Effects of background mutations and single nucleotide polymorphisms (SNPs) on the Disc1 L100P behavioral phenotype associated with schizophrenia in mice.

BACKGROUND: Disrupted-in-schizophrenia 1 (DISC1) is a promising candidate susceptibility gene for psychiatric disorders, including schizophrenia, bipolar disorder and major depression. Several previous studies reported that mice with N-ethyl-N-nitrosourea (ENU)-induced L100P mutation in Disc1 showed some schizophrenia-related behavioral phenotypes. This line originally carried several thousands of ENU-induced point mutations in the C57BL/6 J strain and single nucleotide polymorphisms (SNPs) from the DBA/2 J inbred strain.
METHODS: To investigate the effect of Disc1 L100P, background mutations and SNPs on phenotypic characterization, we performed behavioral analyses to better understand phenotypes of Disc1 L100P mice and comprehensive genetic analyses using whole-exome resequencing and SNP panels to map ENU-induced mutations and strain-specific SNPs, respectively.
RESULTS: We found no differences in spontaneous or methamphetamine-induced locomotor activity, sociability or social novelty preference among Disc1 L100P/L100P, L100P/+ mutants and wild-type littermates. Whole-exome resequencing of the original G1 mouse identified 117 ENU-induced variants, including Disc1 L100P per se. Two females and three males from the congenic L100P strain after backcrossing to C57BL/6 J were deposited to RIKEN BioResource Center in 2008. We genotyped them with DBA/2 J × C57BL/6 J SNPs and found a number of the checked SNPs still remained.
CONCLUSION: These results suggest that causal attribution of the discrepancy in behavioral phenotypes to the Disc1 L100P mutant mouse line existing among different research groups needs to be cautiously investigated in further study by taking into account the effect(s) of other ENU-induced mutations and/or SNPs from DBA/2 J.

Background

Schizophrenia is a devastating mental disorder with a significant genetic component that affects approximately 0.5–1.0% of the general population. Based on genetic epidemiological studies, it was estimated that schizophrenia has a heritability of 60-80% [1, 2]. Disrupted-in-schizophrenia 1 (DISC1) is one of the candidate susceptibility genes for a spectrum of major psychiatric disorders. DISC1 was originally identified on chromosome 1 by analyzing a large Scottish pedigree showing a heavy burden of major psychiatric disorders associated with balanced chromosomal translocation (1:11)(q42.1:q14.3) [3, 4]. DISC1 is implicated in the genetic risk for many aspects of quantitative cognitive traits of psychotic patients [5-8]. Accumulating evidence indicates that DISC1 acts as a scaffold protein that in concert with numerous interacting proteins regulates neurogenesis, neuronal migration, neurite outgrowth, and signal transduction [9-11].

Mouse models for the genetic mutation of Disc1 are of relevance for unraveling the impact of gene disruption on neural integrity associated with schizophrenia. A number of conditional transgenic mouse models overexpressing truncated versions of human DISC1 protein under various exogenous promoters have been reported. In these models, truncated versions of human DISC1 protein are overexpressed either constitutively [12-14] or transiently during early postnatal life [15-18]. A mouse strain carrying a truncating lesion (25-bp deletion in exon 6) in the endogenous Disc1 orthologue has also been reported [19-21]. Although these mouse models display a range of psychiatric disease-related behavioral phenotypes, many have been inconsistent across studies due to differences in behavior tests and experimental design and the specific genetic aberrations.

An alternative method for generation of a mouse Disc1 model is to screen an archive of G1 mice carrying many N-ethyl-N-nitrosourea (ENU)-induced mutations. With this approach, it is feasible to identify an allelic series of ENU-induced mutations in a particular gene, in this case Disc1, as a gene-driven approach based on a priori assumptions of the function of a gene of interest [22, 23]. Genomic DNA libraries from thousands of G1 mice born to ENU-mutagenized C57BL/6 J male mice were screened for a point mutation in Disc1, one of which was subsequently identified as conferring a L100P amino acid substitution in exon 2 [24]. Roder and his collaborators reported that the Disc1 L100P mouse line showed schizophrenia-like behavior, such as increased basal and amphetamine-induced open field activity, and deficits in prepulse inhibition (PPI) [24, 25]. However, other independent research groups observed neither an increase in basal locomotor activity nor abnormal PPI [26, 27].

Because ENU generates multitude, randomly distributed, single-base point mutations throughout the entire mouse genome, ENU mutant mouse lines may harbor unknown functional mutations other than those are assumed to contribute to the phenovariance of interest [22, 23, 28, 29]. Additionally, ENU-mutagenized C57BL/6 J male mice at RIKEN were out-crossed to untreated DBA/2 J female mice to produce the original progeny (G1). Although heterozygous mice resulting from repeated backcrossing to the C57BL/6 J background were finally intercrossed to generate L100P/L100P homozygous mice, remaining ENU-induced mutations outside Disc1 and/or single nucleotide polymorphisms (SNPs) from DBA/2 J background may confound the causal relationship between L100P mutation and the behavioral phenotype. Disclosure of this information would provide a justification for the usage of ENU-mutagenesis to fully explain the phenotypic manifestation of the main causative mutation. Herein, we performed three experiments to better understand behavioral phenotypes of Disc1 L100P mice with reference to our new genetic data. First, we investigated the genetic background of the L100P strain that was reversely-deposited back to the Experimental Animal Division (EAD), RIKEN BioResource Center (BRC) by Roder’s group [24]. The genetic background of the deposited L100P/L100P homozygous pairs and their offspring born via intercrossing between the homozygotes were investigated. Second, L100P/L100P, L100P/+ and wild-type littermates (+/+), which were derived from the deposited L100P/L100P homozygous mice mated with the C57BL/6 J mice (details in Materials and Methods), were examined for schizophrenia-related behavioral phenotypes using open field and social interaction tests. Third, we conducted whole-exome resequencing analysis of the original G1 genome to screen for ENU-induced mutations in addition to the Disc1 L100P mutation. The results obtained were discussed in light of the validity and issue of Disc1 L100P mutant model.

