Scientific Publications

Peer-reviewed published studies

Robinson C1, van den Boom D, Bombard AT. Clin Obstet Gynecol. 2014 Mar;57(1):210-25. doi: 10.1097/GRF.0000000000000016.

Noninvasive prenatal testing (NIPT) uses cell-free fetal DNA from the plasma of pregnant women to provide valuable information about the potential risks for fetal aneuploidy. This article provides a historical overview of both invasive diagnostic testing and serum screening approaches, both biochemical and the newer molecular noninvasive prenatal testing assays, used to identify patients who would be best served by invasive testing.

Lambert-Messerlian G1, Kloza EM1, Williams J 3rd2, Loucky J3, O'Brien B4, Wilkins-Haug L5, Mahoney MJ6, De Biasio P7, Borrell A8, Ehrich M9, van den Boom D9, Bombard AT10, Deciu C11, Palomaki GE1. Genet Med. 2014 May;16(5):419-22. doi: 10.1038/gim.2013.149. Epub 2013 Oct 3.
PURPOSE

We sought to compare measurements of circulating cell-free DNA as well as Down syndrome test results in women with naturally conceived pregnancies with those conceived using assisted reproductive technologies.

METHODS

Data regarding assisted reproductive technologies were readily available from seven enrollment sites participating in an external clinical validation trial of nested case/control design. Measurements of circulating cell-free fetal and total DNA, fetal fraction (ratio of fetal to total DNA), chromosome-specific z-scores, and karyotype results were available for analysis.

RESULTS

Analyses were restricted to 632 euploid (5.2% assisted reproductive technologies) and 73 Down syndrome (13.7% assisted reproductive technologies), including 16 twin pregnancies. No differences were found for fetal or total circulating cell-free DNA, or for the fetal fraction in euploid (P = 0.70) or Down syndrome (P = 0.58) pregnancies by method of conception. There appeared to be systematic z-score reductions for chromosomes 21, 18, and 13 in assisted reproductive technologies versus natural euploid pregnancies (P = 0.048, 0.0032, and 0.36, respectively).

CONCLUSION

Assisted reproductive technologies and naturally conceived pregnancies contribute similar levels of circulating cell-free DNA into maternal circulation. Small differences in the z-scores of pregnancies achieved by assisted reproductive technologies were observed and do not appear to be test-related artifacts. However, the findings need confirmation before any consideration of changes to testing and reporting protocols.

Wong D1, Moturi S, Angkachatchai V, Mueller R, DeSantis G, van den Boom D, Ehrich M. Clin Biochem. 2013 Aug;46(12):1099-104. doi: 10.1016/j.clinbiochem.2013.04.023. Epub 2013 Apr 30.
OBJECTIVES

Fetal mutations and fetal chromosomal abnormalities can be detected by molecular analysis of circulating cell free fetal DNA (ccffDNA) from maternal plasma. This comprehensive study was aimed to investigate and verify blood collection and blood shipping conditions that enable Noninvasive Prenatal Testing. Specifically, the impact of shipping and storage on the stability and concentration of circulating cell-free DNA (ccfDNA) in Streck® Cell-Free DNA™ Blood Collection Tubes (Streck BCTs, Streck, Omaha NE). These BCTs were designed to minimize cellular degradation, and thus effectively prevent dilution of fetal ccf DNA by maternal genomic DNA, was evaluated.

DESIGN AND METHODS

Peripheral venous maternal blood was collected into Streck BCTs to investigate four aspects of handling and processing conditions: (1) time from blood draw to plasma processing; (2) storage temperature; (3) mechanical stress; and (4) lot-to-lot tube variations.

RESULTS

Maternal blood stored in Streck BCTs for up to 7 days at ambient temperature provides stable concentrations of ccffDNA. The amount of fetal DNA did not change over a broad range of storage temperatures (4°C, 23°C, 37°C, 40°C), but the amount of total (largely maternal) DNA increased in samples stored at 23°C and above, indicating maternal cell degradation and genomic DNA release at elevated temperatures. Shipping maternal blood in Streck BCTs, did not affect sample quality.

CONCLUSIONS

Maternal plasma DNA stabilized for 0 to 7 days in Streck BCTs can be used for non-invasive prenatal molecular applications, when temperatures are maintained within the broad parameters assessed in this study.

Jensen TJ1, Zwiefelhofer T, Tim RC, Džakula Ž, Kim SK, Mazloom AR, Zhu Z, Tynan J, Lu T, McLennan G, Palomaki GE, Canick JA, Oeth P, Deciu C, van den Boom D, Ehrich M. PLoS One. 2013;8(3):e57381. doi: 10.1371/journal.pone.0057381. Epub 2013 Mar 6.
BACKGROUND

Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.

METHODS

Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.

RESULTS

Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.

