Hippokratia 2020, 24(2):59-65
Güler S1, Gürkan H2, Demir S2
1Department of Neurology, 2Department of Genetics, Trakya University Faculty of Medicine, Edirne, Turkey
Background: The molecular basis of migraines is still not completely understood. Over the last 30 years, mitochondrial dysfunction has been postulated as a potential mechanism in migraine pathogenesis. This study aimed to determine whether maternal mitochondrial variation was associated with migraines with aura.
Methods: In this cross-sectional study, 50 individuals, who had been diagnosed with migraines with aura between January 2016 and July 2018 in the Neurology Department of the University Medical Faculty, and 50 healthy controls were recruited. Genomic DNA was isolated from the Ethylenediaminetetraacetic acid (EDTA) blood samples of the patients and the controls using the Easy One automated DNA isolation system. Mitochondrial DNA (mtDNA) libraries were prepared according to the Nextera XT DNA library-preparation protocol, and they were sequenced on the MiSeq platform (Illumina Inc., San Diego, CA, USA).
Results: In the patient and control groups’ analysis, 13 mtDNA variations were determined to be significantly different (p <0.05). The CC genotype for NC_012920.1: m.8277T>C variation was found to be higher in the patient group than the control group (p =0.001). The mtDNA NC_012920.1: m.8277T>C variation was significantly associated with the presence of neurological disease in the patient’s family (p =0.043).
Conclusions: The present study is the ﬁrst to demonstrate an association between mitochondrial dysfunction and the susceptibility to migraine with aura in individuals carrying the NC_012920.1: m.8277T>C variation. Knowing the level of cytochrome C oxidase and oxidative phosphorylation corruption in these patients may be predictive in understanding the phenotype/genotype relationship. Thus, mtDNA variations may contribute to the pathogenesis of migraines with aura. HIPPOKRATIA 2020, 24(2): 59-65.
Keywords: Aura, migraine, molecular basis, mitochondrial DNA, mitochondrial dysfunction
Corresponding author: Assoc. Dr. Prof. Dr. Sibel Güler, Department of Neurology, Trakya University Faculty of Medicine, Edirne, TR 22030, Turkey, tel: +902842364981, fax: +902842234203, e-mail: email@example.com
Over the last 30 years, mitochondrial dysfunction has been proposed as a potential mechanism in migraine pathogenesis. The relationship between some subtypes of migraine and mitochondrial DNA (mtDNA) mutations has been demonstrated. It has been suggested that abnormal oxidative metabolism in the brain, due to mutations in mtDNA, may increase sensitivity to migraines1.
Mitochondrial dysfunction is associated with a diversity of nervous system diseases. It has been declared that mitochondrial function is frequently impaired in patients with migraines, and mitochondrial dysfunction is contained in the pathophysiology of migraines2-4. In the 1980s, scientists first hypothesized a connection between migraines and mitochondrial disorders. Recently studies have proposed that at least some subtypes of migraines may be related to mitochondrial disturbance2. Maternal inheritance of migraines also makes mitochondrial etiology especially interesting. Indeed, lactic acidosis develops due to the impaired function of pyruvate in the Krebs cycle. Consequently, the reduction of pyruvate to lactate by lactate dehydrogenase leads to increasing concentrations of lactate5. The increased use of glycogen and glycolytic activity and flawed anaerobic metabolism results in increased lactate levels in tissues. Thus, depending on the degree of oxidative metabolic dysfunction, lactic acidosis is the first sign of generalized metabolic acidosis in patients with migraines and may occur between or during attacks5.
Nuclear DNA and mtDNA have distinctive features. mtDNA is small, circular, and sensitive to oxidative stress. mtDNA and nuclear DNA mutations are seen in diseases such as Leigh syndrome, “mitochondrial encephalopathy, lactic acidosis, stroke-like episodes” (MELAS), and Kearns-Sayre syndrome. Some of these diseases are associated with the migraine phenotype2,6. mtDNA variations (16.519 CT and 3010 G-A) have also been reported to be associated with periodic cyclic vomiting syndrome and migraines. One of the main symptoms of mitochondrial disease is episodic headache2. Therefore, mitochondria are considered to play an important role in the pathogenesis of headaches, particularly migraines. In experimental and clinical studies, mitochondria were shown to play a role in migraines, causing depression and forming the clinical cascade of a migraine. As a result, it is extremely important to identify the potential mtDNA variations of the primary common headaches that cause disability in peoples’ daily lives, such as migraines.
