Cell Free Fetal DNA in Non- Invasive Prenatal Molecular Screening Christopher K. Henry Honors Program Thesis Faculty Mentor: Dr. Pamela Langer Department of Molecular Biology University of Wyoming May 2020 Chris Henry 2 Abstract Prenatal genetic diagnostic testing methods are commonly used for high-risk pregnancies in order to gain information about the embryo or fetus. There are many different technologies that can be used for prenatal testing, each which comes with its own advantages and disadvantages. One screening method that is becoming increasingly reliable is cell free fetal DNA (cffDNA) testing. The cffDNA screen analyzes fetal DNA that is present in the maternal bloodstream during a pregnancy. This cffDNA can be separated from other blood components and sequenced, which will then provide information about the likelihood of a fetus having a chromosomal abnormality or a single gene disorder. There are three main DNA sequencing methods: Massively Parallel Shotgun Sequencing (MPSS), Digital Analysis of Specific Regions (DANSR), and Single Nucleotide Polymorphism (SNP)-based sequencing. The cffDNA screening method has very high accuracy for detecting abnormal numbers of chromosomes (aneuploidies), specifically the three copies of chromosome 21 in Down syndrome, for which this screening method is the most accurate non-invasive prenatal test. Benefits of cffDNA screening include that it is non-invasive and has a zero chance of increasing the risk of spontaneous termination of a fetus, a risk that is more of a concern for invasive prenatal diagnostic procedures such as chorionic villus sampling and amniocentesis. The accuracy of the cffDNA screen will continue to improve as technologies progress; but until that time, it is best used as a preliminary screen with positive results followed by invasive testing procedures if desired. Chris Henry 3 Introduction When a female becomes pregnant, she has the option to go through prenatal genetic diagnosis testing in order to gain information about the fetus or embryo (1). There are many reasons to undergo prenatal genetic diagnosis, many of which are associated with high-risk pregnancies where there is a higher than average likelihood of having a baby with a genetic defect, such as Down syndrome (Trisomy 21) or Cystic Fibrosis. Some causes of high-risk pregnancies include advanced maternal age (usually over 35), having a previous child with a chromosome abnormality, or one of the parents being a carrier of a monogenic disorder (2). The risk of having a child with an abnormal number of chromosomes, a condition called aneuploidy, increases dramatically in females with advanced maternal age (3) (Figure 1). Figure 1. Probability of chromosomal abnormality in the neonate as a function of maternal age. Chris Henry 4 Prenatal genetic diagnosis modalities include both invasive and non-invasive procedures. Invasive procedures include amniocentesis and chorionic villus sampling, where sample collection is followed by fluorescent in situ hybridization (FISH) with colored probes or DNA amplification and sequencing. Chromosomal abnormalities such as Trisomy 12 (Down syndrome) can be detected by FISH analysis (Figure 2.) Figure 2. Spectral karyotyping using FISH analysis revealing a trisomy 21 aneuploidy. Non-invasive testing involves ultrasounds and/or blood biochemical assessments such as the Quad screen. However, these non-invasive methods report risk and likelihood rather than definitive answers, which are current goals of researchers in the field (1). One of the newest and most promising tests is Cell Free Fetal DNA (cffDNA). This test uses fetal DNA that is found in the mother’s bloodstream (Figure 3) to screen for any disorders, such as chromosome aneuploidies or monogenic diseases and can even determine fetal sex and fetal RhD status (3). This wide range of applications demonstrates how promising the cffDNA test is for prenatal diagnosis and explains the dramatic increase in usage, with a total of 4-6 million pregnant women using this screen since its inception as of 2017 (4). Chris Henry 5 Figure 3. Cell free fetal DNA in the maternal bloodstream. Origin and Discovery of Cell Free Fetal DNA Cell free fetal DNA was first discovered in 1997 (5). Previously, researchers had reported that DNA from tumors was present in blood circulation (5). This finding led others to hypothesize that fetal DNA may be circulating in the mother’s bloodstream. To test this, researchers took the serum and plasma of pregnant women and ran a polymerase chain reaction (PCR) to amplify Y chromosome-specific DNA fragments (5). Since females do not have Y chromosomes, any Y chromosomal DNA in the serum or plasma would have to have come from a male fetus. In their testing, they were successful in being able to find Y chromosome DNA fragments in 21 out of 30 maternal serum samples and 24 out of 30 maternal plasma samples from females carrying a male fetus (5). They did not find any Y chromosomal DNA in the samples from the non-pregnant control women nor in the women carrying female fetuses. This study also determined that fetal DNA concentration in maternal circulation increases throughout pregnancy, an observation that has been confirmed by other studies that found it increases at a rate of 0.1 % weekly between 10 and 20 weeks and then increases by 1% weekly until delivery Chris Henry 6 (5,6). Further studies have determined that the fetal DNA comes from the apoptosis of syncytiotrophoblast cells which form the outermost layer of the placenta (6). cffDNA Screening Procedure The procedure for screening the cffDNA begins with a blood draw from the mother. Since there are higher levels of maternal DNA in the bloodstream than the fetal DNA, the draw needs to take place when the fetal fraction is high enough (6) (Figure 4). The fetal fraction is the proportion of total DNA in the bloodstream that is fetal DNA. The fetal fraction is approximately 11 to 13 percent of the total DNA present in the mother’s bloodstream during the late first trimester/early second trimester (6). Fetal fractions less than 4% are too low to be used in this screening method which is why this procedure is not performed until after the 10th week of gestation (4). Figure 4. Fraction of total blood DNA originating from the fetal placenta is the fetal fraction. Chris Henry 7 Once the blood is drawn, the plasma is separated from the platelets and red and white blood cells through centrifugation, and then DNA purification will be performed on the plasma to isolate the fragmented DNA that will be tested (7). Currently, there are many methods that are used when analyzing DNA that is purified from the plasma. One of them is real time PCR, but this method is expensive and time consuming (3). Other methods that are being used more frequently, as studies continuously prove their effectiveness, are random sequencing and targeted sequencing (4). Random sequencing is also known as Massively Parallel Shotgun Sequencing (MPSS) and involves sequencing millions of overlapping DNA fragments from every chromosome (8). Targeted sequencing involves amplifying and sequencing regions of DNA on chromosomes of interest. A promising targeted sequencing method involves sequencing DNA containing single nucleotide polymorphisms (SNPs) on specific chromosomes of interest (4). These methods have been successful and have their own advantages as well as limitations. Random Sequencing: Massively Parallel Shotgun Sequencing (MPSS) In this method, all the DNA fragments that are isolated from the maternal blood will be sequenced, including both fetal and maternal DNA. Most studies used Illumina sequencing as the sequencing method of choice, and then used Solexa to map the sequences to the genome (7). Illumina sequencing is a DNA sequencing method that determines the DNA sequence of fragmented DNA through the use of adaptors, bridge amplification, and sequencing using fluorescently tagged nucleotides. Once the sequencing is finished, there will be DNA sequences for all the DNA fragments in the sample which will then be analyzed using Solexa, which is a Chris Henry 8 computer software that aligns the overlapping DNA sequence reads with their respective chromosome. The number of reads per chromosome are then counted and converted to a percentage which are then compared with the normal percentages of a euploid genome. If the percentages fall in the accepted range, then it can be determined that the fetus is euploid. However, if a percentage