Subchromosomal Abnormalities Research Articles

Application of Genetic Origin Analysis of Copy Number Variations in Non-Invasive Prenatal Testing.

This study aimed to assess the application of origin analysis of copy number variations (CNVs) in non-invasive prenatal testing (NIPT) and provide a basis for expanding the clinical application of NIPT. We enrolled 35,317 patients who underwent NIPT between January 2019 and March 2023. Genome sequencing of copy number variation (CNV-Seq) analysis was performed using the CNV calling pipeline to identify subchromosomal abnormalities in maternal plasma. Genetic origin was determined by comparing the chimaerism ratio of CNV and the concentration of cell-free foetal DNA (cffDNA). All pregnant women with a high risk of CNV, as indicated by the NIPT, were informed of their genetic origins. Amniocentesis was recommended for detecting the CNVs in foetal chromosomes, and pregnancy outcomes were tracked. A total of 109 pregnancies showed clinically significant positive results for CNV after NIPT, including 65 cases of maternal/foetal (M/F)-CNVs and 44 cases of F-CNVs. The occurrence of M/F-CNVs was independent of age, screening (serological or ultrasound) indications for abnormalities, and mode of pregnancy. The incidence of pathogenic/likely pathogenic (P/LP)-F-CNVs was high in cases where serological screening indicated intermediate, high-risk, or abnormal US findings (p<0.05). In the M/F-CNV group, most of the P/LP-CNVs were small fragments with low penetrance; 55 (84.62%) were less than 5Mb in size, and nine (13.85%) were between 5 and 10Mb. In the F-CNV group, foetal P/LP-CNV was detected in 36 of 42 cases undergoing prenatal diagnosis, and no significant bias was noted in the size distribution of P/LP-F-CNV fragments. The prenatal diagnostic rate and positive predictive value in the F-CNV group were 95.45% and 85.71%, respectively, which were significantly different from those in the M/F group (26.15% and 52.95%), respectively (p<0.05). Genetic origin analysis of CNV can effectively improve adherence to prenatal diagnosis in pregnant women and the accuracy of prenatal diagnosis.

Detection of 22q11.2 deletion syndrome by single-nucleotide polymorphism based non-invasive prenatal test

Non-invasive prenatal test (NIPT) has become a popular screening test worldwide for screening common trisomies. In addition, the test can also sex chromosomal aneuploidies (SCAs) with similar sensitivity. In recent years, the scope of NIPT has extended to screen pregnancies for clinically significant microdeletions (MDs), rare autosomal aneuploidies, and subchromosomal abnormalities. The clinical utility of NIPT screening beyond trisomies 21,18,13 and SCAs are still being evaluated because of low positive predictive value which in turn leads to an increase in invasive procedures. Here, we present a case where SNP - NIPT correctly identified a microdeletion syndrome, i.e., 22q11.2DS in a pregnant woman with normal ultrasound findings. This NIPT finding was further confirmed in the chromosomal microarray study and FISH.

Diagnostic yield of chromosomal microarray and trio whole exome sequencing in congenital brain anomalies

