Clinical Genetics Leading the way in genetic issues

NIPT- just fetal trisomy screening or more?

NGS course: genomic resequencing in medical diagnostics 2018 5 september 2018

Diane Van Opstal, laboratory specialist clinical genetics

1. First trimester screening (combined test): risk calculation of trisomy 21, 13 and 18 • Between 9-13+6 wks of GA • Maternal serum concentrations of PAPP-A and β-HCG and US measurement of fetal nuchal translucency (NT) and maternal age: if risk > 1/200 invasive testing.

2. Fetal anomaly ultrasound scan: Between 18-22 wks of GA

Non-invasive prenatal screening

CT: identify fetuses at risk for +13, +18 and +21

Trisomy 21 Down syndrome (prev 1/700)

Trisomy 18 Edwards syndrome Prev 1/5000

Trisomy 13 Patau syndrome Prev 1/16000

o 1 april 2014: start of NIPT in the Netherlands for pregnant women at increased risk:

-Abnormal first trimester combined test results (risk > 1/200, normal NT) -Medical indication (e.g. previous child with T13, T18 or T21, parental robertsonian translocation)

o Trident 1: choice between NIPT or diagnostic testing (CVS/AC)

o 1 april 2017: NIPT for all pregnant women:

o Trident 2: choice between NIPT or CT

TRIDENT

The beginning

“Fetus-derived Y sequences were detected in 24 (80%) of the 30 maternal plasma samples………. may have implications for non-invasive prenatal diagnosis…………” -NIPT with fetal cells: expensive and time-consuming

-presence of tumour DNA in plasma of cancer patient

(cf)-DNA in maternal blood is partially fetal

Fetal fraction: (Wang et al., 2013, Prenat Diagn, 33, 662–666)

mean 10,2 % at 10 wks

in 2% < 4% FF

4% is theoretical cut-off value (<->Fiorentino et al., 2016, Prenat Diagn: 2%)

Half-life very short

Uniformely distributed over the whole genome

Gestational age

BMI

Fetal aneuploidy

Factors that influence the fetal fraction

Wang et al., 2013, Prenat Diagn, 33, 662–666 Taglauer et al., 2014, Placenta, 28, S64-S68

Technique

Shotgun massively parallel sequencing

Gregg et al., 2014, Annu Rev Genomics Hum Genet

Bij een trisomie: extra stukken DNA van dat chromosoom aanwezig

Analyse met WISECONDOR (Straver et al., 2013)

Developped by VU

It relies on a within-sample comparison: bins (1 Mb) on chromosome of interest are compared to look-alike bins on other chromosomes within the sample

On the basis of a reference set of

normal samples, look-alikes are determined

Enables the detection of whole

chromosome aneuploidies, but also

subchromosomal aberrations (> 20 Mb) NB: Bins 1 Mb, coverage 0.5, reads 50nt

10.000 reads/bin, 5% fetal= 500 fetal reads/bin

WIthin SamplE COpy Number aberration DetectOR (Wisecondor)

Analysis method: WISECONDOR

-alle chromosomen m.u.v. geslachtschromosomen

WISECONDOR Straver et al., 2014, Nucleic Acids Research, Vol. 42, No. 5 e31 doi:10.1093/nar/gkt992

-GROTE afwijkingen (resolutie ~ 15Mb): dus GEEN monogene aandoeningen (CF, etc), GEEN microdeleties (22q11 deletie, etc), GEEN erfelijke aanleg voor kanker, alzheimer etc, GEEN triploidie, GEEN geslachtschromosomale afwijkingen (Turner, Klinefelter etc)

Prenatal screening for Down, Edwards en Patau syndrome

“nevenbevindingen”

= chromosome aberrations different from the common

trisomies (trisomy 21, 18 and 13)

Determined by: Technique and analysis method Origin of cfDNA

T13, 18, 21 en “nevenbevindingen”

