Genetic Testing in Pregnancy

1. Genetic Testing in Pregnancy Johanna Warren, MD OAFP Women’s Health Winter Conference January 19, 2014

2. What genetic screening test do you routinely offer your patients? • Quad/Penta screen • Integrated Screening • Nuchal Translucency (NT) only • Stepwise Sequential Screen • NIPT (cffDNA) • none

3. Learning Objectives • Use the concepts of pre-test probability, positive and negative predictive values as they apply to testing for fetal aneuploidy. • Outline advantages and disadvantages of various approaches to first-trimester and second-trimester screening as well as invasive diagnostic testing for Down syndrome. • Discuss emerging cell-free fetal DNA (cffDNA) technology and review indications for use in screening. • Summarize the current recommendations for cystic fibrosis screening.

4. Statistics • Pre-test probability • Prevalence of the disease • Post-test probability after one or more preceding tests • Rough clinical estimation • Positive predictive value (PPV) • the likelihood that an individual with a positive test result truly is affected/has the genetic condition • inherently dependent upon the prevalence • Negative predictive value (NPV) • the likelihood that an individual with a negative test result is truly unaffected/does NOT have the genetic condition • measure of test accuracy

5. What Makes a Good Screening Test? • The condition should be an important health problem. • There should be an acceptable test for the condition. • High sensitivity to detect not yet clinically detectable condition • High specificity to minimize false positives • The test should be available to the population. • The test should be affordable. • There should be acceptable treatment for the condition. • There should be a “latent stage” of the disease, allowing for detection/testing before a critical point, during which treatment would be optimal.

6. What is the purpose of antenatal genetic testing? • Assess risk for chromosome abnormalities • particularly Down syndrome & Trisomy 18 • Determine which pregnancies should be offered invasive prenatal diagnosis

7. Who Should Be Screened? • ACOG Practice Bulletin (2007) • All pregnant women, regardless of their age, should be offered screening and diagnostic testing for aneuploidy. • Women should be counseled regarding the differences between screening and invasive diagnostic testing. • Ideally, patients seen early in pregnancy should be offered screening that combines 1st and 2nd trimester testing • Screening test chosen will depend on availability of NT US as well as CVS

8. Testing for Fetal Aneuploidy – Focus on Down Syndrome (trisomy 21) • Explosion of available screening tests • Detection of Down Syndrome 90+% with non-invasive screening tests • Confirmation diagnosis still requires invasive testing • Amniocentesis • Chorionic villus sampling

9. Quick Review • Several methods for combining first- and second-trimester screening reach higher detection rates for Trisomy 21 than either first- or second-trimester screening alone. • Common options: • Ultrasound: Nuchal Translucency • Integrated Screenings • Quadruple/Penta Marker Screenings • Stepwise Sequential Screening

10. First Trimester - Ultrasound • Nuchal Translucency (NT) • Normal: 1-3mm • Increased NT is an indication of a chromosomal abnormality, single gene defect, or birth defects (commonly cardiac defects) • Nasal Bone • Absent/hypoplastic in 70% T21 • DuctusVenosus – reversed a-wave flow • Detection rate 75%, FPR 5% • Tricuspid Regurgitation • Detection rate 67.3% in T21, FPR 5.2%

11. Integrated Screenings • Integrated Screening • Ultrasound for Nuchal Translucency (NT) + serum PAPP-A/hCG analysis between 10-13.6wks GA; results of these tests are HELD • Serum quad screen test between 15 -19 wks GA • At that time, the results of all the studies, combined with risk assessment due to the patient's age, are used to present a single-risk figure • Serum Integrated Screening • first-trimester serum PAPP-A/hCG test result is combined with a second-trimester quad screen test to provide a single-risk figure (no NT US)

12. Quadruple Marker Screening • Measure raw values of: • AFP (alpha fetoprotein) • ue3 (unconjugatedestriol) • hCG (human chorionic gonadotropin) • DIA (dimericinhibin A) • Compare to median value for the appropriate gestational age (MoM) • Valid between 14-22.9 weeks GA (optimal 16-18wks); risk of NTD not provided for samples collected prior to 15 weeks.

13. Stepwise Sequential Screening • First Trimester • NT US + serum PAPP-A analysis between 10-13.6wks GA • Results combined with the patient's age-associated risk, • Patient is given a risk assessment for aneuploidy • may choose at this time to undergo invasive testing (e.g., amnio or CVS), or add quad screen test at 15-19 wks GA • Second Trimester • Quad screen test at 15-19 wks GA • a new risk is assessed based on the results of patient’s age and both the first- and second-trimester screening test results

14. Sequential Screening • How do you decide when to proceed with invasive testing? • Risk of miscarriage (approximate; operator-specific) • chorionic villus sampling (CVS) ~ 0.5-1/100 • amniocentesis ~1/1000 • After 1st trimester results return: • If risk is greater than ~1 in 50, offer CVS • If risk is less than ~1 in 1,000, advise no further testing is necessary. • If risk is between these two (arbitrary) cutoffs, offer quad screen test after 15 wks GA, and determine a new risk assessment

15. FASTER Trial Data

16. What do you do with… • Normal Ultrasounds • Other Abnormal Serum Studies

17. Normal Ultrasounds • Normal ultrasound: 50-60% decrease in risk for chromosome abnormalities • Remember that at least 30% of fetuses with Down syndrome have NO abnormal ultrasound findings!

