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Mitochondrial DNA T4216C and A4917G variations in Multiple Sclerosis
Journal of the Neurological Sciences (Available online 7 May 2015)
Multiple Sclerosis (MS) affects the brain and spinal cord and long has been the topic of global research; yet there is no commonly accepted cause and no cure for the disease. Mounting evidence supports the role of genetics in susceptibility to MS. From this perspective, a current effort focuses on the neurogenetics of the complex pathogenesis of MS in relation to factors such as mitochondrial DNA (mtDNA) variations. T4216C and A4917G are common mitochondrial gene variations associated with MS. The present study tested whether mtDNA T4216C variation in the NADH Dehydrogenase 1 (ND1) mtDNA gene and A4917G variation in the mtDNA NADH Dehydrogenase 2 (ND2) gene is associated with MS in an Iranian population.
Material and Methods
Blood samples were collected from 100 patients with MS and 100 unrelated healthy controls, and DNA extraction was performed by salting-out. By means of appropriate primers, polymerase chain reaction (PCR) amplification was carried out for the mtDNA fragment. Afterwards, the PCR products were digested using Nla III and Acc I restriction endonuclease enzymes for analysis of Restriction Fragment Length polymorphism (RFLP) in mtDNA T4216C and A4917G variations, respectively. With electrophoresis by means of 3% agarose gel and safe DNA gel stain, we imaged restriction products in a UV transilluminator. The accuracy of genotyping procedure was confirmed by sequencing the mtDNA fragment.
No significant statistical difference in the frequency of the T4216C mtDNA variation was found between the patients (24 %) and the control subjects (21 %) (P = 0.61). Logistic regression analysis showed an OR of 1.1 (95% CI = 0.5-2.4). Moreover, there was no significant statistical difference in the frequency of mtDNA A4917G variation between the cases (11 %) and the controls (9 %) (P = 0.637). Logistic regression analysis revealed an odds ratio (OR) of 1.2 with 95% CI of 0.4-3.5.
The present study revealed no association between MS and T4216C variation in the ND1 mtDNA gene and A4917G variation in the mtDNA ND2 gene in the Iranian population.
- We investigated whether mtDNA T4216C and A4917G variations are associated with MS.
- No association was found between mtDNA T4216C variation and MS.
- No association was found between mtDNA A4917G variation and MS.
Keywords: Multiple Sclerosis, MS, Mitochondrial DNA, mtDNA variation, T4216C, A4917G, Iranian population.
Multiple sclerosis (MS) is complex and unpredictable. An estimated 2,500,000 individuals throughout the world suffer from MS  . Since identification, MS has been the subject of intense and worldwide research; but its cause and cure remain elusive. Individuals aged 20-40 years are the most victims of MS  ; of these, women are more frequent  . Pathologically, MS is characterized by oligodendrocytosis and loss of myelin  and . Not only does MS affect the myelin containing white matter of the CNS  , perhaps owing to blood-derived immune cells invading the CNS  , but it also affects the gray matter  , which would appear to be due to a neurodegenerative mechanism  . The conventional perspective views MS as an inflammatory disorder of the central nervous system (CNS) but recently and increasingly, MS is held to be the result of a neurodegenerative process  and  related to genetic susceptibility. Clinically, MS patients present with a broad spectrum of symptoms, including fatigue and sleepiness  , tremor  , spasticity  and , mood disturbance and depression  and , cognitive dysfunction and memory impairment  and , fear of progression  , dysarthria  and , dysphagia  and , bladder, bowel and sexual dysfunction  and , nystagmus  , and hypoesthesia  , all of which can be ascribed to genetically determined mitochondrial dysfunction. The recent literature includes numerous reports of treatments that allegedly modify the natural history of MS, albeit MS per se remains incurable. The McDonald criteria  and expanded disability status scale (EDSS) are used to assess MS status  . Laboratory tests, viz. Magnetic Resonance Imaging (MRI), Cerebrospinal Fluid (CSF) analysis  , and Optic Coherence Tomography (OCT)  supply crucial information concerning MS. The etiology of MS has not yet been fully clarified but involves genetic factors. Thus, the first- and second-degree families of an MS patient are exposed at higher risk of MS  , and nuclear and mitochondrial DNA (mtDNA) alterations may be involved in this susceptibility as is the case with altered MHC  and HLA , , and  nuclear genes that have been shown to be involved, although mutations of nuclear genes that encode for mitochondrial proteins also may be associated with MS. Gene alterations in the mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α), and nuclear respiratory factor 1 (NRF1) that serve a crucial role in the maintenance of the mitochondrial DNA (mtDNA) content, are known to be associated with MS  and . Mitochondrial dysfunction is associated with axonal loss in MS  