ARTICLE International Journal of Neuropsychopharmacology (2006), 9, 705–712. Copyright f 2005 CINP doi:10.1017/S1461145705006267 An investigation of the dihydropyrimidinase- like 2 (DPYSL2) in schizophrenia: genetic association study and expression analysis

Xinzhi Zhao1,2*, Ruqi Tang1,2*, Zeping Xiao3*, Yongyong Shi1,2, Guoying Feng3, Niufan Gu3, Jianguo Shi4, Yangling Xing4, Lijuan Yan5, Hong Sang5, Shaomin Zhu6, Huijun Liu6, Wuyan Chen1,2, Jixia Liu1,2, Wei Tang1,2, Jing Zhang1,2 and Lin He2,7 1 Bio-X Center, Shanghai Jiao Tong University, Shanghai, China 2 Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 3 Shanghai Institute of Mental Health, Shanghai, China 4 Xi’an Institute of Mental Health, Xi’an, China Downloaded from https://academic.oup.com/ijnp/article/9/6/705/666184 by guest on 01 October 2021 5 Changchun Kaixuan Hospital, Changchun, China 6 JiLin Institute of Mental Health, JiLin, China 7 NHGG, Shanghai Jiao Tong University, Shanghai, China

Abstract

Several linkage studies support a susceptibility for schizophrenia on 8p21-22. In this study, we investigated a gene mapping to 8p21, dihydropyrimidinase-like 2 (DPYSL2). DPYSL2 plays an important role in axonal formation and dysfunction of DPYSL2 may result in neurodevelopmental abnormalities. In previous studies, the expression of the gene has been shown to display alteration in the brain of schizophrenia patients compared with those of healthy controls. Recently, Nakata and colleagues found polymorphisms in the 3k-end of DPYSL2 to be associated with schizophrenia, especially the paranoid type, in a Japanese population. In this study, we genotyped four SNPs in DPYSL2 in 2552 Chinese Han specimens. Case-control and TDT analyses were performed to detect association of DPYSL2 with schizophrenia. However, no allele, genotype or haplotype association was found. We investigated the expression of DPYSL2 in 29 schizophrenia patients and 54 healthy controls using quantitative real-time PCR and no difference was found between the two groups. In a comparative allele-specific expression test, we used two SNPs as markers. Only a small proportion of heterozygotes revealed a significant difference (>20%) in allele representation. The results indicated the mRNA level did not contribute mainly in the altered expression of the gene in schizophrenia patients. Although our results provided no evidence for DPYSL2 itself as a susceptibility gene for schizophrenia, recent findings have indicated that DPYSL2 may interact with other candidate for schizophrenia and be worthy of further studies. Received 12 June 2005; Reviewed 17 July 2005; Revised 19 August 2005; Accepted 23 August 2005; First published online 2 December 2005

Key words: Allele-specific expression, DPYSL2, genetic association, , schizophrenia.

Introduction As a result of a large number of genome-wide linkage studies, many plausible candidate regions for Schizophrenia (MIM 181500) is one of the most severe schizophrenia have been found although only small mental disorders, with a worldwide incidence of 1% parts of them have been well replicated. However, a (Lewis and Lieberman, 2000). Data from twin, family, series of independent studies indicate that the 8p21-22 and adoption studies provide strong evidence that region is a locus associated with genetic susceptibility schizophrenia is predominantly a genetic disorder to schizophrenia (Blouin et al., 1998; Gurling et al., with high heritability (Tsuang, 2000). 2001; Kendler et al., 2000; Stefansson et al., 2002). Several genes located in the 8p21-22 region may Address for correspondence : Dr Lin He, Institute for Nutritional be strong candidates for susceptibility to schizo- Sciences, Shanghai Institutes for Biological Sciences, 294 Taiyuan phrenia. In the present study, we have focused on Road, Chinese Academy of Sciences, Shanghai 200031, China. dihydropyrimidinase-like 2 (DPYSL2, also named as Tel. : 00 86 21 62822491 Fax : 00 86 21 62822491 E-mail : [email protected] DRP2 or CRMP2), which is localized on 8p21 and is * These authors contributed equally to this work. highly expressed in the developing nervous system. 706 X. Zhao et al.

