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Shui Ying Tsang,
Songfa Zhong,
Lingling Mei,
Jianhuan Chen,
Siu-Kin Ng,
Frank W Pun,
Cunyou Zhao,
Bingyi Jing,
Robin Chark,
Jianhua Guo,
Yunlong Tan,
Lijun Li,
Chuanyue Wang,
Soo Hong Chew,
Hong Xue
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ABSTRACT: The occurrence of positive selection in schizophrenia-associated GABRB2 suggests a broader impact of the gene product on population fitness. The present study considered the possibility of cognition-related GABRB2 involvement by examining the association of GABRB2 with psychosis and altruism, respectively representing psychiatric and psychological facets of social cognition. Four single nucleotide polymorphisms (SNPs) were genotyped for quantitative trait analyses and population-based association studies. Psychosis was measured by either the Positive and Negative Syndrome Scale (PANSS) or antipsychotics dosage, and altruism was based on a self-report altruism scale. The minor alleles of SNPs rs6556547, rs1816071 and rs187269 in GABRB2 were correlated with high PANSS score for positive symptoms in a Han Chinese schizophrenic cohort, whereas those of rs1816071 and rs1816072 were associated with high antipsychotics dosage in a US Caucasian schizophrenic cohort. Moreover, strongly significant GABRB2-disease associations were found among schizophrenics with severe psychosis based on high PANSS positive score, but no significant association was observed for schizophrenics with only mild psychosis. Interestingly, in addition to association with psychosis in schizophrenics, rs187269 was also associated with altruism in healthy Han Chinese. Furthermore, parallel to correlation with severe psychosis, its minor allele was correlated with high altruism scores. These findings revealed that GABRB2 is associated with psychosis, the core symptom and an endophenotype of schizophrenia. Importantly, the association was found across the breadth of the psychiatric (psychosis) to psychological (altruism) spectrum of social cognition suggesting GABRB2 involvement in human cognition.
PLoS ONE 01/2013; 8(4):e62322. · 4.09 Impact Factor
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ABSTRACT: To improve the understanding of psychotic abnormalities and their non-Mendelian inheritance patterns, the epigenetic regulation of the psychotic disorder-associated GABRB2, gene for the type A γ-aminobutyric acid receptor β(2)-subunit, was investigated.
Expression of GABRB2, and the epigenetic regulatory enzymes histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) in mouse and postmortem human brains was analyzed using real-time PCR.
Results showed that expression of GABRB2 isoforms significantly increased over time in both mouse and human, especially for the long splicing isoform. In the brains of non-psychiatric controls (CON), a significant positive correlation of GABRB2 expression with age was observed in individuals with MM genotypes of the single nucleotide polymorphisms (SNPs) rs187269 and rs1816072. This was reversed to a significant negative correlation in schizophrenics (SCZ). A similar reversal was also displayed by bipolar disorder (BPD) patients. In parallel, a significant co-variation of HDAC1 with GABRB2 expression observed in CON remained significant in BPD but not in SCZ; comparably, a significant co-variation of HDAC2 with GABRB2 expression observed in CON became non-significant in both SCZ and BPD. Moreover, co-variations of DNMT1 and DNMT3B with GABRB2, not observable in CON, became significant in BPD.
These findings demonstrated that GABRB2 expression was under epigenetic regulation that varied with development, genotype and disease status, and these regulatory mechanisms were observably disrupted in SCZ and BPD. This study provided insight into the complex inheritance patterns of psychiatric disorders, and pointed to the involvement of epigenetic dysregulation in the disease process of major psychotic disorders.
