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.
2Verifitest.com 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
2http://
3http://ncbi.nlm.nih.gov

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.