DOI: 10.1002/pd.4159 NIPT SPECIAL ISSUE SNP-based non-invasive prenatal testing detects sex chromosome aneuploidies with high accuracy Carole Samango-Sprouse1,3*, Milena Banjevic2, Allison Ryan2, Styrmir Sigurjonsson2, Bernhard Zimmermann2, Matthew Hill2, Megan P. Hall2, Margaret Westemeyer2, Jennifer Saucier2, Zachary Demko2 and Matthew Rabinowitz2 1George Washington University School of Medicine and Health Sciences, Washington, D.C., USA 2Natera Inc., San Carlos, CA, USA 3Neurodevelopmental Diagnostic Center for Young Children, Davidsonville, MD, USA *Correspondence to: Carole Samango-Sprouse. E-mail: [email protected] ABSTRACT Objective This study aimed to develop a single-nucleotide polymorphism-based and informatics-based non-invasive prenatal test that detects sex chromosome aneuploidies early in pregnancy. Methods Sixteen aneuploid samples, including thirteen 45,X, two 47,XXY, and one 47,XYY, along with 185 euploid controls, were analyzed. Cell-free DNA was isolated from maternal plasma, amplified in a single multiplex polymerase chain reaction assay that targeted 19 488 polymorphic loci covering chromosomes 13, 18, 21, X, and Y, and sequenced. Sequencing results were analyzed using a Bayesian-based maximum likelihood statistical method to determine copy number of interrogated chromosomes, calculating sample-specific accuracies. Results Of the samples that passed a stringent quality control metric (93%), the algorithm correctly identified copy number at all five chromosomes in all but one of the 187 samples, for 934/935 correct calls as early as 9.4 weeks of gestation. We detected 45,X with 91.7% sensitivity (CI: 61.5–99.8%) and 100% specificity (CI: 97.9–100%), and 47,XXY and 47,XYY. The average calculated accuracy was 99.78%. Conclusion This method non-invasively detected 45,X, 47,XXY, and 47,XYY fetuses from cell-free DNA isolated from maternal plasma with high calculated accuracies and thus offers a non-invasive method with the potential to function as a routine screen allowing for early prenatal detection of rarely diagnosed yet commonly occurring sex aneuploidies. © 2013 John Wiley & Sons, Ltd. Supporting information may be found in the online version of this article. Funding sources: This study was supported in part by a grant from the National Institute of Health, National Institute of Child Health and Human Development (4R44HD062114–02). Conflicts of interest: M. B., A. R., S. S., B. Z., M. H., M. P. H., M. W., J. S., Z. D., and M. R. are employees of Natera. INTRODUCTION nor the sex chromosome trisomies are consistently detected During the last two decades, non-invasive prenatal screening using the current non-invasive screening approaches.2,3 Thus, tests based on biochemical analysis of maternal serum and/ diagnosis of sex chromosome abnormalities generally occurs or ultrasonography have been increasingly adopted into during pregnancy when invasive diagnostic testing is clinical practice to identify women at increased risk of carrying completed, often for other reasons, or after the child is born a fetus with a chromosomal aneuploidy. Until recently, and presents with signs and symptoms, which may not occur screening focused primarily on autosomal trisomies: detection until the teen years or when there are eventual reproductive of trisomy 21 (Down syndrome), which is seen in issues.4 Although there are challenges that accompany the approximately 1 in 600 live births, and to a lesser extent diagnosis, there are now several articles that demonstrate the trisomy 18 (Edwards syndrome) and trisomy 13 (Patau benefits of early detection and treatment, as well as syndrome). However, the combined at-birth prevalence of sex neurodevelopmental progression in Turner and Klinefelter chromosome aneuploidies (aneuploidies involving the X and/ syndromes.5,6 A non-invasive prenatal test that expands or Y chromosomes, including Turner syndrome [45,X], clinical coverage to include the X and Y chromosomes thus Klinefelter syndrome [47,XXY], 47,XYY, and 47,XXX) is higher has the potential to offer significant value to parents and than that of the autosomal aneuploidies, occurring in physicians in the form of early detection and the choice of approximately 1 in 400 live births.1 Neither Turner syndrome possible biological treatments.5,6 Prenatal Diagnosis (2013), 33,1–7 © 2013 John Wiley & Sons, Ltd. C. Samango-Sprouse et al. Recent advances based on genetic analysis of cell-free DNA centrifugation protocol of 2000 g for 20 min, followed by (cfDNA, a mixture of maternal and fetal cfDNA) isolated from 3220 g for 30 min, with supernatant transfer following the first maternal plasma provide the opportunity to non-invasively spin. cfDNA was isolated from 7 to 10 mL plasma by using the detect fetal aneuploidies, including sex