A. T. J. I. Go

Non-invasive prenatal assessment of Trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study

Objectives To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling. Design Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples. Setting Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands. Participants 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. Main outcome measures Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection. Results Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%. Conclusion Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing.

Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing

Significance Noninvasive prenatal testing (NIPT) using fetal DNA in maternal plasma has been rapidly adopted worldwide. Current NIPT for fetal chromosomal disorders are based on the counting of DNA molecules in maternal plasma. Here, we show that plasma DNA-based molecular diagnostics can also be built around DNA fragment size, instead of count. First, we demonstrate that the fetal DNA fraction in maternal plasma can be rapidly measured by size analysis, even simply using microchip-based capillary electrophoresis. Second, we show that plasma DNA size analysis can be used for the detection of multiple types of fetal chromosomal aneuploidies with high accuracy. This strategy has many potential diagnostic applications, e.g., in oncology and transplantation monitoring. Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

Ultrafast Scan Magnetic Resonance in Prenatal Diagnosis

Objective: To determine whether magnetic resonance (MR) can give additional information in prenatal diagnosis of congenital anomalies, when the ultrasound (US) analysis is not conclusive. Methods: Ultrafast MR scanning examined 39 pregnant women with 41 fetuses in whom US was suspicious of fetal congenital abnormalities. Two techniques were used namely (1) HASTE inversion recovery sequence and (2) FISP 2D. Results: Thirty-nine patients with 41 fetuses were referred for MR because of an equivocal US with regard to brain, spine, skeletal and miscellaneous anomalies. In 1 twin pregnancy, 1 co-twin has not been examined with MRI because of its demise. In 22 of them, additional information was obtained by MR. In 9 the MR was confirmative with the US examination. Four were false negative, comparing with the postnatal diagnosis. Three failed because of maternal claustrophobia and in 2 a diagnosis could not be made. From the 40 fetuses in this study, 38 were examined postnatally by MR, US, plain X-ray or autopsy was performed to confirm the prenatal diagnosis. Conclusion: The use of MRI in obstetrics has been limited, until recently. With fast MRI sequences it is not necessary to sedate the fetus. It is advisable in cases where US is equivocal concerning congenital anomalies of the fetus to use MR with fast or ultrafast scan technique, especially when the central nervous system is concerned.

Additional value of prenatal genomic array testing in fetuses with isolated structural ultrasound abnormalities and a normal karyotype: a systematic review of the literature

To establish the prevalence of submicroscopic genetic copy number variants (CNVs) in fetuses with a structural ultrasound anomaly (restricted to one anatomical system) and a normal karyotype. The aim was to determine the diagnostic and prognostic value of genomic array testing in these pregnancies.

Long-term neurodevelopmental outcome of monochorionic and matched dichorionic twins

Background Monochorionic (MC) twins are at increased risk for perinatal mortality and serious morbidity due to the presence of placental vascular anastomoses. Cerebral injury can be secondary to haemodynamic and hematological disorders during pregnancy (especially twin-to-twin transfusion syndrome (TTTS) or intrauterine co-twin death) or from postnatal injury associated with prematurity and low birth weight, common complications in twin pregnancies. We investigated neurodevelopmental outcome in MC and dichorionic (DC) twins at the age of two years. Methods This was a prospective cohort study. Cerebral palsy (CP) was studied in 182 MC infants and 189 DC infants matched for weight and age at delivery, gender, ethnicity of the mother and study center. After losses to follow-up, 282 of the 366 infants without CP were available to be tested with the Griffiths Mental Developmental Scales at 22 months corrected age, all born between January 2005 and January 2006 in nine perinatal centers in The Netherlands. Due to phenotypic (un)alikeness in mono-or dizygosity, the principal investigator was not blinded to chorionic status; perinatal outcome, with exception of co-twin death, was not known to the examiner. Findings Four out of 182 MC infants had CP (2.2%) - two of the four CP-cases were due to complications specific to MC twin pregnancies (TTTS and co-twin death) and the other two cases of CP were the result of cystic PVL after preterm birth - compared to one sibling of a DC twin (0.5%; OR 4.2, 95% CI 0.5–38.2) of unknown origin. Follow-up rate of neurodevelopmental outcome by Griffiths test was 76%. The majority of 2-year-old twins had normal developmental status. There were no significant differences between MC and DC twins. One MC infant (0.7%) had a developmental delay compared to 6 DC infants (4.2%; OR 0.2, 95% 0.0–1.4). Birth weight discordancy did not influence long-term outcome, though the smaller twin had slightly lower developmental scores than its larger co-twin. Conclusions There were no significant differences in occurrence of cerebral palsy as well as neurodevelopmental outcome between MC and DC twins. Outcome of MC twins seems favourable in the absence of TTTS or co-twin death.

Trial by Dutch Laboratories for Evaluation of Non-Invasive Prenatal Testing. Part I - Clinical Impact

To evaluate the clinical impact of nationwide implementation of genome‐wide non‐invasive prenatal testing (NIPT) in pregnancies at increased risk for fetal trisomies 21, 18 and 13 (TRIDENT study).

Decreased plasma levels of metastin in early pregnancy are associated with small for gestational age neonates.

To investigate whether pregnancies with small for gestational age (SGA) neonates, defined as customized birth weight below the 10th centile, are associated with altered levels of metastin in maternal plasma in the first trimester.

Perinatal mortality and mode of delivery in monochorionic diamniotic twin pregnancies >= 32 weeks of gestation: a multicentre retrospective cohort study

Please cite this paper as: Hack K, Derks J, Elias S, van Mameren F, Koopman‐Esseboom C, Mol B, Lopriore E, Schaap A, Arabin B, Duvekot J, Go A, Wieselmann E, Eggink A, Willekes C, Vandenbussche F, Visser G. Perinatal mortality and mode of delivery in monochorionic diamniotic twin pregnancies ≥32 weeks of gestation: a multicentre retrospective cohort study. BJOG 2011;118:1090–1097.

44 Single-Nucleotide Polymorphisms Expressed by Placental RNA: Assessment for Use in Noninvasive Prenatal Diagnosis of Trisomy 21

For noninvasive prenatal diagnosis, markers that directly reflect changes in chromosome dosage are preferred over indirect markers that are associated with epiphenomena(1)(2). The RNA:single-nucleotide polymorphism (SNP) allelic ratio strategy was described recently as a means to directly assess fetal chromosome dosage in maternal plasma(2). Quantitive comparison of the allelic expression ratios of a placentally expressed, chromosome 21–encoded gene, placenta-specific 4 ( PLAC4 ), enabled detection in maternal plasma of the differences between 2 (normal) or 3 copies of chromosome 21(2). The RNA:SNP ratio strategy is currently limited to a subset of the population with heterozygosity of the SNP used. Theoretically, an increase in population coverage can be obtained by inclusion of additional SNPs within PLAC4 or other chromosome 21–encoded transcripts with placental expression and detectability in maternal plasma(2). We therefore tested 44 SNPs expressed by 7 chromosome 21–encoded, placentally expressed genes(2), PLAC4 , collagen, type VI, alpha 2 ( COL6A2 ), collagen, type VI, alpha 1 ( COL6A1 ), BTG family, member 3 ( BTG3 ), ADAM metallopeptidase with thrombospondin type 1 motif, 1 ( ADAMTS1 ), chromosome 21 open reading frame 105 ( C21orf105 ), and amyloid …