Prenatal and fetal diagnosis of trisomy 18 after low-risk cell-free fetal DNA screening: A report of four cases.

INTRODUCTION: Non-Invasive Prenatal Screening (NIPS) is a useful screening method for common aneuploidies that can occur in pregnancies. It yields high sensitivities and specificities for the targeted conditions it tests for. Most commonly, these include Trisomies in chromosomes 21, 18, and 13, as well as aneuploidies in chromosomes X and Y. It does not, however, replace diagnostic testing. We review four cases seen by our institutions of patients who had NIPS performed with low-risk results and subsequently had fetuses affected with trisomy 18. METHODS: All fetal samples were evaluated by level II anatomic ultrasound and tested on amniocytes or products of conception through karyotype or chromosomal microarray following low-risk NIPS. RESULTS: None of the fetuses showed evidence of mosaicism and had features (both on ultrasound and postnatally) consistent with Trisomy 18. Postnatal fluorescence in situ hybridization performed on Formalin-Fixed Paraffin-Embedded tissue from 3 of the affected pregnancies' placentas identified mosaicism of trisomy 18. DISCUSSION: We discuss the possible explanations for the discrepancy between NIPS results and fetal karyotype, including, but not limited to placental mosaicism, placental size, and limitations of NIPS as a screening test.

Reproductive outcomes in individuals with chromosomal reciprocal translocations.

PURPOSE: Patients with reciprocal balanced translocations (RBT) have a risk for recurrent pregnancy losses (RPL), affected child, and infertility. Currently, genetic counseling is based on karyotypes found among the products of conception (POC), although factors influencing the success of assisted reproductive technologies (ART) in RBT couples are not established. METHODS: Cytogenetic results from 261 POC and offspring of the parents (113 women and 90 men) with RBT were evaluated. Chromosome segregation modes and number of euploid embryos were assessed in couples undergoing in vitro fertilization. RESULTS: Patients with translocations involving an acrocentric chromosome have a higher risk of unbalanced gametes caused by a 3:1 segregation. Female RBT patients have a statistically higher risk of aneuploidy due to an interchromosomal effect. The rate of euploid embryos is low due to meiosis I malsegregation of RBT, meiosis II nondisjunction, additional whole chromosome or segmental aneusomies. RBT patients with RPL have a higher rate of miscarriage of euploid fetuses with RBT. CONCLUSION: Chromosome-specific factors, female gender, age, and history of RPL are the risk elements influencing pregnancy and in vitro fertilization success in RBT patients. Chromosomal microarray analysis of POC is necessary to provide an accurate and timely diagnosis for patients with adverse reproductive outcomes.

Cytogenetic signatures of recurrent pregnancy losses.

OBJECTIVES: To investigate the incidence of chromosomal abnormalities in the products of conception (POC) of patients with spontaneous miscarriages (SM) and with recurrent pregnancy losses (RPL) and to determine biological mechanisms contributing to RPL. METHODS: During a 20-year period, 12 096 POC samples underwent classical chromosome analysis. Cytogenetic findings were compared between the SM and RPL cohorts. RESULTS: Analysis of RPL cohort has identified an increased incidence of inherited and de novo structural chromosome abnormalities, recurrent polyploid conceptions, and complex mosaic alterations. These abnormalities are the signature of genomic instability, posing a high risk of genetic abnormalities to offspring independent of maternal age. Predominance of male conceptions in the RPL cohort points toward an X-linked etiology and gender-specific intolerance for certain genetic abnormalities. CONCLUSIONS: Our study showed several possible genetic etiologies of RPL, including parental structural chromosome rearrangements, predisposition to meiotic nondisjunction, and genomic instability. Loss of karyotypically normal fetuses might be attributed to defects in genes essential for fetal development, as well as aberrations affecting the X chromosome. Molecular studies of parental and POC genomes will help to identify inherited defects in genes involved in meiotic divisions and DNA repair to confirm our hypotheses, and to discover novel fetal-essential genes.

Copy number alterations involving 59 ACMG-recommended secondary findings genes.

