Abstract
Epigenetic modifications have been implicated in the development of cognitive impairment. However, the relationship between DNA methylation levels and cognitive impairment can be greatly influenced by environmental factors some blood-based nutrition markers. The present study aims to understand the relationship between global DNA methylation levels and cognitive impairment independently and in light of micronutrient status among North Indian adults. This study was conducted among 614 individuals, aged 30 to 79 years from Palwal, Haryana. Cognitive impairment (CI) was assessed using Mini-Mental State Examination (MMSE). Folate, vitamin B12, and homocysteine levels were estimated using chemiluminescence technique. Estimation of global DNA methylation (5mC) levels was performed using the ELISA-based colorimetric technique. Appropriate comparison tests (based on normality distribution) were applied to compare the levels of global DNA methylation in different study groups. Logistic regression models were run to examine association between global DNA methylation and CI. Median 5mC levels of both mild and moderate/severe CI groups were significantly lower than that of the control group. Individuals in the 1st quartile of 5mC, with those in the 4th quartile as the reference, were at a significantly increased risk of both mild and moderate/severe CI. Vitamin B12, but not folate, appeared to mediate global DNA hypomethylation among CI cases. Cognitive impairment may be associated with Global DNA hypomethylation in the studied North Indian population. Vitamin B12 sufficiency may help improve the methylation levels among the cases of cognitive impairment. There is a need to develop population and context-specific epigenetic markers for cognitive impairment.
Keywords: Mini Mental State Examination, Biological aging, Cognitive impairment, Folate, Vitamin B12, Global DNA methylation
Introduction
Cognitive impairment (CI) ranges from mild cognitive impairment (MCI), a decline in cognitive processes greater than what’s expected for someone’s age but does not interfere with everyday life, to more severe forms of dementia such as Alzheimer’s disease (AD) [1]. There is an increasing global burden of cognitive impairment among adults and older individuals, and it is estimated that 66 million people will be living with AD or other forms of dementia worldwide by the year 2023, and this number may rise to 115 million by the year 2050 [2]. As per an estimate, the global prevalence of CI is 19%, with huge regional variations (ranging between 5.1 and 41%) [3]. The situation is not very different in India. Different studies have reported a prevalence rate of CI in India ranging from 3.5 to 63.2% [4–7].
Given the alarming situation, there has been a steady increase in the number of studies attempting to understand the aetiology of CI in the last two decades. These studies have undoubtedly augmented our understanding of CI, yet the aetiological pathways of CI remain poorly understood. Studies have identified a number of demographic, biochemical, and genetic factors associated with increased risk of CI [8, 9]. However, the degree and nature of the association between these risk factors and CI have been reported to vary across population groups. This is primarily attributed to gene-environment interactions and epigenetic modulations, making it crucial to understand the epigenetic pathways associated with CI. The epigenetics of CI has produced much curiosity also because epigenetic changes are intricately intertwined with biological aging, which in turn is the biggest risk factor for CI [10]
DNA methylation, by far, is the most widely explored epigenetic marker in relation to various health conditions, including CI. Recent advances are underway to explicate cognitive impairment-associated alterations in DNA methylation patterns [11, 12]. However, since several environmental and cultural factors, viz dietary pattern, physical activity, etc., can influence the relationship between DNA methylation and CI, therefore, it is important to undertake such studies in different populations of the world to arrive at a universal finding. Also, studies have mostly focused on region-specific alternations in DNA methylation patterns associated with CI [11], and much less attention has been given to the relationship between global DNA methylation and CI.
Further, since some biochemical nutrition markers, like folate, vitamin B12, and homocysteine, play a crucial role in the one-carbon metabolic pathway, which can directly influence DNA methylation [13, 14], it is important to include them in studies examining the role of DNA methylation on various disorders. Considering these knowledge gaps, the present population-based study attempts to understand the relationship between blood leucocyte global DNA methylation levels and cognitive impairment independently and also in light of folate, vitamin B12, and homocysteine status among North Indian adults.
