13 July 2007. Many DNA variations that associate with disease are found in the areas of the genome that do not code for protein. How most of these variants influence the biology of a gene and contribute to pathology is unclear, but in the case of neuregulin 1 (NRG1), a potential risk gene for schizophrenia (see SchizophreniaGene NRG1 overview), at least one of those variations alters the activity of the gene itself. So report Amanda Law and colleagues in a paper in the Journal of Biological Chemistry. The finding strengthens the case that the protein, specifically the neuregulin IV isoform (NRG1 type IV), may be important to the etiology of the disease. The researchers report that NRG1 type IV is found exclusively in the brain and is more highly expressed in the fetal brain. In addition, “This is the first functional demonstration of a regulatory element in the human NRG1 gene with differential promoter activity associated with a SNP linked to risk for schizophrenia and adult brain function,” said Law in an interview with SRF.
The connection between NRG1 and schizophrenia was originally made in studies of an Icelandic population. In 2002, researchers at deCODE Genetics, Reykjavik, found that a group of single nucleotide polymorphisms (SNPs) lying upstream of the NRG1 coding region was associated with the disease (see Stefannson et al., 2002). Two years later, the same research group discovered novel neuregulin exons that could potentially give rise to three new isoforms, neuregulin 1, types IV, V, and VI. Last year, Law and colleagues at Oxford University, working in collaboration with Daniel Weinberger at the NIH, Bethesda, Maryland, reported that one of those Icelandic SNPs, rs6994992, associated with the expression of type IV NRG1, with the risk allele (thymine) increasing levels of type IV messenger RNA in the human brain (see SRF related news story). This finding led to their suggestion that the risk allele may affect the promoter, or regulatory region of the type IV isoform. The regulation of NRG1 is complicated by the use of nine different promoters that differentially control expression of the different isoforms.
To test that theory and to characterize the full-length type IV neuregulin 1, first author Wei Tan and colleagues set about to clone the entire gene and its promoter. “NRG1 type IV is a relatively recent discovery and only about 10 percent of its structure was known; therefore, full characterization of the transcript and its promoter was important to future research on the schizophrenia associated variant,” explained Law. Tan and colleagues first cloned the mRNA from both adult and fetal brain cDNA libraries. Their analysis reveals that in addition to an immunoglobulin-like domain, the type IV isoform isolated from adult hippocampus and prefrontal cortex also contains a β domain and a cytoplasmic “a” tail, putting it in the “β1a” family of NRG1s. Other NRG1 isoforms contain a cysteine-rich motif instead of the Ig-like domain, and can have the rarer “b” cytoplasmic tail. In fetal samples, most type IV variants had the same structure, but the researchers found four novel isoforms with either slight differences in the spacer region lying downstream of the IgG domain, or lacking the β and “stalk” domains. An additional isoform with a nonsense mutation that codes for truncated isoforms was also detected.
To determine how SNP rs6994992 affects expression of NRG1, Tan and colleagues made gene reporter constructs, splicing the promoter region of NRG1 type IV to a luciferase gene. They fished out a putative promoter from genomic DNA by using the 5’ cDNA sequence as a basis for DNA amplification. By this method, the researchers obtained promoter regions with both thymine (T) and cytosine (C) bases at the polymorphic site, and they found that in cultured cells the former produced 65 percent more protein when used to drive luciferase expression. To test if that difference was solely due to the SNP, Tan mutated the T of that promoter to a C, which reduced luciferase expression by 60 percent. The result shows that that one SNP alone has a profound effect on transcription and helps explain the association of the rs6994992 risk allele (T) with increased NRG1 type IV mRNA expression in the human brain.
This particular SNP has received attention outside of schizophrenia. Last year it was found to be linked to psychosis (see SRF related news story), while earlier this year it was shown to be linked to spatial working memory deficits in normal controls (see Stefanis et al., 2007). How NRG1 type IV contributes to disease pathology is at present unclear, but Tan and colleagues found that expression was approximately 3.5-fold higher in fetal brain, suggesting that it plays an important role in development. “It is true for other classes of NRG1s and other genes that the different isoforms play different roles in the developing brain compared to the adult, but I think it is probably too early to say whether the developmental component of altered NRG1 type IV expression is more critical than its effects in the adult brain. Much work is needed to figure out type IV's biological role before we can answer this,” said Law. In the adult brain, type IV, like other Ig NRG1s, may be involved in regulating some important aspect of cortical function, such as NMDA receptor activity, LTP, or GABAergic function, she added.
Interestingly, in contrast to NRG1 types I, II, and III, Tan and colleagues failed to detect NRG1 type IV expression outside of the brain, suggesting it is specific to the CNS, which may make it a favorable therapeutic target.—Tom Fagan.
Tan W, Wang Y, Gold B, Chen J, Dean M, Harrison PJ, Weinberger DR, Law AJ. Molecular cloning of a brain-specific, developmentally regulated neuregulin 1 (NRG1) isoform and identification of a functional promoter variant associated with schizophrenia. J Biol Chem. 2007 Jun 12; [Epub ahead of print] Abstract