Abstracts

Rationale: Somatic mutation of the lissencephaly-1 gene (LIS1) is a cause of subcortical band heterotopia (SBH; “double cortex”). The severity of the phenotype depends on the frequency of mutation in brain tissue. Detecting and quantifying low frequency somatic mosaic mutations is challenging using traditional molecular assays such as sub-cloning. Methods: Here we utilized droplet digital PCR (ddPCR), an ultra-sensitive method for low level mutation detection, previously used to elucidate and determine allele frequency of tumor mutations. We used this method in concert with classical genotyping techniques – SnapShot assays and pyrosequencing – to detect and characterize the tissue mosaicism of a somatic mutation (LIS1 c.190A>T; p.K64X) in an Australian patient with posterior bilateral SBH and refractory epilepsy. The high sensitivity of ddPCR allowed us to readily detect the mutation at low levels in the patient’s brain tissue. Results: Remarkably, even though the DNA sample derived from formalin-fixed paraffin-embedded (FFPE) brain tissue was low quality, detection at 5% mutant allele frequency was achieved, and even lower frequencies would have been detectable using this approach. This low mutation frequency in brain was consistent with the relatively subtle malformation resolved by magnetic resonance imaging (MRI). The presence of the mutation at low level in other tissues from the patient permitted us to predict the time of mutagenesis. Conclusions: This sensitive methodology will have utility for a variety of other brain malformation syndromes associated with epilepsy where somatic mutations are involved in causation. Funding: This study was supported by National Health and Medical Research Council Program Grant (1091593) to S.F.B, a Project Grant (1129054) to S.F.B, a Project Grant (1079058) to M.S.H, and a R.D Wright Career Development Fellowship (1063799) to M.S.H.