Alterations in CDH15 and KIRREL3 in patients with mild to severe intellectual disability.

Cell-adhesion molecules play critical roles in brain development, as well as maintaining synaptic structure, function, and plasticity. Here we have found the disruption of two genes encoding putative cell-adhesion molecules, CDH15 (cadherin superfamily) and KIRREL3 (immunoglobulin superfamily), by a chromosomal translocation t(11;16) in a female patient with intellectual disability (ID). We screened coding regions of these two genes in a cohort of patients with ID and controls and identified four nonsynonymous CDH15 variants and three nonsynonymous KIRREL3 variants that appear rare and unique to ID. These variations altered highly conserved residues and were absent in more than 600 unrelated patients with ID and 800 control individuals. Furthermore, in vivo expression studies showed that three of the CDH15 variations adversely altered its ability to mediate cell-cell adhesion. We also show that in neuronal cells, human KIRREL3 colocalizes and interacts with the synaptic scaffolding protein, CASK, recently implicated in X-linked brain malformation and ID. Taken together, our data suggest that alterations in CDH15 and KIRREL3, either alone or in combination with other factors, could play a role in phenotypic expression of ID in some patients.

Microdeletion at 4q21.3 is associated with intellectual disability, dysmorphic facies, hypotonia, and short stature.

Chromosomal imbalances are a major cause of intellectual disability (ID) and multiple congenital anomalies. We have clinically and molecularly characterized two patients with chromosome translocations and ID. Using whole genome array CGH analysis, we identified a microdeletion involving 4q21.3, unrelated to the translocations in both patients. We confirmed the 4q21.3 microdeletions using fluorescence in situ hybridization and quantitative genomic PCR. The corresponding deletion boundaries in the patients were further mapped and compared to previously reported 4q21 deletions and the associated clinical features. We determined a common region of deletion overlap that appears unique to ID, short stature, hypotonia, and dysmorphic facial features.

Microarray data integration for genome-wide analysis of human tissue-selective gene expression.

BACKGROUND: Microarray gene expression data are accumulating in public databases. The expression profiles contain valuable information for understanding human gene expression patterns. However, the effective use of public microarray data requires integrating the expression profiles from heterogeneous sources. RESULTS: In this study, we have compiled a compendium of microarray expression profiles of various human tissue samples. The microarray raw data generated in different research laboratories have been obtained and combined into a single dataset after data normalization and transformation. To demonstrate the usefulness of the integrated microarray data for studying human gene expression patterns, we have analyzed the dataset to identify potential tissue-selective genes. A new method has been proposed for genome-wide identification of tissue-selective gene targets using both microarray intensity values and detection calls. The candidate genes for brain, liver and testis-selective expression have been examined, and the results suggest that our approach can select some interesting gene targets for further experimental studies. CONCLUSION: A computational approach has been developed in this study for combining microarray expression profiles from heterogeneous sources. The integrated microarray data can be used to investigate tissue-selective expression patterns of human genes.

Sequence feature-based prediction of protein stability changes upon amino acid substitutions.

BACKGROUND: Protein destabilization is a common mechanism by which amino acid substitutions cause human diseases. Although several machine learning methods have been reported for predicting protein stability changes upon amino acid substitutions, the previous studies did not utilize relevant sequence features representing biological knowledge for classifier construction. RESULTS: In this study, a new machine learning method has been developed for sequence feature-based prediction of protein stability changes upon amino acid substitutions. Support vector machines were trained with data from experimental studies on the free energy change of protein stability upon mutations. To construct accurate classifiers, twenty sequence features were examined for input vector encoding. It was shown that classifier performance varied significantly by using different sequence features. The most accurate classifier in this study was constructed using a combination of six sequence features. This classifier achieved an overall accuracy of 84.59% with 70.29% sensitivity and 90.98% specificity. CONCLUSIONS: Relevant sequence features can be used to accurately predict protein stability changes upon amino acid substitutions. Predictive results at this level of accuracy may provide useful information to distinguish between deleterious and tolerant alterations in disease candidate genes. To make the classifier accessible to the genetics research community, we have developed a new web server, called MuStab (http://bioinfo.ggc.org/mustab/).

