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Non-invasive Prenatal Testing

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Presentation on theme: "Non-invasive Prenatal Testing"— Presentation transcript:

1 Non-invasive Prenatal Testing

2 Aneuploidy Screening Approach: Observed Detection Rates
1960 1980 1990 2005 2011 Detection Rate (%)

3 Cell-free DNA (cfDNA) cfDNA comes from apoptotic cells derived from:
Maternal Circulation Adipocytes White Blood Cells Fetal Placental cells (trophoblasts) in the maternal circulation

4 Differentiating NIPT Methodologies
Massively Parallel Shotgun Sequencing Targeted Sequencing Targeted Sequencing Sequenom MaterniT21TM Verinata Verifi® Ariosa HarmonyTM Natera PanoramaTM COUNTING SNPs

5 Relative Size of Chromosomes

6 Counting Chromosome 21 Chromosome 3

7 Counting Expected Amount: 20% 80% Observed Amount: 25% 75%
Chromosome 21 Chromosome 3 Expected Amount: % % Observed Amount: % %

8 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

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

10 Binning/Counting Method
Method Comparison Binning/Counting Method SNP Method Deletion Euploid

11 Fetal Fraction Matters
0-4% 4-8% 8%+ “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 Musci is from Ariosa No one gets great results on fetal fractions less than 4%. There is too little fetal DNA to get adequate results and all are no-called >8% is the fraction at which you are likely to get very good results no matter the method It is the intermediate 4-8% that causes problems. Counting is the most greatly affected. Fetal Fraction too low to report Intermediate fetal fraction – decreased sensitivity with counting methodology Fetal fraction adequate to achieve best performance

12 Why Counting Suffers at Low Fetal Fractions
Fetal Fraction Matters Why Counting Suffers at Low Fetal Fractions When using the counting methodology, as fetal fraction decreases, there is less distinction between the euploid and aneuploid distributions From Canick, et al. Prenat Diagn 2013, 33, 1–8

13 Trisomy Detection by Counting
How fetal fraction affects sensitivity Fetal Disomy Fetal Trisomy Maternal COUNTING METHOD -Blue is maternal contribution. Fetal fraction is on X axis so when fetal fraction is 15%, maternal(blue) is 85% Green represents the normal reference chromosome, and Red is the trisomic chromosome of interest. It is very easy to see the difference of observed versus expected at 15 and 10% fetal fraction, but as the fetal fraction decreases (left to right) it becomes harder and harder to distinguish this difference and thus are more likely to call the result normal and thus falsely negative “Fraction of cfDNA that is fetal is a key component, with trisomy becoming easier to detect at higher fetal fractions” (Norton, et al. 2013)

14 Trisomy Detection by Counting
No distinction between maternal and fetal DNA It is even harder when the counting method cannot detect which is the maternal versus fetal contribution. This would become even more important for conditions such as Monsomy X, when you cannot tell if mother is mosaic for XXX for example and thus not contributing the expected “blue” amount. Many articles are starting to surface that discuss the lower sensitivities at these intermediate fetal fractions. “Excess maternal DNA could lower the sensitivity of the test” (Futch, et al 2013)

15 Importance of detecting triploidy
Although most miscarry, incidence is 1/1000 at 10 weeks1 Paternal triploidy carries risk for partial molar pregnancy - Up to 5% risk for gestational trophoblastic disease with partial molar pregnancy2,3 - Risk for malignant tumors Maternal triploidy can be recurrent in future pregnancies4 1Snijders, et al. Fetal Diagn Ther 1995; 10:357-9. 2Berkowitz, R. S. and Goldstein, D. P. (1995), Gestational trophoblastic disease. Cancer, 76: 2079–2085. 3Soper, J. (2006) Gestational trophoblastic disease. Obstet Gynecol 108:176–87 4Chromosome Abnormalities and Genetic Counseling, Gardner and Sutherland, 2004.

16 False Positive Rates for Autosomes 21, 18, 131
1Based on cumulative data from 6 trials for shotgun, 6 trials for targeted, and 2 trials for SNP based methods. Benn P., J Clin Med, in press.

