Development of Next Generation Sequencing Based Kit for the Detection of BRCA1 and BRCA2 Mutations, and Validation With Routinely Used Methods

Year 2021, Volume , Issue 13, 1 - 15, 29.04.2021

Gözde GİRGİN ÖZGÜMÜŞ İlter GÜNEY

https://doi.org/10.38079/igusabder.843199

Abstract

Aim: Breast cancer is the most common type of cancer in women, and it is well known that Breast Cancer Susceptibility gene (BRCA1 and BRCA2) mutations are responsible for a substantial portion of the breast and ovarian cancers. Individuals carrying a mutation in one of these genes have an increased substantially lifelong risk of breast, ovarian, pancreatic, and other types of cancers. Identifying individuals with BRCA1/2 gene mutations is vital for increasing screening of family members via genetic counselling, and using potentially life-saving preventive measures. The aim of this study is preparation of next generation sequencing (NGS) libraries of BRCA1/2 genes using real time polymerase chain reaction (RT-PCR), and determination of bioinformatic workflow suitable for NGS analysis.

Methods: Using blood samples previously analyzed with Multiplicom BRCA MASTR™ Dx kit that is widely utilized in routine practice; DNA isolation was consequently followed by preparation of NGS libraries with RT-PCR, marking each sample with 2 different tag sequences, and determination of bioinformatic workflow for NGS analysis.

Results: Compared with the reference method, test limits were: 100% sensitivity, 100% specificity, and 100% validity. The confidence interval was calculated to be between 86 and 100% using the Wilson method.

Conclusion: This study resulted in the successful development and analytic validation of a low-cost NGS test kit capable of accurately determining pathogenic mutations of BRCA1 and BRCA2 genes.

Keywords

Breast Cancer, BRCA1, BRCA2, Next Generation Sequencing, Library Preparation for NGS

BRCA1 ve BRCA2 Mutasyonlarının Tespitine Yönelik Yeni Nesil Dizileme Temelli Kit Geliştirilmesi ve Rutinde Kullanılan Yöntemler ile Valide Edilmesi

Abstract

Amaç: Meme kanseri, kadınlarda en yaygın görülen kanser türü olup, Göğüs Kanseri Duyarlılık gen (BRCA1 ve BRCA2) mutasyonlarının meme ve yumurtalık kanserlerinin önemli bir kısmından sorumlu olduğu bilinmektedir. Bu genlerden birinde mutasyon taşıyan bireylerde yaşam boyu meme, yumurtalık, pankreas ve diğer kanserlere yakalanma riski oldukça yükselmektedir. BRCA1/2 gen mutasyonlarına sahip olan kişilerin belirlenmesi, genetik danışma ile tarama sıklığının artırılması ve potansiyel olarak hayat kurtaran önleyici tedavi stratejilerinin uygulanabilmesi için büyük önem taşımaktadır. Bu çalışmada gerçek zamanlı polimeraz zincir reaksiyonu (RT-PZR) yöntemi kullanılarak BRCA1/2 genlerinin yeni nesil dizi (NGS) analizi kütüphanelerinin hazırlanması ve NGS analizlerine uygun biyoinformatik iş akışının belirlenmesi amaçlanmıştır.

Yöntem: Rutin analizlerde yaygın olarak kullanılan Multiplicom BRCA MASTR™ Dx Kiti ile çalışılmış hastalardan alınan kan örneklerinden, DNA izolasyonu sonrası RT-PZR ile NGS kütüphanelerinin hazırlanması ve her bir örneğin 2 farklı etiket dizi ile işaretlenmesinin ardından NGS analizlerinin biyoinformatik iş akışlarının belirlenmesi gerçekleştirilmiştir.

Bulgular: Referans metoda göre test limitleri; %100 duyarlılık, %100 özgüllük ve %100 doğruluk olarak belirlenmiştir. Wilson yöntemi kullanılarak testin güven aralığı CI: %86-%100 olarak hesaplanmıştır.

Sonuç: BRCA1 ve BRCA2 genlerinin klinik laboratuvarlar değerlendirmesine uygun verilerin elde edildiği, patojenik mutasyon tespitini yüksek verimlilik ve doğrulukla yapabilen uygun maliyetli bir NGS testinin geliştirilmesi ve analitik doğrulaması bu çalışma ile tamamlanmıştır.

Keywords

Meme Kanseri, BRCA1, BRCA2, Yeni Nesil Dizileme, NGS Kütüphane Oluşturması

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