1. Prenatal screening for genetic disorders: best current practice and what’s around the corner
June C Carroll MD CCFP FCFP
Sydney G Frankfort Chair in FM
Associate Professor and Research Scientist
Department of Family & Community Medicine
Mount Sinai Hospital, University of Toronto
OCFP 51st ASA, November 29, 2013

2. Objectives Prenatal screening for genetic disorders
FTS/IPS/MSS
Non-invasive prenatal testing (NIPT)
Use of microarray for prenatal diagnosis
Ethnicity based screening
Consanguinity and genetics

3. Prenatal Screening tests for: DS, T18, T13 and ONTD
Recommendations: any prenatal screen offered to women who present in T1 should have DR of 75% and no more than 3% FPR, if presenting in T2 DR of 75% with no more than 5% FPR
ONTD: MSAFP in T2 + comprehensive U/S screen at weeks
U/S for delayed ossification of fetal nasal bone – T1 (11-14 weeks) or T2
T1 DR 69% FPR depending on maternal ethnicity (9% Afro-Caribbeans, 5% Asians, 2.2% Caucasian)

4. Factors affecting FTS/IPS screening performance…and why you need to record them on the requisition
Gestational dating
FPR lowered by ~2% when GA estimated using scan
Maternal weight
Negative association between levels of maternal serum markers and maternal weight due to dilution effect produced by increased blood volume
Weight adjustment
increases DR by ~1% for a given FPR
reduces FPR by 0.2% for given DR
Ethnic origin
Adjusting tends to equalize the FPR among women of different ethnic groups
SOGC Guidelines 2011

5. Factors affecting FTS/IPS screening performance…and why you need to record them on the requisition
Insulin-dependent diabetes mellitus
Some T2 markers are lower
Smoking
Affects risk calculations – indicate if any smoking during pregnancy (even at time of conception)
Assisted Reproduction
Maternal age used is age of donor or woman at time egg was harvested
IVF pregnancies have higher risk of false positive screen – correction factor used – so important to indicate
SOGC Guidelines 2011

6. Prenatal Genetic Screening
Maternal age
Practice of using solely maternal age at EDD to identify at-risk pregnancies should be abandoned
DR for DS: 44%, FPR 16%
Maternal age should not be used as a basis for recommending invasive testing when non-invasive PN screening is available
Modify age-related risk by multiple biochemical markers with or without first trimester u/s assessment of nuchal translucency
SOGC Guidelines 2011

7. Use of Ultrasound in Screening for Chromosomal Anomalies
Detailed u/s at weeks’ gestation
Most major fetal anatomic abnormalities will be detected
Majority of ONTD
Soft markers
Should not be used alone
Use to modify any a priori risk established by age or prior screening
In absence of soft markers and anomalies – reduction of risk of 0.5 can be applied – only in tertiary level scan centre
See 2007 SOGC Ultrasound Soft Marker Guideline
- Negative likelihood ratio
SOGC Guidelines 2011

8. Prenatal Screening with addition of Non-invasive Prenatal Testing (NIPT)
What is NIPT?
What is the evidence for NIPT?
What do the experts say?
What is current Ontario prenatal screening landscape?

9. What is Non-invasive Prenatal Testing (NIPT)?
Screening test to prenatally detect Down syndrome and other aneuploidies (extra or missing chromosomes)
trisomy 21, 18, 13
trisomy of sex chromosomes (XXX, XXY, XYY)
Turner syndrome (monosomy X)
triploidy (extra copy of all chromosomes)
Aneuploidy: refers to an abnormal chromosome number (extra or missing)

10. NIPT
NIPT measures circulating cell free fetal DNA (ccffDNA) present in maternal blood (from trophoblast)
Comprises ~10% of DNA in maternal blood
Increases with gestational age
ccffDNA analysis determines if normal, higher, or lower than expected quantity of particular DNA sequences found on select chromosomes (13, 18, 21, X, Y)
Performed on maternal blood sample
As early as 9 weeks’ gestation
Dating u/s – viability, accurate GA, exclude multiples

11. What’s the evidence for NIPT?
7 studies of “high risk” women
High risk:
Screen positive
AMA (≥35 yrs)
Ultrasound findings
Family history indicating increased risk
Previous pregnancy with aneuploidy

12. What’s the evidence for NIPT?
By far most accurate performance for T21/18
Benn et al, Ultras Obstet Gynecol 2013, 42: 15-33

13. What’s the evidence for NIPT?
4 studies on unselected pregnancies
Most mixed risk, some after pos screen, AMA, fewer with neg or no screen
> 14,000 pregnancies total, largest (11,000 significant loss to follow up)
Similar performance
Not yet validated in low risk women, triplets or higher, pregnancies conceived with egg donation
Benn et al, Ultras Obstet Gynceol 2013, 42: 15-33

14. What’s the evidence for NIPT?
Failed results
6.1% ( ) untested for insufficient sample quality
2% (436/22,222) no result after testing
Rarely happens with conventional screening
ccffDNA decreases with increased maternal BMI
Benn et al, Ultras Obstet Gynceol 2013, 42: 15-33