Materials and Methods
Study Population
A total of 808 individuals (58.66% females), aged 30–79 years with median age (IQR) of 52 (45–61) years, belonging to the Jat community of Haryana, North India, were recruited in the present cross-sectional study by Kaur et al. 2018 [7]. As per the population size of Jat, Haryana, (aged 30 + years), the samples size was calculated to 768 individuals (95% C.I. interval and 5% allowable error). However, after considering 5% drop out, the sample size was increased upto 808 participants (Krejcie and Morgan, 1970). The participants represented a Mendelian population, sharing a common gene pool and practicing marriages within the same community. The majority of the studied participants were vegetarians (99.5%). Participants were conveniently recruited through door-to-door household surveys. Individuals with chronic diseases (such as cancers and CVDs), those on long-term medications or supplements, pregnant women, and lactating mothers were excluded from the study. Of the recruited participants, 614 individuals (58.79% females) were included in the analyses after removing people with missing methylation data.
Data Collection
The study was approved by the Institutional Ethics Committee of the host institute (Ref No. Anth/2018/2890/1/28-12-2018). Pre-informed voluntary written consent, transcribed in the local language, was obtained from each participant prior to recruitment and data collection. Data pertaining to socio-demographic variables such as age, sex, literacy status, occupation, employment status, marital status, and annual family income were collected using pretested interview schedules.
Cognitive Assessment
Mini-Mental State Examination (MMSE) scale [15] was used to assess the cognitive status of the participants of the present study. Each item of the MMSE scale was administered to the participants as per the instruction manual by trained field investigators, and the responses were carefully noted. Based on the responses, each participant was scored out of 30 (maximum score) to define cognitive impairment (CI). Individuals with scores ≥ 24 were considered to have normal cognition; those with scores between 19 and 23 were considered to have mild CI; those with scores between 10 and 18 were considered to have moderate CI, while those with scores between 0 (minimum score) and 9 were considered as severe CI. Due to the extremely low prevalence of severe CI (n = 2), the severe CI category was merged with the moderate CI category for all the statistical analyses in the present study.
Blood Sample Collection
A sample of 5 ml overnight fasting blood was collected from each participant by a trained technician. Serum and plasma were separated from the blood cells for further analysis. DNA extraction was performed using the salting-out method [16]. Serum, plasma, and DNA samples were stored at -80º Celsius until further analysis. A260/A280 ~ 1.8 was used as a standard for DNA quality checks.
Biochemical Analysis
Folate and vitamin B12 levels were estimated using serum samples, whereas homocysteine was estimated using plasma samples. Immulite 1000 Analyser (Siemens Diagnostic products, USA) employing chemiluminescence technique was used to estimate folate, vitamin B12, and homocysteine. Hyperhomocysteinemia was defined as ≥ 15 μmol/l, vitamin B12 deficiency as ≤ 220 pg/ml, and folate deficiency as < 3 ng/ml [17]. Of the studied participants, Vitamin B12, Folate, and Homocysteine were successfully estimated for 738, 726, and 742 individuals respectively.
Global DNA Methylation
DNA methylation assay was performed in duplicates with 200 ng DNA. Global DNA methylation levels were assessed using the MethylFlash™ Global DNA Methylation (5-mC) ELISA Assay Kit (Colorimetric) by EpiGentek Group Inc. USA. Optical density of methylated DNA samples was measured using a spectrophotometer (Thermo Scientific, MULTISKAN GO) and observations were carefully noted. For all samples included in the study, intra- and inter-assay coefficients of variation were found to be 5%.
