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Overview of Technical
Methods of Prevention
3rd Pan-European Conference on
Haemoglobinopathies & Rare Anaemias
Limassol, 24 – 26 October 2012
Marina Kleanthous
The Cyprus Institute of Neurology & Genetics
Overview
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PD and new approach to therapy
PGD
PGD and HLA (new therapeutic options)
NIPD
Prenatal Diagnosis by CVS or AF
Blood samples from
family members
CVS biopsy
Amniocentesis
Molecular analysis
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Healthy Fetus
Diagnosis
Affected
fetus
Pregnancy
termination
iPS cell therapy
Sick child
Cured Fetus
iPS cell for treatment in the
perinatal period
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Amniotic fluid cells – ideal human somatic cell
resource for patient-specific iPS cells
Reprogramming
β-globin gene corrected by gene therapy
Differentiation into haematopoietic cells
Return to the patient
Chang et al, PNAS:106, 2009
Fan Y. et al, J. Reproduction and Development: 58, 2012
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Preimplantation
Genetic Diagnosis
PGD
STAGES
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Counseling
Induction of ovulation
Oocyte collection
Fertilization by ICSI
Embryo biopsy
Genetic diagnosis
Implantation of 1-2 suitable embryos
Confirmation of pregnancy
Prenatal diagnosis (ESHRE guidelines)
(http://www.eshre.eu/)
Induction of ovulation and
oocyte retrieval
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In order to obtain a large
number of oocytes, the
patients undergo controlled
ovarian stimulation (COS),
with the use of follicle
stimulating hormone (FSH)
Ultrasound-guided transvaginal oocyte retrieval
Fertilization
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Intracytoplasmic
sperm injection (ICSI)
Pronuclear formation
(+2nd polar body)
Pronuclear fusion
Zygote
PGD approaches in
Embryo Biopsy
Polar body analysis
Cleavage stage biopsy
Blastocyst stage biopsy
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Second polar body extrusion and pronuclear formation
following ICSI in a zona-free human oocyte
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
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PCR-based PGD analysis
Blastomere
Biopsy
Freeze
(-200C >30 min)
Lab
Lysis / PK
digestion
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1st round PCR
(external primers)
Real Time PCR
(TaqMan Probes)
STR Fragment analysis
PGD for β-thalassaemia
One copy of each allele
Optimization of first PCR conditions
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Allele Drop Out (ADO) (<10%)
Amplification efficiency (>90%)
Contamination (No)
β-globin gene
Second PCR
Any mutation detection technique
Fragment analysis for STRs
Embryo Transfer
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Transfer 1-2 embryo in the uterus
Confirmation of pregnancy
Prenatal diagnosis (ESHRE guidelines)
Delivery
Cryopreservation of oocytes or embryo
PGD for β-thalassaemia in Cyprus
Results 2004-Oct 2012
143 PGD cases for β-thalassaemia
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1042 embryos tested
1-2 embryos transferred / case
Diagnosis confirmed with PND on 11th week of gestation (50%)
Six cases no embryos where transferred
38 pregnancies (26.5%)
30 successful pregnancies (8 lost)
22 single and 8 twin pregnancies
26 healthy deliveries (~19%)
3 ongoing pregnancies
34 healthy children born (24-26%)
PGD as a therapeutic approach
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Bone marrow transplantation (BMT) – Allogeneic (30%)
Graft rejection
Graft-versus-host disease
HSCT from HLA-identical siblings can cure about 90% of the cases
Reduced complications
Only 30% can find an HLA-identical related donor
PGD with HLA typing an attractive option for treatment
HLA: Human Leucocyte Antigens
HSCT: Haematopoietic Stem Cell Transplantation
PGD for β-thalassaemia and
HLA typing
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PGD for selecting and transferring unaffected embryos who
are HLA-identical with a sibling with a life threatening
disorder (3 out of 16)
At delivery HSC form newborn umbilical cord blood (UCB) are
