Noninvasive prenatal testing (NIPT) for the sex chromosome aneuploidies (45,X, 47,XXY, 47,XXX, and 47,XYY) differs significantly from that for the autosomal aneuploidies (trisomy 13, 18, and 21). As a group, sex chromosome aneuploidies occur more commonly (1/400) than any one isolated autosomal aneuploidy, the phenotypic variation is greater, the role of mosaicism more challenging, and the positive predictive value of a high-risk NIPT result is substantially lower. These considerations should be identified during pretest counseling, the inclusion of sex chromosome testing offered separately, and the differences from autosomal aneuploidy NIPT clearly delineated. <<<

Huilin Wang, Zirui Dong, Rui Zhang, Matthew Hoi Kin Chau, Zhenjun Yang, Kathy Yin Ching Tsang, Hoi Kin Wong, Baoheng Gui, Zhuo Meng, Kelin Xiao, Xiaofan Zhu, Yanfang Wang, Shaoyun Chen, Tak Yeung Leung, Sau Wai Cheung, Yvonne K. Kwok, Cynthia C. Morton, Yuanfang Zhu, Kwong Wai Choy <<<

Carrier screening has historically assessed a relatively small number of autosomal recessive and X-linked conditions selected based on frequency in a specific subpopulation and association with severe morbidity or mortality. Advances in genomic technologies enable simultaneous screening of individuals for several conditions. The American College of Medical Genetics and Genomics recently published a clinical practice resource that presents a framework when offering screening for autosomal recessive and X-linked conditions during pregnancy and preconception and recommends a tier-based approach when considering the number of conditions to screen for and their frequency within the US population in general. This laboratory technical standard aims to complement the practice resource and to put forth considerations for clinical laboratories and clinicians who offer preconception/prenatal carrier screening. <<<

The fetal fraction (FF) is a function of both biological factors and bioinformatics algorithms used to interpret DNA sequencing results. It is an essential quality control component of noninvasive prenatal testing (NIPT) results. Clinicians need to understand the biological influences on FF to be able to provide optimal post-test counseling and clinical management. There are many different technologies available for the measurement of FF. Clinicians do not need to know the details behind the bioinformatics algorithms of FF measurements, but they do need to appreciate the significant variations between the different sequencing technologies used by different laboratories. There is no universal FF threshold that is applicable across all platforms and there have not been any differences demonstrated in NIPT performance by sequencing platform or method of FF calculation. Importantly, while FF should be routinely measured, there is not yet a consensus as to whether it should be routinely reported to the clinician. The clinician should know what to expect from a standard test report and whether reasons for failed NIPT results are revealed. Emerging solutions to the challenges of samples with low FF should reduce rates of failed NIPT in the future. In the meantime, having a "plan B" prepared for those patients for whom NIPT is unsuccessful is essential in today's clinical practice. <<<

Kimberly Martin, Pe’er Dar, Cora MacPherson, Melissa Egbert, Zachary Demko, Sheetal Parmar, Katelyn Hashimoto, Sina Haeri, Fergal Malone, Ronald J. Wapner, Ashley S. Roman, Asma Khalil, Revital Faro, Rajeevi Madankumar, Noel Strong, Robert M. Silver, Nidhi Vohra, Jon Hyett, Matt Rabinowitz, Charlly Kao, Hakon Hakonarson, Bo Jacobsson, Mary E. Norton <<<

Researchers spend a great deal of time reading research papers. However, this skill is rarely taught, leading to much wasted effort. This article outlines a practical and efficient three-pass method for reading research papers. I also describe how to use this method to do a literature survey. <<<

