A CAG repeat threshold for therapeutics targeting somatic instability in Huntington's disease.

The Huntington's disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable and expansions of hundreds of CAGs have been detected in Huntington's disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntington's disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntington's disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown. The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntington's disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterized. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knockout backgrounds to determine the maximum benefit of targeting Msh3 in this model. Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis. Given that the trajectory for somatic CAG expansion in the brains of Huntington's disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.

Benefits of global mutant huntingtin lowering diminish over time in a Huntington's disease mouse model.

We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.

Dynamic single-cell RNA sequencing identifies immunotherapy persister cells following PD-1 blockade.

Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects gene expression pathways beyond cholesterol metabolism in liver cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces degradation of low-density lipoprotein receptor (LDLR) in the liver. It is being pursued as a therapeutic target for LDL-cholesterol reduction. Earlier genome-wide gene expression studies showed that PCSK9 over-expression in HepG2 cells resulted in up-regulation of genes in cholesterol biosynthesis and down-regulation of genes in stress response pathways; however, it was not known whether these changes were directly regulated by PCSK9 or were secondary to PCSK9-induced changes to the intracellular environment. In order to further understand the biological function of PCSK9 we treated HepG2 cells with purified recombinant wild type (WT) and D374Y gain-of-function PCSK9 proteins for 8, 24, and 48 h, and used microarray analysis to identify genome-wide expression changes and pathways. These results were compared to the changes induced by culturing HepG2 cells in cholesterol-free medium, mimicking the intracellular environment of cholesterol starvation. We determined that PCSK9-induced up-regulation of cholesterol biosynthesis genes resulted from intracellular cholesterol starvation. In addition, we identified novel pathways that are presumably regulated by PCSK9 and are independent of its effects on cholesterol uptake. These pathways included "protein ubiquitination," "xenobiotic metabolism," "cell cycle," and "inflammation and stress response." Our results indicate that PCSK9 affects metabolic pathways beyond cholesterol metabolism in HepG2 cells.

HCV genotyping using statistical classification approach.

The genotype of Hepatitis C Virus (HCV) strains is an important determinant of the severity and aggressiveness of liver infection as well as patient response to antiviral therapy. Fast and accurate determination of viral genotype could provide direction in the clinical management of patients with chronic HCV infections. Using publicly available HCV nucleotide sequences, we built a global Position Weight Matrix (PWM) for the HCV genome. Based on the PWM, a set of genotype specific nucleotide sequence "signatures" were selected from the 5' NCR, CORE, E1, and NS5B regions of the HCV genome. We evaluated the predictive power of these signatures for predicting the most common HCV genotypes and subtypes. We observed that nucleotide sequence signatures selected from NS5B and E1 regions generally demonstrated stronger discriminant power in differentiating major HCV genotypes and subtypes than that from 5' NCR and CORE regions. Two discriminant methods were used to build predictive models. Through 10 fold cross validation, over 99% prediction accuracy was achieved using both support vector machine (SVM) and random forest based classification methods in a dataset of 1134 sequences for NS5B and 947 sequences for E1. Prediction accuracy for each genotype is also reported.

Replicable and Coupled Changes in Innate and Adaptive Immune Gene Expression in Two Case-Control Studies of Blood Microarrays in Major Depressive Disorder.

BACKGROUND: Peripheral inflammation is often associated with major depressive disorder (MDD), and immunological biomarkers of depression remain a focus of investigation. METHODS: We used microarray data on whole blood from two independent case-control studies of MDD: the GlaxoSmithKline-High-Throughput Disease-specific target Identification Program [GSK-HiTDiP] study (113 patients and 57 healthy control subjects) and the Janssen-Brain Resource Company study (94 patients and 100 control subjects). Genome-wide differential gene expression analysis (18,863 probes) resulted in a p value for each gene in each study. A Bayesian method identified the largest p-value threshold (q = .025) associated with twice the number of genes differentially expressed in both studies compared with the number of coincidental case-control differences expected by chance. RESULTS: A total of 165 genes were differentially expressed in both studies with concordant direction of fold change. The 90 genes overexpressed (or UP genes) in MDD were significantly enriched for immune response to infection, were concentrated in a module of the gene coexpression network associated with innate immunity, and included clusters of genes with correlated expression in monocytes, monocyte-derived dendritic cells, and neutrophils. In contrast, the 75 genes underexpressed (or DOWN genes) in MDD were associated with the adaptive immune response and included clusters of genes with correlated expression in T cells, natural killer cells, and erythroblasts. Consistently, the MDD patients with overexpression of UP genes also had underexpression of DOWN genes (correlation > .70 in both studies). CONCLUSIONS: MDD was replicably associated with proinflammatory activation of the peripheral innate immune system, coupled with relative inactivation of the adaptive immune system, indicating the potential of transcriptional biomarkers for immunological stratification of patients with depression.