In clinical exome/genome sequencing, the American College of Medical Genetics and Genomics (ACMG) recommends reporting of secondary findings unrelated to a patient's phenotype when pathogenic single-nucleotide variants (SNVs) are observed in one of 59 genes associated with a life-threatening, medically actionable condition. Little is known about the incidence and sensitivity of chromosomal microarray analysis (CMA) for detection of pathogenic copy number variants (CNVs) comprising medically-actionable genes. Clinical CMA has been performed on 8865 individuals referred for molecular cytogenetic testing. We retrospectively reviewed the CMA results to identify patients with CNVs comprising genes included in the 59-ACMG list of secondary findings. We evaluated the clinical significance of these CNVs in respect to pathogenicity, phenotypic manifestations, and heritability. We identified 23 patients (0.26%) with relevant CNV either deletions comprising the entire gene or intragenic alterations involving one or more secondary findings genes. A number of patients and/or their family members with pathogenic CNVs manifest or expected to develop an anticipated clinical phenotype and would benefit from preventive management similar to the patients with pathogenic SNVs. To improve patients' care standardization should apply to reporting of both sequencing and CNVs obtained via clinical genome-wide analysis, including chromosomal microarray and exome/genome sequencing.

Importance of complete phenotyping in prenatal whole exome sequencing.

Whole exome sequencing (WES) is an emerging technique in prenatal diagnosis. In this retrospective study, we examined diagnostic utility and limitations of WES in prenatal cases with structural birth defects. DNA from 20 trios (fetal and parental), with normal karyotype and microarray findings, underwent WES and variant interpretation at a reference laboratory. The WES results were later re-evaluated in our academic center utilizing prenatal and postnatal phenotyping. Initial analysis using only prenatal ultrasound findings revealed no pathogenic or likely pathogenic variants in 20 pregnancies with structural birth defects. Re-analysis of WES variants and combination of prenatal and postnatal phenotyping yielded pathogenic variants in at least 20% of cases including PORCN gene in a fetus with split-hand/foot malformation, as well as variants of uncertain significance in NEB and NOTCH1 in fetuses with postnatal muscle weakness and Adams-Oliver syndrome, respectively. Furthermore, Sanger sequencing in a patient with holoprosencephaly, elucidated by postnatal MRI, revealed a pathogenic 47-base pairs deletion in ZIC2 which was missed by prenatal WES. This study suggests that incomplete prenatal phenotyping and lack of prenatal ultrasound-genotype databases are the limiting factors for current interpretation of WES data in prenatal diagnosis. Development of prenatal phenotype-genotype databases would significantly help WES interpretation in this setting. Patients who underwent prenatal clinical WES may benefit from the re-analysis based on detailed postnatal findings.

Investigating Pregnancy Outcomes After Abnormal Cell-Free DNA Test Results.

Cell-free DNA (cfDNA) testing has increased sensitivity and specificity compared to other prenatal screening methods, but invasive diagnostic testing (IDT) is recommended for confirmation. We performed a retrospective chart review of 39 women with abnormal cfDNA results between March 2012 and September 2015 at an urban academic hospital to evaluate patient choice and pregnancy outcomes. We analyzed data using descriptive statistics, Fisher's exact tests, and Wilcoxon rank-sum tests. Median maternal age was 36.0 years [interquartile range (IQR) 31, 39]; 64.1% of women (25/39) were advanced maternal age and 69.2% (27/39) had abnormal ultrasounds. Median gestational age at time of cfDNA testing was 18 3/7 weeks [IQR 12 2/7, 20 5/7]. cfDNA results included trisomy 21 (89.7%, 35/39), trisomy 18 (7.7%, 3/39), and both trisomy 21/monosomy X (2.6%, 1/39). Of 39 women, 22 (56.4%) continued and 10 (25.6%) terminated the pregnancy; six (15.4%) had fetal demises, and one was lost to follow-up. Of women continuing their pregnancies, 54.6% (12/22) declined further genetic counseling, and 77.3% (17/22) declined IDT. Only 14 women pursued IDT; not pursuing IDT was associated with continuing the pregnancy (Fisher's exact test, p = .001). All women terminating their pregnancy (90.0%, 9/10) pursued IDT or had major anomalies on ultrasound, suggesting that women considering termination undergo more confirmatory tests or already have high suspicion for an abnormal pregnancy.