Methods

Animals

Male and female homozygous Disc1 L100P/L100P mutant mice backcrossed to C57BL/6 J were deposited at the EAD at RIKEN BRC (Tsukuba, Japan, http://www.brc.riken.jp/lab/animal/en/) by another research group [24], and their offspring was bred to maintain the homozygosity (L100P/L100P). These L100P/L100P mutant male mice (Disc1 < Rgsc1390>) were obtained from RIKEN and then backcrossed to an inbred C57BL/6 J female (Japan Clea Co., Tokyo Japan) for one generation. The resultant L100P/+ progeny were intercrossed to generate L100P/L100P, L100P/+ and +/+ littermates. Mice were weaned at postnatal day 25–28 and segregated by sex; were housed 2–4 per cage in a temperature-controlled (25 ± 1°C) and light-controlled room (light on 0600–1800 h) in plastic cages with ad libitum access to food and water. PCR-based genotyping was conducted with a primer pair (common forward primer: 5'-CCTGTCCCAAGGACTGGCATC-3'; reverse primer for L100L wild-type: 5'-CAGGGACAAGGGAGCTCTTCA-3'; reverse primer for L100P mutants: 5'- CAGGGACAAGGGAGCTCTTCG-3') and genomic DNA extracted from tail biopsies using Hot Sodium Hydroxide and Tris (HotSHOT) method [30]. Age-matched male L100P/L100P, L100P/+ and +/+ mice (12–16 weeks old) were compared in behavioral analyses. To compare wild-type littermates with inbred C57BL/6 J mice, 12–16-week-old male C57BL/6 J mice were purchased from Japan Clea Co. (Tokyo, Japan). All experiments were performed in accordance with the Guidelines for Care and Use of Laboratory Animals, Dokkyo University School of Medicine and conformed to all Japanese federal rules and guidelines.

Drug treatment

Methamphetamine hydrochloride (Dainippon Pharmaceutical Co., Osaka, Japan) was dissolved in saline and administered subcutaneously (s.c.) in a volume of 10 ml/kg.

Behavioral analysis

All behavioral analyses were recorded with a CCD camera and analyzed using video tracking software (ANY-maze ver. 4.82; Stoelting Co., USA). The apparatus was illuminated at approximately 300 lux. Mice were transferred to the experimental site at least 30 min before testing.

Spontaneous locomotor activity

Mice were placed individually in grey plastic cages (42 × 42 × 30 cm). Spontaneous locomotor activity was recorded in 5-min sessions during a 90-min test period, with distance traveled as the primary outcome measure.

Methamphetamine-induced locomotor activity

Mice were placed individually in grey plastic cages (42 × 42 × 30 cm) for a 30-min habituation session and then injected subcutaneously with methamphetamine (0.2, 0.5 or 1.0 mg/kg) or saline. Locomotor activity was recorded continuously during the 30-min habituation period and for 60 min after injection of saline or methamphetamine. To verify effects of genetic characteristics on behavioral phenotypes, methamphetamine-induced locomotor activity was compared among mice in the following experiments. In experiment 1, effects of L100P on methamphetamine-induced locomotor activity were compared in age-matched male littermates (L100P/L100P, L100P/+ and +/+). In experiment 2, effects of genetic background on methamphetamine-induced locomotor activity were tested by comparing inbred C57BL/6 J mice with the results of wild-type littermate born to Disc1 L100P mutant line obtained from the experiment 1.

Sociability and social novelty preference tests

Effects of the L100P point mutation on social behavior were assessed according to previously described methods [24, 31] with minor modifications. A three-chamber apparatus was constructed of clear acrylic sheets; each chamber had a size of 19 × 40 × 25 cm (width × depth × height). Each side chamber contained a wire-bar cup (Galaxy Cup, Spectrum Diversified Designs, Inc., OH, USA) placed on either side of the arena. Two dividing walls containing doors allowed access to each of the side chambers from the center chamber. The behavioral test consisted of three sessions: (1) habituation, (2) sociability and (3) social novelty preference. In the habituation session (1), the subject mouse was placed in the center chamber and allowed to freely explore all three chambers for 10 min. In the sociability session (2), following the habituation session, the subject mouse was introduced in the center chamber for 1 min with access to the side chambers blocked by white partitions. An unfamiliar male C57BL/6 J mouse (Stranger 1) was enclosed in the wire-bar cup in one of the side chambers. The location of Stranger 1 alternated among subject mice. On removal of partitions, the subject mouse was allowed to freely explore the entire apparatus for 10 min. Time spent within the interaction zone (an oblong area of 19 × 15 cm containing the wire-bar cup) and the number of entries into each chamber was measured. In the social novelty preference test (3) conducted after the sociability test, the subject mouse was placed in the center chamber for 1 min. An unfamiliar intruder male C57BL/6 J mouse (Stranger 2) was enclosed in the wire-bar cup in the other side chamber. Time spent in the interaction zones and the number of entries into each chamber was measured.

