Contribution of SHANK3 mutations to autism spectrum disorder

Mutations in SHANK3, which encodes a synaptic scaffolding protein, have been described in subjects with an autism spectrum disorder (ASD). To assess the quantitative contribution of SHANK3 to the pathogenesis of autism, we determined the frequency of DNA sequence and copy-number variants in this gene in 400 ASD-affected subjects ascertained in Canada. One de novo mutation and two gene deletions were discovered, indicating a contribution of 0.75% in this cohort. One additional SHANK3 deletion was characterized in two ASD-affected siblings from another collection, which brings the total number of published mutations in unrelated ASD-affected families to seven. The combined data provide support that haploinsufficiency of SHANK3 can cause a monogenic form of autism in sufficient frequency to warrant consideration in clinical diagnostic testing.     

Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: A Gradient of Severity in Cognitive Impairments

SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability—more than 1 in 50—warrant its consideration for mutation screening in clinical practice.     

SHANK1 and autism spectrum disorders

Autism spectrum disorders(ASD) are highly heterogeneous pediatric developmental disorders with estimated heritability more than 70%. Although the genetic factors in ASD are mainly unknown, a large number of gene mutations have been found, especially in genes involved in neurogenesis. The Neurexin-Neuroligin-Shank(NRXN-NLGN-SHANK) pathway plays a key role in the formation, maturation and maintenance of synapses, consistent with the hypothesis of neurodevelopmental abnormality in ASD. Presynaptic NRXNs interact with postsynaptic NLGNs in excitatory glutamatergic synapses. SHANK proteins function as core components of the postsynaptic density(PSD) by interacting with multiple proteins. Recently, deletions and point mutations of the SHANK1 gene have been detected in ASD individuals, indicating the involvement of SHANK1 in ASD. This review focuses on the function of SHANK1 protein, Shank1 mouse models, and the molecular genetics of the SHANK1 gene in human ASD.     

Genetic and functional analyses of SHANK2 mutations suggest a multiple hit model of autism spectrum disorders

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23–4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11–q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the “multiple hit model” for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.     

Rare deletions at the neurexin 3 locus in autism spectrum disorder

The three members of the human neurexin gene family, neurexin 1 (NRXN1), neurexin 2 (NRXN2), and neurexin 3 (NRXN3), encode neuronal adhesion proteins that have important roles in synapse development and function. In autism spectrum disorder (ASD), as well as in other neurodevelopmental conditions, rare exonic copy-number variants and/or point mutations have been identified in the NRXN1 and NRXN2 loci. We present clinical characterization of four index cases who have been diagnosed with ASD and who possess rare inherited or de novo microdeletions at 14q24.3-31.1, a region that overlaps exons of the alpha and/or beta isoforms of NRXN3. NRXN3 deletions were found in one father with subclinical autism and in a carrier mother and father without formal ASD diagnoses, indicating issues of penetrance and expressivity at this locus. Notwithstanding these clinical complexities, this report on ASD-affected individuals who harbor NRXN3 exonic deletions advances the understanding of the genetic etiology of autism, further enabling molecular diagnoses.     

Truncating mutations in NRXN2 and NRXN1 in autism spectrum disorders and schizophrenia

Growing genetic evidence is converging in favor of common pathogenic mechanisms for autism spectrum disorders (ASD), intellectual disability (ID or mental retardation) and schizophrenia (SCZ), three neurodevelopmental disorders affecting cognition and behavior. Copy number variations and deleterious mutations in synaptic organizing proteins including NRXN1 have been associated with these neurodevelopmental disorders, but no such associations have been reported for NRXN2 or NRXN3. From resequencing the three neurexin genes in individuals affected by ASD (n = 142), SCZ (n = 143) or non-syndromic ID (n = 94), we identified a truncating mutation in NRXN2 in a patient with ASD inherited from a father with severe language delay and family history of SCZ. We also identified a de novo truncating mutation in NRXN1 in a patient with SCZ, and other potential pathogenic ASD mutations. These truncating mutations result in proteins that fail to promote synaptic differentiation in neuron coculture and fail to bind either of the established postsynaptic binding partners LRRTM2 or NLGN2 in cell binding assays. Our findings link NRXN2 disruption to the pathogenesis of ASD for the first time and further strengthen the involvement of NRXN1 in SCZ, supporting the notion of a common genetic mechanism in these disorders.     

