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

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

References

1. Robson ME, Storm CD, Weitzel J, Wollins DS, Offit K. American Society of Clinical Oncology policy statement update: genetic and genomic testing for cancer susceptibility. J Clin Oncol. 2010;28(5):893-901.
2. Statement of the American Society of Human Genetics on genetic testing for breast and ovarian cancer predisposition. Am J Hum Genet. 1994;55(5):i-iv.
3. Hampel H, Bennett RL, Buchanan A, Pearlman R, Wiesner GL. A practice guideline from the American College of Medical Genetics and Genomics and the National Society of Genetic Counselors: referral indications for cancer predisposition assessment. Genet Med. 2015;17(1):70-87.
4. Nelson HD, Pappas M, Zakher B, Mitchell JP, Okinaka-Hu L, Fu R. Risk assessment, genetic counseling, and genetic testing for BRCA-related cancer in women: a systematic review to update the U.S. Preventive Services Task Force recommendation. Ann Intern Med. 2014;160(4):255-266.
5. Lancaster JM, Powell CB, Kauff ND, et al. Society of Gynecologic Oncologists Education Committee statement on risk assessment for inherited gynecologic cancer predispositions. Gynecol Oncol. 2007;107(2):159-162.
6. Balmaña J, Díez O, Rubio IT, Cardoso F. BRCA in breast cancer: ESMO Clinical Practice Guidelines. Ann Oncol. 2011;22(Suppl 6):31-34.
7. Petrucelli N, Daly MB, Pal T. BRCA1- and BRCA2-Associated Hereditary Breast and Ovarian Cancer. In: Adam MP, Ardinger HH, Pagon RA, et al., eds. GeneReviews®. Seattle (WA): University of Washington, Seattle;1998.
8. Tung NM, Garber JE. BRCA1/2 testing: therapeutic implications for breast cancer management. Br J Cancer. 2018;119(2):141–152.

9. Solmaz AE, Onay H, Yeniay L, et al. BRCA1-BRCA2 mutation analysis results in 910 individuals: Mutation distribution and 8 novel mutations. Cancer Genet. 2020;241:20-24.
10. Heather JM, Chain B. The sequence of sequencers: The history of sequencing DNA. Genomics. 2016;107(1):1–8.
11. Qin D. Next-generation sequencing and its clinical application. Cancer Biol Med. 2019;16(1):4–10.
12. Bosdet IE, Docking TR, Butterfield YS, et al. A clinically validated diagnostic second-generation sequencing assay for detection of hereditary BRCA1 and BRCA2 mutations. J Mol Diagn. 2013;15(6):796-809. doi: 10.1016/j.jmoldx.2013.07.004
13. Chong HK, Wang T, Lu HM, et al. The validation and clinical implementation of BRCAplus: a comprehensive high-risk breast cancer diagnostic assay. PLoS One. 2014;9(5):e97408.
14. Judkins T, Leclair B, Bowles K, et al. Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk. BMC Cancer. 2015;15:215.
15. Lincoln SE, Kobayashi Y, Anderson MJ, et al. A systematic comparison of traditional and multigene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients. The Journal of Molecular Diagnostics: JMD. 2015;17(5):533-544.
16. Strom CM, Rivera S, Elzinga C, et al. Development and validation of a next-generation sequencing assay for BRCA1 and BRCA2 variants for the clinical laboratory. PLoS One. 2015;10(8):e0136419.
17. Yoonjung Kim, Chi-Heum Cho, Jung-Sook Ha, et al. An optimized BRCA1/2 next-generation sequencing for different clinical sample types. J Gynecol Oncol. 2020;31(1):e9.
18. Chacon-cortes D, Griffiths L, Chacon-Cortes D. Methods for extracting genomic DNA from whole blood samples: current perspectives. Journal of Biorepository Science for Applied Medicine. 2014;2:1–9.
19. Montgomery J, Wittwer CT, Palais R, Zhou L. Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. Nat Protoc. 2007;2(1):59-66.
20. Wilson JR, Bateman AC, Hanson H, et al. A novel HER2-positive breast cancer phenotype arising from germline TP53 mutations. J Med Genet. 2010;47(11):771-774.
21. Tung N, Battelli C, Allen B, et al. Frequency of mutations in individuals with breast cancer referred for BRCA1 and BRCA2 testing using next-generation sequencing with a 25-gene panel. Cancer. 2015;121(1):25-33.
22. Hiraki S, Rinella ES, Schnabel F, Oratz R, Ostrer H. Cancer risk assessment using genetic panel testing: considerations for clinical application. J Genet Couns. 2014;23(4):604-617.
23. Kurian AW, Hare EE, Mills MA, et al. Clinical evaluation of a multiple-gene sequencing panel for hereditary cancer risk assessment. J Clin Oncol. 2014;32(19):2001-2009.
24. Nagahashi M, Shimada Y, Ichikawa H, et al. Next generation sequencing‐based gene panel tests for the management of solid tumors. Cancer Sci. 2019;110(1):6–15.
25. Chen M, Zhao H. Next-generation sequencing in liquid biopsy: cancer screening and early detection. Hum Genomics. 2019;13:34-43.
26. Catana A, Apostu AP, Antemie RG. Multi gene panel testing for hereditary breast cancer - is it ready to be used? Med Pharm Rep. 2019;92(3):220-225.
27. Ladd MK, Peshkin BN, Isaacs C, et al. Predictors of genetic testing uptake in newly diagnosed breast cancer patients. J Surg Oncol. 2020;122(2):134-143.
28. Kang HP, Maguire JR, Chu CS, et al. Design and validation of a next generation sequencing assay for hereditary BRCA1 and BRCA2 mutation testing. PeerJ. 2016;4:e2162.
29. Beck TF, Mullikin JC, Biesecker LG. Systematic evaluation of sanger validation of next-generation sequencing variants. Clinical Chemistry. 2016;62(4):647–654.
30. Rehm HL, Bale SJ, Bayrak-Toydemir P, et al. ACMG clinical laboratory standards for next-generation sequencing. Genet Med. 2013;15(9):733-747.