SNP-typing for genetic background of L100P/L100P mutant mice

We genotyped 117 SNPs, which were openly available in the SNP database of RIKEN BRC (http://ja.brc.riken.jp/lab/jmc/mapping.html), distributed at approximately 15-cM intervals over the entire mouse genome in 8 mice; 3 males and 1 female of the L100P/L100P deposited mice to the RIKEN EAD and 1 male and 3 female progeny from the deposited pairs to distinguish between C57BL/6 J and DBA/2 J genetic backgrounds.

Exome re-sequencing of the original G1 carrying the L100P mutation

We used the SureSelect Mouse All Exon Kit (Agilent, Santa Clara, CA, USA) to enrich whole exons from genomic DNA of the G1 mouse. We performed resequencing using SOLiD4 (Life Technologies, Carlsbad, CA, USA) as reported previously [32].

Statistical analysis

Statistical analysis was conducted using SPSS software (ver. 19, IBM Japan). Data were analyzed using paired t-tests, one-way ANOVA, two-way ANOVA or two-way repeated measures ANOVA as appropriate. The Greenhouse–Geisser correction for repeated measures was applied as necessary.

Results

Spontaneous locomotor activity in the open field declined to a similar extent in all three genotypes (L100P/L100P, L100P/+ and +/+) over 90 min (Figure  1A). Two-way repeated measures ANOVA revealed no significant genotype × time interaction (F(20.169, 554.636) = 0.884, p = 0.609). Moreover, total distance traveled was similar among genotypes (Figure  1B). ANOVA revealed no main effect of genotype on total distance traveled (F(2, 55) = 0.785, p = 0.461).

Figure 1

Comparison of spontaneous locomotor activity in the open field. (A) Distance traveled in 5 min bins during spontaneous locomotion by Disc1 L100P/L100P mutants (n = 20), L100P/+ mutants (n = 18), and wild-type (+/+) littermates (n = 20). Repeated measures ANOVA revealed no significant genotype × time interaction. Data are expressed as mean ± SEM. (B) Total distance traveled over 90 min. ANOVA revealed no significant main effect of genotype. Data are expressed as mean ± SEM.

There was no difference among genotypes in locomotor activity during either the habituation period or post-injection period (Figure  2A–D). ANOVA indicated no significant genotype × drug interaction on total distance traveled (F(6, 117) = 0.214, p = 0.972), no main effect of drug treatment (F(3, 117 = 1.824, p = 0.147) and no main effect of genotype (F(2, 117) = 0.098, p = 0.907) during 30-min habituation (Figure  2E). Similarly, there was no significant genotype × drug interaction on total distance traveled among groups (F(6, 117) = 0.433, p = 0.856) or a main effect of genotype (F(2, 117) = 0.015, p = 0.985) during 60 min after drug injection (Figure  2F). Methamphetamine (0.5 and 1.0 mg/kg) significantly increased total distance traveled in all genotypes. ANOVA revealed a significant effect of drug treatment (F(3, 117) = 19.844, p < 0.001); Tukey post hoc analysis showed a significant effect of 0.5 mg/kg (p < 0.01) and 1.0 mg/kg (p < 0.001) methamphetamine versus saline.

Figure 2

Effect of methamphetamine on locomotor activity in the open field. (A–D) Distance traveled in 5 min bins following methamphetamine or saline treatment. Repeated measures ANOVA revealed no significant genotype × time interactions among Disc1 L100P/L100P (n = 11–12), L100P/+ (n = 11–13), and +/+ littermates (n = 8–9). Data are expressed as mean ± SEM. (E) Total distance traveled during 30 min before drug injection. ANOVA revealed no significant genotype × drug interaction or a main effect of genotype. (F) Total distance traveled during 60 min after drug injection. ANOVA revealed a significant main effect of the drug. Methamphetamine elicited hyperlocomotion in a dose-dependent manner in all three genotypes. (G, H) Effect of methamphetamine on the locomotor activity of inbred C57BL/6 J mice and +/+ littermates derived from Disc1 L100P mutant line in the open field. (G) Total distance traveled for 30 min before drug injection. ANOVA revealed no significant genetic background × drug interaction or a main effect of genetic background. (H) Total distance traveled over 60 min after drug injection. ANOVA revealed a significant main effect of drug. Methamphetamine elicited hyperlocomotion in a dose-dependent manner in both two groups. L100P line +/+ in (G, H) and +/+ in (E, F) were the same mice for each treatment group. Data are expressed as mean ± SEM. **p < 0.01 or ***p < 0.001 compared with the saline-treated group.

To test for effects of putatively existing genetic elements inherited from the DBA/2 J background on spontaneous and methamphetamine-induced locomotion, we compared wild-type progeny born to Disc1 L100P/+ mutants and inbred C57BL/6 J mice. There was no difference in either spontaneous or methamphetamine-induced locomotor activity (Figure  2G–H). ANOVA revealed no significant drug × genetic background interaction in either the 30-min habituation period (F(3,59) = 0.779, p = 0.511) or the 60-min post-injection period (F(3, 59) = 0.198, p = 0.898). There was no main effect of genetic background on locomotor activity in either the habituation period (F(1,59) = 0.806, p = 0.373) or the post-injection period (F(1,59) = 0.195, p = 0.660). There was no main effect of drug treatment on total distance traveled during the habituation period (F(3,59) = 0.244, p = 0.866) (Figure  2G). Methamphetamine (0.5 and 1.0 mg/kg) significantly increased locomotor activity in both wild-type progeny of Disc1 L100P/+ mice and inbred C57BL/6 J mice (Figure  2H). There was a significant main effect of drug treatment on total distance traveled after the drug administration (F(3, 59) = 12.114, p < 0.001) and significant effects of both methamphetamine 0.5 mg/kg (p < 0.01) and 1.0 mg/kg (p < 0.001) versus saline as revealed by Dunnett’s T3 post hoc analysis.

