A Recombination Hotspot in a Schizophrenia-Associated Region of GABRB2

Background: 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 β2 subunit of GABAA receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs) and containing schizophrenia-associated SNPs and haplotypes. Methodology/Principal Findings: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 (Hd) over mutation rate (θ), 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. Conclusions/Significance: 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. © 2010 Ng et al

AluScan: a method for genome-wide scanning of sequence and structure variations in the human genome

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Alternative-Splicing in the Exon-10 Region of GABAA Receptor β2 Subunit Gene: Relationships between Novel Isoforms and Psychotic Disorders

BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSION: 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

Social Cognitive Role of Schizophrenia Candidate Gene GABRB2

10.1371/journal.pone.0062322PLoS ONE84

Positive Selection within the Schizophrenia-Associated GABA(A) Receptor β(2) Gene

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 β(2) subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced β(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 β(2), especially its long isoform. Electrophysiological analysis showed that this long β(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

Alternative splicing isoforms of GABAA̳ receptor [Beta]{208} subunit in schizophrenia and bipolar disorder

Single nucleotide polymorphisms (SNPs) in type A γ-aminobutyric acid (GABAA) receptor β2 subunit gene (GABRB2) were found to be associated with schizophrenia in Chinese, German, Japanese and Portuguese. To explore the potential functional consequences of these DNA sequence polymorphisms, this study examined the expression and electrophysiological properties of alternatively spliced products of GABRB2 along with genotypical disease-association analysis. Real-time quantitative PCR, performed with a US population of 31 schizophrenics, 30 bipolar disorders and 31 controls showed 21.7% reduction in the expression of the long isoform β2L, and 13.4% in the short isoform β2S (dominant isoform) in postmortem schizophrenic brain, but not in bipolar disorder’s brain. Furthermore, two novel short isoforms, β2S1 and β2S2, from human brain were observed and characterized. The expression of β2S2, bearing a frame-shift deletion in the terminal Exon-11, was decreased in schizophrenia (by 17.9%) and bipolar disorders (by 22.5%). The expression of the less abundant β2S1 was marginally increased in both of these mental disorders (by 43.0% in schizophrenics; by 43.7 % in bipolar disorders). Moreover, the relative expressions of β2L over β2S were significantly decreased, suggesting the occurrence of altered splicing, in schizophrenia. In male schizophrenics, the heterozygous genotypes of rs1876071 (T/C) and rs1876072 (A/G) were correlated with reduced expression of β2L and β2S, and the heterozygous of rs2546620 (A/G) and homozygous-minor of rs1876071 (C/C) and rs1876072 (G/G) were correlated with reduced expression of β2S2. In male bipolar disorders, the heterozygous genotypes of rs187269 (T/C) were correlated with reduced expression of β2S2 as well as the β2S2/β2S ratio. Electrophysiologically, the EC50 for neurotransmitter GABA was significantly reduced with β2S1, potentially compensating for the GABA deficit observed in schizophrenia. The results thus revealed genotype-dependent expression of the alternatively spliced isoforms of GABAA receptor β2 subunit, giving rise to electrophysiological consequences that could play an important role in the pathogenesis mechanism of schizophrenia

A simple method for high-throughput quantification of genome-wide DNA methylation by fluorescence polarization

To rapidly determine DNA methylation levels from a large number of biological or clinical samples, we have developed an accurate and sensitive method for high-throughput quantification of global methylation of 5'-Cm(5)CGG-3' sites in the genome, visualized by fluorescence polarization (FP) based measurement of DNA methylation (FPDM). In FPDM, the methyl-sensitive HpaII and methyl-insensitive MspI restriction enzymes were employed to achieve DNA cleavage, followed by incorporation of fluorescent dCMP into the enzyme-cleavage products through polymerase chain extension, yielding an FP-ratio between the HpaII- and MspI-restricted preparations as a measure of methylation. FPDM provided stable estimates of methylation level of submicrograms of lambda or human DNA, and of a 255-bp DNA segment containing a single HpaII/MspI restriction site in accord with, and more accurate than, determination by gel electrophoresis. FPDM was also applied to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA-methyltransferase inhibitor 5-aza-dC

