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Table of Contents    
BRIEF REPORT
Year : 2021  |  Volume : 69  |  Issue : 2  |  Page : 461-465

Mitochondrial DNA Haplogroups and Three Independent Polymorphisms have no Association with the Risk of Parkinson's Disease in East Indian Population


1 Department of Genetics, University of Calcutta, Kolkata, West Bengal, India
2 S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, West Bengal, India
3 Movement Disorders Clinic, Bangur Institute of Neurosciences, Kolkata, West Bengal, India
4 School of Biological Sciences, RKMVERI, Narendrapur, West Bengal, India

Date of Submission09-Apr-2020
Date of Decision21-Jul-2020
Date of Acceptance07-Aug-2020
Date of Web Publication24-Apr-2021

Correspondence Address:
Dr. Mainak Sengupta
35, Ballygunge Circular Road, Kolkata - 700 019
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.314553

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 » Abstract 


Background: Parkinson's disease (PD) is a multifaceted illness affecting ~ 0.3% of the world population. The genetic complexity of PD has not been, fully elucidated. Several studies suggest that mitochondrial DNA variants are associated with PD.
Objective: Here, we have explored the possibility of genetic association between mitochondrial haplogroups as well as three independent SNPs with PD in a representative east Indian population.
Methods and Material: The Asian mtDNA haplogroups: M, N, R, B, D, M7, and 3 other SNPs: 4336 T/C, 9055 G/A, 13708 G/A were genotyped in 100 sporadic PD patients and 100 matched controls via conventional PCR-RFLP-sequencing approach.
Results: The distribution of mtDNA haplogroups, as well as 3 single polymorphisms, did not show any significant differences (P > 0.05) between patients and controls.
Conclusion: This is the first of its kind of study from India that suggests no association of selected mitochondrial DNA variations with PD.


Keywords: Genetic Association, Haplogroups, Mitochondrial DNA, Parkinson's disease, SNP
Key Messages: Several studies suggest mitochondrial DNA variants to be associated with Parkinson's disease. Our genotyping analysis found no significant association between relevant mitochondrial DNA haplogroups and three individual SNPs in representative eastern Indian cases and controls.


How to cite this article:
Saha T, Roy S, Chakraborty R, Biswas A, Das SK, Ray K, Ray J, Sengupta M. Mitochondrial DNA Haplogroups and Three Independent Polymorphisms have no Association with the Risk of Parkinson's Disease in East Indian Population. Neurol India 2021;69:461-5

How to cite this URL:
Saha T, Roy S, Chakraborty R, Biswas A, Das SK, Ray K, Ray J, Sengupta M. Mitochondrial DNA Haplogroups and Three Independent Polymorphisms have no Association with the Risk of Parkinson's Disease in East Indian Population. Neurol India [serial online] 2021 [cited 2021 May 14];69:461-5. Available from: https://www.neurologyindia.com/text.asp?2021/69/2/461/314553




Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder characterized by degeneration and ultimate death of dopaminergic neurons in the substantia nigra of the midbrain. Bradykinesia, tremor, rigidity, and postural instability are some of the classical symptoms of PD. Although PD is broadly considered as a late age disease; nowadays, early-onset Parkinson's disease (EOPD) is not quite rare where PD symptoms appear even before ~ 50 years of age. EOPD is said to account for 3–5% of all PD cases. PD is the second most common neurodegenerative disorder after Alzheimer's disease in the world. In India, the highest prevalence rate (328.3/100,000) of PD has been reported among a population of 14,010 Parsis living in colonies in Mumbai.[1]

PD is a classic example of movement disorder. Any kind of movement requires energy that is mostly produced inside mitochondria in the form of adenosine triphosphate (ATP) through oxidative phosphorylation. Thus, mitochondrial dysfunction is very much evident in classical forms of PD. Published literature showed that Parkinsonism could be induced by a toxin that inhibits mitochondrial respiratory complex I.[2] Also, post-mortem brains from sporadic PD patients showed defects in complex I. Many of the nuclear genes that are linked to PD such as PRKN, PINK1, DJ-1, and HTRA2 are also associated with mitochondrial impairment.[3] Somatic mtDNA deletions,[4] structural alterations, and mutations in mtDNA displacement loop (D-loop)[5] have also been reported in PD. Interestingly, mitochondrial DNA haplogroups that are defined as the common and “non-pathological” mtDNA variations, as well as a few independent Single Nucleotide Polymorphisms (SNPs) have been inconsistently reported to establish differences in oxidative phosphorylation (OXPHOS) performance,[6] complex I activity[2] and susceptibility toward PD[7],[8],[9],[10] among different populations all over the world. However, no such study has, so far, been undertaken in the Indian population.

