Open Access

Deep exon resequencing of DLGAP2 as a candidate gene of autism spectrum disorders

  • Wei-Hsien Chien1,
  • SusanShur-Fen Gau2, 3Email author,
  • Hsiao-Mei Liao2,
  • Yen-Nan Chiu2,
  • Yu-Yu Wu4,
  • Yu-Shu Huang4,
  • Wen-Che Tsai2,
  • Ho-Min Tsai5 and
  • Chia-Hsiang Chen2, 4, 5Email author
Molecular Autism20134:26

https://doi.org/10.1186/2040-2392-4-26

Received: 16 January 2013

Accepted: 26 June 2013

Published: 1 August 2013

Abstract

Background

We recently reported a terminal deletion of approximately 2.4 Mb at chromosome 8p23.2-pter in a boy with autism. The deleted region contained the DLGAP2 gene that encodes the neuronal post-synaptic density protein, discs, large (Drosophila) homolog-associated protein 2. The study aimed to investigate whether DLGAP2 is genetically associated with autism spectrum disorders (ASD) in general.

Methods

We re-sequenced all the exons of DLGPA2 in 515 patients with ASD and 596 control subjects from Taiwan. We also conducted bioinformatic analysis and family study of variants identified in this study.

Results

We detected nine common single nucleotide polymorphisms (SNPs) and sixteen novel missense rare variants in this sample. We found that AA homozygotes of rs2906569 (minor allele G, alternate allele A) at intron 1 (P = 0.003) and CC homozygotes of rs2301963 (minor allele A, alternate allele C) at exon 3 (P = 0.0003) were significantly over-represented in the patient group compared to the controls. We also found no differences in the combined frequency of rare missense variants between the two groups. Some of these rare variants were predicted to have an impact on the function of DLGAP2 using informatics analysis, and the family study revealed most of the rare missense mutations in patients were inherited from their unaffected parents.

Conclusions

We detected some common and rare genetic variants of DLGAP2 that might have implication in the pathogenesis of ASD, but they alone may not be sufficient to lead to clinical phenotypes. We suggest that further genetic or environmental factors in affected patients may be present and determine the clinical manifestations.

Trial registration

ClinicalTrial.gov, NCT00494754

Keywords

Autism spectrum disorders DLGAP2 Common variants Rare variants

Background

Autism spectrum disorders (ASD) are a group of childhood-onset neurodevelopmental disorders characterized by impaired verbal/nonverbal communication, abnormal reciprocal social interaction, and the presence of stereotyped behaviors and restricted interests. Due to increased awareness and clinical sensitivity to ASD, broadening of the diagnostic criteria of ASD by including Asperger’s disorder and Pervasive Developmental Disorder, not otherwise specified in addition to autistic disorder, and other contributing factors, the prevalence of ASD has increased markedly in the past decade. Recent data showed that up to approximately 11 persons per thousand in the USA are affected with ASD, and males are more predominantly affected than females [13]. The literature documents strong evidence of a high degree of genetic influence in the etiology of autism with high heritability estimated to be more than 90% [4]. Genetic approaches such as cytogenetic analysis, genome-wide linkage and association scans, and candidate gene analysis, have been used to dissect the genetic complexity of ASD [5].

Traditional cytogenetic studies [69] and the recent array-based comparative genomic hybridization (array CGH) analysis have shown that chromosome abnormalities and rare copy number variants [10, 11] are associated with ASD. Various chromosome abnormalities such as deletion, duplication, inversion, and translocation were identified in ASD patients. In particular, the advent of array CGH technology has greatly facilitated the detection of formerly undetectable submicroscopic copy number variants that are associated with ASD [1014].

Our group previously identified two novel chromosome deletions in ASD using karyotyping analysis and array CGH [15]. One was a terminal deletion of approximately 2.4 Mb at 8p23.2-pter detected in a male patient with autistic disorder [15]. Several genes with neurobiological functions such as discs, large (Drosophila) homolog-associated protein 2 (DLGAP2), ceroid-lipofuscinosis, neuronal 8 (CLN8), the Rho guanine nucleotide exchange factor 10 (ARHGEF10) and F-box protein 25 (FBXO25), were mapped to this region. It is likely that haploinsufficiency of one or several of these genes might result in the clinical phenotypes of the affected patients. Hence, these genes might be considered as candidate genes of ASD patients in general.

DLGAP2 (GeneID 9298) encodes the discs, large (Drosophila) homolog-associated protein 2, which is also called PSD-95/SAP90-binding protein 2 and SAP90/PSD-95-associated protein 2 (SAPAP2). The protein is one of the membrane-associated guanylate kinases localized at the post-synaptic density that plays a role in the molecular organization of synapses and in neuronal cell signaling [16]. These kinases are a family of signaling molecules expressed at various submembrane areas, and contain the PDZ, SH3 and the guanylate kinase domains. Several studies have suggested that the synapse associated proteins (SAPs) localized at postsynaptic density are involved in the pathophysiology of psychiatric disorders [1721]. Marshall et al. detected 13 loci with recurrent CNV in autism cases; they found that several postsynaptic density genes and synapse complex genes were mapped in these CNVs [22]. They also found that a genomic DNA duplication intersected the DLGAP2 gene in a patient with autism [22]. In addition, Ozgen et al. reported a classical inv dup del(8p) in a female patient with autism whose DLGAP2 gene was located within the 6.9 Mb terminal deletion [23]. Together with the finding in our previous study, these studies suggest that the DLGAP2 gene is an important candidate gene of ASD. To test this hypothesis, we conducted a deep resequencing of all the exons of the DLGAP2 in a sample of ASD from Taiwan. Herein, we present our findings of the genetic analysis of DLGAP2 in this report.

