Fetal fraction-based risk algorithm for non-invasive prenatal testing: screening for trisomies 13 and 18 and triploidy in women with low cell-free fetal DNA.

OBJECTIVE: To identify pregnancies at increased risk for trisomy 13, trisomy 18 or triploidy attributable to low fetal fraction (FF). METHODS: A FF-based risk (FFBR) model was built using data from more than 165 000 singleton pregnancies referred for single-nucleotide polymorphism (SNP)-based non-invasive prenatal testing (NIPT). Based on maternal weight and gestational age (GA), FF distributions for normal, trisomy 13, trisomy 18 and triploid pregnancies were constructed and used to adjust prior risks for these abnormalities. A risk cut-off of ≥ 1% was chosen to define pregnancies at high risk for trisomy 13, trisomy 18 or triploidy (high FFBR score). The model was evaluated on an independent blinded set of pregnancies for which SNP-based NIPT did not return a result, and for which pregnancy outcome information was gathered retrospectively. RESULTS: The evaluation cohort comprised 1148 cases, of which approximately half received a high FFBR score. Compared with rates expected based on maternal age (MA) and GA, cases with a high FFBR score had a significantly increased rate of trisomy 13, trisomy 18 or triploidy combined (5.7% vs 0.7%; P < 0.001) and also of unexplained pregnancy loss (14.7% vs 10.4%; P < 0.001). For cases that did not receive a high FFBR score, the incidence of a chromosomal abnormality or pregnancy loss was not significantly different from that expected based on MA and GA. In this study cohort, the sensitivity of the FFBR model for detection of trisomy 13, trisomy 18 or triploidy was 91.4% (95% CI, 76.9-98.2%) with a positive predictive value of 5.7% (32/564; 95% CI, 3.9-7.9%). CONCLUSIONS: For pregnancies with a FF too low to receive a result on standard NIPT, the FFBR algorithm identified a subset of cases at increased risk for trisomy 13, trisomy 18 or triploidy. For the remainder of cases, the risk of a fetal chromosomal abnormality was unchanged from that expected based on MA and GA. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Ductus Venosus Agenesis as a Marker of Pallister-Killian Syndrome.

The ductus venosus (DV) is a shunt that allows the direct flow of well-oxygenated blood from the umbilical vein (UV) to the coronary and cerebral circulation through the foramen ovale. Its agenesis has been associated with chromosomal abnormalities and rare genetic syndromes, structural defects, intrauterine growth restriction (IUGR) and even antepartum fetal demise. Pallister-Killian Syndrome (PKS) is a rare sporadic disorder with specific tissue mosaic distribution of an extra 12p isochromosome (i(12p)). Its main clinical features are moderate to severe intellectual disability/neuromotor delay, skin pigmentation abnormalities, typical facial appearance, variable association with multiple congenital malformations and epilepsy. Though prenatal findings (including congenital diaphragmatic hernia, ventriculomegaly, congenital heart disease, polyhydramnios, and rhizomelic shortening) have been described in literature, prenatal diagnosis is difficult as there are no associated identification signs no distinctive or pathognomonic signs, and some of these malformations are hard to identify prenatally. The tissue mosaicism linked to this syndrome and the decrease of the abnormal clone carrier of the i(p12) after successive trypsinizations of cultured cells makes the diagnosis even more challenging. We present the case of a 27.5 weeks pregnant woman with a fetal ductus venosus agenesis (DVA) as the main guide marker. To our knowledge this is the first case published in literature reporting a DVA as a guide sign to diagnose a complex condition as Pallister-Killian syndrome. We also underscore the key role of new genetic techniques as microarrays to avoid misdiagnosis when only a subtle sonographic sign is present in complex conditions like this.

Cherchez la femme: maternal incidental findings can explain discordant prenatal cell-free DNA sequencing results.