In this study, 5 SNPs representing the major Asian mtDNA haplogroups (M, N) and their sub-haplogroups (D, M7, R, and B) and 3 independent SNPs: 4336T/C, 9055 G/A, 13708 G/A were selected to explore their relationship with PD susceptibility in an eastern Indian case-control population.


 » Materials and Methods Top


Study subjects

The study consisted of 100 PD cases [50 EOPD (<= 50 years, mean age 39.2 years); 50 LOPD/Late Onset Parkinson's Disease (>50 years, mean age 59.5 years); and 100 healthy controls. The inclusion criteria for PD were to have bradykinesia and at least two of the three cardinal features (i.e., tremor, rigidity, and postural instability) of Parkinsonism. Any individual having drug-induced Parkinsonism and secondary Parkinsonism were excluded from the study. The PD patients of the study were recruited from Bangur Institute of Neurosciences, Kolkata by Professor Jharna Ray's group, under the supervision of expert medical practitioners. All subjects were interviewed, and written consent was obtained as per the guidelines of the Indian Council of Medical Research before sample collection. Only voluntary participation of the patients was considered for the study. The Institutional Ethics Committee of the Bangur Institute of Neurosciences, Kolkata and Ethics Committee of the University of Calcutta has approved the study.

Assignment of haplogroups and SNPs for the study

We have checked 5 SNPs representing the major Asian mtDNA haplogroups (M, N) and their sub-haplogroups (D, M7, R, and B) and 3 independent SNPs: 4336T/C, 9055 G/A, 13708 G/A based on previous research[7],[8],[9] and MITOMAP (Human Mitochondrial Genome Database; http://www.Mitomap.org). The diagnostic SNPs and corresponding haplogroups are represented in [Figure 1].
Figure 1: Classification of major Asian mitochondrial DNA haplogroups (M, N) and their sub-haplogroups (D, M7, R and B). [We did not check for haplogroup L3]

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Genomic DNA isolation and genotyping

For both patient and control subjects, genomic DNA was isolated from peripheral blood sample following the conventional salting-out method using sodium perchlorate followed by isopropanol precipitation. Standard methods for polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) [Table 1] were used to identify the eight mtDNA SNPs, that are, the characteristic markers of different mitochondrial haplogroups as well as the three independent SNPs. Eight pairs of mitochondria-specific primers were designed following the Revised Cambridge Reference Sequences (rCRS) (www.mitomap.org/MITOMAP/HumanMitoSeq) to amplify the desired fragments. The PCR amplicons were digested by relevant restriction enzymes and the digested products were electrophoresed through 9% acrylamide gel and visualized by ethidium bromide staining in Gel documentation (BIO-RAD). Approximately 5% of all the samples representing each genotype were sequenced to determine the accuracy of our genotyping.
Table 1: PCR-RFLP detection of mitochondrial polymorphisms

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Statistical analysis

To assess if any specific mitochondrial haplogroup or the mutually exclusive SNPs, pose any risk toward PD in our representative sample population, we needed to check if there is any significant bias in the distribution of the concerned haplogroups as well as the independent SNPs within the PD cases and controls. For this, we performed a two-tailed Fisher's exact test using VassarStats software (http://vassarstats.net/odds2 × 2.html). Fisher's exact test is a recommended statistical test used to determine if there are non-random associations between two categorical variables, especially when the sample size is small. The level of significance was kept at P < 0.05.