Methods

Subjects and procedures

All subjects were Han Chinese from Taiwan. Patients meeting the diagnostic criteria of either autistic disorder or Asperger’s disorder according to the DSM-IV and ICD-10 were enrolled in this study from the psychiatry department of a university hospital, a private medical center, and schools and early intervention centers in northern Taiwan. Subjects with ASD aged 3 to 25 years old, and with a clinical diagnosis of ASD confirmed by the structured interview using the Chinese version of the Autism Diagnostic Interview-Revised (ADI-R) [24, 25]. They received clinical assessments and provided DNA data at the university hospital, no matter whether they were recruited from this university hospital or referred by other places. Patients with known chromosomal abnormalities and associated medical conditions were excluded from the study. Subjects receiving routine medical check-ups at the Department of Family Medicine of a medical center were recruited as controls. The mental status of the control subjects was screened by a senior psychiatrist. Individuals with major psychiatric disorders including ASD were excluded.

The Research Ethics Committee of the research sites approved this study. The patients and their parents were informed that participation in this study was completely voluntary and that nonparticipation would not influence their treatment. Written informed consent was obtained from the patients if they were able to understand the contents of the study, the parents of all the patients, and all the control subjects after the procedures were fully explained. A total of 515 patients with ASD were recruited into this study, including 449 male patients (mean age ± standard deviation (SD) = 8.9 ± 4.6 years) and 66 female patients (mean age ± SD = 8.5 ± 4.4 years). The control group comprised 596 individuals including 263 males (mean age ± SD = 42.5 ± 15.1 years) and 333 females (mean age ± SD = 45.2 ± 13.4 years).

The ADI-R data of the 515 patients revealed the scores as 21.19 ± 5.78 in the ‘qualitative abnormalities in reciprocal social interaction’ (cut-off = 10), 15.35 ± 4.19 in the ‘qualitative abnormalities in communication, verbal’ (cut-off = 8), 8.58 ± 3.28 in the ‘qualitative abnormalities in communication, nonverbal’ (cut-off = 7), and 7.14 ± 2.42 in the ‘restricted, repetitive and stereotyped patterns of behaviors’ (cut-off = 3). All patients with ASD were noted to have had abnormal development at or before 36 months.

Genomic DNA was prepared from peripheral blood using the Puregene DNA purification system (Gentra Systems Inc. Minneapolis, MI, USA) according to the manufacturer’s instructions.

PCR amplification and sequencing

The genomic sequences of human DLGAP2 are available from the NCBI Reference Sequence: NM_004745.3. The human DLGAP2 comprises twelve exons that span approximately 207 kb on chromosome 8p23.2-23.3 [16]; the schematic genomic structure of the DLGAP2 is shown in Figure 1. Optimal PCR primer sequences were generated to amplify each exon of the DLGAP2 using Primer3 (http://bioinfo.ut.ee/primer3/). All the primer sequences, optimal annealing temperatures and size of each amplicon are listed in Additional file 1: Table S1. After PCR, aliquots of PCR products were processed using a PCR Pre-Sequencing Kit (USB, Cleveland, OH, USA) to remove residual primers and dNTPs following the manufacturer’s protocol. The purified PCR products were subjected to direct sequencing using the ABI Prism™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit Version 3.1, and the ABI autosequencer 3730 (Perkin Elmer Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s protocol. The quality of the sequencing results was visualized using Chromas 2.4.1 software (Technelysium Pty Ltd, South Brisbane, Australia). For variant identification, sequencing results of each subject were aligned and compared with the reference sequences using BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html). To verify the authenticity of mutations identified in this study, repeated PCR from genomic DNA and re-sequencing of the amplicon in both directions were performed. The nomenclature of genetic variants follows the rules of the ‘Nomenclature for description of human sequence variations’ [26].
Figure 1

(A) Schematic genomic structure of the DLGAP2 , and locations of the common single nucleotide polymorphisms (SNPs) and rare variants identified in this study. (B) Linkage disequlibrium (LD) analysis of nine common SNPs identified in this study.

Statistical analysis

For the common single nucleotide polymorphisms (SNPs), the differences in the allele and genotype frequencies between the patients and controls were assessed with the Chi-square test. Fisher's exact test was used to compare the combined frequency of rare variants between the patient and the control groups. It was also used to compare the frequency of damaging and functional rare missense variants between the ASD and control groups. Assessment of haplotype-based association analyses was performed using the SHEsis computer program [27]. A P value of less than 0.05 was considered statistically significant, and Bonferroni correction for multiple comparisons was performed when appropriate. We also calculated the Q value for each test with the false discovery test of 5% using QVALUE software (http://genomics.princeton.edu/storeylab/qvalue/) [28].

