Quantitative Analysis of BRCA1 and BRCA2 Germline Splicing Variants Using a Novel RNA-Massively Parallel Sequencing Assay

Frontiers in Oncology

Hsiao-Mei Lu¹  Dong Xu¹  Bing Li¹  Sitao Wu¹  Huy Vuong¹  Jayne Hoo²  Phillip Gray²  Stephanie Lam²  Aaron Elliott²  Brigette Tippin Davis²  Deborah Toppmeyer³  Sarah Nashed³  Rachid Karam⁴  Tyler Landrith⁴  Suzette Farber-Katz⁴  Ginger Haynes⁴  Vickie Hsuan⁴ 

[1] Department of Bioinformatics, Ambry Genetics, Aliso Viejo, CA, United States;Department of Research and Development, Ambry Genetics, Aliso Viejo, CA, United States;Rutgers Cancer Institute of New Jersey, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States;Translational Genomics Laboratory, Ambry Genetics, Aliso Viejo, CA, United States;

关键词: genetic testing;    hereditary breast and ovarian cancer;    HBOC;    BRCA1;    BRCA2;    RNA;   

DOI  :  10.3389/fonc.2018.00286

来源: DOAJ

【 摘 要 】

Clinical genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming widespread. However, the interpretation of variants of unknown significance (VUS) in HBOC genes, such as the clinically actionable genes BRCA1 and BRCA2, remain a challenge. Among the variants that are frequently classified as VUS are those with unclear effects on splicing. In order to address this issue we developed a high-throughput RNA-massively parallel sequencing assay—CloneSeq—capable to perform quantitative and qualitative analysis of transcripts in cell lines and HBOC patients. This assay is based on cloning of RT-PCR products followed by massive parallel sequencing of the cloned transcripts. To validate this assay we compared it to the RNA splicing assays recommended by members of the ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium. This comparison was performed using well-characterized lymphoblastoid cell lines (LCLs) generated from carriers of the BRCA1 or BRCA2 germline variants that have been previously described to be associated with splicing defects. CloneSeq was able to replicate the ENIGMA results, in addition to providing quantitative characterization of BRCA1 and BRCA2 germline splicing alterations in a high-throughput fashion. Furthermore, CloneSeq was used to analyze blood samples obtained from carriers of BRCA1 or BRCA2 germline sequence variants, including the novel uncharacterized alteration BRCA1 c.5152+5G>T, which was identified in a HBOC family. CloneSeq provided a high-resolution picture of all the transcripts induced by BRCA1 c.5152+5G>T, indicating it results in significant levels of exon skipping. This analysis proved to be important for the classification of BRCA1 c.5152+5G>T as a clinically actionable likely pathogenic variant. Reclassifications such as these are fundamental in order to offer preventive measures, targeted treatment, and pre-symptomatic screening to the correct individuals.

【 授权许可】

Unknown