OBJECTIVE: To investigate the pregnancy outcomes in a cohort of women who failed to obtain a result in non-invasive prenatal testing (NIPT).DESIGN: Historical cohort study.SETTING: A multicentre private practice in Sydney, Australia.POPULATION: Women who failed to obtain a result from NIPT (n = 131).METHODS: The maternal characteristics, antenatal investigations and pregnancy outcomes for these women were compared with those who obtained a result at the same practice and to the general Australian obstetric population.MAIN OUTCOME MEASURES: Antenatal investigations: pregnancy-associated plasma protein-A (PAPP-A), free β-human chorionic gonadotrophin (β-hCG), placental growth factor (PlGF), uterine artery pulsatility index (PI), mean arterial pressure (MAP). Pregnancy outcomes: chromosomal abnormality, pre-eclampsia, gestational diabetes, small-for-gestational-age (SGA), preterm delivery.RESULTS: Only 1.1% of NIPT samples failed to return a result. This cohort was significantly older and had significantly increased weight compared with the general Australian obstetric population. Pregnancy outcomes were available for 94% of the cohort. There were significantly higher rates of chromosomal aneuploidies (6.5% versus 0.2%, P < 0.0001), pre-eclampsia (11% versus 1.5%, P < 0.0001) and gestational diabetes (23% versus 7.5%, P < 0.0001) compared with the general obstetric population. Rates of preterm delivery and SGA were elevated but did not reach significance. Antenatal investigations demonstrated decreased PAPP-A MoM (0.75 versus 1.14, P < 0.0001), decreased free β-hCG (0.71 versus 1.01, P < 0.0001) and increased uterine artery PI (1.79 versus 1.65, P = 0.02).CONCLUSION: Women who fail to obtain a result from NIPT are at increased risk of adverse pregnancy outcomes, in particular chromosomal aneuploidy, gestational diabetes and pre-eclampsia.FUNDING: None received.TWEETABLE ABSTRACT: Women who fail to obtain a result from cell-free DNA NIPT are at increased risk of adverse pregnancy outcomes. <<<

Taila Hartley, Deborah Marshall, Meryl Acker, Katharine Fooks, Meredith K Gillespie, E Magda Price, Ian D Graham, Alexandre White-Brown, Layla MacKay, Stella K Macdonald, Lauren Brady, Angela Y Hui, Joseph D Andrews, Ashfia Chowdhury, Erika Wall, Élisabeth Soubry, Grace U Ediae, Samantha Rojas, Daniel Assamad, David Dyment, Mark Tarnopolsky, Sarah L Sawyer, Caitlin Chisholm, Gabrielle Lemire, Kimberly Amburgey, Joanna Lazier, Roberto Mendoza-Londono, James J Dowling, Tugce B Balci, Christine M Armour, Priya T Bhola, Gregory Costain, Lucie Dupuis, Melissa Carter, Lauren Badalato, Julie Richer, Christie Boswell-Patterson, Peter Kannu, Dawn Cordeiro, Jodi Warman-Chardon, Gail Graham, Victoria Mok Siu, Cheryl Cytrynbaum, Alison Rusnak, Ritu B Aul, Grace Yoon, Hernan Gonorazky, Vanda McNiven, Saadet Mercimek-Andrews, Andrea Guerin, Ashish R Deshwar, Ashish Marwaha, Rosanna Weksberg, Natalya Karp, Maggie Campbell, Sarah Al-Qattan, Andrew Y Shuen, Michal Inbar-Feigenberg, Ronald Cohn, Anna Szuto, Cara Inglese, Myriam Poirier, Lauren Chad, Beth Potter, Kym M Boycott, Robin Hayeems, Care4Rare Canada Consortium <<<

PURPOSE: To evaluate the diagnostic utility of publicly funded clinical exome sequencing (ES) for patients with suspected rare genetic diseases.METHODS: We prospectively enrolled 297 probands who met eligibility criteria and received ES across 5 sites in Ontario, Canada, and extracted data from medical records and clinician surveys. Using the Fryback and Thornbury Efficacy Framework, we assessed diagnostic accuracy by examining laboratory interpretation of results and assessed diagnostic thinking by examining the clinical interpretation of results and whether clinical-molecular diagnoses would have been achieved via alternative hypothetical molecular tests.RESULTS: Laboratories reported 105 molecular diagnoses and 165 uncertain results in known and novel genes. Of these, clinicians interpreted 102 of 105 (97%) molecular diagnoses and 6 of 165 (4%) uncertain results as clinical-molecular diagnoses. The 108 clinical-molecular diagnoses were in 104 families (35% diagnostic yield). Each eligibility criteria resulted in diagnostic yields of 30% to 40%, and higher yields were achieved when >2 eligibility criteria were met (up to 45%). Hypothetical tests would have identified 61% of clinical-molecular diagnoses.CONCLUSION: We demonstrate robustness in eligibility criteria and high clinical validity of laboratory results from ES testing. The importance of ES was highlighted by the potential 40% of patients that would have gone undiagnosed without this test. <<<