In neurons, DPYSL2 binds to tubulin heterodimers Materials and methods to enhance microtubule assembly, which helps in Subjects the elongation of axons (Fukata et al., 2002). The hypofunction of DPYSL2 may inhibit axonal formation For the case-control investigation, 741 unrelated and may result in neurodevelopmental abnormalities patients and 752 controls were recruited. The patient (Inagaki et al., 2001), which may be a factor in the group consisted of 403 males and 338 females with pathogenesis of schizophrenia (Weinberger, 1995). a mean age of 42.74 yr (S.D.=12.58 yr), of whom 549 Two-dimensional gel electrophoresis and sequenc- originated from Shanghai and 192 from Jilin. The ing of proteins have indicated that DPYSL2 displays controls consisted of 359 males and 393 females with disease-specific alterations in the human brain. a mean age of 34.28 yr (S.D.=9.59 yr), of whom 560 Johnston-Wilson et al. (2000) reported that the DPYSL2 were from Shanghai and 192 from Jilin. Among these protein level was significantly lower in the frontal specimens, there were 675 patients with information cortex in schizophrenia, bipolar and major depression on subtypes, including 279 cases of paranoid type, patients; while Edgar et al. (2000) found increased seven cases of catatonic type, 29 cases of disorganized Downloaded from https://academic.oup.com/ijnp/article/9/6/705/666184 by guest on 01 October 2021 levels of DPYSL2 in the hippocampus in schizo- type, 76 cases of residual type and 284 cases of phrenia patients. undifferentiated type. Genotyping data from the Recently, Nakata and colleagues (2003) investigated paranoid-type schizophrenia patients (134 males five polymorphisms within DPYSL2 and performed and 145 females, mean age 45.62 yr, S.D.=13.60) were a case-control analysis in a Japanese population. additionally compared with those from the controls They found that a polymorphism, rs17666, in the (Table 1). Another subgroup of population-based 3k untranslated region of the gene was significantly specimens including 29 patients (15 males and 14 associated with schizophrenia (p=0.0097), especially females, mean age 39.68 yr, S.D.=12.66 yr) and 54 the paranoid-type (p=0.0083). However, more inde- controls (27 males and 27 females, mean age 39.66 yr, pendent replications are needed to confirm this S.D.=11.99 yr) from Shanghai were used to investigate finding. Another allele-specific gene expression study gene expression and allele-specific expression. For indicated that some cis-acting variations may alter the transmission disequilibrium test (TDT) study, 353 the expression of DPYSL2 but may be in low unrelated schizophrenia probands (187 males and 165 heterozygosity: Bray et al. (2003) used the SNP females, mean age 24.88 yr, S.D.=6.82 yr) and their rs708621 as the marker, and found that one of the 20 biological parents were recruited. All subjects were heterozygotes revealed a significant difference (39%) Han Chinese in origin. Subjects with schizophrenia in allele representation. Since polymorphisms in the were strictly diagnosed by two independent psy- untranslated region may alter the gene expression chiatrists according to the DSM-III-R criteria. A (Conne et al., 2000), an allele-specific gene expression standard informed consent in the protocol, which was study may be able to detect the potential function of reviewed and approved by the Shanghai Ethical the SNP rs17666. Committee of Human Genetic Resources, was given In the present study, we analysed the association by the participants, after the nature of study had been between four SNPs (rs327234, rs2289592, rs708621 and fully explained. rs17666) in DPYSL2 and schizophrenia in a Chinese Han population, using a total of 2552 specimens. We Genotyping choose SNPs in the study according to the following criteria: the SNP rs17666, which showed association The genomic structure of DPYSL2 and the location with schizophrenia in the Japanese population, used of the SNPs genotyped in the present study are in the study of Nakata et al. (2003); rs708621 used in shown in Figure 1. All the four SNPs were selected the study of Bray et al. (2003); rs327234 and rs2289592 from the dbSNP (http://www.ncbi.nlm.nih.gov/ with proper localization and heterozygosity. We also SNP). Genomic DNA was prepared from venous investigated the gene expression in the lymphocytes blood using the phenol chloroform extraction method. 1 of 83 samples. After synthesizing cDNA, real-time The four SNPs were genotyped using TaqMan tech- polymerase chain reaction (PCR) was performed to nology on an ABI 7900 system (Applied Biosystems, investigate whether the expression of DPYSL2 was Foster City, CA, USA). All probes and primers were altered in schizophrenia patients compared with designed by the Assay-by-DesignTM or Assay-on- controls. Template-directed primer extension was DemandTM service of Applied Biosystems. The stan- used to detect allele-specific expression of rs17666 dard PCR reactions of 5 ml were carried out using 1 and rs708621. TaqMan Universal PCR Master Mix reagent kits An investigation of the DPYSL2 gene in schizophrenia 707