CONCLUSIONS

These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

Shea JL1, Diamandis EP, Hoffman B, Lo YM, Canick J, van den Boom D. Clin Chem. 2013 Aug;59(8):1151-9. doi: 10.1373/clinchem.2012.201996. Epub 2013 Feb 20.
Mazloom AR1, Džakula Ž, Oeth P, Wang H, Jensen T, Tynan J, McCullough R, Saldivar JS, Ehrich M, van den Boom D, Bombard AT, Maeder M, McLennan G, Meschino W, Palomaki GE, Canick JA, Deciu C. Prenat Diagn. 2013 Jun;33(6):591-7. doi: 10.1002/pd.4127.
OBJECTIVE

Whole-genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes.

METHOD

Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype. A classification algorithm for SCA detection was constructed and trained on this cohort. Another study of 411 maternal samples from women with blinded-to-laboratory fetal karyotypes was then performed to determine the accuracy of the classification algorithm.

RESULTS

In the training cohort, the new algorithm had a detection rate (DR) of 100% (95%CI: 82.3%, 100%), a false positive rate (FPR) of 0.1% (95%CI: 0%, 0.3%), and nonreportable rate of 6% (95%CI: 4.9%, 7.4%) for SCA determination. The blinded validation yielded similar results: DR of 96.2% (95%CI: 78.4%, 99.8%), FPR of 0.3% (95%CI: 0%, 1.8%), and nonreportable rate of 5% (95%CI: 3.2%, 7.7%) for SCA determination

CONCLUSION

Noninvasive prenatal identification of the most common sex chromosome aneuploidies is possible using ccf DNA and massively parallel sequencing with a high DR and a low FPR.

Ronald Lindsay; Daniel Farkas; Allan Bombard. New England Journal of Medicine:Nov-13
Porreco RP1, Garite TJ2, Maurel K3, Marusiak B3; Obstetrix Collaborative Research Network, Ehrich M4, van den Boom D4, Deciu C4, Bombard A4. Am J Obstet Gynecol. 2014 Oct;211(4):365.e1-12. doi: 10.1016/j.ajog.2014.03.042. Epub 2014 Mar 19.
OBJECTIVE

The objective of this study was to validate the clinical performance of massively parallel genomic sequencing of cell-free deoxyribonucleic acid contained in specimens from pregnant women at high risk for fetal aneuploidy to test fetuses for trisomies 21, 18, and 13; fetal sex; and the common sex chromosome aneuploidies (45, X; 47, XXX; 47, XXY; 47, XYY).

STUDY DESIGN

This was a prospective multicenter observational study of pregnant women at high risk for fetal aneuploidy who had made the decision to pursue invasive testing for prenatal diagnosis. Massively parallel single-read multiplexed sequencing of cell-free deoxyribonucleic acid was performed in maternal blood for aneuploidy detection. Data analysis was completed using sequence reads unique to the chromosomes of interest.

RESULTS

A total of 3430 patients were analyzed for demographic characteristics and medical history. There were 137 fetuses with trisomy 21, 39 with trisomy 18, and 16 with trisomy 13 for a prevalence rate of the common autosomal trisomies of 5.8%. There were no false-negative results for trisomy 21, 3 for trisomy 18, and 2 for trisomy 13; all 3 false-positive results were for trisomy 21. The positive predictive values for trisomies 18 and 13 were 100% and 97.9% for trisomy 21. A total of 8.6% of the pregnancies were 21 weeks or beyond; there were no aneuploid fetuses in this group. All 15 of the common sex chromosome aneuploidies in this population were identified, although there were 11 false-positive results for 45,X. Taken together, the positive predictive value for the sex chromosome aneuploidies was 48.4% and the negative predictive value was 100%.

CONCLUSION

Our prospective study demonstrates that noninvasive prenatal analysis of cell-free deoxyribonucleic acid from maternal plasma is an accurate advanced screening test with extremely high sensitivity and specificity for trisomy 21 (>99%) but with less sensitivity for trisomies 18 and 13. Despite high sensitivity, there was modest positive predictive value for the small number of common sex chromosome aneuploidies because of their very low prevalence rate.

Taylor Jensen, Sung K Kim, Zhanyang Zhu, Christine Chin, Claudia Gebhard, Tim Lu, Cosmin Deciu, Dirk van den Boom, Mathias Ehrich. Genome Biology 2015, 16:78
BACKGROUND

Circulating cell free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the genetically distinct maternal and fetal DNA. Current testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA and thus support additional clinical opportunities; however, this depends on knowledge of the methylomes of ccf DNA and its cellular contributors.

RESULTS

Whole genome bisulfite sequencing was performed on a set of unmatched samples including ccf DNA from 8 non-pregnant (NP) and 7 pregnant female donors and genomic DNA from 7 maternal buffy coat and 5 placenta samples. We found CpG cytosines within longer fragments were more likely to be methylated, linking DNA methylation and fragment size in ccf DNA. Comparison of the methylomes of placenta and NP ccf DNA revealed many of the 51,259 identified differentially methylated regions (DMRs) were located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases, regions we termed placenta hypomethylated domains (PHDs). We found PHDs were consistently located within regions exhibiting low CpG and gene density. DMRs identified when comparing placenta to NP ccf DNA were recapitulated in pregnant ccf DNA, confirming the ability to detect differential methylation in ccf DNA mixtures.