Results

After sequencing all the exons of DLGAP2 in 515 patients and 596 control subjects, we identified nine common SNPs (defined as with a minor allele frequency > 0.05), and a total of 49 rare variants (defined as with a frequency < 0.05) in this sample. The locations of these variants are listed in Figure 1.

Case-control association study of common SNPs

The genotype and allele frequencies of nine common variants in the patients and control subjects are listed in Table 1. Two SNPs (rs2906569 and rs2301963) were found to have significant differences in genotype frequency distribution between the patient and control groups, even after correction for multiple comparisons. The rs2906569 (A > G) was located at intron 1. The genotype AA homozygotes (G: minor allele, A: alternate allele) were significantly over-represented in the patient group compared to the control group (odds ratio: 1.46; 95% confidence interval, 1.13 to 1.87, P = 0.003) (Table 2). When the subjects were sub-grouped by gender, the over-representation of genotype AA homozygotes was still observed in male patients but not in female patients (Table 2). The rs2301963 (C > A) was a missense variant (P384Q) located at exon 3 (A: minor allele, C: alternate allele). The CC homozygotes were significantly over-represented in the patient group compared to the control group (odds ratio: 1.30; 95% confidence interval, 0.99 to 1.70; P = 0.0003) (Table 2). When the subjects were sub-grouped by gender, the over-representation of CC homozygotes was observed in male patients but not in female patients (Table 2).
Table 1

Allele and genotype frequencies of common SNPs of the DLGAP2 gene in ASD patients and control subjects (MAF > 5%)

SNP

Location

 

Diagnosis

n

Genotype

P value

Q value

Allele

P value

Q value

Odds ratio (95 % CI)

rs6996621

intron 1

   

C/C

C/A

A/A

  

C

A

  

C versus A

c.-68-61C > A

 

total

autism

454

387 (85.2%)

62 (13.7%)

5 (1.1%)

0.619

0.312

836 (92.1%)

72 (7.9%)

0.401

0.204

1.11 (0.83 to 1.57)

   

control

557

467 (83.8%)

80 (14.4%)

10 (1.8%)

  

1014 (91.0%)

100 (9.0%)

   
  

male

autism

394

336 (85.3%)

55 (14.0%)

3 (0.7%)

0.593

0.203

727 (92.3%)

61 (7.7%)

0.611

0.611

 
   

control

246

208 (84.6%)

34 (13.8%)

4 (1.6%)

  

450 (91.5%)

42 (8.5%)

   
  

female

autism

60

51 (85%)

7 (11.7%)

2 (3.3%)

0.663

0.266

109 (90.8%)

11 (9.2%)

0.956

0.956

 
   

control

311

259 (83.3%)

46 (14.8%)

6 (1.9%)

  

564 (90.7%)

58 (9.3%)

   

rs2906568

intron 1

   

C/C

C/G

G/G

  

C

G

  

C versus G

c.-68-25C > G

 

total

autism

456

188 (41.2%)

169 (37.1%)

99 (21.7%)

0.142

0.112

545 (59.8%)

367 (40.2%)

0.125

0.105

1.15 (0.96 to 1.37)

   

control

557

197 (35.4%)

234 (42.0%)

126 (22.6%)

  

628 (56.4%)

486 (43.6%)

   
  

male

autism

396

171 (43.2%)

138 (34.8%)

87 (22.0%)

0.072

0.107

480 (60.6%)

312 (39.4%)

0.146

0.299

 
   

control

246

86 (35.0%)

106 (43.0%)

54(22.0%)

  

278 (56.5%)

214 (43.5%)

   
  

female

autism

60

17 (28.3%)

31 (51.7%)

12 (20%)

0.316

0.266

65 (54.2%)

55 (45.8%)

0.671

0.897

 
   

control

311

111 (35.7%)

128 (41.1%)

72 (23.2%)

  

350 (56.3%)

272 (43.7%)

   

rs2906569

intron 1

   

A/A

A/G

G/G

  

A

G

  

A versus G

c.-68-4A > G

 

total

autism

458

219 (47.8%)

138 (30.1%)

101 (22.1%)

0.0006

0.095

576 (62.9%)

340 (37.1%)

0.112

0.105

1.16 (0.97 to 1.38)

   

control

557

215 (38.6%)

232 (41.7%)

110 (19.7%)

  

662 (59.4%)

452 (40.6%)

   
  

male

autism

398

199 (50.0%)

109 (27.4%)

90 (22.6%)

0.0001

0.107

507 (63.7%)

289 (36.3%)

0.075

0.299

 
   

control

246

91 (37.0%)

107 (43.5%)

48 (19.5%)

  

289 (58.7%)

203 (41.3%)

   
  

female

autism

60

20 (33.3%)

29 (48.3%)

11 (18.4%)

