NIPT Non-Invasive Prenatal Test By Dr. A. Kollaee (DCLS & PhD in Medical Genetics)

What are the goals of NIPT?

Should NIPS be offered to all pregnant patients? ACMG recommends: - Informing all pregnant women that NIPS is the most sensitive and reliable option for traditionally screened aneuploidies involving chromosomes 13,18 and 21

Cell-Free DNA (cfDNA) Small Fragments (<200 bp) of DNA In Maternal Circulation Maternal Fraction Adipocytes White Blood Cells Fetal Fraction Placental Cells (Trophoblasts) in Maternal Circulation % of Total cfDNA in Maternal Plasma, After 10 Wks of Gestation Which One: cffDNA or cfDNA? AJOG 2015; SMFM Publiation Committee

Available commercial NIPT testing Europe -Prena test -Life Codexx -IONA test -Premaitha Health China - NIFTY Test – BGI USA - Harmony Test - Ariosa Diagnostic, Sanjose California - MaterniT21 Plus Test – Sequenom Laboratories, San Diego - Verifi Test – Verinata Health - Panorama Test – Natera San Carlos California

Differentiating NIPT Methodologies

Trisomy Detection by Counting Fetal Disomy Fetal Trisomy Maternal “Fetal Fraction of cfDNA Is Key In “Counting” => Higher Fetal Fractions => Easier to Detect Trisomy (Norton, Et Al. 2013) How Fetal Fraction Affects Sensitivity Just Visually Compare!

Fetal Fraction Matters for Counting 0-4%4-8%8%+ Fetal Fraction Too Low To Report Intermediate Fetal Fraction – Decreased Sensitivity With Counting Methodology Fetal Fraction Adequate To Achieve Best Performance “An Aneuploidy Sample With A Lower Fetal Fraction Has A Higher Probability Of Resulting In A False Negative Result.” Thomas Musci, MD Prenatal Perspectives. Volume 1, No

Chromosome 3 Chromosome 21 Counting Smaller Chromosome  Lower Fraction (e.g. Chr. 21) Larger Chromosome  Higher Fraction (e.g. Chr. 3)

Chromosome 3Chromosome 21 Expected Amount: 20% 80% Countin g Observed Amount: 25% 75% How Would You Then Assess the Ratios for Chr. 21 vs. Chr. 18 vs. Chr. 13?

SNP = Single Nucleotide Polymorphism A DNA sequence variation occurring when a single base pair (nucleotide) - A, T, C, or G – is changed. These are normal genetic changes that occur in every person

Buffy coat = Maternal DNA Plasma = Maternal + Fetal DNA SNP Sequencing SNP Sequencing Maternal Genotype Maternal + Fetal Genotype Fetal Genotype Maternal blood SNP approach Using the Buffy Coat to Sequence Maternal Genotype Algorithm

NIPT Test procedure The NIPT Test can be performed from week 10+0 of the pregnancy. Before carrying out a NIPT Test, the patient should be given an ultrasound scan to determine the gestational age and to check whether it is a singleton or multiple pregnancy.

Excellent detection rate in singleton pregnancies PanoramaVerifiMaternit21Harmony testDetection ate >99% 99.7% T21 98%97%96.4%98% T18 95%93%92%93% T13

Low false positive rate DANSR method 5 times lower than rmps

Reliable even with heparin treatment [1] Ashoor G, Syngelaki A, Wagner M, Birdir C, Nicolaides KH: Chromosome-selective sequencing of maternal plasma cell-free DNA for first-trimester detection of trisomy 21 and trisomy 18. Am J Obstet Gynecol Apr;206(4):322.e1-5. [2] Nicolaides KH, Syngelaki A, Ashoor G, Birdir C, Touzet G: Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. Am J Obstet Gynecol Nov;207(5):374.e1-6. [3] 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: Non-Invasive Chromosomal Evaluation (NICE) Study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol Aug;207(2):137.e1-8. [4] Sparks AB, Struble CA, Wang ET, Song K, Oliphant A: Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol Apr;206(4):319.e1-9. [5] Verweij EJ, Jacobsson B, van Scheltema PA, de Boer MA, Hoffer MJ, Hollemon D, Westgren M, Song K, Oepkes D: European non-invasive trisomyevaluation (EU-NITE) study: a multicenter prospective cohort study for non-invasive fetal trisomy 21 testing. Prenat Diagn Oct;33: [6] Juneau K, Bogard PE, Huang S, Mohseni M, Wang ET, Ryvkin P, Kingsley C, Struble CA, Oliphant A, Zahn JM: Microarray-based cell-free DNA analysis improves noninvasive prenatal testing. Fetal Diagn Ther. 2014;36: [7] Norton ME, Jacobsson B, Swamy GK, Laurent LC, Ranzini AC, Brar H, Tomlinson MW, Pereira L, Spitz JL, Hollemon D, Cuckle H, Musci TJ and Wapner RJ (Next-Study): Cell free DNA Analysis for Noninvasive Examination of Trisomy. N Engl J Med. 2015, Apr 1, DOI: /NEJMoa [8] Stokowski R, Wang E, White K, Batey A, Jacobsson B, Brar H, Balanarasimha M, Hollemon D, Sparks A, Nicolaides K, Musci TJ..: Clinical performance of non-invasive prenatal testing (NIPT) using targeted cell-free DNA analysis in maternal plasma with microarrays or next generation sequencing (NGS) is consistent across multiple controlled clinical studies. Prenat Diagn Sep 1. PanoramaVerifiMaternit21Harmony test YESNO YES

Positive predictive value (PPV)

which options are possible with different types of NIPT Tr. 21/18/13sex determinationX/Y analysis Singleton pregnancies incl. all IVF pregnancies YES Twin pregnancies incl. all IVF pregnancies/ egg donation YES (new) × More than two fetuses ×××

NIPT results 1. Normal Result : No Specific Follow Up Necessary, Unless Ultrasound Examination Of The Fetus Reveals Anomalies +AFP Level 2. Test Failure : In 1.4 % Pregnancies Not Enough Fetal DNA : NIPT Repeated At No Extra Cost. 3. Abnormal NIPT Result => Diagnostic Tests (E.G. Amniocentesis Or Chorion Biopsy)

Complications With NIPT 1- cfDNA-based methodologies detect Only a subset of chromosomal aberrations 2- Concerning aneuploidy of other chromosomes, molecular or congenital anomalies including neural tube defects are not excluded with NIPT. 3- Single-gene mutations? => No 4- Placental Mosaicism: i.e. Placental T21, While Normal Fetus! Note: This Is An Issue With CVS (In 1 th Trimester) As Well! 5- Still False-positive And False-negative Results (Even If At Very Low Levels) The American College Of Medical Genetics & Genomics (ACMG) Labeled The Testing “Non-invasive Prenatal Screening” Or “NIPS.”Labeled

Thank You for your attention