CONCLUSIONS

We generated methylome maps for four sample types at single base resolution, identified a link between DNA methylation and fragment length in ccf DNA, identified DMRs between sample groups, and uncovered the presence of megabase-size placenta hypomethylated domains. Furthermore, we anticipate these results to provide a foundation to which future studies using discriminatory DNA methylation may be compared.

Grömminger S1, Yagmur E2, Erkan S3, Nagy S4, Schöck U5, Bonnet J6, Smerdka P7, Ehrich M8, Wegner RD9, Hofmann W10, Stumm M11. J Clin Med. 2014 Jun 25;3(3):679-92. doi: 10.3390/jcm3030679.
ABSTRACT

Non-invasive prenatal testing (NIPT) by random massively parallel sequencing of maternal plasma DNA for multiple pregnancies is a promising new option for prenatal care since conventional non-invasive screening for fetal trisomies 21, 18 and 13 has limitations and invasive diagnostic methods bear a higher risk for procedure related fetal losses in the case of multiple gestations compared to singletons. In this study, in a retrospective blinded analysis of stored twin samples, all 16 samples have been determined correctly, with four trisomy 21 positive and 12 trisomy negative samples. In the prospective part of the study, 40 blood samples from women with multiple pregnancies have been analyzed (two triplets and 38 twins), with two correctly identified trisomy 21 cases, confirmed by karyotyping. The remaining 38 samples, including the two triplet pregnancies, had trisomy negative results. However, NIPT is also prone to quality issues in case of multiple gestations: the minimum total amount of cell-free fetal DNA must be higher to reach a comparable sensitivity and vanishing twins may cause results that do not represent the genetics of the living sibling, as described in two case reports.

Bombard AT1, Farkas DH, Monroe TJ, Saldivar JS. Obstet Gynecol. 2014 Aug;124(2 Pt 1):379. doi: 10.1097/AOG.0000000000000400.

ABSTRACT

The identification of circulating cell-free fetal DNA in maternal plasma has led to the introduction of noninvasive prenatal tests with high sensitivity and high specificity for common aneuploidies (trisomy 13, trisomy 18, trisomy 21). A new expanded noninvasive prenatal testing panel that includes five microdeletion syndromes (22q11 deletion syndrome, cri-du-chat [5p minus], Prader Willi or Angelman syndrome, 1p36 deletion syndrome) and two aneuploidies usually associated with nonviable pregnancies (trisomy 16 and trisomy 22) is now available. This expanded panel will be performed unless an opt-out box is checked. Because these disorders are so rare, the positive predictive value is expected to be low. As with all new screening tests and technologies, the expanded panel should be appropriately studied before it is widely adopted.

Spaans VM1, Trietsch MD2, Crobach S2, Stelloo E2, Kremer D3, Osse EM2, Haar NT2, van Eijk R2, Muller S4, van Wezel T2, Trimbos JB5, Bosse T2, Smit VT2, Fleuren GJ2. PLoS One. 2014 Mar 26;9(3):e93451. doi: 10.1371/journal.pone.0093451. eCollection 2014.

Somatic mutations play a major role in tumour initiation and progression. The mutation status of a tumour may predict prognosis and guide targeted therapies. The majority of techniques to study oncogenic mutations requires high quality and quantity DNA or are analytically challenging. Mass-spectrometry based mutation analysis however is a relatively simple and high-throughput method suitable for formalin-fixed, paraffin-embedded (FFPE) tumour material. Targeted gene panels using this technique have been developed for several types of cancer. These current cancer hotspot panels are not focussed on the genes that are most relevant in gynaecological cancers. In this study, we report the design and validation of a novel, mass-spectrometry based panel specifically for gynaecological malignancies and present the frequencies of detected mutations. Using frequency data from the online Catalogue of Somatic Mutations in Cancer, we selected 171 somatic hotspot mutations in the 13 most important genes for gynaecological cancers, being BRAF, CDKN2A, CTNNB1, FBXW7, FGFR2, FGFR3, FOXL2, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A and PTEN. A total of 546 tumours (205 cervical, 227 endometrial, 89 ovarian, and 25 vulvar carcinomas) were used to test and validate our panel, and to study the prevalence and spectrum of somatic mutations in these types of cancer. The results were validated by testing duplicate samples and by allele-specific qPCR. The panel presented here using mass-spectrometry shows to be reproducible and high-throughput, and is usefull in FFPE material of low quality and quantity. It provides new possibilities for studying large numbers of gynaecological tumour samples in daily practice, and could be useful in guided therapy selection.

Jensen TJ1, Kim SK1, van den Boom D2, Deciu C1, Ehrich M3. Clin Chem. 2014 Oct;60(10):1298-305. doi: 10.1373/clinchem.2014.223198. Epub 2014 Jul 16.
BACKGROUND

Massively parallel sequencing of circulating cell free (ccf) DNA from maternal plasma has been demonstrated to be a powerful method for the detection of fetal copy number variations (CNVs). Although the detection of CNVs has been described by multiple independent groups, genomic aberrations resulting in copy number-neutral events including balanced translocations have proven to be more challenging to detect noninvasively from ccf DNA.