15. Summary of evidence for NIPT
Disorder
Sensitivity
FPR
Down syndrome
99-100%
~0.1%
Trisomy 18
97-100%
Trisomy 13
79-92%
Sex chromosome differences
94-99%
Benn et al, Ultras Obstet Gynceol 2013, 42: 15-33

16. What do the experts say?
American College of Obstetricians and Gynecologists 2012
Has been validated in industry sponsored studies on at risk populations
History of prior pregnancy with trisomy
Positive multiple marker test
Parental balanced Robertsonian translocation with increased risk of fetal trisomy 13/21
Maternal age >35
Fetal ultrasound findings with increased risk of aneuploidy
ACOG Committee Opinion: Dec 2012

17. What do the experts say?
Society of Obstetricians & Gynecologists of Canada 2013
Non-invasive prenatal testing using massive parallel sequencing of cell-free fetal DNA to test for trisomies 21, 18, and 13 should be an option available to women at increased risk in lieu of amniocentesis. Pretest counselling of these women should include a discussion of the limitations of non-invasive prenatal testing.
Genetics Ctte Technical Update JOGC: Feb 2013

18. What do the experts say?
Society of Obstetricians & Gynecologists of Canada 2013
No irrevocable obstetrical decision should be made in pregnancies with a positive non-invasive prenatal testing result without confirmatory invasive diagnostic testing.
Genetics Ctte Technical Update JOGC: Feb 2013

19. Recommendation in Ontario
Offer all women prenatal screening using either FTS, IPS or MSS (SIPS or Quad)
IF screen +ve or at high risk for other reasons
Consider NIPT as secondary screen of higher sensitivity if she is willing to pay for the test
High risk:
Advanced maternal age
Previous aneuploidy pregnancy
Personal hx of sex chromosome aneuploidy (Turner mosaic, XXX)
Abnormal u/s
Pregnancy conceived via reproductive technologies

20. Recommendation in Ontario
NIPT is not a replacement for diagnostic PN testing
Positive NIPT should be confirmed by diagnostic testing: amnio or CVS
Expected benefit of NIPT: fewer women having diagnostic tests with associated risk of pregnancy loss

21. Recommendation in Ontario
If result is negative → reassuring
NIPT cannot:
Completely rule out aneuploidy
Detect chromosome differences other than aneuploidy of chromosomes 13, 18, 21, X and Y
Detect single gene conditions
Detect congenital anomalies
Still offer MSAFP and wk ultrasound

22. Recommendation in Ontario
If result is positive:
Genetic counselling
Confirmation by diagnostic testing
No irrevocable obstetrical decisions should be made in pregnancies with abnormal NIPT results without confirmatory invasive testing (CVS or amnio) - SOGC

23. NIPT in Ontario Increasing demand from women (who can pay)
Increasing uptake in most (urban) centres
3 separate companies, 3 separate technologies
No mechanism to evaluate centrally
Has it become mainstream now, become a “standard of care”? (? Only for high risk women)
Heading for 2 tiered prenatal screening
Costs between $795 and $1200
8-10 days for result

24. Where does NIPT fit with respect to the 11 to 14 wk scan?
11 to 14 week scan has value to pregnancy care
NT may shift in importance (importance to other chromosomal, genetic and structural disorders)
Accurate dating/establishment of live fetus
Multiples/chorionicity affects management
Detects structural abnormalities

25. NIPT and counselling
Likely primary providers will not have time/expertise to counsel in detail
Refer to genetic counselling if your centre provides that service (significant impact on counselling demands)
Company websites:
Panorama™ by Lifelabs
Harmony™ by Ariosa
Verifi™ by Verinata Health (Medcan)

26. NIPT vs microarray: 2 diverging philosophies

27. Chromosomal Microarray (CMA)
CMA is a technology used to determine if there are small extra (microduplication) or missing (microdeletion) pieces of genetic information. These gains and losses are called copy number variants (CNVs). A CNV can be: of no medical consequence; pathogenic resulting in physical and/or intellectual consequences; or protective against disease (e.g. HIV infection).
Reference DNA from control labeled Red
Test DNA from patient labeled Green
Glass microarray slide
Denature the DNA (separate the strands) and Hybridize to slide
Computer scans and analyzes signal outputs
Areas of loss (deletion)
Area of gain (duplication)

28. Microarray for Prenatal Diagnosis
Microarray can detect submicroscopic copy number changes that are associated with clinically significant outcomes
Systematic review to calculate utility of PN microarray in presence of normal conventional karyotype
12,362 pregnancies
2.4% overall had copy number changes with associated clinical significance
6.5% if ascertained because of abnormal ultrasound
1.0% if increased maternal age
1.1% for other reasons (parental anxiety, abnormal MSS)
Wapner NEJM 2012, Callaway Prenatal Dx 2013