Table 1. Genotype and allele frequencies of SNPs and association analysis of each SNP in population-based samples

Genotypeb Alleleb

Marker na T/T T/C C/CHWE (p) p value T C p value OR rs327234 Control 733 356 (48.6) 323 (44.1) 54 (7.4) 0.10 1035 (70.6) 431 (29.4) Schizophrenia 730 365 (50.0) 291 (39.9) 74 (10.1) 0.16 0.09 1023 (70.1) 439 (29.9) 0.69 0.97 Paranoid-type 277 149 (53.8) 103 (37.2) 25 (9.0) 0.25 0.13 401 (72.4) 153 (27.6) 0.43 1.09

G/G G/C C/C G C rs2289592

Control 738 325 (44.0) 330 (44.7) 83 (11.2) 0.96 980 (66.4) 496 (33.6) Downloaded from https://academic.oup.com/ijnp/article/9/6/705/666184 by guest on 01 October 2021 Schizophrenia 728 315 (43.3) 310 (42.6) 103 (14.1) 0.06 0.24 940 (64.6) 516 (35.4) 0.30 1.09 Paranoid-type 274 117 (42.7) 125 (45.6) 32 (11.7) 0.87 0.93 359 (65.5) 189 (34.5) 0.71 0.96

T/T T/C C/C T C rs708621 Control 729 235 (32.2) 372 (51.0) 122 (16.8) 0.22 842 (57.8) 616 (42.2) Schizophrenia 733 261 (35.6) 365 (49.8) 107 (14.6) 0.26 0.30 887 (60.5) 579 (39.5) 0.13 1.12 Paranoid-type 279 100 (35.8) 142 (50.9) 37 (13.3) 0.23 0.31 342 (61.3) 216 (38.7) 0.15 1.16

T/T T/C C/C T C rs17666 Control 717 535 (74.6) 167 (23.3) 15 (2.1) 0.65 1237 (86.3) 197 (13.7) Schizophrenia 726 534 (73.8) 169 (23.3) 21 (2.9) 0.09 0.63 1237 (85.2) 211 (14.8) 0.53 0.93 Paranoid-type 273 209 (76.6) 60 (21.9) 4 (1.5) 0.90 0.72 478 (87.5) 68 (12.5) 0.45 1.12

HWE, Hardy–Weinberg equilibrium; OR, odds ratio. a Number of samples which were successfully genotyped. b Frequencies are shown in parentheses (%).

under the guidelines provided. Samples in which Biosciences, Darmstadt, Germany) at 37 xC for 20 min the TaqMan assay failed were genotyped again by to remove residual DNA followed by inactivation at direct sequencing (479 SNPs in 288 samples). After 65 xC for 10 min. RNA samples were further purified 1 cleaning the PCR products, sequencing reactions were by using a HiBind spin column (Omega Bio-Tek, performed using BigDye Terminator cycle sequencing Doraville, GA, USA) according to the manufacturer’s kit (Applied Biosystems), and all the PCR forward instructions. The purified RNA samples (0.5 mg) were primers were used as sequencing primers. The frag- then reverse-transcribed using the SuperScript first- ments of sequencing reactions were separated by strand synthesis system (Invitrogen, Carlsbad, CA, electrophoresis on ABI 3100 instruments (Applied Bio- USA). Random hexamers were used as primers for systems). reverse transcription.

RNA extraction and cDNA synthesis Real-time quantitative PCR

Lymphocytes were isolated from fresh venous blood Real-time PCR was performed using the ABI 7900 by centrifugation on Ficoll gradient. Total RNA and system (Applied Biosystems). Reactions were per- DNA were in turn extracted from the lymphocytes formed in a 10-ml volume including diluted cDNA using Trizol reagent. RNA integrity was confirmed by samples, primers and SYBR Green I Mastermix direct visualization of 18S and 28S rRNA bands after (Applied Biosystems). Diluted cDNA samples pro- agarose gel electrophoresis. RNA were incubated duced from 10 ng total RNA was added to each well. using 10 units of DNase I (Novagen, Merck Real-time PCR data were collected using SEQUENCE 708 X. Zhao et al.

