Association of the Pentanucleotide Repeat Polymorphism in NOS2 Promoter Region with Susceptibility to Migraine in a Chinese Population

Abstract

Genes involved in the production of nitric oxide (NO) have been suggested as genetic factors for migraine. It has been studied whether polymorphisms in the genes encoding for different types of NO synthase (NOS) could be involved in the liability to migraine; however, most studies yield negative results. The pentanucleotide repeat microsatellite in the promoter region of inducible NOS (NOS2) shows highly significant differences in allelic frequencies among ethnically diverse populations. Thus, variation in the number of pentanucleotide repeats may have some significance in the predisposition to migraine among different human populations. The aim of this study was to investigate the possible association between pentanucleotide repeat polymorphism and the risk for migraine in Chinese population. We studied the genotypic and allelic frequencies of the pentanucleotide repeat polymorphism in the promoter region of NOS2 in 504 patients with migraine and 512 healthy controls, using polymerase chain reaction amplification and polyacrylamide gel electrophoresis analyses. Comparison of global allele counts between patients and controls showed that the difference was significant (p = 0.0014). The carriage of 9-repeat and 10-repeat alleles was significantly more common in controls, whereas 11-repeat allele was more common in patients after Bonferroni correction for multiple comparisons. A specific analysis of the different cutoffs for number of repeats showed that allelic and genotypic frequencies for the 9-repeat and 10-repeat cutoff were significantly different between cases and controls (p = 0.007 and p = 0.005 for allelic frequencies, respectively; p = 0.0086 and p = 0.0033 for genotypic frequencies, respectively). Our results implied an association between NOS2 pentanucleotide repeat polymorphism and migraine susceptibility in a Chinese population. Considering the significant allelic frequency differences in ethnically diverse populations, further replication studies, especially in ethnically different groups, were necessary to fully establish the role of NOS2 polymorphism in migraine susceptibility.

Introduction

M igraine is a common neurovascular disorder with a multifactorial mode of inheritance and it affects a significant proportion of the population worldwide (Stovner et al., 2007). The most common forms of this disorder have been classified as migraine with aura (MA) and migraine without aura (MO) (Bär et al., 1997). The precise pathophysiology of this primary headache is still unknown, although “sterile inflammation” or “neurogenic inflammation” seems to play a key role (de Vries et al., 2009). Migraine has a strong genetic component, and identification of genetic factors conferring migraine susceptibility will be important for our understanding of its pathophysiological mechanisms. Many candidate gene association studies have been performed, mainly of genes involved in the serotonin and dopamine pathways and other genes with an already suspected function in migraine pathophysiology (Fernandez et al., 2006; Stam et al., 2008). Genes involved in the production of nitric oxide (NO) have been suggested as genetic factors for migraine, because NO plays an important role in vasodilatation, neurotransmission, and inflammation (Olesen, 2008). NO is synthesized by NO synthase (NOS), which has three different isoforms: inducible NOS (NOS2), neuronal NOS, and endothelial NOS (Bredt, 1999). NOS2 is rarely detectable, but it can be induced by a variety of stimuli unrelated to intracellular calcium. Many cells including neurons and particularly glial cells are capable of expressing NOS2, which, when stimulated, can produce NO in quantities up to 1000 times more than those produced by endothelial NOS and neuronal NOS. It has been studied whether polymorphisms in the genes encoding for the different types of NOS could be involved in the liability to migraine (Griffiths et al., 1997; Lea et al., 2001; Borroni et al., 2006; Toriello et al., 2008). The derived states from most studies have shown negative association of NOS polymorphisms with migraine susceptibility (Stam et al., 2008). A pentanucleotide repeat microsatellite, (CCTTT)n, in the promoter region of NOS2 has been identified (Xu et al., 1997), and the level of interleukin-1β–induced inducible NOS expression has been reported to vary according to the length of this functional polymorphism (Warpeha et al., 1999). It has been the focus of various studies to examine the relation between this microsatellite and diverse diseases (Kun et al., 1998; Morris et al., 2002; Gonzalez-Gay et al., 2004; Liao et al., 2006). Also, a possible role of this polymorphism in cluster headache has been suggested (Sjöstrand et al., 2002). Moreover, a report from Xu et al. (2000) has demonstrated that there are highly significant differences in allelic frequencies for this pentanucleotide repeat among ethnically diverse populations. In view of this, we seek to assess the contribution of the NOS2 pentanucleotide repeat polymorphism to migraine susceptibility in a Chinese population by employing an association study approach in groups of migraine patients and controls.