Global transcriptional program of p53 target genes during the process of apoptosis and cell cycle progression.

The temporal gene expression profile during the entire process of apoptosis and cell cycle progression in response to p53 in human ovarian cancer cells was explored with cDNA microarrays representing 33 615 individual human genes. A total of 1501 genes (4.4%) were found to respond to p53 (approximately 80% of these were repressed by p53) using 2.5-fold change as a cutoff. It was anticipated that most of p53 responsive genes resulted from the secondary effect of p53 expression at late stage of apoptosis. To delineate potential p53 direct and indirect target genes during the process of apoptosis and cell cycle progression, microarray data were combined with global p53 DNA-binding site analysis. Here we showed that 361 out of 1501 p53 responsive genes contained p53 consensus DNA-binding sequence(s) in their regulatory region, approximately 80% of which were repressed by p53. This is the first time that a large number of p53-repressed genes have been identified to contain p53 consensus DNA-binding sequence(s) in their regulatory region. Hierarchical cluster analysis of these genes revealed distinct temporal expression patterns of transcriptional activation and repression by p53. More genes were activated at early time points, while more repressed genes were found after the onset of apoptosis. A small-scale quantitative chromatin immunoprecipitation analysis indicated that in vivo p53-DNA interaction was detected in eight out of 10 genes, most of which were repressed by p53 at the early onset of apoptosis, suggesting that a portion of p53 target genes in the human genome could be negatively regulated by p53 via sequence-specific DNA binding. The approaches and genes described here should aid the understanding of global gene regulatory network of p53.

Comparative promoter analysis and its application in analysis of PTH-regulated gene expression.

Taking advantage of the "working draft" of the human genome and the MIT shotgun assembly of the mouse genome, we performed a comparative promoter analysis of human RefSeq mRNA (sequences from GenBank's RefSeq database). By combining this analysis with a transcription factor (TF) binding site analysis using a TRANSFAC position weight matrix (PWM) search, 86% of non-specific TF sites were removed. Using a set of genes that are regulated by parathyroid hormone (PTH), a statistical analysis was performed on the conserved TF binding sites among a set of eight human and mouse genes. From among the eight genes tested, we obtained a set of 31 TFs, suggesting possible roles for associated genes in PTH-mediated pathways. All three known PTH-responsive TFs (AP1, RUNX2, CREB) were correctly predicted by this analysis as well as two other potential TFs (VDR and CEBP Delta). Additionally, a model was made to describe the TF site characteristic module of PTH-regulated genes. This model was then used to search all human RefSeq gene promoters with established human-mouse ortholog relationships to identify other PTH-regulated genes. This comparative approach combined with statistical analysis proved to be sufficiently specific to decipher critical TFs involved in PTH-regulated pathways.

Analysis of a human brain transcriptome map.

BACKGROUND: Genome wide transcriptome maps can provide tools to identify candidate genes that are over-expressed or silenced in certain disease tissue and increase our understanding of the structure and organization of the genome. Expressed Sequence Tags (ESTs) from the public dbEST and proprietary Incyte LifeSeq databases were used to derive a transcript map in conjunction with the working draft assembly of the human genome sequence. RESULTS: Examination of ESTs derived from brain tissues (excluding brain tumor tissues) suggests that these genes are distributed on chromosomes in a non-random fashion. Some regions on the genome are dense with brain-enriched genes while some regions lack brain-enriched genes, suggesting a significant correlation between distribution of genes along the chromosome and tissue type. ESTs from brain tumor tissues have also been mapped to the human genome working draft. We reveal that some regions enriched in brain genes show a significant decrease in gene expression in brain tumors, and, conversely that some regions lacking in brain genes show an increased level of gene expression in brain tumors. CONCLUSIONS: This report demonstrates a novel approach for tissue specific transcriptome mapping using EST-based quantitative assessment.

