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 features1,2,3,4,5. 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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
Accessions
NCBI Reference Sequence
References
Clayton-Smith, J. et al. Macrocephaly with cutis marmorata, haemangioma and syndactyly—a distinctive overgrowth syndrome. Clin. Dysmorphol. 6, 291–302 (1997).
Mirzaa, G. et al. Megalencephaly and perisylvian polymicrogyria with postaxial polydactyly and hydrocephalus: a rare brain malformation syndrome associated with mental retardation and seizures. Neuropediatrics 35, 353–359 (2004).
Moore, C.A. et al. Macrocephaly-cutis marmorata telangiectatica congenita: a distinct disorder with developmental delay and connective tissue abnormalities. Am. J. Med. Genet. 70, 67–73 (1997).
Mirzaa, G.M. et al. Megalencephaly-capillary malformation (MCAP) and megalencephaly-polydactyly-polymicrogyria-hydrocephalus (MPPH) syndromes: two closely related disorders of brain overgrowth and abnormal brain and body morphogenesis. Am. J. Med. Genet. A. 158A, 269–291 (2012).
Conway, R.L. et al. Neuroimaging findings in macrocephaly-capillary malformation: a longitudinal study of 17 patients. Am. J. Med. Genet. A. 143A, 2981–3008 (2007).
Oduber, C.E. et al. A proposal for classification of entities combining vascular malformations and deregulated growth. Eur. J. Med. Genet. 54, 262–271 (2011).
Happle, R. Lethal genes surviving by mosaicism: a possible explanation for sporadic birth defects involving the skin. J. Am. Acad. Dermatol. 16, 899–906 (1987).
Gripp, K.W. et al. Significant overlap and possible identity of macrocephaly capillary malformation and megalencephaly polymicrogyria-polydactyly hydrocephalus syndromes. Am. J. Med. Genet. A. 149A, 868–876 (2009).
Brodbeck, D., Cron, P. & Hemmings, B.A. A human protein kinase Bγ with regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain. J. Biol. Chem. 274, 9133–9136 (1999).
Hers, I., Vincent, E.E. & Tavare, J.M. Akt signalling in health and disease. Cell. Signal. 23, 1515–1527 (2011).
Otsu, M. et al. Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell 65, 91–104 (1991).
Scarano, E., Iaccarino, M., Grippo, P. & Parisi, E. The heterogeneity of thymine methyl group origin in DNA pyrimidine isostichs of developing sea urchin embryos. Proc. Natl. Acad. Sci. USA 57, 1394–1400 (1967).
Lindhurst, M.J. et al. A mosaic activating mutation in AKT1 associated with the Proteus syndrome. N. Engl. J. Med. 365, 611–619 (2011).
Volinia, S. et al. Molecular cloning, cDNA sequence, and chromosomal localization of the human phosphatidylinositol 3-kinase p110α (PIK3CA) gene. Genomics 24, 472–477 (1994).
Robinson, J.T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
Vanhaesebroeck, B. et al. Synthesis and function of 3-phosphorylated inositol lipids. Annu. Rev. Biochem. 70, 535–602 (2001).
Engelman, J.A., Luo, J. & Cantley, L.C. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat. Rev. Genet. 7, 606–619 (2006).
Mora, A., Komander, D., van Aalten, D.M. & Alessi, D.R. PDK1, the master regulator of AGC kinase signal transduction. Semin. Cell Dev. Biol. 15, 161–170 (2004).
Katso, R. et al. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu. Rev. Cell Dev. Biol. 17, 615–675 (2001).
Samuels, Y. & Ericson, K. Oncogenic PI3K and its role in cancer. Curr. Opin. Oncol. 18, 77–82 (2006).
Sansal, I. & Sellers, W.R. The biology and clinical relevance of the PTEN tumor suppressor pathway. J. Clin. Oncol. 22, 2954–2963 (2004).
Cho, H. et al. Insulin resistance and a diabetes mellitus–like syndrome in mice lacking the protein kinase Akt2 (PKBβ). Science 292, 1728–1731 (2001).
Cho, H., Thorvaldsen, J.L., Chu, Q., Feng, F. & Birnbaum, M.J. Akt1/PKBα is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J. Biol. Chem. 276, 38349–38352 (2001).
