NIPD

non invasive prenatal diagnosis is maternal serum test that can ascertain some genetic fetal disorders without taking fetal sample.

1. NIPD Ahmed El- Habashy A.Lect.OB / GYN.Alex.Univ . Non-Invasive Prenatal Diagnosis The future arrived

2. OUTLINES TERMINOLOGY TICHNIQUE & SAMPLING APPLICATION ACCURACY RECOMMENDATIONS NIPD Habashy

3. What is cffDNA ? 1 st reporting of cffDNA in the Maternal Circulation  1997. From Placental Apoptosis . cffDNA in Mother is 25 times > Fetus . Not contained within cells. Short life 3h. Lo YM et al. Lancet 1997. NIPD Habashy

4. cffDNA  F/M FRACTIONS NIPD Habashy

5. NIPD = NIPT = NIPS Diagnosis Testing Screening. Although cffDNA discovered Since 1997 , FTS  Non-Invasive Prenatal Screening of Fetal Trisomies . Its Implementation in Practice was just since 2011 . NIPD = cffDNA ONLY . NIPD Habashy

6. NIPD GENERATIONS NIPD Habashy K. Nicolaides Prenat Diagn 2013 Random MPSS Selective Directed Single Nucleotide Polymorphism

7. Massively Parallel Sequencing MPS Millions of DNA fragments From All Chromosomes Most Widely Used NIPT Digital ANalysis of Selected Regions DANSR Sparks AB Prenat Diagn 2012 K.Nicolaides AJOG 2012 Ehrich et al Am J Obstset Gynecol 2011 Shotgun MPSS Targeted / Directed MPS NIPD Habashy

8. MPSS VS DANSR ?!?!? WHETHER DANSR or MPSS  MORE ACCURATE RESULS. Need Further Studies to COMPARE BOTH APPROACHES IN the SAME COHORT of PATIENTS . DANSR Use 1/10 cfDNA Fragments in MPSS May ↑ Resampling This ↑Throughput & ↓ Cost . RCOG 2014 NIPD Habashy

9. STEPS of MPS NIPD Habashy

10. Chromosome Mapping to a Reference Disomic Genome NIPD Habashy

11. SEQUENCING Spatial Parallel Segregation F/M cfDNA PROPORTIONAL RELEATIONSHIP NIPD Habashy

12. 2 nd Generation NIPD SNP Also a Targeted Sequencing. Not Need A Disomic Reference Chromosome. Zimmermann Prenat Diag 2012 K.Nicolaides Prenat Diagn2013 NIPD Habashy

13. SNP NIPD Habashy

14. cffDNA SAMPLING >10w GA ( SNP can at 9w). Spontaneous or Assisted Conception. 10-20 cc Maternal Venous Blood. (?! 2cc)* Results within 1-2 w. 5 different Companies ( USA,Germany,China ). PRICE : 800-3000 $ . * K.Nicolaides AJOG 2012 NIPD Habashy

15. cffDNA APPLICATIONS NIPD Habashy

16. Autosomal Aneuploidy Commonest T21 T18 T13 On Request in Some NIPT T16 T22 NIPD Habashy

17. Sex Chromosomal Aneuploidy (Some NIPT) XO (Turner Syndrome) XXX (Triple X) XXY ( Klienfelter Syndrome) XYY (Jacob Syndrome) NIPD Habashy

18. SO NIPD Can Detect ~ 85% of Fetal Chromosomal Aneuploidy NIPD Habashy

19. Deletion Syndromes Some NIPD • 22q11.2 deletion syndrome ( DiGeorge ) • Cri-du-chat syndrome (5p minus) • Prader-Willi / Angelman syndrome (15q) • 1p36 deletion syndrome NIPD Habashy

20. SensiGene RhD – ve Mother / RhD + ve Father. + ve Maternal Indirect Coomb (Sensitized). ± Hetrogenous Rh + ve Father. cffDNA ♀ RhD-ve  Can not Verify the presence of Fetal DNA = Mother RCOG 2014 NIPD Habashy

21. SensiGene - SNP Fetal Identifier (FI) control is performed to Compare M/F genotype. If ≥ 6 Markers are Observed  Verify True RhD-ve ♀ Fetus : No Antenatal RhIG Prophylaxis . No Extra M/F Testing (± Invasive) RCOG 2014 NIPD Habashy

22. Fetal Gender If ♀ Fetus  50% ↓INVASIVE TESTING. If ♀ Fetus Early Dexamethasone ↓ Virilization If ♂ Fetus Early cessation of Dexamethasone. Duchenne Muscle Dystrophy CAH RCOG 2014 NIPD Habashy

23. Single Gene Disorders Paternally Inherited Allele Autosomal Dominant Huntington Disease. Achondroplasia . Thanatophoric Dysplasia. Autosomal Recessive ß Thalassemia. Cystic Fibrosis. ABSENT Paternal Allele  NO INVASIVE TESTING. RCOG 2014 NIPD Habashy

24. NIPD Accuracy SPECIFICITY and SENSETIVITY CLOSE TO 100% (>98%) FOR T21,18; Less for T13. FPR < 0.5%. 1-10% Chance of No Result (Sample Failure- No Call Rate)  Mostly √ in Repeat Sample. (Mostly FREE). RCOG 2014 NIPD Habashy

