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Validacija neinvazivnega presejalnega testa za odkrivanje Downovega sindroma iz polne krvi nosečnic
Redenšek, Sara (Author), Štrukelj, Borut (Mentor) More about this mentor... This link opens in a new window

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Abstract
Downov sindrom je ena izmed najpogostejših prirojenih genetskih motenj in je hkrati najpogostejši vzrok za mentalno zaostalost. Tveganje za Downov sindrom ugotavljamo z neinvazivnimi testi. Če je ugotovljeno tveganje visoko, izvedemo še invaziven test, s katerim potrdimo oz. ovržemo prisotnost Downovega sindroma. Invazivni testi lahko povzročijo različne zaplete, med drugim tudi splav. Slednje je že vrsto let gonilna sila za razvoj novih neinvazivnih testov, s katerimi bi se lahko dokopali do plodovega dednega materiala. Z našo raziskavo smo želeli validirati neinvaziven presejalni test za ugotavljanje prisotnosti oz. odsotnosti Downovega sindroma na podlagi vzorca periferne krvi nosečnice in ga preizkusiti na slovenski populaciji nosečnic. V krvi nosečnice se nahaja fetalna DNA, ki je na kromosomu 21 na določenih regijah hipermetilirana v primerjavi z materino. Z metodo imunoprecipitacije metilirane DNA smo fetalne tarčne regije ločili od materinih. Razliko v številu kopij tarčnih regij med trisomijo in normalnim vzorcem smo zaznali z verižno reakcijo s polimerazo v realnem času. Rezultate smo nato analizirali z določenim algoritmom.Testirali smo 26 vzorcev krvi, od katerih je bila pri enem z amniocentezo dokazana trisomija 21. V našem laboratoriju smo uspeli z nekoliko prilagojenim protokolom pravilno določiti trinajst vzorcev, med katerimi je bil tudi vzorec z Downovim sindromom. Tekom študije smo naleteli na nekaj težav z eno izmed tarčnih regij, zato smo se odločili, da te regije v izračunu ne bomo upoštevali, s čimer smo dobili precej bolj prepričljive in jasne rezultate. Menimo, da ima validirana metoda potencial, da bi se lahko nekoč uporabljala tudi v klinični praksi, vendar jo je potrebno testirati na večjem vzorcu nosečnic v standardiziranih pogojih. Test lahko izvedemo relativno zgodaj v nosečnosti, je enostaven, poceni in hiter.

Language:Slovenian
Keywords:neinvaziven presejalni test DNA različna metilacija imunoprecipitacija neinvazivna diagnostika validacija testov
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:FFA - Faculty of Pharmacy
Year:2015
Publisher:[S. Redenšek]
Number of pages:VI, 58 f.
UDC:616.899.6(079.1)(043.3)
COBISS.SI-ID:3920241 Link is opened in a new window
Views:86
Downloads:30
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Secondary language

Language:English
Title:Validation of a non-invasive screening test for diagnostics of Down syndrome from whole blood samples of pregnant women
Abstract:
Down syndrome is one of the most common congenital genetic disorders and is also the most common reason for mental retardation. Risk for Down syndrome is usually determined by noninvasive tests. If high risk is detected, then invasive tests are performed to confirm or deny the presence of Down syndrome. Invasive tests can cause different complications, including miscarriage. The latter is the main driving force for scientists attempting to find new noninvasive methods to get to fetal genetic material. The goal of our research was to validate a noninvasive screening test for determination of presence or absence of Down syndrome in a pregnant woman’s peripheral blood sample and test it on a small Slovenian population of pregnant women. Fetal DNA, which is also present in maternal blood, is differentally methylated in certain regions on the 21st chromosome. Fetal target regions are hypermethylated in comparison to corresponding maternal regions. We separated fetal target regions from maternal ones with the method of methylated DNA immunoprecipitation. The difference in copy numbers between Down syndrome and healthy cases was detected with real time polymerase chain reaction. The results were then analysed with a certain algorithm. We tested 26 blood samples, of which one had been confirmed positive for Down syndrome with amniocentesis. In our laboratory we managed to correctly classify 13 cases, the positive one as well, with few adjustions to the protocol. During the study we had some problems with a certain target region, which is why we decided to exclude it from the algorithm. After the exclusion more convincing and clearer results were obtained. We believe that the validated method has potential to be used in clinical practice, but still needs to be tested on a larger population under standardized conditions. It can be performed relatively early in pregnancy, is easy to do, cheap and fast.

Keywords:Down syndrome noninvasive screening test DNA differential methylation, immunoprecipititaion.

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