IntroductionThe deductive method: from karyotyping to aCGH and WES is an important aspect in the diagnosis and search for the causes of intellectual disability due to congenital brain anomalies. There is recommendation to exclude the presence of CNV or monogenic variants for patients with a normal karyotype, but with a clinical picture of syndromic disease.ObjectivesImprovement of diagnosis of intellectual disability.MethodsaCGH with 60K Agilent microarrays, WES with SureSelect Human All Exon V8ResultsPathogenic or potentially pathogenic CNVs were excluded previously by aCGH for 10 families (total 32 people, 2 families had 2 children) with intellectual disability and congenital brain anomalies (for example, polymicrogyria, pachygyria, lissencephaly). The WES identified candidate variants for all families that can lead to impaired neurodevelopment, including 3 pathogenic variants in 3 families, 3 likely pathogenic in three other families, and 10 variants with uncertain clinical significance for 4 families. Almost all of these variants were identified de novo, except for one family, where the proband has been a compound heterozygous for two variants in the RELN gene. The first case of pathogenic mutation de novo was detected in a girl with agenesis of the corpus callosum. It was a missense mutation DYNC1H1 (NM_001376.5): c.4868G&gt;A (p.Arg1623Gln), which leads to impaired intellectual development in autosomal dominant type 13 (OMIM 614563). The second variant was detected in a boy with corpus callosum agenesis, pontine hypogenesis, pachygyria in the frontal lobes. It was a missense variant MACF1 (ENST00000567887.5): c.21989A&gt;G(p.Asp7330Gly), which leads to lissencephaly 9 with complex brainstem malformation (OMIM 614563). The third variant was found in a girl with epilepsy and impaired myelination of the white matter of the parietal-occipital areas of the cerebral hemispheres. It was a missense variant CDKL5 (NM_001323289.2):c.404-1G&gt;A that leads to developmental and epileptic encephalopathy 2 (OMIM 300672).ConclusionsSixteen candidate variants potentially responsible for mental health were reported in this study. Most of these variants were missense changes in genes. All except one anomalies arisen de novo. Trio-based WES has been shown to be an important step in making a genetic diagnosis if other chromosomal and subchromosomal abnormalities had been excluded. The clinical description of the patient is the most important step for the correct interpretation of WES results, which allows to establish the exact genetic cause of the disease if several variants with unclear clinical significance were previously identified.This study was supported by the Russian Science Foundation, grant 21-65-00017, https://rscf.ru/project/21-65-00017/Disclosure of InterestNone Declared

Prenatal Diagnosis and Outcomes in Fetuses with Hemivertebra.

Background: There are few studies on the burden of chromosomal abnormalities and single gene disorders in fetal hemivertebra (HV). We aim to investigate the cytogenetic and monogenic risk and evaluate prenatal outcomes of fetal HV. Method: This study included fetuses diagnosed with HV divided into two groups: isolated HV and non-isolated HV. Data on other sonographic structural anomalies, chromosomal and sub-chromosomal abnormalities, monogenic variations detected by WES, and prenatal outcomes are recorded and reviewed. Results: Among 109 fetal HV cases, forty-seven (43.1%) non-isolated HV cases were associated with structural anomalies. Chromosomal test results were available in 58 cases, identifying six (10.3%) chromosomal aberrations involved in four isolated and two non-isolated HV. WES identified four (likely) pathogenic variants in three cases among 16 fetuses with HV, involving three novel variants, 1250G > T and c.1277G> inherited from parents, respectively, in DLL3 and c.7213C > A ** in the FLNB. The live birth rate (LB) was higher in the isolated fetal HV group than in the non-isolated group (67.7% (42/62) vs. 12.5% (12/47), p < 0.001). Conclusion: This study emphasizes the risk of cytogenetic abnormalities in isolated HV. WES yields a diagnostic rate of 18.3% in HV with normal CMA, probably aiding the prenatal counseling and management of fetal HV.

Should prenatal chromosomal microarray analysis be offered for isolated ventricular septal defect? A single-center retrospective study from China.

ObjectiveTo evaluate the utility of chromosomal microarray analysis (CMA) in fetuses with isolated ventricular septal defect (VSD) and to explore the favorable factors for predicting spontaneous closure of defects.MethodsThe study included 436 singleton pregnancies seen at a referral prenatal diagnosis center, between January 2016 and May 2020, of which 168 fetuses with isolated VSD were diagnosed in the prenatal setting. VSD was classified as an isolated VSD whether it had ultrasound soft markers or not. All patients underwent testing employing quantitative fluorescent polymerase chain reaction (QF-PCR) and CMA as the first-line genetic detection strategies, mainly in amniotic fluid and umbilical blood samples. Rates of chromosomal abnormalities were compared by subgroups of isolated VSD (muscular or perimembranous). Binary logistic regression analysis was performed to predict the independent determinants of spontaneous closure by 2 years.ResultsOverall, the CMA identified clinically significant copy number variations (CNVs) in 7/168 (4.2%) fetuses and variants of unknown significance (VOUS) in 15/168 (8.9%). Muscular and perimembranous VSDs were found in 53.6 and 46.4%, respectively. Clinically significant relevant subchromosomal aberrations were revealed in seven (9.0%) perimembranous VSDs compared with none in 90 muscular defects (P < 0.01). The median initial size of the defect in the muscular VSDs was 2.2(1.8–2.7) mm, as compared to that of 2.8 (2.2–3.2) mm in the perimembranous VSDs group (p = 0.000). In muscular vs. perimembranous VSDs, spontaneous closure occurred more frequently and earlier [40.0 vs. 15.4% in utero (p = 0.000), 61.1 vs. 30.8% at 1-year (p = 0.000), and 75.6 vs. 42.3% at 2-year (P = 0.000)]. Postnatal surgical closure was warranted in 4/90 (4.4%) of the infants with muscular VSDs, as compared to 29/71 (40.8%) with perimembranous defects (p = 0.000). Furthermore, isolated muscular type VSD, smaller defect size, and maternal age of less than 35 years are all positive predictors of spontaneous closure of the defects.ConclusionThis study highlighted the value of microarray for unbalanced subchromosomal abnormalities in fetuses with isolated VSD, particularly in the perimembranous defects. The detection of an isolated muscular VSD prenatally may be considered a benign or likely benign finding; in contrast, for perimembranous VSD, a prenatal CMA should be offered.

