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Vincenzo Cirigliano
Molecular Genetics Synlab, Barcelona
cfDNA Based Prenatal Screening for Common Aneuploidies
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0%
20%
40%
60%
80%
100%
Maternal age AFP only Quad MarkerScreen
FirstTrimester
Screen
FullIntegrated
Screen
Detection Rate for Down Syndrome*
1970’s 1980’s 1990’s 2000
*ACOG practice bulletin no. 77, Obstet Gynecol 2007;109:217-27.
Evolution of Prenatal Screening
FPR=5.0%
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Cell-free DNA in Maternal Blood
• Released through apoptosis
Fetal cfDNA from the Cytotrophoblast
• Fetal DNA reliably detected from
7w gestation
• Cleared by the kidney disappears
within hours postpartum
• Maternal Plasma contains both
fetal and maternal cfDNA
1-20% of cfDNA is of fetal origin
• Fetal cfDNA is shorter than maternal
(140-160b.p.)
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Chromosome 21 fragments
Reference chromosome
Fetal cfDNA
Maternal cfDNA
Detecting Fetal Trisomy by cfDNA Analysis
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Fetal cfDNA
Maternal cfDNA
Extra fragments derived
from fetal trisomy 21
Chromosome 21 fragments
Reference chromosome
Detecting Fetal Trisomy by cfDNA Analysis
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Alignment of Fragments
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Fragment Count
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Fetal Fraction
Expected ratio for Trisomy
4% 1.02
10% 1.05
20% 1.10
40% 1.20
Higher Fetal Fraction Easier Detection Fetal cfDNA
Maternal cfDNA
Detecting Fetal Trisomy by cfDNA Analysis
Chromosome 21 fragments
Reference chromosome
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Massively Parallel Shotgun Sequencing (MPSS)
• MPSS is a random sampling of cfDNA fragments
• An arbitrary z-score value is used as a cut-off for trisomy
Palomaki et al.. Genet Med. 2011 Nov;13(11): 913-20.
N=1696
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• “Unclassified” zone for values between 2.5-4
• true and false positives in this zone
Bianchi, DW, et al.. Obstet Gynecol. 2012 May;119(5): 890-901.
MPSS Unclassified Values
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Chr 21, 18, 13, X & Y cfDNA
Other Chr cfDNA
Unmapped cfDNA
cfDNA in blood
MPSS (shotgun)
Selected Tags
Directed
Random analysis of cfDNA
Targeted vs WGS
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Sensitivity is only driven by the number of useful counts (Sequencing Depth)
Specificity increases with the Fetal Fraction (FF)
Fetal fraction
Ch
rom
oso
me 2
1 Z
-sco
re
-3
0
3
6
9
12
15
18
21
0 5 10 15 20 25 30 35
FF 8% FF 2%
24
Fetal fraction
Ch
rom
oso
me
21
Z-s
co
re
-3
0
3
6
9
12
15
18
21
0 5 10 15 20 25 30 35
FF 8% FF 2%
24
a) b)
Fracción fetal
Z-s
core
para
Cro
mosm
a 2
1
FF 2% FF 8%
Cut-off at 4% FF for reportable cases
Aneuploidy detection by MPSS
Wright et al. 2014 UOG 45(1), 48–54
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Detection rate FPR
Trisomy 21 99.7% 0.04%
Trisomy 18 97.9% 0.04%
Trisomy 13 99.0% 0.04%
cfDNA and Aneuploidy Screening
Gil et al, Ultrasound Obstet Gynecol 2017; 50: 302–314
cfDNA not always reflects fetal genotype (placental /maternal mosaicism, vanishing twin…)
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Trisomy 18 and 13 – discordance
Explaining “false positive” and “false negative” NIPT results
*Kalousek DK et al., Am J Hum Genet. 1989 Mar;44(3): 338-43 .
