Nederlandse labtechnische publicaties:

van Beek DM, Straver R, Weiss MM, Boon EMJ, Huijsdens-van Amsterdam K, Oudejans CBM, Reinders MJT, Sistermans EA. Comparing methods for fetal fraction determination and quality control of NIPT samples. Prenatal Diagnosis 2017;37:769-773.

Van Opstal D, et al. False Negative NIPT Results: Risk Figures for Chromosomes 13, 18 and 21 Based on Chorionic Villi Results in 5967 Cases and Literature Review. PLoS One. 2016 Jan 15;11(1):e0146794.

Neveling K, et al. Validation of two-channel sequencing-by-synthesis for noninvasive prenatal testing of fetal whole and partial chromosome aberrations. Prenat Diagn. 2016 Jan 15. doi: 10.1002/pd.4777.

Straver R, et al. Calculating the fetal fraction for noninvasive prenatal testing based on genome-wide nucleosome profiles. Prenat Diagn. 2016;36:614-21.

Van Opstal D, Srebniak MI. Cytogenetic confirmation of a positive NIPT result: evidence-based choice between chorionic villus sampling and amniocentesis depending on chromosome aberration. Expert Rev Mol Diagn. 2016 Feb 10.

Tamminga S, et al. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing. Adv Clin Chem 2016;74:63-102.

Sistermans E, et al. Maternal Malignancies Detected With Noninvasive Prenatal Testing. JAMA. 2015 Nov 24;314(20):2192. [reactie op Bianchi et al]

Thurik FF, et al. Analysis of false-positive results of fetal RHD typing in a national screening program reveals vanishing twins as potential cause for discrepancy. Prenat Diagn. 2015;35(8):754-60.

Mersy E, et al. Cell-Free RNA Is a Reliable Fetoplacental Marker in Noninvasive Fetal Sex Determination. Clin Chem. 2015 Dec;61(12):1515-23.

van den Oever JM, et al. Noninvasive prenatal diagnosis of Huntington disease: detection of the paternally inherited expanded CAG repeat in maternal plasma. Prenat Diagn. 2015;35:945-9.

Hochstenbach R, et al Unexplained False Negative Results in Noninvasive Prenatal Testing: Two Cases Involving Trisomies 13 and 18. Case Rep Genet. 2015;2015:926545.

Hochstenbach R et al. Cell-free fetal DNA in the maternal circulation originates from the cytotrophoblast: proof from an unique case. Clin Case Rep. 2015 Jun;3(6):489-91

Straver R et al. Introducing WISECONDOR for noninvasive prenatal diagnostics.
Expert Rev Mol Diagn. 2014 Jun;14(5):513-5

Thung DT et al. Implementation of whole genome massively parallel sequencing for noninvasive prenatal testing in laboratories. Expert Rev Mol Diagn. 2015 Jan;15(1):111-24

Buysse K et al. Reliable noninvasive prenatal testing by massively parallel sequencing of circulating cell-free DNA from maternal plasma processed up to 24hours after venipuncture. Clin Biochem. 2013 Aug 8

Oever van den J etal.
Noninvasive prenatal diagnosis of Huntington disease: detection of the paternally inherited expanded CAG repeat in maternal plasma.
Prenatal Diagnosis 2015″]

van den Oever JM, Bijlsma EK, Feenstra I, Muntjewerff N, Mathijssen IB, Bakker E, van Belzen MJ, Boon EM.



With a shift towards noninvasive testing, we have explored and validated the use of noninvasive prenatal diagnosis (NIPD) for Huntington disease (HD).


Fifteen couples have been included, assessing a total of n = 20 pregnancies. Fetal paternally inherited CAG repeat length was determined in total cell-free DNA from maternal plasma using a direct approach by PCR and subsequent fragment analysis.


All fetal HD (n = 7) and intermediate (n = 3) CAG repeats could be detected in maternal plasma. Detection of repeats in the normal range (n = 10) was successful in n = 5 cases where the paternal repeat size could be distinguished from maternal repeat patterns after fragment analysis. In all other cases (n = 5), the paternal peaks coincided with the maternal peak pattern. All NIPD results were concordant with results from routine diagnostics on fetal genomic DNA from chorionic villi.


In this validation study, we demonstrated that all fetuses at risk for HD could be identified noninvasively in maternal plasma. Additionally, we have confirmed results from previously described case reports that NIPD for HD can be performed using a direct approach by PCR. For future diagnostics, parental CAG profiles can be used to predict the success rate for NIPD prior to testing.

Prenat Diagn. 2015 Oct;35(10):945-9.


[toggle Title=”Oever van den J etal.
Successful noninvasive trisomy 18 detection using single molecule sequencing.
Clin Chem. 2013″]

van den Oever JM, Balkassmi S, Johansson LF, Adama van Scheltema PN, Suijkerbuijk RF, Hoffer MJ, Sinke RJ, Bakker E, Sikkema-Raddatz B, Boon EM.

Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA), Leiden University Medical Center, Leiden, The Netherlands.



Noninvasive trisomy 21 detection performed by use of massively parallel sequencing is achievable with high diagnostic sensitivity and low false-positive rates. Detection of fetal trisomy 18 and 13 has been reported as well but seems to be less accurate with the use of this approach. The reduced accuracy can be explained by PCR-introduced guanine-cytosine (GC) bias influencing sequencing data. Previously, we demonstrated that sequence data generated by single molecule sequencing show virtually no GC bias and result in a more pronounced noninvasive detection of fetal trisomy 21. In this study, single molecule sequencing was used for noninvasive detection of trisomy 18 and 13.