EP474: Prenatal exome sequencing analysis in the clinical setting of fetuses with structural anomalies or increased nuchal translucency

Although prenatal screening and diagnosis are generally mostly focused on the detection of aneuploidies and subchromosomal abnormalities, testing for monogenic disorders has become an important prenatal diagnostic test for pregnancies complicated by congenital anomalies. Multiple studies using gene panels and exome sequencing of fetal samples have revealed that pathogenic variants in single genes are an important cause of congenital anomalies. In this study, we reviewed the indications and results of prenatal trio-exome sequencing performed in a clinical diagnostic laboratory.

Karyotyping and Chromosomal Microarray Analysis in Women Requesting Amniocentesis for Isolated Sonographic Soft Markers or Advanced Maternal Age.

Background:Chromosomal microarray analysis (CMA) has gained acceptance in prenatal diagnosis, gradually replacing the traditional cytogenetic analysis following amniocentesis or chorionic villi sampling due to its higher resolution than traditional cytogenetics.Objctive:The present study investigated the prevalence of major and sub-chromosomal abnormalities in fetuses with isolated ultrasound findings during routine anatomy scan or pregnancies of advanced maternal age after maternal request without medical indication.Methods:Total number of 126 cases were included in total, consisted of two groups; the first group with isolated sonographic soft markers (84 fetuses) and the second group of advanced maternal age (42 fetuses). The group of isolated sonographic markers was further divided per anatomical system affected. The prevalence of genetic aberrations via QF-PCR and CMA was noted.Results:Clinically significant genetic abnormalities were detected in 12% of the first and 7% of cases in the second group. Interestingly, 40% and 67% of abnormal cases in the first and second group respectively, were identified only after CMA, and they would have missed diagnosis with standard karyotype or QF-PCR alone. Most genetic aberrations were detected in fetuses with findings in the central nervous, craniofacial, cardiovascular, and musculoskeletal system. Sub-microscopic chromosomal aberrations identified only after CMA were gathered in cases with short long bones and in one case with ventriculomegaly.Conclusion:Even in pregnancies with a first trimester screening low risk result, the risk of identifying a clinically significant CMA aberration is considerable, when an isolated sonographic marker is identified later on in pregnancy or maternal age is advanced.

Consistency in rates of diagnosis of embryonic mosaicism, segmental abnormalities, and “no call” results among experienced embryologists performing preimplantation genetic testing for aneuploidy

ObjectiveTo determine whether differences exist in rates of subchromosomal abnormalities, mosaicism, and “no call” results among embryologists performing and loading trophectoderm biopsies for preimplantation genetic testing for aneuploidy (PGT-A).DesignRetrospective cohort.SettingLarge infertility center.Patient(s)All patients undergoing in vitro fertilization with PGT-A.Intervention(s)The NexCCS next generation sequencing platform was used for PGT-A. The χ2 testing assessed differences in rates of primary outcomes between embryologists. Intraclass correlation coefficients evaluated inter-embryologist reliability in rates of abnormal and no call results. Median absolute performance difference (MAPD) scores, which quantify the impact of technical variation on analytical performance, were averaged for individual embryologists. Analysis of variance assessed differences in mean MAPD scores.Main Outcome Measure(s)Interoperator variability in rates of mosaic, segmental, and no call results.Result(s)Four embryologists performed 30,899 biopsies and 6 embryologists loaded specimens into designated tubes. Among individuals performing trophectoderm sampling, rates of mosaicism were 4.3% to 6.1%, segmental errors were 9.0% to 10.7%, and inconclusive results were 1.1% to 2.9%. For those loading, the incidence of mosaicism was 4.2% to 5.9%, subchromosomal abnormalities was 9.7% to 10.4%, and no call results was 1.2% to 2.2%. The intraclass correlation coefficient was 0.978 for embryologists performing biopsies and 0.981 for those loading. Differences in mean MAPD scores were within 0.6% and 0.2% of each other for doing biopsies and loading embryologists, respectively.Conclusion(s)Rates of mosaicism, segmental, and no call PGT-A results are consistent among experienced embryologists. Due to the large sample size included, differences within 1% of the mean were deemed clinically irrelevant despite statistical significance.

