GABA- and acetylcholine-related gene expression in blood correlate with tic severity and microarray evidence for alternative splicing in Tourette syndrome: a pilot study.

Tourette syndrome (TS) is a complex childhood neurodevelopmental disorder characterized by motor and vocal tics. Recently, altered numbers of GABAergic-parvalbumin (PV) and cholinergic interneurons were observed in the basal ganglia of individuals with TS. Thus, we postulated that gamma-amino butyric acid (GABA)- and acetylcholine (ACh)-related genes might be associated with the pathophysiology of TS. Total RNA isolated from whole blood of 26 un-medicated TS subjects and 23 healthy controls (HC) was processed on Affymetrix Human Exon 1.0 ST arrays. Data were analyzed to identify genes whose expression correlated with tic severity in TS, and to identify genes differentially spliced in TS compared to HC subjects. Many genes (3627) correlated with tic severity in TS (p < 0.05) among which GABA- (p = 2.1 × 10⁻³) and ACh- (p = 4.25 × 10⁻8) related genes were significantly over-represented. Moreover, several GABA and ACh-related genes were predicted to be alternatively spliced in TS compared to HC including GABA receptors GABRA4 and GABRG1, the nicotinic ACh receptor CHRNA4 and cholinergic differentiation factor (CDF). This pilot study suggests that at least some of these GABA- and ACh-related genes observed in blood that correlate with tics or are alternatively spliced are involved in the pathophysiology of TS and tics.

Exon expression and alternatively spliced genes in Tourette Syndrome.

Tourette Syndrome (TS) is diagnosed based upon clinical criteria including motor and vocal tics. We hypothesized that differences in exon expression and splicing might be useful for pathophysiology and diagnosis. To demonstrate exon expression and alternatively spliced gene differences in blood of individuals with TS compared to healthy controls (HC), RNA was isolated from the blood of 26 un-medicated TS subjects and 23 HC. Each sample was run on Affymetrix Human Exon 1.0 ST (HuExon) arrays and on 3' biased U133 Plus 2.0 (HuU133) arrays. To investigate the differentially expressed exons and transcripts, analyses of covariance (ANCOVA) were performed, controlling for age, gender, and batch. Differential alternative splicing patterns between TS and HC were identified using analyses of variance (ANOVA) models in Partek. Three hundred and seventy-six exon probe sets were differentially expressed between TS and HC (raw P < 0.005, fold change >|1.2|) that separated TS and HC subjects using hierarchical clustering and Principal Components Analysis. The probe sets predicted TS compared to HC with a >90% sensitivity and specificity using a 10-fold cross-validation. Ninety genes (transcripts) had differential expression of a single exon (raw P < 0.005) and were predicted to be alternatively spliced (raw P < 0.05) in TS compared to HC. These preliminary findings might provide insight into the pathophysiology of TS and potentially provide prognostic and diagnostic biomarkers. However, the findings are tempered by the small sample size and multiple comparisons and require confirmation using PCR or deep RNA sequencing and a much larger patient population.

Blood gene expression correlated with tic severity in medicated and unmedicated patients with Tourette Syndrome.

BACKGROUND: Tourette Syndrome (TS) has been linked to both genetic and environmental factors. Gene-expression studies provide valuable insight into the causes of TS; however, many studies of gene expression in TS do not account for the effects of medication. MATERIALS & METHODS: To investigate the effects of medication on gene expression in TS patients, RNA was isolated from the peripheral blood of 20 medicated TS subjects (MED) and 23 unmedicated TS subjects (UNMED), and quantified using whole-genome Affymetrix microarrays. RESULTS: D2 dopamine receptor expression correlated positively with tic severity in MED but not UNMED. GABA(A) receptor ε subunit expression negatively correlated with tic severity in UNMED but not MED. Phenylethanolamine N-methyltransferase expression positively correlated with tic severity in UNMED but not MED. CONCLUSION: Modulation of tics by TS medication is associated with changes in dopamine, norepinephrine and GABA pathways.

Tourette syndrome: gene expression as a tool to discover drug targets.