Biological Psychiatry 12/2011; 134(2-3):260-6. · 8.28 Impact Factor
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Lingling Mei,
Xiaofan Ding, Shui-Ying Tsang,
Frank W Pun,
Siu-Kin Ng,
Jianfeng Yang,
Cunyou Zhao,
Dezhi Li,
Weiqing Wan,
Chi-Hung Yu,
Tze-Ching Tan,
Wai-Sang Poon,
Gilberto Ka-Kit Leung,
Ho-Keung Ng,
Liwei Zhang,
Hong Xue
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ABSTRACT: To complement next-generation sequencing technologies, there is a pressing need for efficient pre-sequencing capture methods with reduced costs and DNA requirement. The Alu family of short interspersed nucleotide elements is the most abundant type of transposable elements in the human genome and a recognized source of genome instability. With over one million Alu elements distributed throughout the genome, they are well positioned to facilitate genome-wide sequence amplification and capture of regions likely to harbor genetic variation hotspots of biological relevance.
Here we report on the use of inter-Alu PCR with an enhanced range of amplicons in conjunction with next-generation sequencing to generate an Alu-anchored scan, or 'AluScan', of DNA sequences between Alu transposons, where Alu consensus sequence-based 'H-type' PCR primers that elongate outward from the head of an Alu element are combined with 'T-type' primers elongating from the poly-A containing tail to achieve huge amplicon range. To illustrate the method, glioma DNA was compared with white blood cell control DNA of the same patient by means of AluScan. The over 10 Mb sequences obtained, derived from more than 8,000 genes spread over all the chromosomes, revealed a highly reproducible capture of genomic sequences enriched in genic sequences and cancer candidate gene regions. Requiring only sub-micrograms of sample DNA, the power of AluScan as a discovery tool for genetic variations was demonstrated by the identification of 357 instances of loss of heterozygosity, 341 somatic indels, 274 somatic SNVs, and seven potential somatic SNV hotspots between control and glioma DNA.
AluScan, implemented with just a small number of H-type and T-type inter-Alu PCR primers, provides an effective capture of a diversity of genome-wide sequences for analysis. The method, by enabling an examination of gene-enriched regions containing exons, introns, and intergenic sequences with modest capture and sequencing costs, computation workload and DNA sample requirement is particularly well suited for accelerating the discovery of somatic mutations, as well as analysis of disease-predisposing germline polymorphisms, by making possible the comparative genome-wide scanning of DNA sequences from large human cohorts.
BMC Genomics 11/2011; 12:564. · 4.07 Impact Factor
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ABSTRACT: Flavones have been studied for their activities via benzodiazepine site on the type-A γ-aminobutyric acid (GABA(A)) receptors, for which knowledge on structure-efficacy relationships has been rather limited in comparison to that on structure-affinity relationships. The present study focused on flavone 6-substitution, implied in previous studies being relevant to efficacy. Structure analogs, each varying only at position 6, were compared, including 6-fluoroflavone, 6-chloroflavone, 6-bromoflavone, and 2'-hydroxyflavone analyzed in the present study, as well as 6,2'-dihydroxyflavone reported earlier. Radio-ligand binding assays, whole-cell patch-clamp, and mouse behavioral experiments were performed. In consistent with a previous report, the present whole-cell patch-clamp and animal behavior experiments demonstrated 6-bromoflavone to be a positive modulator at GABA(A) receptors acting through flumazenil-sensitive high-affinity benzodiazepine site. In contrast, the other two 6-haloflavones were both neutralizing modulators. In vitro electrophysiological and in vivo animal experiments showed that 2'-hydroxyflavone was a neutralizing modulator, different in efficacy from its structural analog, 6,2'-dihydroxyflavone, a negative modulator of GABA(A) receptors. The fact that flavone analogs differing only at position 6 showed drastically different pharmacological properties clearly points to 6-substitution being an important determinant of efficacy. The results suggest that a large width of the first atom on the 6-substituent favors a high binding affinity of the 6-substituted flavone, whereas a large overall volume of the 6-substituent favors positive modulator activity, which could be modified by, e.g., 2'-hydroxyl substitution. These findings have contributed to the understanding of quantitative structure-efficacy relationships for flavones acting at GABA(A) receptors, and hence facilitation of flavone-based drug development.