Circulating DNA fragments in a pregnant woman's plasma derive from three sources: placenta, maternal bone marrow, and fetus. Prenatal sequencing to noninvasively screen for fetal chromosome abnormalities is performed on this mixed sample; results can therefore reflect the maternal as well as the fetoplacental DNA. Although it is recommended that pretest counseling include the possibility of detecting maternal genomic imbalance, this seldom occurs. Maternal abnormalities that can affect a prenatal screening test result include disorders that affect the size and metabolism of DNA, such as B12 deficiency, autoimmune disease, and intrahepatic cholestasis of pregnancy. Similarly, maternal tumors, both benign and malignant, can release DNA fragments that contain duplications or deletions. Bioinformatics algorithms can subsequently interpret the raw sequencing data incorrectly, resulting in false-positive test reports of fetal monosomies or test failures. Maternal sex-chromosome abnormalities, both constitutional and somatic, can generate results that are discordant with fetal ultrasound examination or karyotype. Maternal copy-number variants and mosaicism for autosomal aneuploidies can also skew interpretation. A maternal etiology should therefore be considered in the differential diagnosis of prenatal cell-free DNA test failures, false-positive and false-negative sequencing results. Further study is needed regarding the clinical utility of reporting maternal incidental findings.

Accuracy and reproducibility of fetal-fraction measurement using relative quantitation at polymorphic loci with microarray.

OBJECTIVES: Various methods of fetal-fraction measurement have been employed in conjunction with different approaches to cell-free DNA testing for fetal aneuploidy. In this study, we determined the accuracy and reproducibility of fetal-fraction measurement using polymorphic assays that are incorporated into the test design as part of the Harmony® prenatal test and evaluated whether the single nucleotide polymorphisms selected for and used in these assays can be applied broadly to all patient populations. METHODS: Clinical maternal plasma samples were assayed using a custom microarray with Digital ANalysis of Selected Regions (DANSR) assays designed to cover non-polymorphic targets on chromosomes of interest for aneuploidy assessment (13, 18, 21, X and Y) and polymorphic targets for fetal-fraction assessment. In a consecutive series of 47 512 maternal plasma samples, fetal-fraction measurements based on polymorphic assays were compared with those from Y-sequence quantitation. Reproducibility was examined between first- and second-tube measurements for the same patient sample in 734 cases. The fraction of informative loci was calculated for 13 988 samples. RESULTS: There was a strong correlation between fetal fractions determined using the polymorphic assays and using Y-chromosome sequence quantitation (r = 0.97). Fetal-fraction measurement between the first and second tubes was highly reproducible (r = 0.98). The fraction of informative loci observed in a clinical series was consistent with predictions based on assay design. CONCLUSIONS: The method based on relative quantitation at polymorphic loci on a microarray is accurate and reproducible for fetal-fraction estimation and is equally informative across global populations. This study provides a useful benchmark for ensuring the reliability and accuracy of fetal-fraction measurement. © 2018 Roche Sequencing Solutions. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Clinical results after the implementation of cell-free fetal DNA detection in maternal plasma.

AIM: Detection of cell-free fetal DNA in maternal blood is a type of noninvasive prenatal diagnosis test (NIPT), which has already been known for some time but has not yet been introduced in most of public hospitals in Spain. How the implementation of cell-free fetal DNA (cffDNA) in a contingent protocol has influenced the aneuploidy screening in our hospital is described. METHODS: Two cohorts of patients with positive combined screening were compared: the first one (years 2012-2013, 5747 patients) from a period of time in which the protocol valid until March 2016 - that included the use of invasive procedures - was applied; and the second one in which the current protocol - that included NIPT versus invasive procedures - was applied (first 7 months after protocol implementation, 898 patients). RESULTS: Comparison of both periods resulted in a 60.5% reduction of invasive procedures (P < 0,001) preserving the same chromosomopathy detection rate. The ratio of positive invasive procedures-indicated invasive procedures was improved by 15% in the first period to 50% in the second period (P = 0.01). CONCLUSION: NIPT introduction has caused a significant reduction of 60.5% of IP in high chromosomopathy risk patients after combined screening without modifying detection rate.

