Local patterns of genetic sharing challenge the boundaries between neuropsychiatric and insulin resistance-related conditions.

The co-occurrence of insulin resistance (IR)-related metabolic conditions with neuropsychiatric disorders is a complex public health challenge. Evidence of the genetic links between these phenotypes is emerging, but little is currently known about the genomic regions and biological functions that are involved. To address this, we performed Local Analysis of [co]Variant Association (LAVA) using large-scale (N=9,725-933,970) genome-wide association studies (GWASs) results for three IR-related conditions (type 2 diabetes mellitus, obesity, and metabolic syndrome) and nine neuropsychiatric disorders. Subsequently, positional and expression quantitative trait locus (eQTL)-based gene mapping and downstream functional genomic analyses were performed on the significant loci. Patterns of negative and positive local genetic correlations (|rg|=0.21-1, pFDR<0.05) were identified at 109 unique genomic regions across all phenotype pairs. Local correlations emerged even in the absence of global genetic correlations between IR-related conditions and Alzheimer's disease, bipolar disorder, and Tourette's syndrome. Genes mapped to the correlated regions showed enrichment in biological pathways integral to immune-inflammatory function, vesicle trafficking, insulin signalling, oxygen transport, and lipid metabolism. Colocalisation analyses further prioritised 10 genetically correlated regions for likely harbouring shared causal variants, displaying high deleterious or regulatory potential. These variants were found within or in close proximity to genes, such as SLC39A8 and HLA-DRB1, that can be targeted by supplements and already known drugs, including omega-3/6 fatty acids, immunomodulatory, antihypertensive, and cholesterol-lowering drugs. Overall, our findings underscore the complex genetic landscape of IR-neuropsychiatric multimorbidity, advocating for an integrated disease model and offering novel insights for research and treatment strategies in this domain.

The role of insulin/IGF1 signalling in neurodevelopmental and neuropsychiatric disorders - Evidence from human neuronal cell models.

Insulin and insulin-like growth factor 1 (IGF1) signalling play a central role in the development and maintenance of neurons in the brain, and human neurodevelopmental as well as neuropsychiatric disorders have been linked to impaired insulin and IGF1 signalling. This review focuses on the impairments of the insulin and IGF1 signalling cascade in the context of neurodevelopmental and neuropsychiatric disorders, based on evidence from human neuronal cell models. Clear evidence was obtained for impaired insulin and IGF1 receptor downstream signalling in neurodevelopmental disorders, while the evidence for its role in neuropsychiatric disorders was less substantial. Human neuronal model systems can greatly add to our knowledge about insulin/IGF1 signalling in the brain, its role in restoring dendritic maturity, and complement results from clinical studies and animal models. Moreover, they represent a useful model for the development of new therapeutic strategies. Further research is needed to systematically investigate the exact role of the insulin/IGF1 signalling cascades in neurodevelopmental and neuropsychiatric disorders, and to elucidate the respective therapeutic implications.

Imbalanced post- and extrasynaptic SHANK2A functions during development affect social behavior in SHANK2-mediated neuropsychiatric disorders.

Mutations in SHANK genes play an undisputed role in neuropsychiatric disorders. Until now, research has focused on the postsynaptic function of SHANKs, and prominent postsynaptic alterations in glutamatergic signal transmission have been reported in Shank KO mouse models. Recent studies have also suggested a possible presynaptic function of SHANK proteins, but these remain poorly defined. In this study, we examined how SHANK2 can mediate electrophysiological, molecular, and behavioral effects by conditionally overexpressing either wild-type SHANK2A or the extrasynaptic SHANK2A(R462X) variant. SHANK2A overexpression affected pre- and postsynaptic targets and revealed a reversible, development-dependent autism spectrum disorder-like behavior. SHANK2A also mediated redistribution of Ca2+-permeable AMPA receptors between apical and basal hippocampal CA1 dendrites, leading to impaired synaptic plasticity in the basal dendrites. Moreover, SHANK2A overexpression reduced social interaction and increased the excitatory noise in the olfactory cortex during odor processing. In contrast, overexpression of the extrasynaptic SHANK2A(R462X) variant did not impair hippocampal synaptic plasticity, but still altered the expression of presynaptic/axonal signaling proteins. We also observed an attention-deficit/hyperactivity-like behavior and improved social interaction along with enhanced signal-to-noise ratio in cortical odor processing. Our results suggest that the disruption of pre- and postsynaptic SHANK2 functions caused by SHANK2 mutations has a strong impact on social behavior. These findings indicate that pre- and postsynaptic SHANK2 actions cooperate for normal neuronal function, and that an imbalance between these functions may lead to different neuropsychiatric disorders.

