Prenatal screening for genetic disorders: best current practice and

what’s around the cornerJune C Carroll MD CCFP FCFP

Sydney G Frankfort Chair in FMAssociate Professor and Research Scientist

Department of Family & Community MedicineMount Sinai Hospital, University of Toronto

OCFP 51st ASA, November 29, 2013

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

Prenatal Screening tests for:DS, T18, T13 and ONTD

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

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

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

Use of Ultrasound in Screening for Chromosomal Anomalies

• Detailed u/s at 18-20 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

SOGC Guidelines 2011

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?

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)

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

• 7 studies of “high risk” women • High risk:

―Screen positive―AMA (≥35 yrs)―Ultrasound findings―Family history indicating increased risk

• Previous pregnancy with aneuploidy

What’s the evidence for NIPT?

What’s the evidence for NIPT?

• By far most accurate performance for T21/18

Benn et al, Ultras Obstet Gynecol 2013, 42: 15-33

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

What’s the evidence for NIPT?

• Failed results―6.1% (0.8-9.9) 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

Summary of evidence for NIPT

Benn et al, Ultras Obstet Gynceol 2013, 42: 15-33

Disorder Sensitivity FPR

Down syndrome 99-100% ~0.1%

Trisomy 18 97-100% ~0.1%

Trisomy 13 79-92% ~0.1%

Sex chromosome differences

94-99%

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

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

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

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

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

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 18-20 wk ultrasound

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

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

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

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)

NIPT vs microarray: 2 diverging philosophies

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)

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

Microarray for PN diagnosis

• Microarray generally detects all aneuploides and unbalanced translocations identified on karyotyping―Does not identify balanced translocations/triploidy

Wapner NEJM 2012

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

Ethnicity based screening

Ethnic Group Disorder Screening testBlack Sickle cell anemia MCV<80% followed by Hb

electrophoresisAshkenazi 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 MCV<80% followed by Hb electrophoresis

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

Ethnicity based screening: Carrier testing for persons of French Canadian background

- Charlevoix/Saguenay-Lac-Saint-Jean area

Disease Carrier frequencyCystic 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 1/23

Screen all Fr Can/Cajun descent for TSD – take a FH/ refer from regions above

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 20-15+% 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

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

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

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

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

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

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

Useful Genetics Resources• GEC-KO website: Genetics Education Website • www.gecko-cegco.ca• NIPT fact sheets:

―http://www.mountsinai.on.ca/care/pdmg/NIPT%20info%20sheet%20for%20parents%2029_11_2012.pdf

―http://www.mountsinai.on.ca/care/pdmg/NIPT%20info%20sheet%20for%20For%20Healthcare%20Providers%2029_11_2012.pdf

• Prenatal Screening Ontario Website―http://www.prenatalscreeningontario.ca/

• morrison@cheo.on.ca, jcarroll@mtsinai.on.ca• Thanks to Shawna Morrison, GEC-KO program manager for

assistance with this presentation