a-simple-cell-based-immunofluorescence-assay-to-detect-autoantibody

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

Chang Gung Memorial Hospital-Linkou

Chang Gung University

We recently reported a terminal deletion of approximately 2.4 Mb at chromosome 8p23.2-pter in a boy with autism. The deleted region contained the DLGAP2 gene that encodes the neuronal post-synaptic density protein, discs, large (Drosophila) homolog-associated protein 2. The study aimed to investigate whether DLGAP2 is genetically associated with autism spectrum disorders (ASD) in general.

Deep exon resequencing of DLGAP2 as a candidate gene of autism spectrum disorders.

Runs of homozygosity (ROH) may play a role in complex diseases. In the current study, we aimed to test if ROHs are linked to the risk of autism and related language impairment. We analyzed 546,080 SNPs in 315 Han Chinese affected with autism and 1,115 controls. ROH was defined as an extended homozygous haplotype spanning at least 500 kb. Relative extended haplotype homozygosity (REHH) for the trait-associated ROH region was calculated to search for the signature of selection sweeps. Totally, we identified 676 ROH regions. An ROH region on 11q22.3 was significantly associated with speech delay (corrected p = 1.73×10(-8)). This region contains the NPAT and ATM genes associated with ataxia telangiectasia characterized by language impairment; the CUL5 (culin 5) gene in the same region may modulate the neuronal migration process related to language functions. These three genes are highly expressed in the cerebellum. No evidence for recent positive selection was detected on the core haplotypes in this region. The same ROH region was also nominally significantly associated with speech delay in another independent sample (p = 0.037; combinatorial analysis Stouffer's z trend = 0.0005). Taken together, our findings suggest that extended recessive loci on 11q22.3 may play a role in language impairment in autism. More research is warranted to investigate if these genes influence speech pathology by perturbing cerebellar functions.

Runs of homozygosity associated with speech delay in autism in a taiwanese han population: evidence for the recessive model.

GABRB3 is a position candidate gene at chromosome 15q12 that has been implicated in the neurobiology of autism spectrum disorders (ASD). The aim of this study was to examine the genetic association of GABRB3 with ASD.

Genetic analysis of GABRB3 as a candidate gene of autism spectrum disorders.

GABRB3 encoding the β3 subunit of GABAA receptor has been implicated in multiple neuropsychiatric disorders, including substance abuse. Previous studies reported that SNPs at the 5' regulatory region of GABRB3 could regulate GABRB3 gene expression and associated with childhood absence epilepsy (CAE). The study aimed to investigate whether SNPs at the 5' regulatory region of GABRB3 were associated with heroin dependence in our population. We first re-sequenced 1.5 kb of the 5'regulatory region of GABRB3 gene to examine the SNP profile in the genomic DNA of 365 control subjects. Then, we conducted a case-control association analysis between 576 subjects with heroin dependence (549 males, 27 females) and 886 controls (472 males, 414 females) by genotyping the rs4906902 as a tag SNP. We also conducted a reporter gene assay to assess the promoter activity of two major haplotypes derived from SNPs at this region. We detected 3 common SNPs (rs4906902, rs8179184 and rs20317) at this region that had strong pair-wise linkage disequilibrium. The C allele of rs4906902 was found to be associated with increased risk of heroin dependence (odds ratio:1.27, p = 0.002). Two major haplotypes (C-A-G and T-G-C) derived from these 3 SNPs accounted for 99% of this sample, and reporter gene activity assay showed that haplotype C-A-G that contained the C allele of the tag SNP rs4906902 had higher activity than haplotype T-G-C. Our data suggest that GABRB3 might be associated with heroin dependence, and increased expression of GABRB3 might contribute to the pathogenesis of heroin dependence.

Association analysis of GABRB3 promoter variants with heroin dependence.