and  and mitochondrial dysfunction may stem from mitochondrial genetic alterations  . Compared to nuclear DNA, mtDNA is more prone to damage and mutation because of the exposure of mtDNA to the locally high mitochondrial oxidizing environment and the relatively poor repair mechanisms  . The damage is associated with inefficient ATP synthesis and generation of toxic waste products  . All cells may be affected by this phenomenon; but neural cells are particularly prone to damage, such that mtDNA mutations lead to neurological disorder as a cause or contributing factor  ; Leber’s Hereditary Optic Neuropathy (LHON)  , Myoclonus Epilepsy with Ragged Red Fibers (MERRF)  , Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like episode (MELAS)  , Alzheimer’s Disease (AD)  , Parkinson’s disease (PD)  , and MS  are among the disorders arising in this manner. Mitochondria are the cytoplasmic energy factories specialized in oxidative phosphorylation and adenosine triphosphate (ATP) production. The genome inside mitochondria, which is inherited only maternally  , is very small circular structure spanning about 16569 base pairs  , and undergoes a great deal of variation as a consequence of divergent evolution. MtDNA embodies 37 intronless genes, all of which are prerequisites of normal mitochondrial function. Thirteen of these genes provide instructions for producing enzymes essential for oxidative phosphorylation; the other genes, however, provide instructions for producing molecules called transfer RNA (tRNA) and ribosomal RNA (rRNA)  . Respiratory chain, which is responsible for oxidative phosphorylation, functions in mitochondria, includes 5 protein complexes. NADH Dehydrogenase (ND) 1, ND2, ND3, ND4, ND4l, ND5, and ND6 in complex I are encoded by mtDNA; however, all 4 subunits of complex II are encoded by nuclear genes. Cytocrome b in complex III, Cytochrome Oxidase (COX) I, COX II, and COX III in complex IV, A6 and A8 proteins in complex V are expressed by mtDNA. Complex I genes are the most vulnerable part of mtDNA  . With this in mind, genetics has emerged to become an important contributing factor in the pathogenesis of neurodegenerative diseases. One of the most significant current discussions in the etiology of MS is the role of mitochondrial dysfunction due to DNA variations or acquired mutations. Gene products of mitochondrial electron transfer chain decline in the brain while axonal damage occurs in MS  . And, cortical mitochondria show a decreased capacity in respiratory chain complex I and II  . In chronic active MS lesions, oxidative damage to mtDNA and impaired activity of complex I have been demonstrated  . Andalib et al.  reviewed mtDNA variations in MS in numerous populations. T4216C in mtDNA ND1 gene and A4917G variation in the mtDNA ND2 gene are common mtDNA variations found in MS patients  . T4216C mtDNA variation was demonstrated to be a predisposing marker of MS in a Bulgarian population  . In an American study, Kalman et al.  sequenced mtDNA in three patients with Devic's neuromyelitis optica (NMO) disease and MS and in spite of the fact that NMO was shown to be without pathogenic mtDNA point mutations, T4216C and A4917G mtDNA variations together were seen only in the one patient, notwithstanding T4216C mDNA variation in the other one. On the contrary, however, Vyshkana et al.  assessed variation candidates of complex I in American MS subjects relative to ethnically matched SLE subjects and controls by means of MassARRAY and found no association between MS and mtDNA T4216C and A4917G variations. And, the frequency of the mtDNA T4216C and A4917G variation was seen to higher than in the controls in German MS subjects  . Moreover, little is known about association of mtDNA T4216C and A4917G variations and MS in Iran. Thus, we designed the present study to test the hypothesis of an association of the mtDNA T4216C and A4917G variations to MS in an Iranian population.
2. Materials and Methods:
2.1. Study design, setting and participants:
A case-control design was used in the present study and a total number of 200 subjects involving 100 patients presenting with relapsing-remitting MS and 100 unrelated healthy controls were recruited. MS subjects were selected from several medical therapeutic centers of Tabriz University of Medical Sciences, Tabriz, Iran to avoid having similar risk factors. MS diagnosis was made according to McDonalad criteria  . Subjects with a family history of neurodegenerative or inherited diseases were excluded from the study. Control subjects were selected from healthy unrelated participants. Informed consent was obtained from each participant. In the present study, a case/control ratio of 1/1 was used; and to restrict the confounding effect, frequency and individual matchings were done in cases and controls for age and gender, respectively. The present study was approved by Ethical Committee of Tabriz University of Medical Sciences.
2.2. Variables, study size
By means of STATA software (version 12) with a test power of 80% and taking into account of study size of the previous studies, the study size was determined.