Statistical Analysis
Statistical analyses were performed using IBM SPSS ver.22 software. The Kolmogorov–Smirnov test was used to determine the normality of continuous variables. Medians and respective interquartile ranges (IQR) have been reported for non-normally distributed continuous variables, and numbers with respective percentages have been reported for categorical variables. Mann–Whitney U-test and Kruskal–Wallis tests were used to determine significant differences between median levels of two and more than two groups, respectively. The chi-square test was used to compare categorical variables. Logistic regression analysis was performed to understand the association between the dependent and independent variables. Confounders such as age, sex, literacy, and employment status were identified and addressed in eth regression models. All statistical tests were considered significant at p-value < 0.05.
Results
General Characteristics of Cases and Controls
More than half of the studied population (62.34%) was observed to have cognitive impairment with 33.06% of participants having mild CI and 29.31% of participants having moderate/severe CI. All the study groups had a similar age group while significant differences in the distribution of other sociodemographic variables were found between the control and the different case groups (Table 1). It was found that females, non-literate individuals, and those engaged in agricultural work were in significantly higher proportions in the mild CI and moderate/severe CI groups than in the normal cognition category. However, marital status and family income did not differ significantly among the study groups.
Table 1.
Distribution of sociodemographic variables with respect to cognitive impairment
| Total (N = 614) |
Control (N = 231) |
Mild CI (N = 203) |
Mod/Sev CI (N = 180) |
p | ||
|---|---|---|---|---|---|---|
| n (%) | n (%) | n (%) | n (%) | |||
| Median Age (IQR) (in Years) | 52 (45–61) | 52 (44–61) | 51 (44–60) | 54 (46–62) | 0.08a | |
| Sex | Male | 253 (41) | 173 (74.9) | 55 (27.1) | 25 (13.9) | < 0.001* |
| Female | 361 (58.8) | 58 (25.1) | 148 (72.9) | 155 (86.1) | ||
| Literacy status | Literate | 290 (47.2) | 207 (89.6) | 66 (32.5) | 17 (9.4) | < 0.001* |
| Non-literate | 324 (52.8) | 24 (10.4) | 137 (67.5) | 163 (90.6) | ||
| Employment status | Employed | 76 (12.4) | 65 (28.1) | 9 (4.4) | 2 (1.1) | < 0.001* |
| Unemployed | 538 (87.6) | 166 (71.9) | 194 (95.6) | 178 (98.9) | ||
| Marital Status | Married | 561 (91.4) | 212 (91.8) | 184 (90.6) | 165 (91.7) | 0.99 |
| Unmarried | 10 (1.6) | 4 (1.7) | 4 (2.0) | 2 (1.1) | ||
| Widow/Widower | 43 (7.0) | 15 (6.5) | 15 (7.4) | 13 (7.2) | ||
| Vitamin B12 | Normal | 281 (38.1) | 101 (36.7) | 88 (34.6) | 92 (44) | 0.100 |
| Low | 457 (61.9) | 174 (67.3) | 166 (65.4) | 117 (56) | ||
| Folate | Normal | 511 (70.4) | 179 (67.5) | 181 (72.1) | 151 (71.9) | 0.446 |
| Low | 215 (29.6) | 86 (32.5) | 70 (27.9) | 59 (28.1) | ||
| Homocysteine | Normal | 209 (28.2) | 70 (25.5) | 78 (30.6) | 61 (28.8) | 0.411 |
| High | 533 (71.8) | 205 (74.5) | 177 (69.4) | 151 (71.2) | ||
| Annual family income (INR) | < 50,000 | 61 (9.9) | 26 (11.3) | 15 (7.4) | 20 (11.1) | 0.33 |
| ≥ 50,000 | 553 (90.1) | 205 (88.7) | 188 (92.6) | 160 (88.9) | ||
*Significant at value of p < 0.05; aMann-whitney p-value; N sample size of the category/sub group; n = count; Control normal cognition; CI cognitive impairment; Mod/Sev moderate/severe; IQR inter quartile range; INR, Indian rupees
Median Global DNA Levels Among Cases and Controls
The median global DNA methylation levels in CI groups, both mild (0.581) and moderate/severe CI (0.700), were found to be significantly lower as compared to normal group (0.904) cognition (Fig. 1).