OBJECTIVE: To compare available analysis methods for determining fetal fraction on single read next generation sequencing data. This is important as the performance of non-invasive prenatal testing (NIPT) procedures depends on the fraction of fetal DNA.METHODS: We tested six different methods for the detection of fetal fraction in NIPT samples. The same clinically obtained data were used for all methods, allowing us to assess the effect of fetal fraction on the test result, and to investigate the use of fetal fraction for quality control.RESULTS: We show that non-NIPT methods based on body mass index (BMI) and gestational age are unreliable predictors of fetal fraction, male pregnancy specific methods based on read counts on the Y chromosome perform consistently and the fetal sex-independent new methods SeqFF and SANEFALCON are less reliable but can be used to obtain a basic indication of fetal fraction in case of a female fetus.CONCLUSION: We recommend the use of a combination of methods to prevent the issue of reports on samples with insufficient fetal DNA; SANEFALCON to check for presence of fetal DNA, SeqFF for estimating the fetal fraction for a female pregnancy and any Y-based method for estimating the fetal fraction for a male pregnancy. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd. <<<

Andrew M. Gross, Subramanian S. Ajay, Vani Rajan, Carolyn Brown, Krista Bluske, Nicole J. Burns, Aditi Chawla, Alison J. Coffey, Alka Malhotra, Alicia Scocchia, Erin Thorpe, Natasa Dzidic, Karine Hovanes, Trilochan Sahoo, Egor Dolzhenko, Bryan Lajoie, Amirah Khouzam, Shimul Chowdhury, John Belmont, Eric Roller, Sergii Ivakhno, Stephen Tanner, Julia McEachern, Tina Hambuch, Michael Eberle, R. Tanner Hagelstrom, David R. Bentley, Denise L. Perry, Ryan J. Taft <<<

Yunli Lai, Xiaofan Zhu, Sheng He, Zirui Dong, Yanqing Tang, Fuben Xu, Yun Chen, Lintao Meng, Yuli Tao, Shang Yi, Jiasun Su, Hongqian Huang, Jingsi Luo, Tak Yeung Leung, Hongwei Wei <<<

To evaluate the performance of noninvasive prenatal screening (NIPS) in the detection of common aneuploidies in a population-based study, a total of 86,262 single pregnancies referred for NIPS were prospectively recruited. Among 86,193 pregnancies with reportable results, follow-up was successfully conducted in 1160 fetuses reported with a high-risk result by NIPS and 82,511 cases (95.7%) with a low-risk result. The screen-positive rate (SPR) of common aneuploidies and sex chromosome abnormalities (SCAs) provided by NIPS were 0.7% (586/83,671) and 0.6% (505/83,671), respectively. The positive predictive values (PPVs) for Trisomy 21, Trisomy 18, Trisomy 13 and SCAs were calculated as 89.7%, 84.0%, 52.6% and 38.0%, respectively. In addition, less rare chromosomal abnormalities, including copy number variants (CNVs), were detected, compared with those reported by NIPS with higher read-depth. Among these rare abnormalities, only 23.2% (13/56) were confirmed by prenatal diagnosis. In total, four common trisomy cases were found to be false negative, resulting in a rate of 0.48/10,000 (4/83,671). In summary, this study conducted in an underdeveloped region with limited support for the new technology development and lack of cost-effective prenatal testing demonstrates the importance of implementing routine aneuploidy screening in the public sector for providing early detection and precise prognostic information. <<<

Within the context of screening tests, it is important to avoid misconceptions about sensitivity, specificity, and predictive values. In this article, therefore, foundations are first established concerning these metrics along with the first of several aspects of pliability that should be recognized in relation to those metrics. Clarification is then provided about the definitions of sensitivity, specificity, and predictive values and why researchers and clinicians can misunderstand and misrepresent them. Arguments are made that sensitivity and specificity should usually be applied only in the context of describing a screening test's attributes relative to a reference standard; that predictive values are more appropriate and informative in actual screening contexts, but that sensitivity and specificity can be used for screening decisions about individual people if they are extremely high; that predictive values need not always be high and might be used to advantage by adjusting the sensitivity and specificity of screening tests; that, in screening contexts, researchers should provide information about all four metrics and how they were derived; and that, where necessary, consumers of health research should have the skills to interpret those metrics effectively for maximum benefit to clients and the healthcare system. <<<

