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 Positive predictive value (PPV)
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
PPV
- The likelihood that an individual with a positive test result truly has the particular gene and/or disease in question.
The probability that an individual is affected with the condition when a positive test result is observed. Predictive values should only be calculated from cohort studies or studies that legitimately reflect the number of people in the population who have the condition of interest at that time since predictive values are inherently dependent upon the prevalence.
NPV
The probability that an individual is not affected with the condition when a negative test result is observed. This measure of accuracy should only be used if the data on the prevalence of condition of interest in given population is available. NPVDT can be determined by calculating: number of true negative results divided by the sum of true negative results plus number of false negative results.
The likelihood that an individual with a negative test result is truly unaffected and/or does not have the particular gene mutation in question.

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
Ductus Venosus – reversed a-wave flow
Detection rate 75%, FPR 5%
Tricuspid Regurgitation
Detection rate 67.3% in T21, FPR 5.2%

11. Integrated Screenings
Ultrasound for Nuchal Translucency (NT) + serum PAPP-A/hCG analysis between wks GA; results of these tests are HELD
Serum quad screen test between 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 (unconjugated estriol)
hCG (human chorionic gonadotropin)
DIA (dimeric inhibin A)
Compare to median value for the appropriate gestational age (MoM)
Valid between weeks GA (optimal wks); risk of NTD not provided for samples collected prior to 15 weeks.
Quad Screen - Detection rate for Down Syndrome 79%, FPR 5%
Penta Screen adds hyperglycosylated hcg (h-hcg)
Penta Screen – Detection rate for Down Syndrome 83%, FPR 5%

13. Stepwise Sequential Screening
First Trimester
NT US + serum PAPP-A analysis between wks 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 wks GA
Second Trimester
Quad screen test at 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 Screening Test Best Detection Rate for Down Syndrome
NT alone
70%
Quad screen alone (2nd trimester)
81%
First Screen (with NT)
87%
Serum Integrated (1st tri PAPP-A/hcg + 2nd tri quad)
88%
Sequential Screen (1st tri PAPP-A + NT + 2nd tri quad)
94%
Integrated Screen (1st tri PAPP-A/hcg + NT + 2nd tri quad); patient does not receive results until 2nd trimester testing complete
96%

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

17. Normal Ultrasounds
Normal ultrasound: % 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
Elevated hcg
Elevated inhibin-A
Low uE3
preeclampsia
steroid sulfatase deficiency
preterm delivery
IUGR
Smith-Lemi-Opitz syndrome
low birth weight infant
congenital adrenal hyperplasia
IUFD
adrenocorticotropin deficiency
placental abruption
hypothalamic corticotropin deficiency
anencephaly

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
Verifi by 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: $
Verifi by Verinata
~80% detection for T13
>90% detection for 45,X
Cost: $
Harmony by Ariosa
Cost: up to $795

24. Cell-free Fetal DNA (cffDNA)
ACOG Committee Opinion, Dec – “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
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.
3 recommendations to take back to your practice; “write down 1 you will commit to” (with strong recommendations of a single listed item)