collected
HSC are transplanted to the affected sibling
Haematopoietic reconstruction was successful in almost all
reported cases
Verlinsky et al, 2001
Velde et al, 2004
PGD for β-thalassaemia and
HLA typing
STRs selected to span the whole MHC region and closely
associated to class I, class II and class III HLA genes
In PGD the STRs and the β-globin gene are co-amplified by
PCR
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Our Approach
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First PCR
Co-amplification of 16 STRs and the β-globin gene
Second PCR
β-thal mutation detection by Real Time-PCR
Three multiplex reactions for nested or hemi-nested
PCR with florescence-labeled primers
Fragment analysis to elucidate haplotypes
Optimization and validation completed
Two families request PGD-HLA
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NON INVASIVE
PRENATAL
DIAGNOSIS
Circulating Nucleic Acids
First report 1948 (Mandel and Metais)
Studies on Circulatory DNA focused on
autoimmune diseases
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Diagnosis and prognosis of cancer 1977
Discovery of fetal DNA in maternal
plasma (Lo et al, 1997)
RNA (Poon et al, 2000)
Development of Non-invasive prenatal
diagnosis
NIPD applications
NIPD for disorders related to sequences
missing from the mother (Y
chromosome, RHD gene) – Clinical tests
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Aneuploidies offered as a clinical test by
Sequenom (Palomaki, 2011, 2012)
Single gene disorders (Thalassaemia,
cystic fibrosis) - Optimization
Pregnancy complications (Preeclampsia,
preterm pregnancies) – Very limited
progress
Properties of fetal DNA
Source placenta (Flori et al, 2004)
Increase in pregnancy-related pathologies (Lo et al, 1999)
Very low concentration (Lo et al, 1998)
Increase during gestation and disappears rapidly after delivery (Lo et al,
1999)
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cf fetal DNA ~10% of total DNA in the plasma (Lun, F.M., et al, 2008)
Fetal fraction varies between pregnancies (Galbiati et al, 2005)
Small, fragmented, not very good quality (<300 bp) (Chan et al, 2004)
Differentially methylated (Chim et al, 2005)
The entire fetal genome is represented in maternal plasma (Lo et al,
2010)
NIPD for single gene disorders
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Limitations
Quality of fetal DNA
The isolated DNA is mainly maternal
Maternal and fetal DNA very similar
Technologies/approaches
Sample enrichment (size fractionation by electrophoresis, micro fluidics)
Preferential amplification (PNA, PAP, Digital PCR)
Microarrays
MALDI-TOF MS
Next generation sequencing
Digital PCR
Cold PCR
Pyrophosphorolysis-activated polymerization (PAP) technique
β-Thalassaemia NIPD by cell free fetal DNA
N/IVS1-110
βthal
Normal
SNP2
SNP3
SNP4
110
SNP1
SNP2
SNP3
SNP4
110
a
c
g
t
a
a
c
c
g
a
ND
-
t
c
g
g
-
c
c
g
g
ND
Mat. plasma
34
THE CYPRUS INSTITUTE OF NEUROLOGY & GENETICS
SNP1
N/IVS1-110
SNP1
SNP2
SNP3
SNP4
(a/-)
c
c
g
t
c
g
110
(a/g)
ND
Normal
Parents have the same mutation
Mutation detection - one point analysis – negative detection - not reliable in
NIPD
Use of SNPs and haplotype analysis – multiple point analysis - positive
detection of paternal alleles of the fetus
Detection of paternally inherited fetal allele (minor allele)
NIPD for β-thalassaemia - progress
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Real Time PCR (Chiu et al, Lancet 2002)
MS (Ding et al, Proc Natl Acad Sci USA 2004 and Tsang et al, Clin. Cem.
2007)
APEX - SNPs (Papasavva et al, Ann N Y Acad Sci 2008 and Chan et al, Eur J
Gynecol Reprod Biol 2010)
Digital PCR (Lo et al Proc Natl Acad Sci USA 2007 and Lun et al, Proc Natl
Acad Sci USA 2008)
PNAs (Galbiati et al, Hematologica 2008)
NGS (Lo et al, Prenatal Diagnosis 2010)
Cold PCR (Galbiati et al, Clin Chem 2011)
Targeted massively parallel sequencing (Lam et al, Clin Chem 2012)
PAP technique (Phylipsen et al, Prenatal Diagnosis, 2012)
Targeted NGS (Papasavva et al, 2012)
49 SNPs on the β-globin gene locus
β-globin gene cluster (NG_000007, 81.7 Kb )
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SNPs with high heterozygosity in the Cyprus population (>6%)
