Genomic subtyping of liver cancers with prognostic application.

BACKGROUND: Cancer subtyping has mainly relied on pathological and molecular means. Massively parallel sequencing-enabled subtyping requires genomic markers to be developed based on global features rather than individual mutations for effective implementation. METHODS: In the present study, the whole genome sequences (WGS) of 110 liver cancers of Japanese patients published with different pathologies were analyzed with respect to their single nucleotide variations (SNVs) comprising both gain-of-heterozygosity (GOH) and loss-of-heterozygosity (LOH) mutations, the signatures of combined GOH and LOH mutations, along with recurrent copy number variations (CNVs). RESULTS: The results, obtained based on the WGS sequences as well as the Exome subset within the WGSs that covered ~ 2.0% of the WGS and the AluScan-subset within the WGSs that were amplifiable by Alu element-consensus primers and covered ~ 2.1% of the WGS, indicated that the WGS samples could be employed with the mutational parameters of SNV load, LOH%, the Signature α%, and survival-associated recurrent CNVs (srCNVs) as genomic markers for subtyping to stratify liver cancer patients prognostically into the long and short survival subgroups. The usage of the AluScan-subset data, which could be implemented with sub-micrograms of DNA samples and vastly reduced sequencing analysis task, outperformed the usage of WGS data when LOH% was employed as stratifying criterion. CONCLUSIONS: Thus genomic subtyping performed with novel genomic markers identified in this study was effective in predicting patient-survival duration, with cohorts of hepatocellular carcinomas alone and those including intrahepatic cholangiocarcinomas. Such relatively heterogeneity-insensitive genomic subtyping merits further studies with a broader spectrum of cancers.

Noninvasive prenatal paternity testing by means of SNP-based targeted sequencing.

OBJECTIVE: To develop a method for noninvasive prenatal paternity testing based on targeted sequencing of single nucleotide polymorphisms (SNPs). METHOD: SNPs were selected based on population genetics data. Target-SNPs in cell-free DNA extracted from maternal blood (maternal cfDNA) were analyzed by targeted sequencing wherein target enrichment was based on multiplex amplification using QIAseq Targeted DNA Panels with Unique Molecular Identifiers. Fetal SNP genotypes were called using a novel bioinformatics algorithm, and the combined paternity indices (CPIs) and resultant paternity probabilities were calculated. RESULTS: Fetal SNP genotypes obtained from targeted sequencing of maternal cfDNA were 100% concordant with those from amniotic fluid-derived fetal genomic DNA. From an initial panel of 356 target-SNPs, an average of 148 were included in paternity calculations in 15 family trio cases, generating paternity probabilities of greater than 99.9999%. All paternity results were confirmed by short-tandem-repeat analysis. The high specificity of the methodology was validated by successful paternity discrimination between biological fathers and their siblings and by large separations between the CPIs calculated for the biological fathers and those for 60 unrelated men. CONCLUSION: The novel method is highly effective, with substantial improvements over similar approaches in terms of reduced number of target-SNPs, increased accuracy, and reduced costs.

Forward and reverse mutations in stages of cancer development.

BACKGROUND: Massive occurrences of interstitial loss of heterozygosity (LOH) likely resulting from gene conversions were found by us in different cancers as a type of single-nucleotide variations (SNVs), comparable in abundance to the commonly investigated gain of heterozygosity (GOH) type of SNVs, raising the question of the relationships between these two opposing types of cancer mutations. METHODS: In the present study, SNVs in 12 tetra sample and 17 trio sample sets from four cancer types along with copy number variations (CNVs) were analyzed by AluScan sequencing, comparing tumor with white blood cells as well as tissues vicinal to the tumor. Four published "nontumor"-tumor metastasis trios and 246 pan-cancer pairs analyzed by whole-genome sequencing (WGS) and 67 trios by whole-exome sequencing (WES) were also examined. RESULTS: Widespread GOHs enriched with CG-to-TG changes and associated with nearby CNVs and LOHs enriched with TG-to-CG changes were observed. Occurrences of GOH were 1.9-fold higher than LOH in "nontumor" tissues more than 2 cm away from the tumors, and a majority of these GOHs and LOHs were reversed in "paratumor" tissues within 2 cm of the tumors, forming forward-reverse mutation cycles where the revertant LOHs displayed strong lineage effects that pointed to a sequential instead of parallel development from "nontumor" to "paratumor" and onto tumor cells, which was also supported by the relative frequencies of 26 distinct classes of CNVs between these three types of cell populations. CONCLUSIONS: These findings suggest that developing cancer cells undergo sequential changes that enable the "nontumor" cells to acquire a wide range of forward mutations including ones that are essential for oncogenicity, followed by revertant mutations in the "paratumor" cells to avoid growth retardation by excessive mutation load. Such utilization of forward-reverse mutation cycles as an adaptive mechanism was also observed in cultured HeLa cells upon successive replatings. An understanding of forward-reverse mutation cycles in cancer development could provide a genomic basis for improved early diagnosis, staging, and treatment of cancers.