SHANK2 mutations impair apoptosis, proliferation and neurite outgrowth during early neuronal differentiation in SH-SY5Y cells.

SHANK2 mutations have been identified in individuals with neurodevelopmental disorders, including intellectual disability and autism spectrum disorders (ASD). Using CRISPR/Cas9 genome editing, we obtained SH-SY5Y cell lines with frameshift mutations on one or both SHANK2 alleles. We investigated the effects of the different SHANK2 mutations on cell morphology, cell proliferation and differentiation potential during early neuronal differentiation. All mutant cell lines showed impaired neuronal differentiation marker expression. Cells with bi-allelic SHANK2 mutations revealed diminished apoptosis and increased proliferation, as well as decreased neurite outgrowth during early neuronal differentiation. Bi-allelic SHANK2 mutations resulted in an increase in p-AKT levels, suggesting that SHANK2 mutations impair downstream signaling of tyrosine kinase receptors. Additionally, cells with bi-allelic SHANK2 mutations had lower amyloid precursor protein (APP) expression compared to controls, suggesting a molecular link between SHANK2 and APP. Together, we can show that frameshift mutations on one or both SHANK2 alleles lead to an alteration of neuronal differentiation in SH-SY5Y cells, characterized by changes in cell growth and pre- and postsynaptic protein expression. We also provide first evidence that downstream signaling of tyrosine kinase receptors and amyloid precursor protein expression are affected.

Generation of two hiPSC lines from a patient with autism spectrum disorder harboring a 120 kb deletion in SHANK2 and two control lines from each parent.

Two human induced pluripotent stem cell lines (hiPSC) were generated by reprogramming fibroblasts isolated from a skin biopsy taken from a female patient diagnosed with autism spectrum disorder (ASD) and intellectual disability (ID). This patient harbors a de novo 120 kb deletion in SHANK2. As controls, four lines were generated in a similar manner from fibroblasts isolated from each of her parents, two clones per parent. All reported hiPSC lines have a normal karyotype, express pluripotency markers and have the ability to differentiate into all three germ layers.

Sex Hormones Regulate SHANK Expression.

Autism spectrum disorders (ASD) have a higher prevalence in male individuals compared to females, with a ratio of affected boys compared to girls of 4:1 for ASD and 11:1 for Asperger syndrome. Mutations in the SHANK genes (comprising SHANK1, SHANK2 and SHANK3) coding for postsynaptic scaffolding proteins have been tightly associated with ASD. As early brain development is strongly influenced by sex hormones, we investigated the effect of dihydrotestosterone (DHT) and 17ß-estradiol on SHANK expression in a human neuroblastoma cell model. Both sex hormones had a significant impact on the expression of all three SHANK genes, which could be effectively blocked by androgen and estrogen receptor antagonists. In neuron-specific androgen receptor knock-out mice (Ar NesCre), we found a nominal significant reduction of all Shank genes at postnatal day 7.5 in the cortex. In the developing cortex of wild-type (WT) CD1 mice, a sex-differential protein expression was identified for all Shanks at embryonic day 17.5 and postnatal day 7.5 with significantly higher protein levels in male compared to female mice. Together, we could show that SHANK expression is influenced by sex hormones leading to a sex-differential expression, thus providing novel insights into the sex bias in ASD.

A direct regulatory link between microRNA-137 and SHANK2: implications for neuropsychiatric disorders.