Fig. 1.
Median (IQR) level of 5-mC in CI status
In the age-wise distribution of median 5mC levels, individuals with CI were found to be having lower levels of methylation in every age group compared to those with normal cognition (Fig. 2). However, no consistent patterns were observed across the age groups regarding median 5mC levels of mild CI and moderate/severe CI groups. In the 30–39 age group, the mild CI category had higher median methylation levels than the moderate/severe CI category; however, that reversed in the 40–49 age group. Interestingly, in the 50–59 age group, the median methylation levels in both CI categories seem to converge, after which there is a slender spike in the 60–69 age group, eventually followed by a substantial decrease in the 70–79 age group.
Fig. 2.
Age-cohort wise distribution of median (IQR) level 5-mC with respect to CI status; x-axis, age cohort categories; y-axis, global DNA methylation
Prevalence and Risk of CI in Various Quartiles of DNA Methylation Levels
The participants were divided into four groups based on the quartiles of global DNA methylation levels, and the prevalence of mild and moderate/severe CI was examined in each quartile of DNA methylation (Table 2). Significant difference was observed in the distribution of cognitive impairment in the different quartiles of global DNA methylation. In general, while the proportion of individuals with normal cognition was higher in the 3rd and the 4th quartile (representing higher levels of global DNA methylation), the proportion of individuals with CI was higher in the 1st and the 2nd quartile (representing lower levels of global DNA methylation).
Table 2.
Prevalence of cognitive impairment in different quartiles of Global DNA methylation (5mC)
| 5mC quartiles | Control (N = 231) | Mild CI (N = 203) | Mod/Sev CI (N = 180) | p |
|---|---|---|---|---|
| n (%) | n (%) | n (%) | ||
| 1st | 42 (18.2) | 61 (30.0) | 50 (27.8) | < 0.01* |
| 2nd | 52 (22.5) | 58 (28.6) | 44 (24.4) | |
| 3rd | 69 (29.9) | 35 (17.2) | 50 (27.8) | |
| 4th | 68 (29.4) | 49 (24.1) | 36 (20.0) |
*Significant at value of p < 0.05; N sample size of the category/sub group; n = count; Control, normal cognition; CI cognitive impairment; Mod/Sev moderate/severe; 5mC Global DNA methylation
Quartile regression analysis was undertaken to assess the risk posed by various levels of global DNA methylation for cognitive impairment (Table 3). After controlling for confounders, viz age, sex, literacy status and employment status, the quartile regression analysis revealed that individuals in the 1st quartile of global DNA methylation, with all other quartiles put together, was at over 2.2-fold (95% confidence interval, 1.271–4.154) significantly increased risk of mild CI. Interestingly, individuals in the 3rd quartile, with other quartiles together as the reference, were at significantly reduced risk for mild CI that requires further investigation. Similar trend of reduced risk was also observed for the 4th quartile, albeit with no statistical significance.
Table 3.
