Intellectual disability (ID) affects approximately 1%-3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%-3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations.

Loss-of-function variants in ANKRD11 were identified as the cause of KBG syndrome, an autosomal dominant syndrome with specific dental, neurobehavioural, craniofacial and skeletal anomalies. We present the largest cohort of KBG syndrome cases confirmed by ANKRD11 variants reported so far, consisting of 20 patients from 13 families. Sixteen patients were molecularly diagnosed by Sanger sequencing of ANKRD11, one familial case and three sporadic patients were diagnosed through whole-exome sequencing and one patient was identified through genomewide array analysis. All patients were evaluated by a clinical geneticist. Detailed orofacial phenotyping, including orthodontic evaluation, intra-oral photographs and orthopantomograms, was performed in 10 patients and revealed besides the hallmark feature of macrodontia of central upper incisors, several additional dental anomalies as oligodontia, talon cusps and macrodontia of other teeth. Three-dimensional (3D) stereophotogrammetry was performed in 14 patients and 3D analysis of patients compared with controls showed consistent facial dysmorphisms comprising a bulbous nasal tip, upturned nose with a broad base and a round or triangular face. Many patients exhibited neurobehavioural problems, such as autism spectrum disorder or hyperactivity. One-third of patients presented with (conductive) hearing loss. Congenital heart defects, velopharyngeal insufficiency and hip anomalies were less frequent. On the basis of our observations, we recommend cardiac assessment in children and regular hearing tests in all individuals with a molecular diagnosis of KBG syndrome. As ANKRD11 is a relatively common gene in which sequence variants have been identified in individuals with neurodevelopmental disorders, it seems an important contributor to the aetiology of both sporadic and familial cases.

Kabuki Syndrome (KS) is a rare syndrome characterized by intellectual disability and multiple congenital abnormalities, in particular a distinct dysmorphic facial appearance. KS is caused by mutations in the MLL2 gene, encoding an H3K4 histone methyl transferase which acts as an epigenetic transcriptional activator during growth and development. Direct sequencing of all 54 exons of the MLL2 gene in 45 clinically well-defined KS patients identified 34 (75.6%) different mutations. One mutation has been described previously, all others are novel. Clinically, all KS patients were sporadic, and mutations were de novo for all 27 families for which both parents were available. We detected nonsense (n=11), frameshift (n=17), splice site (n=4) and missense (n=2) mutations, predicting a high frequency of absent or non-functional MLL2 protein. Interestingly, both missense mutations located in the C-terminal conserved functional domains of the protein.Phenotypically our study indicated a statistically significant difference in the presence of a distinct facial appearance (p=0.0143) and growth retardation (p=0.0040) when comparing KS patients with an MLL2 mutation compared to patients without a mutation. Our data double the number of MLL2 mutations in KS reported so far and widen the spectrum of MLL2 mutations and disease mechanisms in KS.

Recently, de novo heterozygous loss-of-function mutations in beta-catenin (CTNNB1) were described for the first time in four individuals with intellectual disability (ID), microcephaly, limited speech and (progressive) spasticity, and functional consequences of CTNNB1 deficiency were characterized in a mouse model. Beta-catenin is a key downstream component of the canonical Wnt signaling pathway. Somatic gain-of-function mutations have already been found in various tumor types, whereas germline loss-of-function mutations in animal models have been shown to influence neuronal development and maturation. We report on 16 additional individuals from 15 families in whom we newly identified de novo loss-of-function CTNNB1 mutations (six nonsense, five frameshift, one missense, two splice mutation, and one whole gene deletion). All patients have ID, motor delay and speech impairment (both mostly severe) and abnormal muscle tone (truncal hypotonia and distal hypertonia/spasticity). The craniofacial phenotype comprised microcephaly (typically -2 to -4 SD) in 12 of 16 and some overlapping facial features in all individuals (broad nasal tip, small alae nasi, long and/or flat philtrum, thin upper lip vermillion). With this detailed phenotypic characterization of 16 additional individuals, we expand and further establish the clinical and mutational spectrum of inactivating CTNNB1 mutations and thereby clinically delineate this new CTNNB1 haploinsufficiency syndrome.

