ADVANCES IN THE USE OF TROPHOBLASTIC
CELLS FOR PRENATAL NON-INVASIVE
DIAGNOSTICS OF GENETIC DISORDERS
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Disclosure
Inventor/coinventor of ISET patents
Founder ans scientific advisor of Rarecells
Academic tasks:
Teaching (University Paris Descartes)
Developing and implementing new tests (Hôpital Necker)
Research activity (INSERM Unit)
Circulating Fetal Trophoblastic Cells (CFTC)
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Genetic Diagnosis
TC= Trophoblastic cells
MC = Maternal cells
ISET by Rarecells: Patented combination of parameters allowing very sensitive and rapid isolation of CFC
4
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G. VONA, …….PATERLINI-BRÉCHOT. Enrichment
and genetic analyses of fetal cells circulating in
the maternal blood by the ISET technique and
single cell microdissection : a non-invasive tool for
early prenatal diagnosis. Am J Pathol, 160 : 51-58,
2002.
13 pregnant women
11-12 WG
Before CVS
CFTC in all the mothers, Y and STR genotyping
C. BÉROUD, ……..PATERLINI-BRÉCHOT. Prenatal
diagnosis of Spinal Muscular Atrophy (SMA) by
genetic analysis of circulating fetal cells. The
Lancet, 361 :1013-4, 2003.
12 mothers at risk for baby with SMA
10-12 WG before CVS
Blind analysis vs CVS
CFTC in all the mothers, correct diagnosis
A. SAKER,……….., P. PATERLINI-BRÉCHOT.
Genetic characterization of circulating fetal cells
allows Non-Invasive prenatal diagnosis of cystic
fibrosis. Prenat Diagn, 26 : 906-16, 2006.
12 mothers at risk for baby with CF
10-12 WG
Blind analysis vs CVS
CFTC in all the mothers, correct diagnosis
H. MOUAWIA, …… P. PATERLINI-BRECHOT.
Trophoblasts enriched from maternal blood provide
definitive genetic diagnosis in 63 consecutive
fetuses at risk for Cystic Fibrosis or Spinal Muscular
Atrophy, Reproductive Biomedicine Online, 2012
PFEIFER I, ……P. PATERLINI-BRECHOT. Cevical
Trophoblasts for non-invasive single cell
genotyping and prenatal diagnosis, Placenta, 2015
63 mothers at risk for baby with SMA or CF
10-12 WG, before CVS
Blind analysis vs CVS
CFTC in all the mothers, correct diagnosis
21 pregnant women
6 before CVS, 3 CF, 3 SMA, correct diagnosis
15 before TOP
PAP like sampling 8-12 WG
2 to 12 trophoblasts in 2 out of 10 ml
TC in all the mothers, correct diagnosis
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Table 2
Non invasive-prenatal diagnosis for spinal muscular atrophy.
Couple no.
(weeks of
gestation)
STR markers
Microdissected
cells (n)
1 (11)
2 (11)a
3 (10)
4 (10)
5 (9)a
6 (9)
7 (9)
8 (11)
9 (11)
10 (10)
11 (9)
12 (11)
13 (10)
14 (11)a
15 (11)
16 (9)a
17 (11)a
18 (10)a
19 (10)a
20 (11)a
21 (11)
22 (10)a
23 (10)a
24 (10)a
25 (11)a
26 (9)a
27 (11)
28 (10)
29 (11)
30 (11)
31 (11)
D16S539, D16S3018, D21S1435
D7S480, D16S3018, D5S465
D7S486, D7S490, D7S523
D7S486, D21S1435, D21S1437
D16S539, C272, D21S1435, D17S800
D16S539, D5S816, D1S1171
D21S1437, D17S800, D5S816
D16S539, D5S816, D5S1360
D16S539, D5S615, D21S1435
D7S523, D16S539, D5S681
D7S486, D16S539, D21S1435
D7S486, D16S3018, D21S1437
D5S637, D16S539, D5S816
D16S539, D5S1360, C272
D16S539, D21S1437, D5S465
D16S539, D5S615, D5S816
D16S539, D5S346, D21S1435
D7S523, D16S539, D5S681, D17S800
D16S3018, D5S615, D17S800
