Introduction
Patients and methods
Study population
Pairs of subjects | Age (years) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Total | Male | Female | ∼3 | ∼4 | ∼5 | ∼6 | ∼7 | ∼12 | |
MeDIP chip study and validation | 5 | 4 | 1 | 2 | 2 | 1 | |||
Additional BSP of ENO2
| 126 | 96 | 30 | 9 | 28 | 39 | 17 | 16 | 17 |
RT-qPCR of ENO2
| 19 | 14 | 5 | 2 | 2 | 3 | 5 | 3 | 4 |
ELISA analysis of ENO2 | 19 | 14 | 5 | 2 | 2 | 3 | 5 | 3 | 4 |
Preparation of plasma, DNA, RNA, and cDNA
Methylated DNA immunoprecipitation (MeDIP) array-based hybridization
Bisulfite sequencing PCR (BSP)
RT-qPCR
Enzyme-linked immunosorbent assay (ELISA)
Statistical analysis
Results
Genome-wide DNA methylation pattern of autism in peripheral blood
Autism vs. control | Hypermethylation |
p value | Hypomethylation |
p value |
---|---|---|---|---|
5 pairs |
IDUA
| 0.0008 |
RTN3
| 0.0012 |
HEATR6
| 0.0009 |
SLC15A4
| 0.0013 | |
LASS3
| 0.0009 | PODNL1 | 0.0015 | |
TOM1L2
| 0.0012 |
HECW1
| 0.0018 | |
LRRC48
| 0.0013 |
IDH3G
| 0.0019 | |
SNORD105B
| 0.0015 |
SPSB4
| 0.0021 | |
ANGPTL6
| 0.0015 | |||
4 pairs (In addition to genes listed in 5 pairs above) |
METAP1
| 0.0005 |
TMEM110
| 0…09 |
MAP1
| 0.0005 |
PCDH10
| 0.0009 | |
BOLIG3
| 0.0006 |
TBC1D9
| 0.0012 | |
ABCF1
| 0.0006 |
AREG
| 0.0012 | |
BEST2
| 0.0007 |
HES3
| 0.0012 | |
GRIN2
| 0.0009 |
PWWP2
| 0.0013 | |
DKDELR1
| 0.0009 |
BCHST3
| 0.0013 | |
FOXS1
| 0.0011 |
MARCH5
| 0.0013 | |
C20orf149
| 0.0023 |
CPEB3
| 0.0026 | |
ENO2
| 0.0024 |
IGSF9B
| 0.0029 | |
PANX2
| 0.0025 |
AP2A2
| 0.0035 | |
PEAR1
| 0.0026 |
F7
| 0.0038 | |
CCNG2
| 0.0031 |
C14orf73
| 0.0041 | |
BDNF
| 0.0036 |
BRMS1L
| 0.0051 | |
CES2
| 0.0045 |
C14orf162
| 0.0061 | |
FAM96
| 0.0051 |
BCL11B
| 0.0071 | |
BNGEF
| 0.0062 |
SOLH
| 0.0082 | |
FAM19A5
| 0.0068 |
C16orf10
| 0.0081 | |
STOML2
| 0.0075 |
CDK3
| 0.0085 | |
GRIN2D
| 0.0089 | |||
ARHGEF18
| 0.0091 | |||
HNRPLL
| 0.0093 | |||
BFSP1
| 0.0093 | |||
C20orf134
| 0.0095 | |||
TNFRSF6B
| 0.0096 | |||
TBC1D23
| 0.0096 | |||
KIAA1257
| 0.0097 | |||
CDV3
| 0.0098 | |||
CXorf38
| 0.0099 | |||
KIAA2022
| 0.0099 | |||
3 pairs (In addition to genes listed in 5 and 4 pairs above) |
NCOA7
| 0.0004 |
HIST2H3D
| 0.0002 |
TUBB2B
| 0.0004 |
RORC
| 0.0030 | |
CALML3
| 0.0004 |
ADAM15
| 0.0004 | |
PSAP
| 0.0004 |
ECE2
| 0.0005 | |
KCNQ1DN
| 0.0011 |
PPP1R2
| 0.0016 | |
HRAS
| 0.0015 |
LYSMD3
| 0.0017 | |
CDX2