Positive predictive value estimates for cell-free noninvasive prenatal screening from data of a large referral genetic diagnostic laboratory.

BACKGROUND: Since its debut in 2011, cell-free fetal DNA screening has undergone rapid expansion with respect to both utilization and coverage. However, conclusive data regarding the clinical validity and utility of this screening tool, both for the originally included common autosomal and sex-chromosomal aneuploidies as well as the more recently added chromosomal microdeletion syndromes, have lagged behind. Thus, there is a continued need to educate clinicians and patients about the current benefits and limitations of this screening tool to inform pre- and posttest counseling, pre/perinatal decision making, and medical risk assessment/management. OBJECTIVE: The objective of this study was to determine the positive predictive value and false-positive rates for different chromosomal abnormalities identified by cell-free fetal DNA screening using a large data set of diagnostic testing results on invasive samples submitted to the laboratory for confirmatory studies. STUDY DESIGN: We tested 712 patient samples sent to our laboratory to confirm a cell-free fetal DNA screening result, indicating high risk for a chromosome abnormality. We compiled data from all cases in which the indication for confirmatory testing was a positive cell-free fetal DNA screen, including the common trisomies, sex chromosomal aneuploidies, microdeletion syndromes, and other large genome-wide copy number abnormalities. Testing modalities included fluorescence in situ hybridization, G-banded karyotype, and/or chromosomal microarray analysis performed on chorionic villus samples, amniotic fluid, or postnatally obtained blood samples. Positive predictive values and false-positive rates were calculated from tabulated data. RESULTS: The positive predictive values for trisomy 13, 18, and 21 were consistent with previous reports at 45%, 76%, and 84%, respectively. For the microdeletion syndrome regions, positive predictive values ranged from 0% for detection of Cri-du-Chat syndrome and Prader-Willi/Angelman syndrome to 14% for 1p36 deletion syndrome and 21% for 22q11.2 deletion syndrome. Detection of sex chromosomal aneuploidies had positive predictive values of 26% for monosomy X, 50% for 47,XXX, and 86% for 47,XXY. CONCLUSION: The positive predictive values for detection of common autosomal and sex chromosomal aneuploidies by cell-free fetal DNA screening were comparable with other studies. Identification of microdeletions was associated with lower positive predictive values and higher false-positive rates, likely because of the low prevalence of the individual targeted microdeletion syndromes in the general population. Although the obtained positive predictive values compare favorably with those seen in traditional screening approaches for common aneuploidies, they highlight the importance of educating clinicians and patients on the limitations of cell-free fetal DNA screening tests. Improvement of the cell-free fetal DNA screening technology and continued monitoring of its performance after introduction into clinical practice will be important to fully establish its clinical utility. Nonetheless, our data provide valuable information that may aid result interpretation, patient counseling, and clinical decision making/management.

The accuracy of cell-free fetal DNA-based non-invasive prenatal testing in singleton pregnancies: a systematic review and bivariate meta-analysis.

