Schizophrenia-associated HapICE haplotype is associated with increased NRG1 type III expression and high nucleotide diversity

Abstract

Excitement and controversy have followed neuregulin (NRG1) since its discovery as a putative schizophrenia susceptibility gene; however, the mechanism of action of the associated risk haplotype (HapICE) has not been identified, and specific genetic variations, which may increase risk to schizophrenia have remained elusive. Using a postmortem brain cohort from 37 schizophrenia cases and 37 controls, we resequenced upstream of the type I-IV promoters, and the HapICE repeat regions in intron 1. Relative abundance of seven NRG1 mRNA transcripts in the prefrontal cortex were determined and compared across diagnostic and genotypic groups. We identified 26 novel DNA variants and showed an increased novel variant load in cases compared with controls (χ(2)=7.815; P=0.05). The average nucleotide diversity (θ = 10.0 × 10(-4)) was approximately twofold higher than that previously reported for BDNF, indicating that NRG1 may be particularly prone to genetic change. A greater nucleotide diversity was observed in the HapICE linkage disequilibrium block in schizophrenia cases (θ((case)) = 13.2 × 10(-4); θ((control)) = 10.0 × 10(-4)). The specific HapICE risk haplotype was associated with increased type III mRNA (F = 3.76, P = 0.028), which in turn, was correlated with an earlier age of onset (r = -0.343, P = 0.038). We found a novel intronic five-SNP haplotype ~730 kb upstream of the type I promoter and determined that this region functions as transcriptional enhancer that is suppressed by SRY. We propose that the HapICE risk haplotype increases expression of the most brain-abundant form of NRG1, which in turn, elicits an earlier clinical presentation, thus providing a novel mechanism through which this genetic association may increase risk of schizophrenia.

Figure 1

Genomic structure and location of resequenced regions of human NRG1. (a) Human NRG1 is transcriptionally complex, with over 30 known mRNA splicing isoforms that are mainly driven off four alternative promoters (I–IV) and two minor promoters (V–VI). Alternative isoforms include different functional protein domains, with type III isoform containing two transmembrane domains (TMD) and a cysteine-rich domain (CRD). Proteolytic cleavage of type I and III isoforms produces a C-terminal fragment (CTF) and N-terminal fragment (NTF) containing a bioactive EGF domain. Downstream processing by γ-secretase processes the CTF to release the intracellular domain (ICD) that contains the CRD. The position of the schizophrenia-associated HapICE haplotype is shown in the upstream promoter region. (b) The relative locations and sizes of resequenced regions are shown and the positions of SNP and microsatellite markers that constitute the HapICE haplotype are indicated.

Figure 2

Nucleotide diversity in NRG1 regions comparing 37 controls (light gray) with 37 schizophrenia patients (dark gray). The dashed line indicates the average nucleotide diversity from non-coding regions of 75 other human genes at θ=5.4 × 10⁻⁴. The regions that fall within the HapICE LD block are indicated.

Figure 3

The effect of the HapICE and intron 1 haplotypes on NRG1 isoform expression. (a) The HapICE 5 SNP risk haplotype GCGTG has a significant effect on type III isoform expression (ANCOVA F(2,69)=3.763, P=0.028; post-hoc LSD: P=0.013478 or 0.019014 (for 1 or 2 copies compared with 0 copies)) with mean expression values (±s.e.) for individuals (n) with 0 copies (n=34), 1 copy (n=32) or 2 copies (n=8) were 3.446±0.168, 4.048±0.173, 4.349±0.357, respectively. (b) The intron 1 haplotype CCC(14)G has a significant effect on type II expression (ANCOVA F(1,65)=4.051, P=0.048), with mean expression values (±s.d.) for individuals (n) with 0 copies (n=46) or 1 copy (n=26) were 1.18±0.56 and 1.49±0.56, respectively. No homozygotes for the CCC(14)G haplotype were observed.

Figure 4

Age of onset at first diagnosis in postmortem brain cohort. (a) Frequency plot of age distribution in the sample, with age of onset on the x axis and frequency on the y axis. (b) Correlation of age of onset with NRG1 type III expression. Type III expression is presented on the y axis as the standardized residual from the regression equation after accounting for the significant demographic effects of pH and postmortem interval (PMI) on expression. The linear regression line showing a significant correlation (r=−0.343, P=0.038) is shown (black line) along with the mean 95% confidence intervals (black curved lines).

Figure 5

NRG1 intron 1 contains a transcriptional enhancer, which is repressed by the transcription factor SRY. HEK293 cells were transfected with 300 ng reporter constructs containing ∼1 kb of DNA spanning the 478B14-848 and 420M9-1395 HapICE repeats, human SRY was cotransfected at 300 ng (+SRY(1:1)) and 900 ng (+SRY(1:3)). The wild-type and schizophrenia-associated novel 5-SNP haplotypes are represented by the light and dark gray bars, respectively. The normalized average values from two triplicate experiments are given.