Epilepsy is characterized by recurrent and unpredictable seizures and affects 65 million people worldwide. Although the new generation of antiepileptic drugs (AEDs) is widely applied in clinical settings, approximately 30% of these patients are still refractory [
MicroRNAs (miRNAs), a key subset of noncoding RNAs, exhibit biological functions by inhibiting the expression of their targets at the posttranscriptional level. Previous studies have shown that miRNAs extensively participate in the regulation of inflammatory diseases, as shown, for example, for miR-146a in Aronica’s paper and for miR-133
Single-nucleotide polymorphisms (SNPs), the most common type of genetic variations, influence susceptibility to disease by altering the expression of related genes. Because miR-155 is a transcription product of its host gene (MIR155HG), its expression could be affected by genetic variations of the MIR155HG gene as well as of the miR-155 gene. To date, two studies have reported the association of MIR155HG/miR-155 SNPs with multiple sclerosis and atopic eczema, and only the GTT haplotype (rs2829803-rs2282471-rs2829806) has been successfully identified as a genetic susceptibility factor for multiple sclerosis [
In the present study, we aimed to explore the association between miR-155 and epilepsy from the perspective of molecular genetics, including SNPs and CNVs, to clarify the genetic role of miR-155 in the generation of epilepsy.
This study was approved by the Ethics Committees of the Affiliated Hospital of Guangdong Medical College and the First Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from all of the subjects before they were enrolled. All activities involving human subjects were conducted in accordance with the Declaration of Helsinki.
Using the Chinese Han in Beijing (CHB) population of the International HapMap Project, we attempted to deduce tag SNPs of the MIR155HG/miR-155 gene, a group of particular SNPs that might represent all of the SNPs identified in the MIR155HG/miR-155 gene. Using Haploview 4.2 (with the following parameters:
Loci of the MIR155HG/miR-155 gene and its tag SNPs. The three blue vertical lines/box and the two blue lines with arrows represent three exons and two introns of the MIR155HG gene, respectively. The miR-155 gene is encoded in the third exon of its host gene (MIR155HG). Regarding the 4 tag SNPs, rs969885 and rs12483428 are located in the predicted promoter region of the MIR155HG gene, whereas rs987195 and rs4817027 are located in the first intron of the MIR155HG gene.
A total of 249 epileptic patients (male/female: 137/112; mean age:
Peripheral blood samples were collected from all of the subjects. DNA was then extracted using blood Genomic DNA Extraction Kits (Tiangen Biotech, Beijing, China) and stored at −80°C before being used for genotyping. All the samples were genotyped for 4 tag SNPs (rs969885, rs12483428, rs987195, and rs4817027) of the MIR155HG/miR-155 gene using the ABI PRISM SNapShot method (Applied Biosystems, Carlsbad, USA). The forward and reverse primers used in multiple PCR for these SNPs were as follows: rs969885, 5′-GGTGGCAGGGACTGAACCATT-3′ (forward primer) and 5′-AGCATTGCATTTCCTTAAGAGTCTGAG-3′ (reverse primer); rs12483428, 5′-ATATGTCCTGGAGATGGGAGTG-3′ (forward primer) and 5′-ATCCCTACCTCATCACCCTTCA-3′ (reverse primer); rs987195, 5′-CCATCAGCCCTGGAGACACATC-3′ (forward primer) and 5′-GGAGGAACCAGTCCTGCTGACA-3′ (reverse primer); rs4817027, 5′-TCACCAAGCATTGATGACTGATGTC-3′ (forward primer) and 5′-GGAGTGTTCATTGTTCTGTCGTTTTCA-3′ (reverse primer). The primers used in multiple SNapShot PCR for these SNPs were as follows: rs969885, 5′-TTTTTTTTTTTTTTTTAGTGGTACTTACTTTGACTTACTTTGGACATA-3′; rs12483428, 5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGAGAATGTTGTTGAGGTCAAAA-3′; rs987195, 5′-TTTTTTTTTTTTGGTACTCTTGTCACAGCAGCCCTC-3′; and rs4817027, 5′-TGACAATGCAAAACATTTAGTTGGGTG-3′. In addition, 5% of the samples were randomly selected for quality control.