DETECTOR software (version 2.1, Applied Biosystems). p>0.05) in both population- and family-based sam- Expression of DPYSL2 was normalized by GAPDH ples. None of the polymorphisms revealed significant levels (denoted as DCt). Both GAPDH and DPYSL2 allele or genotype association with disease status in were tested four times for each sample. case-control samples. Using diagnostic subtype data for the schizophrenia patients, we compared the allele Allele-specific expression assay and genotype frequencies of the SNPs between Two SNPs (rs708621, rs17666) within the expressed the paranoid-type schizophrenia patients and healthy sequence were used as the markers for mRNA tran- controls, but no significant differences were found. scribed from each chromosomal allele. A SNuPe The detailed results are shown in Table 1. In family- method, based on template-directed primer extension, based samples, no transmission distortions were was used to compare relative allele expression from found in any of the markers (Table 2). individual subjects who were heterozygous for the There was moderate or even high LD between the marker polymorphisms on MegaBACE 1000 instru- four markers (Figure 1). We calculated all the global ments (Amersham Biosciences, Piscataway, NJ, USA). p values from multi-locus haplotypes (two to four) Downloaded from https://academic.oup.com/ijnp/article/9/6/705/666184 by guest on 01 October 2021 In order to normalize the signal from different fluor- consisting of the SNPs (Table 3). No positive results escent dyes, genomic DNA, which is a perfect 1:1 ratio were detected in any of the global tests either in case- of the two alleles, was used to correct allele ratios control samples or in family trios. Although one obtained from each cDNA analysis. The SnuPe pro- common haplotype, ‘CCTC’ revealed weak associ- ducts of DNA and cDNA templates from the same ation in population-based samples (8.8% in patients samples would be electrophorized in the same capil- and 6.3% in controls, p=0.013), the association was laries in one run by multiple injections (Figure 2b). not confirmed in family trios (p=0.11). Considering the high volume of statistical tests carried out in this Statistical analysis study, this positive result is more likely to be a type-I The deviations from Hardy–Weinberg equilibrium of error by chance. all the SNPs were assessed using an online calculator The protein level of DPYSL2 has been shown to be (http://www.kursus.kvl.dk/shares/vetgen/_Popgen/ altered in schizophrenia patients. In order to define genetik/applets/kitest.htm). For case-control analy- whether the alteration is attributable to mRNA levels, sis, the statistical significance of differences of the we used real-time PCR to quantify the expression of SNPs in the allele and genotype frequency distri- DPYSL2. The resulting data is shown in Figure 2a. butions was estimated using the program Clump Compared with the expression in controls, the 2.2 (Sham and Curtis, 1995). Each computation was decrease was only 6.4% in patients. There is no sig- performed with at least 100 000 simulations. The nificant difference between the two groups (t=1.538, haplotype frequencies of the multiple markers were d.f.=81, p=0.13). Neither did the eight patients who estimated using the program EHPLUS (Zhao et al., 2000). were paranoid-type reveal significant alteration in The standardized measure of linkage disequilibrium expression (10% decrease, t=1.494, d.f.=60, p=0.14). (LD) for adjacent pairs of markers, denoted as D’, was The mRNA level of DPYSL2 was not correlated with estimated using the software 2LD (Zapata et al., 2001). gender (p=0.89), but showed a weak negative corre- Power analysis was performed using an online power lation with age (r=x0.228, p=0.039). calculator (http://calculators.stat.ucla.edu/power- The expression of genes may be influenced by calc/). For TDT analysis, the TDTPHASE program of multiple factors. In order to detect the possible UNPHASED set (Dudbridge, 2003) was used for single effects of cis-acting polymorphisms in DPYSL2,48 and multiple marker haplotype transmission analyses, samples who were heterozygous either for rs708621 and LD between the adjacent markers. For expression or for rs17666 were used to perform allele-specific and allele-specific expression analyses, group com- expression tests. 31 samples were heterozygous for parisons were analysed using the t test. Correlations rs708621 while 18 were heterozygous for rs17666, between age or gender and DPYSL2 mRNA level were respectively (one sample was heterozygous for both assessed with Pearson’s correlation test. All tests were markers and was tested twice). When the data from two-tailed and significance was accepted at p<0.05. all the samples were combined, neither of the markers revealed any significant difference between the two alleles (rs708621: T/C=0.97, p=0.11; rs17666: Results T/C=1.07, p=0.10). Several samples revealed allele The genotype distributions of all the four SNPs differences beyond the threshold of 20% employed by accorded with Hardy–Weinberg equilibrium (x2<3.84, Yan et al. (2002), indicating the existence of a cis-acting An investigation of the DPYSL2 gene in schizophrenia 709