is abnormal, then an aneuploidy is expected. For instance, the normal percentage of chromosome 21 fragments is 1.3 percent of total chromosomal DNA (6). If the screen finds that a percentage higher than 1.3 percent is attributed to chromosome 21, then it is expected that the fetus will be aneuploid for chromosome 21 which will cause the higher than expected percentage (Figure 5). The same principle is used for other common aneuploidies, such as trisomy 18, trisomy 13, and sex chromosome aneuploidies. Since this method uses overall percentages of all the DNA sequencing reads together, including the mother with a normal number of chromosome 21, it is not necessary to differentiate maternal and fetal DNA, which makes it easier to use than other uncommon methods (7). Figure 5. Results of Massively Parallel Shotgun Sequencing (MPSS) based on the ratio of sample sequence tags to normal sequence tags for chromosome 21. Chris Henry 9 Targeted Sequencing: Digital Analysis of Specific Regions (DANSR) and Single Nucleotide Polymorphisms (SNPs) In this targeted sequencing method, there are two approaches that are commonly used. One is very similar to the MPSS discussed above, except instead of randomly sequencing fragments from every chromosome, it specifically targets fragments from the chromosomes of interest, such as 21, 18, 13, X, and Y. This method is commonly referred to as DANSR (Digital Analysis of Specific Regions) because it targets loci on specific chromosomes (8). Like MPSS, DANSR requires a comparison to known proportions of chromosomal DNA in order to provide a result. The other approach uses single nucleotide polymorphisms (SNPs) on chromosomes of interest. SNPs on target chromosomes are amplified with PCR and sequenced (4). Then, the ratios between SNPs are determined and compared with the expected ratio. For instance, if the ratio for one chromosome was found to be 1:1, then it would be predicted to be normal and euploid. If the ratio for a different chromosome was 2:1, which is abnormal, then it would be predicted that that chromosome would be aneuploid (4). Since this approach compares the ratios of SNPs, it does not require a euploid reference for percentages as the other methods do (8). This method is also able to detect triploidy as well, which is something the other methods are unable to do (8). Chris Henry 10 Accuracy and Effectiveness of cffDNA screening Not only is the procedure for cffDNA screening simple, it also has provided a useful method for detecting chromosomal aneuploidies. In 2016, a meta-analysis of cffDNA studies was published (9). This meta-analysis had a strict selection process to remove any studies with bias and reviewed 41 different studies that used cffDNA to screen for Down syndrome (T21), Edwards syndrome (T18), and Patau syndrome (T13). The results reported that cffDNA screening had a sensitivity of 99.3% and specificity of 99.9% for Down syndrome. For Edwards syndrome the sensitivity was 97.4% and the specificity was 99.9%. For Patau syndrome the sensitivity was 97.4% and the specificity was 99.9% (Figure 6). T21, T18, and T13 Meta-Analysis Results Sensitivity Specificity Down syndrome (T21) 99.3% 99.9% Edwards syndrome (T18) 97.4% 99.9% Patau syndrome (T13) 97.4% 99.9% Figure 6. Results from a meta-analysis (9) performed on cffDNA screening accuracies in 2016 on trisomy 21, trisomy 18, and trisomy 13. A separate meta-analysis was done for sex chromosome aneuploidies that gave a detection rate of 90.3% and a false positive rate of 0.23% for monosomy X, and a detection rate of 93% and false positive rate of 0.14% for the sex chromosome trisomies (47, XXX; 47, XXY; and 47, XYY) (Figure 7) (10). Chris Henry 11 Sex Chromosome Meta-Analysis Results Detection Rate False Positive Rate Monosomy X 90.3% 0.23% Sex Chromosome trisomies (47, XXX; 93% 0.14% 47, XXY; and 47, XYY) Figure 7. Results from a meta-analysis (10) performed on cffDNA screening in 2015 on sex chromosome aneuploidies. Risks and Limitations of cffDNA Screening While cffDNA screening has very high detection rates for chromosome aneuploidies, especially trisomy 21, there is not 100% accuracy with the test, but other procedures such as amniocentesis and chorionic villus sampling (CVS) have essentially 