Although there have been few studies investigating mtDNA variants and migraines, it is possible that variants in mtDNA could play a signiﬁcant role in the disorder. Systematic screening studies have failed to demonstrate the exact relationship between migraine headaches and mtDNA abnormalities. We, therefore, consider the possibility that mitochondrial mutations may play some role in migraine with prolonged aura. Therefore, we screened the whole mtDNA of all the patients and controls comprising 16,569 nucleotides. The present study aimed to investigate the frequency of the mtDNA variations in with-aura migraine patients in the Edirne population.
Material and Methods
This cross-sectional study was approved by the University of Trakya, Medical Faculty Ethics Committee (decision No 2014/26, date: 13/2/2014). All the procedures complied with the 1964 Declaration of Helsinki and its subsequent amendments or comparable ethical standards. Informed consent was obtained from the participants. A total of 100 individuals were recruited: 50 with migraines with aura [12 (24 %) males, 38 (76 %) females] and 50 healthy controls [10 (20 %) males and 40 (80 %) females]. The participants with migraines had all been referred to the Neurology Polyclinic of the Medical Faculty, Trakya University, Edirne, Turkey, between January 2016 to July 2018, and they had been diagnosed according to the International Classification of Headache Disorders (ICHD-II) criteria7. The need to recruit a total of 96 cases for the patient and control groups was calculated on the basis that the mtDNA variation might be observed as 0.14 in the control group, with an odds ratio (OR) of 2.21, in order to find an mtDNA variation and migraine association at 80 % power and 5 % probability of error (Medcalc v12.12.0). However, considering possible case losses, 100 people (50 patients and 50 controls) were recruited to adhere to our sample size calculation. Patients with cardiovascular, renal, hepatic, gastrointestinal, pulmonary, endocrine, oncologic, autoimmune, respiratory, or inflammatory diseases were excluded. The controls were selected randomly; the individuals in this group have a similar age and gender, and they have no cardiovascular, renal, hepatic, gastrointestinal, pulmonary, endocrine, oncologic, autoimmune, respiratory, inflammatory, or psychiatric diseases.
mtDNA variation determination
Peripheral blood samples from both patients and controls were drawn into 2 mL EDTA tubes. DNA isolation from peripheral blood samples was performed using Qiagen DNA isolation kits (EZ1® DNA Blood 200 µL Kit) with an EZ1 Advanced XL Nucleic Acid Isolation system (Qiagen, Hilden, North Rhine-Westphalia, Germany). Consequently, DNA concentration and purity of isolated DNA samples were measured using a Nano Drop 2000C device (Thermo Fisher Scientific Inc., Wilmington, MA, USA). In the patient and control groups included in the study, mtDNA sequencing was performed using Next Generation Sequencing (NGS) technology. For this purpose, mitochondrial DNA samples were amplified via a long-range polymerase chain reaction (PCR). PCR primers for long-range amplification were optimized using nested sets of primers previously defined by Tanwar et al8. Five single plex long-range PCR reactions were performed for each sample to amplify the whole mitochondrial genome. PCR products were then processed according to Nextera XT DNA Library Preparation (Illumina Inc., San Diego, CA, USA) protocol.
Primary data analysis was performed on Miseq Reporter Software (Illumina Inc., San Diego, CA, USA). Fastq files taken from Miseq Reporter Software were directly submitted to mtDNA server9. Visual evaluation of the data was performed using the Integrative Genomics Viewer (IGV, Broad Institute, Cambridge, USA) program. mtDNA variants were compared according to the literature and MitoMap database.
Statistical evaluation was performed using the IBM SPSS Statistics for Windows, Version 21.0. (IBM Corp., Armonk, NY, USA). One-sample Kolmogorov-Smirnov test was used to assess the eligibility for normal distribution of measured data, and since the data did not show a normal distribution, the Mann Whitney U test and the Kruskal-Wallis analysis of variance were used for comparison between groups. Pearson’s chi-squared test, Fisher’s exact test, and the Kolmogorov-Smirnov two-sample test were used to analyze qualitative data. Median (minimum-maximum) values and mean value ± standard deviation (SD) were determined as descriptive statistics. The significance limit was set at p <0.05 for all statistics.
