Irina Manokhina

Quantification of Cell-Free DNA in Normal and Complicated Pregnancies: Overcoming Biological and Technical Issues

The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal ‘fetal’ markers in maternal plasma, thus overcoming the limitations of gender- or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2–39.5 weeks). Two fetal- or placental- specific duplex assays (RPP30/SRY and RASSF1A/β-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p<0.0001; placental cfDNA (RASSF1A): R = 0.75, p<0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.

Maternal NLRP7 and C6orf221 variants are not a common risk factor for androgenetic moles, triploidy and recurrent miscarriage

Maternal effect genes control early events of embryogenesis. Maternal homozygous and compound mutations in two such genes, NLRP7 and c6orf221, have been detected in the majority of women experiencing recurrent biparental hydatidiform moles. It was suggested that other forms of reproductive wastage, including diploid androgenetic moles, partial moles, polyploidy, recurrent spontaneous abortions and stillbirths of uncertain etiology, may be caused by NLRP7 or c6orf221 mutations in the mother. To elucidate which subpopulations of women with adverse reproductive outcomes should be screened for NLRP7/C6orf221 variants, we sequenced coding sequence and exon/intron boundaries of NLRP7 and C6orf221 in a well-defined group of 17 women with recurrent miscarriage and additional triploidy or complete hydatidiform moles. The major findings for this group were non-synonymous variants of NLRP7, rather than clearly pathogenic mutations. To assess the role of these variants, we genotyped them in a larger group including women with primary recurrent miscarriage (n = 39), paternal triploid conceptions (n = 22) and women with proven fertility after age 37 and no prior history of miscarriage or pregnancy complications (n = 52). No associations between non-synonymous NLRP7 variants and primary recurrent miscarriage or partial hydatidiform molar pregnancies were detected. Our findings suggest that neither mutations nor variants in NLRP7 and C6orf221 are major factors contributing to the risk of these types of pregnancy complications. Further studies in larger groups of patients and controls are needed to specify the impact of NLRP7 rare non-synonymous variants on genetic susceptibility to recurrent reproductive wastage.

Beckwith–Wiedemann syndrome in sibs discordant for IC2 methylation†‡

Genetically heterogeneous imprinting disorders include Beckwith–Wiedemann syndrome (BWS) and multiple maternal hypomethylation syndrome (MMHS). Using DNA sequencing, quantitative PCR, SNuPE, pyrosequencing, and hybridization to the Illumina GoldenGate Methylation Cancer Panel 1 array, we characterized the genomic DNA of two brothers with BWS who were discordant for loss of methylation at several differentially methylated regions (DMR), including imprinting center 2 (IC2) on chromosome band 11p15.5, which is often hypomethylated in BWS. In keeping with MMHS, the elder child had hypomethylation of SGCE and PLAGL1 as well as of IC2, whereas the younger brother demonstrated no loss of methylation at these DMRs. Although this discordance is consistent with the observation that 15–20% of individuals with BWS do not have detectable genetic or epigenetic alterations of 11p15.5, this is the first report of familial recurrence of BWS with discordance for chromosomal 11p15.5 alterations. We hypothesize that this apparent discordance arises either from mosaicism precluding identification of IC2 hypomethylation in blood or buccal mucosa DNA of the younger child, or from hypomethylation at a site not interrogated by our molecular studies.

Noninvasive nucleic acid–based approaches to monitor placental health and predict pregnancy-related complications

During pregnancy, the placenta releases a variety of nucleic acids (including deoxyribonucleic acid, messenger ribonucleic acid, or microribonucleic acids) either as a result of cell turnover or as an active messaging system between the placenta and cells in the maternal body. The profile of released nucleic acids changes with the gestational age and has been associated with maternal and fetal parameters. It also can directly reflect pathological changes in the placenta. Nucleic acids may therefore provide a rich source of novel biomarkers for the prediction of pregnancy complications. However, their utility in the clinical setting depends, first, on overcoming some technical considerations in their quantification, and, second, on developing a better understanding of the factors that influence their function and abundance.