Whole Genome Sequencing Reveals a De Novo SHANK3 Mutation in Familial Autism Spectrum Disorder

IntroductionClinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD). Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS) for the diagnostic approach to ASD.MethodsWe identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents.ResultsThree male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon ({"type":"entrez-nucleotide","attrs":{"text":"NM_033517","term_id":"380748962","term_text":"NM_033517"}}NM_033517:c.3259_3259delC, p.Ser1088Profs*6).ConclusionsWe reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder.     

Pten regulates neuronal arborization and social interaction in mice

CNS deletion of Pten in the mouse has revealed its roles in controlling cell size and number, thus providing compelling etiology for macrocephaly and Lhermitte-Duclos disease. PTEN mutations in individuals with autism spectrum disorders (ASD) have also been reported, although a causal link between PTEN and ASD remains unclear. In the present study, we deleted Pten in limited differentiated neuronal populations in the cerebral cortex and hippocampus of mice. Resulting mutant mice showed abnormal social interaction and exaggerated responses to sensory stimuli. We observed macrocephaly and neuronal hypertrophy, including hypertrophic and ectopic dendrites and axonal tracts with increased synapses. This abnormal morphology was associated with activation of the Akt/mTor/S6k pathway and inactivation of Gsk3beta. Thus, our data suggest that abnormal activation of the PI3K/AKT pathway in specific neuronal populations can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD.     

Communication impairments in mice lacking Shank1: reduced levels of ultrasonic vocalizations and scent marking behavior

Autism is a neurodevelopmental disorder with a strong genetic component. Core symptoms are abnormal reciprocal social interactions, qualitative impairments in communication, and repetitive and stereotyped patterns of behavior with restricted interests. Candidate genes for autism include the SHANK gene family, as mutations in SHANK2 and SHANK3 have been detected in several autistic individuals. SHANK genes code for a family of scaffolding proteins located in the postsynaptic density of excitatory synapses. To test the hypothesis that a mutation in SHANK1 contributes to the symptoms of autism, we evaluated Shank1(-/-) null mutant mice for behavioral phenotypes with relevance to autism, focusing on social communication. Ultrasonic vocalizations and the deposition of scent marks appear to be two major modes of mouse communication. Our findings revealed evidence for low levels of ultrasonic vocalizations and scent marks in Shank1(-/-) mice as compared to wildtype Shank1(+/+) littermate controls. Shank1(-/-) pups emitted fewer vocalizations than Shank1(+/+) pups when isolated from mother and littermates. In adulthood, genotype affected scent marking behavior in the presence of female urinary pheromones. Adult Shank1(-/-) males deposited fewer scent marks in proximity to female urine than Shank1(+/+) males. Call emission in response to female urinary pheromones also differed between genotypes. Shank1(+/+) mice changed their calling pattern dependent on previous female interactions, while Shank1(-/-) mice were unaffected, indicating a failure of Shank1(-/-) males to learn from a social experience. The reduced levels of ultrasonic vocalizations and scent marking behavior in Shank1(-/-) mice are consistent with a phenotype relevant to social communication deficits in autism.     