Intellectual disability, midface hypoplasia, facial hypotonia, and Alport syndrome are associated with a deletion in Xq22.3.

Alport syndrome with intellectual disability (ID) is a contiguous gene deletion syndrome involving several genes on Xq22.3 including COL4A5 and ACSL4. We report on a family with two males with this disorder and a Xq22.3 deletion. Fluorescent in situ hybridization and genomic analyses mapped the deletion region to between exon 1 of COL4A5 and exon 12 of ACSL4. The patients' mother has microscopic hematuria and was found to be heterozygous for the Xq22.3 deletion. Analysis using reverse transcription polymerase chain reaction of lymphoblastoid cell line RNA from an affected male in the family revealed a stable chimeric transcript with the ACSL4 exons 13-17 replaced by a cryptic exon from intron 1 of the COL4A5 gene. A truncated 54 kDa protein was predicted from this transcript but Western blot analysis and ACSL4 enzyme assay both showed functional nullisomy of ACSL4. We also compared the clinical features of the family with three previously reported families with the ACSL4 gene deletion and found that ID with absent or severely delayed speech, midface hypoplasia, and facial hypotonia are consistent features observed in the absence of ACSL4 gene.

Mutations in ionotropic AMPA receptor 3 alter channel properties and are associated with moderate cognitive impairment in humans.

Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (iGluRs) mediate the majority of excitatory synaptic transmission in the CNS and are essential for the induction and maintenance of long-term potentiation and long-term depression, two cellular models of learning and memory. We identified a genomic deletion (0.4 Mb) involving the entire GRIA3 (encoding iGluR3) by using an X-array comparative genomic hybridization (CGH) and four missense variants (G833R, M706T, R631S, and R450Q) in functional domains of iGluR3 by sequencing 400 males with X-linked mental retardation (XLMR). Three variants were found in males with moderate MR and were absent in 500 control males. Expression studies in HEK293 cells showed that G833R resulted in a 78% reduction of iGluR3 due to protein misfolding. Whole-cell recording studies of iGluR3 homomers in HEK293 cells revealed that neither iGluR3-M706T (S2 domain) nor iGluR3-R631S (near channel core) had substantial channel function, whereas R450Q (S1 domain) was associated with accelerated receptor desensitization. When forming heteromeric receptors with iGluR2 in HEK293 cells, all four iGluR3 variants had altered desensitization kinetics. Our study provides the genetic and functional evidence that mutant iGluR3 with altered kinetic properties is associated with moderate cognitive impairment in humans.

Candidate Agtr2 influenced genes and pathways identified by expression profiling in the developing brain of Agtr2(-/y) mice.

Intellectual disability (ID) is a common developmental disability observed in 1 to 3% of the human population. A possible role for the Angiotensin II type 2 receptor (AGTR2) in brain function, affecting learning, memory, and behavior, has been suggested in humans and rodents. Mice lacking the Agtr2 gene (Agtr2(-/y)) showed significant impairment in their spatial memory and exhibited abnormal dendritic spine morphology. To identify Agtr2 influenced genes and pathways, we performed whole genome microarray analysis on RNA isolated from brains of Agtr2(-/y) and control male mice at embryonic day 15 (E15) and postnatal day one (P1). The gene expression profiles of the Agtr2(-/y) brain samples were significantly different when compared to profiles of the age-matched control brains. We identified 62 differently expressed genes (p< or =0.005) at E15 and in P1 brains of the Agtr2(-/y) mice. We verified the differential expression of several of these genes in brain samples using quantitative RT-PCR. Differentially expressed genes encode molecules involved in multiple cellular processes including microtubule functions associated with dendritic spine morphology. This study provides insight into Agtr2 influenced candidate genes and suggests that expression dysregulation of these genes may modulate Agtr2 actions in the brain that influences learning and memory.

