Although there have been major advances in understanding the genetic basis of schizophrenia (SZ) and bipolar disorder (BD) using genome-wide association studies (GWAS), identifying disease causing functional variants in patients remains a problem. An exception is the finding of structural alterations, such as patient-specific copy number variants (CNVs), which have been found a sizeable minority of patients with SZ and autism spectrum disorders (ASD), especially do novo cases. Disease-associated SNPs identified in GWAS are usually not functional variants on their own, but are presumably in linkage disequilibrium (LD) with bona fide disease-causing functional mutations. However, identifying such variants is difficult, especially when associated SNPs are found in non-coding regions - deep within introns or in intergenic regions far removed from the nearest annotated genes, which has been a common theme in GWAS. A reasonable assumption, considering that significant LD is maintained for only ~10-20 kb, is that many disease-causing mutations are occurring in regulatory elements that are far removed from a candidate gene's coding region, such as distal enhancers, suppressors and promoters of non-coding RNAs. A feature of many regulatory elements is sequence conservation. However, sequence conservation alone is not sufficient to establish biological function and human-specific enhancer elements have been observed. What are the regulatory elements that control the regulation of genes involved in psychiatric disorders? Does genetic variation in these regions contribute to disease risk? Are the positive association signals identified in GWAS due to LD with these features? One strategy we successfully implemented in the initial grant period was to use a chromatin immunoprecipitation based method - ChIP-chip - as a screening tool to identify novel regulatory elements in SZ and BD candidate genes. The basic ChIP-chip strategy involves immunoprecipitation of chromatin using antibodies (Ab) to proteins, such as covalently modified histones, that bind to DNA and are enriched in regulatory domains. These regions can then be assayed for biological activity and, if such activity can be demonstrated, resequenced to identify rare patient-specific variants or SNPs that have higher allele frequencies in patients compared with controls. Such potential disease- causing variants can also be subjected to functional analysis to establish biological activity. However, ChIP-chip is limiting by the number of genes and amount of DNA that can be interrogated. A much more comprehensive analysis for regulatory domain screening is ChIP-Seq, the primary advantage of which is that it provides unbiased, genomewide coverage. Thus, ChIP-Seq has the potential to identify regulatory elements in all previously characterized candidate genes, as well as those identified in the future, providing researchers with biologically active sites that can be efficiently screened to identify patient-specific mutations. The ChIP-Seq approach will also be used to identify binding sites, throughout the entire human genome, of transcription factors relevant to SZ and BD pathophysiology.
This proposal describes experiments that will help identify regions of the genome involved in regulating the expression of candidate genes for schizophrenia, bipolar disorder, autism spectrum disorders and other neuropsychiatric disorders. Understanding how these genes are regulated may lead to novel treatment strategies. Furthermore, mutations in these regulatory regions may be responsible for disease susceptibility in a subgroup of patients.
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