Structural variation of chromosomes in autism spectrum disorder

Structural variation (copy number variation [CNV] including deletion and duplication, translocation, inversion) of chromosomes has been identified in some individuals with autism spectrum disorder (ASD), but the full etiologic role is unknown. We performed genome-wide assessment for structural abnormalities in 427 unrelated ASD cases via single-nucleotide polymorphism microarrays and karyotyping. With microarrays, we discovered 277 unbalanced CNVs in 44% of ASD families not present in 500 controls (and re-examined in another 1152 controls). Karyotyping detected additional balanced changes. Although most variants were inherited, we found a total of 27 cases with de novo alterations, and in three (11%) of these individuals, two or more new variants were observed. De novo CNVs were found in approximately 7% and approximately 2% of idiopathic families having one child, or two or more ASD siblings, respectively. We also detected 13 loci with recurrent/overlapping CNV in unrelated cases, and at these sites, deletions and duplications affecting the same gene(s) in different individuals and sometimes in asymptomatic carriers were also found. Notwithstanding complexities, our results further implicate the SHANK3-NLGN4-NRXN1 postsynaptic density genes and also identify novel loci at DPP6-DPP10-PCDH9 (synapse complex), ANKRD11, DPYD, PTCHD1, 15q24, among others, for a role in ASD susceptibility. Our most compelling result discovered CNV at 16p11.2 (p = 0.002) (with characteristics of a genomic disorder) at approximately 1% frequency. Some of the ASD regions were also common to mental retardation loci. Structural variants were found in sufficiently high frequency influencing ASD to suggest that cytogenetic and microarray analyses be considered in routine clinical workup.     

Mutations in the gene encoding CADM1 are associated with autism spectrum disorder

The unified idea on the molecular pathogenesis of Autism Spectrum Disorder (ASD) is still unknown although mutations in genes encoding neuroligins and SHANK3 have been shown in a small part of the patients. RA175/SynCAM1/CADM1(CADM1), a member of immunoglobulin superfamily, is another synaptic cell adhesion molecule. To clarify the idea that impaired synaptogenesis underlies the pathogenesis of ASD, we examined the relationship between mutations in the CADM1 gene and ASD. We found two missense mutations, C739A(H246N) and A755C(Y251S), in the CADM1 gene of male Caucasian ASD patients and their family members. Both mutations were located in the third immunoglobulin domain, which is essential for trans-active interaction. The mutated CADM1 exhibited less amount of high molecular weight with the matured oligosaccharide, defective trafficking to the cell surface, and more susceptibility to the cleavage and or degradation. Our findings provide key support for the unified idea that impaired synaptogenesis underlies the pathogenesis of ASD.     

Lack of Association between NLGN3, NLGN4, SHANK2 and SHANK3 Gene Variants and Autism Spectrum Disorder in a Chinese Population

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication, absence or delay in language development, and stereotyped or repetitive behaviors. Genetic studies show that neurexin-neuroligin (NRXN-NLGN) pathway genes contribute susceptibility to ASD, which include cell adhesion molecules NLGN3, NLGN4 and scaffolding proteins SHANK2 and SHANK3. Neuroligin proteins play an important role in synaptic function and trans-synaptic signaling by interacting with presynaptic neurexins. Shank proteins are scaffolding molecules of excitatory synapses, which function as central organizers of the postsynaptic density. Sequence level mutations and structural variations in these genes have been identified in ASD cases, while few studies were performed in Chinese population. In this study, we examined the copy numbers of four genes NLGN4, NLGN3, SHANK2, and SHANK3 in 285 ASD cases using multiplex fluorescence competitive polymerase chain reaction (PCR). We also screened the regulatory region including the promoter region and 5′/3′ untranslated regions (UTR) and the entire coding region of NLGN4 in a cohort of 285 ASD patients and 384 controls by direct sequencing of genomic DNA using the Sanger method. DNA copy number calculation in four genes showed no deletion or duplication in our cases. No missense mutations in NLGN4 were identified in our cohort. Association analysis of 6 common SNPs in NLGN4 did not find significant difference between ASD cases and controls. These findings showed that these genes may not be major disease genes in Chinese ASD cases.     

A Pilot Feasibility Study of Neurofeedback for Children with Autism

Neurofeedback (NFB) is an emerging treatment for children with autism spectrum disorder (ASD). This pilot study examined the feasibility of NFB for children with ASD. Ten children ages 7–12 with high functioning ASD and attention difficulties received a NFB attention training intervention. A standardized checklist captured feasibility, including focus during exercises and academic tasks, as well as off-task behaviors. Active behaviors and vocalizations were the most frequent off-task behaviors. Positive reinforcement and breaks including calm breathing exercises were the most common supports. Low motivation was associated with higher feasibility challenges, yet parental involvement and accommodations were helpful. This pilot study shows that it is feasible to conduct NFB sessions with children with high functioning autism and attention difficulties.     