BACKGROUND: Mutations in the SHANK genes, which encode postsynaptic scaffolding proteins, have been linked to a spectrum of neurodevelopmental disorders. The SHANK genes and the schizophrenia-associated microRNA-137 show convergence on several levels, as they are both expressed at the synapse, influence neuronal development, and have a strong link to neurodevelopmental and neuropsychiatric disorders like intellectual disability, autism, and schizophrenia. This compiled evidence raised the question if the SHANKs might be targets of miR-137. METHODS: In silico analysis revealed a putative binding site for microRNA-137 (miR-137) in the SHANK2 3'UTR, while this was not the case for SHANK1 and SHANK3. Luciferase reporter assays were performed by overexpressing wild type and mutated SHANK2-3'UTR and miR-137 in human neuroblastoma cells and mouse primary hippocampal neurons. miR-137 was also overexpressed or inhibited in hippocampal neurons, and Shank2 expression was analyzed by quantitative real-time PCR and Western blot. Additionally, expression levels of experimentally validated miR-137 target genes were analyzed in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia and control individuals using the RNA-Seq data from the CommonMind Consortium. RESULTS: miR-137 directly targets the 3'UTR of SHANK2 in a site-specific manner. Overexpression of miR-137 in mouse primary hippocampal neurons significantly lowered endogenous Shank2 protein levels without detectable influence on mRNA levels. Conversely, miR-137 inhibition increased Shank2 protein expression, indicating that miR-137 regulates SHANK2 expression by repressing protein translation rather than inducing mRNA degradation. To find out if the miR-137 signaling network is altered in schizophrenia, we compared miR-137 precursor and miR-137 target gene expression in the DLPFC of schizophrenia and control individuals using the CommonMind Consortium RNA sequencing data. Differential expression of 23% (16/69) of known miR-137 target genes was detected in the DLPFC of schizophrenia individuals compared with controls. We propose that in further targets (e.g., SHANK2, as described in this paper) which are not regulated on RNA level, effects may only be detectable on protein level. CONCLUSION: Our study provides evidence that a direct regulatory link exists between miR-137 and SHANK2 and supports the finding that miR-137 signaling might be altered in schizophrenia.

A novel homozygous ARL13B variant in patients with Joubert syndrome impairs its guanine nucleotide-exchange factor activity.

ARL13B encodes for the ADP-ribosylation factor-like 13B GTPase, which is required for normal cilia structure and Sonic hedgehog (Shh) signaling. Disruptions in cilia structure or function lead to a class of human disorders called ciliopathies. Joubert syndrome is characterized by a wide spectrum of symptoms, including a variable degree of intellectual disability, ataxia, and ocular abnormalities. Here we report a novel homozygous missense variant c.[223G>A] (p.(Gly75Arg) in the ARL13B gene, which was identified by whole-exome sequencing of a trio from a consanguineous family with multiple-affected individuals suffering from intellectual disability, ataxia, ocular defects, and epilepsy. The same variant was also identified in a second family. We saw a striking difference in the severity of ataxia between affected male and female individuals in both families. Both ARL13B and ARL13B-c.[223G>A] (p.(Gly75Arg) expression rescued the cilia length and Shh defects displayed by Arl13b hennin (null) cells, indicating that the variant did not disrupt either ARL13B function. In contrast, ARL13B-c.[223G>A] (p.(Gly75Arg) displayed a marked loss of ARL3 guanine nucleotide-exchange factor activity, with retention of its GTPase activities, highlighting the correlation between its loss of function as an ARL3 guanine nucleotide-exchange factor and Joubert syndrome.

Investigation of SHANK3 in schizophrenia.

The postsynaptic scaffolding protein SHANK3 is essential for the normal function of glutamatergic synapses in the brain. Emerging evidence suggests that impaired plasticity of glutamatergic synapses contributes to the pathology of schizophrenia (SCZ). To investigate whether variants in the SHANK3 gene contribute to the etiology of SCZ, we sequenced SHANK3 in 500 affected individuals (cohort C1). In total, we identified 48 variants and compared them to European controls from the 1000 Genomes Project and the Exome Variant Server. Five variants showed significant differences in frequencies between patients and controls. We were able to follow three of them up in an independent cohort (C2) comprising 993 SCZ patients and 932 German controls. We could not confirm an association for three of these variants (rs140201628, rs1557620, and rs61729471). Two rare variants with predicted functional relevance were identified in further SCZ individuals of cohort C1: c.3032G>T (p.G1011V) and c.*27C>T. The latter variant was found in one additional SCZ individual and the p.G1011V variant was identified in two additional SCZ individuals from cohort C2. The p.G1011V variant was the most interesting variant in our study; together with previous studies this variant has been identified in 4 out of 1,524 SCZ patients and in 4 out of 2,147 individuals with autism spectrum disorder (ASD), but not in 2468 European Sanger-sequenced controls. Therefore, we consider this variant a promising candidate variant for follow-up studies in larger samples and functional investigations. © 2017 Wiley Periodicals, Inc.

