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
Method Comparison Deletion Euploid Binning/Counting Method SNP Method
Fetal Fraction Matters 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
Why Counting Suffers at Low Fetal Fractions From Canick, et al. Prenat Diagn 2013, 33, 1–8 When using the counting methodology, as fetal fraction decreases, there is less distinction between the euploid and aneuploid distributions Fetal Fraction Matters
Trisomy Detection by Counting Fetal Disomy Fetal Trisomy Maternal “Fraction of cfDNA that is fetal is a key component, with trisomy becoming easier to detect at higher fetal fractions” (Norton, et al. 2013) How fetal fraction affects sensitivity
Trisomy Detection by Counting “Excess maternal DNA could lower the sensitivity of the test” (Futch, et al 2013) No distinction between maternal and fetal DNA
Importance of detecting triploidy Although most miscarry, incidence is 1/1000 at 10 weeks 1 Paternal triploidy carries risk for partial molar pregnancy - Up to 5% risk for gestational trophoblastic disease with partial molar pregnancy 2,3 - Risk for malignant tumors Maternal triploidy can be recurrent in future pregnancies 4 3 Soper, J. (2006) Gestational trophoblastic disease. Obstet Gynecol 108:176–87 4 Chromosome Abnormalities and Genetic Counseling, Gardner and Sutherland, Snijders, et al. Fetal Diagn Ther 1995; 10: Berkowitz, R. S. and Goldstein, D. P. (1995), Gestational trophoblastic disease. Cancer, 76: 2079–2085.
False Positive Rates for Autosomes 21, 18, Based 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.
1 Yanlin Wang et al. Clinical Chemistry 2014; v. 60, p Futch, et al. Prenat Diagn 2013 Jun;33(6): Bretelle F, et al. Ultrasound Obstet Gynecol. 2002: 20: Understanding Discordance Maternal contribution Sex chromosome abnormalities in the mother 1 Vanishing twins Vanishing twin with an abnormality can cause incorrect fetal results 2 Gender Incorrect gender calls can cause complicated clinical care and unnecessary concern 3
Maternal Mosaicism on the X Chromosome Figure adapted from Russell LM, et al. X chromosome loss and ageing. Cytogenet Genome,; 116:
Maternal Mosaicism on the X Chromosome Analyzing maternal DNA contribution decreases false positives related 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 By counting method, 8.56% of results positive for sex chromosome aneuploidies were FP due to maternal mosaicism.
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
Company Comparison- Twins Sequenom 1 MaterniT21 Plus TM Verinata 2 Verifi® Ariosa 3 Harmony TM Number of total cases Number of abnormal cases correctly reported 8411 Failure rate Not reported 7% False negatives Not reported 1 1. Canick et al. DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations. Prenatal Diagnosis, August Gil et al. Cell-Free DNA Analysis for Trisomy Risk Assessment in First-Trimester Twin Pregnancies. Fetal Diagnosis and Therapy, 2014
Vanishing Twins Vanishing twin contributes additional SNP haplotype 0.2% of commercial cases 1 Seen up to 8 weeks post-demise More false positives by counting method >15% of discordant commercial results in counting methodology involved vanishing twin 2 1 Natera internal data 2 Futch, et al. Prenat Diagn 2013 Jun;33(6):569-74
Error Rate – Sex Determination 1 Mazloom et. al Prenat Diagn. 4 Nicolaides et. al Prenat Diagn. 2 Verifitest.com 5 Natera manuscript under review. 3 Nicolaides et. al Fetal Diagn Ther. As many as 1/100 cases can have gender discrepancy when using counting methodologies. 13,4 5 2
What Happens With Discrepant Fetal Sex? Large dilemma when ultrasound is discrepant with some potential diagnoses requiring complicated work-up 1 : 1 Bretelle F, et al. Fetal gender: antenatal discrepancy between phenotype and genotype. Ultrasound Obstet Gynecol. 2002: 20: Incorrect NIPT result Ultrasound error/misread Confined placental mosaicism Feto-placental mosaiscism Wrong embryo transfer in PGD Maternal andgrogen exposure Congenital adrenal hyperplasia Partial or complete sex reversal Gonadal dysgenesis, gonadoblastoma Masculinization 45,X or 46, XX, +SRY Campomelic dysplasia Denys-Drash syndrome Smith-Lemli-Optiz Alfi syndrome Swyer syndrome 5 α-Reductase deficiency Androgen insensitivity Frasier syndrome ATR-X syndrome More than just BOY or GIRL
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
High and Low Risk Populations
Sensitivity Versus Positive Predictive Value
Low Risk Versus High Risk Prior Risk (from conventional screening) Final Risk (following MPS test) MPS Test + MPS Test - 1:1029:11:1,100 1:1003:11:11,000 1:2701:11:30,000 “Dr. – does this mean my baby has Down Syndrome?” Benn P, Cuckle H, Pergament E. Prenatal diagnosis for Down syndrome: the paradigm will shift, but slowly. Ultrasound Obstet Gynecol Feb;39(2):
NIPT in a Low Risk Population Studies suggesting FPR and failure rate should be consistent 1 ◦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 category 3 1 Brar, et al J Matern Fetal Neonatal Med. 2 Canick et al Prenat Diagn. 3 Hudecova et al PLoS ONE.
Low Risk - False Positive Rate 1 Bianchi, et al. NEJM. 2 Natera Internal Data. Trisomy 21 FPR Aneuploidy + Microdeletions FPR False positive rate for NIPT is significantly lower than traditional maternal serum screening. NIPT MSS
First trimester screening NIPT Comparison of First Trimester Screening and NIPT Nicolaides et al Prenat Diagn, 2013
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
Common microdeletions included on panels 22q11.2 deletion/DiGeorge 1p36 deletion Angelman Prader-Willi Cri-du-chat Vast majority missed on ultrasound Cardiac indications on ultrasound but many missed
High Incidence Conditions Incidence out of 100,000 Live Births 1 Nussbaum et al Thompson and Thompson Genetics in Medicine (7th edn). Oxford Saunders: Philadelphia 2 3
Microdeletions Maternal Age NIPT Microdeletion Panel 2 Down Syndrome 1 1 Snijders, 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.www.genetests.org More Common Than Down Syndrome in Younger Women
22q11.2 Deletion Syndrome 1 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 1 International 22q11.2 Foundation - Handbook
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
Prenatal Testing Algorithm WOMEN SHOULD BE OFFERED INVASIVE TESTING ACOG PRACTICE BULLETIN #88, 2007 ACMG GUIDELINES 2009 Do not want further information Want further information but do not want risk of invasive procedures Women who want to know everything Amniocentesis or CVS Detailed ultrasound is still recommended for all patients regardless of testing decisions. Nuchal translucency provides information beyond fetal aneuploidy status.