Gómez Massa, Elena

Universitat Autònoma de Barcelona. Facultat de Biociències

Diposit Digital de Documents de la UAB

Non‐Invasive Prenatal Testing In Maternal Plasma: Down Syndrome as a potential applicationElena Gómez Massa, Autonomous University of Barcelona, Biomedical Sciences DegreeNIPT is a potential emerging field in genetic prenatal diagnosis and screening. Thus, NIPT relies on the identification ofDown syndrome (DS) or trisomy 21 is the most common aneuploidy (1/800 live births) and the most commoncause of sever mental delay. DS is compatible with life but it involves some characteristic clinical features (Table1). The high mortality is due to heart disease which is very frequent. In addition, maternal age and previous familyhistory are the main risk factors (Figure 5).DS clinical features‐Hypotonia‐ Smaller nose and ears‐ Slanted eyes‐ Variable intellectual coefficient (45‐70)1. NIPT: an overview3. Down SyndromeAims and MethodsAims‐ To delve in non‐invasive prenatal testing (NIPT) concretely with its applications and techniques. ‐ To compare between NIPT and the current prenatal screening. ‐ To consider the bioethical aspects related to prenatal testing. MethodsScientific literature search on PubMed database: recent papers and reviews, chosen depending on their data ofpublication and quality.gfetal genetic material in the blood of pregnant women without no additional risk of miscarriage. The presence of fetalgenetic material in the maternal bloodstream during the pregnancy is well‐known and it is explained by thebidirectional traffic between the fetus and the mother through the placenta.There are three possible fetal sources for NIPT (Figure 1): intact fetal cells, cell‐free fetal DNA (cffDNA) and cell‐free fetalRNA (cffRNA). Moreover, clinical applications of NIPT can be classified in four groups: sex‐linked diseases, haemolyticdisease of the fetus and newborn (HDFN), monogenic diseases and chromosomal aneuploidies.The detection of universal fetal markers minimizes the interference between maternal and fetal DNA in maternalbloodstream. The different epigenetic pattern is an example of an useful method to detect fetal markers. In addition,PLAC4 mRNA is a placental transcript located on chromosome 21 so it is interesting for Down syndrome (DS) testing.Intact fetal cells‐ Pregnancy specificH lf lif 25 35 dcffDNA‐ 3‐6% of total cfDNAcffRNA‐ Pregnancy specificHalf life: 14 min?intellectual impairment ?developmental delay‐Alzheimer susceptibility ‐ Leukaemia susceptibility (at earlier ages)Owing to its high incidence, DS is the main reason why women undergo prenatal testing. The detection of DSprenatally involves screening and diagnostic testing. Currently, there are three screening algorithms designedwith the aim to estimate a pregnancy‐specific individual risk at carrying a DS fetus. These algorithms are basedon the evaluation of three elements: maternal age, nuchal translucency (NT) and maternal serum biomarkers.Figure 6 only shows the first trimester combined test which is recommended by ISPD (International Society forPrenatal Diagnosis). Depending on the estimated risk level various strategies can be followed (Figure 7).Figure 5. This graphic shows an exponential increase risk of carrying a DS baby as maternal age increases. FIRST TRIMESTER SCREENINGTable 1. It is summarised the main clinical features in DS.2. Techniques‐ a ‐ e: ‐ aysCell type: NRBC2nd trimester2nd trimester‐ Low concentration‐ No identification of specificfetal markers‐ Pregnancy specific‐ Half‐life: 16.3 min‐Background of maternal DNA‐Specific fetal markers arenecessary1st trimester‐ ‐    ‐ Stable and easy isolation‐ tomarkers[cffRNA] is relatedexpression levels in placenta‐ No need of specific fetalDepend on the marker (1st or 2nd trimester)Figure 6. In this scheme is shown the essential information as regards first trimester screening. It allows to estimate risk level which can be low (<1:250) or high (>1:250). Figure 7. The risk level estimation is basedWhat is realized?1. Echography ‐ Nuchal Translucency (NT)2. Blood extraction: ‐ β‐hCG and PAPP‐AWhen?1. Week 11‐132. Week 8‐13Who? All the women (age‐independent)   Combined test (NT + Biochemical markers): TP=75%  and FP<3%Figure 1. Description of the different fetal sources for NIPT. cfDNA means cell‐free DNA. Low