The mean age of the male and female patients in our migraine with aura group was 35.7 (SD: 1.89) and 37.4 (SD: 1.67), respectively. The mean age of the male and female controls was 40.2 (SD: 1.60) and 38.8 (SD: 1.76), respectively (Table 1). There was no significant difference in terms of average age or body mass index (BMI) (p =0.589) between the patient and control groups. Moreover, there was no statistically significant difference between time after migraine diagnosis, frequency of migraines, family history, stroke, and coronary artery disease. Other clinical features of the individuals in the patient and control groups are summarised in Table 1. The genotypes of the patients were evaluated, and haplotype analysis was performed. Unlike other studies, a total of 16,569 mtDNA variations were studied in this research. As a result of the analysis between the patient and the control group, 13 mtDNA variations were determined to be statistically significant (p =0.001, or p =0.012, or p =0.027) (Table 2). Moreover, the CC genotype of the NC_012920.1: m.8277T> C variation in the mt-NC7 locus was found to be higher in the patient group than the control group (Table 2). This result supports the theory that the CC genotype may be associated with migraines with aura. Mutant genotypes of the other variations were higher in the control group than the patient group (Table 2), suggesting that the CC genotype of this variation may protect against migraines with aura. Also, in the human map mtDNA is located in the mt-NC7 locus (molecular location: base pairs 7,586 to 8,269 on mtDNA)10 as shown in Figure 1. The clinical characteristics associated with migraine with aura and the mt-NC7 m.8277T>C DNA variations are summarised in Table 3. There was no statistically significant difference between coronary artery disease presence in the family and the frequency of the mtDNA variation of mt-NC7 m.8277T>C DNA (p =0.056). However, a family history of coronary artery disease may be an important factor in the variation of mt-NC7 m.8277T>C DNA. A statistically significant difference was found between the presence of neurological disease in the patient’s family and the frequency of the mtDNA variation mt-NC7 m.8277T>C DNA (p =0.043), as shown in Table 3. It is important to consider the presence of the mt-NC7 m.8277T>C DNA variation in relation to the etiopathogenesis of other neurological diseases (stroke, myopathy, epilepsy, etc.). Although the HVOe haplotype was found to be higher in the patient group, no statistically significant difference was observed (p =0.076). Although the H2a2, T2b, H5, and T haplotypes were found to be higher in the control group, no statistically significant difference was observed (p =0.362, p =0.357, p =0.242, and p =0.495, respectively). The analysis of mtDNA variations’ results that were considered to be statistically significant revealed 85 haplogroups in the patient and control groups, as shown in Table 4.
Figure 1: This suggests that the CC genotype of this variation may protect against migraines with aura. The mT-NC7 locus (Molecular Location: base pairs 7,586 to 8,269 on mitochondrial DNA) is shown in the map of human mitochondrial DNA.
The mitochondrial component of migraines is suggested to be important in the pathophysiology of migraines with aura11,12. Impaired energy metabolism is a common feature of migraines13. In experimental and clinical studies, it has been shown that mitochondria play a role in migraine’s progress, the cortical spreading of depression, and the generation of the clinical cascade of migraines2. It has been stipulated that abnormal oxidative metabolism in the brain due to variations in mtDNA may increase susceptibility to migraines14. Mitochondrial dysfunction may be one of the underlying reasons that cortical hyperexcitability has been demonstrated in individuals with migraines15.
mtDNA variants have been associated with various disorders, including cancer, diabetes, Alzheimer’s, Parkinson’s, stroke, cardiomyopathy, mental retardation, and other complex diseases4. Although there have been few studies investigating mtDNA variants and migraines, it is possible that variants in mtDNA could play a signiﬁcant role in the disorder. Guo et al16 noticed a high prevalence of migraine in individuals with the mtDNA 3243A>G variation. This evidence suggested a clinical association between a monogenetically inherited disorder of mitochondrial dysfunction and susceptibility to migraine. Also, Guler et al determined significant differences in the distribution of eNOS haplotypes in patients with migraines, with and without aura17. Molecular genetic studies have not detected specific mtDNA variations in patients with migraines, although other studies suggest that particular genetic markers (i.e., neuronal polymorphisms or secondary mtDNA mutations) might be present in some migraine sufferers5. However, we detected some specific mtDNA variations associated with migraine patients with aura.