Preimplantation Genetic Testing for Chromosomal Abnormalities: Aneuploidy, Mosaicism, and Structural Rearrangements.

There is a high incidence of chromosomal abnormalities in early human embryos, whether they are generated by natural conception or by assisted reproductive technologies (ART). Cells with chromosomal copy number deviations or chromosome structural rearrangements can compromise the viability of embryos; much of the naturally low human fecundity as well as low success rates of ART can be ascribed to these cytogenetic defects. Chromosomal anomalies are also responsible for a large proportion of miscarriages and congenital disorders. There is therefore tremendous value in methods that identify embryos containing chromosomal abnormalities before intrauterine transfer to a patient being treated for infertility—the goal being the exclusion of affected embryos in order to improve clinical outcomes. This is the rationale behind preimplantation genetic testing for aneuploidy (PGT-A) and structural rearrangements (-SR). Contemporary methods are capable of much more than detecting whole chromosome abnormalities (e.g., monosomy/trisomy). Technical enhancements and increased resolution and sensitivity permit the identification of chromosomal mosaicism (embryos containing a mix of normal and abnormal cells), as well as the detection of sub-chromosomal abnormalities such as segmental deletions and duplications. Earlier approaches to screening for chromosomal abnormalities yielded a binary result of normal versus abnormal, but the new refinements in the system call for new categories, each with specific clinical outcomes and nuances for clinical management. This review intends to give an overview of PGT-A and -SR, emphasizing recent advances and areas of active development.

Incidence, Origin, and Predictive Model for the Detection and Clinical Management of Segmental Aneuploidies in Human Embryos

Despite next-generation sequencing, which now allows for the accurate detection of segmental aneuploidies from invitro fertilization embryo biopsies, the origin and characteristics of these aneuploidies are still relatively unknown. Using a multifocal biopsy approach (four trophectoderms [TEs] and one inner cell mass [ICM] analyzed per blastocyst; n = 390), we determine the origin of the aneuploidy and the diagnostic predictive value of segmental aneuploidy detection in TE biopsies toward the ICM's chromosomal constitution. Contrary to the prevalent meiotic origin of whole-chromosome aneuploidies, we show that sub-chromosomal abnormalities in human blastocysts arise from mitotic errors in around 70% of cases. As a consequence, the positive-predictive value toward ICM configuration was significantly lower for segmental as compared to whole-chromosome aneuploidies (70.8% versus 97.18%, respectively). In order to enhance the clinical utility of reporting segmental findings in clinical TE biopsies, we have developed and clinically verified a risk stratification model based on a second TE biopsy confirmation and segmental length; this model can significantly improve the prediction of aneuploidy risk in the ICM in over 86% of clinical cases enrolled. In conclusion, we provide evidence of the predominant mitotic origin of segmental aneuploidies in preimplantation embryos and develop a risk stratification model that can help post-test genetic counseling and that facilitates the decision-making process on clinical utilization of these embryos.