17 Understanding Discordance
Maternal contribution Sex chromosome abnormalities in the mother1 Vanishing twins Vanishing twin with an abnormality can cause incorrect fetal results2 Gender Incorrect gender calls can cause complicated clinical care and unnecessary concern3 1Yanlin Wang et al. Clinical Chemistry 2014; v. 60, p 2Futch, et al. Prenat Diagn 2013 Jun;33(6): 3Bretelle F, et al. Ultrasound Obstet Gynecol. 2002: 20:

18 Maternal Mosaicism on the X Chromosome
Figure adapted from Russell LM, et al. X chromosome loss and ageing. Cytogenet Genome ,; 116:

19 Maternal Mosaicism on the X Chromosome
Analyzing maternal DNA contribution decreases false positives related to maternal mosaicism 16/187 = 8.56% Berry Genomics (China) Especially important with 22q11.2 Deletion syndrome By counting method, 8.56% of results positive for sex chromosome aneuploidies were FP due to maternal mosaicism. Table adapted from Yanlin Wang et al. Maternal Mosaicism Is a Significant Contributor to Discordant Sex Chromosomal Aneuploidies Associated with Noninvasive Prenatal Testing. Clinical Chemistry 2014; v. 60, p

20 Twin Gestations More difficult to analyze because each fetus will release different amounts of cfDNA Increased no-call rate Doubled by one counting method (Struble 2014) There is limited data <200 total samples in any study 4-11 positives

21 Company Comparison- Twins
Sequenom1 MaterniT21 PlusTM Verinata2 Verifi® Ariosa3 HarmonyTM Number of total cases 25 115 207 Number of abnormal cases correctly reported 8 4 11 Failure rate Not reported 7% False negatives 1 1. Canick et al. DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations. Prenatal Diagnosis, August 2012 2. 3. Gil et al. Cell-Free DNA Analysis for Trisomy Risk Assessment in First-Trimester Twin Pregnancies. Fetal Diagnosis and Therapy, 2014

22 Vanishing Twins Vanishing twin contributes additional SNP haplotype
0.2% of commercial cases1 Seen up to 8 weeks post-demise More false positives by counting method >15% of discordant commercial results in counting methodology involved vanishing twin2 2/13 of discordant had vanishing twin, addition ½ double aneuploidies without confirmation had vanishing twin noted Landy says 3% of all pregnancies start as twins and 20% are VT. 1Natera internal data 2Futch, et al. Prenat Diagn 2013 Jun;33(6):569-74

23 Error Rate – Sex Determination
As many as 1/100 cases can have gender discrepancy when using counting methodologies. 1 2 3,4 5 1Mazloom et. al Prenat Diagn. 4Nicolaides et. al Prenat Diagn. 5Natera manuscript under review. 3Nicolaides et. al Fetal Diagn Ther.

24 What Happens With Discrepant Fetal Sex?
More than just BOY or GIRL Large dilemma when ultrasound is discrepant with some potential diagnoses requiring complicated work-up1: Incorrect NIPT result 45,X or 46, XX, +SRY Ultrasound error/misread Campomelic dysplasia Confined placental mosaicism Denys-Drash syndrome Feto-placental mosaiscism Smith-Lemli-Optiz Wrong embryo transfer in PGD Alfi syndrome Maternal andgrogen exposure Swyer syndrome Congenital adrenal hyperplasia 5 α-Reductase deficiency Partial or complete sex reversal Androgen insensitivity Gonadal dysgenesis, gonadoblastoma Frasier syndrome ATR-X syndrome Masculinization Patients 1Bretelle F, et al. Fetal gender: antenatal discrepancy between phenotype and genotype. Ultrasound Obstet Gynecol. 2002: 20:

25 Mosaicism Confined placental mosaicism – May not affect fetus
Follow-up diagnostic testing recommended Is amniocentesis preferred over chorionic villus sampling? Fetal mosaicism – May not be detectable by cfDNA Identification of mosaicism will be less effective because the contribution from abnormal is partial (Canick 2013) Maternal Mosaicism – May alter results of counting method Sequencing of buffy coat may determine if maternal chromosome abnormality is confounding the results

26 High and Low Risk Populations

27 Sensitivity Versus Positive Predictive Value

28 Low Risk Versus High Risk
“Dr. – does this mean my baby has Down Syndrome?” Prior Risk (from conventional screening) Final Risk (following MPS test) MPS Test MPS Test - 1:10 29:1 1:1,100 1:100 3:1 1:11,000 1:270 1:1 1:30,000 Benn P, Cuckle H, Pergament E. Prenatal diagnosis for Down syndrome: the paradigm will shift, but slowly. Ultrasound Obstet Gynecol Feb;39(2):

29 NIPT in a Low Risk Population
Studies suggesting FPR and failure rate should be consistent1 Larger studies are coming from multiple companies Fetal fraction seems to have largest impact on results SNP method uses DNA, which shouldn’t change with indication Fetal fraction not correlated with risk category3 1Brar, et al J Matern Fetal Neonatal Med. 2Canick et al Prenat Diagn. 3Hudecova et al PLoS ONE.