Recurrent triploidy due to a failure to complete maternal meiosis II: whole-exome sequencing reveals candidate variants

Triploidy is a relatively common cause of miscarriage; however, recurrent triploidy has rarely been reported. A healthy 34-year-old woman was ascertained because of 18 consecutive miscarriages with triploidy found in all 5 karyotyped losses. Molecular results in a sixth loss were also consistent with triploidy. Genotyping of markers near the centromere on multiple chromosomes suggested that all six triploid conceptuses occurred as a result of failure to complete meiosis II (MII). The probands mother had also experienced recurrent miscarriage, with a total of 18 miscarriages. Based on the hypothesis that an inherited autosomal-dominant maternal predisposition would explain the phenotype, whole-exome sequencing of the proband and her parents was undertaken to identify potential candidate variants. After filtering for quality and rarity, potentially damaging variants shared between the proband and her mother were identified in 47 genes. Variants in genes coding for proteins implicated in oocyte maturation, oocyte activation or polar body extrusion were then prioritized. Eight of the most promising candidate variants were confirmed by Sanger sequencing. These included a novel change in the PLCD4 gene, and a rare variant in the OSBPL5 gene, which have been implicated in oocyte activation upon fertilization and completion of MII. Several variants in genes coding proteins playing a role in oocyte maturation and early embryonic development were also identified. The genes identified may be candidates for the study in other women experiencing recurrent triploidy or recurrent IVF failure.

Are we ready for DNA methylation-based prenatal testing?

as well as undergo dramatic changes throughout gestation and after birth [4]. A consideration in the application of DNA methylation-based technology to pregnancy screening is that the source of cells for some prenatal samples may be unclear. For example, cells within the AF can de derived from a variety of distinct sources (i.e., lung, skin, blood and kidney) and may only be useful for the study of methylation changes that do not vary amongst different fetal tissues. As the source of DNA for methylation ana lysis in evaluation of pregnancy is largely placental, we focus our further comments on the m ethylation profile and variability in this tissue.

Review: placental biomarkers for assessing fetal health

The placenta is a multifunctional organ that regulates key aspects of pregnancy maintenance and fetal development. As the placenta is in direct contact with maternal blood, cellular products (DNA, RNA, proteins, etc.) from the placenta can enter maternal circulation by a variety of ways. The application of serum proteins and circulating placental derived DNA has been well demonstrated for the diagnosis of aneuploidy, and there is great interest in exploring the use of placental biomarkers for the prediction of a range of fetal health parameters. In this review, we discuss how placental biomarkers might be used for the diagnosis and early detection of preeclampsia, fetal growth restriction and inflammation associated with preterm birth. We emphasize how increased understanding of the underlying placental biology can aid in the interpretation of such approaches and development of new biomarkers that can help predict the onset of pregnancy and neonatal health concerns before they manifest.

Cell-Free Placental DNA in Maternal Plasma in Relation to Placental Health and Function

Background: While cell-free placental DNA (cfp-DNA) increases in response to certain pathological conditions, confounding variables, such as placental size, may also contribute to its release. Furthermore, the relationship between cfp-DNA and maternal serum proteins has not been well investigated. Objective: To analyze plasma cfp-DNA levels and correlate with measurable placental parameters, maternal serum proteins, or pathologic conditions reflecting placental dysfunction. Method: Methylated fraction of RASSF1A was quantified in maternal plasma as a measure of cfp-DNA in a cohort of 86 pregnant women. Results: Placental dimensions or weight had no impact on cfp-DNA levels in noncomplicated pregnancies (n = 63). However, an association between β-hCG and cfp-DNA levels (p = 0.0012) was detected. Complications occurred in 23 pregnancies including chromosomal abnormalities, gestational hypertension, intrauterine growth restriction, and preterm birth. There was overall a skewed distribution (<-1 SD or >1 SD from mean) for cfp-DNA in the abnormal group, although due to the small number of samples for each pathology, we provide only descriptive data to assess possible trends in cfp-DNA variation. Conclusion: While cfp-DNA levels outside of the normal range may reflect placental distress, this relationship may be masked by a number of physiological confounders. The independence of cfp-DNA from β-hCG levels commonly assessed in pregnancy need to be further addressed.

