Mitochondrial DNA Haplogroups and Three Independent Polymorphisms have no Association with the Risk of Parkinson's Disease in East Indian Population
Keywords: Genetic Association, Haplogroups, Mitochondrial DNA, Parkinson's disease, SNPKey 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.
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.
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. 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. Somatic mtDNA deletions, structural alterations, and mutations in mtDNA displacement loop (D-loop) 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, complex I activity and susceptibility toward PD,,, 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.
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,, and MITOMAP (Human Mitochondrial Genome Database; http://www.Mitomap.org). The diagnostic SNPs and corresponding haplogroups are represented in [Figure 1].
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.
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.
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. 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 that was also found to have resistance against PD in Spanish and Taiwanese 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. 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].
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 and haplogroup D leads to a higher risk of PD in people younger than 50 years of age in Han Chinese. 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, 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. Female PD patients belonging to R haplogroup has a higher risk of having the disease at a later age (LOPD) among Greek-Cypriots. 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, and haplogroup B5 is also reported to give protection against PD in various populations. 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 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. 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].
Mitochondrial haplotype 9055G-10398A-13708G demonstrated a significant decrease in risk of developing PD in Taiwanese population above 70 years of age. Haplotype analysis did not yield any significant association with PD in our study population [Table 4].
We observed the same results when the patients were categorized into LOPD and EOPD and compared to the controls.
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., 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.
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.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]