Megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndromes are sporadic overgrowth disorders associated with markedly enlarged brain size and other recognizable features1-5. We performed exome sequencing in three families with MCAP or MPPH and confirmed our initial observations in exomes from 7 MCAP and 174 control individuals, as well as in 40 additional megalencephaly subjects 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 two mutations of AKT3, one recurrent mutation of PIK3R2 in 11 unrelated MPPH families, and 15 mostly postzygotic mutations of PIK3CA in 23 MCAP and one MPPH patients. 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.

Different mutations in the same gene often cause distinct disease phenotypes in humans. Generally, such variations in the clinical phenotypes have been considered to be a consequence of the function or dysfunction of mutant proteins. Thus, a primary emphasis in genotype-phenotype correlation studies has been placed on determining the unique functional properties of encoded mutant proteins. But in vitro functional assays of mutant proteins often show discordance between predicted protein function and clinical outcome. Little is known about the many factors that are potentially involved in this discrepancy, but loss-of-function versus gain-of-function effects are often invoked as a possible mechanism.We previously identified two unrelated individuals with an unusual phenotype that combined four distinct syndromesperipheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome and Hirschsprung disease-that are characterized by deficiencies of Schwann cells, oligodendrocytes, melanocytes and enteric ganglia neurons, respectively 1,2 . Here we describe four more individuals and propose that this complex disorder is a newly described neurocristopathy called PCWH.We previously identified mutations in SOX10 in all affected individuals 1,2 . SOX10 is a transcription factor that contains a central high mobility group (HMG) DNA-binding domain and a transactivation domain at its C terminus 3 . SOX10 is essential for the development of cells in the neural crest lineage, including melanocytes and enteric ganglia neurons 4,5 ; it also controls the proliferation and differentiation of Schwann cells and oligodendrocytes [6][7][8] . Notably, some mutations in SOX10 also cause a distinct and more restricted disease that does not involve either the peripheral (PNS) or the central (CNS) nervous systems 9-11 . This less complicated neurocristopathy, called WS4, combines Waardenburg and Hirschsprung diseases 12 . Most SOX10 disease-associated mutations, regardless of whether they cause PCWH or WS4, result in premature termination codons (PTCs).As in SOX10, different mutations in MPZ are responsible for distinct neurological diseases, which each affect the myelin of the PNS. These neuropathies include early onset congenital hypomyelinating neuropathy (CHN; OMIM 605253), Dejerine-Sottas neuropathy (DSN; OMIM 145900) and the less severe, adult onset Charcot-MarieTooth disease type 1B (CMT1B; OMIM 118200; ref. 13). It has been suggested that the severity of alleles in CHN and DSN is due to dominant-negative effects, whereas the reduced severity of alleles in CMT1B is due to loss of function. But although some nonsense and frameshift alleles cause CMT1B, several truncating mutations have been reported that convey either a CHN or a DSN phenotype.We investigated the molecular mechanisms underlying the neurological phenotypes of the PCWH and WS4 neurocristopathies resulting from allelic SOX10 truncating mutations, as well as those underlying the CHN, DSN and CMT1B myelinopathies caused by allelic MPZ truncatin...

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations.

Infantile spasms (ISS) are an epilepsy disorder frequently associated with severe developmental outcome and have diverse genetic etiologies. We ascertained 11 subjects with ISS and novel copy number variants (CNVs) and combined these with a new cohort with deletion 1p36 and ISS, and additional published patients with ISS and other chromosomal abnormalities. Using bioinformatics tools, we analyzed the gene content of these CNVs for enrichment in pathways of pathogenesis. Several important findings emerged. First, the gene content was enriched for the gene regulatory network involved in ventral forebrain development. Second, genes in pathways of synaptic function were overrepresented, significantly those involved in synaptic vesicle transport. Evidence also suggested roles for GABAergic synapses and the postsynaptic density. Third, we confirm the association of ISS with duplication of 14q12 and maternally inherited duplication of 15q11q13, and report the association with duplication of 21q21. We also present a patient with ISS and deletion 7q11.3 not involving MAGI2. Finally, we provide evidence that ISS in deletion 1p36 may be associated with deletion of KLHL17 and expand the epilepsy phenotype in that syndrome to include early infantile epileptic encephalopathy. Several of the identified pathways share functional links, and abnormalities of forebrain synaptic growth and function may form a common biologic mechanism underlying both ISS and autism. This study demonstrates a novel approach to the study of gene content in subjects with ISS and copy number variation, and contributes further evidence to support specific pathways of pathogenesis.

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