Yunli Lai, Xiaofan Zhu, Sheng He, Zirui Dong, Yanqing Tang, Fuben Xu, Yun Chen, Lintao Meng, Yuli Tao, Shang Yi, Jiasun Su, Hongqian Huang, Jingsi Luo, Tak Yeung Leung, Hongwei Wei <<<

To evaluate the performance of noninvasive prenatal screening (NIPS) in the detection of common aneuploidies in a population-based study, a total of 86,262 single pregnancies referred for NIPS were prospectively recruited. Among 86,193 pregnancies with reportable results, follow-up was successfully conducted in 1160 fetuses reported with a high-risk result by NIPS and 82,511 cases (95.7%) with a low-risk result. The screen-positive rate (SPR) of common aneuploidies and sex chromosome abnormalities (SCAs) provided by NIPS were 0.7% (586/83,671) and 0.6% (505/83,671), respectively. The positive predictive values (PPVs) for Trisomy 21, Trisomy 18, Trisomy 13 and SCAs were calculated as 89.7%, 84.0%, 52.6% and 38.0%, respectively. In addition, less rare chromosomal abnormalities, including copy number variants (CNVs), were detected, compared with those reported by NIPS with higher read-depth. Among these rare abnormalities, only 23.2% (13/56) were confirmed by prenatal diagnosis. In total, four common trisomy cases were found to be false negative, resulting in a rate of 0.48/10,000 (4/83,671). In summary, this study conducted in an underdeveloped region with limited support for the new technology development and lack of cost-effective prenatal testing demonstrates the importance of implementing routine aneuploidy screening in the public sector for providing early detection and precise prognostic information. <<<

Kimberly Martin, Pe'er Dar, Cora MacPherson, Melissa Egbert, Zachary Demko, Sheetal Parmar, Katelyn Hashimoto, Sina Haeri, Fergal Malone, Ronald J Wapner, Ashley S Roman, Asma Khalil, Revital Faro, Rajeevi Madankumar, Noel Strong, Robert M Silver, Nidhi Vohra, Jon Hyett, Matt Rabinowitz, Charlly Kao, Hakon Hakonarson, Bo Jacobsson, Mary E Norton <<<

PURPOSE: The aim of this study was to assess the performance of cell-free DNA (cfDNA) screening to detect sex chromosome aneuploidies (SCAs) in an unselected obstetrical population with genetic confirmation.METHODS: This was a planned secondary analysis of the multicenter, prospective SNP-based Microdeletion and Aneuploidy RegisTry (SMART) study. Patients receiving cfDNA results for autosomal aneuploidies and who had confirmatory genetic results for the relevant sex chromosomal aneuploidies were included. Screening performance for SCAs, including monosomy X (MX) and the sex chromosome trisomies (SCT: 47,XXX; 47,XXY; 47,XYY) was determined. Fetal sex concordance between cfDNA and genetic screening was also evaluated in euploid pregnancies.RESULTS: A total of 17,538 cases met inclusion criteria. Performance of cfDNA for MX, SCTs, and fetal sex was determined in 17,297, 10,333, and 14,486 pregnancies, respectively. Sensitivity, specificity, and positive predictive value (PPV) of cfDNA were 83.3%, 99.9%, and 22.7% for MX and 70.4%, 99.9%, and 82.6%, respectively, for the combined SCTs. The accuracy of fetal sex prediction by cfDNA was 100%.CONCLUSION: Screening performance of cfDNA for SCAs is comparable to that reported in other studies. The PPV for the SCTs was similar to the autosomal trisomies, whereas the PPV for MX was substantially lower. No discordance in fetal sex was observed between cfDNA and postnatal genetic screening in euploid pregnancies. These data will assist interpretation and counseling for cfDNA results for sex chromosomes. <<<