100% accuracy due to their respective methods. Due to the lack of 100% accuracy, the cffDNA test is considered only a screen and not a diagnostic test (9). Another problem with this test is that it is reliant on a high fetal fraction of DNA. If the fetal fraction is not high enough, then the cffDNA test is inconclusive. Causes of low fetal fractions include young gestational age (earlier than 10 weeks), maternal obesity, and fetal karyotype (6). That is, Trisomy 21 causes a higher fetal fraction, but trisomy 18, monosomy X, and overall triploidy (69, XXY) have lower than normal fetal fractions and can cause test failures (6). Triploidy rarely results in a live birth, as the chromosome abnormalities are too severe. The fact that one of the causes of test failure is part of what the test is screening for, is not ideal. Chris Henry 12 Another downside to the cffDNA screen is that the DNA it screens comes from the placenta instead of the fetus. The DNA from the placenta and fetus are usually the same since they come from the same fertilized egg, but differences can exist between the two. Approximately 0.1% of clinical cases analyzing cffDNA found that the placental DNA did not match the fetal karyotype (11). A common cause of this is confined placental mosaicism, which is when the placenta has chromosome abnormalities while the fetus does not (11) (Figure 8). CVS also samples cells from the placenta and thus has this same issue. Figure 8. Illustration of confined placental mosaicism, as well as other mosaicisms. Despite the negatives, this cffDNA screen has some distinct advantages over other testing methods. One of these is that there is zero risk of spontaneous termination of the fetus with cffDNA collection. Both amniocentesis and CVS have approximately a 1% risk of spontaneous termination (1). This risk is significant enough that it may prevent many pregnant women from getting invasive testing done, but since cffDNA does not carry that risk, it is a good option for women concerned about the risks of amniocentesis or CVS, especially women with advanced maternal age who have spent a long time trying to conceive. Additionally, the procedure for Chris Henry 13 cffDNA screening is less invasive than amniocentesis and CVS, requiring only a blood draw. Amniocentesis requires extracting amniotic fluid and CVS requires the removal of chorionic villi cells (1,2). These more invasive procedures carry more risks, such as higher risks of infection, risk of spontaneous termination discussed above, and risk of misdiagnosis from confined placental mosaicism which can lead to more complications (2). Another risk associated with any prenatal testing is the emotional and psychological toll that the woman could experience. If the woman is informed that her fetus contains an abnormality then she may begin considering terminating the pregnancy, even if this is something she had not considered an option before. Not knowing either the presence or absence of an abnormality is more comforting to some people, and cffDNA can remove that comfort. However, some people desire to know regardless of if termination is an option for them so they can be better prepared for the baby if an abnormality is present. Emotional and psychological damage is not often assessed when going over the positives and negatives of prenatal testing and should be considered more frequently, although the lack of risk of spontaneous termination already removes some of the potential damage if cffDNA testing is used. Conclusions Cell free fetal DNA analysis has proven to be a reliable and effective screening method for chromosomal aneuploidies. It is most accurate at detecting trisomy 21, but it also has high accuracy for trisomy 18, 13, and sex chromosome aneuploidies (9). It is the best of the non- invasive tests for trisomy 21 detection, although detection of the other aneuploidies is equal to or worse than other testing, such as the combined test, which uses ultrasound as well as a blood Chris Henry 14 biochemical assessment (10). However, since testing for trisomy 21 is the most desired, the testing for other trisomies is usually just a benefit of prenatal testing for trisomy 21 and not the goal, so the superiority of cffDNA screening for