Technique: other chromosome aberrations

Trisomy 16 del(4)(p15.3) ~ Wolf-Hirschhorn

t(4q;12p)

-maternal mosaicism -maternal CNV -maternal tumor/myoma

Origin of cfDNA: ~90% maternal

Maternal CNV

Partial duplication 21q21.1

Array on buffy coat: 2 Mb mat duplication

chr21:20,604,811-22,572,304: VOUS

Maternal mosaic CNV

Microdeletion 2p23

Windowed Bin test Bin

Z-score -4,3

Call chr2:21000000-31000000

chr2:24509507-26490492

Size 10Mb 1,98 Mb

Microdeletion 2p23 UCSC - chr2:24509507-26490492

DNMT3A - Tatton-Brown-Rahman syndrome

Aanwijzing voor pathogene afwijking

Rapporteren

Maternal mosaic microdeletion 2p23

Moeder (buffy coat): arr[hg19]2p23.3(24390347-26090869)x1~2 Size: 1,7 Mb Moeder (haar en huidbiopt – normaal)

Foetus CVS (CTB en MC): normaal

Maternal malignancy

Maternal malignancy

Multidisciplinair team: Klinische genetica Gynaecoloog perinatoloog Gynaecoloog oncoloog Interne geneeskunde oncologie Interne geneeskunde hematologie Radiologie Dermatoloog (MDL)

Advice invasive prenatal diagnosis

Tumorcytogenetica van het lymfoom:

Maternal malignancy

Met dank aan Femke de Vries

Uterus myomatosus

Origin of fetal part of cfDNA: placental

Cf-fetal DNA has placental origin

Confined placental mosaicism

Same karyotype in placenta and fetus

N

N N +21 +21

+21

Types chromosomal mosaicism

Diploid

Trisomy

A B C generalized CPM (“false” positive NIPT)

CFM (“false” negative NIPT)

NIPT: duplication 4q

amniocentesis: deletion 2

Placenta na TOP: deletion 2

Placenta na TOP: duplication 4

o Trident 1 o april 2014-april 2015

o n=3306

o 753 trisomie 21, 18 en 13

o 2553 alle chromosomen

o n=2527 met resultaat

o n=119 afwijkingen (4.7%)

o 78 trisomie 21, 18 of 13 (3.1%)

o 61x +21, 11x +18 en 6x +13

o 41 andere chromosoomafwijkingen (1.6%)

frequency unsolicited findings?

Clinical relevance of genome-wide screening

extra chromosome aberrations in 0.25 -1.6%

Different categories of chromosome aberrations

010203040506070

61 10 22 1 8 11 6

N=78 N=41

Trident 1 (high risk)

Clinical relevance of confined placental mosaicism

trisomy n pregnancy outcome

trisomy 2 1 TOP: fetal growth restriction and US abnormalities (rocker bottom feet, suspicion abnormal spine, suspicion cardiomegaly)

trisomy 3 1 fetal growth restriction

trisomy 7 6 fetal growth restriction (1) congenital anomaly (1) (anusatresia ,abnormal scrotum and horseshoe kidney) normal (4)

trisomy 8 2 Normal (2)

trisomy 9 1 fetal growth restriction (1)

trisomy 16 9 fetal growth restriction (7) Normal (2) congenital anomaly (3) -extended hemihypertrophia and good catch-up growth . At 11 months, neurological investigations normal, but tubefeeding and Silver Russell syndrome. -dysmorphic features (simple helix both ears, simian crease both hands, 2/3 syndactyly toes). -VACTERL association

trisomy 22 1 fetal growth restriction

trisomy 2,20 1 normal

59% (13/22) groeiproblemen: -32% (7/22) SGA (<p10) -27% (6/22) IUGR (<p2.3) 23% (5/22) MCA + SGA/IUGR

o In patients with an elevated risk for trisomy 13,18 or 21, genome wide

cfDNA testing reveals a chromosomal aberration other than trisomy 13,18 or 21 in 1/3 of cases with an abnormal result.