D16S539, D5S1360, D7S523
D16S539, D21S1435, D1S1171
D16S539, D21S1437, D5S465
D16S539, D5S681, D21S1435, D17S800
D16S539, D5S816, D5S1360
D16S539, D5S681, D5S637
D21S1437, D17S800, C272
D16S539, D21S1437, D21S1435
D16S539, D21S1435, D1S1171, D7S486
D21S1435, D7S486, D17S800
D16S539, D21S1435, D16S3018
D21S1435, D1S1171, D7S486
Total SMA
CFTC (n)
CFTC result (n)
13
11
15
17
15
13
14
12
11
19
16
14
11
12
25
24
21
25
21
27
22
27
27
23
23
22
21
24
22
21
6
5
7
8
8
6
6
7
5
9
7
6
5
7
13
11
10
12
10
12
10
13
12
12
11
10
10
10
10
10
0
5
0
0
5
0
0
0
0
0
5
0
5
0
0
0
0
0
0
10
0
0
10
10
0
0
0
0
0
0
0
586
276
50
Test results
Without
gene deletion
With gene
deletion
5
0
5
5
0
5
5
5
5
5
0
5
0
5
5
10
10
10
10
0
10
10
0
0
10
10
10
10
10
10
10
185
NI-PND
Invasive
PND
NA
A
NA
NA
A
NA
NA
NA
NA
NA
A
NA
A
NA
NA
NA
NA
NA
NA
A
NA
NA
A
A
NA
NA
NA
NA
NA
NA
NA
NA
A
NA
NA
A
NA
NA
NA
NA
NA
A
NA
A
NA
NA
NA
NA
NA
NA
A
NA
NA
A
A
NA
NA
NA
NA
NA
NA
NA
A = affected; C = carrier; CFTC = circulating fetal trophoblastic cells; CVS = chorionic-villous sampling; N = normal; NA = not affected; NI-PND = non-invasive prenatal diagnosis; STR = short
tandem repeat.
a
Cases redundantly tested by indirect diagnosis.
Table 1
Non-invasive-prenatal diagnosis for cystic fibrosis.
Couple no.
(weeks of gestation)
STR markers
1 (11)a
2 (9)a
3 (11)
4 (10)
5 (10)
6 (9)a
7 (11)
8 (11)
9 (10)a
10 (9)a
11 (10)b
12 (10)c
13 (11)a
14 (11)a
15 (11)
16 (10)a
17 (10)a
18 (11)
19 (10)a
20 (9)a
21 (11)
22 (11)
23 (11)
24 (11)a
25 (11)
26 (10)b
27 (9)b
28 (10)b
29 (11)a
30 (11)
31 (11)b
32 (10)
D7S486, D16S539, D21S1435
D7S486, D16S3018, D21S1437
D16S539, D21S1435, D21S1437
D16S3018, D21S1437, D21S1435
D16S539, D16S3018, D21S1435
D7S480, D16S539, D16S3018
D16S539, D21S1437, D21S1435
D16S539, D16S3018, D21S1435
D7S480, D16S3018, D21S1435
D7S486, D7S490, D7S523
D7S486, D21S1435, D21S1437
D7S480, D7S486, D7S523
D7S486, D21S1435, D16S3018
D7S480, D21S1435, D21S1435
D7S486, D16S539, D21S1435
D7S486, D16S539, D16S3018
D16S539, D21S1437, D7S486
D16S3018, D21S1435, D1S1171
D16S3018, D7S486, D5S615
D16S3018, D5S816, D7S480
D16S539, D16S3018, D21S1435
D16S539, D21S1437, D5S637
D16S539, D21S1435, C272
D7S480, D16S3018, D16S539
D16S539, D5S816, D21S1437
D7S486, D16S539, D5S816
D7S486, D7S490, D21S1437
D7S523, D21S1435, D16S539
D7S486, D16S539, D5S816
D21S1435, D7S490, D5S681
D7S523, D21S1435, D7S490
D7S523, D21S1435, D7S490
Microdissected
cells (n)
Total CF
CFTC (n)
CFTC result for F508del (n)
Test results
Without
F508del
NI-PND
Invasive
PND
14
12
16
15
14
13
19
15
12
11
13
17
13
13
11
15
23
27
24
26
21
24
21
22
21
30
23
22
25
21
26
27
7
6
7
8
6
6
9
7
6
5
6
8
7
6
5
7
11
12
11
12
10
11
10
10
11
13
11
11
12
10
12
12
0
0
0
0
0
0
0
0
5
0
0
0
0
5
0
5
0
0
0
0
0
0
0
0
0
10
0
0
10
0
10
10
Heterozygous
5
5
0
5
0
0
5
5
0
5
5
0
5
0
5
0
0
10
10
0
10
10
10
0
0
0
0
0
0
10
0
0
0
0
5
0
5
5
0
0
0
0
0
5
0
0
0
0
10
0
0
10
0
0
0
10
10
0
10
10
0
0
0
0
C
C
N
C
N
N
C
C
A
C
C
N
C
A
C
A
N
C
C
N
C
C
C
N
N
A
N
N
A
C
A
A
C
C
N
C
N
N
C
C
A
C
C
N
C
A
C
A
N
C
C
N
C
C
C
N
N
A
N
N
A
C
A
A
606
285
55
105
80
Homozygous
A = affected; C = carrier; CFTC = circulating fetal trophoblastic cells; CVS = chorionic-villous sampling; N = normal; NI-PND = non-invasive prenatal diagnosis; STR = short tandem repeat.
