Subjects
This case-control study was conducted on 192 children who were diagnosed as autism. The patients were fulfilling the criteria for the diagnosis of autism according to the 4th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). The case group comprised 154 males and 38 females. They were recruited from the Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine. Their ages ranged from 2 to 18 years old (mean ± SD = 5.65 ± 3.04). Patients were excluded if they had known mental and physical illness or chromosomal abnormalities. The 192 healthy controls subjects were volunteers recruited by advertisement from Shanghai Mental Health Center and local community, who did not suffer from any severe physical diseases, as well as personal and family history of mental diseases according to the brief interviews by senior psychiatrists. The control group included 147 males and 45 females and their ages ranged from 2 to 65 years old (mean ± SD = 31.59 ± 19.01). All the subjects are Chinese Han descendant. Ethical approval was obtained from the ethics committees of Shanghai Mental Health Center and an informed written consent of participation in the study was signed by the parents or the legal guardians of the studied subjects.
Gene screening
Five millilitres of the whole blood was taken and ethylenediaminetetraacetic acid (EDTA) was used to be anticoagulant. DNA was isolated according to the established laboratory protocols.
Sequencing data were aligned to the annotated human genome sequence (hg19) using the BLAT tool of the UCSC Genome Browser (http://genome.ucsc.edu/). The UBE3A gene sequence was searched from the UCSC Genome Browser database, which showed that there were three spliceosomes (NM_130838, NM_000462 and NM_130839) of UBE3A gene. The sequence information of spliceosome 2 includes spliceosome 1 (NM_130838) and 3 (NM_130839). Therefore, the sequence information of spliceosome 2(NM_000462) was selected as the candidate sequence. UBE3A gene sequence has 14 exons, exon 1, 2, 3, initiation 36 bp of exon 4 and the terminal 1888 bp of exon 14 are non-coding regions (untranslated regions, UTR), the terminal 29 bp of the exon 4, exon 5, 6, 7, 8, 9, 10, 11, 12, 13 and initiation 121 bp of exon 14 are coding sequence (CDS) (Fig. 1). In this study, we performed mutation screening for all coding regions and their adjacent non-coding regions named by CDS1-CDS11.
The two methods high resolution melting (HRM) and Sanger sequencing were performed in our study. Finally, the HRM method was used to detect CDS 3, 8, 9, 10 and 11, and the remaining CDS were directly sequenced by Sanger method after a series of preliminary experiments.
HRM analysis
The CDS 3, 8, 9, 10 and 11 were amplified using polymerase chain reaction (PCR) and the primers information summarized in Table 1. The primers were designed to generate amplicons of 200–400 bp. Then HRM were performed using LightCycler® 96 Instrument (Roche Diagnostics, Roche Instrument Center AG, Rotkreuz, Switzerland) in DNA samples from autism patients and healthy controls. The amplifications were performed in 10 µL volumes containing 10 ng of genomic DNA, 2 µmol/L primers, 2.5 mmol/L MgCl2 and 5 µL 2X LightCycler® 96 High Resolution Melting Master (Roche Diagnostics) buffer. PCR cycling included an initial preincubation at 95℃ for 10 min, followed by 45 cycles of 10 s at 95 ℃, from 65℃ to a “touchdown” at 55℃, and 30 s at 72℃. The melting program included three steps: denaturation at 95℃ for 10 s, renaturation at 65℃ for 1 min, and a subsequent melting cycle consists of a continuous fluorescent reading from 60℃ to 90℃ at a rate of 25 acquisitions per ℃. Mutations were confirmed with an independent PCR and bidirectional sequencing.
Table 1
The primer information for HRM.
CDS
|
|
primer sequence
|
amplicon length(bp)
|
3
|
Forward
|
TCCCACATGGTTTTCAGGCA
|
397
|
|
Reverse
|
GAGAGCTGTACTAATCACTGTGC
|
|
8
|
Forward
|
TTTTGCAGACACCTGCTTTCTTA
|
251
|
|
Reverse
|
GCAGCCCAATAACTTGTGTTTTGT
|
|
9
|
Forward
|
GTCTGAAGCAAAATCACACACCC
|
256
|
|
Reverse
|
ATATGTGGAAGCCGGGTAAGAA
|
|
10
|
Forward
|
ACGAGGAATGCAAGGTTTTCG
|
242
|
|
Reverse
|
ATGAATGCCAAACTGAAACCAGTA
|
|
11
|
Forward
|
GTACTGGGACACTATCACCACC
|
285
|
|
Reverse
|
TTTCCCATGACTTACAGTTTTCCTG
|
|
Sanger Sequencing
CDS 1, 2, 4, 5, 6 and 7 were directly sequenced using Sanger method, which could detect about 800 bp sequence length. The length of CDS4 was 1247 bp, which was sequenced forward and backward. The PCR primers information were listed in Table 2. Each PCR master mix included 2 µM of each primer, 1 µl mix dNTP, 1.5 µl MgCl2, 5 µl of 10 × PCR buffer, (5 U/µl) AmpliTaq Gold DNA polymerase, 3 µl of DNA sample, and double-distilled water to reach the total volume of 50 µl. PCR condition contained initial denaturation at 95 °C for 5 min, 30 cycles of 95 °C for 30 s and 62 °C-65 °C for 30 s and 72 °C for 45 s, then 72 °C for 5 min as final extension was performed. After purification of PCR products using TaKaRa® Shrimp Alkaline Phosphatase (SAP) and ExonucleaseI (ExonI), sequencing was performed by BigDye Terminator Kit (Applied Biosystems, USA) according to the method by Sanger F, et al [16]. The sequence information were read using Applied Biosystems (ABI) 3130 Genetic Analyzer (Applied Biosystems, USA). Mutations were confirmed with an independent PCR and bidirectional sequencing.
Table 2
The primer information for Sanger Sequencing.
CDS
|
|
primer sequence
|
amplicon length(bp)
|
1
|
Forward
|
GGTCTTGATTTGAATCGCAGAAA
|
779
|
|
Reverse
|
CATTGACACCTAATTTGAAGCTTTG
|
|
2
|
Forward
|
ATTTGCTTCTGCATCTTTCACTCT
|
639
|
|
Reverse
|
TGTTGTATGGCCACCTGATCT
|
|
4
|
Forward
|
TCCATGTGTTCCTATGCTATATGGT
|
1466
|
|
Reverse
|
TGAGCCTAGAATGTTTGGCTGT
|
|
5
|
Forward
|
CAGTCATGATGTGTGATTCTGGGT
|
603
|
|
Reverse
|
TTCCATGTCCTGTGTAGTCCAG
|
|
6
|
Forward
|
AGGCACACTCGTTGTAACTACC
|
800
|
|
Reverse
|
CCGATGCCACCAAATTACTTACT
|
|
7
|
Forward
|
GGGCTTTAGTGCCCAACTGTG
|
555
|
|
Reverse
|
GGGACATCACAGTGACTGACAAT
|
|
Association Analysis
For each detected variant, we further expanded the sample size of case and control subjects and case-control genotyping studies and association analysis were performed. Each detected variant of expanded samples were genotyped using HRM method.
For the association analysis, the online software SHEsis [17] (http://analysis2.bio-x.cn/myAnalysis.php) was used to compare the allelic and genotypic frequencies between the case and control groups. Another online software SNPStats [18] (http://bioinfo.iconcologia.net/snpstats/start.htm) was used to calculate the association between the detected variants and the risk of autism under 5 inheritance models, including codominant, dominant, recessive, overdominant and log-additive models. All of the statistical tests were two-sided, and p < 0.05 was defined as statistically significant.