Our findings showed that, as expected, females have a higher risk of migraine than men. Our next aim was then to assess if there was a gender-biased transmission. We found that the mothers of probands were more frequently affected than expected. This biased transmission could be explained by a maternally inherited factor, such as mtDNA18. One study reported that the ratio of affected probands’ fathers is lower than the ratio of affected mothers and siblings, which is evidence in favor of a maternally inherited factor, according to Boles et al18. Although mtDNA by itself may not explain the gender differences found since migraine is a complex disease with several genetic factors variants in mtDNA, or in nuclear genes affecting mitochondrial mechanisms, could influence migraine susceptibility. Also, it has been suggested that impairment of mitochondrial metabolism could lower the threshold for migraine attacks5.
The Mitochondrially Encoded Cytochrome C Oxidase II (mT-CO2) gene has been previously associated with encephalomyopathy, late-onset Alzheimer’s disease19. Leber’s Hereditary Optic Neuropathy LHON20, myopathy21, hypertrophic cardiomyopathy22, Alpers-Huttenlocher-like disease23, pseudoexfoliation glaucoma24, and rhabdomyolysis25. The mt-CO2 gene is a protein-coding gene that encodes for the second subunit of cytochrome C oxidase (complex IV), which is a component of the mitochondrial respiratory chain that catalyzes the reduction of oxygen to water. mt-CO2 is one of the three subunits responsible for forming the functional core of the cytochrome C oxidase mt-CO2. Among its related pathways are gene expression and the epidermal growth factor (EGF)/EGF receptor (EGFR) signaling pathway. Gene Ontology annotations related to this gene include oxidoreductase activity and cytochrome C oxidase activity. mt-CO2 has been described in relation to cytochrome C oxidase subunit II26.
On the other hand, the mt-TK gene provides instructions for a specific form of tRNA designated as tRNALys. The tRNALys molecule is involved in the production of proteins that carry out oxidative phosphorylation. A mutation in the mt-TK gene was found in maternally inherited diabetes and deafness (MIDD)10,27. Several mutations in the mt-TK gene were identified in people with myoclonic epilepsy with ragged-red fibers (MERRF). A small number of subjects with a mutation in the mT-TK gene demonstrate both features of MERRF and some of MELAS. These affected individuals are said to have MERFF/MELAS overlap syndrome28.
In addition, the mt–TK gene has been identified in Leigh Syndrome. In the literature, the Mt-CO2 7896 variation is associated with the lack of cytochrome C oxidase level29. The mt-NC7 locus is located between mt-CO2 and mt-TK loci even though it is defined as a non-coding nucleotide region in mitochondrial DNA function. In our study, the 8277 variation of the mt-NC7 locus was first shown to be associated with migraines with aura. Because it is located between mt-NC7 locus, mt-CO2, and mt-TK loci, the investigation of the cytochrome C oxidase levels in migraine patients with aura is thought to be predictive in terms of the phenotype/genotype relationship. The NC_012920.1: m.8277T> C variation in the mt-NC7 locus associated with migraines with aura, and this is the first study to show the effect of mtDNA variations on aura migraines’ pathogenesis in four different gene variations and 13 different positions. Therefore, it is thought that our work provides significant contributions to the literature.
Until now, no specific mtDNA variation and locus has been associated with aura migraine. In our study, the CC genotype of the NC_012920.1: m.8277T> C variation in the mt-NC7 locus was found to be higher in migraines with aura than in the control group. Hence, it is the first study to indicate the mt-NC7 locus, which is a non-coding nucleotide region in mitochondrial DNA function, as a possible risk factor for aura migraine. In addition, we found 12 different mtDNA variations that protect against with-aura migraines. Our investigation is the first study showing that four loci (mt-NC7, mt-DLOOP1, mt-DLOOP2, mt-ND6) may be important in the pathogenesis of migraines.