METHODS

Data modeling was initially performed to evaluate multiple methods, ultimately leveraging the short length of ccf DNA and paired-end sequencing to construct read-specific mapping characteristics. After testing in a model system, we evaluated the methods on ccf DNA isolated from the plasma of a donor known to be carrying a fetus with a balanced translocation [t(8;11)]. Sequencing was performed with Illumina sequencing technology.

RESULTS

Our methodology identified the known translocation (P = 1.21 × 10(-8)) and discounted the likelihood of others, enabling the base specific identification of the rearrangement positions. In total, 402 unique sequencing reads spanned the putative breakpoints, of which 76 contained the structural rearrangement. In addition, 38 of the chimeric reads were mapped to each of the resulting derivative chromosomes, supporting the presence of a reciprocal translocation. Finally, we identified a 6-bp deletion present within der(8) that was absent from the der(11) reciprocal rearrangement.

CONCLUSIONS

We have developed an algorithm to detect balanced rearrangements and applied our methodology to demonstrate the first proof-of-principle study on the noninvasive detection of a fetal-specific balanced translocation by sequencing ccf DNA from maternal plasma.

McCullough RM1, Almasri EA1, Guan X1, Geis JA1, Hicks SC1, Mazloom AR1, Deciu C1, Oeth P1, Bombard AT1, Paxton B1, Dharajiya N1, Saldivar JS1. PLoS One. 2014 Oct 7;9(10):e109173. doi: 10.1371/journal.pone.0109173. eCollection 2014.
OBJECTIVE

As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT.

STUDY DESIGN

The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA-licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting.

RESULTS

NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥ 35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result.

CONCLUSION

NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.

Rolnik DL1, O'Gorman N, Fiolna M, van den Boom D, Nicolaides KH, Poon LC. Ultrasound Obstet Gynecol. 2015 Jan;45(1):106-11. doi: 10.1002/uog.14671. Epub 2014 Dec 4.
OBJECTIVES

To examine whether maternal plasma concentrations of total cell-free (cf)DNA and fetal fraction at 11-13 and 20-24 weeks' gestation in pregnancies that subsequently develop pre-eclampsia (PE) are different from those without this complication.

METHODS

Total cfDNA and fetal fraction were measured in 20 cases of early PE requiring delivery at < 34 weeks, in 20 cases of late PE with delivery at ≥ 34 weeks and in 200 normotensive controls, at 11-13 and 20-24 weeks' gestation. Total cfDNA and fetal fraction measured at 11-13 weeks were converted to multiples of the median (MoM), corrected for maternal characteristics and gestational age. The distributions of total cfDNA and fetal fraction at 20-24 weeks were expressed as MoM of values at 11-13 weeks. The Mann-Whitney U-test was used to determine the significance of differences in the median values in each outcome group relative to that in the controls.

RESULTS

In the early-PE group at 11-13 weeks, compared with controls, there was a significant increase in median total cfDNA (2104 genome equivalents (GE)/mL vs 1590 GE/mL) and a decrease in median fetal fraction (6.8% vs 8.7%). In the late-PE group at 20-24 weeks, compared with controls, there was a significant decrease in median fetal fraction (8.2% vs 9.6%). These significant differences between groups were not observed when the values were converted to MoM.

CONCLUSION

Measurements of total cfDNA and fetal fraction in maternal plasma at 11-13 and 20-24 weeks are not predictive of PE.

Noehammer C1, Pulverer W, Hassler MR, Hofner M, Wielscher M, Vierlinger K, Liloglou T, McCarthy D, Jensen TJ, Nygren A, Gohlke H, Trooskens G, Braspenning M, Van Criekinge W, Egger G, Weinhaeusel A. Epigenomics. 2014;6(6):603-22. doi: 10.2217/epi.14.43.

DNA methylation is a stable covalent epigenetic modification of primarily CpG dinucleotides that has recently gained considerable attention for its use as a biomarker in different clinical settings, including disease diagnosis, prognosis and therapeutic response prediction. Although the advent of genome-wide DNA methylation profiling in primary disease tissue has provided a manifold resource for biomarker development, only a tiny fraction of DNA methylation-based assays have reached clinical testing. Here, we provide a critical overview of different analytical methods that are suitable for biomarker validation, including general study design considerations, which might help to streamline epigenetic marker development. Furthermore, we highlight some of the recent marker validation studies and established markers that are currently commercially available for assisting in clinical management of different cancers.

Palomaki GE1, Ashwood ER, Weck KE. Ultrasound Obstet Gynecol. 2015 Jan;45(1):117. doi: 10.1002/uog.14739.

Palomaki GE1, Kloza EM, Lambert-Messerlian GM, van den Boom D, Ehrich M, Deciu C, Bombard AT, Haddow JE. Prenat Diagn. 2015 Mar;35(3):289-93. doi: 10.1002/pd.4541. Epub 2015 Jan 8.
OBJECTIVE

The proportion of circulating cell free DNA derived from the feto-placental unit (fetal fraction or FF) correlates with test success and interpretation reliability. Some fetal disorders are associated with systematically lower FF, sometimes resulting in noninformative results.