Cases redundantly tested by indirect diagnosis.
b
One parent with non-F508del mutation.
c
Both parents with non-F508del mutation.
a
1e−02
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
Effect of circulating fetal trophoblastic cell replicated
tests on the overall reliability of the diagnostic
procedure.
●
●
●
●
●
●
●
●
●
●
●
1e−05
●
●
●
●
1e−08
●
1e−11
●
When using 10 CFTC replicates, the protocol of independent diagnostic
analysis (correlation = 0), virtually never gives rise to a global (clinical)
mistake at an error rate of 10%, i.e. the probability of global diagnostic
error rate is 1 in 10 billion. With five CFTC replicates, this
extraordinary performance can be reached if the error rate is 1% or
less.
5 duplicates, Se=0.80
5 duplicates, Se=0.84
5 duplicates, Se=0.88
5 duplicates, Se=0.92
5 duplicates, Se=0.96
10 duplicates, Se=0.80
10 duplicates, Se=0.84
10 duplicates, Se=0.88
10 duplicates, Se=0.92
10 duplicates, Se=0.96
1e−14
Probability of simultaneous error on all duplicates
Successful clinical validation - Mouawia et al, RBMO 2012
0.0
0.2
0.4
0.6
0.8
1.0
Correlation coefficient
SMA + CF: total (31+32) = 63 pregnant women
Consecutive cases
1191 microdissected cells: 475 CFTC
7 + 7 affected fetuses
as compared to CVS
Sensitivity: 100%
Specificity: 100%
Kinetics of Circulating Fetal Trophoblastic Cells in maternal blood
after In Vitro Fertilization (4th to 12th WG) (collab. Prof R Frydman):
CFTC start to circulate at the 5th WG
A
4,5
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
4
Cells/ml
Total
473 CFTC
CFTC/ml
3,5
3
2,5
2
1,5
1
0,5
0
5
6
7
8
9
10
11
12
Weeks of Gestation
B
3,5
3
CFTC/ml
Cells/ml
2,5
2
1,5
1
0,5
0
5
6
7
8
9
10
11
12
Weeks of Gestation
Mouawia et al, RBMO 2012
9
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Acknowledgements
INSERM
and Laboratoire de Biochimie A,
Hôpital Necker-Enfants Malades, Paris,
France, Université Paris Descartes,
Hussein Mouawia
Ali Saker
Ingrid Pfifer
Lucile Broncy
Patrizia Paterlini Bréchot
Department of Pharmaceutics
University of Ghent
Belgium
Philip Van Nieuwerburgh
Lieselot Deleye
Geneva - Switzerland
Dr Laurent Farinelli
Dr Magne Osteras
Service de Gynécologie-Obstétrique,
Hôpital Antoine Béclère, Clamart, France;
Alexandra Benachi
Laboratoire de Génétique Médicale, Hôpital
Necker-Enfants Malades, Paris, France;
Jean-Paul Bonnefont
Service de Gynécologie-Obstétrique,
Hôpital Foch, France;
René Frydman
Biostatistique, Université Paris Descartes,
Jean-Philippe Jais,
Unité de Recherche Clinique Paris Ouest, Hôpital
Ambroise-Paré AP-HP, Boulogne Billancourt
Laurence Bussières