6/20/2014. Noninvasive Prenatal Testing (NIPT): Separate But Not Equal. Normal Male = 46,XY. Normal Female = 46,XX

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1 Noninvasive Prenatal Testing (NIPT): Separate But Not Equal Charles (Buck) Strom MD, PhD, FAAP, FACMG, HCLD Senior Medical Director, Genetics Quest Diagnostics Nichols Institute San Juan Capistrano, CA Normal Male = 46,XY 23 pairs of chromosomes 23 rd pair, sex chromosomes Males: XY Females: XX Each pair 1 maternal 1 paternal Normal Female = 46,XX 3 1

2 Normal Conception 46 Meiosis 23 sperm 46 Definitions Disomy = normal number of chromosomes; 46,XX Trisomy = gain of 1 chromosome; 47,XY,+21 Monosomy: loss of one chromosome, 45,XY,-16 Triploidy: extra entire set of chromosomes: 69,XXY Euploidy: a normal set of chromosomes (46) Aneuploidy: An abnormal number of chromosomes but not an abnormal set of chromosomes (all trisomies and monosomies) 47, XX, +21; Trisomy 21 = Down Syndrome Mental retardation Facial features Short stature Heart problems Intestinal problems Other birth defects 2

3 47,XX, + 18;Trisomy 18 = Edwards Syndrome Reduced survival (<10% beyond first year) Severe to profound mental retardation Rocker bottom feet Heart defects Clenched hands Other birth defects 47,XX, + 13; Trisomy 13 = Patau Syndrome <5-10% survive beyond one year Severe to profound mental retardation Extra fingers/toes Cleft lip Brain malformation Heart defects Other birth defects 45,X; Monosomy X = Turner Syndrome 3

4 NIPT: A New Method for Prenatal Aneuploidy Screening Prenatal Screening Prenatal Diagnostics MSS & Ultrasound NIPT CVS or Amniocentesis Karyotype FISH CGH or SNP prenatal NIPT detects cell-free fetal DNA (cfdna) from the plasma of pregnant women to screen for fetuses affected with trisomy 21, 18 and 13 NIPT currently is considered as screening test; not diagnostic False positives and negatives do occur, meaning an invasive test is still required to confirm any positive result Unlike CVS or amniocentesis, it is a non-invasive test by using the mother s blood to test fetal aneuploidy Cell-Free Fetal DNA in Maternal Circulation Discovery of cell-free fetal DNA in the maternal circulation in 1997* Initial use: RhD blood group Fetal gender Single-gene disorders 2008 publications drove implementation of this new technology into clinical practice * Lo YM, et al., Lancet 1997; 350:485-7 How Does NIPT Work? Maternal blood (1-2 tubes) collected in special (Streck ) tubes Centrifuged to remove cells and cellular debris Free floating DNA is analyzed 4 different methods Quantitative (Z score method) Sequenom» Shotgun whole genome sequencing with fetal fraction assessment Verinata» Shotgun (deeper) whole genome sequencing without fetal fraction assessment Ariosa» Targeted amplification plus fetal fraction by SNP frequency Non-quantitative SNP Based Natera: B allele frequency analysis with fetal fraction determination 4

5 NIPT Approaches and Methodologies MPSS Targeted Sequencing Targeted Sequencing Sequenom (MaterniT21) Verinata (Verifi) PerkinElmer QUANTITATIVE METHOD (Z Score) Ariosa (Harmony) LabCorp Natera (Panorama ) Quest Diagnostics SNP METHOD What is a Z Score? Knowing the size of the chromosomes, one can predict the frequency that any sequence will occur in the genome. For example, how many times a chromosome 21 sequence should occur when compared to a sequence on a non-variant chromosome A Z-score calculates the ratio of observed sequences from a given chromosome versus a non-variant chromosome In a Trisomy 21 conception, the number of times a chromosome sequence is increased, leads to a larger Z- score Different Z Scores for Different Folks Whole genome shotgun sequencing Sequenom and Verinata Targeted sequence analysis (non-polymorphic) Ariosa All Z score (quantitative) suffer from 2 limitations Sequencing bias Chromosome 13 and 18 have high GC content and are underrepresented in Illumina sequencing - leads to lower sensitivity and specificity for those chromosomes With low fetal fractions can give undetected false negative results 5

