Screening of BRCA1/2 deep intronic regions by targeted gene sequencing identifies the first germline BRCA1 variant causing pseudoexon activation in a patient with breast/ovarian cancer

Abstract

BackgroundGenetic analysis of BRCA1 and BRCA2 for the diagnosis of hereditary breast and ovarian cancer (HBOC) is commonly restricted to coding regions and exon-intron boundaries. Although germline pathogenic variants in these regions explain about ~20% of HBOC cases, there is still an important fraction that remains undiagnosed. We have screened BRCA1/2 deep intronic regions to identify potential spliceogenic variants that could explain part of the missing HBOC susceptibility.MethodsWe analysed BRCA1/2 deep intronic regions by targeted gene sequencing in 192 high-risk HBOC families testing negative for BRCA1/2 during conventional analysis. Rare variants (MAF <0.005) predicted to create/activate splice sites were selected for further characterisation in patient RNA. The splicing outcome was analysed by RT-PCR and Sanger sequencing, and allelic imbalance was also determined when heterozygous exonic loci were present.ResultsA novel transcript was detected in BRCA1 c.4185+4105C>T variant carrier. This variant promotes the inclusion of a pseudoexon in mature mRNA, generating an aberrant transcript predicted to encode for a non-functional protein. Quantitative and allele-specific assays determined haploinsufficiency in the variant carrier, supporting a pathogenic effect for this variant. Genotyping of 1030 HBOC cases and 327 controls did not identify additional carriers in Spanish population.ConclusionScreening of BRCA1/2 intronic regions has identified the first BRCA1 deep intronic variant associated with HBOC by pseudoexon activation. Although the frequency of deleterious variants in these regions appears to be low, our study highlights the importance of studying non-coding regions and performing comprehensive RNA assays to complement genetic diagnosis.

Splicing profile by capture RNA-seq identifies pathogenic germline variants in tumor suppressor genes

“…9 Thus, a substantial proportion of patients currently receiving DNA testing are likely to benefit from the addition of RNA genetic testing. Several studies have also identified pathogenic deep-intronic variants across a range of hereditary cancer conditions, including hereditary breast and ovarian cancer (HBOC), 10,11 Lynch syndrome, 12,13 familial adenomatous polyposis, 14 neurofibromatosis, 15 and Li-Fraumeni syndrome. 16 However, the prevalence of cancer-predisposing deep-intronic variants has not been fully explored due to the limited scalability of previous RNA testing methods.…”

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

“…Overall, human BRCA2 variants in mESCs rendered similar mRNA transcript profiles as previously detected in LCLs and minigene analysis with all major aberrant splicing events identified. 12 , 28 – 31 The complementation phenotype of two variants, c.316+5G>C and c.7007G>A, resembled that of high-risk (class 4/5) variants with respect to their inability to rescue the cell lethality imposed by Cre-mediated loss of mBrca2 (Fig. S2 ).…”

“…Also, deep intronic variants can impose aberrant splicing as previously reported for c.6937+594T>G. 28 , 31 Due to the activation of a cryptic splice site an intronic fragment of 95 bases is inserted between exons 12 and 13 leading to an out-of-frame transcript. Molecular analysis revealed that although intron retention seems to be the predominant splicing event (Fig.…”

Functional and in-silico interrogation of rare genomic variants impacting RNA splicing for the diagnosis of genomic disorders

“…Whilst this represents a significant improvement to molecular diagnostic services, we expect that the true impact of such analysis strategies will be more pronounced. The targeted NGS approaches employed within this large cohort ignore deeply intronic regions of genes, and as shown here (Box 1, Case Example) and in other studies, [43][44][45][46][47] variation within these regions can cause aberrant splicing through the production of novel cryptic exons. We expect, therefore, that extending variant prioritization approaches to large cohorts of individuals with whole genome sequencing datasets will enable the identification of clinically relevant deeply intronic variants.…”