De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes

Abstract

Megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndromes are sporadic overgrowth disorders associated with markedly enlarged brain size and other recognizable features. We performed exome sequencing in 3 families with MCAP or MPPH, and our initial observations were confirmed in exomes from 7 individuals with MCAP and 174 control individuals, as well as in 40 additional subjects with megalencephaly, using a combination of Sanger sequencing, restriction enzyme assays and targeted deep sequencing. We identified de novo germline or postzygotic mutations in three core components of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. These include 2 mutations in AKT3, 1 recurrent mutation in PIK3R2 in 11 unrelated families with MPPH and 15 mostly postzygotic mutations in PIK3CA in 23 individuals with MCAP and 1 with MPPH. Our data highlight the central role of PI3K-AKT signaling in vascular, limb and brain development and emphasize the power of massively parallel sequencing in a challenging context of phenotypic and genetic heterogeneity combined with postzygotic mosaicism.

Figure 1

Craniofacial appearance and magnetic resonance imaging (MRI) of the three index patients. Photos and brain MRI of patients LR08-018 (a–c), LR00-016a1 (d–f), and LR09-006 (g–i). Photos of patients LR08-018 (a), LR00-016a1 (d) and LR09-006 (g) were taken at 11 months, 15 years, and 5 months, respectively. Note the prominent forehead and apparent macrocephaly in all three patients (a, d, g), and a midline facial capillary malformation (or nevus flammeus) in LR09-006 (g). Midline sagittal brain images (b, e, h) show prominent forehead, increased cranium-to-face ratio and cerebellar tonsillar ectopia (arrowheads), while axial or coronal images (c, f, i) show bilateral perisylvian polymicrogyria (arrows). Scale bars correspond to 1 cm. Additional photos of patient LR09-006 and a clinical description of the three index patients are provided in the Supplementary Note. We obtained written consent to publish photographs of the patients.

Figure 2

PIP3 levels in lymphoblastoid cell lines derived from an unaffected control, a patient with Cowden disease (GM10080), and four megalencephaly patients. (a) Indirect immunofluorescence staining of PIP3 in exponentially growing lymphoblastoid cell lines using a mouse monoclonal anti-PIP3 antibody (Online Methods). Scale bar corresponds to 10μm. (b) Per-cell quantification of PIP3 levels based on anti-PIP3 signal intensity (a.u., arbitrary units). Levels of PIP3 signal in control cells (WT) are comparable to those of LR08-018 (AKT3 p.Arg465Trp). All other mutant cell lines show increased PIP3 signal compared to control cells. Elevated PIP3 signal is also evident in cells derived from the patient with Cowden disease (PTEN p.Glu261Ter), which served as a positive control. * Statistically significant difference compared to control cells (p<0.05 two-tailed t-test assuming unequal variance, n=30 to 50 cells per cell line). Error bars indicate standard deviation. (c) Levels of PIP3 in cell lines from LR00-016a1 (PIK3R2 p.Gly373Arg) and LR05-204 (PIK3CA p.Glu453del) can be reduced following treatment with the PI3K-inhibitor PI-103 (5μM for 16 hours). Scale bar corresponds to 10μm.

Figure 3

Distribution of mutations in AKT3, PIK3R2, and PIK3CA. The activating Akt3 mutation in mouse is indicated in grey (p.Asp219Val). For recurrent mutations, the number of occurrences is indicated in parentheses. PH: pleckstrin homology domain; C-terminal: carboxyl-terminal domain; SH2 and SH3: Src-homology-2 and -3 domains; Rho-GAP: Rho GTPase-activating protein domain; p85-BD and RAS-BD: p85- and RAS-binding domains; C2: protein-kinase-C-homology-2 domain. The PIK3CA mutations affect a total of 15 residues, mainly localized in the p85-binding, C2, and catalytic lipid kinase domains.