From ectodermal dysplasia to selective tooth agenesis.

The history and the lessons learned from hypohidrotic ectodermal dysplasia (HED) may serve as an example for the unraveling of the cause and pathogenesis of other ectodermal dysplasia syndromes by demonstrating that phenotypically identical syndromes (HED) can be caused by mutations in different genes (EDA, EDAR, EDARADD), that mutations in the same gene (EDA) can lead to different phenotypes (HED and selective tooth agenesis) and that mutations in genes further downstream in the same signaling pathway (NEMO) may modify the phenotype quite profoundly (incontinentia pigmenti (IP) and HED with immunodeficiency). But it also demonstrates that diligent phenotype characterization and classification is extremely helpful in uncovering the underlying genotype. We also present a new mutation in the EDA gene which causes selective tooth agenesis and demonstrates the phenotype variation that can be encountered in the ectodermal dysplasia syndrome (HED) with the highest prevalence worldwide.

Dedicator of cytokinesis 8 is disrupted in two patients with mental retardation and developmental disabilities.

We have identified disruptions in the dedicator of cytokinesis 8 gene, DOCK8, in two unrelated patients with mental retardation (MR). In one patient, a male with MR and no speech, we mapped a genomic deletion of approximately 230 kb in subtelomeric 9p. In the second patient, a female with mental retardation and ectodermal dysplasia and a balanced translocation, t(X;9) (q13.1;p24), we mapped the 9p24 breakpoint to a region overlapping with the centromeric end of the 230-kb subtelomeric deletion. We characterized the DOCK8 gene from the critical 9p deletion region and determined that the longest isoform of the DOCK8 gene is truncated in both patients. Furthermore, the DOCK8 gene is expressed in several human tissues, including adult and fetal brain. Recently, a role for DOCK8 in processes that affect the organization of filamentous actin has been suggested. Several genes influencing the actin cytoskeleton have been implicated in human cognitive function and thus a possibility exists that the rare mutations in the DOCK8 gene may contribute to some cases of autosomal dominant mental retardation.

UBE2A-related X-linked intellectual disability.

UBE2A-related X-linked intellectual disability is characterized by a distinctive facial phenotype (dense eyebrows and eyelashes, synophrys, hypertelorism, upslanted palpebral fissures, wide mouth, and thin lips), generalized hirsutism, hypoplastic genitalia, short stature, hypotonia, seizures, and severe intellectual disability. Five affected males in two families are described here and compared with the previously reported 17 males in eight families. The new cases are notable for the absence of nail dystrophy, previously considered a defining manifestation, and for the presence of hypogammaglobulinemia and adult-onset ataxia.

Evidence that SIZN1 is a candidate X-linked mental retardation gene.

An estimated 1-3% of individuals within the United States are diagnosed with mental retardation (MR), yet the cause is unknown in nearly 50% of the patients. While several environmental, genetic and combined teratogenetic etiologies have been identified, many causative genes remain to be identified. Furthermore, the pathogenetic mechanisms underlying MR are known for very few of these genes. Males have a much higher incidence of MR implicating genes on the X-chromosome. We have recently identified a novel gene, SIZN1, on the X-chromosome and showed that it functions in modulating the BMP signaling pathway. Furthermore, we have shown this gene is necessary for basal forebrain cholinergic neuron (BFCN) specific gene expression. Given that cognitive function is impaired when BFCNs are lost or functionally disrupted, we undertook a screen of cognitively impaired males for SIZN1 mutations. We report on four different sequence variants in SIZN1 in 11 individuals with nonsyndromic X-linked mental retardation (XLMR). Our data implicate SIZN1 as a candidate gene for XLMR and/or as a neurocognitive functional modifier.

Co-expression of long non-coding RNAs and autism risk genes in the developing human brain.