Materials and Methods

Study populations

Migraine patients were outpatients who had made their first visit or a follow-up visit to the headache clinic of the Department of Neurology at No. 1 Affiliated Hospital of Soochow University during the period between October 2008 and March 2010. There were totally 504 unrelated migraine patients recruited in this study. All patients were diagnosed by clinical neurologists in our team based on the International Criteria for Headache Disorders, 2nd edition (Headache Classification Subcommittee of the International Headache Society, 2004), after administration of a structured questionnaire and direct interview. A total of 512 unrelated healthy blood donors without any kind of headache were selected from a community nutritional survey conducted in the same region during the same period of recruitment of migraine patients. Migraine and positive family history of migraine or any type of severe or recurrent headache in first-degree relatives were excluded in all control subjects through personal interview. Ethnic origin was determined by appearance and self-declaration. All individuals included in this study came from Chinese Han ethnic group. To minimize potential bias from population stratification, cases and controls were matched for sex and age. All participants gave their informed consent to participate in the study and the design of the study was approved by the Ethical Committee of Soochow University.

Genotype determination of NOS2 pentanucleotide repeat polymorphism

A Chelex method was used for extracting genomic DNA of blood samples (Walsh et al., 1991). Polymerase chain reaction (PCR) was used to determine the genotypes of the polymorphism from genomic DNA, using specific and previously described primers (Warpeha et al., 1999). PCR was performed in a total volume of 37.5 μL, including 3.75 μL of 10 × PCR buffer, 1.5 mM MgCl₂, 0.25 mM dNTPs, 0.5 mM each primer, 100 ng of genomic DNA, and 1.5 U of Taq DNA polymerase. The PCR conditions were 94°C for 5 min, followed by 35 cycles of 40 s at 94°C, 40 s at 60°C, and 40 s at 72°C, with a final elongation at 72°C for 5 min. The PCR products were analyzed by 7% nondenaturing polyacrylamide gel electrophoresis and visualized by silver staining (Allen et al., 1989). Allele designation was established following the recommendations of the DNA Commission of the International Society for Forensic Haemogenetics (Bär et al., 1997). Genotyping was performed by individuals blinded to the case or control status. A 10% random sample was tested in duplicate by different persons, and the reproducibility was 100%.

Statistical analysis

Genotypic frequencies for patients and controls were compared by χ ² test. Probability values for 2 × K contingency tables, in which allelic frequencies for patients and controls were compared, were calculated by χ ² test. In addition, different cutoffs (9–16 repeats) were studied. For each cutoff, alleles with an equal or fewer repeats were defined as short (S) and alleles with larger number of repeats were defined as long (L). The subjects were classified into three groups: S homozygous (S/S), heterozygous (S/L), and L homozygous (L/L). Monte Carlo simulation and Bonferroni correction for multiple comparisons were provided when required. Conditional logistic regression was used to evaluate the association between the polymorphism and migraine susceptibility. These statistical analyses were implemented in Statistic Analysis System software (version 8.0; SAS Institute, Cary, NC). Probability values of 0.01 or less were set to be a criterion of statistical significance, following recommendations published for association studies of common neurological diseases (Bird et al., 2001).

Results

The characteristics of migraine patients and controls are summarized in Table 1. There were no statistically significant differences between cases and controls in terms of the frequency distribution of sex, age, smoking, and drinking status (p > 0.05). Allelic and genotypic frequencies of the polymorphism are shown in Table 2. Thirteen different alleles were detected corresponding to 7–19 repeats in our samples. There were a total of 48 and 51 different genotypes observed in cases and controls, respectively. To facilitate analysis, nine higher frequency alleles (9–17 repeats) were used. Alleles with frequencies lower than 0.03 were combined with the adjacent alleles. Genotypic distributions had no deviation from Hardy–Weinberg equilibrium in both case and control groups (both p > 0.05). The most frequent allele contained 10 repeats for both groups. For the overall distribution of alleles, a statistical difference was observed between the control and patient group (p = 0.0014). The carriage of 9-repeat and 10-repeat alleles was significantly more common in controls, whereas 11-repeat allele was more common in patients (Table 2). This difference was still significant after Bonferroni correction for multiple comparisons (corrected α level = 0.00138). A specific analysis of different cutoffs for repeat number is shown in Tables 3 and 4. A significant difference was detected in allelic and genotypic frequencies for the 9-repeat and 10-repeat cutoff between cases and controls (p = 0.007 and p = 0.005 for allelic frequencies, respectively; p = 0.0086 and p = 0.0033 for genotypic frequencies, respectively). At 10-repeat cutoff level, conditional logistic regression analysis showed that homozygote S/S genotype was associated with a significantly decreased risk of migraine compared with homozygote L/L (odds ratio = 0.47, 95% confidence interval: 0.26–0.85) (Table 4). Further, we performed stratified analysis based on age, sex, age at onset of migraine, smoking status, drinking status, and family history. The genotypic and allelic frequencies were not influenced by these factors (Supplementary Tables S1 and S2; Supplementary Data are available online at www.liebertonline.com/dna).

Table 1.