European journal of pharmacology 09/2011; 670(1):121-9. · 2.59 Impact Factor
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ABSTRACT: Flavones have been studied for their activities via benzodiazepine site on the type-A γ-aminobutyric acid (GABAA) receptors, for which knowledge on structure–efficacy relationships has been rather limited in comparison to that on structure–affinity relationships. The present study focused on flavone 6-substitution, implied in previous studies being relevant to efficacy. Structure analogs, each varying only at position 6, were compared, including 6-fluoroflavone, 6-chloroflavone, 6-bromoflavone, and 2′-hydroxyflavone analyzed in the present study, as well as 6,2′-dihydroxyflavone reported earlier. Radio-ligand binding assays, whole-cell patch-clamp, and mouse behavioral experiments were performed. In consistent with a previous report, the present whole-cell patch-clamp and animal behavior experiments demonstrated 6-bromoflavone to be a positive modulator at GABAA receptors acting through flumazenil-sensitive high-affinity benzodiazepine site. In contrast, the other two 6-haloflavones were both neutralizing modulators. In vitro electrophysiological and in vivo animal experiments showed that 2′-hydroxyflavone was a neutralizing modulator, different in efficacy from its structural analog, 6,2′-dihydroxyflavone, a negative modulator of GABAA receptors. The fact that flavone analogs differing only at position 6 showed drastically different pharmacological properties clearly points to 6-substitution being an important determinant of efficacy. The results suggest that a large width of the first atom on the 6-substituent favors a high binding affinity of the 6-substituted flavone, whereas a large overall volume of the 6-substituent favors positive modulator activity, which could be modified by, e.g., 2′-hydroxyl substitution. These findings have contributed to the understanding of quantitative structure–efficacy relationships for flavones acting at GABAA receptors, and hence facilitation of flavone-based drug development.
European Journal of Pharmacology - EUR J PHARMACOL. 01/2011; 670(1):121-129.
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Siu-Kin Ng,
Wing-Sze Lo,
Frank W Pun,
Cunyou Zhao,
Zhiliang Yu,
Jianhuan Chen,
Ka-Lok Tong,
Zhiwen Xu, Shui-Ying Tsang,
Qiang Yang,
Weichuan Yu,
Vishwajit Nimgaonkar,
Gerald Stöber,
Mutsuo Harano,
Hong Xue
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ABSTRACT: Schizophrenia is a major disorder with complex genetic mechanisms. Earlier, population genetic studies revealed the occurrence of strong positive selection in the GABRB2 gene encoding the beta(2) subunit of GABA(A) receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs) and containing schizophrenia-associated SNPs and haplotypes.
In the present study, the possible occurrence of recombination in this 'S1-S29' segment was assessed. The occurrence of hotspot recombination was indicated by high resolution recombination rate estimation, haplotype diversity, abundance of rare haplotypes, recurrent mutations and torsos in haplotype networks, and experimental haplotyping of somatic and sperm DNA. The sub-segment distribution of relative recombination strength, measured by the ratio of haplotype diversity (H(d)) over mutation rate (theta), was indicative of a human specific Alu-Yi6 insertion serving as a central recombining sequence facilitating homologous recombination. Local anomalous DNA conformation attributable to the Alu-Yi6 element, as suggested by enhanced DNase I sensitivity and obstruction to DNA sequencing, could be a contributing factor of the increased sequence diversity. Linkage disequilibrium (LD) analysis yielded prominent low LD points that supported ongoing recombination. LD contrast revealed significant dissimilarity between control and schizophrenic cohorts. Among the large array of inferred haplotypes, H26 and H73 were identified to be protective, and H19 and H81 risk-conferring, toward the development of schizophrenia.
The co-occurrence of hotspot recombination and positive selection in the S1-S29 segment of GABRB2 has provided a plausible contribution to the molecular genetics mechanisms for schizophrenia. The present findings therefore suggest that genome regions characterized by the co-occurrence of positive selection and hotspot recombination, two interacting factors both affecting genetic diversity, merit close scrutiny with respect to the etiology of common complex disorders.