2.3. MtDNA genotyping
DNA was extracted by using a salting-out method  form blood sample collected from each subject. Quantitation of the extracted DNA samples, showing optimal DNA extraction, was assessed by the spectrophotometry. MtDNA was amplified by Polymerase Chain Reaction (PCR) using a thermocycler (Peqlab, Germany) for mtDNA by using appropriate forward and reverse primers ( Table 1 ). Using a gradient thermocycler (Peqlab, Germany), temperatures and cycling times were optimized for each DNA template target and primer pair. A previously used standard PCR protocol  and  was applied for all the reactions. The PCR products (10 μm) were digested with suitable restriction endonuclease enzymes which target sequences were influenced by the nucleotide changes (RFLP) ( Table 1 ). Electrophoresis of restriction products were carried out using 3% Agarose gel and DNA safe staining. Electrophoresis results were visualized by using a camera. DNA analyses were conducted blind to the diagnosis, and identities of subjects were known exclusively when the results were completed. PCR-RFLP findings were confirmed by sequencing. In order to confirm the PCR-RFLP results, several samples from each mtDNA variation were selected and purified with QIAquick SpinR Purification Kit and directly sequenced by Macrogen Inc. (Soth Korea), using an automated ABI Prism 3730XL DNA sequencer (Perkin-Elmer).
|MtDNA variation||Forward primer (5′-3′)||Reverse primer (3′-5′)||Endonuclease: Target sequence|
|T4216C||CACCTCTGATTACTCCTGCC||TAGGTTTGAGGGGGAATGCT||Nla III: CATG/|
|A4917G||CCAGAGGTTACCCAAGGCAC||AGTAGTAGGGTCGTGGTGCT||Mae I: C/TAG|
2.4. Statistical methods
Data analysis was carried out using STATA software (Version 12.0). More precisely, Chi-square test was used and a P-value of less than 0.05 was considered statistically significant. Moreover, odds ratio (OR) along with 95% confidence interval (95% CI) was calculated by means of bivariate logistic regression analysis.
We selected 100 cases with MS and 100 healthy unrelated control subjects. A ratio of 1 case per 1 control was applied to this study. To restrict confounding effect of age, frequency matching was carried out in 60% of cases and controls aged 20-35 years. Similarly, individual matching was carried out for gender and consequently 63 females and 37 males were selected in, both, case and control groups. Table 2 tabulates demographic summary of the cases and controls.
|Case group||Control group|
|Age (mean ± SD)||32.47 ± 8.13 y||30.24 ± 15.49 y|
|Gender||F = 63(63%) M = 37(37%)||F = 63(63%) M = 37(37%)|
3.2. Analysis of mtDNA T4216C and A4917G variations
Electrophoretic analysis of the mtDNA T4216C ( Fig. 2 ) showed that the variation was present in 24 out of 100 cases (24%) and 21 out of 100 controls (21%) ( Fig. 3 ). The Chi square analysis showed no significant association between the MS and T4216C variation (P = 0.61) and bivariate logistic regression analysis showed an OR of 1.1 ( Table 3 ). The PCR-RFLP results of mtDNA T4216C variation were confirmed by the sequencing ( Fig. 4 ). Electrophoresis analysis of the mtDNA A4917G variation ( Fig. 5 ) revealed that the variation was present in 11 of 100 the MS subjects (11%), while it was seen in 9 of 100 the healthy controls (9%) ( Fig. 6 ). Consequently, the Chi square analysis revealed no significant association between the MS and A4917G variation (P = 0.637); even so, bivariate logistic regression analysis showed an OR of 1.24 ( Table 1 ). The PCR-RFLP results of mtDNA A4917G variations were confirmed by the sequencing ( Fig. 7 ). No heteroplasmy was observed for both mtDNA variations. (See Fig. 1 .)
|Variation||Cases N (%)||Controls N (%)||P value||OR||95% CI|
|T4216C||24/100 (0.24)||21/100 (0.21)||0.611||1.1||0.5- 2.4|
|A4917G||11/100 (0.11)||9/100 (0.09)||0.637||1.2||0.4-3.5|
We assessed the association of mtDNA T4216C and A4917G variations with MS in an Iranian population and found the variation in 24 of 100 cases (24%) and 21 of 100 controls (21%). Withal, 11 out of 100 MS patients (11%) and 9 out of 100 healthy controls (9%) showed the mtDNA A4917G variation. In the present study, no significant association was seen between mtDNA T4216C and A4917G variations and MS.
