNPs) may improve genomic predictions, since haplotypes are in stronger linkage disequilibrium with the quantitative trait loci than are individual SNPs. It has also been hypothesized that an appropriate selection of a subset of haplotype blocks can result in similar or better predictive ability than when using the whole set of haplotype blocks. This study investigated genomic prediction using a set of haplotype blocks that contained the SNPs with large effects estimated from an individual SNP prediction model. We analyzed protein yield, fertility and mastitis of Nordic Holstein cattle, and used high-density markers (about 770k SNPs). To reach an optimum number of haplotype variables for genomic prediction, predictions were performed using subsets of haplotype blocks that contained a range of 1000 to 50 000 main SNPs.

Results

The use of haplotype blocks improved the prediction reliabilities, even when selection focused on only a group of haplotype blocks. In this case, the use of haplotype blocks that contained the 20 000 to 50 000 SNPs with the highest effect was sufficient to outperform the model that used all individual SNPs as predictors (up to 1.3 % improvement in prediction reliability for mastitis, compared to individual SNP approach), and the achieved reliabilities were similar to those using all haplotype blocks available in the genome data (from 0.6 % lower to 0.8 % higher reliability).

Conclusions

Haplotype blocks used as predictors can improve the reliability of genomic prediction compared to the individual SNP model. Furthermore, the use of a subset of haplotype blocks that contains the main SNP effects from genomic data could be a feasible approach to genomic prediction in dairy cattle, given an increase in density of genotype data available. The predictive ability of the models that use a subset of haplotype blocks was similar to that obtained using either all haplotype blocks or all individual SNPs, with the benefit of having a much lower computational demand.     

Polymorphisms of the Tissue Inhibitor of Metalloproteinase 3 Gene Are Associated with Resistance to High-Altitude Pulmonary Edema (HAPE) in a Japanese Population: A Case Control Study Using Polymorphic Microsatellite Markers

Introduction

High-altitude pulmonary edema (HAPE) is a hypoxia-induced, life-threatening, high permeability type of edema attributable to pulmonary capillary stress failure. Genome-wide association analysis is necessary to better understand how genetics influence the outcome of HAPE.

Materials and Methods

DNA samples were collected from 53 subjects susceptible to HAPE (HAPE-s) and 67 elite Alpinists resistant to HAPE (HAPE-r). The genome scan was carried out using 400 polymorphic microsatellite markers throughout the whole genome in all subjects. In addition, six single nucleotide polymorphisms (SNPs) of the gene encoding the tissue inhibitor of metalloproteinase 3 (TIMP3) were genotyped by Taqman® SNP Genotyping Assays.

Results

The results were analyzed using case-control comparisons. Whole genome scanning revealed that allele frequencies in nine markers were statistically different between HAPE-s and HAPE-r subjects. The SNP genotyping of the TIMP3 gene revealed that the derived allele C of rs130293 was associated with resistance to HAPE [odds ratio (OR) = 0.21, P = 0.0012) and recessive inheritance of the phenotype of HAPE-s (P = 0.0012). A haplotype CAC carrying allele C of rs130293 was associated with resistance to HAPE.

Discussion

This genome-wide association study revealed several novel candidate genes associated with susceptibility or resistance to HAPE in a Japanese population. Among those, the minor allele C of rs130293 (C/T) in the TIMP3 gene was linked to resistance to HAPE; while, the ancestral allele T was associated with susceptibility to HAPE.     

Variation in the Maternal Corticotrophin Releasing Hormone-Binding Protein (CRH-BP) Gene and Birth Weight in Blacks, Hispanics and Whites

Background

Given the unique role of the corticotrophin-releasing hormone (CRH) system in human fetal development, the aim of our study was to estimate the association of birth weight with DNA sequence variation in three maternal genes involved in regulating CRH production, bioavailability and action: CRH, CRH-Binding Protein (CRH-BP), and CRH type 1 receptor (CRH-R1), respectively, in three racial groups (African-Americans, Hispanics, and non-Hispanic Whites).

Methods

Our study was carried out on a population-based sample of 575 mother–child dyads. We resequenced the three genes in mouse–human hybrid somatic cell lines and selected SNPs for genotyping.