Variation of global DNA methylation levels with age and in autistic children.

BACKGROUND: The change in epigenetic signatures, in particular DNA methylation, has been proposed as risk markers for various age-related diseases. However, the course of variation in methylation levels with age, the difference in methylation between genders, and methylation-disease association at the whole genome level is unclear. In the present study, genome-wide methylation levels in DNA extracted from peripheral blood for 2116 healthy Chinese in the 2-97 age range and 280 autistic trios were examined using the fluorescence polarization-based genome-wide DNA methylation quantification method developed by us. RESULTS: Genome-wide or global DNA methylation levels proceeded through multiple phases of variation with age, consisting of a steady increase from age 2 to 25 (r = 0.382) and another rise from age 41 to 55 to reach a peak level of ~80 % (r = 0.265), followed by a sharp decrease to ~40 % in the mid-1970s (age 56 to 75; r = -0.395) and leveling off thereafter. Significant gender effect in methylation levels was observed only for the 41-55 age group in which methylation in females was significantly higher than in males (p = 0.010). In addition, global methylation level was significantly higher in autistic children than in age-matched healthy children (p < 0.001). CONCLUSIONS: The multiphasic nature of changes in global methylation levels with age was delineated, and investigation into the factors underlying this profile will be essential to a proper understanding of the aging process. Furthermore, this first report of global hypermethylation in autistic children also illustrates the importance of age-matched controls in characterization of disease-associated variations in DNA methylation.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

GABRB2 in schizophrenia and bipolar disorder: disease association, gene expression and clinical correlations.

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.

GABA A receptor subtype selectivity underlying selective anxiolytic effect of baicalin.

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.

Gabrb2-knockout mice displayed schizophrenia-like and comorbid phenotypes with interneuron-astrocyte-microglia dysregulation.

Intronic polymorphisms of the GABAA receptor ß2 subunit gene (GABRB2) under adaptive evolution were associated with schizophrenia and reduced expression, especially of the long isoform which differs in electrophysiological properties from the short isoform. The present study was directed to examining the gene dosage effects of Gabrb2 in knockout mice of both heterozygous (HT) and homozygous (KO) genotypes with respect to possible schizophrenia-like and comorbid phenotypes. The KO mice, and HT mice to a lesser extent, were found to display prepulse inhibition (PPI) deficit, locomotor hyperactivity, stereotypy, sociability impairments, spatial-working and spatial-reference memory deficits, reduced depression and anxiety, and accelerated pentylenetetrazol (PTZ)-induced seizure. In addition, the KO mice were highly susceptible to audiogenic epilepsy. Some of the behavioral phenotypes showed evidence of imprinting, gender effect and amelioration by the antipsychotic risperidone, and the audiogenic epilepsy was inhibited by the antiepileptic diazepam. GABAergic parvalbumin (PV)-positive interneuron dystrophy, astrocyte dystrophy, and extensive microglia activation were observed in the frontotemporal corticolimbic regions, and reduction of newborn neurons was observed in the hippocampus by immunohistochemical staining. The neuroinflammation indicated by microglial activation was accompanied by elevated brain levels of oxidative stress marker malondialdehyde (MDA) and the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). These extensive schizophrenia-like and comorbid phenotypes brought about by Gabrb2 knockout, in conjunction with our previous findings on GABRB2 association with schizophrenia, support a pivotal role of GABRB2 in schizophrenia etiology.

GABRB2 association with schizophrenia: commonalities and differences between ethnic groups and clinical subtypes.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

6,2'-Dihydroxyflavone, a subtype-selective partial inverse agonist of GABAA receptor benzodiazepine site.

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.

Analysis of GABRB2 association with schizophrenia in German population with DNA sequencing and one-label extension method for SNP genotyping.

OBJECTIVES: 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. DESIGN AND METHODS: 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. RESULTS AND CONCLUSIONS: 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.

Feature co-localization landscape of the human genome.