Cell-free fetal DNA screening for detection of microdeletion syndromes: a cost-effectiveness analysis

Objective Fetuses with genetic copy number variants are poorly detected through traditional prenatal screening. Microdeletions and duplications are clearly identified with diagnostic testing through chromosomal microarray, and screening of a select number of microdeletions has become available with cell-free DNA (cfDNA). Our study compares the costs and outcomes of cfDNA for five pathogenic microdeletions and aneuploidy to cfDNA for aneuploidy alone in conjunction with ultrasound. Methods A decision-analytic model was constructed using TreeAge software to compare cfDNA with microdeletions versus traditional cfDNA in a theoretical cohort of 4,000,000 pregnancies that would also be screened with ultrasound. Probabilities, costs, and utilities were derived from literature. The primary outcomes were the incremental cost per quality-adjusted life-year (QALY), terminations, and procedure-related losses. Because the microdeletion results are available, but not reported, on all cfDNA testing we set the incremental cost of the cfDNA microdeletion screening test to zero at baseline and varied the cost in sensitivity analysis. Results Screening with cfDNA for microdeletions among all pregnant women would result in 83 fewer anomalous neonates compared to traditional cfDNA with ultrasound. This reduction is due to increased diagnosis and termination of fetuses with microdeletions in this group. Routine use of cfDNA with microdeletions resulted in more procedure-related losses. cfDNA with microdeletions would improve effectiveness by 977 QALYs and decrease costs by $90,991,784. When we varied the specificity of the screening test, we found that it remained cost-effective down to a specificity of 91%. With a threshold of $100,000/QALY, microdeletion screening is cost-effective to an incremental increase in cost over cfDNA for aneuploidy alone of $47.10. Conclusion For detection of fetal subchromosomal abnormalities, use of cfDNA with microdeletions is a cost-effective strategy compared to cfDNA for aneuploidy alone in conjunction with ultrasound. Cell-free DNA for microdeletions is not currently recommended as routine screening for low-risk obstetric populations by the American College of Obstetrics and Gynecologists or the Society for Maternal–Fetal Medicine. The test characteristics of cfDNA with microdeletions require greater examination before being routinely recommended.

885: Cell-free fetal DNA screening for detection of microdeletion syndromes: A cost-effectiveness analysis

Copy number variants including microdeletion syndromes are poorly detected through traditional screening. While they can be diagnosed with invasive testing with chromosomal microarray analysis, cell-free DNA (cf DNA) screening is also currently available for detection of select microdeletions. Our study aims to investigate the costs and outcomes associated with detection of five microdeletion syndromes, using cf DNA to screen for microdeletions and aneuploidy compared to cf DNA for aneuploidy alone but in conjunction with ultrasound. A decision-analytic model was constructed using TreeAge software to compare cf DNA with microdeletions versus cf DNA with aneuploidies plus ultrasound in a theoretical cohort of 4,000,000 pregnancies for detection of 22q11.2, Prader-Willi, Angelman, Cri-du-chat, and 1p36 deletion syndromes. All probabilities, costs, and utilities were derived from literature. The primary outcome was incremental cost per quality-adjusted life year (QALY) in addition to terminations and procedure-related losses. Because the microdeletion results are available on all cf DNA testing, we set the incremental cost of cf DNA microdeletion screening to zero at baseline, but varied the cost in sensitivity analysis. Screening with cf DNA for microdeletions among all pregnant women would result in 75 fewer anomalous neonates as compared to cf DNA with ultrasound (Table). This reduction is due to increased termination rates of fetuses with microdeletions in this group. Routine use of cf DNA with microdeletions resulted in more procedure- related losses and losses of normal fetuses. cf DNA with microdeletions would improve effectiveness by 752 QALYs and decrease cost by $83,524,493. When we varied the specificity of the screening test, we found that it remained cost-effective down to a specificity of 92%. With a threshold of $100,000/QALY, microdeletion screening is cost- effective up until an incremental cost over cf DNA for aneuploidy alone of $39.60 (Figure). For fetal detection of subchromosomal abnormalities in pregnancy, cf DNA with microdeletions is a cost-effective strategy compared to cf DNA for aneuploidies and ultrasound. The test characteristics of cf DNA with microdeletions require greater examination and study before being routinely recommended.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Reliable detection of subchromosomal deletions and duplications using cell-based noninvasive prenatal testing.