George, S. et al. A family with severe insulin resistance and diabetes due to a mutation in AKT2 . Science 304, 1325–1328 (2004).
Hussain, K. et al. An activating mutation of AKT2 and human hypoglycemia. Science 334, 474 (2011).
Ballif, B.C. et al. High-resolution array CGH defines critical regions and candidate genes for microcephaly, abnormalities of the corpus callosum, and seizure phenotypes in patients with microdeletions of 1q43q44. Hum. Genet. 131, 145–156 (2012).
Boland, E. et al. Mapping of deletion and translocation breakpoints in 1q44 implicates the serine/threonine kinase AKT3 in postnatal microcephaly and agenesis of the corpus callosum. Am. J. Hum. Genet. 81, 292–303 (2007).
Tschopp, O. et al. Essential role of protein kinase Bγ (PKBγ/Akt3) in postnatal brain development but not in glucose homeostasis. Development 132, 2943–2954 (2005).
Tokuda, S. et al. A novel Akt3 mutation associated with enhanced kinase activity and seizure susceptibility in mice. Hum. Mol. Genet. 20, 988–999 (2011).
Poduri, A. et al. Somatic activation of AKT3 causes hemispheric developmental brain malformations. Neuron 74, 41–48 (2012).
Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455, 1061–1068 (2008).
Cheung, L.W. et al. High frequency of PIK3R1 and PIK3R2 mutations in endometrial cancer elucidates a novel mechanism for regulation of PTEN protein stability. Cancer Discov. 1, 170–185 (2011).
Samuels, Y. et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 304, 554 (2004).
Thomas, R.K. et al. High-throughput oncogene mutation profiling in human cancer. Nat. Genet. 39, 347–351 (2007).
Jaiswal, B.S. et al. Somatic mutations in p85α promote tumorigenesis through class IA PI3K activation. Cancer Cell 16, 463–474 (2009).
Gymnopoulos, M., Elsliger, M.A. & Vogt, P.K. Rare cancer-specific mutations in PIK3CA show gain of function. Proc. Natl. Acad. Sci. USA 104, 5569–5574 (2007).
Ikenoue, T. et al. Functional analysis of PIK3CA gene mutations in human colorectal cancer. Cancer Res. 65, 4562–4567 (2005).
Oda, K. et al. PIK3CA cooperates with other phosphatidylinositol 3′-kinase pathway mutations to effect oncogenic transformation. Cancer Res. 68, 8127–8136 (2008).
Butler, M.G. et al. Subset of individuals with autism spectrum disorders and extreme macrocephaly associated with germline PTEN tumour suppressor gene mutations. J. Med. Genet. 42, 318–321 (2005).
Liaw, D. et al. Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat. Genet. 16, 64–67 (1997).
Marsh, D.J. et al. Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation. Hum. Mol. Genet. 7, 507–515 (1998).
Kong, D. & Yamori, T. Phosphatidylinositol 3-kinase inhibitors: promising drug candidates for cancer therapy. Cancer Sci. 99, 1734–1740 (2008).
Marone, R., Cmiljanovic, V., Giese, B. & Wymann, M.P. Targeting phosphoinositide 3-kinase: moving towards therapy. Biochim. Biophys. Acta 1784, 159–185 (2008).
Riviere, J.B. et al. De novo mutations in the actin genes ACTB and ACTG1 cause Baraitser-Winter syndrome. Nat. Genet. 44, 440–444, S1–2 (2012).
DePristo, M.A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).
1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
O'Roak, B.J. et al. Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat. Genet. 43, 585–589 (2011).
Cooper, G.M. et al. Single-nucleotide evolutionary constraint scores highlight disease-causing mutations. Nat. Methods 7, 250–251 (2010).
Grantham, R. Amino acid difference formula to help explain protein evolution. Science 185, 862–864 (1974).
Brownstein, M.J., Carpten, J.D. & Smith, J.R. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques 20, 1004–1006, 1008–1010 (1996).
Lynch, M. Rate, molecular spectrum, and consequences of human mutation. Proc. Natl. Acad. Sci. USA 107, 961–968 (2010).