BACKGROUND: Autism Spectrum Disorder (ASD) is the umbrella term for a group of neurodevelopmental disorders convergent on behavioral phenotypes. While many genes have been implicated in the disorder, the predominant focus of previous research has been on protein coding genes. This leaves a vast number of long non-coding RNAs (lncRNAs) not characterized for their role in the disorder although lncRNAs have been shown to play important roles in development and are highly represented in the brain. Studies have also shown lncRNAs to be differentially expressed in ASD affected brains. However, there has yet to be an enrichment analysis of the shared ontologies and pathways of known ASD genes and lncRNAs in normal brain development. RESULTS: In this study, we performed co-expression network analysis on the developing brain transcriptome to identify potential lncRNAs associated with ASD and possible annotations for functional role of lncRNAs in brain development. We found co-enrichment of lncRNA genes and ASD risk genes in two distinct groups of modules showing elevated prenatal and postnatal expression patterns, respectively. Further enrichment analysis of the module groups indicated that the early expression modules were comprised mainly of transcriptional regulators while the later expression modules were associated with synapse formation. Finally, lncRNAs were prioritized for their connectivity with the known ASD risk genes through analysis of an adjacency matrix. Collectively, the results imply early developmental repression of synaptic genes through lncRNAs and ASD transcriptional regulators. CONCLUSION: Here we demonstrate the utility of mining the publically available brain gene expression data to further functionally annotate the role of lncRNAs in ASD. Our analysis indicates that lncRNAs potentially have a key role in ASD due to their convergence on shared pathways, and we identify lncRNAs of interest that may lead to further avenues of study.

Neurodevelopmental disorder-associated ZBTB20 gene variants affect dendritic and synaptic structure.

Dendritic spine morphology and dendritic arborization are key determinants of neuronal connectivity and play critical roles in learning, memory and behavior function. Recently, defects of ZBTB20, a BTB and zinc finger domain containing transcriptional repressor, have been implicated in a wide range of neurodevelopmental disorders, including intellectual disability and autism. Here we show distinct effects of expression of two major isoforms, long and short, of ZBTB20, and its neurodevelopmental disorder-linked variants, on dendritic architecture of cultured rat cortical pyramidal neurons. The N-terminal of ZBTB20 showed a role in regulating dendritic spine morphology. Two ZBTB20 single nucleotide variants, located at the N-terminal and central regions of the protein and potentially conferring autism risk, altered dendritic spine morphology. In contrast, a single nucleotide variant identified in patients with intellectual disability and located at the C-terminus of ZBTB20 affected dendritic arborization and dendritic length but had no effect on dendritic spine morphology. Furthermore, truncation of the extreme C-terminus of ZBTB20 caused spine and dendritic morphological changes that were similar but distinct from those caused by the C-terminal variant. Taken together, our study suggests ZBTB20's role in dendritic and synaptic structure and provide possible mechanisms of its effect in neurodevelopmental disorders.

A Rare De Novo RAI1 Gene Mutation Affecting BDNF-Enhancer-Driven Transcription Activity Associated with Autism and Atypical Smith-Magenis Syndrome Presentation.

Deletions and mutations involving the Retinoic Acid Induced 1 (RAI1) gene at 17p11.2 cause Smith-Magenis syndrome (SMS). Here we report a patient with autism as the main clinical presentation, with some SMS-like features and a rare de novo RAI1 gene mutation, c.3440G > A (p.R1147Q). We functionally characterized the RAI1 p.R1147Q mutant protein. The mutation, located near the nuclear localization signal, had no effect on the subcellular localization of the mutant protein. However, similar to previously reported RAI1 missense mutations in SMS patients, the RAI1 p.R1147Q mutant protein showed a significant deficiency in activating in vivo transcription of a reporter gene driven by a BDNF (brain-derived neurotrophic factor) intronic enhancer. In addition, expression of other genes associated with neurobehavioral abnormalities and/or neurodevelopmental disorders were found to be altered in this patient. These results suggest a likely contribution of RAI1, either alone or in combination of other factors, to social behavior and reinforce the RAI1 gene as a candidate gene in patients with autistic manifestations or social behavioral abnormalities.