Table 2. Genotype and allele frequencies of SNPs and association analysis of each SNP in family-based samples

Genotypeb Alleleb

Marker na T/T T/C C/CHWE (p) T C T/NT p value rs327234 Parents 686 325 (47.3) 300 (43.7) 61 (8.9) 0.48 950 (69.2) 422 (30.8) 137/146 0.59 Probands 347 164 (47.3) 154 (44.4) 29 (8.3) 0.39 482 (69.5) 212 (30.5)

G/G G/C C/C G C rs2289592 Parents 678 304 (44.8) 307 (45.3) 67 (9.9) 0.41 915 (60.7) 441 (39.3) 132/149 0.31

Probands 342 141 (41.2) 169 (49.4) 32 (9.4) 0.06 451 (62.8) 233 (37.2) Downloaded from https://academic.oup.com/ijnp/article/9/6/705/666184 by guest on 01 October 2021

T/T T/C C/C T C rs708621 Parents 684 258 (37.7) 314 (45.9) 112 (16.4) 0.31 830 (60.7) 538 (39.3) 160/140 0.25 Probands 344 137 (39.8) 158 (45.9) 49 (14.2) 0.75 432 (62.8) 256 (37.2)

T/T T/C C/C T C rs17666 Parents 674 510 (75.6) 146 (21.7) 18 (2.7) 0.06 1166 (0.86) 182 (0.14) 69/74 0.68 Probands 344 258 (75.0) 80 (23.3) 6 (1.7) 0.94 596 (0.87) 92 (0.13)

HWE, Hardy–Weinberg equilibrium. a Number of samples which were successfully genotyped. b Frequencies are shown in parentheses (%).

D ′=0.48/0.45 D ′=0.55/0.43 D ′=0.78/0.71 rs327234(C/T) rs2289592(G/C) rs708621(T/C) rs17666(C/T)

Distance 39.7k 26.4k 4.7k

Exon 1 2 3 4 5–7 8 9 10 11 12 13 14

Figure 1. Genomic structure of DPYSL2 and the location of SNPs included in the present study. The distance and the pair-wise linkage disequilibrium (LD) (expressed in D’, LD in case-control specimen are shown ahead of the slash, LD in family trios are shown behind the slash) between adjacent markers are also shown in the figure. polymorphism affecting gene transcription. However, and the relative risk of the susceptibility factors were most of the differences were marginal. The maximum comparatively low (the power to detect allele associ- difference in allele representation observed in the ation in population-based samples is 0.753, assuming a investigation was 50% (Figure 2c). frequency of risk factor of 0.05 and a relative risk of 1.5). However, none of the polymorphisms showed allele, genotype or haplotype association with schizo- Discussion phrenia in either the case-control study or the TDT In this study, we investigated several polymorphisms analysis. in the DPYSL2 gene in a large-size Han Chinese Although we did not replicate the association sample group. Our samples should provide enough detected in the Japanese population, the rejection power to detect association even if the frequencies of a role for DPYSL2 in the pathophysiology of 710 X. Zhao et al.

Table 3. Global p values from multilocus (two to four) (a) association analysis 140 n=54 120 n=29 n=8 Case-control study TDT study 100

2 SNPs 3 SNPs 4 SNPs 2 SNPs 3 SNPs 4 SNPs 80

60 rs327234 0.73 0.77 40 rs2289592 0.41 0.53 20 0.38 0.13 0.24 0.35 0 rs708621 0.51 0.16 Controls Schizophrenia Paranoid 0.25 0.57 patients type rs17666

(b) Downloaded from https://academic.oup.com/ijnp/article/9/6/705/666184 by guest on 01 October 2021

