10/18/2018 N O F I N A N C I A L D I S C LO S U R E S I recommend Cell Free DNA for: OBJECTIVES A. All my patients 34% 34% Background 33% B. Only for women over age 35 When is it the right test? When is it the wrong test? C. For any patient with abnormal state screening What are some new things it might do? All my patients Only for women over age 35 For any patient with abnormal state sc... 1
10/18/2018 P a t i e n t W N 37-year-old G5P4004 at 12 weeks gestation • What testing options should be -3 healthy children -1 son with Wolf-Hirschorn syndrome offered? - Parental testing performed Father is a carrier of a balanced translocation • A. Cell free DNA ** 25% recurrence risk 61% B. Invasive testing with CVS C. Cell free DNA followed by CVS 26% 13% only if abnormal A S N V D C . . . e h f e t i i r w y f n l l l g o e n C i S t V e s C t y e b v s i d a e v w n o I o l l f A N D e e f r l l e C C e l l F r e e D N A - w h a t i s i t ? cfDNA: Common Clinical Applications • Made up of short segments of fetal Aneuploidy DNA (<200 bp) Sex determination • Origin is primarily placental in origin Fetal RH status • Results from apoptosis • Circulate in maternal plasma 2
10/18/2018 Cell Free DNA: Characteristics cfDNA represents ~10-15% of total DNA in maternal plasma 1 Reliably detected after 7 wks gestation 2 Higher concentrations late in gestation Short half life (16 min), undetectable by 2 hrs postpartum 3 JAMA. 2018;320(6):591-592. doi:10.1001/jama.2018.9418 What happens in the lab? Techniques Massively parallel shotgun sequencing (MPSS) Targeted sequencing Single nucleotide polymorphisms (SNP) analysis 3
10/18/2018 Why does the technique matter? Fetal Fraction MPSS Targeted SNP • Amplification and • Sequencing of • Comparison of sequencing of all regions of interest SNPs of maternal cfDNA only origin to fetal • Benefit: Broad, • Benefit: less • Benefit: accuracy looks at cfDNA expensive is equal across from all chromosomes chromosomes • May allow • Limitations: Cost, differentiation requires minimum between triploidy, fetal fraction of 4% uniparental Taglauer, WES, Wilkins-Haug L, Bianchi DW Review: cell-free fetal DNA in maternal circulation as an indicatation of placental health and disease disomy, maternal mosaicism Fetal Fraction Why does fetal fraction matter? The less fetal DNA, the harder to tell Prenatal Diagnosis 2012; 32(13) p 1233-41 normal from abnormal 4
10/18/2018 Cell free DNA screening: Factors that impact fetal fraction Biologic Challenges Maternal BMI - increased BMI associated with False Positives decreased FF Unrecognized or “vanishing”twin Failed to provide a result in 20% of women >250 lb and 50% of women >350 lb. Placental Mosaicism Gestational age Low level maternal mosaicism, esp. sex Increases after 21 weeks chromosomes Aneuploidy Maternal Malignancy Increased FF with infants with T21 Decreased FF with infants with T18 Twins Cell free DNA screening: Cell free DNA screening: Biologic Challenges Biologic Challenges False negatives Failed Results Low level of fetal DNA Placental mosaicism Increased BMI Maternal genetic variation (copy Low level of fetal DNA number variants) Fetal Aneuploidy 5
10/18/2018 CFDNA and aneuploidy: CFDNA and aneuploidy: Perf ormance of cf dna Perf ormance of cf dna It is important to counsel patients about the positive predictive value (PPV) of the test PPV is influenced by prevalence of a disease An individual PPV can be calculated using maternal age or risk generated from prior screening test https://www.perinatalquality.org/vendors/ nsgc/nipt/ PPV calculator Cell Free DNA for Aneuploidy in Twins Unaffected twin may mask an affected twin Although initial studies are reassuring regarding use of cfDNA for T21 in twins, the overall data are limited Not currently recommended by ACOG/SMFM For higher order multiples Maternal age and nuchal translucency measurement for aneuploidy estimation 6