PLoS ONE 01/2010; 5(3):e9547. · 4.09 Impact Factor
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Jianhuan Chen, Shui-Ying Tsang,
Cun-You Zhao,
Frank W Pun,
Zhiliang Yu,
Lingling Mei,
Wing-Sze Lo,
Shisong Fang,
Hua Liu,
Gerald Stöber,
Hong Xue
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ABSTRACT: The SCZ (schizophrenia)-associated GABA(A) receptor (gamma-aminobutyric acid type A receptor) beta(2) subunit gene GABRB2 was recently associated with BPD (bipolar disorder). Although weaker than its association with SCZ, significant association of GABRB2 with BPD was found in both German and Chinese, especially for the haplotypes rs1816071-rs187269 and rs1816072-rs187269 for which the M-M variants showed higher frequency in disease than the control. Significant genotype-dependent reduction in GABRB2 expression was shown for BPD, but to a lesser extent than that for SCZ. Temporal effects on GABRB2 expression were observed. Moreover, for the homozygous major genotypes of rs1816071, rs1816072 and rs187269, expression increased with time in CON but decreased in SCZ and BPD. The genotypes of these three SNPs (single nucleotide polymorphisms) were further correlated with antipsychotics dosage in SCZ cohorts. The findings highlight the importance of GABRB2 in neuropsychiatric disease aetiology, with respect to haplotype association, as well as reduction of and temporal effects on gene expression in both SCZ and BPD, but to a lesser extent in the latter, supporting the suggestion that functional psychosis can be conceptualized as a continuous spectrum of clinical phenotypes rather than as distinct categories.
Biochemical Society Transactions 12/2009; 37(Pt 6):1415-8. · 3.71 Impact Factor
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Cunyou Zhao,
Zhiwen Xu,
Feng Wang,
Jianhuan Chen,
Siu-Kin Ng,
Pak-Wing Wong,
Zhiliang Yu,
Frank W Pun,
Lihuan Ren,
Wing-Sze Lo, Shui-Ying Tsang,
Hong Xue
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ABSTRACT: Non-coding single nucleotide polymorphisms (SNPs) in GABRB2, the gene for beta(2)-subunit of gamma-aminobutyric acid type A (GABA(A)) receptor, have been associated with schizophrenia (SCZ) and quantitatively correlated to mRNA expression and alternative splicing.
Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1) and beta(2S2), bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1) expression and decreased beta(2S2) expression in both SCZ and bipolar disorder (BPD) compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1) and beta(2S2) expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2) expression. Moreover, site-directed mutagenesis indicated that Thr(365), a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down.
This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2)-subunit splicing diversity and the etiologies of SCZ and BPD.
PLoS ONE 01/2009; 4(9):e6977. · 4.09 Impact Factor
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ABSTRACT: The type A γ-aminobutyric acid (GABAA) receptor is a major inhibitory neurotransmitter-gated ion channel. Previously, we identified a membrane-proximal β-rich (MPBR) domain in fragment C166-L296 of GABAA receptor α1 subunit, forming nativelike pentamers. In the present study, another structural domain, the amino-terminal domain, was shown to exist in the fragment Q28-E165. The secondary structures of both fragments were β-rich as measured using FTIR spectroscopy and estimated from the CD spectra to be 42% and 51% β-strand for Q28-E165 and C166-L296, respectively. The CD spectrum of the combined fragment Q28-L296 was additive of the spectra of the two fragments. In addition, denaturation curves of both fragments were characteristic of cooperative transitions, supporting their domainlike nature. C166-L296 required 6.5 M of guanidine chloride for total denaturation, therefore it is extraordinarily stable, more so than Q28-E165. Moreover, effects of detergent on the molecular masses of Q28-E165 and C166-L296, as monitored using laser-scattering spectroscopy, indicated that intermolecular interactions were much more significant in C166-L296 than in Q28-E165. Effects of pH on their molecular masses suggested that ionic forces were involved in these interactions. Together the results show that the two adjacent fragments form independent folding units, MPBR and amino-terminal domains, different in secondary structure content, denaturation profile, and polymerization status, and suggest that the former may play a more important role in receptor assembly and that the extraordinary stability may underlie its intrinsic tendency to form oligomers. More significantly, the present study has provided direct evidence for the long-postulated multidomain nature of this family of receptors.
