, first identified as a schizophrenia susceptibility gene by linkage studies, has a relatively common risk haplotype in the 5′ promoter region; however, the mechanism through which this specific risk haplotype may increase susceptibility to mental illness is controversial. Our study has shown that the 5′ upstream regions of NRG1
have a relatively high nucleotide diversity as compared with the 5′ regions of many other human genes previously investigated,32, 33, 34
in the top 15% of those investigated. This suggests that the regulatory regions of NRG1
may be relatively unstable and a hotspot for genetic change. Furthermore, people with schizophrenia demonstrate a higher novel variant load than controls, particularly in the HapICE LD block; however, the high variant load was not linked to the specific risk haplotype. Observations of a higher mutational load may fit with other mechanistic studies that suggest the genomes of people who develop schizophrenia are more susceptible to DNA damage or mistakes in DNA repair.35
Indeed a recent study, suggests that rare de novo
mutations were more common in schizophrenia genomes.36
However, as peripheral tissue samples were not collected alongside the brain tissue used in this study, we are unable to determine whether the high novel variant load we observed in NRG1
in schizophrenia is brain specific. The presence of the novel 5-SNP haplotype in DNA from peripheral blood in an independent cohort implies a germline rather than somatic origin for this particular haplotype, nevertheless future studies sampling additional tissue sources from individual patients would be beneficial in determining the mechanism of the increased nucleotide diversity in NRG1
Importantly, this study provides the first demonstrated link between the HapICE risk haplotype and increased
NRG1 type III mRNA expression levels in the human frontal cortex. A previous study linking rs7014762 genotype with type III expression reported that the minor allele (designated T) was associated with schizophrenia, and that the minor allele resulted in a reduced
expression of type III mRNA.15
In support of this finding, Nicodemus et al.15
incorrectly stated that the same minor allele was also overtransmitted in bipolar disorder in the study by Georgieva et al.37
; whereas it appears that it was the major allele that was overtransmitted. We show the same direction of effect of the minor allele (designated A) on type III expression, with reduced expression of minor allele homozygotes compared with major allele homozygotes. However, our data and that of others,37
shows that it is the major allele
of rs7014762 that lies on the schizophrenia-associated HapICE risk haplotype and the major allele
that is significantly overtransmitted to bipolar patients.37
Hence, we propose a novel mechanism of risk for psychosis, whereby an increase, rather than a decrease, in type III mRNA is linked to NRG1 genetic risk. Consistent with this observation is that increased type III mRNA levels correlate with an earlier age of onset and hence a more severe phenotype. Interestingly, a study by Papiol et al.38
indicates an age of onset effect of the ‘protective' alleles at SNP8NRG221533 and SNP8NRG243177, where homozygous individuals (TT and CC, respectively) show a later age of onset. This is conceptually consistent with our finding that the HapICE risk alleles lead to an earlier age of onset, by increasing type III expression.
Our finding raises the need to more fully understand the role of NRG1 type III, the most brain-abundant and functionally unique form of NRG1. NRG1 type III acts in a contact-dependant manner, is expressed higher in deeper cortical layers and can be found on axons of projection neurons.39, 40
Furthermore, NRG1 type III is membrane bound and thought to be uniquely capable of back signaling to the nucleus by proteolytic processing and release of a transcriptionally active intracytoplasmic domain that increases neuronal survival.41
We previously found evidence of increased NRG1-intracytoplasmic domain domain in the DLPFC of people with schizophrenia.42
Recently, an amino-acid substitution within the intramembranous domain of NRG1 was associated with schizophrenia43
and this change abolishes intracytoplasmic domain cleavage and leads to decreased branching and growth of cortical neuronal dendrites.3
Commonly employed NRG1 type III mouse models use a heterozygous transmembrane domain knockout8
or a cystine-rich domain knockout,44
both of which model a reduction in type III NRG1 expression while maintaining expression of the soluble isoforms. Our data, which links type III overexpression with disease risk, suggests that a NRG1 type III hypermorphic mouse45
may help to elucidate the molecular mechanism of NRG1-related disease risk. Further work is also needed to clarify how increased type III mRNA may further be linked to changes in protein levels, NRG1-intracytoplasmic domain formation and cortical function.
Another unique finding of our study was the discovery of a novel 5-SNP haplotype in intron 1 that was present in 10% of cases with schizophrenia (4/37 individuals vs 0/37 controls). This haplotype was detected in peripheral blood from one Australian control from a wider population sample, indicating that it is both rare, and may not be exclusive to the brain or disease. However, without longitudinal follow-up of this subject, we cannot rule out that this haplotype is unique to schizophrenia, as some individuals can be diagnosed later in life.46
As this novel SNP haplotype was found in DNA derived from both blood and brain, it is likely that these changes do not reflect somatic changes; however, further studies comparing genetic diversity across different tissue types from the same individual are necessary to more directly address the issue of how ubiquitous unique and rare variants in the NRG1 gene promoter may be.