De novo mutations in specific mTOR pathway genes cause brain overgrowth in the context of intellectual disability (ID). By analyzing 101 mMTOR-related genes in a large ID patient cohort and two independent population cohorts, we show that these genes modulate brain growth in health and disease. We report the mTOR activator gene RHEB as an ID gene that is associated with megalencephaly when mutated. Functional testing of mutant RHEB in vertebrate animal models indicates pathway hyperactivation with a concomitant increase in cell and head size, aberrant neuronal migration, and induction of seizures, concordant with the human phenotype. This study reveals that tight control of brain volume is exerted through a large community of mTOR-related genes. Human brain volume can be altered, by either rare disruptive events causing hyperactivation of the pathway, or through the collective effects of common alleles.

Rett syndrome is one of the most common causes of complex disability in girls. It is characterized by early neurological regression that severely affects motor, cognitive and communication skills, by autonomic dysfunction and often a seizure disorder. It is a monogenic X-linked dominant neurodevelopmental disorder related to mutation in MECP2, which encodes the methyl-CpG-binding protein MeCP2. There are several mouse models either based on conditional knocking out of the Mecp2 gene or on a truncating mutation. We discuss the clinical aspects with special emphasis on the behavioral phenotype and we review current perspectives in clinical management alongside with perspectives in altering gene expression.

A partial deletion of chromosome band 2p25.3 (2pter) is a rarely described cytogenetic aberration in patients with intellectual disability (ID). Using microarrays we identified deletions of 2p25.3, sized 0.37-3.13 Mb, in three adult siblings and three unrelated patients. All patients had ID, obesity or overweight and/or a square-shaped stature without overt facial dysmorphic features. Combining our data with phenotypic and genotypic data of three patients from the literature we defined the minimal region of overlap which contained one gene, i.e., MYT1L. MYT1L is highly transcribed in the mouse embryonic brain where its expression is restricted to postmitotic differentiating neurons. In mouse-induced pluripotent stem cell (iPS) models, MYT1L is essential for inducing functional mature neurons. These resemble excitatory cortical neurons of the forebrain, suggesting a role for MYT1L in development of cognitive functions. Furthermore, MYT1L can directly convert human fibroblasts into functional neurons in conjunction with other transcription factors. MYT1L duplication was previously reported in schizophrenia, indicating that the gene is dosage-sensitive and that shared neurodevelopmental pathways may be affected in ID and schizophrenia. Finally, deletion of MYT1, another member of the Myelin Transcription Factor family involved in neurogenesis and highly similar to MYT1L, was recently described in ID as well. The identification of MYT1L as candidate gene for ID justifies further molecular studies aimed at detecting mutations and for mechanistic studies on its role in neuron development and on neuropathogenic effects of haploinsufficiency.

Noonan syndrome (NS) is an autosomal dominant disorder caused by mutations in PTPN11, KRAS, SOS1, and RAF1. We performed SOS1, RAF1, BRAF, MEK1, and MEK2 mutation analysis in a cohort of 102 PTPN11- and KRAS-negative NS patients and found pathogenic SOS1 mutations in 10, RAF1 mutations in 4, and BRAF mutations in 2 patients. Three novel SOS1 mutations were found. One was classified as a rare benign variant and the other remains unclassified. We confirm a high prevalence of pulmonic stenosis and ectodermal abnormalities in SOS1-positive patients. Three patients with SOS1 mutations presented with tumors (embryonal rhabdomyosarcoma, Sertoli cell testis tumor, and granular cell tumors of the skin). One patient with a RAF1 mutation had a lesion suggestive for a giant cell tumor. This is the first report describing different tumor types in NS patients with germ line SOS1 mutations.