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High-resolution Studies of mRNA Expression in Brain: A Search for Genes Differently Expressed in Schizophrenia
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Evolutionary Biology.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gene expression differences between patients and controls can be used to find susceptibility genes and drug targets for a disease. High-resolution strategies are required because the differences between the investigated groups may be small and numerous factors may affect the mRNA quantity. This thesis is based on the use of real-time RT-PCR combined with a new statistical approach, developed to detect small differences between patients and controls and differences due to patient subgroups.

Comparisons between human brain biopsy and autopsy samples showed that post-mortem tissue can be used to make conclusions on the relative mRNA levels in the living brain.

Power analysis based on human brain mRNA expression from 14 genes adjusted with two reference genes, revealed that a sample size of 50 patients and 50 controls was required to detect a 2-fold difference with a power and a confidence of 95%. A similar study in rats revealed that approximately the same sample size was required for rat brain mRNA expression studies.

The mRNA levels of several genes were studied in 55 schizophrenia and 55 control prefrontal brain autopsies, using a novel and more powerful statistical analysis. The serotonin receptor 2C gene (HTR2C) showed a significant 1.5-fold decrease in the patients as compared to controls, and the monoamine oxidase B gene (MAOB) a 1.2-fold increase.

The mechanism behind the decrease of HTR2C mRNA levels was investigated by studying the correlation of drug treatment and HTR2C promoter polymorphisms to the HTR2C expression levels. The observed decrease was present in untreated patients, suggesting that the HTR2C mRNA decrease is correlated with the disease and not the treatment. There was no association between promoter polymorphisms and HTR2C expression levels. Thus, the molecular mechanism for the decreased expression remains unclear. Nevertheless, the results support a role for monoaminergic synapses in schizophrenia.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2003. , p. 47
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 894
Keywords [en]
Genetics, mRNA, gene expression, real-time RT-PCR, schizophrenia, 5-HT (serotonin) receptor 2C, brain, psychiatric genetics
Keywords [sv]
Genetik
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-3605ISBN: 91-554-5755-X (print)OAI: oai:DiVA.org:uu-3605DiVA, id: diva2:163422
Public defence
2003-11-07, Lindahlsalen, EBC, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2003-10-14 Created: 2003-10-14 Last updated: 2018-01-13Bibliographically approved
List of papers
1. High-resolution quantification of specific mRNA levels in human brain autopsies and biopsies
Open this publication in new window or tab >>High-resolution quantification of specific mRNA levels in human brain autopsies and biopsies
2000 (English)In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 10, no 8, p. 1219-29Article in journal (Refereed) Published
Abstract [en]

Quantification of mRNA levels in human cortical brain biopsies and autopsies was performed using a fluorogenic 5' nuclease assay. The reproducibility of the assay using replica plates was 97%-99%. Relative quantities of mRNA from 16 different genes were evaluated using a statistical approach based on ANCOVA analysis. Comparison of the relative mRNA levels between two groups of samples with different time postmortem revealed unchanged relative expression levels for most genes. Only CYP26A1 mRNA levels showed a significant decrease with prolonged time postmortem (p = 0.00004). Also, there was a general decrease in measured mRNA levels for all genes in autopsies compared to biopsies; however, on comparing mRNA levels after adjusting with reference genes, no significant differences were found between mRNA levels in autopsies and biopsies. This observation indicates that studies of postmortem material can be performed to reveal the relative in vivo mRNA levels of genes. Power calculations were done to determine the number of individuals necessary to detect differences in mRNA levels of 1.5-fold to tenfold using the strategy described here. This analysis showed that samples from at least 50 individuals per group, patients and controls, are required for high-resolution ( approximately twofold changes) differential expression screenings in the human brain. Experiments done on ten individuals per group will result in a resolution of approximately fivefold changes in expression levels. In general, the sensitivity and resolution of any differential expression study will depend on the sample size used and the between-individual variability of the genes analyzed.