METHODS

We analyzed results from pregnancies tested in a nested case/control study derived from a cohort of 4664 high-risk pregnancies. Low FF was defined before and after adjusting for maternal weight and gestational age.

RESULTS

Compared with euploid pregnancies, the median FF was significantly higher in Down syndrome pregnancies (ratio 1.17) and significantly lower in trisomy 18 and triploid pregnancies (ratios 0.71 and 0.19, respectively). Among 2157 pregnancies tested, 13 (0.6%) had FF <3.0% (all noninformative), including three trisomy 18 and three triploidy fetuses. After adjustment, 16 pregnancies (0.7%) had FF <0.3 multiples of the median (six informative), including one trisomy 18 and three triploidy fetuses. Modeled positive predictive values for low and high-risk populations were 7% and 30%, respectively.

CONCLUSION

Among women with noninformative results attributable to low FF, trisomy 18 and/or triploidy risk are sufficiently high to warrant offering additional assessments (e.g. ultrasound). If the testing indication is ultrasound abnormality, amniocentesis and karyotype/microarray should be considered.

Dharajiya N1, Zwiefelhofer T, Guan X, Angkachatchai V, Saldivar JS. Curr Protoc Hum Genet. 2015 Jan 20;84:8.15.1-8.15.20. doi: 10.1002/0471142905.hg0815s84.

Noninvasive prenatal testing (NIPT) represents an outstanding example of how novel scientific discoveries can be quickly and successfully developed into hugely impactful clinical diagnostic tests. Since the introduction of NIPT to detect trisomy 21 in late 2011, the technology has rapidly advanced to analyze other autosomal and sex chromosome aneuploidies, and now includes the detection of subchromosomal deletion and duplication events. Here we provide a brief overview of how noninvasive prenatal testing using next-generation sequencing is performed.

Zhao C1, Tynan J1, Ehrich M2, Hannum G1, McCullough R1, Saldivar JS1, Oeth P1, van den Boom D3, Deciu C4. Clin Chem. 2015 Apr;61(4):608-16. doi: 10.1373/clinchem.2014.233312. Epub 2015 Feb 20.
BACKGROUND

The development of sequencing-based noninvasive prenatal testing (NIPT) has been largely focused on whole-chromosome aneuploidies (chromosomes 13, 18, 21, X, and Y). Collectively, they account for only 30% of all live births with a chromosome abnormality. Various structural chromosome changes, such as microdeletion/microduplication (MD) syndromes are more common but more challenging to detect. Recently, several publications have shown results on noninvasive detection of MDs by deep sequencing. These approaches demonstrated the proof of concept but are not economically feasible for large-scale clinical applications.

METHODS

We present a novel approach that uses low-coverage whole genome sequencing (approximately 0.2×) to detect MDs genome wide without requiring prior knowledge of the event's location. We developed a normalization method to reduce sequencing noise. We then applied a statistical method to search for consistently increased or decreased regions. A decision tree was used to differentiate whole-chromosome events from MDs.

RESULTS

We demonstrated via a simulation study that the sensitivity difference between our method and the theoretical limit was <5% for MDs ≥9 Mb. We tested the performance in a blinded study in which the MDs ranged from 3 to 40 Mb. In this study, our algorithm correctly identified 17 of 18 cases with MDs and 156 of 157 unaffected cases.

CONCLUSIONS

The limit of detection for any given MD syndrome is constrained by 4 factors: fetal fraction, MD size, coverage, and biological and technical variability of the event region. Our algorithm takes these factors into account and achieved 94.4% sensitivity and 99.4% specificity.

Julie Jesiolowski, Thomas Monroe, Courtney Fitch, Jenna Wardrop, Theresa Boomer, Juan-Sebastian Saldivar. Contemporary OBGYN: March 5, 2015
INTRODUCTION

Noninvasive prenatal testing (NIPT) has rapidly changed the prenatal landscape for pregnant women at increased risk of fetal aneuploidy. This technology provides high sensitivity and specificity for Trisomy 21, 18, and 13. Overrepresentation of a chromosome can be detected by an increased Z-score in comparison with a normal euploid genome. Here we report a case involving a partial chromosome 13 duplication that assisted in identifying a maternal balanced translocation and revealed the limitations of suboptimal karyotype resolution.

METHODS

Maternal plasma samples were subjected to DNA extraction and library preparation followed by massively parallel sequencing as described by Palomaki et al. Sequencing data were analyzed using a novel algorithm to detect trisomies and other subchromosomal events as described by Chen et al.