Sociability and social preference test

In the sociability session, two-way ANOVA revealed a significant interaction between genotype and the chambers in time spent in the interaction zones (F(2, 50 = 3.495, p < 0.05). Post hoc analyses revealed no significant main effect of genotype for either side, but did show a significant main effect of preference for the stranger mouse (Stranger 1) for all genotypes (+/+: p < 0.01, L100P/+: p < 0.001, L100P/L100P: p < 0.05) on stay duration (Figure  3A). Two-way repeated measures ANOVA showed neither a significant genotype × chamber interaction for the number of entries nor a main effect of genotype, but did show a significant main effect of the chamber (F(1.533, 76.638) = 403.012, p < 0.001). Post hoc analyses revealed a significant main effect of preference for the center zone for the combined group (Empty: p < 0.001, Stranger 1: p < 0.001), but the number of transitions into the chamber containing Stranger 1 did not differ from the number of transitions into the empty chamber. In the social novelty preference session, two-way repeated measures ANOVA showed neither a significant genotype × side interaction in time spent in the interaction zones nor a significant main effect of genotype, but did reveal a significant main effect of both sides (F(1, 50) = 17.247, p < 0.001). Post hoc analyses revealed a significant main effect of preference for the unfamiliar intruder (Stranger 2) compared with the by-then familiar mouse (Stranger 1) for the combined group (p < 0.001) (Figure  3B). Two-way repeated measures ANOVA showed neither a significant genotype × chamber interaction for the number of entries nor a significant main effect of genotype, but did show a significant main effect of the chamber (F(1.669, 83.456) = 346.789, p < 0.001). Post hoc analyses revealed a significant main effect of preference for the center zone for the combined group (Stranger 1: p < 0.001, Stranger 2: p < 0.001), but the number of transitions into the chamber containing Stranger 1 did not differ from the number of transitions into the chamber containing Stranger 2.

Figure 3

Comparison of sociability and social novelty preference among L100P/L100P, L100P/+, and +/+ littermates. (A) Sociability: mean time spent near the empty cage (Empty) or the cage containing the unfamiliar mouse (Stranger 1) (left), and the number of entries into each chamber (right). (B) Mean time spent near the cage containing the first unfamiliar mouse (Stranger 1) or a new unfamiliar mouse (Stranger 2) (left), and the number of entries into each chamber (right). All values are expressed as mean ± SEM for each group. * p < 0.05, ** p < 0.01 or *** p < 0.001 versus time spent near the unfamiliar mouse (Stranger 1). ### p < 0.001 versus the number of entries into the center chamber.

SNP typing for genetic background of L100P/L100P mutant mouse line (Disc1 < Rgsc1390>)

Despite backcrosses, all the genotyped eight L100P/L100P homozygotes harbored DBA/2 J-derived SNP alleles, which were either homozygous or heterozygous variant (Additional file 1: Figure S1). Genome-wide SNP panel analysis revealed that the numbers of SNPs indicative of homozygosity or heterozygosity for DBA/2 J ancestry varied among individual mice. Each mouse harbored 1–5 homozygous and 8–12 heterozygous loci for DBA/2 J-derived SNP alleles (Additional file 2: Table S1).

Exome resequencing of the original G1 genome carrying L100P mutation

There was no single nucleotide mutation in the Disc1 exons other than L100P. In addition to the L100P mutation, exome resequencing analysis revealed 117 single nucleotide mutations in the exome and exome-flanking regions of the genomic DNA from the original G1 mouse, the F1 progenitor born to an ENU-treated G0 mouse (Table  1). Disc1 L100P and two other mutations (in Rab11fip1, known as Rab-coupling protein and Nr3c2) were found in chromosome 8. Other variants included synonymous coding variants (5), nonsynonymous coding (27 including ryr3, encoding the intracellular Ca2+ release channel ryanodine receptor 3 and Rab11fip1), knockout-equivalent nonsense (Als2 and Ttll1), essential splice site variants (3), 5′ UTR (3, including Dyx1c1, a susceptibility gene for dyslexia), 3′ UTR (9), and intronic mutations (49). Base substitution spectra of the 117 ENU-induced mutations were A/T to T/A transversions (26.5%), A/T to G/C transitions (39.3%), G/C to A/T transitions (18.8%), A/T to C/G transversions (6.8%), and G/C to T/A transversions (8.5%).