Results

A significant association was observed in each race between birth weight and maternal CRH-BP SNP genotypes. Estimates of linkage disequilibrium and haplotypes established three common haplotypes marked by the rs1053989 SNP in all three races. This SNP predicted significant birth weight variation after adjustment for gestational age, maternal BMI, parity, and smoking. African American and Hispanic mothers carrying the A allele had infants whose birth weight was on average 254 and 302 grams, respectively, less than infants having C/C mothers. Non-Hispanic White mothers homozygous for the A allele had infants who were on average 148 grams less than those infants having A/C and C/C mothers.

Conclusions

The magnitudes of the estimates of the birth weight effects are comparable to the combined effects of multiple SNPs reported in a recent meta-analysis of 6 GWAS studies and is quantitatively larger than that associated with maternal cigarette smoking. This effect was persistent across subpopulations that vary with respect to ancestry and environment.     

A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection

Objective

The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure.

Methods

Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction.

Results

A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B.

Conclusion

A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability.     

Noninvasive Prenatal Detection for Pathogenic CNVs: The Application in α-Thalassemia

Background

The discovery of cell free fetal DNA (cff-DNA) in maternal plasma has brought new insight for noninvasive prenatal diagnosis. Combining with the rapidly developed massively parallel sequencing technology, noninvasive prenatal detection of chromosome aneuploidy and single base variation has been successfully validated. However, few studies discussed the possibility of noninvasive pathogenic CNVs detection.

Methodology/Principal Findings

A novel algorithm for noninvasive prenatal detection of fetal pathogenic CNVs was firstly tested in 5 pairs of parents with heterozygote α-thalassemia of Southeast Asian (SEA) deletion using target region capture sequencing for maternal plasma. Capture probes were designed for α-globin (HBA) and β-globin (HBB) gene, as well as 4,525 SNPs selected from 22 automatic chromosomes. Mixed adaptors with 384 different barcodes were employed to construct maternal plasma DNA library for massively parallel sequencing. The signal of fetal CNVs was calculated using the relative copy ratio (RCR) of maternal plasma combined with the analysis of R-score and L-score by comparing with normal control. With mean of 101.93× maternal plasma sequencing depth for the target region, the RCR value combined with further R-score and L-score analysis showed a possible homozygous deletion in the HBA gene region for one fetus, heterozygous deletion for two fetus and normal for the other two fetus, which was consistent with that of invasive prenatal diagnosis.

Conclusions/Significance

Our study showed the feasibility to detect pathogenic CNVs using target region capture sequencing, which might greatly extend the scope of noninvasive prenatal diagnosis.     

Fetal Gender and Several Cytokines Are Associated with the Number of Fetal Cells in Maternal Blood – An Observational Study

Objective

To identify factors influencing the number of fetal cells in maternal blood.

Methods

A total of 57 pregnant women at a gestational age of weeks 11–14 were included. The number of fetal cells in maternal blood was assessed in 30 ml of blood using specific markers for both enrichment and subsequent identification.

Results

Participants carrying male fetuses had a higher median number of fetal cells in maternal blood than those carrying female fetuses (5 vs. 3, p = 0.04). Certain cytokines (RANTES, IL-2 and IL-5) were significantly associated with the number of fetal cells in maternal blood.

Conclusion

The number of fetal cells in maternal blood is associated with certain cytokines and fetal gender.     

High-Throughput Massively Parallel Sequencing for Fetal Aneuploidy Detection from Maternal Plasma

Background

Circulating cell-free (ccf) fetal DNA comprises 3–20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.

Methods

Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.

Results

Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.

Conclusions

These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.     

Maternal Smoking during Pregnancy and Fetal Organ Growth: A Magnetic Resonance Imaging Study

Objective

To study whether maternal cigarette smoking during pregnancy is associated with alterations in the growth of fetal lungs, kidneys, liver, brain, and placenta.

Design

A case-control study, with operators performing the image analysis blinded.

Setting

Study performed on a research-dedicated magnetic resonance imaging (MRI) scanner (1.5 T) with participants recruited from a large teaching hospital in the United Kingdom.

Participants

A total of 26 pregnant women (13 current smokers, 13 non smokers) were recruited; 18 women (10 current smokers, 8 nonsmokers) returned for the second scan later in their pregnancy.

Methods

Each fetus was scanned with MRI at 22–27 weeks and 33–38 weeks gestational age (GA).

Main outcome measures

Images obtained with MRI were used to measure volumes of the fetal brain, kidneys, lungs, liver and overall fetal size, as well as placental volumes.