Acknowledgements
We wish to thank all of the children and families in this study, their referring physicians and the M-CM Network (see URLs) for their help with this project over many years. We thank the members of the Northwest Genomics Center and the McGill University and Genome Quebec Innovation Centre for their excellent technical assistance. We also thank the Finding of Rare Disease Genes (FORGE) Canada Consortium, especially J. Marcadier for her contribution to the infrastructure.
This work was funded by the US National Institutes of Health under National Institute of Neurological Disorders and Stroke (NINDS) grant NS058721 (to W.B.D.), National Institute of Child Health & Human Development (NICHD) grant HD36657 and National Institute of General Medical Sciences (NIGMS) grant 5-T32-GM08243 (to J.M.G.), the Government of Canada (to FORGE) through Genome Canada, the Canadian Institutes of Health Research (CIHR) and the Ontario Genomics Institute (OGI-049). Additional funding was provided to FORGE by Genome Quebec and Genome British Columbia. J.-B.R. is supported by a Banting Postdoctoral Fellowship from the CIHR. K.M.B. is supported by a Clinical Investigatorship Award from the CIHR Institute of Genetics. The laboratory of M.O. is funded by Cancer Research UK (CR-UK), the Medical Research Council (UK) and Leukaemia Lymphoma Research (UK). M.O. is a Senior CR-UK Research Fellow.
We would like to thank the Simons Foundation Autism Research Initiative (SFARI) for providing control exome data (grant 191889 to J.S.). We also thank the NIEHS Environmental Genome Project (contract HHSN273200800010C) and the NHLBI GO Exome Sequencing Project and its ongoing studies—Lung GO (HL-102923), Broad GO (HL-102925), Seattle GO (HL-102926), Heart GO (HL-103010) and the Women's Health Institute (WHI; HL-102924) Sequencing Projects—for providing exome variant calls for comparison.
Author information
Authors and Affiliations
Consortia
Contributions
J.-B.R., G.M.M., K.M.B. and W.B.D. designed the study. J.-B.R., B.J.O. and J.S.-O. designed and performed the genetics experiments. M.B., T.W., C.T.S. and T.R.W. contributed to the genetics experiments. J.-B.R., J.A.S. and B.J.O. performed the bioinformatics experiments. D.A. performed the experiments in lymphoblastoid cell lines. G.M.M., R.L.C., K.W.G., S.M.N., B.A., C.M.A., L.A., O.C., C.C., B.A.D., A.M.I., J.L.L., A.E.L., G.M.S.M., W.S.M., J.D.R., A.K.S., T.L.-S., G.U., R.W., B.Z., J.M.G., K.M.B. and W.B.D. recruited and evaluated the study subjects. H.E.B., N.A.K. and C.L.B. provided administrative support and recruited the study subjects. J.M., D.E.B., M.O., J.S., K.M.B. and W.B.D. supervised the study. J.-B.R., G.M.M. and W.B.D. wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Additional information
Membership of the Steering Committee for the Consortium is provided in the Supplementary Note.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5, Supplementary Tables 1–12 and Supplementary Note (PDF 1017 kb)
Rights and permissions
About this article
Cite this article
Rivière, JB., Mirzaa, G., O'Roak, B. et al. De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes. Nat Genet 44, 934–940 (2012). https://doi.org/10.1038/ng.2331
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ng.2331
This article is cited by
-
Delineation of the phenotypes and genotypes of facial infiltrating lipomatosis associated with PIK3CA mutations
Orphanet Journal of Rare Diseases (2023)
-
Somatic mutation spectrum of a Chinese cohort of pediatrics with vascular malformations
Orphanet Journal of Rare Diseases (2023)
-
An expanded clinical spectrum of hypoinsulinaemic hypoketotic hypoglycaemia
Orphanet Journal of Rare Diseases (2023)
-
Targeted next-generation sequencing for detection of PIK3CA mutations in archival tissues from patients with Klippel–Trenaunay syndrome in an Asian population
Orphanet Journal of Rare Diseases (2023)
-
AKT inhibition in the central nervous system induces signaling defects resulting in psychiatric symptomatology
Cell & Bioscience (2022)