BACKGROUND: Cell-free fetal DNA (cffDNA) non-invasive prenatal testing (NIPT) is rapidly expanding, and is being introduced at varying rates depending on country and condition. OBJECTIVES: Determine accuracy of cffDNA-based NIPT for all conditions. Evaluate influence of other factors on test performance. SEARCH STRATEGY: Medline, Embase, CINAHL, Cochrane Library, from 1997 to April 2015. SELECTION CRITERIA: Cohort studies reporting cffDNA-based NIPT performance in singleton pregnancies. DATA COLLECTION AND ANALYSIS: Bivariate or univariate meta-analysis and subgroup analysis performed to explore influence of test type and population risk. MAIN RESULTS: A total of 117 studies were included that analysed 18 conditions. Bivariate meta-analysis demonstrated sensitivities and specificities, respectively, for: fetal sex, 0.989 (95% CI 0.980-0.994) and 0.996 (95% CI 0.989-0.998), 11 179 tests; rhesus D, 0.993 (95% CI 0.982-0.997) and 0.984 (95% CI 0.964-0.993), 10 290 tests; trisomy 21, 0.994 (95% CI 0.983-0.998) and 0.999 (95% CI 0.999-1.000), 148 344 tests; trisomy 18, 0.977 (95% CI 0.952-0.989) and 0.999 (95% CI 0.998-1.000), 146 940 tests; monosomy X, 0.929 (95% CI 0.741-0.984) and 0.999 (95% CI 0.995-0.999), 6712 tests. Trisomy 13 was analysed by univariate meta-analysis, with a summary sensitivity of 0.906 (95% CI 0.823-0.958) and specificity of 1.00 (95% CI 0.999-0.100), from 134 691 tests. False and inconclusive results were poorly reported across all conditions. Although the test type affected both sensitivity and specificity, there was no evidence that population risk had any effect. CONCLUSION: Performance of cffDNA-based NIPT is affected by condition under investigation. For fetal sex and rhesus D status, NIPT can be considered diagnostic. For trisomy 21, 18, and 13, the lower sensitivity, specificity, and disease prevalence, combined with the biological influence of confined placental mosaicism, designates it a screening test. These factors must be considered when counselling patients and assessing the cost of introduction into routine care. TWEETABLE ABSTRACT: cffDNA NIPT accuracy high, can be diagnostic for fetal sex and rhesus D, but only screening test in aneuploidy.

Clinical utility of non-invasive prenatal testing in pregnancies with ultrasound anomalies.

OBJECTIVE: To evaluate the application of non-invasive prenatal testing (NIPT) as an alternative to invasive diagnostic prenatal testing in pregnancies with abnormal ultrasound findings. METHODS: This was a retrospective analysis of 251 singleton and multiple pregnancies at high risk for fetal chromosomal abnormality based on findings at sonographic examination, in which NIPT was performed as a first-tier genetic test. NIPT was performed by massively parallel sequencing of cell-free DNA in maternal plasma, allowing genome-wide detection of whole-chromosome, as well as partial, autosomal aneuploidy. Sex chromosomes were not analyzed, according to the current protocol in Dutch laboratories. RESULTS: NIPT was performed at a median gestational age of 20 weeks, indicated by the presence of multiple congenital anomalies (n = 13), isolated structural anomalies (n = 57), increased nuchal translucency ≥ 3.5 mm (n = 58), soft markers (n = 73), growth restriction (n = 40) and other anomalies (n = 10). NIPT results were normal in 224 (89.2%) pregnancies, inconclusive in one (0.4%) and abnormal in 26 (10.4%). Most genetic aberrations detected by NIPT were common whole-chromosome aneuploidies: trisomy 21 (n = 13), trisomy 18 (n = 6) and trisomy 13 (n = 3). Four further NIPT results were abnormal; one was suspected of being confined placental mosaicism and one was of maternal origin. In those with normal NIPT results, sonographic follow-up or examination of the newborn indicated the need for diagnostic genetic testing in 33/224 (14.7%) pregnancies. Clinically relevant genetic aberrations were revealed in 7/224 (3.1%) cases, two of which were whole-chromosome aneuploidies: trisomy 13 and monosomy X. As sex chromosomal aberrations are not included in NIPT analysis, the latter cannot be considered a false-negative result. Other discordant findings were subchromosomal aberrations (< 20 megabases, n = 2) and monogenic aberrations (n = 3). CONCLUSIONS: NIPT should not be recommended for genetic evaluation of the etiology of ultrasound anomalies, as both resolution and sensitivity, or negative predictive value, are inferior to those of conventional karyotyping and microarray analysis. Nonetheless, some pregnant women consider NIPT to be an acceptable alternative to invasive diagnostic testing. © 2016 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex.