0.491

0.266

69 (57.5%)

51 (42.5%)

0.614

0.897

 
   

control

311

124 (39.9%)

125 (40.2%)

62 (19.9%)

  

373 (60.0%)

249 (40.0%)

   

rs60089073

exon 2

   

C/C

C/T

T/T

  

C

T

  

C versus T

His73

 

total

autism

466

401 (86.1%)

54 (11.6%)

11 (2.3%)

0.989

0.388

856 (91.8%)

76 (8.2%)

0.891

0.363

0.98 (0.71 to 1.35)

   

control

557

481 (86.4%)

63 (11.3%)

13 (2.3%)

  

1025 (92.0%)

89 (8.0%)

   
  

male

autism

404

346 (85.6%)

48 (11.9%)

10 (2.5%)

0.236

0.133

740 (91.6%)

68 (8.4%)

0.093

0.299

 
   

control

246

219 (89.0%)

25 (10.2%)

2 (0.8%)

  

463 (94.1%)

29 (5.9%)

   
  

female

autism

62

55 (88.7%)

6 (9.7%)

1 (1.6%)

0.607

0.266

116 (93.5%)

8 (6.5%)

0.259

0.897

 
   

control

311

262 (84.3%)

38 (12.2%)

11 (3.5%)

  

562 (90.4%)

60 (9.6%)

   

rs2301963

exon 3

   

C/C

C/A

A/A

  

C

A

  

C versus A

Pro384Gln

 

total

autism

513

171 (33.3%)

231 (45.1%)

111 (21.6%)

0.0005

0.095

573 (55.8%)

453 (44.2%)

0.026

0.105

1.21 (1.02 to 1.43)

   

control

593

139 (23.4%)

328 (55.%)

126 (21.2%)

  

606 (51.1%)

580 (48.9%)

   
  

male

autism

448

150 (33.5%)

202 (45.1%)

96 (21.4%)

0.068

0.107

502 (56.0%)

394 (44.0%)

0.102

0.299

 
   

control

260

66 (25.4%)

136 (52.3%)

58 (22.3%)

  

268 (51.5%)

252 (48.5%)

   
  

female

autism

65

21 (32.3%)

29 (44.6%)

15 (23.1%)

0.124

0.266

71 (54.6%)

59 (45.4%)

0.420

0.897

 
   

control

333

73 (21.9%)

192 (57.7%)

68 (20.4%)

  

338 (50.8%)

328 (49.2%)

   

rs6995760

intron 5

   

A/A

A/G

G/G

  

A

G

  

A versus G

c.1570 + 14A > G

 

total

autism

500

346 (69.2%)

141 (28.2%)

13 (2.6%)

0.913

0.388

833 (83.3%)

167 (16.7%)

0.925

0.363

1.01 (0.80 to 1.27)

   

control

531

368 (69.3%)

147 (27.7%)

16 (3.0%)

  

883 (83.1%)

179 (16.9%)

   
  

male

autism

432

305 (70.6%)

115 (26.6%)

12 (2.8%)

0.502

0.193

725 (83.9%)

139 (16.1%)

0.245

0.299

 
   

control

234

155 (66.3%)

71 (30.3%)

8 (3.4%)

  

381 (81.4%)

87 (18.6%)

   
  

female

autism

68

41 (60.3%)

26 (38.2%)

1 (1.5%)

0.103

0.266

108 (79.4%)

28 (20.6%)

0.148

0.897

 
   

control

297

213 (71.7%)

76 (25.6%)

8 (2.7%)

  

502 (84.5%)

92 (15.5%)

   

rs2235112

exon 6

   

A/A

A/G

G/G

  

A

G

  

A versus G

Glu598

 

total

autism

509

95 (18.8%)

233 (45.7%)

181 (35.5%)

0.028

0.095

424 (41.6%)

595 (58.4%)

0.012

0.105

1.29 (1.05 to 1.50)

   

control

513

66 (12.9%)

239 (46.6%)

208 (40.5%)

  

371 (36.2%)

655 (63.8%)

   
  

male

autism

444

85 (19.1%)

205 (46.2%)

154 (34.7%)

0.049

0.107

375 (42.2%)

513 (57.8%)

0.014

0.299

 
   

control

224

28 (12.5%)

102 (45.5%)

94 (42.0%)

  

158 (35.3%)

290 (64.7%)

   
  

female

autism

66

11 (16.7%)

28 (42.4%)

27 (40.9%)

0.671

0.266

50 (37.9%)

82 (62.1%)

0.825

0.928

 
   

control

289

38 (13.2%)

137 (47.4%)

114 (39.4%)

  

213 (36.9%)

365 (63.1%)

   

rs2235113

intron 6

   

C/C

C/G

G/G

  

C

G

  

C versus G

c.1920 + 19C > G

 

total

autism

510

156 (30.6%)

233 (45.7%)

121 (23.7%)

0.050

0.095

545 (53.4%)

475 (46.6%)

0.030

0.105

1.21 (1.02 to 1.44)

   

control

509

121 (23.8%)

253 (49.7%)

