Maternal serumdan doğrudan-PCR ile fetüsün cinsiyetinin belirlenmesi

Year 2024, Volume: 17 Issue: 2, 311 - 322

Uğur Akpulat Çiğdem Eresen Yazıcıoğlu

Abstract

Amaç: Fetüsün sağlık durumunu ciddi ölçüde etkileyebilecek anöploidiler ve cinsiyete bağlı geçiş gösteren genetik hastalıklar için, günümüzde maternal dolaşımdaki fetüse ait serbest DNA’nın incelenmesine dayanan analizler, girişimsel yöntemlere bağlı komplikasyonları en aza indirmek amacıyla yaygın olarak tercih edilmektedir. Bu analizlerin güvenirliğini etkileyen başlıca etken analiz edilebilir miktarda fetüse ait DNA’nın elde edilebilirliğidir. Maternal dolaşımdaki serbest DNA’nın izolasyonu sırasında kaynaktaki miktara göre her zaman bir kayıp yaşanmaktadır. Bu gibi durumlarda alternatif bir yaklaşım da kaynağın herhangi bir izolasyon işlemi yapılmadan kalıp olarak kullanıldığı doğrudan-PCR yöntemi ile analiz edilmesidir. Çalışmamızda PCR analizi ile fetüsün cinsiyetini belirlemede maternal serumun doğrudan kullanılabilirliği test edilmiştir. Yöntem: Fetüsün cinsiyetini belirlemek amacıyla Y kromozomu üzerindeki DYS14 ve SRY genlerine özgü PCR reaksiyonları tasarlanmıştır. Analiz edilmek üzere 40 gebe kadının serum örneği araştırmaya dahil edilmiştir. Serum örneklerinin Y kromozomu için tasarlanan reaksiyonlarında özgün amplifikasyon görülmesi durumunda fetüsün cinsiyeti erkek, görülmemesi durumunda dişi olarak kabul edilmiştir. Özgün amplifikasyonun görülmediği durumlarda DNA’nın varlığı GAPDH’e özgü PCR ile teyit edilmiştir. Bulgular: PCR analizi ile elde edilen sonuçlar ile klinik sonuçlar karşılaştırıldığında, doğrudan-PCR analizi ile fetal cinsiyeti belirleme duyarlılığının %87.5, özgünlüğünün %81.8 ve pozitif tahmin değerinin (PPV) %91.3 olduğunu ortaya koymuştur. Sonuç: Elde edilen sonuçlar, fetüsün genetik durumunu ortaya koymak amacıyla, gebe kadınların serum örneklerinin doğrudan moleküler analizlerde kullanılabileceğini göstermektedir.

Keywords

Serbest fetal DNA, maternal serum, doğrudan-PCR, DYS14, SRY

Ethical Statement

Başvurusu yapılan makale, “Maternal Periferik Kanda Fetal Genomik DNA’nın Belirlenmesi” başlıklı tez çalışmasından hazırlanmıştır. Tez çalışması, Dokuz Eylül Üniversitesi Sağlık Bilimleri Enstitüsü yönetim kurulu tarafından 22/08/2006 tarihinde 29 nolu toplantı sayısı ile kabul edilmiştir. İlgili tez, Yükseköğretim Kurulu Başkanlığı Ulusal Tez Merkezinde “193214” kayıt numarası ile bulunmaktadır.

Supporting Institution

Dokuz Eylül Üniversitesi Araştırma Fon Saymanlığı tarafından “2005.KB.SAG.004” proje numarası ile desteklenmiştir.

Project Number

2005.KB.SAG.004

Direct PCR-based determination of fetal gender from maternal serum

Abstract

Aim: Current analyses based on the examination of cell-free fetal DNA in maternal circulation are widely preferred to minimize complications associated with invasive methods, particularly for detecting aneuploidies and genetic disorders with sex-dependent inheritance that could significantly affect the fetal health. One of the primary factors influencing the reliability of these analyses is the availability of analyzable amounts of fetal DNA. During the isolation of cell-free DNA in maternal circulation, it is not always possible to isolate the entirety of the DNA present in the source material. In such cases, an alternative approach is to directly analyze the source material without any isolation process by using a direct PCR method. In our study, the feasibility of using maternal serum directly in PCR analysis for determining fetal sex was tested. Method: PCR reactions specific to the DYS14 and SRY genes on the Y chromosome were designed to determine the fetal sex. Serum samples from 40 pregnant women were included in the study for analysis. In the reactions designed for the Y chromosome, the sex of the fetus was assumed to be male if specific amplification was observed and female if not. In cases where specific amplification was not observed, the presence of DNA was confirmed with PCR specific to GAPDH. Results: When comparing the results obtained from PCR analysis with clinical outcomes, it was determined that the sensitivity, specificity, and positive predictive value (PPV) of determining fetal sex via direct-PCR analysis were 87.5%, 81.8%, and 91.3%, respectively. Conclusion: The obtained results demonstrate that serum samples from pregnant women can be directly utilized in molecular analyses to reveal the genetic status of the fetus.

Keywords

Cell-free fetal DNA, maternal serum, direct-PCR, DYS14, SRY

Project Number

2005.KB.SAG.004

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