Meta-analysis of SHANK Mutations in Autism Spectrum Disorders: a gradient of severity in cognitive impairments.

Mutations in the synaptic gene SHANK3 lead to a neurodevelopmental disorder known as Phelan-McDermid syndrome (PMS). PMS is a relatively common monogenic and highly penetrant cause of autism spectrum disorder (ASD) and intellectual disability (ID), and frequently presents with attention deficits. The underlying neurobiology of PMS is not fully known and pharmacological treatments for core symptoms do not exist. Here, we report the production and characterization of a Shank3-deficient rat model of PMS, with a genetic alteration similar to a human SHANK3 mutation. We show that Shank3-deficient rats exhibit impaired long-term social recognition memory and attention, and reduced synaptic plasticity in the hippocampal-medial prefrontal cortex pathway. These deficits were attenuated with oxytocin treatment. The effect of oxytocin on reversing non-social attention deficits is a particularly novel finding, and the results implicate an oxytocinergic contribution in this genetically defined subtype of ASD and ID, suggesting an individualized therapeutic approach for PMS.DOI: http://dx.doi.org/10.7554/eLife.18904.001

Chapter 4.7 - SHANK2 and SHANK3 Mutations Implicate Glutamate Signaling Abnormalities in Autism Spectrum Disorders

Many studies have found a variety of SHANK3 mutants in Autism spectrum disorder (ASD) patients, and SHANK3 mutants are believed to be closely related to ASD. SHANK3 is a gene on chromosome 22 that encodes a postsynaptic density scaffold protein and is often considered to play an indispensable role in glutamatergic synapses. ASD has a rather complex mechanism and is not fully understood. It is related to many genes, and SHANK3 is a strong candidate gene. Many studies have found that a variety of SHANK3 mutant genes, especially de novo SHANK3 gene, can cause decreased excitability and even spinal dysplasia, which becomes one of the important contributing genes of ASD. ASD symptoms have also been observed by direct modification of SHANK3 gene using CRISPR in various experimental animals. At present, there are many treatments for ASD, but none of them can cure ASD. Improving knowledge of the association between the SHANK3 gene and ASD and additional research on this subject may be one of the potential therapy modalities.

Copy number variants (CNVs) have a major role in the etiology of autism spectrum disorders (ASD), and several of these have reached statistical significance in case–control analyses. Nevertheless, current ASD cohorts are not large enough to detect very rare CNVs that may be causative or contributory (that is, risk alleles). Here, we use a tiered approach, in which clinically significant CNVs are first identified in large clinical cohorts of neurodevelopmental disorders (including but not specific to ASD), after which these CNVs are then systematically identified within well-characterized ASD cohorts. We focused our initial analysis on 48 recurrent CNVs (segmental duplication-mediated ‘hotspots’) from 24 loci in 31 516 published clinical cases with neurodevelopmental disorders and 13 696 published controls, which yielded a total of 19 deletion CNVs and 11 duplication CNVs that reached statistical significance. We then investigated the overlap of these 30 CNVs in a combined sample of 3955 well-characterized ASD cases from three published studies. We identified 73 deleterious recurrent CNVs, including 36 deletions from 11 loci and 37 duplications from seven loci, for a frequency of 1 in 54; had we considered the ASD cohorts alone, only 58 CNVs from eight loci (24 deletions from three loci and 34 duplications from five loci) would have reached statistical significance. In conclusion, until there are sufficiently large ASD research cohorts with enough power to detect very rare causative or contributory CNVs, data from larger clinical cohorts can be used to infer the likely clinical significance of CNVs in ASD.

To explore possible relationship between copy-number variations (CNVs) in 15q11-13, 16p11 and SHANK3 gene by using multiplex ligation-dependent probe amplification (MLPA) and the phenotypes in children with autism and to further explore the clinical application of MLPA to make an etiological diagnosis of Autism. The diagnosed of autism was made according to the criteria of the ICD-10 and DSM-IV, with typical cluster of symptoms comprise social disability, communication impairments and repetitious behaviors. MLPA KIT P343-C1 AUTISM-1 was used to detect and describe the incidence of CNVs in these three domains. Among 109 cases collected from 102 autistic pedigrees, 2 individuals had SHANK3 microdeletion, accounting for approximately 2% (2/109) of cases, suggesting the proportion of SHANK3 microdeletion might contribute to typical autism. The phenotypic traits of patients with SHANK3 microdeletions showed homogenicity in severe core symptoms and mental retardation. SHANK3 microdeletion is an important genetics component for autism, which may explain 2% typical autism cases. SHANK3 microdeletion might explain autistic core symptoms and mental retardation. MLPA is a sensitive and a high throughput technique to detect CNVs in specific DNA segments, which is beneficial for further investigation of etiology of autism.

