21 April 2006. Since single nucleotide polymorphisms (SNPs) in the gene for neuregulin 1 (NRG1) were linked to increased risk for schizophrenia, scientists have struggled to explain exactly how those SNPs might relate to the disease at a biological level. The problem is that the SNPs do not lie in the coding region of the gene, but upstream. This has led to suggestions that the nucleotide changes may alter expression of neuregulin. Now, in an April 25 PNAS paper, currently available online (and first reported in our Neurosciences 2005 coverage), researchers report that they have addressed that theory in a large sample of postmortem brains. Amanda Law and colleagues from Oxford University, England, and the National Institutes of Health, Bethesda, Maryland, have analyzed SNPs and measured NRG1 isoform expression in postmortem hippocampal samples from more than 80 controls and 40 schizophrenia patients. So does aberrant expression of NRG1 explain why these SNPs increase risk for the disease? The answer appears to be yes…and no.
Law and colleagues measured hippocampal expression of four major isoforms of neuregulin (type I – IV). In a subset of samples (53 controls and 38 patients), they found that mRNA for the type I isoform is slightly, though significantly higher in patient tissue. On average, that slight increase is 34 percent, though deviations from the mean are quite large. The authors report finding a similar relationship when they examined samples from the full cohort (n = 84 for controls, n = 44 for patients). None of the other three isoforms showed any expression differences between patient and controls. Researchers from the same group have previously reported that type I NRG1 is also increased slightly in the prefrontal cortex in the disease (see Hashimoto et al., 2004). Both the prefrontal cortex and the hippocampus have been linked to the psychopathology of the disease.
When Law and colleagues examined the effects of the risk alleles on type I NRG1 expression, they found that the relationship was quite complex and not easily reconciled with disease susceptibility. The authors found that one of the original five SNPs identified as increasing risk for schizophrenia (see Stefansson et al., 2002) did seem to alter gene expression but not in the same way in patients and controls. Patients homozygous for the risk allele of this SNP (SNP8NRG221132) had slightly higher type I expression than patients who were heterozygous for the allele—this difference was not significant. But in control cases the trend was in the opposite direction—those homozygous for the high-risk allele actually had significantly lower expression of type I NRG1. “At present, the relevance of and an explanation for these findings is obscure,” write the authors in their supplementary discussion, which can be found at the PNAS website. But one possibility that they put forth is that other factors driving increased expression of NRG1 in schizophrenia patients may be masking the effect of the high-risk allele seen in normal controls. If this turns out to be true, then the risk allele (a guanine nucleotide instead of adenine) would be predicted to decrease NRG1 expression.
In the case of type IV NRG1, the opposite seems to be the case. The authors found that increased expression of the type IV isoform was associated with the risk allele for a second SNP, SNP8NRG243177. The presence of the high-risk thymine instead of cytosine at this position was correlated with higher levels of type IV NRG1 in both patients and controls, and there is evidence of a dose effect: T/T homozygotes have highest expression followed by C/T heterozygotes and C/C homozygotes. Though this trend appeared more pronounced in patients, the authors did not find any strong relationship between SNP genotype and diagnosis of schizophrenia. Nevertheless, in the total cohort (patients and controls), the difference between type IV NRG1 expression in the two homozygote groups reached statistical significance, indicating that the risk allele does increase type IV NRG1 expression. Given that the risk allele has similar effects in both patients and normal controls, additional factors must contribute to pathology in patients.
How might these SNPs affect expression of the different isoforms of neuregulin? The authors note that both lie in nucleotide motifs that are recognized by a variety of transcription factors. SNP8NRG221132, for example, lies in a region recognized by serum response factor (SRF), and the high-risk allele is predicted to remove binding of this transcription factor and therefore reduce expression of neuregulin. The presence of SNP8NRG243177, on the other hand, is predicted to increase binding of high-mobility group box protein-1, which would fit with the increased expression of type IV NRG1 that appears to be elicited by this SNP. The differential effects of the SNPs on different isoforms may be related to the complex alternative splicing of this gene, which is governed by alternative promoter usage.
But the situation is slightly more complex, because SNP8NRG243177 also lies in a second SRF-binding motif and is predicted to abolish SRF binding also. All told, the interplay among high-risk SNPs, differential promoter usage, NRG1 expression, and schizophrenia is turning out to be extremely complex. Nonetheless, the authors conclude “…that the mechanism behind the clinical association of NRG1 with schizophrenia is altered transcriptional regulation, which modifies, probably to a small degree and in an isoform-limited fashion, the efficiency of NRG1 signaling effects on neural development and plasticity.”—Tom Fagan.
Law AJ, Lipska BK, Weickert CS, Hyde TM, Straub RE, Hashimoto R, Harrison PJ, Kleinman JE, Weinberger DR. Neuregulin 1 transcripts are differentially expressed in schizophrenia and regulated by 5’ SNPs associated with the disease. PNAS April 25, 2006;103:6747-6752. Abstract