Although feature co-localizations could serve as useful guide-posts to genome architecture, a comprehensive and quantitative feature co-localization map of the human genome has been lacking. Herein we show that, in contrast to the conventional bipartite division of genomic sequences into genic and inter-genic regions, pairwise co-localizations of forty-two genomic features in the twenty-two autosomes based on 50-kb to 2,000-kb sequence windows indicate a tripartite zonal architecture comprising Genic zones enriched with gene-related features and Alu-elements; Proximal zones enriched with MIR- and L2-elements, transcription-factor-binding-sites (TFBSs), and conserved-indels (CIDs); and Distal zones enriched with L1-elements. Co-localizations between single-nucleotide-polymorphisms (SNPs) and copy-number-variations (CNVs) reveal a fraction of sequence windows displaying steeply enhanced levels of SNPs, CNVs and recombination rates that point to active adaptive evolution in such pathways as immune response, sensory perceptions, and cognition. The strongest positive co-localization observed between TFBSs and CIDs suggests a regulatory role of CIDs in cooperation with TFBSs. The positive co-localizations of cancer somatic CNVs (CNVT) with all Proximal zone and most Genic zone features, in contrast to the distinctly more restricted co-localizations exhibited by germline CNVs (CNVG), reveal disparate distributions of CNVTs and CNVGs indicative of dissimilarity in their underlying mechanisms.

Topology characterization of a benzodiazepine-binding beta-rich domain of the GABAA receptor alpha1 subunit.

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.

Neuroactive flavonoids interacting with GABAA receptor complex.

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.

SAMSVM: A tool for misalignment filtration of SAM-format sequences with support vector machine.

Sequence alignment/map (SAM) formatted sequences [Li H, Handsaker B, Wysoker A et al., Bioinformatics 25(16):2078-2079, 2009.] have taken on a main role in bioinformatics since the development of massive parallel sequencing. However, because misalignment of sequences poses a significant problem in analysis of sequencing data that could lead to false positives in variant calling, the exclusion of misaligned reads is a necessity in analysis. In this regard, the multiple features of SAM-formatted sequences can be treated as vectors in a multi-dimension space to allow the application of a support vector machine (SVM). Applying the LIBSVM tools developed by Chang and Lin [Chang C-C, Lin C-J, ACM Trans Intell Syst Technol 2:1-27, 2011.] as a simple interface for support vector classification, the SAMSVM package has been developed in this study to enable misalignment filtration of SAM-formatted sequences. Cross-validation between two simulated datasets processed with SAMSVM yielded accuracies that ranged from 0.89 to 0.97 with F-scores ranging from 0.77 to 0.94 in 14 groups characterized by different mutation rates from 0.001 to 0.1, indicating that the model built using SAMSVM was accurate in misalignment detection. Application of SAMSVM to actual sequencing data resulted in filtration of misaligned reads and correction of variant calling.

GABRB2 Haplotype Association with Heroin Dependence in Chinese Population.

Substance dependence is a frequently observed comorbid disorder in schizophrenia, but little is known about genetic factors possibly shared between the two psychotic disorders. GABRB2, a schizophrenia candidate gene coding for GABAA receptor ß2 subunit, is examined for possible association with heroin dependence in Han Chinese population. Four single nucleotide polymorphisms (SNPs) in GABRB2, namely rs6556547 (S1), rs1816071 (S3), rs18016072 (S5), and rs187269 (S29), previously associated with schizophrenia, were examined for their association with heroin dependence. Two additional SNPs, rs10051667 (S31) and rs967771 (S32), previously associated with alcohol dependence and bipolar disorder respectively, were also analyzed. The six SNPs were genotyped by direct sequencing of PCR amplicons of target regions for 564 heroin dependent individuals and 498 controls of Han Chinese origin. Interestingly, it was found that recombination between the haplotypes of all-derived-allele (H1; OR = 1.00) and all-ancestral-allele (H2; OR = 0.74) at S5-S29 junction generated two recombinants H3 (OR = 8.51) and H4 (OR = 5.58), both conferring high susceptibility to heroin dependence. Additional recombination between H2 and H3 haplotypes at S1-S3 junction resulted in a risk-conferring haplotype H5 (OR = 1.94x109). In contrast, recombination between H1 and H2 haplotypes at S3-S5 junction rescued the risk-conferring effect of recombination at S5-S29 junction, giving rise to the protective haplotype H6 (OR = 0.68). Risk-conferring effects of S1-S3 and S5-S29 crossovers and protective effects of S3-S5 crossover were seen in both pure heroin dependent and multiple substance dependence subgroups. In conclusion, significant association was found with haplotypes of the S1-S29 segment in GABRB2 for heroin dependence in Han Chinese population. Local recombination was an important determining factor for switching haplotypes between risk-conferring and protective statuses. The present study provide evidence for the schizophrenia candidate gene GABRB2 to play a role in heroin dependence, but replication of these findings is required.