Homozygous missense mutation in the LMAN2L gene segregates with intellectual disability in a large consanguineous Pakistani family.

BACKGROUND: Intellectual disability (ID) is a neurodevelopmental disorder affecting 1%-3% of the population worldwide. It is characterised by high phenotypic and genetic heterogeneity and in most cases the underlying cause of the disorder is unknown. In our study we investigated a large consanguineous family from Baluchistan, Pakistan, comprising seven affected individuals with a severe form of autosomal recessive ID (ARID) and epilepsy, to elucidate a putative genetic cause. METHODS AND RESULTS: Whole exome sequencing (WES) of a trio, including a child with ID and epilepsy and its healthy parents that were part of this large family, revealed a homozygous missense variant p.R53Q in the lectin mannose-binding 2-like (LMAN2L) gene. This homozygous variant was co-segregating in the family with the phenotype of severe ID and infantile epilepsy; unaffected family members were heterozygous variant carriers. The variant was predicted to be pathogenic by five different in silico programmes and further three-dimensional structure modelling of the protein suggests that variant p.R53Q may impair protein-protein interaction. LMAN2L (OMIM: 609552) encodes for the lectin, mannose-binding 2-like protein which is a cargo receptor in the endoplasmic reticulum important for glycoprotein transport. Genome-wide association studies have identified an association of LMAN2L to different neuropsychiatric disorders. CONCLUSION: This is the first report linking LMAN2L to a phenotype of severe ARID and seizures, indicating that the deleterious homozygous p.R53Q variant very likely causes the disorder.

Inherited and de novo SHANK2 variants associated with autism spectrum disorder impair neuronal morphogenesis and physiology.

Mutations in the postsynaptic scaffolding gene SHANK2 have recently been identified in individuals with autism spectrum disorder (ASD) and intellectual disability. However, the cellular and physiological consequences of these mutations in neurons remain unknown. We have analyzed the functional impact caused by two inherited and one de novo SHANK2 mutations from ASD individuals (L1008_P1009dup, T1127M, R462X). Although all three variants affect spine volume and have smaller SHANK2 cluster sizes, T1127M additionally fails to rescue spine volume in Shank2 knock-down neurons. R462X is not able to rescue spine volume and dendritic branching and lacks postsynaptic clustering, indicating the most severe dysfunction. To demonstrate that R462X when expressed in mouse can be linked to physiological effects, we analyzed synaptic transmission and behavior. Principal neurons of mice expressing rAAV-transduced SHANK2-R462X present a specific, long-lasting reduction in miniature postsynaptic AMPA receptor currents. This dominant negative effect translates into dose-dependent altered cognitive behavior of SHANK2-R462X-expressing mice, with an impact on the penetrance of ASD.

Mutations in the SHANK2 synaptic scaffolding gene in autism spectrum disorder and mental retardation.

Using microarrays, we identified de novo copy number variations in the SHANK2 synaptic scaffolding gene in two unrelated individuals with autism-spectrum disorder (ASD) and mental retardation. DNA sequencing of SHANK2 in 396 individuals with ASD, 184 individuals with mental retardation and 659 unaffected individuals (controls) revealed additional variants that were specific to ASD and mental retardation cases, including a de novo nonsense mutation and seven rare inherited changes. Our findings further link common genes between ASD and intellectual disability.

Poorly differentiated small cell carcinoma of the pancreas. A case report and review of the literature.

Small cell carcinoma (SCC) of the pancreas is a rare malignancy with an extremely poor prognosis. We present the case of a 74-year-old man with a 2-month history of upper abdominal discomfort who was diagnosed with SCC of the pancreas tail, involvement of peripancreatic and mesenteric lymph nodes and multiple liver metastases (extended disease). A CT scan and a positive somatostatin receptor scintigraphy showed no evidence of a primary lung tumour. The diagnosis of a SCC was confirmed by biopsy. Local tumour control could be achieved by gemcitabine once a week and a long-acting somatostatin analogue once a month, but liver metastasis showed progress. Thus, 5-fluorouracil on a weekly basis was started. The patient died 8 months after diagnosis and had not been hospitalised in the meantime.