Svetlana Madjunkova RCGEB “Georgi D Efremov ”, MASA MACPROGEN Final Conference Ohrid 29 March – 1 April 2012
Svetlana Madjunkova RCGEB “Georgi D Efremov”, MASA
MACPROGEN Final Conference Ohrid 29 March – 1 April 2012
Provide information to prospective parents regarding fetal diagnosis
Counseling and support for prospective parents for reproductive decisions
Fetal therapy and prevention of postnatal medical complications
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
Prevention – the only option for pregnancies affected by genetic conditions for which there is no cure (therapeutic abortion)
Chromosomal abnormalities (numerical, structural) (1/160 live births)
Monogenetic disorders (2% live births)
◦ deep personal problems (physical/psychological)
◦ high managing expenses
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
Invasive procedures to obtain fetal material:
Chorionic villus sampling (10-14 GW)
Amniocentesis (after 15GW)
1% risk for miscarriage
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
Rapid detection of common chromosomal aneuploidies
Molecular diagnosis of monogenetic disorders
◦ Multiplex quantitative fluorescent (QF)-PCR
◦ Multiplex ligation probe amplification (MLPA) (MRC Holland)
All subtelomeric regions
Common microdeletion/duplication syndromes
◦ Array comparative genome hybridization (aCGH) 180K Human SurePrint (Agilent)
◦ β-Thalassaemia ◦ Cystic fibrosis ◦ Spinal muscular atrophy ◦ Duchenne/Becker muscular
dystrophy ◦ Friedriech’s ataxia ◦ Hemophilia A ◦ Lech Nichan syndrome ◦ Rett syndrome ◦ Phenylketonuria ◦ Galactosemia ◦ Pseudohipoaldosteronizam
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
One tube multiplex QF-PCR testing for most common aneuploidies
22 highly polymorphic STRs on chromosomes 13, 18, 21, X and Y
Chromosome dosage: STR relative peak area ratio (capillary electrophoresis)
Chr 13 Chr 18 Chr 21 X Y
STR marker
D13S631 D18S535 D21S1414 AMEL X/Y* AMEL X/Y*
D13S305 D18S391 D21S1446 DXYS218* DXYS218*
D13S258 D18S386 D21S1411 TAF9B** MYPT2Y***
D13S1817 D18S390 D21S1435 XHPRT SRY
DXS6803 DYS448
*Locus on chr X and Y, **Locus on chr X and chr 3, *** Locus on chr Y and chr 2
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
1:2
1:1:1
2:1 1:1:1
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
94,03 %
3,77 %
3,775 %
Normal
Maternal contamination
Trisomy 21 (n=26)
Trisomy 18 (n=18)
Trisomy 13 (n=5)
45,X0 (n=5)
47, XXY (n=4)
Triploidy (n=2)
47, XYY (n=1)
47, XXX (n=1)
Trisomy 21+ 47,XXY (n=1)
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
2200 prenatal samples
No discordant results with cytogenetic analysis
Polymorphic duplications of STRs: D13S631, D21S1441, D18S978 and D18S535
0
20
40
60
80
100
Trisomy 21
(n=39)Trisomy 18
(n=13)Trisomy 13
(n=3)45,X0
(n=5)47,XXY
(n=4)
%
Maternal
Paternal
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
Fetal material in maternal plasma ◦ Cell free fetal (cff) DNA ◦ Cell free fetal (cff) RNA ◦ Fetal proteins (placental) ◦ Fetal cells (erythroblasts)
Challenge-low amount (6-12% cffDNA) of fetal material in vast background of maternal one
New approaches in screening (new protein biomarkers) and
PD (new diagnostic tests)
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
Noninvasive determination of paternally inherited traits ◦ fetal sex: Y chromosome specific loci (Davalieva et al. J Matern
Fetal Neonatal Med 2006)
X-linked disorders (Duchenne/Becker muscular dystrophy, Hemophilia А, X-linked mental retardation, etc)
◦ fetal RhD status in RhD (-) mothers
RHD feto-maternal incompatibility
Targeted immunoprophilaxis
◦ fetal specific mutant alleles
monogenic diseases (β-thalassemia, cystic fibrosis)
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
Pregnancy n GW AV±STDEV I
trimester 5 5-12 7±2
II trimester 19 15-25 20,375±2,6
III trimester 48 31-42 39,24±1,84
0
10
20
30
40
50
60
70
Positive Negative
33,8
16,9
32,4
16,9
fetal sex Male
fetal sex Female
Real time PCR using DYS14 –multi copy marker for fetal sex determination Real time PCR using RHD gene ex5 and ex7 probes for determination of fetal RHD status in RhD negative pregnant women
Fetal RHD staus
Study performed in Prof. Neil Avent laboratory, School of Biological and Biomedical Sciences, Plymouth University, UK
100% correct fetal sex determination 100% concordance with results from fetal material (CVS/amniocentesis)
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
%
Translation of non-invasive based approach for PD into clinical practise ◦ fetal sex determination using cffDNA in pregnancies at risk for
X-linked disorders
◦ fetal RHD status determination and targeted anti-RhD prophylaxis
◦ Determination of paternal mutant alleles in pregnancies at risk for monogenetic diseases
Non-invasive approach for PD of trisomy 21
New protein biomarker discovery for chromosomal aneuploidies
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
RCGEB “Georgi D. Efremov” Project collaborators
Prof. Dr. Dijana Plaseska-Karanfilska
Dr. Svetlana Kocheva, PhD
Ivana Maleva, Msc
Sanja Kiprijanovska,
Predrag Noveski, Msc
Marija Mirchevska, Msc
Clinic for gynecology and obstetrics, Medical Faculty, University “Stt. Cyril and Methodius”, Macedonia Dr. Mitko Madjunkov, Dr. Sasa Jovcevski Obstetrics department staff School of Biological and Biomedical Sciences, Plymouth University, UK Prof. Dr. Neil Avent Dr. Tracey Madgett PHO “Sistina”- Skopje, Macedonia Dr. Pavle Dimchev and al. PHO “Dr.Ina”-Skopje, Macedonia Dr. Katerina Stankova PHO “Re-Medika”- Macedonia Dr. Elena Patrova and al.
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012
Kariotip FISH QF-PCR Aneuploidii da da da Poliploidii da da triploidii
Balans. hr. rearan`. da da ne Nebalans. hr. rearan`. Visoka rezol. da da Mozaicizam da da nad 15%
Uniparentna disomija ne ne da Kontaminacija od majka ma{ki ma{ki da* Poteklo od roditel ne ne da Mejotsko poteklo ne ne da Potrebno vreme 2-3 nedeli 4-5 dena 1-2 dena Cena na ~inewe visoka visoka niska
MACPROGEN Final Conference, Ohrid 29 March - 1 April 2012