Table 1

List of ENU-induced mutations in G1 mouse of Disc1

Gene symbol	Entrez Gene ID	Chromosome	Position (MM9)	Base change	Spectrum	Classification	Amino acid substitution
Pkhd1	241035	1	20,530,613	T → A	AT to TA transversions	Intronic
Trak2	70827	1	58,975,614	A → G	AT to GC transitions	Essential splice site
Als2	74018	1	59,235,933	A → T	AT to TA transversions	Stop gained	Y- > X
Ngef	53972	1	89,379,202	A → T	AT to TA transversions	Nonsysnonymous	I- > N
2310035C23Rik	227446	1	107,592,127	A → G	AT to GC transitions	Intronic
Ikbke	56489	1	133,172,385	C → A	GC to TA transversions	Nonsysnonymous	A- > S
Cdc73	214498	1	145,542,523	A → T	AT to TA transversions	Nonsysnonymous	S- > T
Nmnat2	226518	1	154,936,083	A → T	AT to TA transversions	Intronic
Atf6	226641	1	172,724,783	G → A	GC to AT transitions	Intronic
Gm5706	435657	1	184,322,753	A → G	AT to GC transitions	Genic
Iars2	381314	1	187,127,260	A → T	AT to TA transversions	Nonsysnonymous	I- > N
Dhtkd1	209692	2	5,831,429	A → G	AT to GC transitions	Synonymous
Cacna1b	12287	2	24,506,396	A → G	AT to GC transitions	Intronic
Dolk	227697	2	30,140,520	A → G	AT to GC transitions	Synonymous
Lrp1b	94217	2	40,854,333	A → C	AT to CG transversions	Intronic
Neb	17996	2	52,114,402	C → A	GC to TA transversions	Nonsysnonymous	R- > L
Gm13559	674940	2	58,593,845	A → G	AT to GC transitions	Genic
Ttn	22138	2	76,553,805	C → T	GC to AT transitions	Nonsysnonymous	M- > I
Olfr1270	258987	2	89,989,253	T → C	AT to GC transitions	Synonymous
Ext2	14043	2	93,558,064	C → T	GC to AT transitions	Intronic
Ryr3	20192	2	112,701,983	C → A	GC to TA transversions	Nonsysnonymous	G- > W
Fmn1	14260	2	113,528,228	T → A	AT to TA transversions	Intronic
Sel1l2	228684	2	140,071,126	C → T	GC to AT transitions	Nonsysnonymous	A- > T
Setd7	73251	3	51,334,212	T → C	AT to GC transitions	Intronic
Ift80	68259	3	68,794,587	A → T	AT to TA transversions	Intronic
Asb17	66772	3	153,516,522	G → A	GC to AT transitions	3' UTR
		4	8,394,708	T → C	AT to GC transitions	Intergenic
Esrp1	207920	4	11,277,315	G → T	GC to TA transversions	Intronic
Gm136	214568	4	34,699,557	C → T	GC to AT transitions	Nonsysnonymous	A- > T
Nfx1	74164	4	40,952,108	T → A	AT to TA transversions	Intronic
Gm12608	664785	4	89,109,036	T → A	AT to TA transversions	Genic
Tek	21687	4	94,516,374	T → A	AT to TA transversions	Nonsysnonymous	S- > T
Dnajc6	72685	4	101,279,103	C → T	GC to AT transitions	Intronic
Txndc12	66073	4	108,533,802	T → A	AT to TA transversions	Intronic
Gm12901	194197	4	122,935,965	T → C	AT to GC transitions	Genic
Tmprss11e	243084	5	87,156,236	C → T	GC to AT transitions	Intronic
Ccng2	12452	5	93,697,763	T → C	AT to GC transitions	Nonsysnonymous	I- > T
Calcr	12311	6	3,650,125	A → G	AT to GC transitions	Essential splice site
C130060K24Rik	243407	6	65,406,380	A → G	AT to GC transitions	Nonsysnonymous	K- > E
Wnk1	232341	6	119,956,300	A → T	AT to TA transversions	Intronic
Vwf	22371	6	125,576,322	G → A	GC to AT transitions	Nonsysnonymous	M- > I
Klrk1	27007	6	129,566,794	G → A	GC to AT transitions	Intronic
Olfr1336	258917	7	6,413,462	A → T	AT to TA transversions	Nonsysnonymous	T- > S
Nlrp9a	233001	7	27,358,923	A → C	AT to CG transversions	Nonsysnonymous	E- > A
Arhgap33	233071	7	31,315,480	C → T	GC to AT transitions	Synonymous
Gucy2d	14918	7	105,600,256	A → T	AT to TA transversions	Exonic
		7	146,105,909	T → C	AT to GC transitions	Intergenic
Rab11fip1	75767	8	28,263,563	G → A	GC to AT transitions	Nonsysnonymous	P- > L
Nr3c2	110784	8	79,741,251	T → C	AT to GC transitions	Intronic
Disc1	244667	8	127,611,597	T → C	AT to GC transitions	Nonsysnonymous	L- > P
Prdm10	382066	9	31,142,994	T → C	AT to GC transitions	Intronic
Olfr909	100043200	9	38,331,305	T → C	AT to GC transitions	Upstream
Olfr44	258716	9	39,291,849	T → G	AT to CG transversions	3' UTR
Pou2f3	18988	9	42,984,368	C → T	GC to AT transitions	Essential splice site
Dyx1c1	67685	9	72,806,667	G → A	GC to AT transitions	5' UTR
Unc13c	208898	9	73,582,186	T → C	AT to GC transitions	Intronic
