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. 2014 Nov 20;5(1):61-72.
doi: 10.1534/g3.114.015636.

Functional Variants in DPYSL2 Sequence Increase Risk of Schizophrenia and Suggest a Link to mTOR Signaling

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Free PMC article

Functional Variants in DPYSL2 Sequence Increase Risk of Schizophrenia and Suggest a Link to mTOR Signaling

Yaping Liu et al. G3 (Bethesda). .
Free PMC article

Abstract

Numerous linkage and association studies by our group and others have implicated DPYSL2 at 8p21.2 in schizophrenia. Here we explore DPYSL2 for functional variation that underlies these associations. We sequenced all 14 exons of DPYSL2 as well as 27 conserved noncoding regions at the locus in 137 cases and 151 controls. We identified 120 variants, eight of which we genotyped in an additional 729 cases and 1542 controls. Several were significantly associated with schizophrenia, including a three single-nucleotide polymorphism (SNP) haplotype in the proximal promoter, two SNPs in intron 1, and a polymorphic dinucleotide repeat in the 5'-untranslated region that alters sequences predicted to be involved in translational regulation by mammalian target of rapamycin signaling. The 3-SNP promoter haplotype and the sequence surrounding one of the intron 1 SNPs direct tissue-specific expression in the nervous systems of Zebrafish in a pattern consistent with the two endogenous dpysl2 paralogs. In addition, two SNP haplotypes over the coding exons and 3' end of DPYSL2 showed association with opposing sex-specific risks. These data suggest that these polymorphic, schizophrenia-associated sequences function as regulatory elements for DPYSL2 expression. In transient transfection assays, the high risk allele of the polymorphic dinucleotide repeat diminished reporter expression by 3- to 4-fold. Both the high- and low-risk alleles respond to allosteric mTOR inhibition by rapamycin until, at high drug levels, allelic differences are eliminated. Our results suggest that reduced transcription and mTOR-regulated translation of certain DPYSL2 isoforms increase the risk for schizophrenia.

Keywords: CRMP2; RNA translation; brain development; gene regulation; mTOR.

Figures

Figure 1
Figure 1
Identification of two sex-specific haplotypes with SZ susceptibility in 729 AJ SZ and 1542 AJ controls. (A) A 2-SNP (rs5029306, rs17088251) haplotype for female- specific SZ risk in AJ. (B) A 3-SNP (rs57045236, rs12155555, rs73229635) haplotype for male-specific SZ risk in AJ. (C) The same five SNPs shown in the UCSC genome browser (genome.ucsc.edu). The LD blocks, indicated by black lines, show that the SZ associated SNPs are in LD with DPYSL2 but not ADRA1A. AJ, Ashkenazi Jewish; LD, linkage disequilibrium.
Figure 2
Figure 2
DPYSL2 Pr3SNP luciferase constructs containing low risk (LR) vs. high risk (HR) haplotypes drive different levels of reporter expression in vitro. (A) Schematic representation of the human DPYSL2 promoter region encompassing the 3-SNP haplotype (LR or HR) and a dinucleotide repeat (DNR) of fixed size (n = 11 repeats) (blue bar). (B) HR haplotype-containing constructs displayed lower luciferase expression than their LR counterparts when assayed in both cell types (HEK293 and primary cortical neurons). Each bar on the chart represents data derived from 6 technical replicates (HEK293) or 12 technical replicates (primary cortical neurons) and is consistent across two independent biological replicates. Error bar: standard error of the mean
Figure 3
Figure 3
A dinucleotide repeat (DNR) in the DPYSL2 5′-UTR influences gene expression in in vitro reporter assays: Luciferase results from series of constructs containing LR/HR 3-SNP haplotype plus various DNR in HEK293 cells (A) and primary cortical neurons.* Risk 3-SNP haplotype with 13 DNRs (most common allele in DNR risk alleles) showed ~1.5- to 2-fold decrease in expression compared with construct having Risk 3-SNP haplotype with 11 DNRs in HEK293 (B) and primary cortical neurons (C). (D) these data were verified in HEK293 using semiquantitative expression level of transfected luciferase and endogenous Beta-actin mRNA were determined by reverse-transcription polymerase chain reaction (25 cycles).
Figure 4
Figure 4
Expression pattern of ZF dpysl2 genes and DPYSL2_I1-1and DPYSL2_ShortProm elements in transgenic ZF. (A): A, B, C, D, E, F: dpysl2a and dpysl2b whole-mount in situ hybridizations at 24, 48, and 72 hpf. (B): G, H, I, J, K, L: Transgenic ZF line for DPYSL2_I1-1 element driving EGFP reporter expression at 20, 48 and 72 hpf. (C): M, N, O, P, Q, R: Transgenic ZF line for DPYSL2_ShortProm element driving EGFP expression at 20, 48, and 72 hpf; Cb, cerebellum; Dc, diencephalon; GCL, retinal ganglion cell layer; Hb, hindbrain; Mb, midbrain; MO, medulla oblongata; Tc, telencephalon. White arrowheads, anterior and posterior lateral line; black arrowheads, Rohon-Beard neurons; anterior is to the left. A, B, D, E, J, K, L, P, Q, and R: lateral view; C, F, G, H, I, M, N, and O: dorsal view.
Figure 5
Figure 5
(A) Representative polysome profile of the 260-nm ultraviolet absorption through the sucrose gradient. Cytoplasmic extracts from HEK293 cells transfected with DNR luciferase constructs were layered over a 15–50% sucrose gradient. A total of 12 fractions (850 μL) were collected from the top of the gradient, and absorbance was measured at 260 nm to identify fractions containing monosomes and polysomes. Fraction 1 was devoid of any ribosomes, and fraction 2s−4 were nonpolysomes containing ribosome subunits and monosomes. Polysomes were present in fractions 5−12. (B and C) Distribution of endogenous beta-actin mRNA and exogenous luciferase mRNA in polysomes separated into 12 fractions by sucrose gradient. Blue line: cell lysates from HEK293 cells transfected with DNR 11 repeats; red line: HEK293 cell lysates from HEK293 cells transfected with DNR 13 repeats. (B) Endogenous beta-actin mRNA was found mostly on polysome fractions in both samples indicating active translation. (C) Polysome distribution of luciferase mRNA generated from transfected cells differed between DNR11 and DNR13—with more mRNA on polysomal fractions from DNR11 (47%) than DNR13 (15%).
Figure 6
Figure 6
Effect of the mTOR inhibitor rapamycin on the DNR-driven expression of luciferase in HEK293 cells. The effects on the empty vector and the two 11 (WT) and 13 (Risk) DNR alleles are shown for a range of rapamycin concentrations. Rapamycin decreases luciferase activity for both alleles and reaches a plateau after ~30 nM. At this concentration the difference between the two alleles is no longer significant.

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