BACKGROUND: There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). METHODS: We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. RESULTS: Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. CONCLUSIONS: The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

Screening for trisomies by cell-free DNA testing of maternal blood: consequences of a failed result.

OBJECTIVES: First, to report the distribution of the fetal fraction of cell-free (cf) DNA and the rate of a failed cfDNA test result in trisomies 21, 18 and 13, by comparison with pregnancies unaffected by these trisomies, second, to examine the possible effects of maternal and fetal characteristics on the fetal fraction, and third, to consider the options for further management of pregnancies with a failed result. METHODS: This was a cohort study of 10 698 singleton pregnancies undergoing screening for fetal trisomies 21, 18 and 13 by cfDNA testing at 10-14 weeks' gestation. There were 160 cases of trisomy 21, 50 of trisomy 18, 16 of trisomy 13 and 10 472 were unaffected by these trisomies. Multivariate regression analysis was used to determine significant predictors of fetal fraction and a failed cfDNA test result amongst maternal and fetal characteristics. RESULTS: Fetal fraction decreased with increasing body mass index and maternal age, was lower in women of South Asian racial origin than in Caucasians and in assisted compared to natural conceptions. It increased with fetal crown-rump length and higher levels of serum pregnancy-associated plasma protein-A and free ß-human chorionic gonadotropin. The median fetal fraction was 11.0% (interquartile range (IQR), 8.3-14.4%) in the unaffected group, 10.7% (IQR, 7.8-14.3%) in trisomy 21, 8.6% (IQR, 5.0-10.2%) in trisomy 18 and 7.0% (IQR, 6.0-9.4%) in trisomy 13. There was a failed result from cfDNA testing after first sampling in 2.9% of the unaffected group, 1.9% of trisomy 21, 8.0% of trisomy 18 and 6.3% of trisomy 13. In the cases with a failed result, 7% of women had invasive testing, mainly because of high risk from the combined test and/or presence of sonographic features suggestive of trisomies 18 and 13. All cases of trisomies were detected prenatally. CONCLUSIONS: In cases of a failed cfDNA test, the rate of trisomies 18 and 13, but not trisomy 21, is higher than in those with a successful test. In the management of such cases, the decision in favor of invasive testing should depend on the risk of prior screening and the results of detailed ultrasound examination. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Calculating the fetal fraction for noninvasive prenatal testing based on genome-wide nucleosome profiles.

OBJECTIVE: While large fetal copy number aberrations can generally be detected through sequencing of DNA in maternal blood, the reliability of tests depends on the fraction of DNA that originates from the fetus. Existing methods to determine this fetal fraction require additional work or are limited to male fetuses. We aimed to create a sex-independent approach without additional work. METHODS: DNA fragments used for noninvasive prenatal testing are cut only by natural processes; thus, influences on cutting by the packaging of DNA in nucleosomes will be preserved in sequencing. As cuts are expected to be made preferentially in linker regions, the shorter fetal fragments should be enriched for reads starting in nucleosome covered positions. RESULTS: We generated genome-wide nucleosome profiles based on single end sequencing of cell-free DNA. We found a difference between DNA digestion of fetal cell-free DNA and maternal cell-free DNA and used this to calculate the fraction of fetal DNA in maternal plasma for both male and female fetuses. CONCLUSION: Our method facilitates cost-effective noninvasive prenatal testing, as the fetal DNA fraction can be estimated without the need for expensive paired-end sequencing or additional tests. The methodology is implemented as a tool, which we called SANEFALCON (Single reAds Nucleosome-basEd FetAL fraCtiON). It is available for academic and non-profit purposes under Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License. github.com/rstraver/sanefalcon. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Macrophage colony-stimulating factor (M-CSF) in first trimester maternal serum: correlation with pathologic pregnancy outcome.