6 Counting Z Score Method Chromosome % of genome Chromosome 3 6.2% of genome Counting Chromosome % of genome Chromosome 3 6.2% of genome Expected Ratio: 20% 80% Observed Ratio: 25% 75% Counting Z Score Method Reasons for a normal count for chromosome 13 Diploid (normal) fetus Trisomy 13 fetus Under amplification / sequencing of chromosome 13 Low fetal fraction Reasons for an increased count for chromosome 13 Trisomy 13 Over correction for sequencing bias for chromosome 13 6

7 Accuracy of Z Score Falls with Decreasing Fetal Fraction 120 Relative amount of DNA mapping to chromosome of interest Z Score Fetal Disomy Fetal Trisomy Maternal % 15% 10% 10% 8% 8% 5% 5% 3% 3% Low Fetal cfdna Fractions Decrease Detection Rates In this data set (average gestational age 15 weeks), 10-15% of women had fetal fractions between 4-8% In our internal data, we found that 25% of 35 women at gestational ages between 9-14 weeks had fetal fractions between 4-8% % Fetal DNA Fraction Counting Down Syndrome Detection Rate 1 Panorama Down Syndrome Detection Rate 3 >10% >99% >99% 4-8% 75% >99% Fetal Fraction (%) Gestational Age (weeks) 1. Palomaki GE et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med Nov; 13(11): Natera internal data 3. Zimmermann, et al. "Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y using targeted sequencing at polymorphic loci." Prenatal Diagnosis, Natera Panorama Non-Quantitative Amplifies ~ 20,000 POLYMORPHIC SNP s Mother s DNA is genotyped using buffy coat When mother is AA or BB and fetus is AB Fetal fraction is directly measurable by frequency of non-maternal allele in sample Mother AA, fetus AB, 10% fetal fraction A allele: 1000 (maternal) (fetal) = 1,100 B allele: 100 (fetal, paternal) % fetal fraction = 100 / ) = 0.10 = 10% 7

8 Natera Panorama Non-Quantitative For each locus Maternal genotype known Fetal genotype AA, AB, or BB Hypothesis test performed for trisomy, disomy, and monosomy X Advantages of non-quantitative technique Sequencing bias irrelevant because looking at the same SNP for maternal and fetal B allele frequency, so accuracy of test does not decrease for trisomy 13 and trisomy 18 Accurate measurement of fetal fraction Paternal genotyping can rescue case with low fetal fraction Can provide accurate results with fetal fractions as low as 4% -6% Advantages of a SNP-based Approach SNP = Single nucleotide polymorphism SNP methodology with 19.5k Multiplex PCR probes Produces higher quality data Ability to achieve high accuracy results at lower fetal fractions How Does Panorama Work? A simplification of Natera s non-invasive prenatal aneuploidy test SNP Plasma = Sequencing Maternal + Fetal DNA Maternal + Fetal Genotype Target Fetal DNA Signal Maternal blood Buffy coat = Maternal DNA SNP Sequencing Maternal Genotype Fetal Genotype 8

9 Next-generation Aneuploidy Test Using SNPs (NATUS) Genotypic data from Mom (+/- Dad) Multiple hypotheses for each chromosome Data from Human Genome Project (HapMap) + Each hypothesis describes the expected sequencing data for that case Sub-hypotheses with different crossover points What is Fetal Fraction? Why is it Important? Fetal fraction is the percent of the cell-free DNA in the mother s blood that is from the fetus. At 13 weeks gestation, over 20% of pregnancies have a fetal DNA fraction <10% 1 Low fetal DNA fractions decrease detection rates of counting technologies Reporting of fetal fraction allows clinician to assess accuracy of the test result % Fetal DNA Fraction Counting Down Syndrome Detection Rate 2 Natera Down Syndrome Detection Rate 3 >10% >99% >99% <10% 91% >99% <8% 75% >99% 1. Lo et al. BMJ, Fetal DNA concentration found in maternal plasma among 314 pregnancies with euploid male fetuses. 2011;342:c Palomaki GE et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med Nov; 13(11): Zimmermann, et al. "Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y using targeted sequencing at polymorphic loci." Prenatal Diagnosis, Fetal Fraction vs. Gestational Age Fetal Fraction 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% Gestational Age (weeks) FF+1SD FF-1SD 9