Binary logistic regression showing the risk of CI in quartiles of global DNA methylation
| 5mC risk quartile | Reference quartile | Mild CI (N = 203) | p | Mod/Sev CI (N = 180) | p |
|---|---|---|---|---|---|
| OR# (95% conf. int.) | OR (95% conf. int.) | ||||
| 1st | 2nd + 3rd + 4th | 2.298 (1.271–4.154) | < 0.01* | 1.861 (0.800–4.328) | 0.14 |
| 2nd | 1st + 3rd + 4th | 1.239 (0.703–2.183) | 0.45 | 1.205 (0.534–2.722) | 0.65 |
| 3rd | 1st + 2nd + 4th | 0.458 (0.251–0.837) | 0.01* | 1.016 (0.476–2.166) | 0.96 |
| 4th | 1st + 2nd + 3rd | 0.787 (0.455–1.359) | 0.39 | 0.460 (0.204–1.039) | 0.06 |
| 1st | 4th | 2.301 (1.119–4.731) | 0.02* | 3.604 (1.050–12.382) | 0.04* |
| 1st + 2nd | 3rd + 4th | 2.133 (1.292–3.522) | < 0.01* | 1.767 (0.876–3.565) | 0.11 |
*Significant at value of p < 0.05; N sample size of the category/sub group; n count; Control, normal cognition; CI cognitive impairment; Mod/Sev moderate/severe; 5mC, Global DNA methylation; OR#, adjusted odds ratio; conf. int. confidence interval
Further, considering hypomethylation as the risk, odds for CI in the 1st quartile with 4th as reference, and 1st + 2nd with 3rd + 4th as reference were estimated. Individuals in the 1st quartile, with the 4th quartile as the reference, were at 2.3-fold (95% confidence interval, 1.119–4.731) and 3.6-fold (95% confidence interval, 1.050–12.382) significantly increased risk for mild and moderate/severe CI, respectively. Further, individuals in 1st + 2nd quartiles, with 3rd + 4th as a reference, showed significantly over two fold increased risk for mild CI.
Global DNA Methylation and CI in Light of Biochemical Nutrition Parameters
Participants were stratified according to normal and deficient levels of folate, vitamin B12, and homocysteine, and then differences in median methylation levels were observed between different cognition categories at the same biochemical level and between two biochemical levels within the same cognition category (Table 4). This analysis revealed a significant lower global DNA methylation in the mild CI group with respect to the control group among those with normal folate and in the moderate/severe CI group with respect to the control group among those with Vitamin B12 deficiency (Table 4). Further, individuals with moderate/severe CI were globally hypomethylated as compared to controls in the normal homocysteine category (p = 0.05).
Table 4.
Median global DNA Methylation levels with respect to normal and abnormal levels folate, Vitamin B12, and homocysteine in cases and controls of CI
| Cognition status | Control (N = 231) | Mild CI (N = 203) | Mod/Sev CI (N = 180) | pq | pb | |
|---|---|---|---|---|---|---|
| Median 5mC (IQR) | Median 5mC (IQR) | Median 5mC (IQR) | ||||
| Folate | Normal | 0.910 (0.516–1.670) | 0.606 (0.334–1.250) | 0.727 (0.401–1.300) | 0.01* | 0.29 |
| Deficient | 0.791 (0.439–1.382) | 0.563 (0.341–1.323) | 0.563 (0.344–1.311) | |||
| p | 0.42 | 0.78 | 0.59 | |||
| Vitamin B12 | Normal | 0.810 (0.473–1.135) | 0.564 (0.339–1.059) | 0.825 (0.422–1.369) | 0.11 | 0.02* |
| Deficient | 0.970 (0.471–1.712) | 0.617 (0.369–1.604) | 0.581 (0.332–1.239) | |||
| p | 0.19 | 0.16 | 0.26 | |||
| Homocysteine | Normal | 0.904 (0.509–1.558) | 0.575 (0.330–1.212) | 0.575 (0.435–1.203) | 0.15 | 0.05 |
| High | 0.877 (0.468–1.662) | 0.579 (0.355–1.450) | 0.751 (0.369–1.318) | |||
| p | 0.87 | 0.74 | 0.60 | |||
*Significant at value of p < 0.05; aNormal levels of biochemicals, bAbnormal levels of biochemicals; N sample size of the category/sub group; Control normal cognition; CI cognitive impairment; Mod/Sev moderate/severe; 5mC, Global DNA methylation; IQR inter quartile range
To further validate this observation, the combined effect of folate and vitamin B12 repletion and deficiency in global DNA methylation was examined (Table 5). In this analysis, a significantly lower global DNA methylation was observed in the mild CI group, with respect to other groups, among those with normal folate + normal vitamin B12. Interestingly, among those with normal folate but vitamin B12 deficiency, the highest median methylation level was found in the normal cognition group, followed by the mild CI group and the least in the moderate/severe CI group (p = 0.06).