If this novel 5-SNP haplotype is causal, then it should show direct functional effects. In support of this, the 420M9-1395 region—containing 3/5 novel SNPs—was a potent driver of gene expression in vitro
. Furthermore, we found that SRY, a sox-like male specific transcription factor, was able to downregulate activity of the 420M9-1395 enhancer and that repression was more extreme with the novel schizophrenia-associated haplotype. Although we were not able to directly link the novel 5-SNP haplotype to NRG1 isoform expression in the brain because of the rarity of the haplotype, we did find a more common haplotype that spanned the 420M9-1395 enhancer region that significantly decreased NRG1 type II expression. Further examination of the transcriptional effects of this enhancer may elucidate whether it is important in driving dysregulation of NRG1 transcripts in certain individuals, particularly the increased type I and decreased type II ratio, which has been reported previously.11
In this study, we did not find large changes in any NRG1 isoform with diagnosis alone, however, it may be that heterogeneity in cause and course of schizophrenia or laterality effects may contribute to the difficulties in reproducing mRNA expression changes across independent cohorts.
Postmortem studies such as this do not aim to establish genetic association with increased risk for schizophrenia, but rather aim to explore the link between risk haplotype and changes in gene expression and mutational diversity. Thus an intrinsic limitation of our study is the power of the cohort for detecting significant genetic effects, and hence the findings presented herein should be considered preliminary and require replication. This is illustrated by the lack of significant association of the 5-SNP HapICE risk haplotype in our cohort, despite a similar frequency difference between cases and controls in the Australian postmortem cohort and the Icelandic population8
(5.4% vs 7.0%, respectively). In addition, the high nucleotide diversity observed here should be interpreted with caution, as we have not surveyed the entire gene, and have observed a relatively small number of chromosomes. The significance of this increased nucleotide diversity will become apparent with forthcoming next-generation sequencing studies in larger population-based cohorts. Although our postmortem sample is limited to detect statistically significant differences at the genetic level, it is one of the larger ethnically matched Caucasian postmortem brain studies published to date. The earlier studies of Nicodemus et al.15
and Law et al.13
used a cohort of mixed ethnicity: 44 schizophrenia cases (55% African American; 45% Caucasian) and 88 controls (60% African American; 28% Caucasian). The Hashimoto et al.11
cohort was largely African American with 19 schizophrenia cases (74% African American; 26% Caucasian) and 20 controls (85% African American, 15% Caucasian). The Parlapani et al.
used a European Caucasian cohort that included 8 controls and 11 cases for BA10. Given the high genetic diversity in the African American genome as compared with European Caucasians, it is possible that genetic effects observed in the African American population will not be replicated in Caucasians and vice versa.
Although there is still ongoing debate as to the robustness of the genetic association implicating NRG1
as a schizophrenia susceptibility gene, functionally NRG1/ErbB3 is critical for multiple stages of Schwann cell development,47, 48, 49
and its role in promoting the development of myelin forming cells is now recognized to include oligodendrocytes. Not only are NRG1 and various ErbB receptors expressed in oligogenic zones,2, 50
but NRG1 can induce the division51
and/or promote the differentiation of oligodendrocyte and neuronal precursors in vitro.52, 53, 54, 55
In particular, cell specific overexpression of NRG1 type III leads to increased myelination in mice,45, 56
although how this relates to findings in schizophrenia, which shows deficits in molecular markers of myelin or disrupted integrity of white matter, is unclear.
Interestingly, the NRG1-ErbB4 pathway appears critical to the proper development and migration of cortical inhibitory interneurons.2, 4, 5
Soluble NRG1 (types I and II) act as cortical chemoattractants, whereas the membrane bound form (type III) acts in a contact dependant way to help guide migration along certain paths.5
Once developing interneurons reach the cortex, axons expressing NRG1 type III can contact postsynaptic ErbB4 and regulate the formation of excitatory synapses from pyramidal neurons onto inhibitory neuron dendrites.4
Cortical interneurons are consistently found to be deficient in the prefrontal cortex of people with schizophrenia57
and are also abnormal in our cohort.58, 59
Furthermore, NRG1-induced migration was found to be altered in lymphoblasts from patients with schizophrenia.60
More studies are needed to link the pathology of interneuron migration, differentiation and survival to abnormalities in NRG1 in the brains of patients with schizophrenia.
Our findings offer further evidence that abnormal expression of NRG1 isoforms in DLPFC may be related to the pathophysiology of schizophrenia. We suggest a novel mechanism of risk, involving increased NRG1 type III expression in schizophrenia. Furthermore, we suggest that the mechanism by which sequence variation within NRG1 imparts risk to schizophrenia is not solely driven by one SNP, but may relate more to diversity in the genetic neighborhood and accumulation of nucleotide changes. This is fueled by our observation that sequence variation within NRG1 is high and varies even more in the disease state. The implications of our results at the phenotypic level are unknown, but they are at least conceptually consistent with evidence that schizophrenia involves genetic abnormalities in developmental/plasticity-related processes involving NRG1.