Association between TNFSF10 polymorphism and migraine susceptibility in a Chinese population

Abstract

Objective

To investigate the correlation between migraine in a Chinese population and a 4 base pair (GAGT) insertion/deletion polymorphism (rs35975099) localized near the 3' end of the tumour necrosis factor superfamily 10 gene, TNFSF10.

Methods

Ethnically Han Chinese patients with migraine and healthy control subjects were recruited. TNFSF10 genotype and allele frequencies were determined via polymerase chain reaction and polyacrylamide gel electrophoresis.

Results

Rs35975099 was in Harvey–Weinberg equilibrium in patients with migraine (n = 269) and control subjects (n = 374). There were no significant relationships between allele or genotype frequency and migraine.

Conclusion

There was no functional significance of the TNFSF10 gene polymorphism rs35975099 in migraine pathogenesis.

Keywords

Migraine gene polymorphisms susceptibility

Introduction

Migraine is a polygenically inherited chronic neurovascular disorder that can negatively impact work, study, daily activities and health-related quality-of-life. The lifetime prevalence of migraine is between 14% and 16%, and it is three times more common in women than in men.^1,2 Migraine is clinically classified between two subtypes based on the presence or absence of aura, symptoms of which include hallucinations, paraesthesia, disturbed vision and weakness. Although the pathological mechanism of migraine is unclear, evidence suggests inflammation may be involved.^3,4

Tumour necrosis factor (TNF) is a key cytokine involved in inflammatory reactions and inflammation-related hyperalgesia.⁵ TNF may play a pivotal role in migraine via activation of meningeal nociceptors, reducing threshold cerebral excitability and propagating neuronal hyperexcitability.^6,7 The TNF superfamilies (TNFSF) and TNF receptor superfamilies (TNFRSF) have extensive actions in cell differentiation and survival, and in controlling expression of inflammatory cytokines.⁸ TNFSF/TNFRSF gene polymorphisms have now been recported in migraine. These include TNF-α 308 G>A,⁹ TNF-β 252 A>G,¹⁰ TNFSF7¹¹ and TNFRSF1B.¹² TNFSF10 has been shown to exert therapeutic efficacy for inflammatory diseases in experimental animal models,¹³ suggesting a role in the progression of inflammation. TNFSF10 polymorphisms are also correlated with cancer susceptibility,^14,15 but there is no direct evidence that TNFSF10 polymorphism is associated with migraine.

The aim of this study was to investigate the correlation between migraine in a Chinese population and a 4 base pair (GAGT) insertion/deletion polymorphism (rs35975099) localized near the 3' end of TNFSF10.

Patients and methods

Study population

The study enrolled unrelated ethnically Han Chinese patients with migraine, attending the Neurological Outpatient Service, First Affiliated Hospital of Soochow University, Suzhou, China, for diagnosis or follow-up between October 2008 and December 2010. Migraine was diagnosed and classified according to the International Classification of Headache Disorders-II criteria, as recommended by the International Headache Society.¹⁶ Ethnically Han Chinese control subjects were recruited from individuals completing a nutritional survey in the same geographical area as the patients. Subjects with migraine, a family history of migraine, or any severe or recurrent headache in direct relatives were excluded. Data regarding tobacco use (more than one cigarette per day for >1 year) and alcohol use (at least one alcoholic drink per week) were recorded for patients and control subjects.

The ethics committee of Soochow University, Suzhou, China approved the study, and all subjects provided written informed consent prior to enrolment.

Genotyping

Peripheral blood (3 ml) was taken from each subject and stored at –20° until use. DNA was extracted from whole blood¹⁷ and polymerase chain reaction (PCR) for TNFSF10 was carried out as described.¹⁸ Primer sequences were: forward 5′- GGCCCTATGTCCAAATAAGGTT-3′; reverse 5′- GTTCTGGGTTGAGTTGTGCTG-3′. PCR cycling conditions were 94℃ for 7 min followed by 35 cycles of 94℃ for 40 s, 60℃ for 40 s and 72℃ for 40 s, and a final extension step at 72℃ for 5 min. PCR products were separated by 7% nondenaturing polyacrylamide gel electrophoresis and visualized by silver staining. Genotype was determined by number and size of bands on the gel. In order to confirm the reproducibility of PCR genotyping, 10% of samples were randomly selected and re-examined by another technician.

Statistical analyses

Hardy–Weinberg Equilibrium (HWE) was analysed by χ²-test. Between-group comparisons of genotypic and allelic distribution were made by χ²-test. Logistic regression analysis (adjusted for sex, age, alcohol and tobacco use, and clinical characteristics) was conducted to obtain odds ratios (ORs) and 95% confidence intervals (CIs) for the correlation between rs35975099 and migraine risk. All statistical analyses were two-sided and performed using SAS statistical software, version 8.0 (SAS Institute Inc., Cary, NC, USA). P-values ≤0.05 were considered statistically significant.

Results

The study enrolled 269 patients with migraine (78 male/191 female; mean age 34.3 ± 11.7 years; age range 14–65 years) and 374 healthy control subjects (106 male/268 female; mean age 35.4 ± 10.6 years; age range 18–70 years). There were no significant between-group differences in age, sex distribution, tobacco use (patient group 50/269 [18.6%] vs control group 59/374 [15.8%]) or alcohol use (patient group 55/269 [20.4%] vs control group 76/374 [20.3%]). Clinical characteristics of the patient group are shown in Table 1.

Table 1.

Clinical characteristics of Chinese patients with migraine, included in a study investigating the correlation between the occurrence of migraine and rs35975099, a 4 base pair (GAGT) insertion/deletion polymorphism localized near the 3' end of the tumour necrosis factor superfamily 10 gene (TNFSF10) (n = 269).