cfDNA originates from placenta ● May be similar to “Direct prep” of chorionic villi
Chromosomal makeup of placenta and fetus can be different
Occurs more frequently with chromosomes 13 and 18
● T13 and T18 (Kalousek et al*) — Only 30% of trophoblast showed trisomy when other fetal tissue
100% for trisomy 13 and 18 (viable)
● Case report of discordance between NIPT and fetus for trisomy 13
— NIPT: “positive” for T13
— CVS: mosaic 47,XY,+13[10]/46,XY[ 12]
— Amnio normal, fetus normal
— Placental biopsies = 2/4 mosaic trisomy 13
**Hall AL, Drendel HM, Verbrugge JL, Reese AM, Schumacher KL, Griffith CB, Weaver DD, Abernathy MP, Litton CG, Vance GH, Genetics in Medicine ( 2013)
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Detection rate FPR
Trisomy 21 99.7% 0.04%
Trisomy 18 97.9% 0.04%
Trisomy 13 99.0% 0.04%
cfDNA and Aneuploidy Screening
It’s a screening test
Gil et al, Ultrasound Obstet Gynecol 2017; 50: 302–314
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0%
20%
40%
60%
80%
100%
Maternal age AFP only Quad MarkerScreen
FirstTrimester
Screen
FullIntegrated
Screen
Detection Rate for Down Syndrome* FPR < 0.1%
Cell-free
DNA
analysis
1970’s 1980’s 1990’s 2000 *ACOG practice bulletin no. 77, Obstet Gynecol 2007;109:217-27.
FPR=5.0%
Evolution of Prenatal Screening
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0
50
100
150
200
250
300
350
2012 2013
ANXIETY
RISK
N 314
N 96
142
29
Invasive Procedures
Pedregosa et. al. 2015 Progresos de Obstetricia y Ginecología 01;58(3).
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18
Paired-end Sequencing More information, maintained accuracy
CGCTAGAAG
ATTTCCGCGATCTTCCCGTTCGACTGCAGACCTTCAGCGCGCATATATCGCTAGCATACCGTTATAC
GAAGTCGCG
Human Genome
Alignment Better accuracy
2 x 36 bp
Fragment Size determination
Fragment length
Count:
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Paired End Sequencing More information, maintained accuracy
Improved Allignment, Increased Depth and Resolution
2xMPSS
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Size Comparison – Maternal and Fetal Fragments
Lo et al. Sci Transl Med 2010 Vol 2 Issue 61 61ra91
Maternal and fetal cfDNA size distribution
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Fragment size distribution
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Cromosoma de interés
Cromosoma(s) de referencia
Cromosoma(s) de referencia
Cromosoma de interés
a) b)
Cromosoma de interés
Cromosoma(s) de referencia
Cromosoma(s) de referencia
Cromosoma de interés
a) b)
Cromosoma de interés
Cromosoma(s) de referencia
Cromosoma(s) de referencia
Cromosoma de interés
a) b)
Short Fragments Long Fragments
Standard methodology: counting statistics to determine if more than the expected number of reads are mapped to the target chromosome
New methodology: fragment size statistics, determine if the fraction of short fragments was higher on the target chromosome.
Paired-end MPSS: new analysis method
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Cromosoma de interés
Cromosoma(s) de referencia
Cromosoma(s) de referencia
Cromosoma de interés
a) b)
Cromosoma de interés
Cromosoma(s) de referencia
Cromosoma(s) de referencia
Cromosoma de interés
a) b)
Cromosoma de interés
Cromosoma(s) de referencia
Cromosoma(s) de referencia
Cromosoma de interés
a) b)
Cromosoma de interés
Cromosoma(s) de referencia
Cromosoma(s) de referencia
Cromosoma de interés
a) b)
Paired-end MPSS: new analysis method
New methodology: fragment size statistics, determine if the fraction of short fragments was higher on the target chromosome.