135 (26.5%)

  

495 (48.6%)

523 (51.4%)

   
  

male

autism

443

137 (30.9%)

205 (46.3%)

101 (22.8%)

0.303

0.139

479 (54.1%)

407 (45.9%)

0.243

0.299

 
   

control

222

56 (25.2%)

113 (50.9%)

53 (23.9%)

  

225 (50.7%)

219 (49.3%)

   
  

female

autism

67

19 (28.3%)

28 (41.8%)

20 (29.9%)

0.513

0.266

66 (49.3%)

68 (50.7%)

0.644

0.897

 
   

control

287

65 (22.6%)

140 (48.8%)

82 (28.6%)

  

270 (47.0%)

304 (53.0%)

   

rs2293909

intron 11

   

T/T

T/C

C/C

  

T

C

  

T versus C

c.2709 + 71T > C

 

total

autism

460

255 (55.4%)

166 (36.1%)

39 (8.5%)

0.012

0.095

676 (73.5%)

244 (26.5%)

0.012

0.105

1.28 (1.06 to 1.55)

   

control

573

265 (46.2%)

254 (44.3%)

54 (9.4%)

  

784 (68.4%)

362 (31.6%)

   
  

male

autism

403

230 (57.1%)

138 (34.2%)

35 (8.7%)

0.117

0.107

598 (74.2%)

208 (25.8%)

0.165

0.299

 
   

control

256

127 (49.6%)

108 (42.2%)

21 (8.2%)

  

362 (70.7%)

150 (29.3%)

   
  

female

autism

57

25 (43.9%)

28 (49.1%)

4 (7.0%)

0.719

0.266

78 (68.4%)

36 (31.6%)

0.698

0.897

 
   

control

317

138 (43.5%)

146 (46.1%)

33 (10.4%)

  

422 (66.6%)

212 (33.4%)

   

ASD, autistic spectrum disorders; MAF, minor allele frequency; SNPs, single nucleotide polymorphisms.

Table 2

Odds ratio of rs2906569 and rs2301963 of the DLGAP2 gene in patients with autism and control subjects

SNP

Location

 

Diagnosis

n

Odds ratio (95% CI)

P value

rs2906569

intron 1

total

  

A/A versus A/G + G/G

 
  

autism

458

1.46 (1.13 to1.87)

0.003a

  

control

557

  
  

male

autism

398

1.70 (1.23 to 2.36)

0.001a

   

control

246

  
  

female

autism

60

0.75 (0.42 to 1.35)

0.341

   

control

311

  

rs2301963

exon 3

total

  

C/C versus C/A + A/A

P value

  

autism

513

1.63 (1.25 to 2.13)

0.0003a

  

control

593

  
  

male

autism

448

1.48 (1.05 to 2.08)

0.024a

   

control

260

  
  

female

autism

65

1.70 (0.95 to 3.04)

0.071

   

control

333

  

aP value < 0.05; SNP, single nucleotide polymorphism.

Further linkage disequilibrium (LD) analysis showed strong LD among rs6996621, rs2906568, rs2906569, and rs60089073. In addition, rs2235112 and rs2235113 also showed strong LD (Figure 1). In haplotype-based association analysis derived from nine SNPs, we found significant difference in the haplotype distribution of ACACAAGGT and CCACCAACT between the ASD patients and controls, but only haplotype CCACCAACT was sustained after correction for multiple comparisons (Table 3).
Table 3

The distributions of haplotypes in ASD and controls

 

Haplotype

Cases (frequency)

Controls (frequency)

Chi2

Fisher's P

Pearson's P

Odds ratio (95%CI)

1

A C A C A A G G T

8.84 (0.012)

28.20 (0.036)

7.081

0.007814a

0.007806a

0.367 (0.170 to 0.790)

2

C C A C C A A C C

41.17 (0.057)

63.13 (0.080)

1.548

0.213443

0.213365

0.768 (0.506 to 1.165)

3

C C A C C A A C T

84.15 (0.117)

60.19 (0.076)

11.981

0.000542a

0.000541a

1.880 (1.310 to 2.698)

4

C C A C C A G G C

12.99 (0.018)

26.04 (0.033)

2.331

0.126865

0.126793

0.592 (0.300 to 1.168)

5

C C A C C A G G T

86.66 (0.121)

89.80 (0.114)

1.380

0.240147

0.240071

1.218 (0.876 to 1.694)

6

C C A T C A A C T

27.74 (0.039)

25.95 (0.033)

0.973

0.324045

0.323985

1.319 (0.759 to 2.292)

7

C G G C A A A C T

31.96 (0.045)

32.97 (0.042)

0.465

0.495147

0.495131

1.192 (0.719 to 1.976)

8

C G G C A A G G C

30.14 (0.042)

45.29 (0.057)

0.902

0.342270

0.342214

0.792 (0.490 to 1.282)

9

C G G C A A G G T

60.91 (0.085)

81.38 (0.103)

0.381

0.537097

0.537090

0.892 (0.622 to 1.281)

10

C G G C A G G C T

19.59 (0.027)

37.66 (0.048)

2.960

0.085418

0.085359

0.613 (0.349 to 1.076)

ASD, autistic spectrum disorders; Global result: total control = 788.0, total case = 718.0; global chi2 is 26.804893 while df = 9 (frequency < 0.03 in both control and case has been dropped). Fisher's P value is 0.001537. Pearson's P value is 0.001507. Note: aP ≤ value 0.05.