29. Microarray for PN diagnosis
Microarray generally detects all aneuploides and unbalanced translocations identified on karyotyping
Does not identify balanced translocations/triploidy
Wapner NEJM 2012

30. Microarray for PN diagnosis
Summary
Consider when doing invasive test like CVS or amnio
Added diagnostic value of microarray especially when karyotype gives normal chromosome result
Genetic consult will assist with which test is most appropriate depending on clinical presentation
karyotype, microarray or both
Challenges:
Variants of unknown significance
Incidental findings
Future:
Next generation sequencing, whole genome sequencing
Wapner NEJM 2012, Callaway Prenatal Dx 2013

31. Ethnicity based screening
Ethnic Group
Disorder
Screening test
Black
Sickle cell anemia
MCV<80% followed by Hb electrophoresis
Ashkenazi Jewish
8 disorders
DNA analysis
French Canadian
Tay-Sachs disease
DNA analysis for selected alleles
Mediterranean
β-thalassemia
MCV<80% followed by HB electrophoresis (r/o iron def)
SE Asian
α-thalassemia

32. Carrier frequency in AJ Population
Ethnicity based screening: Carrier testing for persons of Ashkenazi Jewish background
Disease
Carrier frequency in AJ Population
Bloom Syndrome
1/102
Canavan Disease
1/57
Familial Dysautonomia
1/32
Fanconi Anemia group C
1/89
Mucolipidosis IV
1/100
Niemann Pick (A&B)
1/90
Tay-Sachs Disease
1/30
Canavan, Familial dysautonomia, Tay-Sachs – progressive neurological disorders
Fanconi anemia – anemia and increased cancer risk
Mucolipidosis and Niemann Pick – storage disorder with dev delay

33. Ethnicity based screening: Carrier testing for persons of French Canadian background - Charlevoix/Saguenay-Lac-Saint-Jean area
Disease
Carrier frequency
Cystic Fibrosis
1/15
Tyrosinemia type 1
1/21
Spastic Ataxia of Charlevoix-Saguenay
1/22
Hereditary Sensory-Motor Polyneuropathy +/- Agenesis of Corpus Callosum
1/23
Cytochrome Oxidase Deficiency
Canavan, Familial dysautonomia, Tay-Sachs – progressive neurological disorders
Fanconi anemia – anemia and increased cancer risk
Mucolipidosis and Niemann Pick – storage disorder with dev delay
Screen all Fr Can/Cajun descent for TSD – take a FH/ refer from regions above

34. Consanguinity
Deeply rooted cultural trend among 1/5 of world population
Mostly residing in Middle East, West Asia, North Africa and emigrants from these communities
Intra-familial unions account for % of all marriages
First cousins 1/3 of all marriages
Def’n: union between 2 people who are related as second cousins (5th degree relatives) or closer
First cousins, first cousins once removed, second cousins
Hamamy J Community Genetics 2012

36. Consanguinity Mainly cultural reasons for consanguineous marriages
Strategy to preserve transmission of cultural values and continuity
Maintenance of familial structure and property
Financial advantage relating to dowry, keeping property within family
Sometimes thought to be more stable unions
Same social relationships before and after marriage

37. Consanguinity
Genetic Risk: first cousin marriages compared to non-consanguineous marriages
Fertility rate slightly higher
Miscarriage rate not different
Stillbirths and infant mortality rates slightly higher
Congenital anomalies 2-3% higher than background population risk (2-3%)
Increased likelihood of autosomal recessive conditions
Increased risk for almost all multifactorial birth defects including congenital heart defects, clefting, club feet, etc
Complex diseases – really not known – could be significant if high susceptibility gene present in family
Hamamy J Community Genet 2012

38. Consanguinity Management Premarital/preconception counselling
For carrier status
May change marriage plans
3 to 4 generation FH
Appropriate testing based on FH and ethnic background
i.e. screen for common autosomal recessive conditions in pop and community
Refer to genetics if FH points to presence of genetic disorder

39. Consanguinity Concerns Fear of stigmatization
Belief that inherited disorders can only arise through cousin marriages on paternal side – specifically inquire re shared biologic relationships on mother’s side
Need to balance risks and benefits

40. Prenatal Screening Summary
Offer all pregnant women, regardless of age, PN screening for fetal aneuploidy (trisomy 13, 18, 21) through FTS, IPS, SIPS or Quad screening
+ second trimester ultrasound for dating, assessment of fetal anatomy and detection of multiples
Maternal age should not be used as a basis for recommending invasive testing when non-invasive PN screening is available
Consider offering NIPT to high risk women or following a positive PN screen for aneuploidy

41. Prenatal Screening Summary
Take a FH to identify familial and/or ethnically related disorders and screen accordingly
Consider consanguinity and screen and test accordingly
Refer or consult genetics when in doubt

42. Useful Genetics Resources
GEC-KO website: Genetics Education Website
NIPT fact sheets:
Prenatal Screening Ontario Website
Thanks to Shawna Morrison, GEC-KO program manager for assistance with this presentation