Clustering Pattern and Functional Effect of SNPs in Human miRNA Seed Regions

miRNAs are a class of noncoding RNAs important in posttranscriptional repressors and involved in the regulation of almost every biological process by base paring with target genes through sequence in their seed regions. Genetic variations in the seed regions have vital effects on gene expression, phenotypic variation, and disease susceptibility in humans. The distribution pattern of genetic variation in miRNA seed regions might be related to miRNA function and is worth paying more attention to. We here employed computational analyses to explore the clustering pattern and functional effect of SNPs in human miRNA seed regions. A total of 1879 SNPs were mapped to 1226 human miRNA seed regions. We found that miRNAs with SNPs in their seed region are significantly enriched in miRNA clusters. We also found that SNPs in clustered miRNA seed regions have a lower functional effect than have SNPs in nonclustered miRNA seed regions. Additionally, we found that clustered miRNAs with SNPs in seed regions are involved in more pathways. Overall, our results demonstrate that SNPs in clustered miRNA seed regions can take part in more intricate and complex gene-regulating networks with lower functional cost by functional complementarity. Moreover, our results also broaden current knowledge on the genetic variation in human miRNA seed regions

Fluorescence polarization-based method with bisulfite conversion-specific one-label extension for quantification of single CpG dinucleotide methylation

To quantify the methylation at individual CpG dinucleotide sites in large biological or clinical samples, we develop a bisulfite conversion-specific one-label extension (BS-OLE) method using visualization by fluorescence polarization (FP) measurement of methylation at single CpG site in small amounts of genomic DNA. Genomic DNA is treated with sodium bisulfite to convert unmethylated cytosine to uracil leaving 5-methylcytosine unaltered and BS-PCR is used to generate DNA template containing target CpG sites. BS-OLE uses a BS-primer hybridized immediately upstream of the target CpG site being examined and then fluorescent dCTP or dUTP is incorporated into the methylated (CpG) or unmethylated (TpG) form of the target site through single-nucleotide chain extension, yielding an FP ratio between the fluorescent dCTP- and dUTP-incorporated products as a measure of methylation. This provides stable estimates of the methylation level of human genomic DNA and of a 250 bp plasmid DNA segment containing a single TCGAThe accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Beneficial Effects of Biochar Application with Nitrogen Fertilizer on Soil Nitrogen Retention, Absorption and Utilization in Maize Production

The irrational use of nitrogen (N) fertilizer has become a major threat to soil quality and food security, resulting in serious ecological and environmental problems. Holistic approaches to N fertilizer application are required to maintain a high N utilization efficiency (NUE) and sustainable agriculture development. Biochar is an efficient carbon-rich material for amending soil quality and promoting crop N uptake, but knowledge pertaining to the promoting effects of biochar application on N fertilizers is still limited. In this study, a field plot experiment was designed to detect the combined effects of biochar (0, 15 and 30 t ha−1) and N fertilizer (204, 240 and 276 kg N ha−1) on the soil nutrient levels, NUE, plant growth performance and crop production of maize. The results demonstrated that the combined application of N fertilizer and biochar can significantly decrease the soil pH and increase the contents of soil organic carbon, mineral N, available phosphorus and potassium. The crop N uptake and N content were largely promoted by the addition of N fertilizer and biochar, resulting in higher leaf photosynthetic efficiency, dry matter accumulation and grain yields. The highest yields (14,928 kg ha−1) were achieved using 276 kg N ha−1 N fertilizer in combination with 15 t ha−1 biochar, and the highest NUE value (46.3%) was reached with 204 kg N ha−1 N of fertilizer blended with 30 t ha−1 of biochar. According to structural equation modeling, the beneficial effects of N fertilizer and biochar on the plant biomass of maize were attributed to the direct effects related to soil chemical properties and plant growth parameters. In conclusion, N fertilizer combined with biochar application is an effective strategy to enhance the utilization of N fertilizer and crop production for maize by increasing soil fertility, improving plant crop uptake and promoting plant growth