Glioma Association and Balancing Selection of ZFPM2.

ZFPM2, encoding a zinc finger protein and abundantly expressed in the brain, uterus and smooth muscles, plays important roles in cardiac and gonadal development. Abnormal expression of ZFPM2 in ovarian tumors and neuroblastoma has been reported but hitherto its genetic association with cancer and effects on gliomas have not been studied. In the present study, the hexamer insertion-deletion polymorphism rs71305152, located within a large haplotype block spanning intron 1 to intron 3 of ZFPM2, was genotyped in Chinese cohorts of glioma (n = 350), non-glioma cancer (n = 354) and healthy control (n = 463) by direct sequencing and length polymorphism in gel electrophoresis, and ZFPM2 expression in glioma tissues (n = 69) of different grades was quantified by real-time RT-PCR. Moreover, potential natural selection pressure acting on the gene was investigated. Disease-association analysis showed that the overall genotype of rs71305152 was significantly associated with gliomas (P = 0.016), and the heterozygous genotype compared to the combined homozygous genotypes was less frequent in gliomas than in controls (P = 0.005) or non-glioma cancers (P = 0.020). ZFPM2 mRNA expression was negatively correlated with the grades of gliomas (P = 0.002), with higher expression levels in the low-grade gliomas. In the astrocytoma subtype, higher ZFPM2 expression was also correlated with the rs71305152 heterozygous genotype (P = 0.028). In addition, summary statistics tests gave highly positive values, demonstrating that the gene is under the influence of balancing selection. These findings suggest that ZFPM2 is a glioma susceptibility gene, its genotype and expression showing associations with incidence and severity, respectively. Moreover, the balancing selection acting on ZFPM2 may be related to the important roles it has to play in multiple organ development or associated disease etiology.

Massive interstitial copy-neutral loss-of-heterozygosity as evidence for cancer being a disease of the DNA-damage response.

BACKGROUND: The presence of loss-of-heterozygosity (LOH) mutations in cancer cell genomes is commonly encountered. Moreover, the occurrences of LOHs in tumor suppressor genes play important roles in oncogenesis. However, because the causative mechanisms underlying LOH mutations in cancer cells yet remain to be elucidated, enquiry into the nature of these mechanisms based on a comprehensive examination of the characteristics of LOHs in multiple types of cancers has become a necessity. METHODS: We performed next-generation sequencing on inter-Alu sequences of five different types of solid tumors and acute myeloid leukemias, employing the AluScan platform which entailed amplification of such sequences using multiple PCR primers based on the consensus sequences of Alu elements; as well as the whole genome sequences of a lung-to-liver metastatic cancer and a primary liver cancer. Paired-end sequencing reads were aligned to the reference human genome to identify major and minor alleles so that the partition of LOH products between homozygous-major vs. homozygous-minor alleles could be determined at single-base resolution. Strict filtering conditions were employed to avoid false positives. Measurements of LOH occurrences in copy number variation (CNV)-neutral regions were obtained through removal of CNV-associated LOHs. RESULTS: We found: (a) average occurrence of copy-neutral LOHs amounting to 6.9% of heterologous loci in the various cancers; (b) the mainly interstitial nature of the LOHs; and (c) preference for formation of homozygous-major over homozygous-minor, and transitional over transversional, LOHs. CONCLUSIONS: The characteristics of the cancer LOHs, observed in both AluScan and whole genome sequencings, point to the formation of LOHs through repair of double-strand breaks by interhomolog recombination, or gene conversion, as the consequence of a defective DNA-damage response, leading to a unified mechanism for generating the mutations required for oncogenesis as well as the progression of cancer cells.

Recombinant extracellular domain of the three major subunits of GABAA receptor show comparable secondary structure and benzodiazepine binding properties.

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.

Two beta-rich structural domains in GABA(A) receptor alpha(1) subunit with different physical properties: Evidence for multidomain nature of the receptor.

The type A gamma-aminobutyric acid (GABA(A)) receptor is a major inhibitory neurotransmitter-gated ion channel. Previously, we identified a membrane-proximal beta-rich (MPBR) domain in fragment C166-L296 of GABA(A) receptor alpha(1) 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 beta-rich as measured using FTIR spectroscopy and estimated from the CD spectra to be 42% and 51% beta-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.