Integrative genomic analyses for identification and prioritization of long non-coding RNAs associated with autism.

Genetic studies have identified many risk loci for autism spectrum disorder (ASD) although causal factors in the majority of cases are still unknown. Currently, known ASD risk genes are all protein-coding genes; however, the vast majority of transcripts in humans are non-coding RNAs (ncRNAs) which do not encode proteins. Recently, long non-coding RNAs (lncRNAs) were shown to be highly expressed in the human brain and crucial for normal brain development. We have constructed a computational pipeline for the integration of various genomic datasets to identify lncRNAs associated with ASD. This pipeline utilizes differential gene expression patterns in affected tissues in conjunction with gene co-expression networks in tissue-matched non-affected samples. We analyzed RNA-seq data from the cortical brain tissues from ASD cases and controls to identify lncRNAs differentially expressed in ASD. We derived a gene co-expression network from an independent human brain developmental transcriptome and detected a convergence of the differentially expressed lncRNAs and known ASD risk genes into specific co-expression modules. Co-expression network analysis facilitates the discovery of associations between previously uncharacterized lncRNAs with known ASD risk genes, affected molecular pathways and at-risk developmental time points. In addition, we show that some of these lncRNAs have a high degree of overlap with major CNVs detected in ASD genetic studies. By utilizing this integrative approach comprised of differential expression analysis in affected tissues and connectivity metrics from a developmental co-expression network, we have prioritized a set of candidate ASD-associated lncRNAs. The identification of lncRNAs as novel ASD susceptibility genes could help explain the genetic pathogenesis of ASD.

Rai1 Haploinsufficiency Is Associated with Social Abnormalities in Mice.

Background: Autism is characterized by difficulties in social interaction, communication, and repetitive behaviors; with different degrees of severity in each of the core areas. Haploinsufficiency and point mutations of RAI1 are associated with Smith-Magenis syndrome (SMS), a genetic condition that scores within the autism spectrum range for social responsiveness and communication, and is characterized by neurobehavioral abnormalities, intellectual disability, developmental delay, sleep disturbance, and self-injurious behaviors. Methods: To investigate the relationship between Rai1 and social impairment, we evaluated the Rai1+/- mice with a battery of tests to address social behavior in mice. Results: We found that the mutant mice showed diminished interest in social odors, abnormal submissive tendencies, and increased repetitive behaviors when compared to wild type littermates. Conclusions: These findings suggest that Rai1 contributes to social behavior in mice, and prompt it as a candidate gene for the social behaviors observed in Smith-Magenis Syndrome patients.

Carriers and patients with muscle-eye-brain disease can be rapidly diagnosed by enzymatic analysis of fibroblasts and lymphoblasts.

We report a new fibroblast and lymphoblast based protein O-mannosyl beta-1,2-N-acetylglucosaminyltransferase 1 enzymatic assay, which allows rapid and accurate diagnosis of carriers and patients with muscle-eye-brain type of congenital muscular dystrophy. Seven patients with genetically confirmed muscle-eye-brain disease were assayed for protein O-mannosyl beta-1,2-N-acetylglucosaminyltransferase 1 enzyme activity. In three patients and their heterozygous parents, the assays were done on EBV-transformed lymphoblasts, in another three patients they were done on cultured fibroblasts and in the last patient on both fibroblasts and lymphoblasts. Cultured fibroblasts and lymphoblasts from the muscle-eye-brain patients showed a highly significant decrease in protein O-mannosyl beta-1,2-N-acetylglucosaminyltransferase 1 activity relative to controls. The residual protein O-mannosyl beta-1,2-N-acetylglucosaminyltransferase 1 level in fibroblasts (average 0.11 nmoles/h per mg) was about 13% of normal controls. The ratio of protein O-mannosyl beta-1,2-N-acetylglucosaminyltransferase 1 activity to the activity of a glycosyltransferase control (N-acetylglucosaminyltransferase 1; GnT1) in fibroblasts was on average 0.006 in muscle-eye-brain patients and 0.045 in controls. The average residual protein O-mannosyl beta-1,2-N-acetylglucosaminyltransferase 1 level in lymphoblasts was 15% of normal controls. The average ratio of protein O-mannosyl beta-1,2-N-acetylglucosaminyltransferase 1/GnT1 activity was 0.007 in muscle-eye-brain patients, 0.026 in heterozygous carriers and 0.046 in normal controls. Assay of protein O-mannosyl beta-1,2-N-acetylglucosaminyltransferase 1 activity in fibroblasts and lymphoblasts from muscle-eye-brain carriers and patients provides a rapid and relatively simple diagnostic test for this disease and could be used as a screening test in carriers and patients with complex congenital muscular dystrophy.