risk Normal pregnancy controlSingle counting molecular techniques‐The main advantage: analysis of total DNA (fetal plus maternal)‐ The main inconvenient: coston the screening results. A low riskpregnancy involves a normal pregnancycontrol with routine ecographies.Nevertheless, a high risk pregnancy requiresto undergo a definitive diagnosis throughinvasive procedures (CVS and amniocentesis)Digital PCR Technical steps (Figure 2):1. Dilution of the sample until <1 template DNA/well2. Real‐time PCR of each template DNA3. Counting the number of positive reactions4. Measure of the total DNA dosageMassively parallel sequencing (MPS)Technical steps (Figure 4):1. Generation of millions of tags across the whole genome2. Alignment and mapping of tags3. Identification of chromosome origin4. Counting of tagsScreeningHigh risk     Definitive diagnosis: ‐ Chorionic villus sampling (CVS)‐ AmniocentesisNIPT: screening or diagnosis?Sensitivity and FPR are the variables that indicate if a test should be used for screening or diagnosis. NIPTpresents high sensitivity and low false positive rate (FPR) (Table 2). Currently, NIPT cannot be considereddiagnostic despite being a highly accurate screening test. Thus, invasive testing is also recommended in order toconfirm a positive NIPT result (Figure 8). Improving sensitivity and specificity is crucial to reduce the number ofinvasive diagnostic tests offered to pregnant women and as a result reducing the number of miscarriages inaffected and unaffected pregnancies.4. Bioethical aspectsSensitivity (%) FPR (%)Currentscreening85‐90 5NIPT 79.1‐100 0‐2.1Definitivediagnosis100 0NIPT cannot be considered a diagnostic testTwo possible applications for NIPT:‐ First‐line screening‐ After a positive result from the current screening testAdvantages (comparing with Real‐time PCR)‐ Standards are no necessary ‐ Regulation of precision:It depends on fetal DNA concentrationApplication? Relative chromosome dosage (RCD)Useful for Aneuploidy detection (Figure 3).5. Comparison of the number of tags from eachchromosome to its reference valueAlternative approach:Digital analysis of selected regions (DANSR)Only it is analysed the chromosome of interest instead towhole genomeApplication:? Aneuploidy detection Table 2. Comparison of sensitivity and FPR values among the different testing strategies.  Figure 8. Currently NIPT is considered a screening test but it is not validated for definitive diagnosis.  • Absence of risk for the mother• Early and easy to performPros• Cost• Detection of genomicaberrations withunknown pathogenicConsMore women undergo prenatal testingVariable insurance coverage ? NIPT only afforded byhigh‐income community‐Genetic counseling difficultiesThe development of NIPT has raised numerous ethical, social and legal issues so it is essential to evaluate the prosand cons (Figure 9). Some ethical concerns should be taken into account such as the right of “not to know” andthe abortion.Figure 2. The main technical steps of Digital PCR.15. Conclusions 6. Referencesimplication ‐Unnecessary patient anxiety1. http://www.lifetechnologies.com/es/en/home/life‐science/pcr/digital‐pcr.html2. DENNIS LO YM, et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidydetection. Nature Medicine (2007) (13): 23. SWANSON A, et al. Non‐invasive Prenatal Testing: Technologies, Clinical Assays and Implementation Strategies forWomen’s Healthcare Practitioners. Genet Med Rep (2013) 1:113–121Figure 3. RCD scheme.2 Figure 4. MPS scheme.3Figure 9. Summary of the pros and the cons of NIPT and their respective consequences.   ‐ NIPT is performed with the aim to identify fetal genetic material in maternal bloodstream so as to detect geneticabnormalities. The absence of invasiveness allows to avoid additional risks caused by the procedure.‐ NIPT can be applied for screening but not for definitive diagnosis.‐ Economic, ethical, social and legal issues should be considered due to the ease and absence of risk performing NIPT.

Non-invasive prenatal testing in maternal plasma : Down syndrome as a potential application

http://ddd.uab.cat/pub/tfg/2014/128287/TFG_ElenaGomezMassa.pdf

Non-invasive prenatal testing in maternal plasma : Down syndrome as a potential application

Abstract

Similar works

Full text

Available Versions

Diposit Digital de Documents de la UAB