Identification of rare genetic variants of DLGAP2

In this study, we found a total of 16 rare missense mutations in our ASD patients and control subjects. The locations of these variants are illustrated in Figure 1. These missense variants are novel and have not been reported before in the literature. Distributions of these rare missense variants are listed in Table 4. There was no significant difference in the combined frequency of rare missense mutations between the two groups (P = 1.00). Those individuals who carried these rare missense mutations had only one missense mutation; we found no individual who carried two rare missense variants simultaneously.
Table 4

Unique nonsynonymous variants of the DLGAP2 gene identified in ASD patients and controls and their functional predictions

Location

  

In silico analysis

    
 

Nucleotide position

Variants

PolyPhen-2

SIFT

Autism

Control

rs2906569

rs2301963

exon2

c.44 C > T

S15F

probably damaging

affect protein function

U1974

 

A/A

C/A

c.277 C > A

R93S

probably damaging

tolerated

U1843

 

A/G

C/A

c.545 G > A

R182Q

probably damaging

tolerated

U173

 

A/G

C/A

c.574 G > T

A192S

benign

tolerated

U396

 

A/A

C/C

c.797 G > T

V234L

benign

tolerated

 

ZN4215

G/G

C/C

c.797 G > T

V234L

benign

tolerated

 

HN616

A/A

C/A

c.841 C > G

P281A

probably damaging

tolerated

U323

 

A/G

C/A

c.841 C > G

P281A

probably damaging

tolerated

U1519

 

G/G

A/A

c.841 C > G

P281A

probably damaging

tolerated

U1988

 

G/G

C/A

c.841 C > G

P281A

probably damaging

tolerated

 

ZN4014

A/G

missing

c.841 C > G

P281A

probably damaging

tolerated

 

HN449

A/G

C/A

c.841 C > G

P281A

probably damaging

tolerated

 

HN581

A/G

C/A

c.970 A > T

R324W

probably damaging

affect protein function

U1803

 

A/A

C/A

exon4

c.1262 C > T

A421V

benign

tolerated

 

ZN4205

G/G

A/A

exon5

c.1516 T > C

C506R

probably damaging

affect protein function

U1247

 

missing

C/A

exon9

c.2135 C > T

T712M

benign

tolerated

U2096

 

G/G

C/A

 

c.2135 C > T

T712M

benign

tolerated

 

HN576

missing

C/A

 

c.2135 C > T

T712M

benign

tolerated

 

HN278

A/A

C/A

 

c.2311 C > G

P771A

benign

tolerated

 

ZN4182

A/A

C/C

 

c.2386 C > G

H796D

probably damaging

affect protein function

 

ZN4053

A/G

C/A

 

c.2392 G > C

E798Q

probably damaging

affect protein function

U1082

 

A/A

C/C

exon11

c.2650 G > A

D884N

benign

tolerated

 

HN526

G/G

C/A

 

c.2676 C > A

N892K

possibly damaging

affect protein function

 

ZN4107

missing

missing

exon12

c.2750 C > T

P917L

benign

tolerated

U1000

 

A/G

C/A

 

c.2750 C > T

P917L

benign (0.002)

tolerated

U2098

 

G/G

C/A

 

c.2750 C > T

P917L

benign (0.002)

tolerated

 

HN444

A/A

C/C

PolyPhen-2

autism

control

P (Fisher’s test)

SIFT

autism

control

P (Fisher’s test)

 

damaging

9

5

0.12

functional

4

2

0.32

 

benign

4

8

 

tolerated

9

11

  

Family study and functional prediction of rare variants

A total of 10 missense variants (S15F, R93S, R182Q, A192S, P281A, R324W, C506R, T712M, E798Q and P917L) were detected in 13 patients (Table 4). All the patients carrying these rare variants were heterozygotes. Four of the 13 patients (A, B, J, K) inherited the variant from their mothers and 6 (C, D, E, F, G, H) from their fathers (Figure 2). Three patients did not have enough genetic information from the parents. Among these ten missense variants, seven (S15F, R93S, R182Q, A192S, R324W, C506R and E798Q were found in the patient group only, while the other three (P281A, T712M and P917L) were detected also in the control group. Aside from these three variants that overlapped with the patient group, six missense variants (V234L, A421V, P771A, H796D, D884N, and N892K) were unique in the control group. The inheritance mode of missense variants found in the control group cannot be traced because we did not collect their parents’ DNA. In the analysis of functional prediction of these 16 rare missense variants, S15F, R93S, R182Q, P281A, R324W, C506R, H796D, E798Q, and N892K were predicted to have functional impact on the protein using the PolyPhen-2 or SIFT computer program (Table 4).
Figure 2

(A) Pedigree of S15F mutation, (B) Pedigree of R93S mutation, (C) Pedigree of R182Q mutation. (D) Pedigree of A192S mutation. (E,F) Pedigrees of P281A mutation. (G) Pedigree of R324W mutation. (H) Pedigree of C506R mutation. (I) Pedigree of T712M mutation. (J) Pedigree of E798Q mutation. (K) Pedigree of P917L mutation.