| 0.0016 |
RAD50
| 0.0021 | |
SLC24A2
| 0.0018 |
PITX1
| 0.0029 | |
MLNR
| 0.0021 |
IRX1
| 0.0032 | |
LEO1
| 0.0026 |
LOC25845
| 0.0043 | |
CHRNA3
| 0.0029 | |||
KRT19
| 0.0031 | |||
TBX21
| 0.0035 | |||
FLJ32065
| 0.0039 | |||
CBX4, | 0.0041 | |||
NRXN1
| 0.0045 | |||
FAM59A
| 0.0059 | |||
GTPBP3
| 0.0067 | |||
PNMAL2
| 0.0073 | |||
DPP10
| 0.0075 | |||
CYP2D7P1
| 0.0081 | |||
SEC61G
| 0.0086 | |||
RIMS2
| 0.0087 |
Validation of hypermethylation of ENO2 gene
Decreased transcription and translation of ENO2
The relation between the hypermethylation of ENO2 gene and environmental factors
Characteristics | Value (%) |
p value | |
---|---|---|---|
Autism | Control | ||
Maternal characteristics | |||
Age at pregnancy (years) | |||
<30 | 51.5 | 65.4 | 0.05 |
30–35 | 37.6 | 29.7 | |
>35 | 10.9 | 4.9 | |
Occupation (%) | |||
Housewife | 58.4 | 50.2 | 0.12 |
Working mother | 41.6 | 49.8 | |
Education (%) | |||
Tertiary education | 36.7 | 38.2 | 0.35 |
Secondary school | 39.2 | 34.3 | |
Primary school | 24.1 | 27.5 | |
Medical history | |||
Caught a cold during the 1st trimester | 16.8 | 14.9 | |
Mental illness | 4.9 | 0 | |
Paternal characteristics | |||
Smoking (%) | 60.2 | 62.1 | 0.25 |
Alcohol consumption (%) | 74.5 | 70.1 | |
Mental illness (%) | 1.8 | 0.9 | |
Family characteristics | |||
Monthly income (%) | |||
>10,000 | 30.5 | 34.2 | 0.08 |
5,000–10,000 | 36.8 | 35.3 | |
3,000–<5,000 | 22.1 | 21.0 | |
<3,000 | 10.6 | 9.5 | |
Child characteristics | |||
Gestational weeks (%) | |||
<37 | 21.8 | 11.9 | 0.04 |
37–40 | 68.3 | 80.2 | |
>40 | 9.9 | 7.9 | |
Apgar score at 5 min (%) | |||
8–10 | 83.2 | 96.5 | 0.03 |
4–7 | 16.8 | 3.5 | |
Length of breast feeding | |||
<4 months | 21.1 | 19.7 | 0.06 |
4–6 months | 42.3 | 38.9 | |
>6 months | 36.6 | 41.4 | |
Child raised by | |||
Parents | 12.9 | 20.8 | 0.03 |
Grandparents | 59.4 | 49.5 | |
Nanny | 27.7 | 29.7 | |
Daily TV time | |||
<1 h | 39.6 | 50.5 | 0.04 |
1–2 h | 32.7 | 29.7 | |
>2 h | 27.7 | 19.8 | |
Medication (%) | |||
Ritalin | 20.8 | 0 | 0.04 |
Risperdal | 7.9 | 0 | |
No specific medication | 71.3 | 100 |
Hypermethylation (n = 19) | Non-hypermethylation (n = 112) |
t
|
p
| |
---|---|---|---|---|
Length of breast-fed (months) | 4.72 ± 1.19 | 7.21 ± 1.91 | 2.539 | 0.019 |
Apgar score at 5 min | 7.12 ± 2.19 | 9.21 ± 1.83 | 2.419 | 0.026 |
Daily TV time (h) | 2.06 ± 0.82 | 1.15 ± 0.35 | 2.149 | 0.032 |