A reference database for tumor-related genes co-expressed with interleukin-8 using genome-scale in silico analysis.

BACKGROUND: The EST database provides a rich resource for gene discovery and in silico expression analysis. We report a novel computational approach to identify co-expressed genes using EST database, and its application to IL-8. RESULTS: IL-8 is represented in 53 dbEST cDNA libraries. We calculated the frequency of occurrence of all the genes represented in these cDNA libraries, and ranked the candidates based on a Z-score. Additional analysis suggests that most IL-8 related genes are differentially expressed between non-tumor and tumor tissues. To focus on IL-8's function in tumor tissues, we further analyzed and ranked the genes in 16 IL-8 related tumor libraries. CONCLUSIONS: This method generated a reference database for genes co-expressed with IL-8 and could facilitate further characterization of functional association among genes.

Hepatitis C virus whole genome position weight matrix and robust primer design.

BACKGROUND: The high degree of sequence heterogeneity found in Hepatitis C virus (HCV) isolates, makes robust nucleic acid-based assays difficult to generate. Polymerase chain reaction based techniques, require efficient and specific sequence recognition. Generation of robust primers capable of recognizing a wide range of isolates is a difficult task. RESULTS: A position weight matrix (PWM) and a consensus sequence were built for each region of HCV and subsequently assembled into a whole genome consensus sequence and PWM. For each of the 10 regions, the number of occurrences of each base at a given position was compiled. These counts were converted to frequencies that were used to calculate log odds scores. Using over 100 complete and 14,000 partial HCV genomes from GenBank, a consensus HCV genome sequence was generated along with a PWM reflecting heterogeneity at each position. The PWM was used to identify the most conserved regions for primer design. CONCLUSIONS: This approach allows rapid identification of conserved regions for robust primer design and is broadly applicable to sets of genomes with all levels of genetic heterogeneity.

Genome wide in silico SNP-tumor association analysis.

BACKGROUND: Carcinogenesis occurs, at least in part, due to the accumulation of mutations in critical genes that control the mechanisms of cell proliferation, differentiation and death. Publicly accessible databases contain millions of expressed sequence tag (EST) and single nucleotide polymorphism (SNP) records, which have the potential to assist in the identification of SNPs overrepresented in tumor tissue. METHODS: An in silico SNP-tumor association study was performed utilizing tissue library and SNP information available in NCBI's dbEST (release 092002) and dbSNP (build 106). RESULTS: A total of 4865 SNPs were identified which were present at higher allele frequencies in tumor compared to normal tissues. A subset of 327 (6.7%) SNPs induce amino acid changes to the protein coding sequences. This approach identified several SNPs which have been previously associated with carcinogenesis, as well as a number of SNPs that now warrant further investigation CONCLUSIONS: This novel in silico approach can assist in prioritization of genes and SNPs in the effort to elucidate the genetic mechanisms underlying the development of cancer.

[IRE_FINDER-computational search of iron response element in human and mouse UTRs].

The iron response element (IRE) is a highly conserved RNA stem loop structure. It is the binding site of iron regulatory protein (IRP). IRP binding to IRE is regulated by cellular iron. When cells are derived of iron, IRP binds IRE. If IRE is located at 5'UTR, IRP binding will inhibit translation initiation, else if IRE is at 3'UTR, IRP binding will stabilize mRNA and prevent it from degradation. So far all known IREs have C at the 1 position and G at the 5 position of the loop (C1G5 type). In vitro studies suggest that the U1A5 type IRE, which has U and A at the 1 and 5 loop position respectively, binds well to IRP. However, U1A5 type's in vivo existence is still elusive. IRE-IRP binding is involved in the regulation of iron metabolism, oxidative stress and possibly aging. Here we use an improved computation method performing a comprehensive search of IRE in human and mouse genes. We try to catalog potential human and mouse IRE containing genes, at the same time identify potential U1A5 IREs.

P518/Qrfp sequence polymorphisms in SAMP6 osteopenic mouse.