Protein Science 12/2008; 11(8):2052 - 2058. · 2.80 Impact Factor
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ABSTRACT: Baicalin, a naturally occurring flavonoid, was previously reported to induce anxiolytic-like effect devoid of sedation and myorelaxation in mice, acting through type A gamma-aminobutyric acid (GABA(A)) receptor benzodiazepine (BZ) site. The present study further expanded the behavioral pharmacology profile of baicalin and subtype selectivity was explored as a possible mechanism underlying its in vivo effects on mice. Baicalin was characterized using convulsion, memory, and motor function related animal tests; and its selectivity towards recombinant GABA(A) receptor subtypes expressed in HEK 293T cells was determined by radioligand binding assay and electrophysiological studies. In the picrotoxin-induced seizure, step-through passive avoidance and rotarod tests, the anticonvulsant, amnesic and motor incoordination effects commonly associated with classical BZs were not observed when baicalin was administered at effective anxiolytic doses, demonstrating a separation of the anticonvulsant, amnesic and motor incoordination effects from the anxiolytic-like effect. Although baicalin exhibited higher binding affinity for the alpha1-containing GABA(A) subtype compared with alpha2-, alpha3-, and alpha5-containing subtypes, this was not statistically significant. In contrast to the classical BZ diazepam, baicalin showed significant preference for alpha2- and alpha3-containing subtypes compared to alpha1- and alpha5-containing subtypes in whole-cell patch clamp studies (P < 0.01). Its subtype selectivity suggested that baicalin exerted its in vivo anxiolytic-like effect mainly through the alpha2- and alpha3-containing subtypes. Therefore, the present study revealed an underlying mechanism for the selective anxiolytic profile of baicalin, suggesting alpha2- and alpha3-containing subtypes were important drug targets for flavonoid-based anxiolytics.
Neuropharmacology 09/2008; 55(7):1231-7. · 4.81 Impact Factor
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ABSTRACT: Neuroactivity of a number of flavonoids is mediated by modulation of type A gamma-aminobutyric acid (GABA(A)) receptor function via benzodiazepine sites, mostly as partial agonists. In the present study, 6,2'-dihydroxyflavone (DHF) was characterized for potential inverse agonistic activity, and its mechanism of action was explored for receptor subtype selectivity. In whole-cell patch clamp studies on neuroblastoma IMR-32 cells expressing native GABA(A) receptors, DHF decreased GABA-induced currents, to an extent similar to that induced by the partial inverse agonist FG-7142, which could be blocked by flumazenil, a BZ site antagonist. In mouse behavioral models, DHF elicited significant anxiogenic-like effects in the elevated plus-maze test, and enhanced cognitive performance in the step-through passive avoidance test, as expected for an inverse agonist. However, DHF did not exhibit any proconvulsant effects, a typical action of inverse agonists. In electrophysiological studies on subtypes of recombinant GABA(A) receptors expressed in HEK 293T cells, DHF decreased GABA-induced currents in alpha(1)beta(3)gamma(2), alpha(2)beta(3)gamma(2), or alpha(5)beta(3)gamma(2), but not alpha(3)beta(3)gamma(2) receptors. The results demonstrated DHF as a partial inverse agonist-like modulator of GABA(A) receptors with selectivity in receptor subtypes as well as behavioral effects. The DHF subtype-selectivity suggested that alpha(3)-containing subtypes could be a mediator of the convulsion activities of GABA(A) receptor inverse agonists. Moreover, the pharmacological profile displayed in mouse behavioral models supported DHF as a useful lead compound for the development of cognition-enhancing agents devoid of convulsion side effects.