TDT, Transmission disequilibrium test. 6000

5000 schizophrenia in the Chinese population may be premature. In addition to the genetic and phenotypic 4000 heterogeneity in complex diseases or potential strati- 3000 fication, Marchini et al. (2005) showed recently that plausible variations in allele frequencies across Allele Allele Allele Allele Allele 2000 Allele C T C T C populations among interacting loci could markedly T affect the power to detect their marginal effects, 1000 which may account in part for the well-known difficulties in replicating association results. The 0 cDNA Genomic DNA cDNA allele frequency of rs17666 in the Chinese population was different from that in the Japanese population. (c) More importantly, recent studies have indicated that IndividualsAverage Individuals Average 1.6 some other genes may regulate the activation of 1.4 DPYSL2 protein via phosphorylation. The interaction between the loci within those genes and DPYSL2 may 1.2 reduce the power to detect their effects in association 1.0 study. 0.8 In neurons, about 30% of the DPYSL2 protein 0.6 will be phosphorylated at Thr514 by glycogen Allelic ratio: T/C 0.4 synthase kinase-3b (GSK-3b) and then be inactivated 0.2 (Yoshimura et al., 2005). GSK-3b is largely regulated 0 by inhibition through AKT1-dependent phosphoryla- rs708621 (n=31) rs17666 (n=18) tion at Ser9 (Vanhaesebroeck and Alessi, 2000). AKT1 Figure 2. Expression analyses of DPYSL2 in the lymphocytes protein levels are lower in schizophrenia patients from schizophrenia patients and healthy controls. (a) mRNA compared with controls and reduce the phosphoryla- level of DPYSL2 measured by real-time PCR. The average tion of GSK-3b (Emamian et al., 2004). Moreover, level of DPYSL2 in healthy controls was defined as 100. No brain-derived neurotrophic factor (BDNF), which disease-specific alteration was detected. (b) SNuPe method has been reported to be in abnormal expression in was used to compare allele-specific expression. The SnuPe the corticolimbic system of schizophrenia patients products of DNA and cDNA templates from the same heterozygous samples would be electrophorized in the same (Takahashi et al., 2000), inhibits GSK-3b via the phos- capillaries in one run by multiple injections. For each sample, phatidylinositol-3-kinase (PI3-kinase)/AKT pathway. cDNA template was tested four times while the DNA Interestingly, both AKT1 (Emamian et al., 2004; Ikeda template was tested twice. (c) Relative allele-specific et al., 2004) and BDNF (Muglia et al., 2003; Neves- expression of the SNPs rs708621 and 17666. Data are Pereira et al., 2005) have been reported to be associated expressed as allele ratios of T/C. Several samples revealed with schizophrenia in different populations. The allele difference in excess of 20%. The maximum allele genotyping of the polymorphisms in AKT1, GSK-3b difference was 50%. An investigation of the DPYSL2 gene in schizophrenia 711 and BDNF are now being performed using the same other genes, and their potential roles in the patho- samples as in this study. As a continuing study, we physiology of schizophrenia. might be able to detect and characterize interactions among the loci in these genes using statistical methods Acknowledgements such as multifactor-dimensionality reduction (MDR; Hahn et al., 2003). This work was supported by grants from the We used the lymphocytes isolated from fresh blood National 863 projects (2001AA224011) and 973 to investigate gene expression of DPYSL2 but no projects (2001CB510304), the National tackle-key- expression difference was detected between schizo- problem project (2002BA711A07-01), the National phrenia patients and healthy controls in mRNA levels. Natural Science Foundation of China, the Shanghai Lymphocytes may serve as an easily accessible probe Municipal commission for Science and Technology to detect cellular function of the brain, including gene (030Z14025), and the Key grant project (No. 10414) of expression (Gladkevich et al., 2004). However, given the Ministry of Education, PRC. the difference between the studies of Johnston-Wilson Downloaded from https://academic.oup.com/ijnp/article/9/6/705/666184 by guest on 01 October 2021 et al. (2000) and Edgar et al. (2000), the regulatory Statement of Interest mechanism of DPYSL2 in gene expression may be complex, and the expressing alteration of the gene None. in schizophrenia patients may be different in the lymphocytes, cortex or hippocampus. One other possible explanation is that the disease-specific alter- References ation of DPYSL2 may be mainly generated in protein Blouin JL, Dombroski BA, Nath SK, Lasseter VK, Wolyniec level by AKT1/GSK-3b-mediated phosphorylation. 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