Prenatal maternal plasma DNA screening for cystic fibrosis: A computer modelling study of screening performance.

Background: Prenatal cystic fibrosis (CF) screening is currently based on determining the carrier status of both parents. We propose a new method based only on the analysis of DNA in maternal plasma. Methods: The method relies on the quantitative amplification of the CF gene to determine the percentage of DNA fragments in maternal plasma at targeted CF mutation sites that carry a CF mutation. Computer modelling was carried out to estimate the distributions of these percentages in pregnancies with and without a fetus affected with CF. This was done according to the number of DNA fragments counted and fetal fraction, using the 23 CF mutations recommended by the American College of Medical Genetics for parental carrier testing. Results: The estimated detection rate (sensitivity) is 70% (100% of those detected using the 23 mutations), the false-positive rate 0.002%, and the odds of being affected given a positive screening result 14:1, compared with 70%, 0.12%, and 1:3, respectively, with current prenatal screening based on parental carrier testing. Conclusions: Compared with current screening practice based on parental carrier testing, the proposed method would substantially reduce the number of invasive diagnostic procedures (amniocentesis or chorionic villus sampling) without reducing the CF detection rate. The expected advantages of the proposed method justify carrying out the necessary test development for use in a clinical validation study.The author(s) declared that no grants were involved in supporting this work

Aneuploidy and Confined Chromosomal Mosaicism in the Developing Human Brain

BACKGROUND: Understanding the mechanisms underlying generation of neuronal variability and complexity remains the central challenge for neuroscience. Structural variation in the neuronal genome is likely to be one important mechanism for neuronal diversity and brain diseases. Large-scale genomic variations due to loss or gain of whole chromosomes (aneuploidy) have been described in cells of the normal and diseased human brain, which are generated from neural stem cells during intrauterine period of life. However, the incidence of aneuploidy in the developing human brain and its impact on the brain development and function are obscure. METHODOLOGY/PRINCIPAL FINDINGS: To address genomic variation during development we surveyed aneuploidy/polyploidy in the human fetal tissues by advanced molecular-cytogenetic techniques at the single-cell level. Here we show that the human developing brain has mosaic nature, being composed of euploid and aneuploid neural cells. Studying over 600,000 neural cells, we have determined the average aneuploidy frequency as 1.25-1.45% per chromosome, with the overall percentage of aneuploidy tending to approach 30-35%. Furthermore, we found that mosaic aneuploidy can be exclusively confined to the brain. CONCLUSIONS/SIGNIFICANCE: Our data indicates aneuploidization to be an additional pathological mechanism for neuronal genome diversification. These findings highlight the involvement of aneuploidy in the human brain development and suggest an unexpected link between developmental chromosomal instability, intercellural/intertissular genome diversity and human brain diseases

Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse

The first 8 weeks of pregnancy is a critical time, with the majority of pregnancy losses occurring during this period. Abnormal chromosome number (aneuploidy) is a common finding in human miscarriage, yet is rarely reported in domestic animals. Equine early pregnancy loss (EPL) has no diagnosis in over 80% of cases. The aim of this study was to characterise aneuploidies associated with equine EPL. Genomic DNA from clinical cases of spontaneous miscarriage (EPLs; 14–65 days of gestation) and healthy control placentae (various gestational ages) were assessed using a high density genotyping array. Aneuploidy was detected in 12/55 EPLs (21.8%), and 0/15 healthy control placentae. Whole genome sequencing (30X) and digital droplet PCR (ddPCR) validated results. The majority of these aneuploidies have never been reported in live born equines, supporting their embryonic/fetal lethality. Aneuploidies were detected in both placental and fetal compartments. Rodents are currently used to study how maternal ageing impacts aneuploidy risk, however the differences in reproductive biology is a limitation of this model. We present the first evidence of aneuploidy in naturally occurring equine EPLs at a similar rate to human miscarriage. We therefore suggest the horse as an alternative to rodent models to study mechanisms resulting in aneuploid pregnancies

Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development