The ADI-R data of these 13 patients revealed scores of 22.82 ± 7.15 (range, 8 to 28) in the ‘qualitative abnormalities in reciprocal social interaction’ (cut-off = 10), 15.00 ± 4.84 (range, 5 to 22) in the ‘qualitative abnormalities in communication, verbal’ (cut-off = 8), 9.55 ± 4.39 (range, 4 to 14) in the ‘qualitative abnormalities in communication, nonverbal’ (cut-off = 7), and 6.00 ± 2.68 (range, 1 to 12) in the ‘restricted, repetitive and stereotyped patterns of behaviors’ (cut-off = 3). The average age at which the 13 patients said their first word was 37.1 ± 17.4 (range, 15 to 66) months old and at which they said their first phrase was 44.8 ± 18.5 (range, 19 to 78) months. Their current average intelligence quotients (IQ) were 82.8 ± 34.0 (range 40 to 126) for full-scale IQ, 84.9 ± 32.1 (range, 41 to 123) for performance IQ, and 82.3 ± 33.1 (range, 44 to 122 ) for verbal IQ, as assessed by the Wechsler Intelligence Scale for Children-third edition. Due to the lack of psychometric data for the control subjects who carried the rare missense mutations, we were not able to determine their clinical characteristics.

Clinical assessments of the parents of the 10 patients carrying rare missense mutations found that none of them achieved the clinical diagnosis of ASD. Their reports on the Chinese version of the Autism Spectrum Quotient [29] also revealed no evidence of an overt autistic trait.

Discussion

In this study, we found that rs2906569 at intron 1 and rs2301963 (P384Q) at exon 3 of DLGAP2 were associated with ASD. These two SNPs did not form significant LD in our genetic analysis. The functional significance of rs2906569 was difficult to infer, because it was located at intron 1. As to rs2301963 (C > A, P384Q), the A allele (Q allele) was the minor allele in our population, and was predicted to be probably damaging using the PolyPhen-2 computer program, but tolerated by SIFT. Based on the finding of significant over-representation of the CC homozygotes in the patient group, we suggest that the Q384 variant of the DLGAP2 might confer an increased risk of ASD, but the real mechanism and meaning of this association remain to be clarified. The small sample size of this study may have led to a false positive, which is also a limitation of this study. In a recent review of the role of common variants in autism, Devlin and colleagues reported their study on three large genome-wide association (GWA) studies of autism, each of which showed a single, non-overlapping risk locus. They found that there was no significant finding when all the data were combined. In their analysis, they found no definitive, replicated results, and they could not be certain that there was a role for common variants in autism risk [30]. Hence, an independent replication study is needed to verify our finding.

We also detected a total of 16 novel missense rare variants in the patient and control groups in this study. Given that some of these missense mutations were predicted to have functional impact on DLGAP2, we found no differences in their combined frequency between the two groups. In addition, most of these rare missense mutations found in the patients were inherited from their unaffected parents. Hence, the clinical relevance of these rare missense mutations to ASD is not straightforward, and needs further elucidation.

We attempted to assess the interactions between rs2906569 and rs2301963 and the missense rare variants in this study, but could not detect interactions between them. The small sample size of this study might be a major limiting factor. There were eight AA homozygotes of rs2906569 carrying rare missense variants, including four patients and four controls, and there were five CC homozygotes of rs2301963 carrying rare missense variants, including two patients and three controls. The genotype of rs2906569 and rs2301963 in subjects carrying the rare missense variants are listed in the Table 4.

To assess the phenotypic significance of two common SNPs associated with ASD, we compared the three core symptoms of ASD measured by the ADI-R and the Chinese version of Social Communication Questionnaire (SCQ) between patients with A/A and patients with A/G + G/G of rs2906569 (Additional file 2: Table S2), between patients with C/C and patients with C/A + A/A of rs2301963 (Additional file 3: Table S3). We also compared the three core symptoms of ASD measured by the ADI-R and SCQ between patients who carried rare missense mutations and those did not carry rare missense mutations (Additional file 4: Table S4). The Chinese SCQ is a screening tool based on the Autism Diagnostic Interview-Revised (ADI-R) algorithm which corresponds to DSM-IV diagnostic criteria. It examines the three functional domains of reciprocal social interaction, communication, and restricted, repetitive, and stereotyped patterns. The Chinese SCQ was translated under the approval of Western Psychological Services and was validated by the research group led by Gau and Wu [31]. The results showed that there were no statistical significant differences in all the comparisons (all P values > 0.1), regardless of gender with the following two exceptions. Patients with A/A of rs2906569 had less severe social interaction impairment than patients with A/G + G/G of rs2906569 (P = 0.0219) and such significant difference was only noted in male patients (P = 0.0285). But, the statistical significance did not sustain after correcting for multiple testing.