Neuropharmacology 10/2007; 53(4):574-82. · 4.81 Impact Factor
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Wing-Sze Lo,
Mutsuo Harano,
Micha Gawlik,
Zhiliang Yu,
Jianhuan Chen,
Frank W Pun,
Ka-Lok Tong,
Cunyou Zhao,
Siu-Kin Ng, Shui-Ying Tsang,
Naohisa Uchimura,
Gerald Stober,
Hong Xue
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ABSTRACT: Single nucleotide polymorphisms (SNPs) and haplotypes in intron 8 of type A gamma-aminobutyric acid (GABA(A)) receptor beta2 subunit gene (GABRB2) were initially found to be associated with schizophrenia in Chinese. This finding was subjected to cross-validation in this study with Japanese (JP) and German Caucasian (GE) subjects.
Single nucleotide polymorphisms discovery and genotyping were carried out through resequencing of a 1839 base pair (bp) region in GABRB2. Tagging SNPs (tSNPs) were selected based on linkage disequilibrium (LD), combinations of which were analyzed with Bonferroni correction and permutation for disease association. Random resampling was applied to generate size- and gender-balanced cases and control subjects.
Out of the 17 SNPs (9.2/kilobase [kb]) revealed, 6 were population-specific. Population variations in LD were observable, and at least two low LD points were identified in both populations. Although disease association at single SNP level was only shown in GE, strong association was demonstrated in both JP (p = .0002 - .0191) and GE (p = .0033 - .0410) subjects, centering on haplotypes containing rs1816072 and rs1816071. Among different clinical subtypes, the most significant association was exhibited by systematic schizophrenia.
Cross-population validation of GABRB2 association with schizophrenia has been obtained with JP and GE subjects, with the genotype-disease correlations being strongest in systematic schizophrenia, the most severe subtype of the disease.
Biological Psychiatry 04/2007; 61(5):653-60. · 8.28 Impact Factor
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ABSTRACT: The inhibitory ligand-gated ion channel family of receptors, including the type A gamma-aminobutryic acid (GABA(A)) and glycine receptors, mediates inhibitory neurotransmissions in the central nervous system. In this study, GABA receptor (GABR) evolution was explored through comparative genomics using genomes that span divergent lineages. GABA(A)/Gly receptor-like (GRL) gene sequences were retrieved from the genomes of various species ranging from mammal to fish to worm and subjected to cross-species comparison. All vertebrate GRL gene sets in the study but no invertebrate ones exhibit the extensive and conserved pattern of gene clustering that is characteristic of human GABR genes, indicating that the gene clusters were established early in vertebrate evolution, after divergence from the invertebrates. Moreover, the vertebrate gene structure is highly conserved with a basic 9-coding exon structure, whereas, as well as being diverse in copy numbers and chromosomal loci, the invertebrate GRL genes display a variety of gene structures. Remarkably, the invertebrates each possess a unique GRL gene pair that lies in neighboring loci within their respective genomes: zc482.5 and zc482.1 in roundworm, CG8916 and CG17336 in fruitfly, Ci4249 and Ci4254 in Ciona, and these were revealed by phylogenetic analysis to be homologous to human GABR alpha and beta subunits, respectively. The phylogenetic classification of these genes is also corroborated by experimental ligand-binding measurements using recombinant gene products. Furthermore, the 3 invertebrate gene pairs harbor characteristic key residues and exhibit similarities in intron positions to their vertebrate counterparts. The results strongly indicate that such a gene pair originally existed in the bilaterian ancestor from which all 3 phyla evolved and suggest that the extant GABR clusters arose from an ancestral alpha-beta subunit gene pair gave rise to the extant GABR clusters.