Summary of the Characteristics Among Migraine Patients and Controls

	Case		Control
Characteristics	n = 504	Frequencies (%)	n = 512	Frequencies (%)
Age (mean ± SD)	34.5 ± 11.2		34.4 ± 10.2
Gender
Male	145	28.8	148	28.9
Female	359	71.2	364	71.1
Pain severity^a
Low	76	15.1
Moderate	81	16.1
Severe	347	68.8
Frequency of attacks
Up to 1/month	93	18.5
2–4/month	221	43.8
More than 5/month	190	37.7
Smoking status^b
Smoker	56	11.1	64	12.5
Nonsmoker	448	88.9	448	87.5
Drinking status^c
Drinker	94	18.7	101	19.7
Nondrinker	410	81.3	411	80.3
Age at onset of migraine	24.7 ± 9.5
Family history of migraine
Positive	211	41.9
Negative	293	58.1
Classification of migraine
Migraine with aura	33	6.5
Migraine without aura	471	93.5
Duration of the migraine (years)	9.8 ± 7.9

Pain severity was graded as follows: low pain did not interfere with daily activities; moderate pain interferes with daily activities; severe pain impedes daily activities.

The subjects who smoked more than one cigarette per day for >1 year were classified as smokers.

Subjects were considered as alcohol drinkers, if they drank at least once per week.

SD, standard deviation.

Table 2.

Genotypic and Allelic Frequencies of NOS2 Pentanucleotide Repeat in Migraine Patients and Controls

	Genotypic frequency					Allelic frequency ^a
Repeat number	Patients n = 504	%	Controls n = 512	%	p-Value	Patients n = 1008	%	Controls n = 1024	%	p-Value
9^b	39	7.7	64	12.5	NS	40	4.0	68	6.6	0.0073
10^b	189	37.5	217	42.4	NS	205	20.3	238	23.2	NS
11^b	174	34.5	133	26.0	0.0068	194	19.2	140	13.7	0.0006
12	159	31.5	171	33.4	NS	169	16.8	190	18.6	NS
13	130	25.8	117	22.9	NS	139	13.8	127	12.4	NS
14	82	16.3	68	13.3	NS	83	8.2	69	6.7	NS
15	62	12.3	52	10.2	NS	65	6.4	53	5.2	NS
16	67	13.3	81	15.8	NS	67	6.6	81	7.9	NS
17	44	8.7	58	11.3	NS	46	4.6	58	5.7	NS

A probability value for a 2 × 9 contingency table was calculated by χ ² test for comparison of global allele counts in patients and controls. The comparison was significant (p = 0.0014).

After Bonferroni correction for multiple comparisons (corrected α level = 0.00138), significant differences were observed at 9 versus 11, as well as 10 versus 11 between patient and control groups (p = 0.00014, OR = 0.42, 95% CI: 0.26–0.68 and p = 0.0011, OR = 0.62, 95% CI: 0.46–0.84, respectively).

CI, confidence interval; NS, nonsignificant; OR, odds ratio.

Table 3.

Allelic Frequencies of Pentanucleotide Repeat Polymorphism for Different Repeat Cutoffs in Patients and Controls

	Case				Control
Repeat	S	%	L	%	S	%	L	%	Monte Carlo ^a p-value	OR (95% CI)
9	40	4.0	968	96.0	68	6.6	956	93.4	0.007	0.58 (0.38–0.88)
10	245	24.3	763	75.7	306	29.9	718	70.1	0.005	0.75 (0.62–0.92)
11	439	43.6	569	56.4	446	43.6	578	56.4	NS	—
12	608	60.3	400	39.7	636	62.1	388	37.9	NS	—
13	747	74.1	261	25.9	763	74.5	261	25.5	NS	—
14	830	82.3	178	17.7	832	81.2	192	18.8	NS	—
15	895	88.8	113	11.2	885	86.4	139	13.6	NS	—
16	962	95.4	46	4.6	966	94.3	58	5.7	NS	—

Monte Carlo p-value after 10⁴ simulations.

Table 4.

Genotypic Frequencies of Pentanucleotide Repeat Polymorphism for Different Repeat Cutoffs and the Association with Migraine