As elegant structures designed for neural communication, synapses are the building bricks of our mental functions. Recently, many studies have pointed out that synaptic protein-associated mutations may lead to dysfunctions of social cognition. Dlgap2, which encodes one of the main components of scaffold proteins in postsynaptic density (PSD), has been addressed as a candidate gene in autism spectrum disorders. To elucidate the disturbance of synaptic balance arising from Dlgap2 loss-of-function in vivo, we thus generated Dlgap2 (-/-) mice to investigate their phenotypes of synaptic function and social behaviors.

Autism-associated gene Dlgap2 mutant mice demonstrate exacerbated aggressive behaviors and orbitofrontal cortex deficits.

Autism spectrum disorder is a heterogeneous neurodevelopmental disorder with strong genetic basis. To identify common genetic variations conferring the risk of ASD, we performed a two-stage genome-wide association study using ASD family and healthy control samples obtained from East Asian populations. A total of 166 ASD families (n = 500) and 642 healthy controls from the Japanese population were used as the discovery cohort. Approximately 900,000 single nucleotide polymorphisms (SNPs) were genotyped using Affymetrix Genome-Wide Human SNP array 6.0 chips. In the replication stage, 205 Japanese ASD cases and 184 healthy controls, as well as 418 Chinese Han trios (n = 1,254), were genotyped by TaqMan platform. Case-control analysis, family based association test, and transmission/disequilibrium test (TDT) were then conducted to test the association. In the discovery stage, significant associations were suggested for 14 loci, including 5 known ASD candidate genes: GPC6, JARID2, YTHDC2, CNTN4, and CSMD1. In addition, significant associations were identified for several novel genes with intriguing functions, such as JPH3, PTPRD, CUX1, and RIT2. After a meta-analysis combining the Japanese replication samples, the strongest signal was found at rs16976358 (P = 6.04 × 10(-7)), which is located near the RIT2 gene. In summary, our results provide independent support to known ASD candidate genes and highlight a number of novel genes warranted to be further investigated in a larger sample set in an effort to improve our understanding of the genetic basis of ASD.

Genome-wide Association Study of Autism Spectrum Disorder in the East Asian Populations.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with strong genetic components. Several recent genome-wide association (GWA) studies in Caucasian samples have reported a number of gene regions and loci correlated with the risk of ASD--albeit with very little consensus across studies.

Genome-Wide Association Study for Autism Spectrum Disorder in Taiwanese Han Population.

Genetic factor plays a critical role in the etiology of autism spectrum disorder (ASD). Both common variants with a small effect and rare mutations with a large effect contribute toward the genetic basis of ASD, showing the high genetic heterogeneity of ASD. Genomic rearrangements account for around 10-15% of its genetic landscape. However, they are highly individualized and each of them has a very rare frequency.

Complex genomic variants contribute toward the genetic architecture of autism spectrum disorder.

Microdeletion at 22q11.2, a common copy number variation (CNV) noted in neurodevelopmental disorders, may be associated with cognitive impairment. However, cognitive function in individuals with microduplication remains unclear. This work presents the genetic, clinical, and brain structural data of two men out of 335 probands with autistic spectrum disorder (ASD) who had different CNV dosages at 22q11.2, and comparison with their siblings, 55 ASD probands, and 73 controls. Both showed severe autistic symptoms, but the proband with microduplication demonstrated better cognitive functions. Furthermore, different cingulate gyrus volume changes were noted, indicating that the proband with 22q11.2 microduplication had a different pattern of cingulate gyrus structure. Our comprehensive characterization of the behavioral, cognitive, and imaging phenotypes of ASD probands with different CNV dosage at 22q11.2 contribute to how copy number changes at 22q11.2 mediate the phenotypes in ASD, and pave the way for future clinical and functional study on these variants.

Dosage of copy number variation at 22q11.2 mediates changes in cognition, social function and brain structure in autism spectrum disorder.

Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer's disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories.

Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood.

