The use of subtraction libraries for the identification of RNA species upregulated in the brains of individuals with schizophrenia

Summary

Schizophrenia is a brain disease of unknown etiology. It is likely that individuals with schizophrenia express RNAs within their brain tissues which differ qualitatively and quantitatively from the RNAs expressed by unaffected individuals. We developed a series of brain subtraction libraries which are enriched for RNAs which are differentially expressed by individuals with schizophrenia. Analysis of these libraries identified a number of RNA species associated with brain function and the inflammatory response. In addition, we identified a number of novel RNAs which have not been previously characterized. The identification of brain RNAs which are disease associated might lead to new modalities for the diagnosis and treatment of schizophrenia.

Schizophrenia is a complex neuropsychiatric disorder with worldwide prevalence. While the specific etiopathogenesis of schizophrenia is not known with certainty, numerous studies have indicated that schizophrenia is associated with abnormalities of brain structure and function. Furthermore, numerous studies have identified familial clusters of cases, indicating possible genetic determinants of disease susceptibility These findings have led to extensive searches for genetic determinants of schizophrenia utilizing linkage analyses and other positional cloning techniques. These analyses have identified genomic regions which display increased rates of inheritance as compared to unaffected controls. However, despite extensive studies of large numbers of individuals and family kindreds, specific genetic defects associated with schizophrenia have not been identified [2,3,4]. This failure has led to the search for environmental factors which might modulate gene expression in the absence of inherited alterations in coding sequences. The role of infection and other environmental factors in the etiology of schizophrenia is supported by a number of studies which have addressed the epidemiology and pathophysiology of this disease [5,6].

Viruses are obligate intracellular parasites which can infect a wide range of host cells. In many cases, viral infection of host cells results in rapid cytolysis with the subsequent release of large numbers of progeny viruses. However, viral genetic material can also integrate into the host genome and alter RNA transcription in the absence of cytopathology. This altered transcription can result in modulation of a range of cellular functions without pathologic evidence of infection or inflammatory response. The effect of viral infection on the host can also be modulated by genetic factors. Genetic determinants of infection include genes which modulate the immune response, the expression of viral receptors, and the susceptibility of cells to soluble factors generated during the course of viral infection [7.8]. It is thus possible that viral infection of neuronal cells can, in combination with genetic determinants of susceptibility, result in the profound alteration of brain function typical of human neuropsychiatriC diseases [9].

The characterization of the role of genetic and environmental effects on RNA transcription in neuronal cells requires the unbiased measurement of differentially expressed RNAs in the brains of affected individuals. Recently, molecular biological technologies have been devised which allow for the precise characterization of RNA derived from human brain tissue. These methods are based on the ability of polymerase chain reaction (PCR) to amplify low levels of RNA in a quantitative fashion and to distinguish levels of specific RNAs in cases and controls [10]. Several of these methods involve the “subtraction” of RNAs amplified from individuals with a defined disease process by RNAs derived from matched controls. These procedures result in the generation of pool (or library) or RNA sequences which is enriched for RNA species which are expressed at a different level in affected individuals as compared to unaffected controls. We have applied the method of subtractive hybridization to characterize RNAs which are differentially expressed in the frontal cortex of individuals with schizophrenia.