30 Low Risk - False Positive Rate
False positive rate for NIPT is significantly lower than traditional maternal serum screening. NIPT MSS Trisomy 21 FPR Aneuploidy + Microdeletions FPR 1Bianchi, et al. NEJM. 2 Natera Internal Data.

31 Nicolaides et al Prenat Diagn, 2013
Comparison of First Trimester Screening and NIPT First trimester screening NIPT Panorama had less variability in risk scores reported out , although this is the data from this study, larger samples sizes with NIPT show that there are going to be some in between along with FP/FN but it is no question a clearer result than FTS Euploid is black and Aneuploid is red Nicolaides et al Prenat Diagn, 2013

32 Microdeletions

33 What is a microdeletion?
1MB (megabase) = 1 million base pairs Microdeletions are 100kb to several MB Karyotype can usually only visually detect >7-10 MB Outcome will depend on the size & the genes involved

34 Common microdeletions included on panels
22q11.2 deletion/DiGeorge 1p36 deletion Angelman Prader-Willi Cri-du-chat Cardiac indications on ultrasound but many missed Vast majority missed on ultrasound Majority of cases are caused by de novo deletions, but some can be inherited (point mutations, undiagnosed parents, translocations). 1. Segmental duplications (chromosome specific low-copy repeats) are responsible for recurrent microdeletions (22q, Angelman, PW). When recombination occurs between homologous chromosomes these duplicon regions don’t always line up exactly and del/dups can occur 2.  Terminal deletions (cri du chat, wolf-hirshhorn, phelan-mcdermid) are 60-90% of the time paternal in origin, meaning occur on the paternally inherited copy, not that they are passed down from father) because of increased # of divisions in sperm versus egg (more divisions = more chance of error.) Terminal ends are most likely to be lost. 3.  FYI 1p36 is exception and is is more often maternally inherited chromosome (60% of the time) 4. Terminal deletions – most often happen in sperm, one extra cycle of meiosis in sperm (# of cell divisions in sperm compared to eggs)

35 High Incidence Conditions
Incidence out of 100,000 Live Births T21 1/600 22q 1/1000-1/4000 CF 1/2500 T18 1/3000 T13 1/5000 FRAX 1/5000 1p36 1/5000 SMA 1/6000-1/10000 Angelman 1/ /20000 PWS Cri-Du-Chat 1/ /50000 1Nussbaum et al Thompson and Thompson Genetics in Medicine (7th edn). Oxford Saunders: Philadelphia 2 3

36 More Common Than Down Syndrome in Younger Women
Microdeletions More Common Than Down Syndrome in Younger Women Down Syndrome1 Risk of T21 increases with maternal age. Risk of Microdeletions is consistent across all ages and younger women have a higher risk of a microdel than T21 b/c microdels are so common Makes it appropriate to screen in all pregnancies, both high and low risk NIPT Microdeletion Panel2 Maternal Age 1Snijders, et al. Ultrasound Obstet Gynecol 1999;13:167– Combined prevalence using higher end of published ranges from Gross et. al., Prenatal Diagnosis 2011; 39, ; and Total prevalence may range from 1/ /2206.

37 Microdeletion Comparison
Counting Methods1,2 SNP Method3 Sensitivity for 22q deletion (3MB) 60-87% 95.7% Specificity Range >99% Detects uniparental disomy NO YES Distinguish maternal/paternal Considers mother’s status 1Verinata Marketing Sheet 2Sequenom Marketing Sheet 3Natera Internal Data

38 22q11.2 Deletion Syndrome1 Population incidence 1/2,000, though NEJM shows 2x higher Several other clinical names in the past: DiGeorge, VCFS Often undiagnosed at birth (25% have no heart defect) Common symptoms 75% with immune deficiencies 30% with feeding difficulties requiring feeding tube 35% with malformed or missing kidney 25% develop schizophrenia in young adulthood Hypocalcemia Developmental delay and learning disabilities 1International 22q11.2 Foundation - Handbook

39 22q: Early Intervention Matters
Prepare to deliver baby at tertiary care center No live vaccines Calcium to avoid seizures and intellectual disability secondary to hypocalcaemia Check palate for clefting

40 Prenatal Testing Algorithm
WOMEN SHOULD BE OFFERED INVASIVE TESTING ACOG PRACTICE BULLETIN #88, 2007 ACMG GUIDELINES 2009 Want further information but do not want risk of invasive procedures Women who want to know everything Do not want further information Amniocentesis or CVS Detailed ultrasound is still recommended for all patients regardless of testing decisions. Nuchal translucency provides information beyond fetal aneuploidy status.

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