svetlana madjunkova

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


Invasive procedures to obtain fetal material:

Chorionic villus sampling (10-14 GW)

Amniocentesis (after 15GW)

1% risk for miscarriage


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


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


1:2

1:1:1

2:1 1:1:1


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)


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


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)


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)


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)


%

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


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.


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


svetlana madjunkova

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