ObjectiveTo gather additional data on the ability to detect subchromosomal abnormalities of various sizes in single fetal cells isolated from maternal blood, using low‐coverage shotgun next‐generation sequencing for cell‐based noninvasive prenatal testing (NIPT).MethodFetal trophoblasts were recovered from approximately 30 mL of maternal blood using maternal white blood cell depletion, density‐based cell separation, immunofluorescence staining, and high‐resolution scanning. These trophoblastic cells were picked as single cells and underwent whole genome amplification for subsequent genome‐wide copy number analysis and genotyping to confirm the fetal origin of the cells.ResultsApplying our fetal cell isolation method to a series of 125 maternal blood samples, we detected on average 4.17 putative fetal cells/sample. The series included 15 cases with clinically diagnosed fetal aneuploidies and five cases with subchromosomal abnormalities. This method was capable of detecting findings that were 1 to 2 Mb in size, and all were concordant with the microarray or karyotype data obtained on a fetal sample. A minority of fetal cells showed evidence of genome degradation likely related to apoptosis.ConclusionWe demonstrate that this cell‐based NIPT method has the capacity to reliably diagnose fetal chromosomal abnormalities down to 1 to 2 Mb in size.

Does Maternal Body Mass Index Affect the Quantity of Circulating Fetal Cells Available to Use for Cell-Based Noninvasive Prenatal Test in High-Risk Pregnancies?

We present the first study that investigates the effect of maternal body mass index (BMI) on the quantity of circulating fetal cells available to use in cell-based noninvasive prenatal test (cbNIPT). cbNIPT has been proposed as a superior alternative to noninvasive prenatal test from cell-free fetal DNA. Kølvraa et al. [Prenat Diagn. 2016 Dec; 36(12): 1127–34] established that cbNIPT can be performed on as few as one fetal cell, and Vestergaard et al. [Prenat Diagn. 2017 Nov; 37(11): 1120–4] demonstrated that these fetal trophoblast cells could be used successfully in cbNIPT to detect chromosomal and sub-chromosomal abnormalities. This study on 91 pregnant women with high-risk pregnancies suggests that cbNIPT should not be hampered by an increased BMI because every pregnancy, irrespective of the BMI, has rendered fetal cells for downstream genetic analysis. The mean number of fetal cells per sample was 12.6, with a range of 1–43 cells in one sample. ANOVA showed that increasing maternal BMI tends to decrease the number of fetal cells, but not significantly.

Increased Positive Predictive Value for a Single-Nucleotide Polymorphism-Based Non-invasive Prenatal Test for the 22q11.2 Deletion

AbstractNon-invasive prenatal testing (NIPT) has expanded its coverage beyond the common aneuploidies to include subchromosomal abnormalities such as the 22q11.2 deletion through the analysis of cell-free DNA. We previously reported a positive predictive value (PPV) of 18% and false-positive rate (FPR) of 0.38% for a single-nucleotide polymorphism (SNP)-based NIPT for fetal 22q11.2 deletion in a clinical cohort. Herein, we assess the performance of the test following methodology changes that include sequencing high-risk calls at high depth of read (HDOR) and raising the confidence threshold of the algorithm from 0.90 to 0.95. At the original confidence cut-off, a PPV of 42.3% upon reflex-sequencing of high-risk samples at a HDOR was reported. Raising the algorithm’s confidence threshold further increased the PPV to 52.4% and reduced the FPR to 0.07%, with no loss in test sensitivity. The PPV was 100% for high a priori risk cases, and 20% for low a priori risk cases. The improved assay performance documented with the updated methodology supports the efficacy of the SNP-based NIPT in the detection of fetal 22q11.2 deletion.

Validation of a SNP-based non-invasive prenatal test to detect the fetal 22q11.2 deletion in maternal plasma samples.

IntroductionNon-invasive prenatal testing (NIPT) for aneuploidy using cell-free DNA in maternal plasma has been widely adopted. Recently, NIPT coverage has expanded to detect subchromosomal abnormalities including the 22q11.2 deletion. Validation of a SNP-based NIPT for detection of 22q11.2 deletions demonstrating a high sensitivity (97.8%) and specificity (99.75%) has been reported. We sought to further demonstrate the performance of a revised version of the test in a larger set of pregnancy plasma samples.MethodsBlood samples from pregnant women (10 with 22q11.2-deletion‒affected fetuses and 390 negative controls) were successfully analyzed using a revised SNP-based NIPT for the 22q11.2 deletion. The sensitivity and specificity of the assay were measured.ResultsSensitivity of the assay was 90% (9/10), and specificity of the assay was 99.74% (389/390), with a corresponding false positive-rate of 0.26%.DiscussionThe data presented in this study add to the growing body of evidence demonstrating the ability of the SNP-based NIPT to detect 22q11.2 deletions with high sensitivity and specificity.

