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 DNAMaternal + Fetal GenotypeSNPSequencingFetal GenotypeMaternal bloodAlgorithmSNPSequencingMaternal GenotypeBuffy coat =Maternal DNA
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, MDPrenatal Perspectives. Volume 1, NoMusci is from AriosaNo 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 methodIt is the intermediate 4-8% that causes problems. Counting is the most greatly affected.Fetal Fraction toolow to reportIntermediate fetal fraction – decreased sensitivity with counting methodologyFetal fraction adequate to achieve best performance
12 Why Counting Suffers at Low Fetal Fractions Fetal Fraction MattersWhy Counting Suffers at Low Fetal FractionsWhen using the counting methodology,as fetal fraction decreases, there is less distinction between the euploid and aneuploid distributionsFrom Canick, et al. Prenat Diagn 2013, 33, 1–8
13 Trisomy Detection by Counting How fetal fraction affects sensitivityFetal DisomyFetal TrisomyMaternalCOUNTING 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 DNAIt 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 weeks1Paternal triploidy carries risk for partial molar pregnancy- Up to 5% risk for gestational trophoblastic disease with partial molar pregnancy2,3- Risk for malignant tumorsMaternal triploidy can be recurrent in future pregnancies41Snijders, et al. Fetal Diagn Ther 1995; 10:357-9.2Berkowitz, R. S. and Goldstein, D. P. (1995), Gestational trophoblasticdisease. Cancer, 76: 2079–2085.3Soper, J. (2006) Gestational trophoblastic disease. Obstet Gynecol 108:176–874Chromosome 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 contributionSex chromosome abnormalities in the mother1Vanishing twinsVanishing twin with an abnormality can cause incorrect fetal results2GenderIncorrect gender calls can cause complicated clinical care and unnecessary concern31Yanlin Wang et al. Clinical Chemistry 2014; v. 60, p2Futch, 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 mosaicism16/187 = 8.56%Berry Genomics (China)Especially important with 22q11.2 Deletion syndromeBy 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 GestationsMore difficult to analyze because each fetus will release different amounts of cfDNAIncreased no-call rateDoubled by one counting method (Struble 2014)There is limited data<200 total samples in any study4-11 positives
21 Company Comparison- Twins Sequenom1MaterniT21 PlusTMVerinata2Verifi®Ariosa3HarmonyTMNumber of totalcases25115207Number of abnormalcases correctly reported8411Failure rateNot reported7%False negatives11. Canick et al. DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations. Prenatal Diagnosis, August 20122.3. 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 cases1Seen up to 8 weeks post-demiseMore false positives by counting method>15% of discordant commercial results in counting methodology involved vanishing twin22/13 of discordant had vanishing twin, addition ½ double aneuploidies without confirmation had vanishing twin notedLandy says 3% of all pregnancies start as twins and 20% are VT.1Natera internal data2Futch, 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.123,451Mazloom 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 GIRLLarge dilemma when ultrasound is discrepant with some potential diagnoses requiring complicated work-up1:Incorrect NIPT result45,X or 46, XX, +SRYUltrasound error/misreadCampomelic dysplasiaConfined placental mosaicismDenys-Drash syndromeFeto-placental mosaiscismSmith-Lemli-OptizWrong embryo transfer in PGDAlfi syndromeMaternal andgrogen exposureSwyer syndromeCongenital adrenal hyperplasia5 α-Reductase deficiencyPartial or complete sex reversalAndrogen insensitivityGonadal dysgenesis, gonadoblastomaFrasier syndromeATR-X syndromeMasculinizationPatients1Bretelle 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 recommendedIs amniocentesis preferred over chorionic villus sampling?Fetal mosaicism – May not be detectable by cfDNAIdentification of mosaicism will be less effective because the contribution from abnormal is partial (Canick 2013)Maternal Mosaicism – May alter results of counting methodSequencing of buffy coat may determine if maternal chromosome abnormality is confounding the results
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 -1:1029:11:1,1001:1003:11:11,0001:2701:11:30,000Benn 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 consistent1Larger studies are coming from multiple companiesFetal fraction seems to have largest impact on resultsSNP method uses DNA, which shouldn’t change with indicationFetal fraction not correlated with risk category31Brar, 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.NIPTMSSTrisomy 21 FPR Aneuploidy +Microdeletions FPR1Bianchi, et al. NEJM.2 Natera Internal Data.
31 Nicolaides et al Prenat Diagn, 2013 Comparison of First Trimester Screening and NIPTFirst trimester screeningNIPTPanorama 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 FTSEuploid is black and Aneuploid is redNicolaides et al Prenat Diagn, 2013
33 What is a microdeletion? 1MB (megabase) = 1 million base pairsMicrodeletions are 100kb to several MBKaryotype can usually only visually detect >7-10 MBOutcome will depend on the size & the genes involved
34 Common microdeletions included on panels 22q11.2 deletion/DiGeorge1p36 deletionAngelmanPrader-WilliCri-du-chatCardiac indications on ultrasound but many missedVast majority missed on ultrasoundMajority 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 occur2. 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 BirthsT21 1/60022q 1/1000-1/4000CF 1/2500T18 1/3000T13 1/5000FRAX 1/50001p36 1/5000SMA 1/6000-1/10000Angelman 1/ /20000PWSCri-Du-Chat 1/ /500001Nussbaum et al Thompson and Thompson Genetics in Medicine (7th edn). Oxford Saunders: Philadelphia2http://www.genetests.org.3http://ncbi.nlm.nih.gov
36 More Common Than Down Syndrome in Younger Women MicrodeletionsMore Common Than Down Syndrome in Younger WomenDown Syndrome1Risk 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 commonMakes it appropriate to screen in all pregnancies, both high and low riskNIPTMicrodeletion Panel2Maternal Age1Snijders, 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.
38 22q11.2 Deletion Syndrome1Population incidence 1/2,000, though NEJM shows 2x higherSeveral other clinical names in the past: DiGeorge, VCFSOften undiagnosed at birth (25% have no heart defect)Common symptoms75% with immune deficiencies30% with feeding difficulties requiring feeding tube35% with malformed or missing kidney25% develop schizophrenia in young adulthoodHypocalcemiaDevelopmental delay and learning disabilities1International 22q11.2 Foundation - Handbook
39 22q: Early Intervention Matters Prepare to deliver baby at tertiary care centerNo live vaccinesCalcium to avoid seizures and intellectual disability secondary to hypocalcaemiaCheck palate for clefting
40 Prenatal Testing Algorithm WOMEN SHOULD BE OFFERED INVASIVE TESTINGACOG PRACTICE BULLETIN #88, 2007 ACMG GUIDELINES 2009Want further information but do not want risk of invasive proceduresWomen who want to know everythingDo not wantfurther informationAmniocentesisor CVSDetailed ultrasound is still recommended for all patients regardless of testing decisions. Nuchal translucency provides information beyond fetal aneuploidy status.