SU16 - THE IMPACT OF CNVs ON ASD/ADHD RISK IN MULTIPLEX FAMILIES

There is a high rate of comorbidity between autism spectrum disorder (ASD), obsessive-compulsive disorder (OCD) and eating disorders (EDs). Furthermore, neuropsychological studies and genetic studies support the presence of shared background among these disorders.1-4 All of these findings suggest shared pathophysiology of these disorders. NBEA encodes neurobeachin and has been strongly implicated in ASD risk by the identification of de novo loss-of-function variants of this gene in multiple unrelated cases.5-7 Neurobeachin regulates recycling of GluN2B-NMDA receptors, and is involved in neurotransmission, synaptic formation and plasticity by a haploinsufficient genetic mechanism.8 Although we also identified an ASD patient with rare deletion disrupting this gene in a study of copy number variation (CNV),2 only limited knowledge was available about the clinical implications of NBEA deletion. It is important to accumulate high-quality case reports to improve the clinical management of patients with such CNVs. Therefore, we report a 10-year clinical course of an ASD patient with NBEA deletion, who had comorbidities of OCD, ED, and intellectual disability (ID). The patient was a 41-year-old Japanese female. Her father had a broad autism phenotype. During infancy, language developmental delay, insistence on the same clothes and foods, tactile hypersensitivity, and impairments in communication and social interactions were noticed. She had very little social contact with her peers throughout her school career. After she graduated from junior college, she had several part-time jobs but soon quit because of interpersonal conflicts in the workplace. Her developmental history is summarized in Table S1. At the age of 31, she had recurrent episodes of overeating and rapidly gained weight. In response to this sudden weight gain, she began to restrict her food intake. Thereafter, her body mass index (BMI) decreased to 12.3 from 21.1 kg/m2 in 6 months. She was hospitalized with a diagnosis of unspecified feeding or eating disorder according to the Diagnostic and Statistical Manual of Mental Disorders-Fifth Edition (DSM-5) diagnostic criteria. Additionally, during the hospitalization, she was diagnosed with ASD. Psychoeducation about ED and ASD and supportive psychotherapy were provided. Although she did not meet the diagnostic criteria for major depressive disorders, pharmacotherapy was initiated to target her depressive symptoms. Gradually, her food intake increased, her BMI recovered to 15.4 kg/m2, and she was discharged in 3 months. After regaining weight, overeating was observed, but improved after discontinuing olanzapine medication. The patient continued to receive outpatient treatment, but obsessive-compulsive symptoms with obsessive thoughts appeared. She developed compulsive behaviors such as hand or body washing, and she spent a lot of time performing such behaviors. She was diagnosed with OCD and received medication (selective serotonin reuptake inhibitors and atypical antipsychotics) and cognitive behavioral therapy, but the symptoms were intractable. Since her last hospitalization, her symptoms have been under control. The clinical time course is summarized in Fig. 1a. Her intelligence quotient score was 62 (Table S2). Array comparative genomic hybridization using NimbleGen 720k Whole-Genome Tiling Array detected a 447-kb deletion spanning exons 11–41 of NBEA2 (Fig. 1b,c). Parental CNV analysis revealed that NBEA deletion was paternally inherited (Fig. 1d). Gene expression analysis revealed that the NBEA mRNA expression level was reduced below the 15th percentile of the control group (N = 29) (Fig. 1e, Appendix S1). We reported an ASD patient with OCD, ED, ID, and NBEA deletion. Neuropsychological studies support the presence of shared traits among ASD, OCD, and EDs.1 As shown in Table S2, ASD patients with NBEA deletion have been reported.6, 7 This is the first case report of an adult with NBEA deletion. Though previously reported cases did not mention OCD and EDs, these patients had not reached the peak age of onset of these disorders. Heterozygous Nbea knockout mice exhibit not only autistic behaviors but also impaired extinction of contextual fear memory, which is associated with OCD, and abnormal feeding behavior, episodically overconsuming tastants.9, 10 Thus, the clinical phenotypes of the patient were similar to those of heterozygous Nbea knockout mice, raising the possibility that exonic NBEA deletion was responsible for the symptoms in this patient. As Nbea knockout mice show hyperphagia, patients with NBEA deletion may be more prone to episodic overeating. The patient we reported showed rapid weight gain during olanzapine medication. Choosing medicines carefully and monitoring weight are important. In conclusion, NBEA deletion may be a shared genetic underpinning of ASD, OCD, and EDs. The accumulation of clinical data of patients with NBEA deletion including the present case may contribute to establishment of appropriate clinical management of these patients in the future. We thank the patient and her family members for participating in this study. We also thank Mami Yoshida, Kiyori Monta, and Yukari Mitsui for their technical assistance. This research was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and the Ministry of Health, Labour and Welfare of Japan; the Japan Agency for Medical Research and Development (AMED) under Grant Nos. JP20dm0107087, JP21wm0425007, JP21dm0207075, JP21ak0101113, JP21dk0307075, JP20dk0307081, JP21dk0307103, JP21ek0109488, JP21km0405216, and JP21ek0109411; the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Nos. 17H05090, 15K19720, 18H04040, 21K07543, 21H00194, 21H04815, and 20K20602; the Uehara Memorial Foundation; and the SENSHIN Medical Research Foundation. H.K., I.K., A.Y., K.I., K.I., and B.A. declare no conflict of interest. N.T. has received research support or speakers' honoraria from, or has served as a consultant to, Otsuka, Shionogi, Takeda, Janssen, and Pfizer, outside the submitted work. T.O. has received lecture fees from Shionogi and Mochida, outside the submitted work. N.O. has received research support or speakers' honoraria from, or has served as a consultant to, Sumitomo Dainippon, Eisai, Otsuka, KAITEKI, Mitsubishi Tanabe, Shionogi, Eli Lilly, Mochida, DAIICHI SANKYO, Nihon Medi-Physics, Takeda, Meiji Seika Pharma, EA Pharma, Pfizer, MSD, Lundbeck Japan, Taisho Pharma, outside the submitted work. This study was approved by the ethics committee of the Nagoya University Graduate School of Medicine and this study complied with all the provisions of the Declaration of Helsinki. The patient and her parents gave written informed consent for genetic testing and publishing of this report. Appendix S1. Supplementary Appendix. Methods of gene expression analysis Appendix S2. Author Contributions Table S1. Time course of the patient's developmental history. Table S2. Clinical summary of cases with NBEA deletion Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The genetic heterogeneity of neuropsychiatric disorders is high, but some pathways emerged, notably synaptic functioning. A large number of mutations have been described in genes such as neuroligins, neurexins, and SHANK that play a role in the formation and the maintenance of synapses. This review focuses on the disorders associated with mutations in SHANK3 and the other members of its family, SHANK1 and SHANK2. SHANKs are scaffolding proteins of the postsynaptic density of glutamatergic synapses. SHANK3 has been described in the Phelan-McDermid syndrome (PMS), but also in autism spectrum disorders (ASD) and schizophrenia associated to moderate to severe intellectual disability (ID) and poor language. The evolution of patients with PMS includes symptoms of bipolar disorder and regression. SHANK2 has been identified in patients with ASD with mild to severe ID. SHANK1 has been associated with high-functioning autism in male patients, while carrier females only display anxiety and shyness. Finally, based on neuropathological findings in animal models and patients, a possible role of SHANK in Alzheimer's disease is discussed. Altogether, this review describes the clinical trajectories associated with different mutations of the SHANK genes and provides information to further investigate the role of the SHANK genes in neuropsychiatric disorders.