Haplotype analysis of genetic markers’ combinations within a chromosome cluster location is valuable because it provides a more powerful approach to genetic studies. Because only the analysis of a single nucleotide can be performed each time, it is possible to eliminate inconsistencies30. Our study found that the H2a2, T2b, H5, and T haplotypes to be high in the control group. However, no statistically significant difference was found between the patient group. It may be shown in future studies whether the presence of these haplotypes is protective against the formation of migraines with aura or not.
One of our study’s limitations is that the number of patients was low due to the kit’s high cost. Our study found a variation related to migraines with aura, but these results need to be studied in different populations with larger numbers of patients. Similarly, no statistically significant difference was found between haplotype frequencies. Also, the study design was cross-sectional rather than longitudinal. Following up the subjects’ clinical progression with the NC_012920.1: m.8277T>C variation CC genotype would be of value to assess whether they developed more severe aura migraines over time. One of our study’s strengths is the genetic study’s geographic extent for mitochondrial variations associated with aura migraines, as it contains the Edirne provincial center and all of its districts. Edirne is located in a region that has not experienced a massive population displacement or migration. This situation is thought to provide homogeneity of genetic studies. Another strength of our study is that it showed that mainly four loci were important in the pathogenesis of migraines with aura, and three of them were protective against migraines with aura. This study is extremely important because it is the first clinical study that shows that the mT-NC7 locus may play a role in the pathogenesis of migraine with aura.
In the future, a study that conducts full mitochondrial genome sequencing should prove useful in determining the role of mitochondrial and mitochondria-related genes in the formation of migraines. Moreover, sequencing would determine all the nuclear encoding genes and non-coding nucleotide regions affecting mitochondrial function in a large migraine patient cohort. Such a study would contribute to current knowledge about migraine pathogenesis and identify genetic variants that affect the risk of developing migraines.
Conflict of interest
The authors report no conflicts of interest.
This research was backed by the Trakya University Scientific Research Projects Unit, Edirne, Turkey.
1. Shimomura T, Kowa H, Nakano T, Kitano A, Marukawa H, Urakami K, et al. Platelet superoxide dismutase in migraine and tension-type headache. Cephalalgia. 1994; 14: 215-218; discussion 181.
2. Yorns WR Jr, Hardison HH. Mitochondrial dysfunction in migraine. Semin Pediatr Neurol. 2013; 20: 188-193.
3. Colombo B, Saraceno L, Comi G. Riboflavin and migraine: the bridge over troubled mitochondria. Neurol Sci. 2014; 35 Suppl 1: 141-144.
4. Stuart S, Griffiths LR. A possible role for mitochondrial dysfunction in migraine. Mol Genet Genomics. 2012; 287: 837-844.
5. Sparaco M, Feleppa M, Lipton RB, Rapoport AM, Bigal ME. Mitochondrial dysfunction and migraine: evidence and hypothe-ses. Cephalalgia. 2006; 26: 361-372.
6. Zaki EA, Freilinger T, Klopstock T, Baldwin EE, Heisner KR, Adams K, et al. Two common mitochondrial DNA polymor-phisms are highly associated with migraine headache and cyclic vomiting syndrome. Cephalalgia. 2009; 29: 719-728.
7. Headache Classification Subcommittee of the International Headache Society. The International Classification of Headache Disorders: 2nd edition. Cephalalgia. 2004; 24 Suppl 1: 9-160.
8. Tanwar M, Dada T, Sihota R, Dada R. Mitochondrial DNA analysis in primary congenital glaucoma. Mol Vis. 2010; 16: 518-533.
9. Weissensteiner H, Forer L, Fuchsberger C, Schöpf B, Kloss-Brandstätter A, Specht G, et al.mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud. Nucleic Acids Res. 2016; 44: W64-W69.
10. Berkovic SF, Shoubridge EA, Andermann F, Andermann E, Carpenter S, Karpati G. Clinical spectrum of mitochondrial DNA mutation at base pair 8344. Lancet. 1991; 338: 457.
11. Montagna P. Magnetic resonance spectroscopy in migraine. Cephalalgia. 1995; 15: 323-327.
12. Okada H, Araga S, Takeshima T, Nakashima K. Plasma lactic acid and pyruvic acid levels in migraine and tension-type headache. Headache. 1998; 38: 39-42.