PURPOSE: To determine correlations between macrophage colony-stimulating factor (MCSF) levels in maternal blood during first trimester screening with respect to normal and pathological pregnancies. METHODS: This was a prospective single centre study. First trimester screening was performed according to FMF London certificates. Nuchal translucency, PAPP-A and free ß-HCG were obtained as well as M-CSF serum levels in maternal blood. Fetal karyotyping was achieved by chorionic villi sampling. RESULTS: 125 patients were enrolled in this study. 21 pregnancies had confirmed aberrant karyotypes. Trisomy 21 cases showed significantly elevated M-CSF levels of 270 ± 91 pg/ml (p = 0.032), whereas cases of trisomy 13 (183 ± 68 pg/ml) and trisomy 18 (143 ± 40 pg/ml) had low M-CSF levels. Furthermore M-CSF levels tended to be low in preterm deliveries, placental insufficiency and nicotine consumption. In cases with gestational diabetes M-CSF tended to be elevated. Furthermore we found a positive correlation between high free ß-human chorionic gonadotropin (hcg) and MCSF values. There was no correlation between pregnancy associated plasma protein (PAPP-A) and M-CSF. CONCLUSIONS: M-CSF is a cytokine promoting placental growth and differentiation. M-CSF is known to be involved in the process of implantation in pregnancy. The role of M-CSF with respect to disturbed pregnancy outcomes such as placental insufficiency in normal or aberrant karyotypes, for example, is yet subject to further research.

Detection of fetal subchromosomal abnormalities by sequencing circulating cell-free DNA from maternal plasma.

BACKGROUND: The development of sequencing-based noninvasive prenatal testing (NIPT) has been largely focused on whole-chromosome aneuploidies (chromosomes 13, 18, 21, X, and Y). Collectively, they account for only 30% of all live births with a chromosome abnormality. Various structural chromosome changes, such as microdeletion/microduplication (MD) syndromes are more common but more challenging to detect. Recently, several publications have shown results on noninvasive detection of MDs by deep sequencing. These approaches demonstrated the proof of concept but are not economically feasible for large-scale clinical applications. METHODS: We present a novel approach that uses low-coverage whole genome sequencing (approximately 0.2×) to detect MDs genome wide without requiring prior knowledge of the event's location. We developed a normalization method to reduce sequencing noise. We then applied a statistical method to search for consistently increased or decreased regions. A decision tree was used to differentiate whole-chromosome events from MDs. RESULTS: We demonstrated via a simulation study that the sensitivity difference between our method and the theoretical limit was <5% for MDs ≥9 Mb. We tested the performance in a blinded study in which the MDs ranged from 3 to 40 Mb. In this study, our algorithm correctly identified 17 of 18 cases with MDs and 156 of 157 unaffected cases. CONCLUSIONS: The limit of detection for any given MD syndrome is constrained by 4 factors: fetal fraction, MD size, coverage, and biological and technical variability of the event region. Our algorithm takes these factors into account and achieved 94.4% sensitivity and 99.4% specificity.

Comparison of long-term outcomes between children with aplastic anemia and refractory cytopenia of childhood who received immunosuppressive therapy with antithymocyte globulin and cyclosporine.

