Non-invasive prenatal screening (NIPS) methodologies and possible unanticipated test results
Welcome to Part 2 of our blog series on how NIPS has changed the world of prenatal screening. This installment will focus on NIPS methodologies, and possible unanticipated test results that can be received.
Methodologies for non-invasive prenatal screening (NIPS)/cell-free fetal DNA screening
The methodologies of testing used for NIPS can be roughly divided into Massively Parallel Shotgun Sequencing (MPSS) and targeted DNA SNP testing.¹ ² MPSS technology sequences millions of maternal and fetal DNA fragments across the genome and uses this data to provide a Z-score for the chromosomes of interest. The Z-score indicates to what degree the data differs from expected ratios. For example, if chromosome 21 makes up 2% of the DNA in the genome, are we seeing 2% or 3% in the sample? This technology means that a lot of data from other chromosomes is collected that may not be used in the final analysis. However, sometimes having this additional data can help resolve confusing cases.
Targeted DNA SNP testing sequences DNA SNP fragments on the chromosomes of interest from both the maternal and fetal genomes and uses the maternal SNP data to predict the fetal status. Both technologies can be used for most pregnancies. However, if you have a pregnancy from an egg donor, a surrogate, a bone marrow transplant recipient, a consanguineous (blood related) couple, or a pregnancy with multiples, it is important to contact the laboratory you plan to use prior to submitting the sample to confirm that they will be able to issue a result.
For either methodology above, the data is then analyzed by a complex bioinformatics process to provide results. Note that just because two laboratories use a similar NIPS methodology does not mean they will have the same capabilities, or provide the same results. As most clinicians will use multiple laboratories for their patients’ testing depending upon health insurance, test cost and other factors, it is important to understand the capabilities, limitations and the reporting methods for each lab individually so that clinicians and patients can make the best testing choice for their unique situation.
Sometimes NIPS can yield results that were not expected by the clinician. The unanticipated results that can be received from NIPS can be subdivided into three main categories: no calls (or partial no calls), false positives, and gender discordance.
No calls may be the most frustrating for a clinician and their patient. The failure to get results back for a patient may be due to a few factors: low fetal fraction, uninterpretable data, maternal neoplasm, and maternal incidental findings. In these cases, the laboratory should provide guidance on appropriate follow up testing choices.
Low fetal fraction: If there is not enough fetal DNA in the sample (versus maternal DNA), there will not be enough data to provide a result.
Maternal neoplasm: Circulating tumor DNA from the mother can cause unusual test results for more than one chromosome, with results showing a risk for not just one trisomy, but for many, leading to concern about a maternal neoplasia (cancer). These results may be reported out as uninterpretable. A publication from 2018 summarizes the findings in 55 no call cases with such complex results, using the MPSS methodology.³ Out of the 55 cases analyzed, 40 cases were confirmed to have a maternal neoplasm. These cases included 20 benign uterine fibroids, 18 confirmed malignancies, and 2 malignancies that were confirmed radiologically but with no pathology.
Maternal incidental findings are biological conditions present in the mother that may confound the NIPS result. If the mother has mosaic Turner syndrome (45 X/46 XX), or 47 XXX, it may result in confusion regarding the status of the fetal X chromosomes. The mother may also have a previously undiagnosed 22q microdeletion, as this phenotype is extremely variable. Either of these may result in a partial or no call report, with guidance given for follow up testing to clarify the fetal and maternal status.
False positive test results may be caused by biological factors, including a vanished twin during the pregnancy, confined placental mosaicism, and benign maternal duplications. The possible occurrence of false positives is why NIPS is referred to as a screening test. With a positive result, the lab should provide a PPV (positive predictive value) on the report. This will help the clinician in counseling the patient to understand the actual risk to the pregnancy, and will help in the discussion of follow up diagnostic testing options.
Vanishing twin is used to describe the spontaneous loss of a twin, seen early in pregnancy. Depending on the timing between that loss and the blood draw for NIPS, some of that DNA may still be present in the mother’s blood.
Confined placental mosaicism is a known entity from the early days of chorionic villus sampling (CVS). As the cell-free DNA that is studied in NIPS is from the placenta and membranes, NIPS can detect a trisomy that may be present only in those tissues, and not in the fetus. This is one reason why diagnostic testing is always recommended for confirmation, prior to any decision making after receiving a positive NIPS screen.
Benign maternal duplications: A benign maternal duplication on chromosome 18 has been reported as one probable cause for false positive trisomy 18 results on NIPS.⁴ This type of copy number variation does not affect the mother’s health, but it may cause the bioinformatic analysis to “think” that there is more of chromosome 18 present than there should be in the fetus. Some laboratories may adjust their bioinformatic analysis to account for these maternal duplications.⁵
Gender Discordance refers to a difference between the NIPS predicted gender, and the actual fetal status as seen on ultrasound. When a potential discordance is recognized, and after a repeat of the NIPS to confirm the result, additional studies may be needed to sort out the discrepancy. These studies may include looking more carefully at the maternal history, the fetal and/or maternal karyotypes, and/or a detailed ultrasound. This type of discrepancy may not be ultimately reconciled until conducting further testing after birth.
Please return to Metis on the Move in the future to read the final installment of our blog series on how NIPS has changed the world of prenatal screening. Our next blog will discuss what patients need to know when being counseled about the option of NIPS.
References:
1. ACMG Statement: Noninvasive prenatal screening for fetal aneuploidy,2016 update: a position statement of the American College of Medical Genetics and Genomics. Anthony R. Gregg, et al. Genet in Med,2016,18(10):1056-1065.
2. Prenatal Screening and Diagnosis, An Issue of the Clinics in Laboratory Medicine, E-Book, Anthony O. Odibo, David A. Krantz, Elsevier Health Sciences, Jun 11, 2016.
3. Incidental Detection of Maternal Neoplasia in Noninvasive Prenatal Testing. Dharajiya NG et al. Clin Chem, 2018 Feb;64(2):329-335.
4. Copy-Number variation and false positive prenatal aneuploidy screening results. Snyder MW et al. N Engl J Med. 2015;372: 1639-1645
5. Improving the Positive Predictive Value of Non-Invasive Prenatal Screening (NIPS). Strom CM, et al. PLoS One, 2017; 12(3): doi: 10.1371/journal.pone.0167130
Joy Redman, MS, CGC, MBA
Joy is a Trusted Advisor for Metis Genetics. She is a board-certified genetic counselor with a long time interest in the translation of genetic information between the laboratory and healthcare providers. She has practiced clinically in both the prenatal and ophthalmology settings; educating patients about the risks and benefits of genetic testing to help them make decisions regarding their own healthcare. She has worked in a variety of laboratory settings; each one focusing on education about genetic test offerings as the field has evolved so that clinicians are able to provide the best testing options to each patient.