13. Lodi R, Tonon C, Testa C, Manners D, Barbiroli B. Energy metabolism in migraine. Neurol Sci. 2006; 27 Suppl 2: S82-S85.
14. Sangiorgi S, Mochi M, Riva R, Cortelli P, Monari L, Pierangeli G, et al. Abnormal platelet mitochondrial function in patients affected by migraine with and without aura. Cephalalgia. 1994; 14: 21-23.
15. Aurora SK, Welch KM, Al-Sayed F. The threshold for phosphenes is lower in migraine. Cephalalgia. 2003; 23: 258-263.
16. Guo S, Esserlind AL, Andersson Z, Frederiksen AL, Olesen J, Vissing J, et al. Prevalence of migraine in persons with the 3243A>G mutation in mitochondrial DNA. Eur J Neurol. 2016; 23: 175-181.
17. Güler S, Gürkan H, Tozkir H, Turan N, Çelik Y. An Investigation of the Relationship between the eNOS Gene Polymorphism and Diagnosed Migraine. Balkan J Med Genet. 2015; 17: 49-59.
18. Boles RG, Gardner A. Sex ratios and mitochondrial genetics in migraine. Cephalalgia 2008; 28: 1001-1002; author reply 1002.
19. Clark KM, Taylor RW, Johnson MA, Chinnery PF, Chrzanowska-Lightowlers ZM, Andrews RM, et al. An mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II gene results in lower levels of the protein and a mitochondrial encephalomyopathy. Am J Hum Genet.1999; 64: 1330-1339.
20. Abu-Amero KK, Bosley TM. Mitochondrial abnormalities in patients with LHON-like optic neuropathies. Invest Ophthalmol Vis Sci. 2006; 47: 4211-4220.
21. Rahman S, Taanman JW, Cooper JM, Nelson I, Hargreaves I, Meunier B, et al. A missense mutation of cytochrome oxidase subunit II causes defective assembly and myopathy. Am J Hum Genet. 1999; 65: 1030-1399.
22. Wei YL, Yu CA, Yang P, Li AL, Wen JY, Zhao SM, et al. Novel mitochondrial DNA mutations associated with Chinese fa-milial hypertrophic cardiomyopathy. Clin Exp Pharmacol Physiol. 2009; 36: 933-939.
23. Uusimaa J, Finnilä S, Vainionpää L, Kärppä M, Herva R, Rantala H, et al. A mutation in mitochondrial DNA-encoded cyto-chrome c oxidase II gene in a child with Alpers Huttenlocher-like disease. Pediatrics. 2003; 111: e262-e268.
24. Abu-Amero KK, Bosley TM, Morales J. Analysis of nuclear and mitochondrial genes in patients with pseudoexfoliation glaucoma. Mol Vis. 2008; 14: 29-36.
25. McFarland R, Taylor RW, Chinnery PF, Howell N, Turnbull DM. A novel sporadic mutation in cytochrome c oxidase subunit II as a cause of rhabdomyolysis. Neuromuscul Disord. 2004; 14: 162-166.
26. MT-CO2 Gene (Protein Coding). Available at: https://www.genecards.org/cgi-bin/carddisp.pl?gene=MT-CO2, date accessed: 20/09/2018.
27. Kameoka K, Isotani H, Tanaka K, Azukari K, Fujimura Y, Shiota Y, et al. Novel mitochondrial DNA mutation in tRNA(Lys) (8296A–>G) associated with diabetes. Biochem Biophys Res Commun.1998; 245: 523-537.
28. Lorenzoni PJ, Scola RH, Kay CS, Arndt RC, Silvado CE, Werneck LC.MERRF: Clinical features, muscle biopsy and molecular genetics in Brazilian patients. Mitochondrion. 2011; 11: 528-532.
29. Campos Y, García-Redondo A, Fernández-Moreno MA, Martínez-Pardo M, Goda G, Rubio JC, et al. Early-onset multisystem mitochondrial disorder caused by a nonsense mutation in the mitochondrial DNA cytochrome C oxidase II gene. Ann Neu-rol. 2001; 50: 409-413.
30. Crawford DC, Nickerson DA. Definition and clinical importance of haplotypes. Annu Rev Med. 2005; 56: 303-320.