National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-90900 (URN)10.1101/gr.10.8.1219 (DOI)10958640 (PubMedID)
Available from: 2003-10-14 Created: 2003-10-14 Last updated: 2017-12-14Bibliographically approved
2. Analysis of gene expression in the rat hippocampus using Real Time PCR reveals high inter-individual variation in mRNA expression levels
Open this publication in new window or tab >>Analysis of gene expression in the rat hippocampus using Real Time PCR reveals high inter-individual variation in mRNA expression levels
Show others...
2002 (English)In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 67, no 2, p. 225-34Article in journal (Refereed) Published
Abstract [en]

In mammals, gene transcription is a step subjected to tight regulation mechanisms. In fact, changes in mRNA levels in the central nervous system (CNS) can account for numerous phenotypic differences in brain function. We performed a high-resolution analysis of mRNA expression levels for 37 genes selected from a normal rat hippocampus cDNA library. mRNA amounts were quantified using a Real Time PCR SYBR Green assay. We found that, in general, individuals from an inbred rat population (n = 20) have shown 2-3 times differences in the basal level of expression of the genes analyzed. Up to several fold differences among individuals were observed for certain genes. These inter-individual differences were obtained after correction for the different amounts of mRNA in each sample. Power calculations were performed to determine the number of individuals required to detect reliable differences in expression levels between a control and an experimental group. These data indicated that, depending on the variability of the candidate gene selected, it was necessary to analyze from five to 135 individuals in each group to detect differences of 50% in the levels of mRNA expression between two groups investigated. The comparison of mRNA abundance from different genes revealed a wide range of expression levels for the 37 genes, showing a 26,000-fold difference between the highest and lowest expressed gene.

National Category
Genetics Physiology
Identifiers
urn:nbn:se:uu:diva-90901 (URN)10.1002/jnr.10105 (DOI)
Available from: 2003-10-14 Created: 2003-10-14 Last updated: 2018-01-13Bibliographically approved
3. Decrease of serotonin receptor 2C in schizophrenia brains identified by high-resolution mRNA expression analysis
Open this publication in new window or tab >>Decrease of serotonin receptor 2C in schizophrenia brains identified by high-resolution mRNA expression analysis
2003 (English)In: Biological Psychiatry, ISSN 0006-3223, E-ISSN 1873-2402, Vol. 54, no 11, p. 1212-1221Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: RNA expression profiling can provide hints for the selection of candidate susceptibility genes, for formulation of hypotheses about the development of a disease, and/or for selection of candidate gene targets for novel drug development. We measured messenger RNA expression levels of 16 candidate genes in brain samples from 55 schizophrenia patients and 55 controls. This is the largest sample so far used to identify genes differentially expressed in schizophrenia brains. METHODS: We used a sensitive real-time polymerase chain reaction methodology and a novel statistical approach, including the development of a linear model of analysis of covariance type. RESULTS: We found two genes differentially expressed: monoamine oxidase B was significantly increased in schizophrenia brain (p =.001), whereas one of the serotonin receptor genes, serotonin receptor 2C, was significantly decreased (p =.001). Other genes, previously proposed to be differentially expressed in schizophrenia brain, were invariant in our analysis. CONCLUSIONS:The differential expression of serotonin receptor 2C is particularly relevant for the development of new atypical antipsychotic drugs. The strategy presented here is useful to evaluate hypothesizes for the development of the disease proposed by other investigators.

National Category
Physiology Genetics
Identifiers
urn:nbn:se:uu:diva-90902 (URN)10.1016/S0006-3223(03)00526-2 (DOI)
Available from: 2003-10-14 Created: 2003-10-14 Last updated: 2018-01-13Bibliographically approved
4. Statistical methodology in case-control 5'-nuclease assays: statistical design, modelling and inference for identification of differentially expressed genes
Open this publication in new window or tab >>Statistical methodology in case-control 5'-nuclease assays: statistical design, modelling and inference for identification of differentially expressed genes
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90903 (URN)
Available from: 2003-10-14 Created: 2003-10-14 Last updated: 2010-02-11Bibliographically approved
5. Serotonin receptor 2C (HTR2C) and schizophrenia: effect of medication and genetic variants on expression levels
Open this publication in new window or tab >>Serotonin receptor 2C (HTR2C) and schizophrenia: effect of medication and genetic variants on expression levels
Show others...
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90904 (URN)
Available from: 2003-10-14 Created: 2003-10-14 Last updated: 2010-02-11Bibliographically approved

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