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.

22q11 Deletion Syndrome (22q11DS) is a common microdeletion syndrome with multisystem expression. Phenotypic features vary with age, ascertainment, and assessment. We systematically assessed 78 adults (36 M, 42 F; mean age 31.5, SD 10.5 years) with a 22q11.2 deletion ascertained through an adult congenital cardiac clinic (n = 35), psychiatric-related sources (n = 39), or as affected parents of subjects (n = 4). We recorded the lifetime prevalence of features requiring attention, with 95% confidence intervals (CI) not overlapping zero. Subtle learning difficulties, hypernasality and facial gestalt were not included. We investigated ascertainment effects using non-overlapping subgroups ascertained with tetralogy of Fallot (n = 31) or schizophrenia (n = 31). Forty-three features met inclusion criteria and were present in 5% or more patients, including several of later onset (e.g., hypothyroidism, cholelithiasis). Number of features per patient (median 9, range 3-22) correlated with hospitalizations (P = 0.0002) and, when congenital features were excluded, with age (P = 0.02). Adjusting for ascertainment, 25.8% (95% CI, 9.5-42.1%) of patients had cardiac anomalies and 22.6% (95% CI, 7.0-38.2%) had schizophrenia. Ascertainment subgroups were otherwise similar in median number and prevalence of features. Non-characteristic features are common in 22q11DS. Adjusting for ascertainment effects is important. Many treatable conditions may be anticipated and features may accumulate over time. The results have implications for clinical assessment and management, genetic counseling and research into pathophysiological mechanisms.

Epigenetic dysregulation has emerged as a recurring mechanism in the etiology of neurodevelopmental disorders. Two such disorders, CHARGE and Kabuki syndromes, result from loss of function mutations in chromodomain helicase DNA-binding protein 7 (CHD7) and lysine (K) methyltransferase 2D (KMT2D), respectively. Although these two syndromes are clinically distinct, there is significant phenotypic overlap. We therefore expected that epigenetically driven developmental pathways regulated by CHD7 and KMT2D would overlap and that DNA methylation (DNAm) alterations downstream of the mutations in these genes would identify common target genes, elucidating a mechanistic link between these two conditions, as well as specific target genes for each disorder. Genome-wide DNAm profiles in individuals with CHARGE and Kabuki syndromes with CHD7 or KMT2D identified distinct sets of DNAm differences in each of the disorders, which were used to generate two unique, highly specific and sensitive DNAm signatures. These DNAm signatures were able to differentiate pathogenic mutations in these two genes from controls and from each other. Analysis of the DNAm targets in each gene-specific signature identified both common gene targets, including homeobox A5 (HOXA5), which could account for some of the clinical overlap in CHARGE and Kabuki syndromes, as well as distinct gene targets. Our findings demonstrate how characterization of the epigenome can contribute to our understanding of disease pathophysiology for epigenetic disorders, paving the way for explorations of novel therapeutics.

Sotos syndrome (SS) represents an important human model system for the study of epigenetic regulation; it is an overgrowth/intellectual disability syndrome caused by mutations in a histone methyltransferase, NSD1. As layered epigenetic modifications are often interdependent, we propose that pathogenic NSD1 mutations have a genome-wide impact on the most stable epigenetic mark, DNA methylation (DNAm). By interrogating DNAm in SS patients, we identify a genome-wide, highly significant NSD1+/−-specific signature that differentiates pathogenic NSD1 mutations from controls, benign NSD1 variants and the clinically overlapping Weaver syndrome. Validation studies of independent cohorts of SS and controls assigned 100% of these samples correctly. This highly specific and sensitive NSD1+/− signature encompasses genes that function in cellular morphogenesis and neuronal differentiation, reflecting cardinal features of the SS phenotype. The identification of SS-specific genome-wide DNAm alterations will facilitate both the elucidation of the molecular pathophysiology of SS and the development of improved diagnostic testing.

Dysregulation of imprinted genes on human chromosome 11p15 has been implicated in Beckwith-Wiedemann syndrome (BWS), an overgrowth syndrome associated with congenital malformations and tumor predisposition. The molecular basis of BWS is complex and heterogeneous. The syndrome is associated with alterations in two distinct imprinting domains on 11p15: a telomeric domain containing the H19 and IGF2 genes and a centromeric domain including the KCNQ1OT1 and CDKNIC genes. It has been postulated that disorders of imprinting in the telomeric domain are associated with overgrowth and cancer predisposition, whereas those in the centromeric domain involve malformations but not tumor development. In this study of 125 BWS cases, we confirm the association of tumors with constitutional defects in the 11p15 telomeric domain; six of 21 BWS cases with uniparental disomy (UPD) of 11p15 developed tumors and one of three of the rare BWS subtype with hypermethylation of the H19 gene developed tumors. Most importantly, we find that five of 32 individuals with BWS and imprinting defects in the centromeric domain developed embryonal tumors. Furthermore, the type of tumors observed in BWS cases with telomeric defects are different from those seen in BWS cases with defects limited to the centromeric domain. Whereas Wilms' tumor was the most frequent tumor seen in BWS cases with UPD for 11p15 or H19 hypermethylation, none of the embryonal tumors with imprinting defects at KCNQ1OT1 was a Wilms' tumor. This suggests that distinct tumor predisposition profiles result from dysregulation of the telomeric domain versus the centromeric domain and that these imprinting defects activate distinct genetic pathways for embryonal tumorigenesis.