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

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 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 18-20 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)

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 - Negative likelihood ratio 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) Aneuploidy: refers to an abnormal chromosome number (extra or missing)

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

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

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 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

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 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

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

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

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