chromosome QIAGEN QIAamp Circulating Nucleic Acid kit and eluted in – aneuploidies, early in pregnancy.7 9 Current methods detect 50 mL 10% DNA Suspension Buffer (Teknova, Hollister, CA). trisomy 21 and trisomy 18 with good accuracy; however, Maternal genomic DNA was isolated from the buffy coat trisomy 13 and sex chromosome aneuploidy detection rates obtained following the first centrifugation, and paternal genomic – and sensitivities are decreased;10 22 for example, reported DNA was prepared from either a blood or buccal swab sample. Monosomy X sensitivities for commercially available methods Samples were pre-amplified for 15 cycles by using PCR and have reached 94.4%, less than the up to >99% reported for 19 488 target-specific assays. Then, an aliquot was transferred – trisomies 18 and 21.13,18,23 26 This is thought to be partially to a second nested 15-cycle PCR reaction. Finally, samples due to highly variable amplification of these chromosomes.27,28 were prepared for sequencing by adding barcoded tags in a Recent reports indicated that guanosine–cytosine (GC) bias third round of 12-cycle PCR reaction. Thus, 19 488 targets were correction techniques may correct for these issues. However, amplified in a single reaction; targets included SNPs from this has only proven effective for trisomy 13 detection,15 chromosomes 13, 18, 21, X, and Y. SNPs targeted on the Y whereas accurate detection of X chromosome copy number chromosome are in the homologous non-recombining regions continues to prove problematic. This is exacerbated at early of the X and Y chromosomes where the loci are common to the gestational ages (GAs) when the fetal cfDNA fraction in two chromosomes. Amplicons were sequenced using an maternal plasma is more likely to be low, and the signal- Illumina HiSeq 2000 sequencer. Parental samples were to-noise ratio is smaller. measured using the same multiplex PCR protocol but We developed a novel non-invasive prenatal aneuploidy test sequenced at a lower depth-of-read (approximately 20%) than – that interrogates single-nucleotide polymorphisms (SNPs).29 32 that for cfDNA analysis. The method involves massively multiplexed polymerase chain reaction (PCR) amplification of cfDNA isolated from maternal NATUS methodology and data analysis plasma, targeting 19 488 SNPs, followed by high-throughput Sequence alignment to the genome was performed using a sequencing. This SNP-based method employs a novel proprietary algorithm adapted from the Novoalign (Novocraft, algorithm called Next-generation Aneuploidy Test Using SNPs Selangor, Malaysia) commercial software package. A (NATUS), which incorporates parental genotypic data, known chromosome copy number classification algorithm was inheritance patterns, and complex data models to correctly implemented in MATLAB (MathWorks, Natic, MA, USA) interpret abnormally distributed data; this is especially leveraging a proprietary statistical algorithm termed Next- relevant for the X chromosome, resulting in consistently generation Aneuploid Test Using SNPs (NATUS). The NATUS ™ accurate copy number determination across all interrogated algorithm is an advanced version of the Parental Support chromosomes.32,33 algorithm.29,31,32 Briefly, NATUS considers parental genotypes This report focuses on the use of a SNP-based non-invasive and crossover frequency data35,36 to calculate in silico the method to identify sex chromosome aneuploidies, which have expected allele distributions for 19 488 SNPs and billions of proven difficult to accurately detect. possible fetal genotypes based on recombination sites in the parent chromosomes. It compares these predicted METHODS distributions with actual allelic distributions as measured from the cfDNA sample, employing a Bayesian-based maximum Subjects and sample collection likelihood approach to determine the relative likelihood of The cohort of 201 pregnancies analyzed here included thirteen each hypothesis and fetal fraction given the observed data: it 45,X, two 47,XXY, one 47,XYY, and 185 euploid samples. calculates the likelihood of each copy number hypothesis Pregnant couples were enrolled at selected prenatal care (monosomy, disomy, or trisomy, for which there are numerous centers under an Institutional Review Board-approved or sub-hypotheses based on recombination sites), sums the National Health Service Research Ethics Committee-approved likelihoods of each copy number sub-hypothesis, and calls protocol.34 Women were at least 18 years of age, had singleton the hypothesis with the maximum likelihood as the copy pregnancies, and signed an informed consent. All samples with number and fetal fraction. The maximum likelihood represents known mosaicism, autosomal trisomy, or triploidy were the sample-specific calculated accuracy. NATUS takes into excluded