Beyond Down syndrome phenotype: Paternally derived isodicentric chromosome 21 with partial monosomy 21q22.3.

Inverted isodicentric chromosome 21 is a rare form of chromosomal rearrangement that may result in trisomy 21; sometimes this rearrangement may also lead to segmental monosomy of the terminal long arm of chromosome 21. In this report, we describe the prenatal diagnosis and neonatal follow-up of a child with a paternally derived, de novo isodicentric chromosome 21 and a concurrent ∼1.2 Mb deletion of the 21q22.3 region [46,XX,idic(21)(q22.3)]. This child presented with unusual phenotype of Down syndrome and additional defects including esophageal atresia and tethered cord syndrome. The resulting phenotype in this infant might be a coalescence of the partial trisomy and monosomy 21, as well as homozygosity for idic (21). The utilization of chromosomal microarray in this case enabled accurate characterization of a rare chromosome abnormality, potentially contributes to future phenotype-genotype correlation and produced evidence for a molecular mechanism underlying this rearrangement.

Genotype-phenotype correlation and pregnancy outcomes of partial trisomy 14q: A systematic review.

Over the last decade, several advances in ultrasound techniques, increasing availability of whole genome microarray testing, and overall expansion of our knowledge about the human genome have drastically enhanced our ability to detect chromosomal abnormalities prenatally. Despite that, genotype-phenotype correlation is difficult to establish for many chromosomal aberrations, particularly for those that are rare, as it requires thorough analysis of a significant number of cases. This in turn increases the burden of the obstetric provider to appropriately counsel a patient regarding prognosis and pregnancy options in these complicated situations. Our experience in prenatal diagnosis and management of a fetus with multiple anomalies and partial trisomy for the 14q11-q24.2 prompted a comprehensive analysis of the relevant literature. Although complete non-mosaic trisomy 14 is associated with first trimester miscarriages, partial trisomy 14q is a rare condition with undefined genotype-phenotype correlation, preventing accurate prenatal counseling, and informed decision making. We performed a systematic literature review, that aimed to summarize prenatal and postnatal findings of individual case reports on 51 patients with partial trisomy 14q in order to elucidate genotype-phenotype correlation, and to supply healthcare professionals with recommendation on essential fetal and parental testing for accurate diagnosis, pregnancy outcomes, and proper family counseling. Comparison of the clinical findings among the patients with partial 14q trisomy suggest that the resulting phenotype is likely to be influenced by the extent of the 14q trisomy segment, associated chromosomal imbalances, parental origin of the rearrangement, and dosage of the genes within the imprinted 14q32 cluster. © 2016 Wiley Periodicals, Inc.

Expansion of phenotype and genotypic data in CRB2-related syndrome.

Sequence variants in CRB2 cause a syndrome with greatly elevated maternal serum alpha-fetoprotein and amniotic fluid alpha-fetoprotein levels, cerebral ventriculomegaly and renal findings similar to Finnish congenital nephrosis. All reported patients have been homozygotes or compound heterozygotes for sequence variants in the Crumbs, Drosophila, Homolog of, 2 (CRB2) genes. Variants affecting CRB2 function have also been identified in four families with steroid resistant nephrotic syndrome, but without any other known systemic findings. We ascertained five, previously unreported individuals with biallelic variants in CRB2 that were predicted to affect function. We compiled the clinical features of reported cases and reviewed available literature for cases with features suggestive of CRB2-related syndrome in order to better understand the phenotypic and genotypic manifestations. Phenotypic analyses showed that ventriculomegaly was a common clinical manifestation (9/11 confirmed cases), in contrast to the original reports, in which patients were ascertained due to renal disease. Two children had minor eye findings and one was diagnosed with a B-cell lymphoma. Further genetic analysis identified one family with two affected siblings who were both heterozygous for a variant in NPHS2 predicted to affect function and separate families with sequence variants in NPHS4 and BBS7 in addition to the CRB2 variants. Our report expands the clinical phenotype of CRB2-related syndrome and establishes ventriculomegaly and hydrocephalus as frequent manifestations. We found additional sequence variants in genes involved in kidney development and ciliopathies in patients with CRB2-related syndrome, suggesting that these variants may modify the phenotype.