 » Results and Discussion Top


We have selected 5 Asian mtDNA haplogroups for this study among which, M, N, and R are frequently found among the Indians. All non-African mtDNA belongs either to the haplogroup N or its sibling haplogroup M.[11] The representative sequencing results of the polymorphisms of each haplogroup and also the independent SNPs are shown in [Figure 2]. Haplogroup M represented by m.10398A>G is thought to result in an alteration in the structure of complex I in the mitochondrial electron transport chain[12] that was also found to have resistance against PD in Spanish[8] and Taiwanese[9] population. On the other hand, haplogroup cluster N (xR) and supercluster LMN are assumed to give protection from PD among the females in a Greek-Cypriot population.[13] In our study population, however, the distribution of haplogroup M and N were found to be 56% and 44% in healthy controls and 60% and 40% in PD patients, respectively, with no statistically significant difference between the two groups. The distribution of all the mtDNA haplogroups and their sub-haplogroups among the study subjects are listed in [Table 2].
Figure 2: Sequencing results of the mitochondrial DNA fragments to confirm the alleles of each single nucleotide polymorphism and deletion. Forward sequencing for C5178A, G9055A, T4336C and reverse sequencing for A10398G + C10400T, C12705T, 8280-8290A [delCCCCCTCTA] G, T9824C, G13708A variants are shown

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Table 2: Distribution of mtDNA haplogroups between PD patients and controls, n (%)

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D and M7 (represented by m. 5178A and m. 9824T) are sub-haplogroups of the major haplogroup M. Previous literature showed that haplogroup D5 influence expression of mitochondrial genes that are involved in cellular energetics[6] and haplogroup D leads to a higher risk of PD in people younger than 50 years of age in Han Chinese.[7] In our study, the sub-haplogroup D was found in 3.6% in healthy controls and 1.7% in PD patients revealing no association with PD. Haplogroup M7a is closely associated with Japanese PD patients,[14] but not a single individual either from control or cases represent M7 haplogroup in our population.

R haplogroup (sub-haplogroup of the major haplogroup N) has a wide geographical distribution within the Indian subcontinent. It is represented by m.12705 C>T and is thought to influence the function of the complex I in electron leakage and reactive oxygen species (ROS) production.[15] Female PD patients belonging to R haplogroup has a higher risk of having the disease at a later age (LOPD) among Greek-Cypriots.[13] In our study population, the frequency of R haplogroup was 77.3% in controls and 60% in PD cases and revealed a lack of any association with PD. The result holds true when tested only among the female patients.

Haplogroup B is derived from an internal node of the haplogroup R in the human mtDNA phylogenetic tree that is represented by a nine-base pair (BP) deletion (8280-8290 = A (delCCCCCTCTA) G). Haplogroup B confers lower risk for EOPD in Han Chinese,[7] and haplogroup B5 is also reported to give protection against PD in various populations.[5] We have not found any individual from haplogroup B in our study population.

A higher risk of PD with m. 4336C allele has been reported[8] in Spanish women. Similarly, m. 9055A and m. 13708A have been reported to have a protective effect on European women and PD patients of ≥70 years of age, respectively.[9] We did not find any individual with allele C in m. 4336 position in our study population. The frequency of m. 9055A and m. 13708A is 6% and 4% in control and 4% and 2% in patients, respectively. Thus, no significant association with the disease was found. The distribution of these three mtSNPs among the study subjects is listed in [Table 3].
Table 3: Distributions of mtDNA SNPs 9055G/A, 13708G/A and 4336T/C between PD patients and controls, n (%)

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Mitochondrial haplotype 9055G-10398A-13708G demonstrated a significant decrease in risk of developing PD in Taiwanese population above 70 years of age.[9] Haplotype analysis did not yield any significant association with PD in our study population [Table 4].
Table 4: Distributions of mtDNA 9055-10398-13708 haplotype between PD patients and controls, n (%)

Click here to view


We observed the same results when the patients were categorized into LOPD and EOPD and compared to the controls.


 » Conclusion Top


PD has a multifactorial etiology where its complex nature is governed by both genetic as well as environmental factors. Different populations are characterized by different patterns of genetic predisposition as well as environmental exposures for the disease. The association status of different mitochondrial haplogroups and individual SNPs with PD as reported in the literature is inconsistent throughout the world.[7],[10] Thus, our finding of absence of any association of the selected haplogroups in the eastern Indian dataset might be due to their ethnic differences in the population structure, as evident in case of other complex diseases, also.