RESULTS

: A 38-year-old G8P3043 presented for NIPT due to advanced maternal age and a previous pregnancy history of Trisomy 13, confirmed by low-resolution karyotype on peripheral blood. Ultrasound at 12 weeks, 6 days was suspicious for micrognathia. NIPT studies were ordered and results were positive for Trisomy 13. Sequencing data were reviewed based on the clinical history and revealed a 24.3Mb duplication of 13q31.2 and an apparent 27.85Mb deletion of 4q32.2. A fetal demise was noted on a 15 week, 0 day ultrasound. Products of conception (POC) studies were performed at 450–500 band resolution resulting in a normal female karyotype 46,XX, discordant from the NIPT results. Follow-up maternal studies at high-resolution karyotype analysis detected a balanced translocation: 46,XX,t(4;13)(q32;q31). After informing the tissue analysis laboratory of the translocation events, discordance between NIPT and POC studies was attributed to low karyotype resolution.

CONCLUSION

This case demonstrates the power of NIPT sequencing technology and optimized bioinformatics. Clinicians should be conscious that standard karyotyping does not have sufficient resolution to detect subchromosomal events detected by NIPT. Accurate clinical information provided to the laboratory may aid in additional interpretation. In these cases, microarray studies or high-resolution karyotype should be used to confirm a suspected abnormality.

Jensen TJ1, Kim SK2, Zhu Z3, Chin C4, Gebhard C5, Lu T6, Deciu C7, van den Boom D8, Ehrich M9. Genome Biol. 2015 Apr 15;16:78. doi: 10.1186/s13059-015-0645-x.
BACKGROUND

Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors.

RESULTS

We perform whole genome bisulfite sequencing on a set of unmatched samples including circulating cell-free DNA from non-pregnant and pregnant female donors and genomic DNA from maternal buffy coat and placenta samples. We find CpG cytosines within longer fragments are more likely to be methylated. Comparison of the methylomes of placenta and non-pregnant circulating cell-free DNA reveal many of the 51,259 identified differentially methylated regions are located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases. We find these placenta hypomethylated domains are consistently located within regions exhibiting low CpG and gene density. Differentially methylated regions identified when comparing placenta to non-pregnant circulating cell-free DNA are recapitulated in pregnant circulating cell-free DNA, confirming the ability to detect differential methylation in circulating cell-free DNA mixtures.

CONCLUSIONS

We generate methylome maps for four sample types at single-base resolution, identify a link between DNA methylation and fragment length in circulating cell-free DNA, identify differentially methylated regions between sample groups, and uncover the presence of megabase-size placenta hypomethylated domains.

Clark-Ganheart CA1, Fries MH, Leifheit KM, Jensen TJ, Moreno-Ruiz NL, Ye PP, Jennings JM, Driggers RW. Obstet Gynecol. 2015 Jun;125(6):1321-9. doi: 10.1097/AOG.0000000000000863

OBJECTIVE

To estimate whether cell-free DNA is present in nonviable pregnancies and thus can be used in diagnostic evaluation in this setting.

METHODS

We conducted a prospective cohort study of 50 participants at MedStar Washington Hospital Center, Washington, DC, between June 2013 and January 2014. Included were women with pregnancies complicated by missed abortion or fetal demise. All gestational ages were considered for study participation. Participants with fetal demise were offered the standard workup for fetal death per the American College of Obstetricians and Gynecologists. Maternal blood samples were processed to determine the presence of cell-free DNA, the corresponding fetal fractions, and genetic abnormalities.

RESULTS

Fifty samples from nonviable pregnancies were analyzed. The average clinical gestational age was 16.9 weeks (standard deviation 9.2). The mean maternal body mass index was 30.3 (standard deviation 9.1). Seventy-six percent (38/50) of samples yielded cell-free DNA results, that is, had fetal fractions within the detectable range of 3.7-65%. Among the 38, 76% (29) were classified as euploid, 21% (8) as trisomies, and 3% (1) as microdeletion. A cell-free DNA result was obtained more frequently at ultrasonographic gestational ages of 8 weeks or greater compared with less than 8 weeks (87.9% [n=29/33, 95% confidence interval (CI) 72.7-95.2; and 52.9%, n=9/17, 95% CI 31.0-73.8] of the time, respectively, P=.012). Time from demise was not associated with obtaining a result.

CONCLUSION

Among nonviable pregnancies, cell-free DNA is present in the maternal plasma with fetal fractions greater than 3.7% in more than three fourths of cases after an ultrasonographic gestational age of 8 weeks.

CLINICAL TRIAL REGISTRATION

ClinicalTrials.gov, www.clinicaltrials.gov, NCT01916928.

LEVEL OF EVIDENCE

III

Kim SK1, Hannum G1, Geis J1, Tynan J1, Hogg G1, Zhao C1, Jensen TJ1, Mazloom AR1, Oeth P1, Ehrich M1, van den Boom D1, Deciu C1. Prenat Diagn. 2015 Aug;35(8):810-5. doi: 10.1002/pd.4615. Epub 2015 Jun 3.
OBJECTIVE

This study introduces a novel method, referred to as SeqFF, for estimating the fetal DNA fraction in the plasma of pregnant women and to infer the underlying mechanism that allows for such statistical modeling.

