Yair J. Blumenfeld

Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood

We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes.

Non-invasive prenatal measurement of the fetal genome

The vast majority of prenatal genetic testing requires invasive sampling. However, this poses a risk to the fetus, so one must make a decision that weighs the desire for genetic information against the risk of an adverse outcome due to hazards of the testing process. These issues are not required to be coupled, and it would be desirable to discover genetic information about the fetus without incurring a health risk. Here we demonstrate that it is possible to non-invasively sequence the entire prenatal genome. Our results show that molecular counting of parental haplotypes in maternal plasma by shotgun sequencing of maternal plasma DNA allows the inherited fetal genome to be deciphered non-invasively. We also applied the counting principle directly to each allele in the fetal exome by performing exome capture on maternal plasma DNA before shotgun sequencing. This approach enables non-invasive exome screening of clinically relevant and deleterious alleles that were paternally inherited or had arisen as de novo germline mutations, and complements the haplotype counting approach to provide a comprehensive view of the fetal genome. Non-invasive determination of the fetal genome may ultimately facilitate the diagnosis of all inherited and de novo genetic disease.

Analysis of the Size Distributions of Fetal and Maternal Cell-Free DNA by Paired-End Sequencing

BACKGROUND Noninvasive prenatal diagnosis with cell-free DNA in maternal plasma is challenging because only a small portion of the DNA sample is derived from the fetus. A few previous studies provided size-range estimates of maternal and fetal DNA, but direct measurement of the size distributions is difficult because of the small quantity of cell-free DNA. METHODS We used high-throughput paired-end sequencing to directly measure the size distributions of maternal and fetal DNA in cell-free maternal plasma collected from 3 typical diploid and 4 aneuploid male pregnancies. As a control, restriction fragments of lambda DNA were also sequenced. RESULTS Cell-free DNA had a dominant peak at approximately 162 bp and a minor peak at approximately 340 bp. Chromosome Y sequences were rarely longer than 250 bp but were present in sizes of <150 bp at a larger proportion compared with the rest of the sequences. Selective analysis of the shortest fragments generally increased the fetal DNA fraction but did not necessarily increase the sensitivity of aneuploidy detection, owing to the reduction in the number of DNA molecules being counted. Restriction fragments of lambda DNA with sizes between 60 bp and 120 bp were preferentially sequenced, indicating that the shotgun sequencing work flow introduced a bias toward shorter fragments. CONCLUSIONS Our results confirm that fetal DNA is shorter than maternal DNA. The enrichment of fetal DNA by size selection, however, may not provide a dramatic increase in sensitivity for assays that rely on length measurement in situ because of a trade-off between the fetal DNA fraction and the number of molecules being counted.

Noninvasive prenatal diagnosis: pregnant women's interest and expected uptake

To investigate pregnant womens level of future interest in noninvasive prenatal diagnosis (NIPD) and what factors might affect expected uptake of this testing.

Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy

OBJECTIVE The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy. STUDY DESIGN Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes. RESULTS Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested. CONCLUSION Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours.

Noninvasive in vivo monitoring of tissue-specific global gene expression in humans.

Significance Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape of circulating RNA in human subjects. By focusing on tissue-specific genes, we were able to identify the relative contributions of these tissues to circulating RNA and monitor changes during tissue development and neurodegenerative disease states. Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape circulating RNA in a cohort of human subjects. By focusing on genes whose expression is highly specific to certain tissues, we were able to identify the relative contributions of these tissues to circulating RNA and to monitor changes in tissue development and health. As one application of this approach, we performed a longitudinal study on pregnant women and analyzed their combined cell-free RNA transcriptomes across all three trimesters of pregnancy and after delivery. In addition to the analysis of mRNA, we observed and characterized noncoding species such as long noncoding RNA and circular RNA transcripts whose presence had not been previously observed in human plasma. We demonstrate that it is possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample. We also studied the role of neuron-specific transcripts in the blood of healthy adults and those suffering from the neurodegenerative disorder Alzheimer’s disease and showed that disease specific neural transcripts are present at increased levels in the blood of affected individuals. Characterization of the cell-free transcriptome in its entirety may thus provide broad insights into human health and development without the need for invasive tissue sampling.