Acknowledgement

We acknowledge Debmalya Sengupta for critically reviewing this manuscript.

Financial support and sponsorship

DST-PURSE (partial support for infrastructural facilities of the Department) and Teachers Research Grant (BI), University of Calcutta.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

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Radhakrishnan DM, Goyal V. Parkinson's disease. A review. Neurology India 2018;66:S26-35.  Back to cited text no. 1
    
2.
Giannoccaro MP, La Morgia C, Rizzo G, Carelli V. Mitochondrial DNA and primary mitochondrial dysfunction in Parkinson's disease. Mov Disord 2017;32:346-63.  Back to cited text no. 2
    
3.
Larsen SB, Hanss Z, Krüger R. The genetic architecture of mitochondrial dysfunction in Parkinson's disease. Cell Tissue Res 2018;373:21-37.  Back to cited text no. 3
    
4.
Bender A, Krishnan KJ, Morris CM, Taylor GA, Reeve AK, Perry RH, et al. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genet 2006;38:515-7.  Back to cited text no. 4
    
5.
Wu HM, Li T, Wang ZF, Huang SS, Shao ZQ, Wang K, et al. Mitochondrial DNA variants modulate genetic susceptibility to Parkinson's disease in Han Chinese. Neurobiol Dis 2018;114:17-23.  Back to cited text no. 5
    
6.
Hwang S, Kwak SH, Bhak J, Kang HS, Lee YR, Koo BK, et al. Gene expression pattern in transmitochondrial cytoplasmic hybrid cells harboring type 2 diabetes-associated mitochondrial DNA haplogroups. PLoS One 2011;6:3-10.  Back to cited text no. 6
    
7.
Chen YF, Chen WJ, Lin XZ, Zhang QJ, Cai JP, Liou CW, et al. Mitochondrial DNA haplogroups and the risk of sporadic Parkinson's disease in Han Chinese. Chin Med J 2015;128:1748-54.  Back to cited text no. 7
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8.
Huerta C, Castro MG, Coto E, Blázquez M, Ribacoba R, Guisasola LM, et al. Mitochondrial DNA polymorphisms and risk of Parkinson's disease in Spanish population. J Neurol Sci 2005;236:49-54.  Back to cited text no. 8
    
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Chen CM, Kuan CC, Lee-Chen GJ, Wu YR. Mitochondrial DNA polymorphisms and the risk of Parkinson's disease in Taiwan. J Neural Transm (Vienna) 2007;114:1017-21.  Back to cited text no. 9
    
10.
Fachal L, Mosquera-Miguel A, Pastor P, Ortega-Cubero S, Lorenzo E, Oterino-Durán A, et al. No evidence of association between common European mitochondrial DNA variants in Alzheimer, Parkinson, and migraine in the Spanish population. Am J Med Genet B Neuropsychiatr Genet 2015;168B: 54-65.  Back to cited text no. 10
    
11.
Raule N, Sevini F, Santoro A, Altilia S, Franceschi C. Association studies on human mitochondrial DNA: Methodological aspects and results in the most common age-related diseases. Mitochondrion 2007;7:29-38.  Back to cited text no. 11
    
12.
Qi Y, Wei Y, Wang Q, Xu H, Wang Y, Yao A, et al. Heteroplasmy of mutant mitochondrial DNA A10398G and analysis of its prognostic value in non-small cell lung cancer. Oncol Lett 2016;12:3081-8.  Back to cited text no. 12
    
13.
Georgiou A, Demetriou CA, Heraclides A, Christou YP, Leonidou E, Loukaides P, et al. Mitochondrial superclusters influence age of onset of Parkinson's disease in a gender specific manner in the Cypriot population: A case-control study. PLoS One 2017;12:1-11.  Back to cited text no. 13
    
14.
Takasaki S. Mitochondrial SNPs associated with Japanese centenarians, Alzheimer's patients, and Parkinson's patients. Comput Biol Chem 2018;32:332-7.  Back to cited text no. 14
    
15.
Yang Y, Shou Z, Zhang P, He Q, Xiao H, Xu Y, et al. Mitochondrial DNA haplogroup R predicts survival advantage in severe sepsis in the Han population. Genet Med 2008;10:187-92.  Back to cited text no. 15
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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