Development of arrayed primer extension microarray assays for molecular diagnostic applications
Date
2013-12-11
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Abstract
Inimese pärilike haiguste põhjuseid on uuritud juba mitmeid aastakümneid ja nende haiguste tuvastamiseks on kasutusel küllaltki muljetavaldav hulk erinevaid molekulaardiagnostilisi teste. Paljudel juhtudel võib aga olemasolevate testide edukat rakendamist rutiinses kliinilises praktikas takistada näiteks liiga suur hulk testimist vajavaid DNA muutusi haiguse kohta, haruldaste haiguste korral liiga väike analüüside arv teatud perioodi vältel, analüüsiks kuluv aeg või testi juurutamise ja läbiviimise kõrge hind. Seega sõltub pärilike haiguste molekulaarse diagnostika areng ja kättesaadavus uutest meetoditest, mis võimaldaksid lahendada nimetatud kitsaskohti.
Käesoleva töö eesmärgiks oli väljatöötada ja hinnata DNA kiibitehnoloogial põhineva APEX (Arrayed Primer Extension) metoodika sobivust haruldaste geneetiliste haiguste molekulaardiagnostikaks ja mutatsioonikandluse määramiseks ning APEX-2 metoodika sobivust Downi sündroomi tuvastamiseks. Antud töös keskenduti nelja uue testi väljatöötamisele. Tsüstilise fibroosi (CF) test võimaldab võrreldes teiste rutiinses kliinilises diagnostikas kasutusel olevate samaotstarbeliste paneelidega kuluefektiivsemalt määrata üle kahe korra rohkem CF seoselisi mutatsioone. Esindatud on sagedasemad CF mutatsioonid, mis on kirjeldatud nii kaukasoididel kui ka mitte-kaukasoidsetes populatsioonides ja segunenud etnilise taustaga indiviididel. Mittesündroomse sensorineuraalse kuulmislanguse (SNHL) testi abil saab kuulmislanguse geneetilisi põhjuseid tuvastada nii vastsündinute kuulmise sõeluuringu järelkontrollis kui ka kuulmispuudega täiskasvanutel ning seeläbi parandada nii patsientide ravi kui nende perekondade geneetilist nõustamist. BBS-ALMS test võimaldab piisavalt tõhusa ja kuluefektiivse esmase diagnostilise testina teostada mutatsioonianalüüsi Bardet-Biedl (BBS) ja Alströmi sündroomiga (ALMS) patsientidel. Uue Downi sündroomi testi kasutamine geneetilise nõustamise skeemis võimaldaks kiiret (kuni 30 tundi) loote eeluuringut Downi sündroomi suhtes, mis võrreldes standardse karüotüpiseerimise ajakuluga (2-3 nädalat) lühendaks oluliselt vanemate pingerohket vastuse ootamise aega.
Genetic causes of inherited diseases have been extensively studied over the past several decades and a wide variety of molecular tests have been developed for diagnosing genetic disorders. However, the successful introduction of currently available tests into routine clinical practice is limited due to different reasons, for example a large number of DNA variations per disease, a low number of samples per rare disease per laboratory, turnaround time, high cost of setup and/or maintenance. Thus, the advances in molecular diagnostic medicine depend on the development and availability of new methods with the potential to solve above-mentioned shortcomings. The aims of this thesis were to develop and evaluate the suitability of APEX (Arrayed Primer Extension) and APEX-2 microarray-based assays for the diagnosis and carrier screening of rare genetic diseases and for rapid molecular diagnosis of Down syndrome in clinical practice. The main advantages of the APEX/APEX-2 assay include its flexibility and single-step hybridization of the template, followed by primer extension, which make the assay relatively fast, simple, and robust with fewer components, pipetting steps, and manipulations in the workflow. The main outcomes of the current thesis include: i) a novel cost-effective cystic fibrosis (CF) test with a higher mutation detection capability for CF diagnosis and carrier screening in Caucasians, non-Caucasians, and individuals of mixed ethnicity, compared to assays currently available in diagnostic laboratories; ii) a novel diagnostic APEX microarray for simultaneous multigene mutation detection in nonsyndromic sensorineural hearing loss (SNHL) patients. The SNHL test allows the detection of genetic causes of deafness in children (and adults), improving medical management for patients and genetic counseling for families; iii) a novel BBS-ALMS test which is an efficient and cost-effective first-pass diagnostic screening tool for molecular analysis of Bardet-Biedl (BBS) and Alström syndrome (ALMS) patients, and iv) a novel T21 APEX-2 assay that could be used as a rapid pre-test to reduce parental anxiety during genetic counseling before final karyotyping.
Genetic causes of inherited diseases have been extensively studied over the past several decades and a wide variety of molecular tests have been developed for diagnosing genetic disorders. However, the successful introduction of currently available tests into routine clinical practice is limited due to different reasons, for example a large number of DNA variations per disease, a low number of samples per rare disease per laboratory, turnaround time, high cost of setup and/or maintenance. Thus, the advances in molecular diagnostic medicine depend on the development and availability of new methods with the potential to solve above-mentioned shortcomings. The aims of this thesis were to develop and evaluate the suitability of APEX (Arrayed Primer Extension) and APEX-2 microarray-based assays for the diagnosis and carrier screening of rare genetic diseases and for rapid molecular diagnosis of Down syndrome in clinical practice. The main advantages of the APEX/APEX-2 assay include its flexibility and single-step hybridization of the template, followed by primer extension, which make the assay relatively fast, simple, and robust with fewer components, pipetting steps, and manipulations in the workflow. The main outcomes of the current thesis include: i) a novel cost-effective cystic fibrosis (CF) test with a higher mutation detection capability for CF diagnosis and carrier screening in Caucasians, non-Caucasians, and individuals of mixed ethnicity, compared to assays currently available in diagnostic laboratories; ii) a novel diagnostic APEX microarray for simultaneous multigene mutation detection in nonsyndromic sensorineural hearing loss (SNHL) patients. The SNHL test allows the detection of genetic causes of deafness in children (and adults), improving medical management for patients and genetic counseling for families; iii) a novel BBS-ALMS test which is an efficient and cost-effective first-pass diagnostic screening tool for molecular analysis of Bardet-Biedl (BBS) and Alström syndrome (ALMS) patients, and iv) a novel T21 APEX-2 assay that could be used as a rapid pre-test to reduce parental anxiety during genetic counseling before final karyotyping.
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Keywords
pärilikud haigused, molekulaardiagnostika, DNA mikrokiibid, Downi sündroom, kuulmispuuded, hereditary diseases, molecular diagnostics, DNA chips, Down syndrome, hearing deficiancy