Application of MALDI-TOF mass spectrometry in molecular diagnostics – MEN2 mutation detection as an example

Abstract

The diagnostic and clinical importance of gene variations and mutations is continuously increasing. In many genes single and/or multiple variants and mutations within the coding or regulating sequences are causative for different diseases. In most cases large genomic regions have to be tested resulting in a high number of sub-analysis, which are the main factors for cost-, time- and personal-intensive procedures. Therefore, fast and accurate detection as well as characterization of the relevant mutations is of diagnostic relevance. Simultaneous identification of already known and possible new mutations will be beneficial for diagnosis. Furthermore, it has to be ensured that known non-pathogenic polymorphisms do not interfere with the diagnostic result. Here we present the results of our development of a diagnostic application based on the MassCLEAVE™ method to detect MEN2 causing mutations in the RET proto-oncogene using chip-based MALDI-TOF mass spectrometry (MALDI-TOF MS). The MassCLEAVE™ assay relies on 4 independent base-specific cleavage reactions analyzed by MALDI-TOF MS. The relevant sequence portions are amplified by PCR. Products are simultaneously transcribed in-vitro into RNA and cleaved base specifically. Obtained fragmentation products are conditioned and analyzed by MALDI-TOF MS. Detected signal pattern will be indicative for presence or absence of mutations and/or variants as well as allow the determination of zygousity status. We investigated a set of 213 anonymized patient DNA samples and 148 control DNAs completed by 23 negative controls (no DNA). The 6 regions known to carry one or more of the common mutations are processed and analyzed as described above. The chosen setup resulted in about 12,800 and 22,400 analytical questions, when 40 common and 70 further 'possible' but rare mutations will be screened. We present our findings in comparison to results of double-strand fluorescent dideoxy-sequencing, which was applied as reference method.