In disease studies, family-based designs have become an attractive approach to analyzing next-generation sequencing (NGS) data for the identification of rare mutations enriched in families. Substantial research effort has been devoted to developing pipelines for automating sequence alignment, variant calling, and annotation. However, fewer pipelines have been designed specifically for disease studies. Most of the current analysis pipelines for family-based disease studies using NGS data focus on a specific function, such as identifying variants with Mendelian inheritance or identifying shared chromosomal regions among affected family members. Consequently, some other useful family-based analysis tools, such as imputation, linkage, and association tools, have yet to be integrated and automated. We developed FamPipe, a comprehensive analysis pipeline, which includes several family-specific analysis modules, including the identification of shared chromosomal regions among affected family members, prioritizing variants assuming a disease model, imputation of untyped variants, and linkage and association tests. We used simulation studies to compare properties of some modules implemented in FamPipe, and based on the results, we provided suggestions for the selection of modules to achieve an optimal analysis strategy. The pipeline is under the GNU GPL License and can be downloaded for free at http://fampipe.sourceforge.net.

FamPipe: An Automatic Analysis Pipeline for Analyzing Sequencing Data in Families for Disease Studies.

Chromosome 22q13 is a hot region of genomic rearrangements that may result in deletion, duplication, and translocation, and that may lead to neurodevelopmental disorders in affected patients.

Clinical and molecular characterization of three genomic rearrangements at chromosome 22q13.3 associated with autism spectrum disorder.

Autoimmune encephalopathy caused by autoantibodies against neuronal cell-surface proteins in the brain is a newly discovered disease category associated with psychiatric disorders. Correct diagnosis of this condition relies on the detection of specific autoantibodies in the blood or cerebral spinal fluid in addition to the clinical presentations. The study aimed to understand the seroprevalence of selective anti-neuronal autoantibodies in our patients with schizophrenia. First, we screened for six anti-neuronal autoantibodies in an archived blood sample collected from patients with the first-episode schizophrenia. The six autoantibodies including antibodies against N-methyl-d-aspartate (NMDA) receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors 1 and 2, γ-butyric acid receptor type B1 (GABARB1), leucine-rich glioma inactivated-1 (LGI1) protein, and contactin-associated protein-like 2 (CASPR2) protein. A total of 78 plasma samples (46 males and 32 females) were investigated; however, no positive case was identified. In this second study, we screened anti-NMDA receptor autoantibodies in a blood sample of 234 patients with chronic schizophrenia (133 females and 101 males) including 48 patients defined as treatment resistance. None of this sample was detected as positive. The negative findings in this study suggest that the seroprevalence of autoantibodies against neuronal surface proteins might be low in patients diagnosed with schizophrenia.

Seroprevalence survey of selective anti-neuronal autoantibodies in patients with first-episode schizophrenia and chronic schizophrenia.

Rare genomic copy number variations (CNVs) (frequency <1%) contribute a part to the genetic underpinnings of autism spectrum disorders (ASD). The study aimed to understand the scope of rare CNV in Taiwanese patients with ASD. We conducted a genome-wide CNV screening of 335 ASD patients (299 males, 36 females) from Taiwan using Affymetrix Genome-Wide Human SNP Array 6.0 and compared the incidence of rare CNV with that of 1093 control subjects (525 males, 568 females). We found a significantly increased global burden of rare CNVs in the ASD group compared to the controls as a whole or when the rare CNVs were classified by the size and types of CNV. Further analysis confirmed the presence of several rare CNVs at regions strongly associated with ASD as reported in the literature in our sample. Additionally, we detected several new private pathogenic CNVs in our samples and five patients carrying two pathogenic CNVs. Our data indicate that rare genomic CNVs contribute a part to the genetic landscape of our ASD patients. These CNVs are highly heterogeneous, and the clinical interpretation of the pathogenic CNVs of ASD is not straightforward in consideration of the incomplete penetrance, varied expressivity, and individual genetic background.

High resolution analysis of rare copy number variants in patients with autism spectrum disorder from Taiwan.