10 Counting Versus SNPs Z Score More false negatives at fetal fractions < 10% Sequencing bias issue for chromosomes 13 and 18 Requires reference chromosome(s) Does not detect triploidy SNP (NATUS) Sensitivity not affected by fetal fraction down to 4% Not affected by sequencing bias Fetal fraction accurately determined and reported Detects triploidy Comparison of NIPT Technology Claims Sensitivity False Positive Rate Sequenom MaterniT21 plus Verinata Verifi Ariosa Harmony Natera Panorama TM Methodology MarterniT21 plus MPSS MPSS with SAFeR Targeted Sequencing with FORTE Targeted Sequencing with SNPs Trisomy 21 (Down Syndrome) >99.9% 0.2% >99.9% 0.2% >99% <0.1% >99% 0% Trisomy 18 (Edwards Syndrome) >99.9% 0.3% 97.4% 0.4% >98% <0.1% >99% 0% Trisomy 13 (Patau Syndrome) 91.7% 0.9% 87.5% 0.1% 80% <0.1% >99% 0% 45,X (Monosomy X) Not evaluated 95.0% 1.0% Not evaluated >99% 0% (1) Zimmermann et al. Prenat Diag 2012 (2) Natera Internal Data (3) Rabinowitz et al. Presented at ASHG 2012 (2) Downloaded on February 5, /Performance-Data (3) Downloaded on February 5, (4) Downloaded on February 5, Limitations of NIPT Testing Accuracy Reduced for Mosaic Fetuses (All) Not applicable to twins (All but Sequenom) Not applicable to gestations >=3 (All) Confined Placental Mosaicism Potential Problem All: Similar to CVS Can t use with donor egg pregnancy (Natera) (donor will be detected) 10

11 Professional Recommendations for NIPT (1) ACOG / Society for MFM Joint Statement December 2012 NIPT should be an informed patient choice after pretest counseling and should not be part of routine prenatal laboratory assessment. It should not be offered to low-risk women or women with multiple gestations because it has not been sufficiently evaluated in these groups. A negative test result does not ensure an unaffected pregnancy. A patient with a positive test should be referred for genetic counseling and should be offered invasive prenatal diagnosis for confirmation. Professional Recommendations for NIPT (2) NSGC 2012 Supports NIPT as an option for patients at an increased risk for certain chromosome abnormalities. NSGC urges that NIPT only be offered in the context of informed consent, education, and counseling by a qualified provider. Patients whose NIPT results are abnormal, or who have other factors suggestive of a chromosome abnormality, should receive genetic counseling and be given the option of standard confirmatory diagnostic testing. Professional Recommendations for NIPT (3) ISPD October 2011 Accepts that, with suitable genetic counseling, NIPT can be helpful for women who may have been determined to be high risk by one of the previously recommended screening strategies. The ISPD does not endorse the ad hoc use of MPS testing in women at lower risk, outside a formal protocol that considers the overall best combination of tests, their impact on screening performance, and patient acceptability. 11

12 NIPT: Current Status Recommendations that NIPT can be offered as an alternative to invasive prenatal diagnosis to couples at risk for aneuploidies Advanced maternal age (AMA, >35 yrs at delivery) High-risk maternal serum screening test Abnormality on ultrasound Previous child or fetus with aneuploidy Robertsonian translocation carriers Recommend invasive prenatal diagnosis for all positives Positive Predictive Value Varies with Specificity and Prevalence Assume 99.9% Specificity Tri 21*: Prevalence 1: 270: PPV = 75% Tri 18*: Prevalence 1: 470: PPV = 67% Tri 13*: Prevalence 1: 1,500 PPV = 37% * Gardner et al, Chromosomal Abnormalities and Genetic Counseling, 4 th Edition, Oxford Press, NY, 2012 Expanding Content - Microdeletions Natera and Sequenom have added DiGeorge and other microdeletions to NIPT Ariosa and Verinata do not offer Rapidly moving target. 12

13 Thank Your Attention 13