Ultrasonography estimates of fetal growth in fetuses affected by trisomy 21.

OBJECTIVE: To construct growth curves specific for fetuses with trisomy 21 (T21) and to compare them with the reference-based standard. METHODS: A retrospective cohort study was conducted of ultrasonography examinations from women with singleton pregnancies with a confirmed diagnosis of T21 who sought care at an academic tertiary-care center in the USA between January 1, 2003, and December 31, 2013. Growth curves were constructed using linear regression and compared with the Hadlock standard. RESULTS: The study included 425 ultrasonography examinations from 235 women. The head circumference and femur length were smaller than the reference standards at all gestational ages (head circumference: P=0.017; femur length: P<0.001). The abdominal circumference was larger than the reference standard from 29weeks onward (P<0.001). The biparietal diameter was smaller in the second trimester and in the late third trimester (P<0.001). The overall estimated fetal weight was not different from the reference standard. CONCLUSION: The T21-specific growth curves indicate anthropometric differences between T21 fetuses and the general population. Once validated, such individual growth curves could allow for more accurate prenatal assessment and management of fetuses affected by T21.

Prenatal whole-exome sequencing: parental attitudes.

OBJECTIVE: The aim of this study was to survey the opinions of expectant parents regarding prenatal whole-exome sequencing. METHODS: The study used a questionnaire that focused on acceptability of prenatal whole-exome sequencing to individuals who pursued first-trimester prenatal screening in a tertiary academic medical center. A total of 186 expectant individuals completed the questionnaire. The results of the questionnaire were analyzed using descriptive statistics and logistic regression models. RESULTS: Eighty-three percent of the participants answered that prenatal whole-exome sequencing should be offered, 14.8% were neutral, and only 2.2% disagreed. Fifty-four percent of the participants were interested in having prenatal whole-exome sequencing for their fetus, 40.1% were neutral, and 6.6% disagreed. The majority of participants expressed a desire to know about treatable (96.2%) and non-treatable (86.3%) childhood conditions, and most said the same for treatable (76.0%) and non-treatable (74.3%) adult-onset conditions. Over half of the participants (59.7%) indicated a maximum acceptable turnaround time of 3 weeks or less for prenatal whole-exome sequencing. CONCLUSIONS: The majority of respondents felt prenatal whole-exome sequencing should be offered. Moreover, the majority wanted to know prenatally about treatable and non-treatable childhood and adult conditions.

Prenatal diagnosis of trisomy 6q25.3-qter and monosomy 10q26.12-qter by array CGH in a fetus with an apparently normal karyotype.

We present the prenatal case of a 12.5-Mb duplication involving 6q25-qter and a 12.2-Mb deletion encompassing 10q26-qter diagnosed by aCGH, while conventional karyotype showed normal results. The genotype-phenotype correlation between individual microarray and clinical findings adds to the emerging atlas of chromosomal abnormalities associated with specific prenatal ultrasound abnormalities.

Maternal cell-free DNA-based screening for fetal microdeletion and the importance of careful diagnostic follow-up.