Table 5.
Median global DNA methylation levels with respect to normal and abnormal levels of folate and vitamin B12 categories among cases and controls of CI
| Control (N = 231) | Mild CI (N = 203) | Mod/Sev CI (N = 180) | p | |
|---|---|---|---|---|
| Median 5mC (IQR) | 5mC Median (IQR) | 5mC Median (IQR) | ||
| Normal Folate + Normal Vitamin B12 | 0.811 (0.516–1.626) | 0.524 (0.340–1.012) | 0.846 (0.449–1.368) | 0.03* |
| Abnormal Folate + Abnormal Vitamin B12 | 0.827 (0.446–1.905) | 0.550 (0.386–1.211) | 0.563 (0.366–1.120) | 0.23 |
| Abnormal Folate + Normal Vitamin B12 | 0.630 (0.434–1.089) | 0.594 (0.177–1.898) | 0.628 (0.255–1.805) | 0.93 |
| Normal Folate + Abnormal Vitamin B12 | 1.035 (0.480–1.727) | 0.710 (0.341–1.675) | 0.581 (0.304–1.229) | 0.06 |
*Significant at value of p < 0.05; N sample size of the category/sub group; Control normal cognition; CI cognitive impairment; Mod/Sev moderate/severe; 5mC Global DNA methylation; IQR inter quartile range
Discussion
The present study examined the relationship between global DNA methylation and cognitive impairment independently and also in light of normal and abnormal levels of selected biochemical nutrition markers. The present study reveals a significant hypomethylation among individuals with CI (both mild and moderate/severe categories) than those with normal cognition. This observation is in concordance with some of the previous reports of DNA hypomethylation in cognitive impairment or decline [18, 19] however, contradictory observations have also been reported [20, 21] in literature.
The role of DNA methylation in biological aging and age-related cognitive decline is a matter of active scientific inquiry [12, 22]. Aging is the biggest risk factor for cognitive impairment, and cognitive impairment is a hallmark of neurological aging [10, 23]. An emerging body of evidence suggests that epigenetic alterations are among the most dramatic molecular changes associated with aging in the brain [10]. Several brain functions, viz. synaptic plasticity, learning and memory, and adult neurogenesis, are regulated by epigenetic mechanisms [24, 25]. Recent studies have shown that DNA methylation-based measures of biological aging can be associated with or predict the risk for cognitive decline [12, 24]. Though results are inconsistent, global DNA hypomethylation, at least among older individuals, has been reported to be associated with aging [26]. Significant hypomethylation among CI cases than their healthy counterparts in the present study can be due to accelerated biological aging among them.
This inference is further validated through age-stratified analysis (Fig. 2). In this analysis, regardless of the age group, cases were found to be relatively (though not significantly) hypomethylated than the controls. The difference was particularly evident in the youngest (30–39 years) age group. Also, the variations in median global DNA methylation levels, as we moved from one age group to the other, were inconsistent and jerky in the case groups when compared to that of the control group, where it was rather steady. Further, in quartile comparison and regression analyses (Table 2 and 3), those in the first quartile of DNA methylation (least DNA methylation levels) were at significantly higher risk of CI than those in the 4th quartile (highest DNA Methylation levels). These observations point towards difference in the global DNA methylation profile of the CI cases than what is expected during normal aging. It is likely that apparent hypomethylation among cases is due to accelerated biological aging, which, in turn, is associated with cognitive decline.
Apart from accelerated biological aging, another possible explanation of hypomethylation among the cases is the CI-induced DNA demethylation. Though studies have not widely investigated the effect of CI on DNA methylation, some health conditions, like obesity (Wu and Yin, 2022) and hypertension (Hong et al., 2023), have been reported to alter DNA methylation patterns. Further, cognitive impairment has been found to be associated with environmental factors like poor nutrition [27, 28], reduced physical activity [29, 30], low education [31] and low socioeconomic status [32]. These factors are also independently associated with alterations in DNA methylation [33–35]. The observed DNA hypomethylation among cases can also be because of these associated risk factors.