METHODS

Autosomal regional read counts from whole-genome massively parallel single-end sequencing of circulating cell-free DNA (ccfDNA) from the plasma of 25,312 pregnant women were used to train a multivariate model. The pretrained model was then applied to 505 pregnant samples to assess the performance of SeqFF against known methodologies for fetal DNA fraction calculations.

RESULTS

Pearson's correlation between chromosome Y and SeqFF for pregnancies with male fetuses from two independent cohorts ranged from 0.932 to 0.938. Comparison between a single-nucleotide polymorphism-based approach and SeqFF yielded a Pearson's correlation of 0.921. Paired-end sequencing suggests that shorter ccfDNA, that is, less than 150 bp in length, is nonuniformly distributed across the genome. Regions exhibiting an increased proportion of short ccfDNA, which are more likely of fetal origin, tend to provide more information in the SeqFF calculations.

CONCLUSION

SeqFF is a robust and direct method to determine fetal DNA fraction. Furthermore, the method is applicable to both male and female pregnancies and can greatly improve the accuracy of noninvasive prenatal testing for fetal copy number variation.

Helgeson J1, Wardrop J1, Boomer T1, Almasri E1, Paxton WB1, Saldivar JS1, Dharajiya N1, Monroe TJ2, Farkas DH3,4, Grosu DS1, McCullough RM1. Prenat Diagn. 2015 Jul 27. doi: 10.1002/pd.4640. [Epub ahead of print]

OBJECTIVE

A novel algorithm to identify fetal microdeletion events in maternal plasma has been developed and used in clinical laboratory-based noninvasive prenatal testing. We used this approach to identify the subchromosomal events 5pdel, 22q11del, 15qdel, 1p36del, 4pdel, 11qdel, and 8qdel in routine testing. We describe the clinical outcomes of those samples identified with these subchromosomal events.

METHODS

Blood samples from high-risk pregnant women submitted for noninvasive prenatal testing were analyzed using low coverage whole genome massively parallel sequencing. Sequencing data were analyzed using a novel algorithm to detect trisomies and microdeletions.

RESULTS

In testing 175,393 samples, 55 subchromosomal deletions were reported. The overall positive predictive value for each subchromosomal aberration ranged from 60% to 100% for cases with diagnostic and clinical follow-up information. The total false positive rate was 0.0017% for confirmed false positives results; false negative rate and sensitivity were not conclusively determined.

CONCLUSION

Noninvasive testing can be expanded into the detection of subchromosomal copy number variations, while maintaining overall high test specificity. In the current setting, our results demonstrate high positive predictive values for testing of rare subchromosomal deletions.



Scientific Posters

Sequenom Laboratories' posters

Jenna Wardrop1, Theresa Boomer1, Eyad Almasri1, Samantha Caldwell1, Sidra Boshes1, Ron McCullough1 1Sequenom Laboratories, San Diego, CA

Samantha Caldwell, Sidra Boshes, Jenna Wardrop, Theresa Boomer, Phillip Cacheris, Hany Magharyous, Ron McCullough.

Sequenom Laboratories.

Roy B. Lefkowitz, John A. Tynan, Tong Liu, Yijin Wu, Amin R. Mazloom, Eyad Almasri, Grant Hogg, Theresa Boomer, Jenna Wardrop, Ron McCollough, Marcos Gonzales, Mathias Ehrich. 

Sequenom Laboratories.

Jenna Wardrop, Marcos Gonzales, Theresa Boomer, Phillip Cacheris, Eyad Almasri, Ron McCullough.

Sequenom Laboratories.

Amin R. Mazloom, Tong Liu, Roy Lefkowitz, Yijin Wu, Grant Hogg, John Tynan, Timothy Burcham, Dirk van den Boom, Mathias Ehrich. Sequenom Laboratories.

  • We previously demonstrated genome-wide detection of 3-40 Mb microdeletions and micro -duplications in maternal plasma at approximately ~16M reads per sample.+++
  • Using a purely theoretic model, we find that ~16M reads per sample can theoretically provide a >95% sensitivity for events ≥7 Mb. 
  • Using whole genome sequencing results from plasma DNA, we were able to model in-silico genome-wide events. These simulations show >90% sensitivity for ≥7 Mb and >95% for events ≥10 Mb.

+++Zhao C, et al. Detection of fetal subchromosomal abnormalities by sequencing circulating cell-free DNA from maternal plasma. Clin Chem, 2015. 61(4), 608-616

Theresa Boomer, Judy Fisher Newell, Patricia Santiago-Munoz, Christina Settler, Michelle N Strecker, Jenna Wardrop, Julie Jesiolowski, Ron McCullough, Juan-Sebastian Saldivar, William B Paxton, Thomas Monroe4, Nilesh Dharajiya. Sequenom Laboratories, University of Texas Southwestern Medical Center, Department of Obstetrics and Gynecology, Dallas, TX and Combimatrix, Irvine, CA.

The emerging contribution of NIPT genomic data offers a unique view into the earliest forming layer of placenta (trophoblast). Providing a snapshot in an embryology timeline, NIPT can assist with deciphering the timing and origin of complex de novo events such as ring chromosomes. This invaluable insight can help explain chromosomal inconsistencies that result among tissue types, screening tests, and diagnostic technologies.