The copy numbers of the MIR155HG/miR-155 gene were determined using Multiplex AccuCopy Kits (Genesky Biotechnologies Inc., Shanghai, China), and 4 reference genes (POP1, RPP14, POLR2A, and TBX15) were used for normalization. The basic principle is shown at
Age is displayed as the mean ± standard deviation (SD) and was compared using Student’s
This study enrolled a total of 249 epileptic patients and 289 healthy individuals, and the subject information is shown in Table
Subject information.
Cases | Controls |
|
|
---|---|---|---|
Gender (male/female, |
137/112 | 157/132 | 0.872 |
Age (mean ± SD, years) | 26.51 ± 15.25 | 27.32 ± 21.24 | 0.325 |
Age of onset ( |
|||
Early-onset epilepsy | 145 | — | — |
Late-onset epilepsy | 104 | — | — |
TLE ( |
174 | — | — |
DRE ( |
67 | — | — |
The frequency distributions of each tag SNP (rs969885, rs12483428, rs987195, and rs4817027) of the MIR155HG/miR-155 gene in the case group and the control group complied with Hardy-Weinberg equilibrium (all
The frequency of the AA genotype at rs4817027 was significantly higher in the male cases than in the male controls (
Frequency distributions of the 4 tag SNPs between the male cases and the male controls.
Male cases |
Male controls |
OR (95% CI) |
|
|
---|---|---|---|---|
rs969885 C>T | ||||
C/T | 238 (86.86)/36 (13.14) | 266 (84.71)/48 (15.29) | 0.80 (0.44–1.45) | 0.463 |
CC/CT/TT | 103 (75.18)/32 (23.36)/2 (1.46) | 111 (70.70)/44 (28.03)/2 (1.27) | 0.457 | |
CC/CT+TT | 103 (75.18)/34 (24.82) | 111 (70.70)/46 (29.30) | 0.82 (0.42–1.60) | 0.564 |
CC+CT/TT | 135 (98.54)/2 (1.46) | 155 (98.73)/2 (1.27) | 2.40 (0.29–19.94) | 0.417 |
rs12483428 T>C | ||||
T/C | 241 (87.96)/33 (12.04) | 270 (85.99)/44 (14.01) | 1.40 (0.76–2.58) | 0.276 |
TT/TC/CC | 107 (78.10)/27 (19.71)/3 (2.19) | 115 (73.25)/40 (25.48)/2 (1.27) | 0.273 | |
TT/TC+CC | 107 (78.10)/30 (21.90) | 115 (73.25)/42 (26.75) | 1.51 (0.77–2.98) | 0.232 |
TT+TC/CC | 134 (97.81)/3 (2.19) | 155 (98.73)/2 (1.27) | 0.95 (0.09–9.60) | 0.962 |
rs987195 C>G | ||||
C/G | 167 (60.95)/107 (33.05) | 184 (58.60)/130 (41.40) | 1.04 (0.68–1.59) | 0.853 |
CC/CG/GG | 51 (37.23)/65 (47.45)/21 (15.33) | 55 (35.03)/74 (47.13)/28 (17.83) | 0.858 | |
CC/CG+GG | 51 (37.23)/86 (62.77) | 55 (35.03)/102 (64.97) | 0.86 (0.47–1.57) | 0.619 |
CC+CG/GG | 116 (84.67)/21 (15.33) | 129 (82.17)/28 (17.83) | 1.46 (0.68–3.13) | 0.335 |
rs4817027 G>A | ||||
G/A | 197 (71.90)/77 (28.10) | 249 (79.30)/65 (20.70) | 1.30 (0.80–2.11) | 0.287 |
GG/GA/AA | 70 (51.09)/57 (41.61)/10 (7.30) | 94 (59.87)/61 (38.85)/2 (1.27) | 0.252 | |
GG/GA+AA | 70 (51.09)/67 (48.91) | 94 (59.87)/63 (40.13) | 1.18 (0.66–2.13) | 0.575 |
GG+GA/AA | 127 (92.70)/10 (7.30) | 155 (98.73)/2 (1.27) | 9.40 (1.17–75.31) | 0.035 |
OR: odds ratio; 95% CI: 95% confidence interval. OR (95% CI) and
This study further explored the haplotypes of the 4 tag SNPs between the male cases and the male controls and found a haplotype block composed of 3 tag SNPs (rs12483428, rs987195, and rs4817027). The frequency of the TCA haplotype was higher in the male cases than in the male controls (28.1% versus 20.7%,
Frequency distributions of the differential haplotype blocks of the MIR155HG/miR-155 gene.