Noura S Abul-Husn, Priya N Marathe, Nicole R Kelly, Katherine E Bonini, Monisha Sebastin, Jacqueline A Odgis, Avinash Abhyankar, Kaitlyn Brown, Miranda Di Biase, Katie M Gallagher, Saurav Guha, Nicolette Ioele, Volkan Okur, Michelle A Ramos, Jessica E Rodriguez, Atteeq U Rehman, Amanda Thomas-Wilson, Lisa Edelmann, Randi E Zinberg, George A Diaz, John M Greally, Vaidehi Jobanputra, Sabrina A Suckiel, Carol R Horowitz, Melissa P Wasserstein, Eimear E Kenny, Bruce D Gelb <<<

PURPOSE: Adoption of genome sequencing (GS) as a first-line test requires evaluation of its diagnostic yield. We evaluated the GS and targeted gene panel (TGP) testing in diverse pediatric patients (probands) with suspected genetic conditions.METHODS: Probands with neurologic, cardiac, or immunologic conditions were offered GS and TGP testing. Diagnostic yield was compared using a fully paired study design.RESULTS: A total of 645 probands (median age 9 years) underwent genetic testing, and 113 (17.5%) received a molecular diagnosis. Among 642 probands with both GS and TGP testing, GS yielded 106 (16.5%) and TGPs yielded 52 (8.1%) diagnoses (P < .001). Yield was greater for GS vs TGPs in Hispanic/Latino(a) (17.2% vs 9.5%, P < .001) and White/European American (19.8% vs 7.9%, P < .001) but not in Black/African American (11.5% vs 7.7%, P = .22) population groups by self-report. A higher rate of inconclusive results was seen in the Black/African American (63.8%) vs White/European American (47.6%; P = .01) population group. Most causal copy number variants (17 of 19) and mosaic variants (6 of 8) were detected only by GS.CONCLUSION: GS may yield up to twice as many diagnoses in pediatric patients compared with TGP testing but not yet across all population groups. <<<

Noninvasive prenatal testing (NIPT) consists of determining fetal aneuploidies by quantifying copy number alteration from the sequencing of cell-free DNA (cfDNA) from maternal blood. Due to the presence of cfDNA of fetal origin in maternal blood, in silico approaches have been developed to accurately predict fetal aneuploidies. Although NIPT is becoming a new standard in prenatal screening of chromosomal abnormalities, there are no integrated pipelines available to allow rapid, accurate and standardized data analysis in any clinical setting. Several tools have been developed, however often optimized only for research purposes or requiring enormous amount of retrospective data, making hard their implementation in a clinical context. Furthermore, no guidelines have been provided on how to accomplish each step of the data analysis to achieve reliable results. Finally, there is no integrated pipeline to perform all steps of NIPT analysis. To address these needs, we tested several tools for performing NIPT data analysis. We provide extensive benchmark of tools performances but also guidelines for running them. We selected the best performing tools that we benchmarked and gathered them in a computational pipeline. NiPTUNE is an open source python package that includes methods for fetal fraction estimation, a novel method for accurate gender prediction, a principal component analysis based strategy for quality control and fetal aneuploidies prediction. NiPTUNE is constituted by seven modules allowing the user to run the entire pipeline or each module independently. Using two cohorts composed by 1439 samples with 31 confirmed aneuploidies, we demonstrated that NiPTUNE is a valuable resource for NIPT analysis. <<<

Anna Fowler, Shazia Mahamdallie, Elise Ruark, Sheila Seal, Emma Ramsay, Matthew Clarke, Imran Uddin, Harriet Wylie, Ann Strydom, Gerton Lunter, Nazneen Rahman <<<