18. Low 1st Trimester PAPP-A • Pregnancies with PAPP-A of ≤ 5%tile (0.4MoM) are at increased risk for: • Spontaneous fetal loss < 24 wks GA • Low birth weight • Preeclampsia • Gestational HTN • Preterm birth and stillbirth • Preterm premature rupture of membranes • Placental abruption

19. Abnormal 2nd Trimester Serum Values

20. What’s next?

21. Cell-free Fetal DNA (cffDNA) • Screening or Diagnosis? • Known as “Noninvasive Prenatal Testing” or “NIPT” • Technology uses circulating cell free fetal DNA found in the maternal plasma • Thought to be derived primarily from placenta • New recognition of limitations of screening with pregnancies with placental mosacisms • Unclear data for egg donor pregnancies • Available as early as 10th week of pregnancy • Cleared from maternal blood almost immediately after childbirth

22. Cell-free Fetal DNA (cffDNA) • 2012 publications (Sparks et al., Ashoor et al., Bianchi et al.) • targeted (chromosome-selective) sequencing of chromosomes 18 and 21 • highly accurate • potentially more cost-effective • Technology can be expected to identify 98% of cases of T21 with a false-positive rate of < 0.5%. • Multiple different labs • MaterniT21plus by Sequenom • Verifiby Verinata • Harmony by Ariosa

23. Cell-free Fetal DNA (cffDNA) Labs • MaterniT21plus by Sequenom • >99% detection for T21, T18 • ~90% detection for T13 • <1% false positive rate • Cost: $235-2700 • Verifi by Verinata • >99% detection for T21, T18 • ~80% detection for T13 • >90% detection for 45,X • <1% false positive rate • Cost: $200-1200 • Harmony by Ariosa • >99% detection for T21, T18 • ~80% detection for T13 • <1% false positive rate • Cost: up to $795

24. Cell-free Fetal DNA (cffDNA) • ACOG Committee Opinion, Dec. 2012 – “Noninvasive Prenatal Testing for Fetal Aneuploidy” • cffDNA testing should be an informed patient choice after counseling and should not be part of routine prenatal laboratory assessment. • cffDNA testing should not be offered to low-risk women or women with multiple gestations (has not been studied). • A negative cffDNA test result does not ensure an unaffected pregnancy. • A patient with a positive test result should be referred for genetic counseling and should be offered invasive prenatal diagnosis for confirmation of test results.

25. Indications for Considering Use of cffDNA for Screening • Maternal Age ≥ 35 years at delivery • Fetal ultrasound findings indicating increased risk of aneuploidy • CPCs? • IEF? • Clinodactyly? • Absent/hypoplastic nasal bone? • History of a prior pregnancy with a trisomy • Positive test result for aneuploidy (any serum test) • Parental balanced translocation with increased risk of fetal trisomy 13 or 21.

27. Considerations • Primary insurers are generally reimbursing for first-trimester screenings, including NT ultrasounds as well as NIPT. • Are there referral sites available to your patients for appropriate genetic counseling and NT US measurements? What about for CVS and/or amniocentesis? • What testing strategy makes the most sense for your patients? • How do you see it evolving in the next 1-2 years?

28. Testing for Cystic Fibrosis (CF) • Site of genetic defect = CF Transmembrane Regulator (CFTR) gene, a chloride channel protein • ~ 1700 mutations of CFTR gene have been described • Disease incidence: 1 in 2500 in the non-Hispanic white population • Carrier frequencies: • 1/24-25 Caucasians of European descent or Ashkenazi Jews • 1/58 Hispanic American • 1/61 African American • 1/94 Asian American

29. CF Genetic Testing • Difficult to assign a single ethnicity to individuals • ACOG 2011 Recommendation – offer CF carrier screening to all women of reproductive age • Need to be screened only once

30. CF Parental Genetic Testing • Sequential testing • Test mother for carrier state • If positive, test father • Concurrent testing • Test mother and father simultaneously • Advantage: can be done prior to conception • Limitation: depends on accurate ID of father • If both parents are unaffected but family hx of CF exists: • Genetic counseling • Identify if CFTR mutation analysis in affected family member is available

31. Positive CF Testing • If both parents are carriers… • and prior to conception • offer ART options for diagnosis of embryo • and currently pregnant • offer CVS or amnio to confirm status of fetus • No in-utero treatments exist • Variable clinical scenarios, with median survival for patients 37yrs

32. “Residual Risk” • Potential risk of having an affected child with CF after testing is completed and is negative • Varies by race and current testing panel for gene abnormalities • Will vary over time and by laboratory • Newborn screening panels that include CF screening do not replace maternal carrier testing

33. Summary Recommendations • Genetic screening in pregnancy (and pre-conception!) is rapidly getting more complex. • OHSU Online Course (FREE!) • 0.5 CME credits available • www.ohsu.edu/prenatal-screening • Understand your patient population and your local capabilities, specifically as they relate to genetic counseling, ultrasound expertise, and diagnostic testing. • Develop practice workflows that allow women to access early genetic screening should they desire.