Haplotype | Frequency (%) | Case ratio (%) | Control ratio (%) |
|
|
---|---|---|---|---|---|
Male cases versus male controls | |||||
rs12483428-rs987195-rs4817027 | TGG | 40.31 | 39.05 | 41.40 | 0.562 |
TCA | 24.15 | 28.10 | 20.70 | 0.036 | |
TCG | 22.45 | 20.80 | 23.89 | 0.372 | |
CCG | 13.10 | 12.04 | 14.01 | 0.480 | |
Early-onset cases versus controls | |||||
rs969885-rs987195 | CC | 46.67 | 52.79 | 43.60 | 0.011 |
CG | 39.62 | 35.48 | 41.70 | 0.079 | |
TC | 13.58 | 11.34 | 14.71 | 0.177 | |
DRE cases versus controls | |||||
rs969885-rs987195 | CC | 47.33 | 58.96 | 44.64 | 0.003 |
CG | 40.73 | 29.85 | 43.25 | 0.004 | |
TC | 11.94 | 11.19 | 12.11 | 0.768 |
No significant differences in the alleles and genotypes were observed between the early-onset/late-onset cases and the controls for the 4 tag SNPs (all
No significant differences in the alleles and genotypes were observed between the TLE cases and the controls for the 4 tag SNPs (all
The frequency of the AA genotype at rs4817027 was significantly higher in the DRE cases than in the controls (
Frequency distributions of the 4 tag SNPs between the DRE cases and the controls.
DRE cases |
Controls |
OR (95% CI) |
|
|
---|---|---|---|---|
rs969885 C>T | ||||
C/T | 119 (88.81)/15 (11.19) | 499 (86.33)/79 (13.67) | 0.79 (0.36–1.72) | 0.548 |
CC/CT/TT | 53 (79.10)/13 (19.40)/1 (1.49) | 214 (74.05)/71 (24.57)/4 (1.38) | 0.536 | |
CC/CT+TT | 53 (79.10)/14 (20.90) | 214 (74.05)/75 (25.95) | 0.85 (0.36–2.04) | 0.717 |
CC+CT/TT | 66 (98.51)/1 (1.49) | 285 (98.62)/4 (1.38) | 5.07 (0.35–73.13) | 0.233 |
rs12483428 T>C | ||||
T/C | 106 (79.10)/28 (20.90) | 496 (85.81)/82 (14.19) | 1.27 (0.63–2.57) | 0.511 |
TT/TC/CC | 44 (65.67)/18 (26.87)/5 (7.46) | 213 (73.70)/70 (24.22)/6 (2.08) | 0.516 | |
TT/TC+CC | 44 (65.67)/23 (34.33) | 213 (73.70)/76 (26.30) | 1.29 (0.57–2.90) | 0.542 |
TT+TC/CC | 62 (92.54)/5 (7.46) | 283 (97.92)/6 (2.08) | 1.55 (0.16–14.83) | 0.705 |
rs987195 C>G | ||||
C/G | 94 (70.15)/40 (29.85) | 332 (57.44)/246 (42.56) | 1.59 (0.92–2.75) | 0.097 |
CC/CG/GG | 34 (50.75)/26 (38.81)/7 (10.45) | 100 (34.60)/132 (45.67)/57 (19.72) | 0.118 | |
CC/CG+GG | 34 (50.75)/33 (49.25) | 100 (34.60)/189 (65.40) | 1.98 (1.92–4.28) | 0.081 |
CC+CG/GG | 60 (89.55)/7 (10.45) | 232 (80.28)/57 (19.72) | 1.48 (0.54–4.03) | 0.448 |
rs4817027 G>A | ||||
G/A | 96 (71.64)/38 (28.36) | 455 (78.72)/123 (21.28) | 1.72 (0.91–3.24) | 0.094 |
GG/GA/AA | 34 (50.75)/28 (41.79)/5 (7.46) | 174 (60.21)/107 (37.02)/8 (2.77) | 0.074 | |
GG/GA+AA | 34 (50.75)/33 (49.25) | 174 (60.21)/115 (39.79) | 1.63 (0.76–3.52) | 0.213 |
GG+GA/AA | 62 (92.54)/5 (7.46) | 281 (97.23)/8 (2.77) | 13.13 (1.40–123.83) | 0.024 |
OR: odds ratio; 95% CI: 95% confidence interval. OR (95% CI) and
This study further explored the haplotypes of the 4 tag SNPs between the DRE cases and the controls and found a haplotype block composed of 2 tag SNPs (rs969885 and rs987195). The frequency of the CC haplotype was higher in the DRE cases compared with the controls (59.0% versus 44.6%,
A total of 3 epileptic cases with CNVs of the MIR155HG/miR-155 gene were observed in the case group, including 1 case with deletion (copy number: 1; 0.40%) and 2 cases with duplication (copy numbers: 3 and 4; 0.80%), but no CNVs were observed in the control group (Figure
Copy numbers of the MIR155HG/miR-155 gene in a total of 538 individuals. Cases
In recent years, a large number of experimental and human studies of epilepsy have performed global expression profiling of miRNAs, and more than one hundred miRNAs have been found to be abnormally expressed in epileptic brains [
