Hidden SVA retrotransposon insertion in BRCA1 revealed by nanopore targeted sequencing causes hereditary breast and ovarian cancer.

In Japan, germline BRCA1/2 genetic testing is extensively used for the diagnosis of hereditary breast and ovarian cancer syndrome (HBOC). However, inconclusive results sometimes complicate clinical management. In this study, we identified an intronic SINE-VNTR-Alu (SVA) insertion in BRCA1 of a proband and her mother, both of whom had inconclusive conventional BRCA1/2 genetic test results, by targeted long-read sequencing (LRS) through the application of nanopore adaptive sampling and Flongle genome amplicon sequencing. We further confirmed splicing aberrations using cDNA quantitative PCR with TaqMan probes and Flongle cDNA amplicon sequencing. Our findings highlighted that, in addition to conventional BRCA1/2 genetic testing, structural variation analysis using targeted LRS is indispensable for the accurate diagnosis of HBOC in certain cases. Furthermore, Flongle amplicon sequencing was demonstrated to be effective for sequencing regions refractory to conventional PCR and Sanger sequencing, particularly repetitive and GC-rich regions, such as retrotransposons.