Molecular Biology and Evolution 03/2007; 24(2):599-610. · 5.55 Impact Factor
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Wing-Sze Lo,
Zhiwen Xu,
Zhiliang Yu,
Frank W Pun,
Siu-Kin Ng,
Jianhuan Chen,
Ka-Lok Tong,
Cunyou Zhao,
Xiaojing Xu, Shui-Ying Tsang,
Mutsuo Harano,
Gerald Stöber,
Vishwajit L Nimgaonkar,
Hong Xue
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ABSTRACT: The gamma-aminobutyric acid type-A (GABA(A)) receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABA(A) receptor beta(2) subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced beta(2) isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D) alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of beta(2), especially its long isoform. Electrophysiological analysis showed that this long beta(2) isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABA(A) receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.
PLoS ONE 02/2007; 2(5):e462. · 4.09 Impact Factor
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ABSTRACT: Schizophrenia (SCZ) is a complex mental disease that affects approximately 1% of the population. In this study, six SNPs in GABRB2 were genotyped for a case-control association study with the cycloid psychosis subtype of SCZ in the German population using two methods for SNP genotyping.
The SNPs were genotyped by direct DNA sequencing, as well as a novel one-label extension method. The results were analyzed for association with SCZ.
Significant association was found for SNPs rs1816071 and rs1816072 with SCZ susceptibility. This is consistent with our previous finding of association of SNPs in GABRB2 with SCZ susceptibility in Han Chinese. There was a total agreement between the genotyping results from one-label extensions and the results of direct DNA sequencing, thus validating the accuracy of the one-label extension method of SNP genotyping.
Clinical Biochemistry 04/2006; 39(3):210-8. · 2.08 Impact Factor
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ABSTRACT: Baicalin, a naturally occurring flavonoid, was previously reported to exert anxiolytic-like effects in the Vogel conflict test. In the present study, the anxiolytic effects of baicalin alone and in combination with other anxiolytics were tested in mice using the elevated plus-maze (EPM). Baicalin treatment (7.5 - 30 mg/kg) significantly increased entries into and time spent in open arms, indicative of an anxiolytic-like effect. Motor-depressive and myorelaxant side effects commonly associated with anxiolytics were not observed with baicalin at effective anxiolytic doses in the hole-board and horizontal wire tests, respectively. Co-administration of baicalin (3.75 mg/kg) with dl-tetrahydropalmatine ( dl-THP; 0.25 mg/kg), an anxiolytic-hypnotic alkaloid, both at sub-effective doses, induced an additive effect resulting in considerable anxiolysis. Similarly, an additive anxiolytic-like effect was observed with baicalin (3.75 mg/kg) and diazepam (DZ; 0.5 mg/kg). Results obtained from this study demonstrate the potential of baicalin as a candidate anxiolytic and its possible application in multidrug therapy. Abbreviations. BZS:benzodiazepine-binding site EPM:elevated plus-maze DZ:diazepam GABA (A):type A gamma-aminobutyric acid dl-THP: dl-tetrahydropalmatine.
Planta Medica 03/2006; 72(2):189-92. · 2.15 Impact Factor
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ABSTRACT: Classical benzodiazepines (BZs) are the most widely prescribed drugs acting on the central nervous system (CNS). They exert their therapeutic effects via binding to the BZ-site of GABAA receptors, and allosterically modulating the chloride flux through the ion channel complex. Given the multiple actions of classical BZs, the serious limitations to their usefulness have directed much research into development of novel ligands for the BZ-site with retained therapeutic effectiveness and minimal side effects. From the studies of CNS-active chemical constituents of medicinal herbs, some members of the family of flavonoids were demonstrated to have moderate binding affinities for the BZ-site. In vivo studies revealed that these compounds were mostly partial agonists of GABAA receptors, and only a few flavonoids were shown to possess antagonistic activities. At effective anxiolytic doses, the actions of partial agonistic flavonoids were often not accompanied by sedative and myorelaxant side effects. Based on structure-activity relationship (SAR) studies, incorporation of electronegative groups to the C6 and C3' on the flavone backbone was found to yield significant increases in the binding affinities for the BZ-site. It was also shown that 2'-hydroxyl was a critical moiety on flavonoids with regard to BZ-site binding. These have guided the identification of several synthetic flavonoids with high BZ-site binding affinity and in vivo activity, and further quantitative SAR studies resulted in the development of several pharmacophore models. This review attempts to summarize these findings, which has led to the establishment of flavonoids as potential therapeutics for GABAA receptor-mediated disorders.