o Besides fetal chromosome aberrations, also CPM associated with an increased risk MCA (23%) and/or growth problems (27% <p2.3)

o these data show a significant risk for obstetric pathology and fetal anomalies

o we advise close obstetric surveillance in all cases of abnormal NIPT even if the fetal karyotype as determined by diagnostic testing is normal

o It seems to be clinically relevant to do genome-wide NIPT

Conclusions from Trident 1

Since April 2017 TRIDENT-2: choice CT or NIPT

Combinatietest blijft beschikbaar: Het betreft een proefimplementatie van NIPT waarbij vrouwen het recht hebben te kiezen voor de standaard screeningtest (combinatietest)

Choice with and without “nevenbevindingen”

Trident 2

-N=73.000 pregnant women chose NIPT ~ 42% of all pregnant women in the Netherlands (before the introduction of NIPT: 34% chose for screening with the CT (figures 2016)) -78% chose “with nevenbevindingen” -~ 3% of all pregnant women did a CT -0.3% T21 (PPV estimated: 75% practice > 90%) -0.1% T18 (PPV estimated: 24% practice > 90%) -0.1% T13 (PPV estimated: 23% practice ~50%) (compare to CT: 5%) -0.3% “nevenbevindingen”

Eerste resultaten 1ste jaar Trident 2

o Genome-wide NIPT (instead of targeted testing) is clinically useful in pregnancies with abnormal CT results (risk T21, T18, T13 > 1:200, normal NT)

o Also clinical utility in a general obstetric population?

o Further development of test to higher resolution will enable the detection of submicroscopic chromosome aberrations as well (background risk 1/100-1/200)

o NIPT for selecting pregancies at risk for “all” chromosome aberrations

Conclusion

Srebniak et al., 2018, Ultrasound Ostet Gtynecol, 51(4):445-452

acknowledgements

Clinical geneticists: Merel van Maarle, AMC Klaske Lichtenbelt, UMCU Lutgarde C.P. Govaerts, Erasmus MC Gita Tan, VUMC Nicolette den Hollander, LUMC Ilse Feenstra, RUNMC Lean Beulen, RUNMC

All Trident consortium members of all disciplines The core group of the TRIDENT1 and 2 study: Erik Sistermans, VUMC Lidewij Henneman, VUMC Lieve Page-Christiaens, UMCU* Robert-Jan Galjaard, Erasmus MC and many others

De TRIDENT studies are sponsored by: Nederlandse Organisatie voor Gezondheidsonderzoek en Zorginnovatie (ZonMw) & Ministry of Health, Welfare and Sport

Clinical cytogeneticists: Shama Bhola, VUMC Karin Huijsdens, AMC Mariëtte Hoffer, LUMC Merryn Macville, MUMC Angelique Kooper,/Brigitte Faas RUNMC Heleen Schuring-Blom, UMCU

Erasmus MC, Rotterdam: -Laboratory of the dpt of Clinical Genetics Marjan Boter, Hasna Kariouh, Iris Hollink, Fernanda Jehee, Frank Sleutels, Walter de Valk, Raoul van de Graaf, Erwin Wauters, Wout Deelen, Genesis-team, Lies Hoefsloot, and many others -Prenatal Multidisciplinary Expertise Group: -Laboratory specialists Clinical Genetics: Gosia Srebniak, Femke de Vries -Clinical Geneticists: Marieke Joosten, Lutgarde Govaerts, Karin Diderich, Robert-Jan Galjaard -Psychologists: Sam Riedijk, Sanne van der Steen -Gynaecologists: of Erasmus MC, Rotterdam, Albert Schweitzer Hospital (Dordrecht), Reinier de Graaf hospital (Voorburg), Amphia hospital (Breda)

Bioinformaticians: Stijn Ghesquiere Rutger W.W. Brouwer Sander Bollen Martin G. Elferink Roy Straver