Intellectual Disability Is Associated with Increased Runs of Homozygosity in Simplex Autism

Intellectual disability (ID), often attributed to autosomal-recessive mutations, occurs in 40% of autism spectrum disorders (ASDs). For this reason, we conducted a genome-wide analysis of runs of homozygosity (ROH) in simplex ASD-affected families consisting of a proband diagnosed with ASD and at least one unaffected sibling. In these families, probands with an IQ ≤ 70 show more ROH than their unaffected siblings, whereas probands with an IQ > 70 do not show this excess. Although ASD is far more common in males than in females, the proportion of females increases with decreasing IQ. Our data do support an association between ROH burden and autism diagnosis in girls; however, we are not able to show that this effect is independent of low IQ. We have also discovered several autism candidate genes on the basis of finding (1) a single gene that is within an ROH interval and that is recurrent in autism or (2) a gene that is within an autism ROH block and that harbors a homozygous, rare deleterious variant upon analysis of exome-sequencing data. In summary, our data suggest a distinct genetic architecture for participants with autism and co-occurring intellectual disability and that this architecture could involve a role for recessively inherited loci for this autism subgroup.     

CACNA1H mutations in autism spectrum disorders

Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impaired social interaction, communication skills, and restricted and repetitive behavior. The genetic causes for autism are largely unknown. Previous studies implicate CACNA1C (L-type Ca(V)1.2) calcium channel mutations in a disorder associated with autism (Timothy syndrome). Here, we identify missense mutations in the calcium channel gene CACNA1H (T-type Ca(V)3.2) in 6 of 461 individuals with ASD. These mutations are located in conserved and functionally relevant domains and are absent in 480 ethnically matched controls (p = 0.014, Fisher's exact test). Non-segregation within the pedigrees between the mutations and the ASD phenotype clearly suggest that the mutations alone are not responsible for the condition. However, functional analysis shows that all these mutations significantly reduce Ca(V)3.2 channel activity and thus could affect neuronal function and potentially brain development. We conclude that the identified mutations could contribute to the development of the ASD phenotype.     

Sensory impairment in mental retardation: a potential role for NGF

Sensory impairment is defined as the inability to interpret outside stimuli such as visual, auditory, verbal, sense of touch, taste or smell or feelings of pain. This leads to absence of sensation and neuronal coordination. The impairment may be caused by ageing and other physiological changes, accident or injuries or can be found in some cases of mental retardation (MR) also referred to as intellectual disability. Known cases of MR involving inability to accurately interpret an outside source or stimuli are: Fragile-X syndrome; Tuberous sclerosis complex (TSC) with associated autism spectrum disorder (ASD); Rett syndrome; Autism and ASD with or without MR; Chromosome 22q13.3 deletion syndrome; familial dysautonomia, Prader-Willi's syndrome, Williams syndrome. In this review we will discuss in particular form of ASD and altered sensory sensitivity. The role of NGF in causing pronociceptive activity and its role in peripheral sensitisation is discussed under the light of its involvement in forms of MR where loss of pain perception is a main feature due to mutations to NGF receptors or NGF genes during development. Other forms of MR with altered sensory impairment will be considered as well as additional potential mechanisms involved.     

Brother/sister pairs affected with early-onset, progressive muscular dystrophy: molecular studies reveal etiologic heterogeneity.

An autosomal recessive (AR) form of muscular dystrophy that clinically resembles Duchenne/Becker types exists, but its frequency is unknown. We have studied three unrelated affected brother/sister pairs and their families for deletions and polymorphisms with the entire dystrophin cDNA and other DNA probes from the Xp21 region to test for involvement of the DMD locus. In family 1 a large intragenic deletion was found in the affected male. The affected sister was heterozygous for this deletion, but the mother was not, implying germinal mosaicism. In family 2, no deletion was detected in the affected male. RFLP analysis revealed that the affected male and an unaffected sister shared a complete Xp21 haplotype while the affected sister had inherited a recombinant Xp21 region resulting from a crossover between pERT 87-15 and J-Bir. Only the 5' region of the dystrophin gene was shared with the affected boy. X-inactivation studies using a polymorphism in the 5'-flanking region of the HPRT gene, in conjunction with methylation-sensitive enzymes, revealed random X inactivation in the affected girl's leukocytes. In a muscle biopsy from the affected male, the dystrophin protein was present in normal amount and size. Family 3 was informative for four RFLPs detected with dystrophin cDNA probes which span the entire gene. The affected male was found to share the complete dystrophin RFLP haplotype with his unaffected brother, while his affected sister had inherited the other maternal haplotype. It is concluded that the clinical presentation of early-onset, progressive muscular dystrophy in a male and in his karyotypically normal sister can be caused by mutations at different loci. While in family 1 a deletion in the dystrophin gene is responsible, this gene does not appear to be involved in families 2 and 3.     