BACKGROUND: Noninvasive prenatal screening (NIPS) by next-generation sequencing of cell-free DNA (cfDNA) in maternal plasma is used to screen for common aneuploidies such as trisomy 21 in high risk pregnancies. NIPS can identify fetal genomic microdeletions; however, sensitivity and specificity have not been systematically evaluated. Commercial companies have begun to offer expanded panels including screening for common microdeletion syndromes such as 22q11.2 deletion (DiGeorge syndrome) without reporting the genomic coordinates or whether the deletion is maternal or fetal. Here we describe a phenotypically normal mother and fetus who tested positive for atypical 22q deletion via maternal plasma cfDNA testing. METHODS: We performed cfDNA sequencing on saved maternal plasma obtained at 11 weeks of gestation from a phenotypically normal woman with a singleton pregnancy whose earlier screening at a commercial laboratory was reported to be positive for a 22q11.2 microdeletion. Fluorescence in situ hybridization and chromosomal microarray diagnostic genetic tests were done postnatally. CONCLUSION: NIPS detected a 22q microdeletion that, upon diagnostic workup, did not include the DiGeorge critical region. Diagnostic prenatal or postnatal testing with chromosomal microarray and appropriate parental studies to determine precise genomic coordinates and inheritance should follow a positive microdeletion NIPS result.

A checklist for timeout on labor and delivery: a pilot study to improve communication and safety.

OBJECTIVE: To assess the impact on staff communication of a standardized checklist for timeout for patients undergoing a trial of labor after cesarean section and/or elective induction at term. STUDY DESIGN: A comparison of presurvey and postsurvey questionnaire results for labor and delivery personnel assessing communication before and after checklist implementation. RESULTS: From October 2011 through March 2012, 52.9% (N=37) of 70 eligible patients had the standardized checklist for timeout performed. Prior to implementation of the checklist, 66% of respondents (48.8% of nurses, 100% of residents, 90% of attendings) slightly or strongly agreed that their opinions were heard versus 83% of respondents during the study period (73.7% of nurses, 100% of residents, 100% of attendings). Following the intervention, nurses reported that they were more likely to feel as though their opinions were heard (p = 0.05). CONCLUSION: Implementation of a formalized obstetric timeout improved the subjective perception of communication among obstetric staff. This tool has the potential to improve patient safety in labor and delivery.

Screening for fetal aneuploidy: is maternal age relevant?

The process of genetic screening has evolved from a simple notation of maternal age to a complex algorithm incorporating age, maternal serum screening, and sonographic findings. The extent to which each of these variables should contribute to the overall screening result is much debated and deserves continued research. It is clear that maternal age provides useful information when used as part of this equation but should not represent the sole screening modality. The use of genetic screening in a general population should be examined in terms of cost effectiveness without sacrificing patient preference and autonomy.

Genetic screening in a university clinic: impact of primary language.

OBJECTIVE: To contrast Spanish-speaking (S) with English-speaking (E) obstetric patients regarding utilization of genetic screening, motivation for undergoing/declining screening, pregnancy-related anxiety, knowledge about genetic conditions, and printed information as an adjunct to counseling. METHOD: Paper surveys were given to patients (n = 121) in an academic OB/GYN clinic or placed in charts (n = 271) over a 4-week period. Comparisons were evaluated with Chi-square and Fisher's exact tests. RESULTS: Completed surveys were returned from 245 gravidas (response rate 63%, S 48%, and E 67%). Uptake of genetic screening was similar between the groups (S 69% vs. E 57%, p = 0.13). No significant differences were noted in patients' motivation regarding screening, source of screening information, or self-assessed pregnancy-related anxiety. Familiarity of genetic disorders other than Down syndrome differed between the S and E groups (p < 0.003). Perceived positive utility of printed information differed significantly when groups were analyzed by language (S 85% vs. E 47%, p < 0.001) and by uptake of screening(screened 62% vs. not screened 44%, p = 0.006). CONCLUSION: A majority of study participants (n = 147, 60%) chose genetic screening; uptake and motivation were similar across language groups. Familiarity with genetic conditions was deficient and screening terminology confusing regardless of primary language. The perceived positive utility of printed information (S > E) highlights the importance of clear and early counseling.

Patient preferences for screening in the first trimester.