The 2008 World Health Organization classification proposed a new entity in childhood myelodysplastic syndrome, refractory cytopenia of childhood. However, it is unclear whether this morphological classification reflects clinical outcomes. We retrospectively reviewed bone marrow morphology in 186 children (median age 8 years; range 1-16 years) who were enrolled in the prospective study and received horse antithymocyte globulin and cyclosporine between July 1999 and November 2008. The median follow-up period was 87 months (range 1-146 months). Out of 186 patients, 62 (33%) were classified with aplastic anemia, 94 (49%) with refractory cytopenia of childhood, and 34 (18%) with refractory cytopenia with multilineage dysplasia. Aplastic anemia patients received granulocyte colony-stimulating factor more frequently and for longer durations than other patients (P<0.01). After six months, response rates to immunosuppressive therapy were not significantly different among the 3 groups. Acquisition of chromosomal abnormalities was observed in 5 patients with aplastic anemia, 4 patients with refractory cytopenia of childhood, and 3 patients with refractory cytopenia with multilineage dysplasia. Although the cumulative incidence of total clonal evolution at ten years was not significantly different among the 3 groups, the cumulative incidence of monosomy 7 development was significantly higher in aplastic anemia than in the other groups (P=0.02). Multivariate analysis revealed that only granulocyte colony-stimulating factor administration duration of 40 days or more was a significant risk factor for monosomy 7 development (P=0.02). These findings suggest that even the introduction of a strict morphological distinction from hypoplastic myelodysplastic syndrome cannot eradicate clonal evolution in children with aplastic anemia.

Elevation of insulin-like growth factor binding protein-2 level in Pallister-Killian syndrome: implications for the postnatal growth retardation phenotype.

Pallister-Killian syndrome (PKS) is a multi-system developmental disorder caused by tetrasomy 12p that exhibits tissue-limited mosaicism. Probands with PKS often demonstrate a unique growth profile consisting of macrosomia at birth with deceleration of growth postnatally. We have previously demonstrated that cultured skin fibroblasts from PKS probands have significantly elevated expression of insulin-like growth factor binding protein-2 (IGFBP2). To further evaluate the role of IGFBP2 in PKS, the amount of IGFBP2 secreted from cultured skin fibroblast cell lines and serum IGFBP2 levels were measured in probands with PKS. Approximately 60% of PKS fibroblast cell lines secreted higher levels of IGFBP2 compared to control fibroblasts, although the remaining 40% of PKS samples produced comparable level of IGFBP2 to that of control fibroblasts. Serum IGFBP2 levels were also measured in PKS probands and were elevated in 40% of PKS probands. PKS probands with elevated IGFBP2 manifested with severe postnatal growth retardation. IGFBPs are the family of related proteins that bind IGFs with high affinity and are typically thought to attenuate IGF action. We suggest that elevated IGFBP2 levels might play a role in the growth retardation phenotype of PKS.

Impact of fetal chromosomal disorders on maternal blood metabolome: toward new biomarkers?

OBJECTIVE: This study aimed at determining the relationship between fetal chromosomal disorders (CDs), including trisomy 21 (T21), and on first- and second-trimester maternal blood plasma, to identify the time-course metabolic adaptations to the conditions and the possible new plasma biomarkers. Furthermore, a definition of a joint circulatory (plasma) and excretory (urine) metabolic description of second-trimester CDs was sought. STUDY DESIGN: Plasma was obtained for 119 pregnant women: 74 controls and 45 CD cases, including 22 T21 cases. Plasma and lipid extracts (for T21 only) were analyzed by nuclear magnetic resonance spectroscopy, and data were handled by variable selection and multivariate analysis. Correlation analysis was used on a concatenated plasma/urine matrix descriptive of second-trimester CD, based on previously obtained urine data. RESULTS: CD cases were accompanied by enhanced lipid ß-oxidation (increased ketone bodies) and underutilization of glucose, pyruvate, and citrate. Lower circulating high-density lipoprotein levels were noted, along with changes in the proline and methanol in the first trimester, and also the urea, creatinine, acetate, and low-density lipoprotein plus very low-density lipoprotein in the second trimester and the different urea and creatinine levels, suggesting fetal renal dysfunction. In terms of plasma composition, T21 cases were indistinguishable from other CDs in the first trimester, whereas in the second trimester, increased methanol and albumin may be T21 specific. Furthermore, first-trimester lipid extracts of T21 showed decreased levels of 18:2 fatty acids, whereas in the second trimester, lower levels of 20:4 and 22:6 fatty acids were noted, possibly indicative of inflammation mechanisms. In both trimesters, high classification rates for CDs (88-89%) and T21 (85-92%) generally relied on variable selection of nuclear magnetic resonance data. Plasma/urine correlations confirmed most metabolic deviations and unveiled possible new ones regarding low-density lipoprotein plus very low-density lipoprotein, sugar, and gut-microflora metabolisms. CONCLUSION: This work partially confirmed previously reported data on first-trimester T21 and provided additional information on time-course metabolic changes accompanying CD and T21, in particular regarding plasma lipid composition. These results demonstrate the potential of plasma metabolomics in monitoring and characterizing CD cases; however, validation in larger cohorts is desirable.