Integrated Model of De Novo and Inherited Genetic Variants Yields Greater Power to Identify Risk Genes

De novo mutations affect risk for many diseases and disorders, especially those with early-onset. An example is autism spectrum disorders (ASD). Four recent whole-exome sequencing (WES) studies of ASD families revealed a handful of novel risk genes, based on independent de novo loss-of-function (LoF) mutations falling in the same gene, and found that de novo LoF mutations occurred at a twofold higher rate than expected by chance. However successful these studies were, they used only a small fraction of the data, excluding other types of de novo mutations and inherited rare variants. Moreover, such analyses cannot readily incorporate data from case-control studies. An important research challenge in gene discovery, therefore, is to develop statistical methods that accommodate a broader class of rare variation. We develop methods that can incorporate WES data regarding de novo mutations, inherited variants present, and variants identified within cases and controls. TADA, for Transmission And De novo Association, integrates these data by a gene-based likelihood model involving parameters for allele frequencies and gene-specific penetrances. Inference is based on a Hierarchical Bayes strategy that borrows information across all genes to infer parameters that would be difficult to estimate for individual genes. In addition to theoretical development we validated TADA using realistic simulations mimicking rare, large-effect mutations affecting risk for ASD and show it has dramatically better power than other common methods of analysis. Thus TADA''s integration of various kinds of WES data can be a highly effective means of identifying novel risk genes. Indeed, application of TADA to WES data from subjects with ASD and their families, as well as from a study of ASD subjects and controls, revealed several novel and promising ASD candidate genes with strong statistical support.     

Dysregulation of translational control signaling in autism spectrum disorders

Deviations from the optimal level of mRNA translation are linked to disorders with high rates of autism. Loss of function mutations in genes encoding translational repressors such as PTEN, TSC1, TSC2, and FMRP are associated with autism spectrum disorders (ASDs) in humans and their deletion in animals recapitulates many ASD-like phenotypes. Importantly, the activity of key translational control signaling pathways such as PI3K-mTORC1 and ERK is frequently dysregulated in autistic patients and animal models and their normalization rescues many abnormal phenotypes, suggesting a causal relationship. Mutations in several genes encoding proteins not directly involved in translational control have also been shown to mediate ASD phenotypes via altered signaling upstream of translation. This raises the possibility that the dysregulation of translational control signaling is a converging mechanism not only in familiar but also in sporadic forms of autism. Here, we overview the current knowledge on translational signaling in ASD and highlight how correcting the activity of key pathways upstream of translation reverses distinct ASD-like phenotypes.     

Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and core autism-related deficits

Although many genes predisposing to autism spectrum disorders (ASD) have been identified, the biological mechanism(s) remain unclear. Mouse models based on human disease-causing mutations provide the potential for understanding gene function and novel treatment development. Here, we characterize a mouse knockout of the Cntnap2 gene, which is strongly associated with ASD and allied neurodevelopmental disorders. Cntnap2(-/-) mice show deficits in the three core ASD behavioral domains, as well as hyperactivity and epileptic seizures, as have been reported in humans with CNTNAP2 mutations. Neuropathological and physiological analyses of these mice before the onset of seizures reveal neuronal migration abnormalities, reduced number of interneurons, and abnormal neuronal network activity. In addition, treatment with the FDA-approved drug risperidone ameliorates the targeted repetitive behaviors in the mutant mice. These data demonstrate a functional role for CNTNAP2 in brain development and provide a new tool for mechanistic and therapeutic research in ASD.