Results

Exposed fetuses showed lower brain volumes, kidney volumes, and total fetal volumes, with this effect being greater at visit 2 than at visit 1 for brain and kidney volumes, and greater at visit 1 than at visit 2 for total fetal volume. Exposed fetuses also demonstrated lower lung volume and placental volume, and this effect was similar at both visits. No difference was found between the exposed and nonexposed fetuses with regards to liver volume.

Conclusion

Magnetic resonance imaging has been used to show that maternal smoking is associated with reduced growth of fetal brain, lung and kidney; this effect persists even when the volumes are corrected for maternal education, gestational age, and fetal sex. As expected, the fetuses exposed to maternal smoking are smaller in size. Similarly, placental volumes are smaller in smoking versus nonsmoking pregnant women.     

Paternal Genetic Contribution Influences Fetal Vulnerability to Maternal Alcohol Consumption in a Rat Model of Fetal Alcohol Spectrum Disorder

Background

Fetal alcohol exposure causes in the offspring a collection of permanent physiological and neuropsychological deficits collectively termed Fetal Alcohol Spectrum Disorder (FASD). The timing and amount of exposure cannot fully explain the substantial variability among affected individuals, pointing to genetic influences that mediate fetal vulnerability. However, the aspects of vulnerability that depend on the mother, the father, or both, are not known.

Methodology/Principal Findings

Using the outbred Sprague-Dawley (SD) and inbred Brown Norway (BN) rat strains as well as their reciprocal crosses, we administered ethanol (E), pair-fed (PF), or control (C) diets to the pregnant dams. The dams'' plasma levels of free thyroxine (fT4), triiodothyronine (T3), free T3 (fT3), and thyroid stimulating hormone (TSH) were measured to elucidate potential differences in maternal thyroid hormonal environment, which affects specific aspects of FASD. We then compared alcohol-exposed, pair fed, and control offspring of each fetal strain on gestational day 21 (G21) to identify maternal and paternal genetic effects on bodyweight and placental weight of male and female fetuses.

Conclusions

SD and BN dams exhibited different baseline hypothalamic-pituitary-thyroid function. Moreover, the thyroid function of SD dams was more severely affected by alcohol consumption while that of BN dams was relatively resistant. This novel finding suggests that genetic differences in maternal thyroid function are one source of maternal genetic effects on fetal vulnerability to FASD. The fetal vulnerability to decreased bodyweight after alcohol exposure depended on the genetic contribution of both parents, not only maternal contribution as previously thought. In contrast, the effect of maternal alcohol consumption on placental weight was consistent and not strain-dependent. Interestingly, placental weight in fetuses with different paternal genetic contributions exhibited opposite responses to caloric restriction (pair feeding). In summary, these novel findings demonstrate both maternal and paternal genetic contributions to in utero vulnerability to alcohol, refining our understanding of the genetically-based heterogeneity seen in human FASD.     

GTP Cyclohydrolase I Gene Polymorphisms Are Associated with Endothelial Dysfunction and Oxidative Stress in Patients with Type 2 Diabetes Mellitus

Background

The genetic background of atherosclerosis in type 2 diabetes mellitus (T2DM) is complex and poorly understood. Studying genetic components of intermediate phenotypes, such as endothelial dysfunction and oxidative stress, may aid in identifying novel genetic components for atherosclerosis in diabetic patients.

Methods

Five polymorphisms forming two haplotype blocks within the GTP cyclohydrolase 1 gene, encoding a rate limiting enzyme in tetrahydrobiopterin synthesis, were studied in the context of flow and nitroglycerin mediated dilation (FMD and NMD), intima-media thickness (IMT), and plasma concentrations of von Willebrand factor (vWF) and malondialdehyde (MDA).

Results

Rs841 was associated with FMD (p = 0.01), while polymorphisms Rs10483639, Rs841, Rs3783641 (which form a single haplotype) were associated with both MDA (p = 0.012, p = 0.0015 and p = 0.003, respectively) and vWF concentrations (p = 0.016, p = 0.03 and p = 0.045, respectively). In addition, polymorphism Rs8007267 was also associated with MDA (p = 0.006). Haplotype analysis confirmed the association of both haplotypes with studied variables.

Conclusions

Genetic variation of the GCH1 gene is associated with endothelial dysfunction and oxidative stress in T2DM patients.