Single molecule sequencing was performed on the Helicos platform with free DNA isolated from maternal plasma from 11 weeks of gestation onward (n = 17). Relative sequence tag density ratios were calculated against male control plasma samples and results were compared to those of previous karyotyping.


All trisomy 18 fetuses were identified correctly with a diagnostic sensitivity and specificity of 100%. However, low diagnostic sensitivity and specificity were observed for fetal trisomy 13 detection.


We successfully applied single molecule sequencing in combination with relative sequence tag density calculations for noninvasive trisomy 18 detection using free DNA from maternal plasma. However, noninvasive trisomy 13 detection was not accurate and seemed to be influenced by more than just GC content.

Clin Chem. 2013 Apr;59(4):705-9


[toggle Title=”Norton ME etal.
Non-Invasive Chromosomal Evaluation (NICE) Study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18.
AJOG 2012“]

Norton ME, Brar H, Weiss J, Karimi A, Laurent LC, Caughey AB, Rodriguez MH, Williams J 3rd, Mitchell ME, Adair CD, Lee H, Jacobsson B, Tomlinson MW, Oepkes D, Hollemon D, Sparks AB, Oliphant A, Song K.

Department of Obstetrics and Gynecology, Stanford University/Lucile Packard Children’s Hospital, Stanford, CA.

We sought to evaluate performance of a noninvasive prenatal test for fetal trisomy 21 (T21) and trisomy 18 (T18).
A multicenter cohort study was performed whereby cell-free DNA from maternal plasma was analyzed. Chromosome-selective sequencing on chromosomes 21 and 18 was performed with reporting of an aneuploidy risk (High Risk or Low Risk) for each subject.
Of the 81 T21 cases, all were classified as High Risk for T21 and there was 1 false-positive result among the 2888 normal cases, for a sensitivity of 100% (95% confidence interval [CI], 95.5-100%) and a false-positive rate of 0.03% (95% CI, 0.002-0.20%). Of the 38 T18 cases, 37 were classified as High Risk and there were 2 false-positive results among the 2888 normal cases, for a sensitivity of 97.4% (95% CI, 86.5-99.9%) and a false-positive rate of 0.07% (95% CI, 0.02-0.25%).
Chromosome-selective sequencing of cell-free DNA and application of an individualized risk algorithm is effective in the detection of fetal T21 and T18.

AJOG June 2012. [Epub ahead of print]


[toggle Title=”Oever van den JM etal.
Single Molecule Sequencing of Free DNA from Maternal Plasma for Noninvasive Trisomy 21 Detection
Clinical Chemistry 2012“]

van den Oever JM, Balkassmi S, Verweij EJ, van Iterson M, Adama van Scheltema PN, Oepkes D, van Lith JM, Hoffer MJ, den Dunnen JT, Bakker E, Boon EM.

Center for Human and Clinical Genetics, Laboratory for Diagnostic Genome Analysis.


Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in amplification bias in GC-rich areas of the human genome. To eliminate this bias, and thereby experimental noise, we have used single molecule sequencing as an alternative method.

For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform.

Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses.

This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.

Clin Chem. 2012 Jan 25. [Epub ahead of print]

[toggle Title=”Faas BH etal.
Non-invasive prenatal diagnosis of fetal aneuploidies using massively parallel sequencing-by-ligation and evidence that cell-free fetal DNA in the maternal plasma originates from cytotrophoblastic cells
Expert Opin Biol There 2012“]

Faas BH, de Ligt J, Janssen I, Eggink AJ, Wijnberger LD, van Vugt JM, Vissers L, Geurts van Kessel A.
Radboud University Nijmegen Medical Centre, Department of Human Genetics


Blood plasma of pregnant women contains circulating cell-free fetal DNA (ccffDNA), originating from the placenta. The use of this DNA for non-invasive detection of fetal aneuploidies using massively parallel sequencing (MPS)-by-synthesis has been proven previously. Sequence performance may, however, depend on the MPS platform and therefore we have explored the possibility for multiplex MPS-by-ligation, using the Applied Biosystems SOLiD(™) 4 system. DNA isolated from plasma samples from 52 pregnant women, carrying normal or aneuploid fetuses, was sequenced in multiplex runs of 4, 8 or 16 samples simultaneously. The sequence reads were mapped to the human reference genome and quantified according to their genomic location. In case of a fetal aneuploidy, the number of reads of the aberrant chromosome is expected to be higher or lower than in normal reference samples. To statistically determine this, Z-scores per chromosome were calculated as described previously, with thresholds for aneuploidies set at > +3.0 and < -3.0 for chromosomal over- or underrepresentation, respectively. All samples from fetal aneuploidies yielded Z-scores outside the thresholds for the aberrant chromosomes, with no false negative or positive results. Full-blown fetal aneuploidies can thus be reliably detected in maternal plasma using a multiplex MPS-by-ligation approach. Furthermore, the results obtained with a sample from a pregnancy with 45,X in the cytotrophoblastic cell layer and 46,XX in the mesenchymal core cells show that ccffDNA originates from the cytotrophoblastic cell layer. Discrepancies between the genetic constitution of this cell layer and the fetus itself are well known, and therefore, care should be taken when translating results to the fetus itself.

Expert Opin Biol Ther. 2012 Apr 16. [Epub ahead of print]

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