A Preliminary Genome-Wide Association Study of Acute Mountain Sickness Susceptibility in a Group of Nepalese Pilgrims Ascending to 4380 m

There is significant interindividual variation in acute mountain sickness (AMS) susceptibility in humans. To identify genes related to AMS susceptibility, we used a genome-wide association study (GWAS) to simultaneously test associations between genetic variants dispersed throughout the genome and the presence and severity of AMS. DNA samples were collected from subjects who ascended rapidly to Gosainkunda, Nepal (4380 m), as part of the 2005, 2010, and 2012 Janai Purnima festivals. The Lake Louise Score was used to measure AMS severity. The primary analysis was based on 99 male subjects (43 with AMS; 56 without AMS). Genotyping for the GWAS was performed using Infinium Human Core Exome Bead Chips (542,556 single-nucleotide polymorphisms were assayed), and validation genotyping was performed with pyrosequencing in two additional cohorts (n = 101 for each). In total, 270,389 single nucleotide polymorphisms (SNPs) passed quality control, and 4 SNPs (one intronic, three nonsynonymous) in the FAM149A gene were associated with AMS severity after correcting for multiple hypothesis testing (p = 1.8E-7); however, in the validation cohorts, FAM149A was not associated with the presence or severity of AMS. No other genes were associated with AMS susceptibility at the genome-wide level. Due to the large influence of environmental factors (i.e., ascent rate and altitude attained) and the difficulties associated with the AMS phenotype (i.e., low repeatability, nonspecific symptoms, potentially independent ailments), we suggest that future studies addressing the variation in the acute human hypoxia response should focus on objective responses to acute hypoxia instead of AMS.

Contents Vol. 41, 2017

R. Achiron, Tel Hashomer N.S. Adzick, Philadelphia, PA L. Allan, London A.A. Baschat, Baltimore, MD K.J. Blakemore, Baltimore, MD T.-H. Bui, Stockholm F.A. Chervenak, New York, NY T. Chiba, Tokyo R. Chmait, Los Angeles, CA F. Crispi, Barcelona J.E. De Lia, Milwaukee, WI J.A. Deprest, Leuven G.C. Di Renzo, Perugia J.W. Dudenhausen, Berlin N.M. Fisk, Brisbane, QLD A.W. Flake, Philadelphia, PA U. Gembruch, Bonn M.R. Harrison, San Francisco, CA J.C. Hobbins, Denver, CO L.K. Hornberger, Edmonton, AB E.R.M. Jauniaux, London M.P. Johnson, Philadelphia, PA J.-M. Jouannic, Paris P.M. Kyle, London O. Lapaire, Basel S. Lipitz, Tel Hashomer E. Llurba, Barcelona G. Malinger, Tel Aviv G. Mari, Detroit, MI M. Martinez-Ferro, Buenos Aires A. McLennan, Sydney, NSW K.J. Moise, Houston, TX F. Molina, Granada K.H. Nicolaides, London L. Otaño, Buenos Aires Z. Papp, Budapest R.A. Quintero, Miami, FL G. Ryan, Toronto, ON J. Rychik, Philadelphia, PA H. Sago, Tokyo W. Sepulveda, Santiago P. Stone, Auckland D.V. Surbek, Bern B.J. Trudinger, Westmead, NSW Y. Ville, Paris J.M.G. van Vugt, Nijmegen Clinical Advances and Basic Research