Current controversies in prenatal diagnosis 1: NIPT for chromosome abnormalities should be offered to women with low a priori risk.

In its successful annual cycle of controversies and debates, the International Society of Prenatal Diagnosis and Therapy once again addressed non-invasive prenatal testing (NIPT) by following up on the 2013 controversy, 'Should non-invasive DNA testing be the standard screening test for Down syndrome in all pregnant women'? with the proposition, 'NIPT for chromosomel abnormalities should be offered to women with low a priori risk'.

Women's views and the impact of noninvasive prenatal testing on procedures in a managed care setting.

OBJECTIVE: To prospectively determine the impact of noninvasive prenatal testing (NIPT) on invasive procedure utilization in a managed care setting and to elucidate women's views. METHODS: Pregnant women at 10- 20 weeks' gestation with high-risk indications for fetal aneuploidy in the Kaiser Permanente Southern California organization were eligible. Enrolled patients received routine prenatal counseling, completed a questionnaire and were offered the option of NIPT by a genetic counselor. Downstream data through 28 weeks' gestation were collected from the electronic medical record (EMR). The EMR was also used to identify a matched historical cohort from 1 year prior to NIPT availability. Rates of invasive prenatal procedures were compared using McNemar's test. RESULTS: Two hundred women completed the questionnaire and underwent NIPT. Twenty-two subjects (11%) in the prospective cohort underwent an invasive prenatal procedure compared with 58 (29%) in the historical cohort (p<0.0001). Safety and accuracy were the most important factors in considering NIPT. At the time of survey, only 12% indicated being very comfortable with the possibility of undergoing amniocentesis. CONCLUSION: This prospective study demonstrates a 62% reduction in invasive prenatal procedures after NIPT testing and finds safety, accuracy, and personal beliefs key to women's decision-making.

Ten years of experience with first-trimester screening for fetal aneuploidy employing biochemistry from gestational weeks 6+0 to 13+6.

OBJECTIVES: To validate the performance of first-trimester screening for fetal aneuploidy employing blood samples drawn in gestational weeks 6-13. METHODS: Prospective combined first-trimester screening for fetal aneuploidy in Denmark was validated in two large datasets: (1) a dataset from the Central Denmark Region including 147,768 pregnancies from October 2003 to October 2013, and (2) a national dataset including 220,739 pregnancies from January 2008 to August 2011. RESULTS: For trisomy 21, the weekly median multiple of the median (MoM) increased from 0.37 in week 6 to 0.70 in week 13 (pregnancy-associated plasma protein-A), and from 0.99 in week 6 to 2.68 in week 13 (free ßhCG). The overall detection rate (DR) for fetal trisomy 21 was 91.2%. Employing blood samples from gestational week 9, the DR was 97% (p = 0.05). For fetal trisomy 18, trisomy 13 and triploidy, the overall DRs after first-trimester screening were 79.5, 86 and 85%. In the national dataset, the overall DR for trisomy 21 was 86.3% ranging from 89 (weeks 9 and 10) to 80% (weeks 12 and 13). CONCLUSION: The results from both datasets show that blood sampling in gestational weeks 9-10 is a robust and high-performance strategy, which can be applied for routine first-trimester screening in clinical practice. © 2014 S. Karger AG, Basel.