Gene expression microarrays are a high-throughput, cost-effective method for measuring the expression of all genes in a sample. By comparing the expression patterns of healthy controls to diseased subjects, the genetic regulatory pathways underlying and affected by the disease can be elucidated. Furthermore, dysregulated genes are possible candidates for pharmaceutical therapy. Here, we consider the possibility of applying this approach to Tourette syndrome. We also review current theories of Tourette syndrome etiology and pharmacology.

Recombinant Fv-Hsp70 protein mediates neuroprotection after focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: This study investigated the effects of intravenous recombinant Fv-Hsp70 protein on infarction volume and behavior after experimental ischemic stroke. METHODS: Focal cerebral ischemia was produced by occluding the middle cerebral artery using the intraluminal suture technique. Rats subjected to 2 hours of focal ischemia were allowed to survive 24 hours. At 2(1/4) hours and 3 hours after onset of ischemia, Fv-Hsp70 recombinant protein (0.5 mg/kg) or saline was injected through the tail vein. Sensorimotor function and infarction volume were assessed at 24 hours after ischemia. RESULTS: Administration of Fv-Hsp70 after focal cerebral ischemia significantly decreased infarct volume by 68% and significantly improved sensorimotor function compared with the saline-treated control group. Western blots showed Fv-Hsp70 in ischemic but not in control brain; and Fv-Hsp70 suppressed endogenous Hsp70. CONCLUSIONS: Fv-Hsp70 protected the ischemic brain in this experimental stroke model.

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood.

BACKGROUND: Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. METHODS: Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. RESULTS: Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder). CONCLUSION: The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

Gene expression in blood of subjects with Duchenne muscular dystrophy.

The objective of this study was to examine RNA expression in blood of subjects with Duchenne muscular dystrophy (DMD). Whole blood was collected into PAX gene tubes and RNA was isolated for 3- to 20-year-old males with DMD (n = 34) and for age- and gender-matched normal healthy controls (n = 21). DMD was confirmed by genetic testing in all subjects. RNA expression was measured on Affymetrix whole-genome human U133 Plus 2.0 GeneChips. Using a Benjamini-Hochberg false discovery rate of 0.05 to correct for multiple comparisons, an unpaired t test for DMD versus controls yielded 10,763 regulated probes with no fold change cutoff, 1,467 probes with >|1.5|-fold change, 191 probes with >|2.0|-fold change, and 59 probes with a >|2.5|-fold change. These genes (probes) separated DMD from controls using cluster analyses. Almost all of the genes regulated in peripheral blood were different from the genes reported to be regulated in diseased muscle of subjects with DMD. It is proposed that the genes regulated in blood of subjects with Duchenne muscular dystrophy are indicative, at least in part, of the immune response to the diseased DMD muscle. The regulated genes might be used to monitor therapy or provide novel targets for immune-directed therapy for DMD.

Empirical Bayes accomodation of batch-effects in microarray data using identical replicate reference samples: application to RNA expression profiling of blood from Duchenne muscular dystrophy patients.

BACKGROUND: Non-biological experimental error routinely occurs in microarray data collected in different batches. It is often impossible to compare groups of samples from independent experiments because batch effects confound true gene expression differences. Existing methods can correct for batch effects only when samples from all biological groups are represented in every batch. RESULTS: In this report we describe a generalized empirical Bayes approach to correct for cross-experimental batch effects, allowing direct comparisons of gene expression between biological groups from independent experiments. The proposed experimental design uses identical reference samples in each batch in every experiment. These reference samples are from the same tissue as the experimental samples. This design with tissue matched reference samples allows a gene-by-gene correction to be performed using fewer arrays than currently available methods. We examine the effects of non-biological variation within a single experiment and between experiments. CONCLUSION: Batch correction has a significant impact on which genes are identified as differentially regulated. Using this method, gene expression in the blood of patients with Duchenne Muscular Dystrophy is shown to differ for hundreds of genes when compared to controls. The numbers of specific genes differ depending upon whether between experiment and/or between batch corrections are performed.