Targeted RNA‐seq successfully identifies normal and pathogenic splicing events in breast/ovarian cancer susceptibility and Lynch syndrome genes

Abstract

A subset of genetic variants found through screening of patients with hereditary breast and ovarian cancer syndrome (HBOC) and Lynch syndrome impact RNA splicing. Through target enrichment of the transcriptome, it is possible to perform deep‐sequencing and to identify the different and even rare mRNA isoforms. A targeted RNA‐seq approach was used to analyse the naturally‐occurring splicing events for a panel of 8 breast and/or ovarian cancer susceptibility genes (BRCA1, BRCA2, RAD51C, RAD51D, PTEN, STK11, CDH1, TP53), 3 Lynch syndrome genes (MLH1, MSH2, MSH6) and the fanconi anaemia SLX4 gene, in which monoallelic mutations were found in non‐BRCA families. For BRCA1, BRCA2, RAD51C and RAD51D the results were validated by capillary electrophoresis and were compared to a non‐targeted RNA‐seq approach. We also compared splicing events from lymphoblastoid cell‐lines with those from breast and ovarian fimbriae tissues. The potential of targeted RNA‐seq to detect pathogenic changes in RNA‐splicing was validated by the inclusion of samples with previously well characterized BRCA1/2 genetic variants. In our study, we update the catalogue of normal splicing events for BRCA1/2, provide an extensive catalogue of normal RAD51C and RAD51D alternative splicing, and list splicing events found for eight other genes. Additionally, we show that our approach allowed the identification of aberrant splicing events due to the presence of BRCA1/2 genetic variants and distinguished between complete and partial splicing events. In conclusion, targeted‐RNA‐seq can be very useful to classify variants based on their putative pathogenic impact on splicing.

Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene

“…Fluorescent capillary electrophoresis of the RT-PCR products also offered high resolution and sensitivity, being capable of distinguishing isoforms that differ only in a few nucleotides [53], such as the full-length and (E8p3)-a,b,c transcripts that just contain a 3-nt insertion. Interestingly, 12 transcripts ( (E2q27), ∆(E2q175), ∆(E2q22), ∆(E2), ∆(E3), ∆(E4), ∆(E4_5), ∆(E5), ∆(E7), ∆(E7_8), ∆(E8), and (E8p3)) had been previously characterized as naturally occurring isoforms of RAD51C [54], suggesting that physiological alternative events may somehow predict variant splicing profiles [55][56][57]. Moreover, minigene assays are capable of mimicking pathological patterns of variants.…”

Alternative mRNA splicing can attenuate the pathogenicity of presumed loss-of-function variants in BRCA2

“… 11 Up to this date, no tissue-specific transcripts have been observed in nonmalignant breast epithelia, ovarian epithelia, or ovarian fimbria. 11 , 15

Fig. 1 Expression of the major naturally occurring alternative BRCA2 transcripts relative to full-length transcript.

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“…11 Up to this date, no tissue-specific transcripts have been observed in nonmalignant breast epithelia, ovarian epithelia, or ovarian fimbria. 11,15 Functional characterization of exon deletions in BRCA2…”

“…For many BRCA1 and BRCA2 variants (both intronic and exonic) an effect on mRNA splicing has been reported using either patient RNA or minigene analysis. [12][13][14][15][16][17][18][19][20][21] The analysis of patient RNA is however often hampered by the inability to determine allele-specific transcript expression. It then remains unclear if and to what extent wild type (WT) mRNA is still produced from the variant allele.…”

A six-mRNA signature-based model for the prognosis prediction of breast cancer

“…Therefore, identifying new biomarkers to predict the prognosis of BC patients is helpful for the customization of personalized treatment. High-throughput RNA-seq contributes to exploring various biomarkers for diagnosis and prognosis prediction of many cancers, including BC, along with the development of technology [20][21][22][23] .…”