Neuroligin 2 (NLGN2) is a postsynaptic adhesion protein that plays an essential role in synaptogenesis and function of inhibitory neuron. We previously identified a missense mutation R215H of the NLGN2 in a patient with schizophrenia. This missense mutation was shown to be pathogenic in several cell-based assays. The objective of this study was to better understand the behavioral consequences of this mutation in vivo. We generated a line of transgenic mice carrying this mutation using a recombinant-based method. The mice were subjected to a battery of behavioral tests including open field locomotor activity assay, prepulse inhibition (PPI) assay, accelerated rotarod test, novel location and novel recognition tests, elevated plus-maze (EPM) test, and Morris water maze test. The transgenic animals were viable and fertile, but the Nlgn2 R215H knock-in (KI) homozygous mice showed growth retardation, anxiety-like behavior, increased PPI, and impaired spatial learning and memory. There was no significant interaction between sex and genotype in most behavioral tests; however, we observed a significant interaction between sex and genotype in EPM test in this study. Also, we found that the Nlgn2 R215H homozygous KI mice did not express the NLGN2 protein, resembling Nlgn2 knockout mice. Our results demonstrate that Nlgn2 R215H KI homozygous mice manifest several behavioral abnormalities similar to those found in psychiatric patients carrying NLGN2 mutations, indicating that dysfunction of NLGN2 contributes to the pathogenesis of certain psychiatric symptoms commonly present in various mental disorders, not limited to schizophrenia.

Article	Year
Deep exon resequencing of DLGAP2 as a candidate gene of autism spectrum disorders. Molecular autism\| PubMed ID: 23915500	2013
Runs of homozygosity associated with speech delay in autism in a taiwanese han population: evidence for the recessive model. PloS one\| PubMed ID: 23977206	2013
Genetic analysis of GABRB3 as a candidate gene of autism spectrum disorders. Molecular autism\| PubMed ID: 24999380	2014
Association analysis of GABRB3 promoter variants with heroin dependence. PloS one\| PubMed ID: 25025424	2014
Autism-associated gene Dlgap2 mutant mice demonstrate exacerbated aggressive behaviors and orbitofrontal cortex deficits. Molecular autism\| PubMed ID: 25071926	2014
Genome-wide Association Study of Autism Spectrum Disorder in the East Asian Populations. Autism research : official journal of the International Society for Autism Research\| PubMed ID: 26314684	2016
Genome-Wide Association Study for Autism Spectrum Disorder in Taiwanese Han Population. PloS one\| PubMed ID: 26398136	2015
Complex genomic variants contribute toward the genetic architecture of autism spectrum disorder. Psychiatric genetics\| PubMed ID: 26901793	2016
Dosage of copy number variation at 22q11.2 mediates changes in cognition, social function and brain structure in autism spectrum disorder. Journal of the Formosan Medical Association = Taiwan yi zhi\| PubMed ID: 27026301	2016
Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood. PloS one\| PubMed ID: 27078154	2016
FamPipe: An Automatic Analysis Pipeline for Analyzing Sequencing Data in Families for Disease Studies. PLoS computational biology\| PubMed ID: 27272119	2016
Clinical and molecular characterization of three genomic rearrangements at chromosome 22q13.3 associated with autism spectrum disorder. Psychiatric genetics\| PubMed ID: 27846046	2017
Seroprevalence survey of selective anti-neuronal autoantibodies in patients with first-episode schizophrenia and chronic schizophrenia. Schizophrenia research\| PubMed ID: 28341002	2017
High resolution analysis of rare copy number variants in patients with autism spectrum disorder from Taiwan. Scientific reports\| PubMed ID: 28931914	2017
Neuroligin 2 R215H Mutant Mice Manifest Anxiety, Increased Prepulse Inhibition, and Impaired Spatial Learning and Memory. Frontiers in psychiatry\| PubMed ID: 29230184	2017

Chia-Hsiang Chen

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