Unusual phenotypic expression of an XLRS1 mutation in X-linked juvenile retinoschisis.

X-linked juvenile retinoschisis is a rare progressive vitreoretinal degenerative process that appears in early childhood, results in decreased visual acuity and blindness (if severe), and is caused by various mutations within the XLRS1 gene at Xp22.2. We report an affected family of Western European ancestry with X-linked juvenile retinoschisis. The family was found to carry a 304C-->T substitution in exon 4 of the XLRS1 gene, resulting in an Arg102Trp amino acid substitution. Two of the four available clinical cases in this family were found to carry the mutation. All available mothers of affected males were found to be unaffected carriers of the mutation, a typical feature of X-linked diseases. Two new female carriers, sisters of affected males, were identified and counseled accordingly. Questionnaires on visual functioning were given to the affected family members to examine the psychologic and sociologic impact of X-linked juvenile retinoschisis, which documented an associated stigma even when affected with a "mild" phenotype.

Autism and Intellectual Disability-Associated KIRREL3 Interacts with Neuronal Proteins MAP1B and MYO16 with Potential Roles in Neurodevelopment.

Cell-adhesion molecules of the immunoglobulin superfamily play critical roles in brain development, as well as in maintaining synaptic plasticity, the dysfunction of which is known to cause cognitive impairment. Recently dysfunction of KIRREL3, a synaptic molecule of the immunoglobulin superfamily, has been implicated in several neurodevelopmental conditions including intellectual disability, autism spectrum disorder, and in the neurocognitive delay associated with Jacobsen syndrome. However, the molecular mechanisms of its physiological actions remain largely unknown. Using a yeast two-hybrid screen, we found that the KIRREL3 extracellular domain interacts with brain expressed proteins MAP1B and MYO16 and its intracellular domain can potentially interact with ATP1B1, UFC1, and SHMT2. The interactions were confirmed by co-immunoprecipitation and colocalization analyses of proteins expressed in human embryonic kidney cells, mouse neuronal cells, and rat primary neuronal cells. Furthermore, we show KIRREL3 colocalization with the marker for the Golgi apparatus and synaptic vesicles. Previously, we have shown that KIRREL3 interacts with the X-linked intellectual disability associated synaptic scaffolding protein CASK through its cytoplasmic domain. In addition, we found a genomic deletion encompassing MAP1B in one patient with intellectual disability, microcephaly and seizures and deletions encompassing MYO16 in two unrelated patients with intellectual disability, autism and microcephaly. MAP1B has been previously implicated in synaptogenesis and is involved in the development of the actin-based membrane skeleton. MYO16 is expressed in hippocampal neurons and also indirectly affects actin cytoskeleton through its interaction with WAVE1 complex. We speculate KIRREL3 interacting proteins are potential candidates for intellectual disability and autism spectrum disorder. Moreover, our findings provide further insight into understanding the molecular mechanisms underlying the physiological action of KIRREL3 and its role in neurodevelopment.