P16.02: Detecting aneuploidies and subchromosomal abnormalities by use of circulating fetal trophoblast cells in maternal blood

P16.02: Detecting aneuploidies and subchromosomal abnormalities by use of circulating fetal trophoblast cells in maternal blood

Accuracy of a Limited vs Expanded cfDNA Platform for the Detection of Fetal Aneuploidy in a High Risk Population [30Q

INTRODUCTION: Two types of cell free DNA (cfDNA) platforms are available to screen for fetal chromosome abnormalities. The “limited” platform screens for a fixed number of chromosomal abnormalities while the “expanded” platform screens for abnormalities throughout the entire genome. We sought to compare the accuracy of these two platforms. METHODS: We performed a retrospective review of pregnancies undergoing cfDNA, invasive genetic testing, or both, between 2015-2016. Singleton high risk pregnancies were included. Maternal and newborn charts were reviewed to determine which cfDNA platform was performed and whether prenatal or postnatal genetic testing was performed. Appropriate statistical tests were performed for parametric and non-parametric data. Significant chromosome abnormalities were defined as whole chromosome aneuploidy, subchromosomal abnormalities &gt;7 megabases and 22q deletion. RESULTS: 790 cfDNA assays were performed: 384 limited, 406 expanded. 44 chromosomal abnormalities were found (34 prenatally, 10 postnatally). 75% were common trisomies or sex chromosome aneuploidies while 25% were uncommon chromosomal abnormalities (deletions, duplications, unbalanced translocations, triploidy, rare autosomal trisomy). The expanded platform could potentially have detected 94% of the total number of abnormalities while the limited platform could potentially have detected only 75% (p=.03). CONCLUSION: We found that that 25% of chromosomal abnormalities in a high risk population cannot be detected by a limited cfDNA platform. Clinicians should consider usage of an expanded genome wide cfDNA platform that screens for pathogenic, clinically relevant, actionable chromosome abnormalities in patients who prefer non-invasive screening as first line testing.

Cell-free DNA fragment-size distribution analysis for non-invasive prenatal CNV prediction.

Non-invasive detection of aneuploidies in a fetal genome through analysis of cell-free DNA circulating in the maternal plasma is becoming a routine clinical test. Such tests, which rely on analyzing the read coverage or the allelic ratios at single-nucleotide polymorphism (SNP) loci, are not sensitive enough for smaller sub-chromosomal abnormalities due to sequencing biases and paucity of SNPs in a genome. We have developed an alternative framework for identifying sub-chromosomal copy number variations in a fetal genome. This framework relies on the size distribution of fragments in a sample, as fetal-origin fragments tend to be smaller than those of maternal origin. By analyzing the local distribution of the cell-free DNA fragment sizes in each region, our method allows for the identification of sub-megabase CNVs, even in the absence of SNP positions. To evaluate the accuracy of our method, we used a plasma sample with the fetal fraction of 13%, down-sampled it to samples with coverage of 10X-40X and simulated samples with CNVs based on it. Our method had a perfect accuracy (both specificity and sensitivity) for detecting 5 Mb CNVs, and after reducing the fetal fraction (to 11%, 9% and 7%), it could correctly identify 98.82-100% of the 5 Mb CNVs and had a true-negative rate of 95.29-99.76%. Our source code is available on GitHub at https://github.com/compbio-UofT/FSDA CONTACT: : brudno@cs.toronto.edu.

Limited Clinical Utility of Non-invasive Prenatal Testing for Subchromosomal Abnormalities

The use of massively parallel sequencing of maternal cfDNA for non-invasive prenatal testing (NIPT) of aneuploidy is widely available. Recently, the scope of testing has increased to include selected subchromosomal abnormalities, but the number of samples reported has been small. We developed a calling pipeline based on a segmentation algorithm for the detection of these rearrangements in maternal plasma. The same read depth used in our standard pipeline for aneuploidy NIPT detected 15/18 (83%) samples with pathogenic rearrangements > 6 Mb but only 2/10 samples with rearrangements 6 Mb) chromosomal rearrangements and requires knowledge of fetal fraction, we consider that it is not yet ready for routine clinical implementation.