Attempts to establish the association of the mtDNA T4216C and A4917G variations with MS. The published correlations in different populations are rather controversial. The present study produced results which corroborate those of Yu et al.  , assessing six European MS case-control cohorts including 5,000 participants and three well-matched cohorts. The authors assessed seven common and potentially functional mtDNA variations and found no association between MS and the mtDNA T4216C variation. The authors found mtDNA T4216C variation in 17.7% of the cases (n = 417) and 13.7% of the controls (n = 510) in the Spanish cohort and consequently no significant association (P = 0.101, OR = 1.36, 95% CI = 0.93–1.97). In the Norwegian cohort, mtDNA T4216C variation was present in 21.5% of the cases (n = 390) and 15.8% of the controls (190) with no significant association (P = 0.119, OR = 1.46, 95% CI = 0.91–2.40). In the German cohort, the mtDNA T4216C variation was demonstrated in 22.8% of cases (n = 285) and 23% of the controls (n = 382) and no significant association (P = 1, OR = 0.98, 95% CI = 0.67–1.44) was found. The pooled analysis also failed to show any association of mtDNA T4216C variation with MS (P = 0.078, OR = 1.06, 95% CI = 0.70–1.01)  . Vyshkina et al.  analyzed several mtDNA variations in Caucasian MS subjects and controls by using Sequenom MassARRAY System and reported no association between mtDNA T4216C variation and MS; however, mtDNA A4917G variation was associated with MS (P = 0.006). Chalmers et al.  investigated 175 unrelated MS patients and 233 healthy control subjects for mtDNA T4216C and A4917G variations by using RFLP analysis and demonstrated that these variations were at a similar frequency in the case and control subjects in UK and therefore not associated with MS.
T4216C mtDNA variation was seen to be a predisposing marker for MS in a study by Mihailova et al.  . The authors analyzed 58 unrelated Bulgarian subjects with relapsing–remitting MS and 104 healthy controls for secondary LHON mutations (np 4216, 14798, and 13708) and 14 main mtDNA variations showing European haplogroups. The common haplogroup-associated variations were assessed by RFLP and a significant difference was seen in T4216C base change between the MS subjects and the controls. It was also demonstrated that 21 out of the 58 subjects (36.2%) carried the T4216C variation, notwithstanding 11.3% in the controls (P = 0.01; OR = 4.38). No association was seen between G13708A, T14798C variations and MS. G13708A was seen in 4 controls and 3 cases. T14798C variations was found to be less in MS subjects (10%) than in the controls (6.9%)  . Penissen-Besnier et al.  studied mtDNA T4216C and A4917G variations in 75 unrelated Caucasian subjects with the relapsing–remitting or primary progressive MS, and 75 age-, gender- and ethnic origin-matched controls by using RFLP and sequencing and the proportion of people having these secondary LHON mutations was equal (27%) in the two groups  . In a German study, Mayr-Wohlfart et al.  studied mtDNA in 100 MS subjects with failures in visual evoked potential (VEP) and 100 controls by RFLP and sequencing and found that mtDNA T4216C variation frequency in MS subjects (18%) was higher than in the controls (11%); even so, there was a high frequency of the A4917T variation in MS cases (11%), compared to controls (4%)  . Elsewhere, an American study demonstrated certain sets of mtDNA variations with association with MS  . The authors assessed 8 variations in MS by RFLP and sequencing and found that class I LHON mutations with primary pathogenic significance for blindness were not present in the MS subjects. Class II LHON mutations with unknown pathogenic significance were also demonstrated to be more in the MS subjects than in the controls, particularly at np 4216 and its associated simultaneous mutations  . Eleven out of the 53 cases, constituting 20.8% of the subjects, carried at least two (4216 and 4917 or 13,708) or three (4216, 13,708, 15,257) simultaneous class II LHON mutations; albeit 7 out of the 74 controls, constituting 9.5% of the controls, carried simultaneous mutations (P = 0.036)  . Furthermore, the mutations were not found to be correlated with the severity of visual loss in either LHON or MS; however, such mutations were as a consequence of random selection in the MS subjects  . Another American study analyzed mtDNA variations within patients with Devic's neuromyelitis optica (NMO) disease and MS in three spinal cord specimens and found that NMO lacked pathogenic mtDNA point mutations  . Nonetheless, mtDNAT4216C and T14798C variations was found in the one of the patients, notwithstanding mtDNA T4216C in the other one  .
In conclusion, the present study suggests lack of association of the mtDNA T4216C variation in mitochondrial ND1 gene and mtDNA A4917T in mitochondrial ND2 gene with MS. Altogether, inasmuch as the degree of involvement of such mtDNA changes in pathogenesis of MS is a less divulged topic, these results need to be cautiously interpreted and more local investigations in different populations would be of value.
Conflict of Interest
We thank all the staffs involved in this study.
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a Neurosciences Research Center, Imam Reza Hospital, Tabriz University of Medical Sciences, Tabriz, Iran
b Division of Regenerative Medicine, School of Medicine, Faculty of Medical and Human Sciences, the University of Manchester, UK & Department of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
c Department of Public Health Sciences, Karolinska Institute, Stockholm, Sweden & Trauma Epidemiology and Road Traffic Injury Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
d Department of Neuroscience and Pharmacology, University of Copenhagen, Denmark
⁎ Corresponding author. Tel./fax: + 98 4133340730.
⁎⁎ Corresponding author. Tel./fax: + 98 4133370684.
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