Elucidating the Paternal Age Effect with BEA: a method to count rare mutations
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7th ÖGMBT Annual Meeting ?Salzburg goes Science?
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Observations have been made that also the probability of having an affected offspring increases exponentially as a function of the father?s age, a phenomenon known as the paternal age effect (PAE). Two well-known examples involve specific activating point mutations in the fibroblast growth factor receptor 3 (FGFR3), achondroplasia (ACH) and thanatophoric dysplasia type II (TDII), with each mutation presenting a different degree of signaling activation. Thus, comparing the distribution of these two mutations in the male germline could provide important insights about the mutation mechanisms driving the PAE in FGFR3. For this purpose we have developed an in-house method that can count simultaneously these two rare mutations (ACH & TDII) in DNA of sperm from different aged donors. Our method is based on the amplification of millions of single molecules in parallel on microscopic magnetic beads contained in PCR compartments of an emulsion. Both the ACH and TDII loci are analyzed in a multiplex reaction, where the clonal amplification products derived from an initial single molecule are captured on the beads, which are then subsequently analyzed with mutant- or wild type specific fluorescent probes on an array. Our results show that this method renders ~150,000 distinct beads each for the ACH or the TDII fragment. In sperm DNA, we measured mutation frequencies for ACH and TDII within the expected frequencies corresponding to the incidence frequency of 1/15,000 - 1/30,000 and 1/33,000 ? 1/47,000, respectively, observed in epidemiological data. In order to determine the specificity of the method, we determined the frequency of false-positives, using an E.Coli plasmid with a mutation-free 2264 bp insert mimicking the assay conditions used for sperm DNA. In conclusion, we can use this method to measure rare ACH and TDII mutations with high accuracy in the male germline to characterize the PAE.