ASD is a complex disease with highly heterogeneous genetic underpinnings, and genotype-phenotype correlation remains a challenging task. According to the common variant hypothesis of complex psychiatric disorders, the effect size of common variants is usually considered small or modest. Moreover, it is not unusual to find inconsistent results among different studies, and the common variants can only explain a small proportion of the clinical variance. In a recent report on stage two of the autism genome project genome-wide association study, Anney and colleagues found that no single SNP showed a significant association with ASD or selected phenotypes at a genome-wide level after genotyping over a million SNPs, and the clinical variance explained by common variants en masse was small [32].

In contrast, the ‘rare variant hypothesis’ of complex psychiatric disorders suggests that rare variants are likely to have large effect sizes and to be de novo in their origin [33]. Given that several studies have provided evidence to support the large effect size of de novo rare mutations associated with ASD [3436], emerging evidence suggests the multiple-hits model may be more appropriate to explain the incomplete penetrance and varied expressivity of genetic underpinnings of ASD [37, 38]. Girirajan and colleagues proposed a ‘two-hit’ model in which a first hit may act in concert with some factors as a second hit, such as mutations in a single gene, micro-deletions/duplications, epigenetic factors, or environmental insults, and result in variable expressivity of phenotypes in complex neuropsychiatric disorders [38]. Furthermore, in a genetic study of high-functioning, idiopathic ASD, Schaaf and colleagues reported that in addition to de novo rare mutations, patients with ASD had a significantly higher proportion of multiple events of oligogenic heterozygosity than control subjects, suggesting oligogenic heterozygosity is a new potential mechanism in the pathogenesis of ASD [39]. In our previous study, we also reported a boy with ASD who carried two CNVs that were inherited respectively from his unaffected parents [40]. Our previous case report also lent support to the two-hit and compound heterozygosity models of ASD.

In a recent study examining the patterns and rates of exonic de novo mutations in ASD, Neale and colleagues found only a small increase in the rate of de novo events in ASD. They suggested an important but limited role for de novo point mutations in ASD, and supported polygenic models of ASD [41]. In the present study, we found that the putative deleterious missense variants occurred in both patient and control groups with equal chance, and that most of the rare missense mutations were inherited from their unaffected parents. We suggest that the missense mutations of DLGAP2 alone may not be sufficient for the clinical presentations of ASD, and additional hits such as environmental insults or further genetic mutations in the affected patients may be needed for the clinical presentations, which support ASD as likely a multifactorial disease. Hence, it is difficult to find a strong association with a single gene, like DLGAP2 in this study.

Conclusions

We identified two common SNPs and some rare missense mutations of DLGAP2 that might be implicated in the pathogenesis of ASD. However, their clinical relevance is not straightforward. We suggest additional genetic or environmental factors in the affected patients might be present to determine the clinical presentations. The findings from this study can only be considered as preliminary as it has only a limited sample size. Further independent replication studies are needed to verify our findings in the present study.

Web resources

Abbreviations

ADI-R: 

Autism diagnostic interview-revised

ARHGEF10: 

Rho guanine nucleotide exchange factor 10

Array CGH: 

Array-based comparative genomic hybridization

ASD: 

Autism spectrum disorders

CI: 

Confidence interval

CLN8: 

Ceroid-lipofuscinosis, neuronal 8

DSM-IV: 

Diagnostic and statistical manual of mental disorders, fourth edition

DLGAP2: 

Disc, large (Drosophila) homolog-associated protein 2

FBXO25: 

F-box protein 25

GWA: 

Genome-wide association

IQ: 

Intelligence quotient

LD: 

Linkage disequilibrium

MAF: 

Minor allele frequency

PCR: 

Polymerase chain reaction

SAPAP2: 

SAP90/PSD-95-associated protein 2

SAPs: 

Synapse associated proteins

SNP: 

Single nucleotide polymorphism

SD: 

Standard deviation.

Declarations

Acknowledgments

This work was supported by grants from National Science Council (NSC96-3112-B-002-033, NSC97-3112-B-002-009, NSC98-3112-B-002-004, and NSC 99-3112-B-002-036 to SS Gau), National Taiwan University (AIM for Top University Excellent Research Project:10R81918-03101R892103, 102R892103 to SS Gau), and an intramural grant from National Health Research Institutes (to CH Chen), Taiwan.

Authors’ Affiliations

(1)
Department of Occupational Therapy, College of Medicine, Fu Jen Catholic University
(2)
Department of Psychiatry, National Taiwan University Hospital and College of Medicine
(3)
Department of Psychology, School of Occupational Therapy, Graduate Institute of Brain and Mind Sciences, and Graduate Institute of Epidemiology and Preventive Medicine, National Taiwan University
(4)
Department of Psychiatry, Chang Gung Memorial Hospital-Linkou Medical Center, Chang Gung University College of Medicine
(5)
Institute of Medical Sciences, Tzu-Chi University

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© Chien et al.; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.