Current Drug Targets - CNS & Neurological Disorders 11/2005; 4(5):575-85.
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Zhiwen Xu,
Shisong Fang,
Haifeng Shi,
Hoiming Li,
Yiqun Deng,
Yinglei Liao,
Jiun-Ming Wu,
Hui Zheng,
Huaimin Zhu,
Hueih-Min Chen, Shui Ying Tsang,
Hong Xue
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ABSTRACT: Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal beta-rich (MPB) domain in the C139-L269 segment of the receptor alpha1 subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad "pin" of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel beta-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold.
Protein Science 11/2005; 14(10):2622-37. · 2.80 Impact Factor
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ABSTRACT: The search for flavonoids with novel therapeutic effects has been intense. Wogonin, as a naturally existing monoflavonoid, has been shown to have therapeutic potential in vitro and in vivo. Methods for its extraction from herbs and its chemical synthesis have been developed. Pharmacokinetic studies have shown a rapid tissue distribution and prolonged plasma elimination phase of wogonin. It has been shown experimentally that wogonin exerts anti-oxidant activity, which may, in part, underlie its antiinflammatory, anti-cancer, antiviral and neuroprotective actions. The recent discovery of its anxiolytic activity suggests a new mechanism of action, involving interaction with the benzodiazepine (BZD) binding site of the GABA(A) receptor and modulation of this receptor activity. Although the safety record of wogonin is remarkable and voluminous literature about its pharmacological effects is available, it has not been used in Western medicine in the form of a pure chemical. In this article we review its therapeutic effects, its sources and pharmacokinetic profile to highlight its therapeutic potential.
CNS Drug Reviews 02/2005; 11(2):141-50. · 4.92 Impact Factor
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ABSTRACT: The three most widely expressed subunits of the GABAA receptor are alpha(1), beta(2), and gamma(2) subunits, and the major isoform in the human brain is a pentameric receptor composed of 2alpha(1)2beta(2)1gamma(2). Previously, we overexpressed the extracellular domain Q28-R248 of GABAA receptor alpha(1) subunit. In the present study, the homologous extracellular domains Q25-G243 of GABAA receptor beta(2) subunit and Q40-G273 of gamma(2) subunit were also obtained through overexpression in Escherichia coli. Successful production of recombinant beta(2) and gamma(2) subunit receptor protein domains facilitates the comparison of structural and functional properties of the three subunits. To this end, the secondary structures of the three fragments were measured using CD spectroscopy and the beta-strand contents calculated to be >30%, indicating a beta-rich structure for all three fragments. In addition, the benzodiazepine (BZ)-binding affinity of the recombinant fragments were measured using fluorescence polarization to be 2.16 microM, 3.63 microM, and 1.34 microM for the alpha(1), beta(2), and gamma(2) subunit fragments, respectively, indicating that all three homomeric assemblies, including that of the beta(2) subunit, generally not associated with BZ binding, can bind BZ in the micromolar range. The finding that the BZ binding affinity of these recombinant domains was highest for the gamma(2) subunit and lowest for the beta(2) subunit is consistent with results from previous binding studies using hetero-oligomeric receptors. The present results exemplify the effective approach to characterize and compare the three major subunits of the GABAA receptor, for two of which the overexpression in E. coli is reported for the first time.
Protein Science 11/2003; 12(11):2642-6. · 2.80 Impact Factor