Characteristic	Total
Age, years	34.3 ± 11.7
Sex, male/female	78/191 (29.0/71.0)
Tobacco use^a	50 (18.6)
Alcohol use^b	55 (20.4)
Pain severity^c
Low	41 (15.2)
Moderate	49 (18.2)
Severe	179 (66.6)
Frequency of attacks/month
≤1	51 (19.0)
2–4	140 (52.0)
≥5	78 (29.0)
Age at migraine onset, years	25.5 ± 9.1
Family history of migraine	170 (63.2)
Migraine type
With aura	17 (6.3)
Without aura	252 (93.7)
Time since diagnosis, years	8.8 ± 3.7

Data presented as mean ± SD or n (%).

More than one cigarette per day for >1 year.

At least one drink per week.

^cLow, no effect on daily activity; moderate, slight effect on daily activity; severe, severe effect on daily activity.

Repeat PCR confirmed that PCR genotyping was 100% reproducible. Genotype frequency was in HWE, in patients and in control subjects. Data regarding allele and genotype frequencies are presented in Table 2. There were no statistically significant between-group differences in allele or genotype distribution, with or without adjustment for sex, age, tobacco and alcohol use.

Table 2.

Logistic regression analysis of genotype and allele frequencies of rs35975099, a 4 base pair (GAGT) insertion/deletion polymorphism localized near the 3' end of the tumour necrosis factor superfamily 10 gene (TNFSF10), in patients with migraine and in healthy control subjects.

Genotype/Allele	Patient group n = 269	Control group n = 374	Crude OR (95% CI)	Adjusted OR^a (95% CI)
Insertion/insertion	48 (17.8)	47 (12.6)	1.00 (reference)	1.00 (reference)
Insertion/deletion	128 (47.6)	189 (50.5)	0.66 (0.41, 1.08)	0.61 (0.38, 1.07)
Deletion/deletion	93 (34.6)	138 (36.9)	0.66 (0.40, 1.10)	0.63 (0.39, 1.06)
Insertion	224 (41.6)	283 (37.8)	1.00 (reference)	–
Deletion	314 (58.4)	465 (62.2)	0.85 (0.68, 1.08)	–

Data presented as n (%).

OR, odds ratio; CI, confidence interval.

Adjusted for age, sex, and smoking and drinking status.

Results of stratification analysis for the patient group are shown in Table 3. There were no significant differences in allele or genotype distribution when patients were stratified by sex, age of disease onset, family history of migraine or migraine type.

Table 3.

Genotype and allele frequencies of rs35975099, a 4 base pair (GAGT) insertion/deletion polymorphism localized near the 3' end of the tumour necrosis factor superfamily 10 gene (TNFSF10), in patients with migraine stratified according to sex, age at onset, family history and migraine subtype (n = 269).

Characteristic	Genotype			Allele
Characteristic	Ins/Ins	Ins/Del	Del/Del	Ins	Del
Sex
Male, n = 78	14 (17.9)	37 (47.4)	27 (34.6)	65 (41.7)	91 (58.3)
Female, n = 191	34 (17.8)	91 (47.6)	66 (34.6)	159 (41.6)	223 (58.4)
Age at onset of migraine, years
<26, n = 144	28 (19.4)	64 (44.4)	52 (36.1)	120 (41.7)	168 (58.3)
≥26, n = 125	20 (16.0)	64 (51.2)	41 (32.8)	104 (41.6)	146 (58.2)
Family history of migraine
Positive, n = 170	31 (18.2)	79 (46.5)	60 (35.3)	141 (41.5)	199 (58.5)
Negative, n = 99	17 (17.2)	49 (49.5)	33 (33.3)	83 (41.9)	115 (58.1)
Migraine type
With aura, n = 17	5 (29.4)	9 (52.9)	3 (17.6)	19 (55.9)	15 (44.1)
Without aura, n = 252	43 (17.1)	119 (47.2)	90 (35.7)	205 (40.7)	299 (59.3)

Data presented as n (%).

Ins, insertion; Del, deletion.

No statistically significant between-group differences (P > 0.05; χ²-test).

Discussion

Our study found no association between genotype or allele distribution of the TNFSF10 gene polymorphism rs35975099 and migraine in Chinese patients with migraine.

Migraine is a primary headache disorder with an unclear pathogenesis, although evidence suggests that ∼ 40% of migraine susceptibility is due to multiple genetic factors.^19,20 Studies support the involvement of proinflammatory cytokines (especially TNF) in migraine.^21,22 Our earlier study revealed a relationship between TNFRSF1B polymorphism and an augmented risk of migraine.¹² TNFSF10 is involved in acute and chronic inflammatory central nervous system disorders²³ and has many functions in the peripheral and central nervous system, including cell death and immune modulation.²⁴ There is no evidence for a correlation between altered plasma or cerebral spinal fluid TNFSF10 concentrations and migraine, however. The findings of the present study indicate no functional significance for TNFSF10 rs35975099 in migraine susceptibility in a Chinese population, suggesting that TNFSF10 may not play a major role in migraine, in this demographic group.

Our study has several limitations. First, the rate of migraine with aura was 6.3% in the present study, which is significantly lower than the 20% reported by others.^25,26 This may be due to between-study differences in population demographics and the possibility of selection bias in our study. Secondly, our study had a relatively small sample size, and further larger scale studies are required.

In conclusion, we have found no major role for the TNFSF10 gene polymorphism rs35975099 in the pathogenesis of migraine.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This study was funded by Research and Innovation Project for College Graduates in Jiangsu Province (CX09B_032Z).

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