Detection of exon skipping events in BRCA1 RNA using MLPA kit P002

Abstract

A rapid and easy method to screen for aberrant cDNA would be a very useful diagnostic tool in genetics since a fraction of the DNA variants found affect RNA splicing. The currently used RT-PCR methods require new primer combinations to study each variant that might affect splicing. Since MLPA is routinely used to detect large genomic deletions and successfully detected exon skipping events in Duchenne muscular dystrophy in cDNA, we performed a pilot study to evaluate its value for BRCA1 cDNA. The effect of puromycin, DNase I and two different DNA cleaning protocols were tested in the RNA analysis of lymphocyte cultures. We used two samples from unrelated families with two different BRCA1 exon deletion events, two healthy unrelated controls and six samples from hereditary breast/ovarian cancer syndrome (HBOC) patients without BRCA1/2 mutations. Using RNA treated with DNase I and cleaned in a column system from puromycin-treated fractions, we were able to identify the two BRCA1 deletions. Additional HBOC patients did not show additional splice events. However, we were not able to get reproducible results. Therefore, the cDNA-MLPA technique using kit BRCA1 P002 is in our hands currently not reliable enough for routine RNA analysis and needs further optimization.

High Incidence of Somatic BAP1 Alterations in Sporadic Malignant Mesothelioma

“…Abnormal splicing forms were detected in one MM biopsy –in which both Sanger sequencing and MLPA had not found any alteration- by RNA sequencing. Introns contain splicing enhancers and silencers 34–36 , which may partially explain the presence of some aberrant splicing variants in these MM samples.…”

Frequency of BRCA1 Mutations in B&H Breast and Ovarian Cancer Patients

“…Early studies for identification of these mutations used protein truncation test (PTT), single strand conformation polymorphism (SSCP), southern blotting and long-range PCR, but technical difficulties, in most cases, have limited screening for large genomic rearrangements. Multiplex ligasedependent probe amplification (MLPA) for screening large genomic rearrangements of BRCA1 gene gave a new perspective on diagnosis, prognosis, therapeutical approaches and prevention of breast cancer (Schouten et al, 2002;Brandão et al, 2012;McVeigh et al, 2017;Meisel et al, 2017). MLPA is simple, robust and cost-efficient method for basic screening for BRCA1 mutations (Hogervorst et al, 2003;Cho et al, 2014;Maistro et al, 2016;Eid et al, 2017;Jakimovska et al, 2018).…”