Senp6	215351	9	79,969,034	T → G	AT to CG transversions	Intronic
Cep63	28135	9	102,521,076	T → C	AT to GC transitions	Intronic
Heca	380629	10	17,635,364	T → C	AT to GC transitions	Nonsysnonymous	N- > S
Olig3	94222	10	19,077,712	A → G	AT to GC transitions	3' UTR
Sobp	109205	10	42,742,835	A → T	AT to TA transversions	Intronic
Serinc1	56442	10	57,245,307	A → G	AT to GC transitions	Intronic
Sf3a2	20222	10	80,263,982	A → G	AT to GC transitions	Nonsysnonymous	D- > G
Cyp27b1	13115	10	126,485,434	T → C	AT to GC transitions	Nonsysnonymous	V- > A
Tns3	319939	11	8,449,105	T → A	AT to TA transversions	5' UTR
Il5	16191	11	53,537,612	C → T	GC to AT transitions	3' UTR
4930404A10Rik	74847	11	54,185,249	T → A	AT to TA transversions	Nonsysnonymous	S- > R
Ulk2	29869	11	61,615,130	A → T	AT to TA transversions	Intronic
Slc13a5	237831	11	72,061,131	C → T	GC to AT transitions	Intronic
Ap2b1	71770	11	83,183,124	G → A	GC to AT transitions	Intronic
Lrrc46	69297	11	96,902,597	T → A	AT to TA transversions	5' UTR
Erbb2	13866	11	98,297,347	T → C	AT to GC transitions	Synonymous
Krt40	406221	11	99,401,666	A → G	AT to GC transitions	Intronic
Syngr2	20973	11	117,674,256	T → A	AT to TA transversions	Intronic
Gm9229	668539	12	18,193,251	C → A	GC to TA transversions	Genic
Mark3	17169	12	112,885,660	T → C	AT to GC transitions	Intronic
Igh	111507	12	115,853,922	T → A	AT to TA transversions	Genic
Tcrg-V4	21638	13	19,282,580	A → C	AT to CG transversions	Genic
Aoah	27052	13	21,003,081	A → T	AT to TA transversions	Intronic
Slc6a3	13162	13	73,682,459	T → C	AT to GC transitions	Intronic
Gtf2h2	23894	13	101,240,804	T → A	AT to TA transversions	Intronic
Ccdc125	76041	13	101,449,272	A → G	AT to GC transitions	Nonsysnonymous	D- > G
3425401B19Rik	100504518	14	33,476,075	A → G	AT to GC transitions	Nonsysnonymous	I- > T
Mcpt1	17224	14	56,637,381	G → T	GC to TA transversions	Intronic
Atp8a2	50769	14	60,266,756	A → G	AT to GC transitions	Nonsysnonymous	Y- > H
Sorbs3	20410	14	70,586,596	A → T	AT to TA transversions	Intronic
Epsti1	108670	14	78,397,889	G → T	GC to TA transversions	Intronic
Diap3	56419	14	87,384,824	G → T	GC to TA transversions	Intronic
Vps13b	666173	15	35,570,473	A → G	AT to GC transitions	Intronic
Ubr5	70790	15	37,898,017	A → G	AT to GC transitions	3' UTR
Micall1	27008	15	78,960,399	G → A	GC to AT transitions	Intronic
Cyp2d12	380997	15	82,387,610	A → T	AT to TA transversions	Intronic
Ttll1	319953	15	83,320,020	G → A	GC to AT transitions	Stop gained	R- > X
Prkag1	19082	15	98,643,692	A → G	AT to GC transitions	3' UTR
Gm7638	665450	16	11,185,895	C → T	GC to AT transitions	Genic
Myh11	17880	16	14,269,121	A → G	AT to GC transitions	Intronic
Gm6931	628903	16	49,425,860	T → A	AT to TA transversions	Genic
		16	80,727,008	A → C	AT to CG transversions	Intergenic
Olfr125	258287	17	37,972,895	T → C	AT to GC transitions	Nonsysnonymous	I- > T
Enpp5	83965	17	44,222,094	T → C	AT to GC transitions	Intronic
Gm7334	654432	17	50,838,499	T → C	AT to GC transitions	Genic
Sult1c1	20888	17	54,101,361	T → C	AT to GC transitions	3' UTR
2700099C18Rik	77022	17	95,163,195	C → T	GC to AT transitions	Genic
Fhod3	225288	18	25,248,741	A → G	AT to GC transitions	Nonsysnonymous	N- > S
Kdm3b	277250	18	34,982,966	T → G	AT to CG transversions	Intronic
Pcdhb6	93877	18	37,493,679	A → G	AT to GC transitions	Upstream
Pcdhga1	93709	18	37,912,242	C → T	GC to AT transitions	Intronic
Spink10	328971	18	62,820,759	G → T	GC to TA transversions	3' UTR
Cst6	73720	19	5,344,061	A → T	AT to TA transversions	Intronic
Ganab	14376	19	8,987,570	T → A	AT to TA transversions	Intronic
Cpsf7	269061	19	10,607,448	G → T	GC to TA transversions	Intronic
Ms4a5	269063	19	11,358,379	A → T	AT to TA transversions	Upstream
Ms4a14	383435	19	11,382,160	A → T	AT to TA transversions	Genic
Tjp2	21873	19	24,194,331	T → C	AT to GC transitions	Intronic
Cyp2c29	13095	19	39,405,010	T → C	AT to GC transitions	3' UTR
Tll2	24087	19	41,257,593	T → C	AT to GC transitions	Intronic
Sorcs3	66673	19	48,768,473	A → C	AT to CG transversions	Nonsysnonymous	I- > L

