Robust, reliable, and reproducible endpoints are essential for decision making in early stage drug discovery and development programs. Given the high level of understanding of molecular events in the pathogenesis of DM, assays that focus on careful quantification of disease-causing molecules are essential for go/no-go decision making in drug screening programs. Thus far, methodology for reliable quantification of mutant RNA copy number, although a critically important tool for drug developers, has been a question mark. A recent study utilized a battery of techniques (Northern blotting, reverse transcriptase-quantitative polymerase chain reaction, RNA-sequencing and fluorescent in situ hybridization analyses) to demonstrate a very low abundance—one to a few dozen molecules per cell—Dmpk/DMPK mRNA in mouse model and patient samples (Gudde et al., 2016).
Dr. David Brook (University of Nottingham) and colleagues have reported out on studies of methodology to precisely quantify (a) the absolute numbers of mutant RNA transcripts and (b) alternative splicing in DM1 and DM1 patient cell samples (Wojciechowska et al., 2018). In these studies, they evaluated the potential value of medium throughput technologies—Multiplex Ligation-Dependent Probe Amplification (MLPA) and droplet digital PCR (ddPCR)—that could be feasibly applied in drug development programs.
To quantitatively assess alternative splicing, the research team designed assay methodology that would evaluate eight key splicing events in DM1 and DM2 patient skeletal muscle biopsy samples. MLPA used two probes for each exon (exon-On and exon-Off). Likewise, ddPCR utilized dual TaqMan probes for each splicing event, specific to exon-On and exon-Off. Transcripts evaluated met specific selection criteria and represented known changes at different disease stages. Both MLPA and ddPCR met rigorous criteria for splicing quantification (sensitivity, reliability) and results showed strong correlation across the two methods.
To achieve absolution quantification of DMPK mRNA copy number, the group optimized ddPRC assays and used them to characterize DM1 skeletal muscle biopsies and cultured fibroblasts. Mutant and wild type DMPK transcripts were differentiated on the basis of the rs527221 G>C SNP in exon 10. Results suggested that highly sensitive, reproducible, and accurate quantification was achievable even with the low mutant mRNA copy number (15-20 molecules/cell) that characterize DM.
In addition to assessing the appropriateness of MLPA and ddPCR as biomarker assays for myotonic dystrophy, the research team demonstrated that the absolute abundance of mutant DMPK transcripts in the nuclear foci of DM1 patient fibroblasts was very low. Thus, sensitive and reliable assays that can avoid floor effects are important for the field.
Finally in situ fluorescence hybridization was used to estimate mutant DMPK mRNA in individual nuclear foci. The median number of mutant CUG transcripts per nuclear foci was between two and four, with between four and six total foci detected per DM1 patient fibroblast.
Taken together, both MLPA and ddPCR technologieswere shown to produce highly reproducible and simultaneous assessment of genes aberrantly spliced in DM, and ddPCR was an effective measure of absolute mutant DMPK transcript copy numbers per cell. Validation of this methodology addresses an important gap in tools available for the therapy development pipeline for DM.
A low absolute number of expanded transcripts is involved in myotonic dystrophy type 1 manifestation in muscle.
Gudde AE, González-Barriga A, van den Broek WJ, Wieringa B, Wansink DG.
Hum Mol Genet. 2016 Apr 15;25(8):1648-62. doi: 10.1093/hmg/ddw042. Epub 2016 Feb 16.
Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy.
Wojciechowska M, Sobczak K, Kozlowski P, Sedehizadeh S, Wojtkowiak-Szlachcic A, Czubak K, Markus R, Lusakowska A, Kaminska A, Brook JD.
Sci Rep. 2018 Apr 12;8(1):5885. doi: 10.1038/s41598-018-24156-x.