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
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11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
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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%)
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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
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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
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Source placenta (Flori et al, 2004)
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Increase in pregnancy-related pathologies (Lo et al, 1999)
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Very low concentration (Lo et al, 1998)
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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)
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Fetal fraction varies between pregnancies (Galbiati et al, 2005)
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Small, fragmented, not very good quality (<300 bp) (Chan et al, 2004)
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Differentially methylated (Chim et al, 2005)
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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
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SNP1
N/IVS1-110
SNP1
SNP2
SNP3
SNP4
(a/-)
c
c
g
t
c
g
110
(a/g)
ND
Normal
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Parents have the same mutation
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Mutation detection - one point analysis – negative detection - not reliable in
NIPD
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Use of SNPs and haplotype analysis – multiple point analysis - positive
detection of paternal alleles of the fetus
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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
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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
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Eleni Kalogirou
UNIVERSITY OF ATHENS, GREECE
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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