Discussion

The present study compared schizophrenia-related behavior among L100P mutant mice (L100P/L100P and L100P/+) and their wild-type littermates, and inbred C57BL/6 J mice by testing spontaneous locomotor activity, methamphetamine-induced locomotor activity in the open field [33], and sociability/social novelty preference in the social interaction [34]. All of behavior was comparable among L100P/L100P, L100P/+ and wild-type littermates; the results were partially inconsistent with previous studies using mice originated from the same G1 founder [24, 25]. To assess effects of genetic background (C57BL/6 J vs DBA/2 J), we conducted comprehensive genotyping of the original G1 mouse and its homozygous offspring obtained by backcrossing to C57BL/6 J, the same strain used for backcrossing in the previous report characterizing the L100P mutant [24]. Our genetic analyses demonstrated that both the L100P/L100P homozygous mice deposited by Roder’s group [24] into the EAD at RIKEN and their homozygous progeny still harbored a small number of SNPs inherited from the DBA/2 J strain (the mother of the G1 founder). Whole-exome resequencing of the original G1 genome also revealed an additional 116 single nucleotide variants induced by ENU.

Disc1 L100P was originally identified in one G1 progeny of several thousand screened for Disc1 mutants from a G1 male genomic DNA archive produced by breeding ENU-mutagenized C57BL/6 J males and untreated DBA/2JJcl females [24]. Live heterozygous mice carrying the L100P mutation were recovered by in vitro fertilization of C57BL/6 J eggs with the cryopreserved sperm of the corresponding G1 progeny. In the study reported by Clapcote et al. [24], mutant mice were then backcrossed to the C57BL/6 J background for at least six generations before intercrossing L100P/+ mice to generate homozygous mutants for behavioral tests. In theory, the backcrossed congenic mutant strain should have lost most of the other ENU-induced mutations and SNPs derived from DBA/2 J; however, the rate of SNP and mutant loss per generation has not been examined experimentally.

DBA/2 J and C57BL/6 J inbred strains were shown to exhibit differential sensitivity to psychostimulants [35, 36] without significant differences in basal locomotor activity or sociability [31]. Thus, residual DBA/2 J SNPs, if any still exist in the analyzed mice, could conceivably influence the behavioral test results. Genomic analysis revealed a significant number of DBA/2 J SNPs remaining in the backcrossed Disc1 L100P mice. Among 117 loci tested, nineteen were still polymorphic in the eight L100P/L100P mice deposited at RIKEN and their progeny (Additional file 2: Table S1). The average frequency of DBA/2 J SNP alleles in the eight L100P/L100P mice was 6.63% (5.56%–8.12%, Additional file 2: Table S1); a rate of genetic vestige was 4.25-fold higher than the theoretical estimate of 1.56% [24]. The question arises as to whether any single DBA/2 J SNP or combination influences Disc1 L100P locomotor activity in the open field compared with the congenic background strain. An important feature of the present study was the inclusion of commercially available inbred C57BL/6 J mice in addition to wild-type littermates derived from Disc1 L100P mutants to assess effects of genetic background on behavioral phenotype. This experimental design could enhance the sensitivity for detecting effects of genetic background and ENU-induced mutations. However, our behavioral analyses showed that neither basal nor methamphetamine-induced locomotor activity differed between wild-type mice derived from Disc1 L100P mutants and inbred C57BL/6 J mice. This finding suggests that locomotor activity is not measurably affected by residual DBA/2 J SNPs. Subsequently, using the exome resequencing analysis, we identified 116 previously unreported mutations in the exome of the original G1 male, although L100P was the only single mutation found in the coding sequence of the Disc1 gene. It has been estimated that approximately 64%, 26% and 10% of ENU-induced single base pair mutations in coding regions are missense, synonymous, and nonsense mutations, respectively [28, 37, 38]. Intriguingly, the frequencies of base substitutions in the G1 exome decreased in the rank order of A/T to G/C transition > AT/TA transversion > G/C to A/T transition > G/C to T/A transversion > A/T to C/G transversion, consistent with estimates for the gene-driven approach [39]. Furthermore, the distribution of single base pair mutations in the G1 exome predicted to generate amino acid variants was approximately consistent with previous studies [37, 38].

Locomotor activity in the open field did not differ between L100P mutant mice (homozygotes and heterozygotes) and wild-type littermates, consistent with the report by Shoji et al. [27]. In contrast, Clapcote et al. [24] and Lipina et al. [25] reported higher locomotor activity in L100P/L100P mice than in wild-type littermates during the first 30 min, after introduction into the open field (but not thereafter). The higher locomotor activity of L100P mice may represent slower habituation to a novel environment. In addition, although Lipina et al. [25] reported higher locomotor activity in L100P mice following injection of amphetamine compared with that of wild-type littermates, we did not observe enhanced locomotion after an acute challenge with methamphetamine. Although the reasons for this discrepancy with previous reports remains to be explored, one possibility is that critical mutation(s) other than L100P may have been missed during maintenance of the strain after submission to the RIKEN EAD and/or the single backcrossing of homozygous (L100P/L100P) mutant males obtained from RIKEN to female inbred C57BL/6 J mice. As shown by the distribution of SNPs, the genetic background in the deposited L100P homozygotes was heterozygous; therefore, such heterogeneity may be fixed to either allele in the offspring, even after many generations of maintenance.