Combine FF, counts and frag size statistics for final scoring (t-score)
Short fragments analysis boosts resolution improving counting statistics
Increased specificity / confident analysis at low FF
V. Cirigliano et al UOG 2017 doi:10.1002/uog.173
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The Fetal Medicine
Foundation
Trisomy 21 n = 47
Trisomy 18 n = 30
Trisomy 13 n = 10
Stored plasma samples 1mL from 1000 singleton pregnancies at 11-13 wks
* Failed result n=12 (1.2%)
K. H. Nicolaides, Neobona Presentation, Madrid Feb. 2016
V. Cirigliano et al UOG 2017 doi:10.1002/uog.173
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Total cfDNA Failed
Normal 1609 1592 17
Trisomy 21 66 63 3
Trisomy 18 34 33 0
Trisomy 13 13 13 0
45, X 5 5 0
47, XXY 3 3 0
Total 1730 1709 20
Results of testing 1730 plasma samples
117 aneuploidies identified, 6 cases with FF between 0.8 and 3%.
Performance evaluation study
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-10
-5
0
5
10
15
20
25
30
35
40
0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000FF
NCV
NCV vs FF
V. Cirigliano et al UOG 2017 doi:10.1002/uog.173
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-100
-80
-60
-40
-20
0
20
40
60
80
100
0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000FF
T18 Score
T-score vs FF
Cut off at 4% FF no longer needed V. Cirigliano et al UOG 2017 doi:10.1002/uog.173
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98% of Trisomies and 53% SCA confirmed by fetal karyotype
All pregnancies to term
1stT Screening /
Streck BCT Blood
Paired-end MPSS: Clinical Application
23749 GA > 10w - 728 Twins
Test performed within 5 Days from sampling 1ml Plasma TAT 2-5 Days All samples tested for common trisomies SCAs included in 54% of cases
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27%
41%
14%
4% 2%
13%
MaternalAge
Anxiety ScreeningPotitive
Ultrasound History Others
Indications
Paired-end MPSS: Clinical Application
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23749 Samples
No result 430 (1.7%)
Not suitable (n=81)
Redraw 406
No result 27 (6.5%)
Normal n= 22767 (98%)
Trisomy 21-18-13 n= 471
XY Chr. n= 11021 (47%)
No Result 0.2%
SCA n= 36
Tested 23668 (730 Twins)
Result 23238 (98.3%)
Result 379 (93.5%)
Result in 99.8%
XXY+18/21 n=3
Paired-end MPSS: Clinical Application
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FPR 0.04% *1 T21, 3 T13 miscarried before confirmation
Results of screening for autosomal trisomies
T21* 363 1 5 99.7% 0.02%
T18 72 - 2 100% 0.01%
T13* 37 - 3 100% 0.01%
Low Risk 23238
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FPR 0.05%
45,X 20 9 3 0.05%
47,XXY 9 5 0 0.00%
47,XXX 5 3 0 0.00%
47,XYY 2 2 0 0.00%
Mat 3
Total 36 19 1
Results of screening for XY aneuploidies
- 11021 consecutive clinical cases - Invasive procedures in 53% high risk results - All pregnancies to term
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T21 14 - 2 100% 0.1%
T18 13 - - 100% 0.0%
T13 7 - - 100% 0.0%
Low Risk 1109
Autosomal trisomies at Low FF
FF<4% n= 1362 (5.5%) 84% reported, 24 Twins (48 Redraw) 20% of total T13 and T18
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• Paired-end MPSS already costs effective
• New generation algorithm (Tscore) High DR, Low FPR
• Improved efficiency, no fixed lower limit for FF to report
• All Trisomies with FF <4% correctly scored
• Including low FF didn’t impact DR while only increasing FPR
by 0.005%
• Increased T13 and T18 prevalence at low FF
• Extends cfDNA screening to a larger proportion of pregnancies
Conclusions
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Dept. of Molecular Genetics Synlab, Barcelona