Trends in Cesarean Delivery for Twin Births in the United States: 1995–2008

OBJECTIVE: To estimate trends and risk factors for cesarean delivery for twins in the United States. METHODS: This was a cross-sectional study in which we calculated cesarean delivery rates for twins from 1995 to 2008 using National Center for Health Statistics data. We compared cesarean delivery rates by year and for vertex compared with breech presentation. The order of presentation for a given twin pair could not be determined from the available records and therefore analysis was based on individual discrete twin data. Multivariable logistic regression was used to estimate independent risk factors, including year of birth and maternal factors, for cesarean delivery. RESULTS: Cesarean delivery rates for twin births increased steadily from 53.4% to 75.0% in 2008. Rates rose for the breech twin category (81.5%–92.1%) and the vertex twin category (45.1%–68.2%). The relative increase in the cesarean delivery rate for preterm and term neonates was similar. After risk adjustment, there was an average increase noted in cesarean delivery of 5% each year during the study period (risk ratio 1.05, 95% confidence interval 1.04–1.05). CONCLUSION: Cesarean delivery rates for twin births increased dramatically from 1995 to 2008. This increase is significantly higher than that which could be explained by an increase in cesarean delivery for breech presentation of either the presenting or second twin. LEVEL OF EVIDENCE: II

Genomic microarray in fetuses with increased nuchal translucency and normal karyotype: a systematic review and meta‐analysis

To estimate the incremental yield of detecting copy number variants (CNVs) by genomic microarray over karyotyping in fetuses with increased nuchal translucency (NT) diagnosed by first‐trimester ultrasound.

Array comparative genomic hybridization and fetal congenital heart defects: a systematic review and meta‐analysis

Array comparative genomic hybridization (aCGH) is a molecular cytogenetic technique that is able to detect the presence of copy number variants (CNVs) within the genome. The detection rate of imbalances by aCGH compared to standard karyotyping and 22q11 microdeletion analysis by fluorescence in‐situ hybridization (FISH), in the setting of prenatally‐diagnosed cardiac malformations, has been reported in several studies. The objective of our study was to perform a systematic literature review and meta‐analysis to document the additional diagnostic gain of using aCGH in cases of congenital heart disease (CHD) diagnosed by prenatal ultrasound examination, with the aim of assisting clinicians to determine whether aCGH analysis is warranted when an ultrasonographic diagnosis of CHD is made, and to guide counseling in this setting.

Antenatal Steroid Administration for Premature Neonates in California

OBJECTIVES: To estimate risk factors for premature neonates not receiving antenatal steroids in a population-based cohort and to determine whether the gains of a quality-improvement collaborative project on antenatal steroid administration were sustained long-term. METHODS: Clinical data for premature neonates born in 2005–2007 were obtained from the California Perinatal Quality Care Collaborative, which collects data on more than 90% of neonatal admissions in California. Eligible neonates had a birth weight of less than 1,500 g or gestational age less than 34 weeks and were born at a Collaborative hospital. These data were linked to administrative data from California Vital Statistics. Sociodemographic and medical risk factors for not receiving antenatal steroids were determined. We also examined the effect of birth hospital participation in a previous quality-improvement collaborative project. A random effects logistic regression model was used to determine independent risk factors. RESULTS: Of 15,343 eligible neonates, 23.1% did not receive antenatal steroids in 2005–2007. Hispanic mothers (25.6%), mothers younger than age 20 (27.6%), and those without prenatal care (52.2%) were less likely to receive antenatal steroids. Mothers giving birth vaginally (26.8%) and mothers with a diagnosis of fetal distress (26.5%) were also less likely to receive antenatal steroids. Rupture of membranes before delivery and multiple gestations were associated with higher likelihood of antenatal steroid administration. Hospitals that participated in a quality-improvement collaborative in 1999–2000 had higher rates of antenatal steroid administration (85% compared with 69%, P<.001). CONCLUSION: A number of eligible mothers do not receive antenatal steroids. Quality-improvement initiatives to improve antenatal steroid administration could target specific high-risk groups. LEVEL OF EVIDENCE: II