Autism spectrum disorder (ASD) is heterogeneous in symptom and etiology. Rare copy number variations (CNVs) are important genetic factors contributing to ASD. Currently chromosomal microarray (CMA) detecting CNVs is recommended as a first-tier diagnostic assay, largely based on research in North America and Europe. The feature of rare CNVs has not been well characterized in ASD cohorts from non-European ancestry. In this study, high resolution CMA was utilized to investigate rare CNVs in a Chinese cohort of ASD (n = 401, including 177 mildly/moderately and 224 severely affected individuals), together with an ancestry-matched control cohort (n = 197). Diagnostic yield was about 4.2%, with 17 clinically significant CNVs identified in ASD individuals, of which 12 CNVs overlapped with recurrent autism risk loci or genes. Autosomal rare CNV burden analysis showed an overrepresentation of rare loss events in ASD cohort, whereas the rate of rare gain events correlated with the phenotypic severity. Further analysis showed rare losses disrupting genes highly intolerant of loss-of-function variants were enriched in the ASD cohort. Among these highly constrained genes disrupted by rare losses, RIMS2 is a promising candidate contributing to ASD risk. This pilot study evaluated clinical utility of CMA and the feature of rare CNVs in Chinese ASD, with candidate genes identified as potential risk factors.

Autism spectrum disorder (ASD) is a developmental disorder with a prevalence of around 1% children worldwide and characterized by patient behaviour (communication, social interaction, and personal development). Data on the efficacy of diagnostic tests using copy number variations (CNVs) in candidate genes in ASD is currently around 10% but it is overrepresented by patients of Caucasian background. We report here that the diagnostic success of de novo candidate CNVs in Vietnamese ASD patients is around 6%. We recruited one hundred trios (both parents and a child) where the child was clinically diagnosed with ASD while the parents were not affected. We performed genetic screening to exclude RETT syndrome and Fragile X syndrome and performed genome-wide DNA microarray (aCGH) on all probands and their parents to analyse for de novo CNVs. We detected 1708 non-redundant CNVs in 100 patients and 118 (7%) of them were de novo. Using the filter for known CNVs from the Simons Foundation Autism Research Initiative (SFARI) database, we identified six CNVs (one gain and five loss CNVs) in six patients (3 males and 3 females). Notably, 3 of our patients had a deletion involving the SHANK3 gene-which is the highest compared to previous reports. This is the first report of candidate CNVs in ASD patients from Vietnam and provides the framework for building a CNV based test as the first tier screening for clinical management.