Indeed, a systematic review of forty clinical reports verified the higher incidence of epilepsy in males compared with females [
Early-onset epilepsy, an age-dependent epileptic syndrome, primarily results from genetic defects and inborn errors. Many of the types of early-onset epilepsy display overlapping clinical features, indicating that they share pathologies, such as metabolic and structural brain abnormalities [
Generally, the mechanisms of DRE involve the transporter hypothesis, the target hypothesis, and hypotheses involving drugs that fail to affect the real targets [
Normally, two copies of human genes are present in autosomal regions. Duplication/deletion of DNA fragments can lead to significant increase/decrease in gene expression levels due to dosage effects of their copy numbers, which plays a key role in the generation of some genetic diseases [
In addition, 1 case with deletion of the MIR155HG/miR-155 gene was observed. This observation was inconsistent with the increased expression of miR-155 in epileptic patients, suggesting that miR-155 may be involved in other biological processes. The gene encoding methyl CpG binding protein 2 (Mecp2) is another predicted target of miR-155, and any gain or loss in the expression of this gene over a narrow threshold level leads to neurological impairment [
Several limitations of this study should be acknowledged. First, relatively few subjects were enrolled in this study, which possibly give rise to nonspecific results. However, the epileptic patients enrolled in this study were relatively young (mean age: 26.51 ± 15.25 years); thus almost all of them did not suffer from other related comorbidities, which limits the risk of nonspecific results. Second, although rs12483428 and rs969885 are located in the predicted promoter region of the MIR155HG/miR-155 gene, this study did not involve additional experiments to assess the expression of miR-155 and its predicted targets, such as SOCS1 and Mecp2, or their association with the potential functional haplotypes, including the TCA haplotype (rs12483428-rs987195-rs4817027) and the CC/CG haplotype (rs969885-rs987195) because obtaining tissue samples of epileptogenic foci is difficult. Moreover, the brain is a specialized tissue with its own resident immune system. The brain also differs from peripheral tissues due to the presence of the blood-brain barrier. Thus, this study did not include functional experiments based on peripheral blood samples. Third, all of the subjects in the study were Han Chinese, and caution should be used when generalizing these findings to different ethnic populations.
This study is the first to demonstrate that MIR155HG/miR-155 tag SNPs are associated with epilepsy. It also showed that rare CNVs were found exclusively in epileptic patients. These findings clarify the role of miR-155 in epilepsy from the perspective of molecular genetics.
No conflict of interests is reported.
Hua Tao, Lili Cui, and You Li contributed equally to this work.
This study was supported by the Research Foundation of Guangdong Medical College (Grant no.: M2014022), the Youth Foundation of the Affiliated Hospital of Guangdong Medical College (Grant no.: QK1321), the National Nature Science Foundation of China (Grant nos.: 31171219, 81271213, 81471294, 81271214, and 81210108045), and the National “Twelfth Five-Year” Plan for Science & Technology Support (Grant no.: 2011BAI08B02).