TY - JOUR T1 - CRISPR–Cas9/long-read sequencing approach to identify cryptic mutations in <em>BRCA1</em> and other tumour suppressor genes JF - Journal of Medical Genetics JO - J Med Genet DO - 10.1136/jmedgenet-2020-107320 SP - jmedgenet-2020-107320 AU - Tom Walsh AU - Silvia Casadei AU - Katherine M Munson AU - Mary Eng AU - Jessica B Mandell AU - Suleyman Gulsuner AU - Mary-Claire King Y1 - 2020/10/15 UR - http://jmg.bmj.com/content/early/2020/10/15/jmedgenet-2020-107320.abstract N2 - Current clinical approaches for mutation discovery are based on short sequence reads (100–300 bp) of exons and flanking splice sites targeted by multigene panels or whole exomes. Short-read sequencing is highly accurate for detection of single nucleotide variants, small indels and simple copy number differences but is of limited use for identifying complex insertions and deletions and other structural rearrangements. We used CRISPR-Cas9 to excise complete BRCA1 and BRCA2 genomic regions from lymphoblast cells of patients with breast cancer, then sequenced these regions with long reads (&gt;10 000 bp) to fully characterise all non-coding regions for structural variation. In a family severely affected with early-onset bilateral breast cancer and with negative (normal) results by gene panel and exome sequencing, we identified an intronic SINE-VNTR-Alu retrotransposon insertion that led to the creation of a pseudoexon in the BRCA1 message and introduced a premature truncation. This combination of CRISPR–Cas9 excision and long-read sequencing reveals a class of complex, damaging and otherwise cryptic mutations that may be particularly frequent in tumour suppressor genes replete with intronic repeats. ER -