trisomy 21 testing is most important (10). The application of cffDNA testing for all the known chromosome aneuploidies proves that it is useful for prenatal genetic testing, and the lack of significant risks associated with this screen make it an option for a greater population compared with the invasive testing procedures. This screening method is continuously gaining improvements through new discoveries that make it both less expensive and more accurate (3). As this process improves it will overcome some of the limitations it currently has, but until that time, this method is best used as a preliminary screen for high-risk pregnancies (cost permitting) with positive results being followed up by invasive testing if desired by the parent(s) (6). Chris Henry 15 References (1) Wieacker, Peter, and Johannes Steinhard. “The Prenatal Diagnosis of Genetic Diseases.” Deutsches Aerzteblatt Online, 2010, doi:10.3238/arztebl.2010.0857. (2) Ghidini, Alessandro. “Chorionic Villus Sampling” In: UpToDate, Post TW (ed), UpToDate, Waltham, MA. (Accessed on April 14, 2020.) (3) Xu, Xu-Ping, et al. “A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection.” Plos One, vol. 11, no. 1, 2016, doi:10.1371/journal.pone.0146997. (4) Bianchi, Diana W, and Rossa WK Chiu. “Sequencing of Circulating Cell-Free DNA During Pregnancy.” Obstetric Anesthesia Digest, vol. 39, no. 1, 2019, pp. 43–44., doi:10.1097/01.aoa.0000552918.35824.84. (5) Lo, Y M Dennis, et al. “Presence of Fetal DNA in Maternal Plasma and Serum.” The Lancet, vol. 350, no. 9076, 1997, pp. 485–487., doi:10.1016/s0140-6736(97)02174-0. (6) Palomaki, Glenn E., et al. “Prenatal screening for common aneuploidies using cell-free DNA” In: UpToDate, Post TW (ed), UpToDate, Waltham, MA. (Accessed on April 14, 2020) (7) Fan, H. C., et al. “Noninvasive Diagnosis of Fetal Aneuploidy by Shotgun Sequencing DNA from Maternal Blood.” Proceedings of the National Academy of Sciences, vol. 105, no. 42, 2008, pp. 16266–16271., doi:10.1073/pnas.0808319105. Chris Henry 16 (8) Nicolaides, K. H., et al. “Validation of Targeted Sequencing of Single-Nucleotide Polymorphisms for Non-Invasive Prenatal Detection of Aneuploidy of Chromosomes 13, 18, 21, X, and Y.” Prenatal Diagnosis, vol. 33, no. 6, 2013, pp. 575–579., doi:10.1002/pd.4103. (9) Taylor-Phillips, Sian, et al. “Accuracy of Non-Invasive Prenatal Testing Using Cell-Free DNA for Detection of Down, Edwards and Patau Syndromes: A Systematic Review and Meta-Analysis.” BMJ Open, vol. 6, no. 1, 2016, doi:10.1136/bmjopen-2015-010002. (10) Gil, M. M., et al. “Analysis of Cell-Free DNA in Maternal Blood in Screening for Aneuploidies: Updated Meta-Analysis.” Ultrasound in Obstetrics and Gynecology, vol. 50, no. 3, 2017, pp. 302–314., doi:10.1002/uog.17484. (11) Taglauer, E.s., et al. “Review: Cell-Free Fetal DNA in the Maternal Circulation as an Indication of Placental Health and Disease.” Placenta, vol. 35, Feb. 2014, doi:10.1016/j.placenta.2013.11.014. References for Figures Figure 1. Wieacker, Peter, and Johannes Steinhard. “The Prenatal Diagnosis of Genetic Diseases.” Deutsches Aerzteblatt Online, 2010, doi:10.3238/arztebl.2010.0857. Figure 2. https://www.dsupnorth.org/what-is-down-syndrome.html Accessed on 4/27/2020 Figure 3. http://www.nipt.se/technology/ Accessed on 4/27/2020 Figure 4. https://www.stgeorges.nhs.uk/wp-content/uploads/2018/08/Module-6-FINAL.pdf Accessed on 4/27/2020 Chris Henry 17 Figure 5. Fan, H. C., et al. “Noninvasive Diagnosis of Fetal Aneuploidy by Shotgun Sequencing DNA from Maternal Blood.” Proceedings of the National Academy of Sciences, vol. 105, no. 42, 2008, pp. 16266–16271., doi:10.1073/pnas.0808319105. Figure 6. Taylor-Phillips, Sian, et al. “Accuracy of Non-Invasive Prenatal Testing Using Cell- Free DNA for Detection of Down, Edwards and Patau Syndromes: A Systematic Review and Meta-Analysis.” BMJ Open, vol. 6, no. 1, 2016, doi:10.1136/bmjopen-2015-010002. Figure 7. Gil, M. M., et al. “Analysis of Cell-Free DNA in Maternal Blood in Screening for Aneuploidies: Updated Meta-Analysis.” Ultrasound in Obstetrics and Gynecology, vol. 50, no. 3, 2017, pp. 302–314., doi:10.1002/uog.17484. Figure 8. https://www.stgeorges.nhs.uk/wp-content/uploads/2018/08/Case-Scenario-10- FINAL.pdf Accessed on 4/30/2020