Megalencephaly Syndromes: Exome Pipeline Strategies for Detecting Low-Level Mosaic Mutations

<div><p>Two megalencephaly (MEG) syndromes, megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyriapolydactyly-hydrocephalus (MPPH), have recently been defined on the basis of physical and neuroimaging features. Subsequently, exome sequencing of ten MEG cases identified <i>de-novo</i> postzygotic mutations in <i>PIK3CA</i> which cause MCAP and <i>de-novo</i> mutations in <i>AKT</i> and <i>PIK3R2</i> which cause MPPH. Here we present findings from exome sequencing three unrelated megalencephaly patients which identified a causal <i>PIK3CA</i> mutation in two cases and a causal <i>PIK3R2</i> mutation in the third case. However, our patient with the <i>PIK3R2</i> mutation which is considered to cause MPPH has a marked bifrontal band heterotopia which is a feature of MCAP. Furthermore, one of our patients with a <i>PIK3CA</i> mutation lacks syndactyly/polydactyly which is a characteristic of MCAP. These findings suggest that the overlap between MCAP and MPPH may be greater than the available studies suggest. In addition, the <i>PIK3CA</i> mutation in one of our patients could not be detected using standard exome analysis because the mutation was observed at a low frequency consistent with somatic mosaicism. We have therefore investigated several alternative methods of exome analysis and demonstrate that alteration of the initial allele frequency spectrum (AFS), used as a prior for variant calling in samtools, had the greatest power to detect variants with low mutant allele frequencies in our 3 MEG exomes and in simulated data. We therefore recommend non-default settings of the AFS in combination with stringent quality control when searching for causal mutation(s) that could have low levels of mutant reads due to post-zygotic mutation.</p>