25. Possible Sources of ERRORS FALSE- ve ↓ Fetal cffDNA Fraction Early GA :<10w  <4-5%. Maternal Obesity . FALSE+ve Confined Placental Mosaicism (CPM ): Trisomic Placental Cells BUT a NORMAL FETUS. Maternal Conditions: Chromosomal Aberrations. Malignancy. RARELY  DISCORDANT RESULTS TWIN Still Evolving RCOG 2014 NIPD Habashy

26. Maternal Weight VS cffDNA NIPD Habashy

27. cffDNA in Twining MC Not only Possible BUT MORE Effective. DC CAN Calculate % Fetal cfDNA Fraction of EACH FETUS. If 1 Miscarries  ?!? Its Fraction Change Still Evolving cffDNA Dx Zygocity * * Qu JZ.et al. Clin Chem 2013 RCOG 2014 NIPD Habashy

28. Comparison with Screening Tests Combined T21 FTS = NT,ßHCG,PAPPA  90% DR with 3% FPR Sensitivity & Specificity of cffDA for T21≈100% . ↓INVASIVE TESTING : COST LOSSES ↓ MORE↓ K.Nicolaides Ultrasound Obstet Gynecol 2007 NIPD Habashy

29. Prenatal Triosomy Detection Rate DR FTS NIPD Invasive (Karyotyping) NIPD Habashy

30. ACOG , SMFM , ACMG , ISPD cffDNA is some FORM of CONTINGENT Approach offering testing to HIGH RISK Pregnancies: Maternal Age ≥35Ys. Previous T21. Parental Balanced Translocation. US Aneuploidy Marker(s ). + ve Biochemical Screening test. THE MOST EFFECTIVE ANEUPLOIDY SCREENING IN HIGH RISK PREG. ASOG,SMFM 2012 ACMG,ISPD 2013 NIPD Habashy

31. SOGC >98% Detection Rate. <0.5 FPR. NOT A DIAGNOSTIC TEST. Can Not Replace US (NT). + ve NIPS  SHOULD do CONFIRMATORY INVASIVE TEST BEFORE TOP. NIPT May REPLACE the CURRENT MATERNAL SCREENING APPROACHES IF:* ↓ COST. ↑ Studies in AVERAGE RISK PREG. Sholud Included in Pre-Test Pt. COUNSILING * * ACOG 2012 *SOGC 2013 NIPD Habashy

32. NIPT IMPLEMENTATION ↑ COST : (COMMERCIALISATION and INTELLECTUAL PROPERTY). FTS also Predict PET,FGR. FTS also Predict Many Non-Chromosomal Structural Anomalies. If Contingent (done for + ve FTS)  MISS the FALSE – ve of FTS . LIMITED STUDIES IN AVERAGE RISK* AND TWINS. ↓ INVASIVE TESTING  ↓ Fetal Medicine Trainee SKILLS. Limited Geographical Lab. Distribution. AGAINST * K.Nicolaides AJOG 2012 NIPD Habashy

33. NIPT IMPLEMENTATION NO F/M Harm. ↓ False – ve . (~ 38%*) ↓ False + ve of FTS  ↓ INVASIVE TESTING COST & ↓ INVASIVE TESTING Losses . (~ 66%*) IF REPLACE ßHCG,PAPPA  SAVE THERE COST FOR NIPT. Multiplexing many patient samples in a SINGLE SEQUENCING RUN ↓ Individual TEST COST. WITH *Garfield S J Manag Care Med. 2012 NIPD Habashy

34. NIPD CAN ADJUST FTS RISK + ve NIPT  x 290 ↑ FTS Risk of T21 - ve NIPT  x 110 ↓ FTS Risk of T21 Benn et al.,Ultrasound Obstet Gynecol ;2012 Benn Calculator NIPD Habashy

35. ACMG 2013 No Doubt NIPS Costs WILL ↓ . NIPS is Likely the 1 st of major steps toward the eventual application of WHOLE FETAL GENOME SEQUENCING . NIPD Habashy

36. NIPT Market Competition Intellectual Property . Non-Profit Funding . ↓ Lab. Geographic Distribution. Companies Litigation None of them FDA Approved Yet ( in Progress). No Insurance Coverage. Stick-holders Should Pay Attention to NIPD Commercialization  ↑ Patient Access. Ashwin A . Prenat Diagnos 2013 NIPD Habashy MARKET MONOPOLY ↑ COST

37. Trials Maternal Urine : cffDNA (MPS)*. Maternal Blood : cffRNA (more Fetus Specific) Fetal Proteins. Fetal CELLS. NIPD Habashy Lo YM , K.Nicolaides Nat Med 2007

38. Take Home Message NIPD  High Accuracy + NO F/M Harm  REVOLUTION in FETAL MEDICINE . NIPD MAJOR PROBLEM  ↑ COST  FALLING  May REPLACE BIOCHEMICAL MARKERS BUT CAN NOT REPLACE US . NIPD are NOT DIAGNOSTIC  SHOULD BE CONFERMED BY INVASIVE TEST. NIPD Habashy

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