Cutoff	Genotypes	Patients n = 504	Controls n = 512	OR	95% CI	p-Value
9	L/L	465	448	1.00
	L/S	38	60	0.61	0.39–0.95	NS
	S/S	1	4	0.24	0.00–2.45	NS
	p _trend					0.0086
10	L/L	280	245	1.00
	L/S	203	228	0.78	0.60–1.01	NS
	S/S	21	39	0.47	0.26–0.85	0.007
	p _trend					0.0033
11	L/L	155	168	1.00
	L/S	259	242	1.16	0.87–1.55	NS
	S/S	90	102	0.96	0.66–1.39	NS
	p _trend					0.99
12	L/L	81	75	1.00
	L/S	238	238	0.93	0.63–1.35	NS
	S/S	185	199	0.86	0.58–1.27	NS
	p _trend					0.41
13	L/L	40	43	1.00
	L/S	181	175	1.11	0.67–1.84	NS
	S/S	283	294	1.03	0.64–1.68	NS
	p _trend					0.84
14	L/L	20	26	1.00
	L/S	138	140	1.28	0.65–2.52	NS
	S/S	346	346	1.30	0.69–2.47	NS
	p _trend					0.54
15	L/L	11	12	1.00
	L/S	91	115	0.86	0.34–2.22	NS
	S/S	402	385	1.14	0.46–2.80	NS
	p _trend					0.12
16	L/L	2	0	—	—
	L/S	42	58	—	—	NS
	S/S	460	454	—	—	NS
	p _trend					0.25

Discussion

NO appears to be one of the important mediators in the pathophysiology of vascular headache (Olesen et al., 1994). The NOS2 pentanucleotide repeat polymorphism has been investigated in several population groups to evaluate its association with diverse diseases. However, the microsatellite allelic distribution shows significant worldwide variation and the significant interethnic variation highlights the necessity to delineate the molecular epidemiology of disease susceptibility in different populations (Xu et al., 2000). Thus, there is a sound scientific basis for us to perform the present genetic association study. As far as we know, the present study is the first attempt to assess the implication of NOS2 pentanucleotide repeat polymorphism in the susceptibility to migraine in Chinese populations.

Our data confirmed the trend of interethnic variation of this locus. The allelic frequencies in our Han ethnic Chinese were significantly different from the figures published in other populations (Xu et al., 2000). In this study, 10-repeat allele was the most common for this polymorphism, which was different from five specific population groups including African-Gambian, African-Caribbean, Caucasian, Japanese, and Indian Gujaratis. However, similar allelic distribution pattern was observed between our data and that from Hong Kong of China, which implied regional homogeneity (Leung et al., 2006).

It has been suggested that migraine may be caused by increased amounts and/or affinity of an enzyme in the NO-triggered cascade of reactions (Olesen et al., 1994). Also, electrophoretic mobility shift assay had shown that the long allele of the microsatellite exhibited specific binding of nuclear proteins, emphasizing that long forms of this polymorphism could act as an enhancer (Motallebipour et al., 2005). Increased spacing by a tandem repeat may create flexibility in the DNA chain, facilitating the interactions that flank the repeat and, thus, increasing the NOS2 promoter activity. Therefore, the enhancement of NOS2 expression promoted by these NOS2 promoter polymorphisms could actually contribute to migraine susceptibility. However, different sites in NOS2 promoter that regulated gene transcription in response to different stimuli have been described (Neufeld and Liu, 2003). Thus, the genotyping of only one polymorphism may not be sufficient in elucidating the association between migraine and NOS2 polymorphism. In addition, the analyses of subgroups between MA and MO were not adequately addressed in our study because of the small sample size of MA (33 MA cases). Perspective studies are needed to clarify this issue. Finally, it was noteworthy that our case samples were composed of a larger proportion of patients suffering from MO (93.5%) compared with other studies for migraine, especially in western countries. This variation may be due to ethnic and/or geographic differences of the study samples. Alternatively, our case samples were collected in the last 2 years in one local comprehensive hospital. Thus, we may not be able to exclude any potential selection bias during sampling process.

Studying the genetics of migraine may provide valuable insights in molecular mechanisms involved in diseases that are comorbid with this disease. Meanwhile, understanding the association of migraine with other health conditions is an important part in providing optimal care. For instance, the nature of comorbidity between migraine and hypertension is an old issue (Diener et al., 2008). It has been reported that the CCTTT repeat polymorphism is associated with the susceptibility to pulmonary arterial hypertension with systemic sclerosis (Kawaguchi et al., 2006). In addition, a bidirectional relation exists between migraine and asthma because inflammatory pathway plays an important role in the pathophysiology of these two disorders. However, no significant association between NOS2 pentanucleotide repeat polymorphism and asthma was detected in Chinese children (Leung et al., 2006).

In summary, our results implied an association between the NOS2 pentanucleotide repeat polymorphism and migraine susceptibility in our population. Individuals carrying shorter alleles (e.g., <11 repeats) seemed to have decreased risk for migraine. However, considering the significant allelic frequency differences in ethnically diverse populations, further replication studies, especially in ethnically different groups, are necessary to fully establish the role of NOS2 polymorphism in migraine and their relationships with other genes implicated in migraine susceptibility.

Footnotes

Acknowledgments

This study was supported by grants from the National Science Foundation of China (No. 30800621) and the China Postdoctoral Science Foundation (No. 20080431121, No. 200902530).

Disclosure Statement

No competing financial interests exist.

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