Patients diagnosed with chromosome microdeletions or duplications, known as copy number variants (CNVs), present a unique opportunity to investigate the relationship between patient genotype and cell phenotype. CNVs have high genetic penetrance and give a good correlation between gene locus and patient clinical phenotype. This is especially effective for the study of patients with neurodevelopmental disorders (NDD), including those falling within the autism spectrum disorders (ASD). A key question is whether this correlation between genetics and clinical presentation at the level of the patient can be translated to the cell phenotypes arising from the neurodevelopment of patient induced pluripotent stem cells (iPSCs).Here, we examine how iPSCs derived from ASD patients with an associated CNV inform our understanding of the genetic and biological mechanisms underlying the aetiology of ASD. We consider selection of genetically characterised patient iPSCs; use of appropriate control lines; aspects of human neurocellular biology that can capture in vitro the patient clinical phenotype; and current limitations of patient iPSC-based studies. Finally, we consider how future research may be enhanced to maximise the utility of CNV patients for research of pathological mechanisms or therapeutic targets.

Reciprocal Duplication of the Williams-Beuren Syndrome Deletion on Chromosome 7q11.23 Is Associated with Schizophrenia

Although autism spectrum disorder (ASD) occurs worldwide, most genomic studies on ASD were performed on those of Western ancestry. We hypothesized ASD-related copy number variations (CNVs) of Japanese individuals might be different from those of Western individuals. Subjects were recruited from the Hirosaki 5-year-old children's developmental health check-up (HFC) between 2013 and 2016 (ASD group; n = 68, control group; n = 124). This study conducted CNV analysis using genomic DNA from peripheral blood of 5-year-old Japanese children. Fisher's exact test was applied for profiling subjects and CNV loci. Four ASD-related CNVs: deletion at 12p11.1, duplications at 4q13.2, 8p23.1 and 18q12.3 were detected (P = 0.015, 0.024, 0.009, 0.004, respectively). Specifically, the odds ratio of duplication at 18q12.3 was highest among the 4 CNVs (odds ratio, 8.13). Four CNVs: microdeletion at 12p11.1, microduplications at 4q13.2, 8p23.1 and 18q12.3 were detected as ASD-related CNVs in Japanese children in this study. Although these CNVs were consistent with several reports by Western countries at cytoband levels, these did not consistent at detailed genomic positions and sizes. Our data indicate the possibility that these CNVs are characteristic of Japanese children with ASD. We conclude that Japanese individuals with ASD may harbor CNVs different from those of Western individuals with ASD.

There now exist multiple lines of evidence pointing to a significant genetic component underlying the aetiology of autism spectrum disorders (ASDs). The advent of methodologies for scanning the human genome at high resolution, coupled with the recognition of copy number variation (CNV) as a prevalent source of genomic variation, has led to new strategies in the identification of clinically relevant loci. Balanced genomic changes, such as translocations and inversions, also contribute to ASD, but current studies have shown that screening with microarrays has up to fivefold increase in diagnostic yield. Recent work by our group and others has shown unbalanced genomic alterations that are likely pathogenic in upwards of 10% of cases, highlighting an important role for CNVs in the genetic aetiology of ASD. A trend in our empirical data has shifted focus for discovery of candidate loci towards individually rare but highly penetrant CNVs instead of looking for common variants of low penetrance. This strategy has proven largely successful in identifying ASD-susceptibility candidate loci, including gains and losses at 16p11.2, SHANK2, NRXN1, and PTCHD1. Another emerging and intriguing trend is the identification of the same genes implicated by rare CNVs across neurodevelopmental disorders, including schizophrenia, attention deficit hyperactivity disorder, and intellectual disability. These observations indicate that similar pathways may be involved in phenotypically distinct outcomes. Although interrogation of the genome at high resolution has led to these novel discoveries, it has also made cataloguing, characterization, and clinical interpretation of the increasing amount of CNV data difficult. Herein, we describe the history of genomic structural variation in ASD and how CNV discovery has been used to pinpoint novel ASD-susceptibility loci. We also discuss the overlap of CNVs across neurodevelopmental disorders and comment on the current challenges of understanding the relationship between CNVs and associated phenotypes in a clinical context.

Advances in Our Understanding of Genetic Risk Factors for Autism Spectrum Disorders