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.
Highlights
Genetic screening of the BRCA1 and BRCA2 genes is offered to families with high risk of breast and ovarian cancer
After the report of Kesari et al [9], who were able to detect skipping events on cDNA from the Duchenne muscular dystrophy (DMD) gene using the respective genomic multiplex ligation probe amplification (MLPA) kit, we sought to evaluate the use of a commercially available BRCA1 MLPA kit [10] for the detection of exon skipping in cDNA instead of genomic DNA
There are a few commercial RT-MLPA kits, these are designed to test the expression of genes associated with certain biological processes, MRC-Holland has not developed RT-MLPA kits to test splice events
Summary
Genetic screening of the BRCA1 and BRCA2 genes is offered to families with high risk of breast and ovarian cancer. Besides clear pathogenic mutations and polymorphisms, unclassified variants (UVs) of unclear clinical relevance are found. Some of these UVs may result in aberrant splicing, by affecting the donor or acceptor splice sites, or exonic splice site enhancer (ESE) sites [1] as predicted in silico. One example of a deep intronic pathogenic variant is the variant CDKN2A IVS2-105A[G, which causes retention of intronic sequence [2]. Another example is the mutation c.903?409T[C in the MTRR (methionine synthase reductase) gene, which activates a pseudoexon, causing a frameshift insertion that leads to a premature stop codon [3]. BRCA1 MLPA is a multiplex assay based on the hybridization of a large set of primers throughout the entire coding part of the BRCA1 gene
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