OBJECTIVE: We theorized that a significant number of women would choose integrated screening (IS) over first trimester screening (FTS) and that demographic characteristics and baseline anxiety levels might predict which patients would choose each test. We also hypothesized that screening results might alter patients' future preferences. METHOD: Patients underwent non-directive genetic counselling and were offered FTS or IS. Prior to undergoing nuchal translucency (NT) ultrasound, patients were given surveys. These questionnaires were repeated in the second trimester and postpartum. They focussed on the patients' background, motivations for screening and anxiety level. RESULTS: Out of 110 patients surveyed, 81 returned their initial questionnaire. 60% of these patients chose FTS and 40% chose IS. There were no demographic differences between the groups. FTS patients desired early reassurance while IS patients wanted the 'most information' and the lowest chance of needing diagnostic testing. In all, 47 women returned second trimester questionnaires and 35 women returned postpartum surveys. These surveys demonstrated that women's motivations remained constant over time. Of the five screen positive results, only one woman regretted her screening choice. CONCLUSION: One cannot predict patients' screening preferences, thus both FTS and IS should be offered. Given non-directive counselling, patients are generally satisfied with their screening choices.

Current methods of prenatal screening for Down syndrome and other fetal abnormalities.

Prenatal diagnosis of Down syndrome is widely available, but the determination of which patients should undergo prenatal diagnosis is changing. With the recent acceptance of first-trimester and integrated screening as a part of routine clinical practice, there are now a variety of accepted screening protocols for Down syndrome and other aneuploidies. These choices can be confusing both to both patients and providers. The following discussion is meant to outline the various options in prenatal screening, and their individual advantages and disadvantages.

Prenatal screening for Down syndrome: current and future methods.

Second-trimester serum screening for Down syndrome has had a relatively long clinical life, beginning in the mid-1980s and continuing to the present day. In the past few years, however, new screening methods that involve testing just a few weeks earlier and the integration of first-trimester and second-trimester markers have been proposed and are being used. These improved methods have begun the transition to better and, hopefully, safer prenatal screening. In the past, as many as 1 in 10 pregnant women learned that they were at increased risk of having a baby with a serious birth defect and had to decide whether to have an invasive diagnostic procedure. Now, screening methods are at the point where as few as 1 in 50 or 1 in 100 pregnant women are found to be at increased risk. The ultimate goal in screening is to make noninvasive testing methods so safe that only those few women who are found to be at the very highest risk will need to face the uncertainty of invasive procedures. In the next few years, that goal will probably be achieved.

Second-trimester maternal serum placental growth factor and vascular endothelial growth factor for predicting severe, early-onset preeclampsia.

OBJECTIVE: To determine whether alterations in second-trimester maternal serum cytokine concentrations can identify women at risk for developing severe, early-onset preeclampsia. METHODS: Patients with severe preeclampsia requiring delivery prior to 34 weeks (n = 20) were each matched by gestational age, gravidity, parity, and sample freezing time with three healthy controls who delivered at term (n = 60). By using second-trimester maternal sera originally collected for fetal aneuploidy screening, the concentrations of placental growth factor, vascular endothelial growth factor, granulocyte colony-stimulating factor, endothelin-1, and human chorionic gonadotropin were compared between patients and controls. Logistic regression analysis was used to estimate odds ratios for high versus low (median split) cytokine concentrations with respect to the development of severe, early-onset preeclampsia. Receiver operating characteristic (ROC) curves based on a second logistic regression, using actual cytokine values, were plotted to illustrate reciprocal impact on sensitivity and specificity. RESULTS: Placental growth factor and vascular endothelial growth factor levels were significantly lower in patients than in controls. No significant differences were observed for the other cytokines. The odds ratios (with 95% confidence intervals) were 15.54 (3.29, 73.40) for vascular endothelial growth factor and 4.20 (1.35, 13.06) for placental growth factor. Receiver operating characteristic analysis of placental growth factor and vascular endothelial growth factor confirmed that both were useful in discriminating between patients and controls. Models combining both vascular endothelial growth factor and placental growth factor provided the best performance for identifying patients at risk for developing severe, early-onset preeclampsia, according to both odds ratios and ROC analyses. CONCLUSION: Combined analysis of placental growth factor and vascular endothelial growth factor is potentially useful as a tool for early identification of patients at risk for developing severe, early-onset preeclampsia.