Mice lacking GPR103A expression display osteopenia. Analysis of mouse quantitative trait loci literature associated with bone mineral density suggested GPR103A ligand P518/Qrfp (chromosome 2qB) as a candidate osteoporosis gene. Promoter and coding regions of mouse P518/Qrfp were sequenced from genomic DNA obtained from the osteoporosis-prone strain SAMP6 and control strains SAMR1, A/J, AKR/J, BALB/c, C3H/HeJ, C57BL/6J, and DBA/2J. Four single-nucleotide polymorphisms (SNPs) were identified in only SAMP6 genomic DNA, g.-1773 T-->C, g.110 A-->G (N37S), g.188 G-->A (R63K), and g.135 T-->C (H45H). The promoter SNP generated a novel neuron-restrictive silencing factor binding site, a repressor that decreases gene expression in nonneuronal tissues. TaqMan analysis demonstrated fivefold lower P518/Qrfp liver expression in SAMP6 versus SAMR1 or C57BL/6J control strains. Tissue distribution of human, mouse, and rat P518/Qrfp and its receptors showed expression in bone and spinal cord. A direct role for P518/Qrfp function in maintaining bone mineral density is suggested.

Computational analysis of composite regulatory elements.

Combinatorial regulation is a powerful mechanism for generating specificity in gene expression, and it is thought to play a pivotal role in the formation of the complex gene regulatory networks found in higher eukaryotes. The term "Composite Element" (CE) refers to a minimal functional unit where protein-DNA and protein-protein interactions contribute to a highly specific pattern of gene transcriptional regulation. Identification of composite elements will help to better understand gene regulation networks. Experimentally identified CEs are limited in number, and the currently available CE database COMPEL is based on such published information. Here, based on the statistical analysis of over-represented adjacent transcription factor binding sites, we describe a computational method to predict composite regulatory elements in genomic sequences. The algorithm proved to be efficient for extracting composite elements that had been experimentally confirmed and documented in the COMPEL database. Furthermore, putative new composite elements are predicted based on this method, and we have been able to confirm some of our predictions which are not included in the COMPEL database by searching published information.

Quantification of single nucleotide polymorphisms by automated DNA sequencing.

Single nucleotide polymorphisms (SNPs) are linked to phenotypes associated with diseases and drug responses. Many techniques are now available to identify and quantify such SNPs in DNA or RNA pools, although the information on the latter is limited. The majority of these methodologies require prior knowledge of target sequences, normally obtained through DNA sequencing. Direct quantitation of SNPs from DNA sequencing raw data will save time and money for large amount sample analysis. A high throughput DNA sequencing assay, in combination with a SNP quantitative algorithm, was developed for the quantitation of a SNP present in HCV RNA sequences. For a side-by-side comparison, a Pyrosequencing assay was also developed. Quantitation performance was evaluated for both methods. The direct DNA sequencing quantitation method was shown to be more linear, accurate, sensitive, and reproducible than the Pyrosequencing method for the quantitation of the SNP present in HCV RNA molecules.

Posaconazole is a potent inhibitor of sterol 14alpha-demethylation in yeasts and molds.

Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata. In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C. albicans (both azole-sensitive and azole-resistant strains), C. glabrata, Aspergillus fumigatus, and Aspergillus flavus. Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy. Ergosterol was the major sterol in all but one of the strains; C. glabrata strain C110 synthesized an unusual sterol in place of ergosterol. Exposure to POS led to a decrease in the total sterol content of all the strains tested. The decrease was accompanied by the accumulation of 14alpha-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14alpha-demethylase enzyme. The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested. POS retained activity against C. albicans isolates with mutated forms of the 14alpha-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR. In addition, POS was a more potent inhibitor of sterol synthesis in A. fumigatus and A. flavus than either ITZ or VOR.

PepPat, a pattern-based oligopeptide homology search method and the identification of a novel tachykinin-like peptide.

UNLABELLED: PepPat, a hybrid method that combines pattern matching with similarity scoring, is described. We also report PepPat's application in the identification of a novel tachykinin-like peptide. PepPat takes as input a query peptide and a user-specified regular expression pattern within the peptide. It first performs a database pattern match and then ranks candidates on the basis of their similarity to the query peptide. PepPat calculates similarity over the pattern spanning region, enhancing PepPat's sensitivity for short query peptides. PepPat can also search for a user-specified number of occurrences of a repeated pattern within the target sequence. We illustrate PepPat's application in short peptide ligand mining. As a validation example, we report the identification of a novel tachykinin-like peptide, C14TKL-1, and show it is an NK1 (neuokinin receptor 1) agonist whose message is widely expressed in human periphery. AVAILABILITY: PepPat is offered online at: http://peppat.cbi.pku.edu.cn.