Clinicians need to be mindful of the biological strengths and limitations of NIPT, especially in regard to chromosomal mosaicism and complex structural rearrangements. Such awareness is invaluable when presented with seemingly discrepant prenatal results and promotes accurate clinical assessment, counseling, and overall case interpretation.
Furthermore, clinicians should also be aware of NIPT’s ability to identify maternal subchromosomal abnormalities. Accurate clinical information provided to the laboratory may aid in additional interpretation.

Jennifer Helgeson, Sharon Namaroff, Mary-Frances Garber, Lisa Dunn-Albanese, Daniel Katz, Yael Hoffman-Sage, Marney Brillinger. Sequenom Laboratories & Newton-Wellesley Hospital, Newton, MA.

The performance of the MaterniT21® Plus LDT in this high risk population, representative of patients typically treated in a Maternal Fetal Medicine setting, continues to show a very low overall false positive rate (0.13%) compared with traditional maternal serum screening modalities. Recently, it has been suggested that samples with a non-reportable ccfDNA result may be enriched for fetal aneuploidy++. This has not been the experience with this cohort.

++Pergament E, et al. Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort; Obstet Gynecol. 2014;0:1-9.

Jenna Wardrop, Julie Jesiolowski, Nilesh Dharajiya, Thomas J Monroe, Theresa Boomer, Eyad Almasri, Ron McCullough. Sequenom Laboratories.

Initially used for detecting trisomy 21 only, MaterniT21® PLUS has advanced into a LDT for also detecting trisomy 18, trisomy 13, and sex chromosome aneuploidies. Here, we demonstrate continued expansion of the technological capabilities to effectively detect microdeletions and two additional rare trisomies.

Penn Whitley, John Tynan, Adam Simpson, Tim Burcham, Ali Torkamani, Ashley Van Zeeland, Mathias Ehrich, Ron M McCullough, Juan-Sebastian Saldivar, Tom Monroe, Paul Oeth, and Dirk van den Boom. Sequenom Laboratories and Cypher Genomics, Inc.

  • The manual curation of Copy Number Variation in a NIPT setting is time consuming (~0.5-4hr/CNV/curator), subjective and potentially subject to error.
  • Data provenance and standards are also difficult to maintain in an environment with manual curation and interpretation.
  • The Cypher Genomics Mantis™ System provided highly concordant results with those from the working group when the initial call was pathogenic or likely pathogenic.
  • Manual VOUS calls were less concordant with the automated results. This finding is likely reflective of the different data sources and scoring systems used by the methods.
  • Discrepancy analysis resulted in several scoring reversals when the working groups re-evaluated the CNV calls. The reasons for these reversals may have been a result of updated data sources or more consistent scoring methodology used during the re-evaluation.
  • These results suggest that an automated interpretation and curation system can provide an excellent ‘first line of defense’ in a clinical setting.
  • The combination of the standardized output and interpretation generated by the Mantis™ system, along with the experience and judgment of a lab director, should provide excellent results to clinicians

Thomas Monroe, Nilesh Dharajiya, Julie Jesiolowski, Theresa Boomer, Ron McCullough, Chen Zhao, Paul Oeth, Juan-Sebastian Saldivar. Sequenom Laboratories.

NIPT has rapidly become a part of the standard of care for aneuploidy testing in high-risk pregnancies. We demonstrated initial expansion of this technology by effectively detecting certain microdeletions. We also continue to augment the number of conditions we are screening, which now includes Wolf-Hirshhorn and other microdeletions of significant clinical relevance. This abstract provides evidence and further supports broadening the capabilities of noninvasive testing to detect subchromosomal deletion events, as well as the potential to derive fetal karyotypes in the future.

Julie Jesiolowski, Thomas Monroe, Courtney Fitch, Jenna Wardrop, Theresa Boomer, Juan-Sebastian Saldivar. Sequenom Laboratories and Lehigh Valley Maternal Fetal Medicine, Allentown, PA.
This case demonstrates the unique insight NIPT data can provide, especially in relation to compromised POC samples. Clinicians should be aware that standard karyotyping may not have sufficient resolution to detect subchromosomal events detectable by NIPT. Accurate clinical information provided to the laboratory may aid in additional interpretation. In these cases, microarray studies or high resolution karyotyping are the preferred methods for confirming a suspected abnormality.

Ron M McCullough, Juan-Sebastian Saldivar, Thomas Monroe, Theresa Boomer, Jenna Wardrop, Julie Jesiolowski, Jennifer Hume, Nilesh Dharajiya. Sequenom Laboratories.

It is imperative when testing for rare conditions, such as 22q11 deletions, that NIPT tests perform with the utmost specificity to result in high positive predictive values. By using a whole genome sequencing approach, we have demonstrated that this objective is achievable. This abstract provides further evidence and support to broaden the scope of noninvasive testing to detect sub-chromosomal deletion/ duplication events and the potential to derive fetal karyotypes in the future.