The enhanced release of amphetamine-evoked dopamine consistently reported in neuroimaging studies of schizophrenia [40-42] may be of relevance to this model of schizophrenia. However, Lipina et al. [25] did not find greater striatal release of dopamine following amphetamine injection in L100P mice. In the adult brain, Disc1 is expressed in the hippocampus and not in the nucleus accumbens [43, 44]. The hyperactivity of subcortical dopaminergic neurons is believed to be conveyed by glutamatergic afferents from the hippocampus to the nucleus accumbens, which in turn regulates release of dopamine and the activity of dopaminergic neurons in the ventral tegmental area [45, 46]. Another possible mechanism relates to disruption of the interaction between the hippocampus and prefrontal cortex [47]. Although subtle cytoarchitectonic changes have been reported in frontal cortical neurons [48], Disc1 L100P mice lack the reduced number of parvalbumin-positive interneurons observed in Disc1 transgenic models [12, 18].

Sociability and social novelty preference tests used in the present study are used frequently to investigate disrupted social interactions in mouse models of schizophrenia [34]. The impoverished social interaction in Disc1 mice is believed to be a relevant quantitative trait of social withdrawal in schizophrenia [14]. Consistent with Clapcote et al. [24], we found that the three L100P genotypes did not differ in sociability or social novelty preference. Intriguingly, sociability was markedly impaired in conditional transgenic lines with either constitutive expression of [14] or inducible expression of a mutant DISC1 C-terminal fragment during the early postnatal period [15]. We hypothesize that transgenic mice overexpressing human truncated DISC1 protein exhibit a dominant-negative effect that may lead to diminished binding between the DISC1 interacting proteins and relevant domains of DISC1 protein, thus altering the behavioral phenotype [15]. Based on these results, the relevance of Disc1 L100P as a mouse model of schizophrenia should be re-evaluated.

A recent review stressed that ENU-mutagenized mice are useful for establishing novel models of complex human diseases, including neuropsychiatric diseases [49]. A range of ENU-generated mutations based on a phenotype-driven approach has an advantage over a complete loss-of-function mutation in recapturing the pleiotropic nature of human neuropsychiatric diseases and the subtlety of manifestation [49]. Thus, both disease-causative and other unexpected variants should be made publicly available [28, 29]. This phenotype-driven approach requires high-throughput behavioral screening to link mutation with function. One promising candidate screening methodology is the identification of outliers by neuroimaging. For example, only a few studies on developmental mouse models of psychiatric illness, including those focusing on DN-DISC1 models, have highlighted enlargement of the lateral ventricle without a marked change in overall brain size [16–18, 50]. Phenotype screening based on standardized behavioral tests [31, 51] combined with neuroimaging [50] to detect enlargement of the lateral ventricle may be an efficacious approach for the identification of robust schizophrenia models.

Several limitations of this study should be considered. First, we did not conduct a mouse comprehensive battery of behavioral tests including cognitive tasks and PPI but only tests of locomotor activity in a novel environment, psychostimulant-induced behavior and sociability in the three-chamber test. Therefore, our results do not clarify effects of the L100P amino acid substitution in exon 2 on other behavioral phenotypes related to psychiatric disorders including schizophrenia. Second, handling, experimental protocols, rearing environment and other environmental factors may influence behavioral test results and contribute to variability across studies [51, 52]. Third, L100P mice used in the current study were not genotyped for all ENU-induced mutations found in the exome and vicinity of the G1 genome, some of which may have been transmitted to progeny despite numerous backcrosses [24]. Given that 117 ENU-induced mutations in the G1 mouse identified with the whole-exome resequencing, it is plausible that 50-fold excess or more than 5,000 of ENU-induced mutations should exist in the entire G1 genome. Although detailed genetic information on models relevant to human disease should be freely available, routine implementation of whole-genome sequencing awaits measures for improved cost-effectiveness. However, as most of the ENU-induced mutations in G1 map to chromosomes other than chromosome 8 (containing Disc1), it is likely that they are transmitted to progeny to some extent regardless of the breeding process used to select mice harboring Disc1 100P. Although it may not be possible to investigate whether genetic background or other ENU-induced mutations have any effects on behavior because of a generalised lack of information on genotype differences among any of their mouse lines or offspring, further behavioral and genetic studies are warranted. For example, generation and breeding of another mouse line harboring Disc1 L100P, which can be accomplished by in vitro fertilization by the cryopreserved sperm of the G1 progeny owned by RIKEN BRC, may facilitate full elucidation of a relationship between genetic components, including Disc1 L100P mutation per se, and behavioral phenotypes, related with schizophrenia in this mouse line.

Conclusion

The present study using behavioral genetic approaches provides an insight into the role of Disc1 L100P and other single nucleotide variants in behavioral phenotypes associated with psychiatric disorders such as schizophrenia. Our present findings suggest that causal attribution of the discrepancy in behavioral phenotypes of the Disc1 L100P mutant mouse line existing among different research groups, including our own, needs to be cautiously investigated in further studies by taking into account the effect(s) of other ENU-induced mutations and/or SNPs from DBA/2 J. Further behavioral genetic analyses are needed to elucidate the cause of behavioral variance associated with Disc1 L100P strain. Using a polygenic animal model including ENU mutagenesis provides an efficacious approach to explore the relationship of variants to behavioral phenotypes associated with polygenic and multifactorial disorders such as psychiatric disorders.

Additional file 1: Figure S1: Pedigree of Disc1 < Rgsc1390 > on RIKEN BRC. (PDF 29 KB)

Additional file 2: Table S1: Characteristics of mouse strain SNPs in Disc1. (PDF 116 KB)