Targeted next generation sequencing (NGS) panels are increasingly being used in clinical genomics to increase capacity, throughput and affordability of gene testing. Identifying whole exon deletions or duplications (termed exon copy number variants, 'exon CNVs') in exon-targeted NGS panels has proved challenging, particularly for single exon CNVs. We developed a tool for the Detection of Exon Copy Number variants (DECoN), which is optimised for analysis of exon-targeted NGS panels in the clinical setting. We evaluated DECoN performance using 96 samples with independently validated exon CNV data. We performed simulations to evaluate DECoN detection performance of single exon CNVs and to evaluate performance using different coverage levels and sample numbers. Finally, we implemented DECoN in a clinical laboratory that tests and with the TruSight Cancer Panel (TSCP). We used DECoN to analyse 1,919 samples, validating exon CNV detections by multiplex ligation-dependent probe amplification (MLPA). In the evaluation set, DECoN achieved 100% sensitivity and 99% specificity for BRCA exon CNVs, including identification of 8 single exon CNVs. DECoN also identified 14/15 exon CNVs in 8 other genes. Simulations of all possible BRCA single exon CNVs gave a mean sensitivity of 98% for deletions and 95% for duplications. DECoN performance remained excellent with different levels of coverage and sample numbers; sensitivity and specificity was >98% with the typical NGS run parameters. In the clinical pipeline, DECoN automatically analyses pools of 48 samples at a time, taking 24 minutes per pool, on average. DECoN detected 24 BRCA exon CNVs, of which 23 were confirmed by MLPA, giving a false discovery rate of 4%. Specificity was 99.7%. DECoN is a fast, accurate, exon CNV detection tool readily implementable in research and clinical NGS pipelines. It has high sensitivity and specificity and acceptable false discovery rate. DECoN is freely available at www.icr.ac.uk/decon. <<<

Desheng Liang, David S Cram, Hu Tan, Siyuan Linpeng, Yingdi Liu, Huaiyu Sun, Yu Zhang, Feng Tian, Hongmin Zhu, Mengnan Xu, Hua Wang, Fuli Yu, Lingqian Wu <<<

PURPOSE: To assess the clinical performance of an expanded noninvasive prenatal screening (NIPS) test ("NIPS-Plus") for detection of both aneuploidy and genome-wide microdeletion/microduplication syndromes (MMS).METHODS: A total of 94,085 women with a singleton pregnancy were prospectively enrolled in the study. The cell-free plasma DNA was directly sequenced without intermediate amplification and fetal abnormalities identified using an improved copy-number variation (CNV) calling algorithm.RESULTS: A total of 1128 pregnancies (1.2%) were scored positive for clinically significant fetal chromosome abnormalities. This comprised 965 aneuploidies (1.026%) and 163 (0.174%) MMS. From follow-up tests, the positive predictive values (PPVs) for T21, T18, T13, rare trisomies, and sex chromosome aneuploidies were calculated as 95%, 82%, 46%, 29%, and 47%, respectively. For known MMS (n = 32), PPVs were 93% (DiGeorge), 68% (22q11.22 microduplication), 75% (Prader-Willi/Angleman), and 50% (Cri du Chat). For the remaining genome-wide MMS (n = 88), combined PPVs were 32% (CNVs ≥10 Mb) and 19% (CNVs <10 Mb).CONCLUSION: NIPS-Plus yielded high PPVs for common aneuploidies and DiGeorge syndrome, and moderate PPVs for other MMS. Our results present compelling evidence that NIPS-Plus can be used as a first-tier pregnancy screening method to improve detection rates of clinically significant fetal chromosome abnormalities. <<<

The purpose of this study was to determine the deep sequencing and analytic conditions needed to detect fetal subchromosome abnormalities across the genome from a maternal blood sample. Cell-free (cf) DNA was isolated from the plasma of 11 pregnant women carrying fetuses with subchromosomal duplications and deletions, translocations, mosaicism, and trisomy 20 diagnosed by metaphase karyotype. Massively parallel sequencing (MPS) was performed with 25-mer tags at approximately 10(9) tags per sample and mapped to reference human genome assembly hg19. Tags were counted and normalized to fixed genome bin sizes of 1 Mb or 100 kb to detect statistically distinct copy-number changes compared to the reference. All seven cases of microdeletions, duplications, translocations, and the trisomy 20 were detected blindly by MPS, including a microdeletion as small as 300 kb. In two of these cases in which the metaphase karyotype showed additional material of unknown origin, MPS identified both the translocation breakpoint and the chromosomal origin of the additional material. In the four mosaic cases, the subchromosomal abnormality was not demonstrated by MPS. This work shows that in nonmosaic cases, it is possible to obtain a fetal molecular karyotype by MPS of maternal plasma cfDNA that is equivalent to a chromosome microarray and in some cases is better than a metaphase karyotype. This approach combines the advantage of enhanced fetal genomic resolution with the improved safety of a noninvasive maternal blood test. <<<