Kvantitativna fluorescenčna verižna reakcija s polimerazo (QF-PCR) kot alternativni test za hitro prenatalno genetsko testiranje / Quantitative fluorescent polimerase chain reaction (QF-PCR) as an alternative test for rapid prenatal genetic testing

Alenka Erjavec Škerget, Špela Stangler Herodež, Boris Zagradišnik in Nadja Kokolj Vokač
 
Anali PAZU, 1 (2011), št. / No. 1, strani / pages 62-66
Celotno besedilo / Full text (PDF)
 
Povzetek: Kvantitativna verižna reakcija s polimerazo (QF-PCR) z uporabo markerjev specifičnih za kratka ponavljajoča se zaporedja (STR-short tandem repeat), značilna za določen kromosom, je uspešna metoda za hitro diagnosticiranje najpogostejših kromosomskih aneuploidij v prenatalnem obdobju. Namen našega prispevka je predstaviti omenjeno metodo in analizirati naše rezultate po treh letih uporabe QF-PCR kot hitrega testa pri iskanju najpogostejših prenatalnih kromosomskih sprememb:  trisomije kromosoma 21, 18, in 13. Genomsko DNA za QF-PCR analizo smo pripravili iz vzorcev amnijskih celic, horionskih resic ali placentarnih celic, ki smo jih pridobili od nosečnic s povišanim tveganjem za rojstvo otroka z katero od najpogostejših prenatalnih kromosomopatij. Za testiranje s QF-PCR metodo smo uporabili 20 različnih STR začetnih oligonukleotidov, specifičnih za kromosome 13, 18, 21, Y ter amelogeninski X/Y alel.  Pri 63.8% vzorcev je obenem bila opravljena še citogenetska analiza v našem laboratoriju zato imamo možnost primerjave obojih rezultatov. V triletnem obdobju smo opravili 243 analiz prenatalnih vzorcev: iz amnijskih celic 72.3%, horionskih resic 27.2%, v dveh primerih smo kot vzorec uporabili celice placente. Pri 7.8% vzorcev smo s QF-PCR našli kromosomsko anevploidijo, najpogostejša je bila trisomija kromosoma 21 (3.7%). Zaradi omejitve metode QF-PCR, je ostalo 4.4% kromosomskih sprememb nezaznanih, od le-teh je bila tretjina s slabo klinično prognozo. Senzitivnost metode QF-PCR v naši študiji je bila 95.4%, specifičnost 100% in učinkovitost 98.8%. Dosedanji rezultati kažejo, da je QF-PCR zanesljiva metoda, ki učinkovito pripomore k uspešnemu kliničnemu vodenju nosečnosti kot hitra metoda za zgodnje zaznavanje najpogostejših kromosomskih sprememb pri nosečnicah z visokim tveganjem za fetalno kromosomsko aneuploidijo. Kariotipizacija po invazivni prenatalni diagnostiki se v Sloveniji opravlja pri vseh vzorcih, v nekaterih evropskih državah pa že razmišljajo o uporabi hitrih presejalnih testov kot samostojnih testov pri interpretaciji rezultatov po invazivni prenatalni genetski diagnostiki v določenih primerih.
Ključne besede: kvantitativna fluorescenčna verižna reakcija s polimerazo; QF-PCR; invazivna prenatalna diagnostika; hitri prenatalni test; kromosomske anevploidije; prenatalno testiranje

Abstract: Diagnosis of common chromosome aneuploidies have been successful through quantitative fluorescent PCR (QF-PCR) assays and small tandem repeats (STR) markers. Our objective was to present and to analyze the results of the first three years of a QF-PCR testing strategy for the prenatal diagnosis of common chromosome aneuploidy (trisomy of chromosome 21, 18 and 13) and to discuss about the advantages and disadvantages of methodology. Amniotic fluid or chorionic villus samples were collected from mother undergoing prenatal invasive testing for fetal abnormalities on ultrasonic examination or abnormal maternal serum aneuploidy screening results. Rapid diagnoses were performed using QF-PCR analysis with several STRs markers specific for chromosomes13, 18, 21, Y and amelogenin X/Y alleles. One QF-PCR testing consisted of  six multiplexes reactions.  Of the 243 samples received (amniotic cells 72.3%, chorionic villi 27.2%, placentocentesis 0.5%) 7.8% had a chromosome abnormality detected by QF-PCR testing. All cases with numerical chromosome abberations involving chromosomes 21, 18, 13 were correctly diagnosed (100%). Of 66 samples 4.5% of samples received a normal QF-PCR result but subsequently had an abnormal karyotype because the present QF-PCR assay was not designed to detect them. Of these samples only 1/3 had a chromosome abnormality associated with a poor prognosis. Based on our result the sensitivity of the QF-PCR assay was 95.4%, the specificity 100% and the efficiency 98.8%, respectively. Our study demonstrates that QF-PCR is a reliable technique that aids clinical management of pregnancy as rapid method for the detection of common numerical chromosome disorders by woman at high risk for fetal aneuploidy. While karyotyping is still required for all samples in Slovenia, using QF-PCR as a stand-alone prenatal test for pregnancies without ultrasound abnormalities reduces costs, provides rapid delivery of results, and avoids ambiguous and uncertain karyotype results, reducing parental anxiety.
Key words: quantitative fluorescent polimerase chain reaction; QF-PCR; prenatal diagnosis; rapid aneuploidy detection; chromosomal aneuploidy; prenatal testing

Vsi prispevki avtorja: 
Alenka Erjavec Škerget
Špela Stangler Herodež
Boris Zagradišnik
Nadja Kokolj Vokač
Številka revije: 
ANALI PAZU 1/ 2011/ 1
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