10/18/2018 Cell free DNA for ultrasound Cell Free DNA for fetal sex? abnormalities? Negative cfDNA and isolated ‘soft marker’ False positive rate is high for monosomy X Diagnostic testing is not recommended solely for Some studies report rates as high as 91% this indication Patients may learn about possible sex Structural abnormality on ultrasound chromosome aneuploidy even when they Offer diagnostic testing using microarray are primarily interested in fetal sex The majority of congenital structural abnormalities In some cases, may be abnormal if the are due to microdeletions or single gene disorders mother has an undiagnosed sex that cannot reliably be detected by cell free DNA chromosome aneuploidy Invasive testing is recommended Cell Free DNA Vs. Serum screening Cell free DNA for Rh blood typing? What if CF DNA is normal followed by Lo and colleagues accurately assess fetal Rh status abnormal serum screening using cfDNA using PCR Positive predictive value reported as 93% Raises concern for other chromosomal Negative predictive value as high as 98% abnormality Moise and colleagues (2016) reported false negative in I n a study by Norton et al. the residual risk 1 in 520 cases and inconclusive results in 5-6% of cases was 2% The lowest detection rate for cfDNA in women who were <25 years old 7
10/18/2018 Microdeletions are rare Cell Free DNA for Microdeletions? Chromosomal microdeletions account for many syndromes May have developmental delay and medical issues There are approximately 40 well described microdeletions Low baseline prevalence of each microdeletion syndrome PPV is signficantly impacted Microdeletions are More Common Should all wome n be Than Down Syndrome for Women offere d s cree ning for Under 40 microde letions? Adapted from: Snijders, et al. Ultrasound Obstet Gynecol 1999;13:167–170 8
10/18/2018 Prevalence of many microdeletion syndromes is unknown Many such clinical syndromes are caused by different molecular mechanisms • Microdeletions can be detected by cfDNA, but… • Difficult to validate Smaller deletions more difficult to detect • Much of the data on in vitro samples 22q syndrome (~1/4000) • Clinical testing includes small number of syndromes 85% have 3Mb deletion , 15% smaller • 22q, 1q36, 5p-, 4p-, 15q11-13 (Prader- “97% detection” refers to only 3Mb deletion Willi/Angelman) • Low positive predictive value C e l l f r e e D N A t e s t i n g f o r s i n g le g e n e d i s o r d e rs Disorders that are a result of DNA changes in a single gene May affect up to 1% of all pregnancies Requires identifying fetal DNA that is unique from maternal These results should not be impacted by placental Screening for these microdeletions has not been mosaicism validated in clinical studies. Current clinically available for limited conditions Achondroplasia Routine cell-free DNA screening for microdeletion Thanatophoric dysplasia syndromes should not be performed. Apert syndrome Cystic fibrosis 9
10/18/2018 Case #2: 38 yo G2P1 at 12 weeks What is the next step? A. No further testing needed 82% B. Repeat cell free DNA C. Recommend invasive testing 11% 7% A g d N n d e i e D t e e s e e t n r e g f v n l i i e l s t s c a e t v t a n r e i e p d h e n t R e u r m f m o N o c e R T he F u t u r e : I s m o re N o n i n v a s i v e al ways W h ol e G e no m e be tt e r ? S e qu en ci n g • OBTAIN INFORMATION ABOUT THE ENTIRE FETAL GENOME • Coding and non coding • RAISES BOTH PRACTICAL portions AND ETHICAL ISSUES • Single nucleotide variants • MAY GIVE • Deletions and Duplications INFORMATION REGARDING RISK FOR • Copy number variants ADULT ONSET CONDITIONS THAT ARE NOT RELEVANT 10
10/18/2018 How to choose a lab Back to case #1… Our patient underwent cell free DNA testing with Technique micro deletions Her results were normal so she declined any Cost further testing Reporting system Level II ultrasound was notable for unilateral cleft lip and palate and abnormal profile Genetic counseling support services Amniocentesis confirmed deletion of 8.9 MB deletion on 4p consistent with Wolf-Hirshorn Current guidelines ACOG/SMFM guidelines Conventional screening is most appropriate first line screen for most patients Ethically any patient may choose cfDNA screening, but should be counseled regarding limitations and benefits Diagnostic testing is required to confirm abnormal results before irreversible decisions Not recommended in twin pregnancies Microdeletion/expanded panels for cell free DNA are not recommended 11
10/18/2018 Special thanks to Mary Norton, MD & Meg Autry, MD 12
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