80% of the families have at least 3 informative SNPs
Papasavva et al, Ann Hum Genet, 2012
NGS for the analysis of
cell free fetal DNA in mat plasma
Illumina Solexa Platform (HiSeq2000) in collaboration with Erasmus
Medical Centre (Rotterdam)
~1 million reads/sample (5% minor allele
50 000 molecules)
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Method
Primer design for 4 SNPs (150-250bp)
PCR Amplification (3 replicates)
PCR product purification
Cluster generation by bridge amplification
Sequencing by synthesis
Bioinformatics analysis
NGS-Illumina Results
Whole blood genomic DNA
SNP1
-/T
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SNP2
T/C
SNP3
G/C
SNP4
C/A
READ
R1
R2
R3
100% T/T
R4
R1
GCATTTAACATTTGCCTTAAAGGTGGTGACAGTTGA
2280896
1989313
GCATTTAACATTTGCCTTAAAGGTGGTGACAGT_GA
11
7
% of reference sequence
100.00
2075229
1714959
1734372
1992984
1816358
2200381
21
49
75563
84364
80019
111577
100.00
95.83
95.94
95.78
90% C/C + 10% T/C
GTGAATAAATGCATGACACATGCTTGCTGACTAATC
1972133
1582060
2502668
2118741
1615931
1551738
1452447
1659819
GTGAATAAATGCATGACACATGCTTGTTGACTAATC
1758
1226
2079
1573
104641
98015
96636
110047
% of reference sequence
99.91
99.92
99.92
100% G/G
99.93
93.92
CCATAGAAAAGAAGGGGAAAGAAAACATCAAGGGTC
1666708
2058647
2196255
2034668
1774046
1873629
1781723
1993217
CCATAGAAAAGAAGGGGAAAGAAAACATCAAGCGTC
207
76
106
99
114245
118662
116282
129296
% of reference sequence
99.99
100.00
93.95
TCCCATTCTAAACTGTACCCTGTTACTTCTCCCCTT
1738020
2099298
1819746
1757466
1416532
1358573
TCCCATTCTAAACTGTACCCTGTTACTTATCCCCTT
1154
1848
1478
1893
116472
115466
% of reference sequence
99.93
99.91
99.92
99.89
92.40
92.17
100.00
100.00
100% C/C
100.00
100.00
100% C/C
R2
R3
90% T/T + 10% -/T
94.06
93.76
90% G/G + 10% G/C
94.04
93.87
90% C/C + 10% A/C
R4
95.17
93.78
93.91
NGS-Illumina Results
Family No
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1
2
3
4
5
6
7
8
9
10
SNP1 (-/a)
Mat
CVS
plasma
-/a
a/a
a/a
a/a
-/a
-/a
a/a
-/a
NA
NA
-/-/a
a/a
a/a
-/a
-/a
-/a
-/a
a/a
a/a
1 false positive
2 false negatives
SNP2 (c/t)
Mat
CVS
plasma
c/c
c/c
c/t
c/c
c/t
c/t
c/t
c/t
c/t
c/t
c/t
c/t
c/t
c/c
c/t
c/t
c/c
c/t
c/c
c/c
2 false positives
1 false negative
SNP3 (c/g)
Mat
CVS
plasma
c/c
c/c
c/g
c/c
c/c
c/c
c/c
c/c
c/c
c/c
NA
NA
c/c
c/c
c/c
c/c
c/c
c/c
NA
NA
1 false positive
SNP4 (g/t)
Mat
CVS
plasma
NA
NA
g/g
NA
g/g
g/t
g/g
g/g
g/g
t/t
NA
NA
g/g
NA
g/g
g/t
g/g
g/g
g/g
t/t
NIPD with haplotype analysis
10 families tested
NIPD was successfully applied to 8 families
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4 families: all SNPs were correctly analyzed
4 families: 2/3 or 3/ 4 SNPs were correctly analyzed
NIPD was inconclusive for 2 families (more than 1 SNP was
incorrect)
Papasavva et al, submitted
Haplotype Analysis_NIPD Fam # 5
Family No
5
SNP1 (-/a)
Mat plasma
CVS
NA
SNP2 (c/t)
Mat plasma
CVS
NA
c/t
SNP3 (c/g)
Mat plasma
CVS
c/t
c/c
c/c
N/IVS1-6
SNP1
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βthal
Normal
t
g/g
N/IVS1-110
SNP2
c
SNP4 (g/t)
Mat plasma
CVS
SNP3
SNP4
SNP1
SNP2
SNP3
SNP4
g
t
c
c
g
ND
c
g
c
c
g
ND
SNP1
Mat plasma
NIPD: normal or β-thal trait
SNP2
SNP3
SNP4
c
c
g
t
c
g
ND
Normal
g/g
Conclusions
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CVS is the most common approach for PD
iPSC from AF and CVS is a possible curative treatment for
thalassaemia
PGD is an alternative option for PD
PGD-HLA a therapeutic approach
NIPD under development for thalassaemia
Use of new technologies
Limited progress in the last 10 years
No clinical test was developed for single gene disorders
The most promising technique for NIPD is NGS
Acknowledgements
THE CYPRUS INSTITUTE OF
NEUROLOGY & GENETICS, NICOSIA CYPRUS
Marina Kleanthous
Thessalia Papasavva
George Christopoulos
ERASMUS MEDICAL CENTRE, ROTTERDAM, NETHERLANDS
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Frank Grosvelt
Wilfred Van Ijcken
Christel Kockx
MAKARIOS HOSPITAL, CYPRUS
Eleni Kalogirou
UNIVERSITY OF ATHENS, GREECE
Emmanuel Kanavakis
Ariadni Mavrou
Joanne Traeger-Synodinos
The project is jointly funded by The Cyprus Institute of Neurology & Genetics and
The Erasmus Medical Centre, Cluster of Medical Genetics