Coming to the role of selected biochemical nutrition markers in modulating the relationship between CI and global DNA methylation, despite folate repletion, individuals with mild CI were significantly hypomethylated as compared to controls. This implies that hypomethylation in mild CI may be independent of folate. However, individuals with moderate/severe CI were significantly hypomethylated as compared to controls in case of vitamin B12 deficiency. In fact, among those with normal folate but vitamin B12 deficiency, CI groups were hypomethylated as compared to the control group. This trend had moderate statistical significance. Together these observations suggest that vitamin B12, but not folate, may be associated with DNA hypomethylation among CI cases.
A vast body of literature has implicated suboptimal levels of B vitamins (viz folate, B6 and B12) in cognitive decline among elderlies [36]. One pathway that is often referred to while explaining the relationship between the deficiency of B vitamins and CI is vitamin deficiency-mediated hyperhomocysteinemia [13]. High homocysteine is one of the most crucial risk factors for CI [13]. However, in the present study, individuals with moderate/severe CI and normal homocysteine levels were hypomethylated as compared to controls, implying that hyperhomocysteinemia may not be correlated with hypomethylation. Therefore, it is likely that vitamin B12 deficiency may be associated with CI epigenetically.
Notably, a recent meta-analysis reported folate deficiency, but not low vitamin B12 or B6, to be a risk factor for CI [36]. In fact, a higher intake of folate, but not vitamin B12 or B6, significantly reduced the risk of incident dementia in older adults [36]. This is, in a way, contrary to the findings of the present study, where hypomethylation among CI cases has been observed in vitamin B12 deficient but folate-replete groups. This contradiction, in fact, is an apt illustration to emphasize the need for population- and context-specific intervention programs. The present study has been conducted on a sample coming from a vegetarian population. Due to a vegetarian dietary pattern, the deficiency of vitamin B12 in the population is rampant, but the prevalence of folate deficiency is low. This is in contrast to the prevalence rates of folate and B12 deficiencies in Western countries, where people are generally non-vegetarians. Since most studies included in the meta-analysis have been conducted on non-vegetarian populations (generally vitamin B12 replete but may suffer from lower folate intake) [36], the conclusion of the meta-analysis is, rather, expected. However, in the studied population, or for that matter, in vegetarian communities, promoting a higher intake of vitamin B12 may be more beneficial than promoting folate intake.
There are a few limitations of the present study that should be stated. The cross-sectional nature of the study precludes causality. Further, CI has been screened using MMSE, which is not the gold standard test for diagnosing cognitive impairment. However, clinical diagnosis of CI was not feasible in this population-based study. Lastly, being a single-site/population study, the study findings should be replicated in larger sample size using multiple sites and populations for generalizability.
To conclude, the present study suggests an association between cognitive impairment and global DNA hypomethylation in a North Indian population. In addition, Vitamin B12 deficiency appears to play a role in the relationship between global DNA hypomethylation and CI. Promoting vitamin B12-rich food in this population may help improve global DNA methylation levels among CI cases. The present study highlights the need for context-specific models to understand the role of epigenetics in CI and the need for designing more focussed population specific interventions to improve the disease burden.
Acknowledgements
The authors would like to acknowledge all the kind participants for their invaluable time and cooperation in the present study. We also thank the Department of Anthropology, University of Delhi for providing the required facilities to conduct the study.
Authors’ contributions
KNS and NB supervised the project and data collection. KNS conceptualised the manuscript. DB and AK performed lab analyses for global DNA methylation. KK performed data analyses and wrote the first draft. VC, GKW, NKD and KNS critically revised the manuscript for intellectual content. All the authors approved the final version of the manuscript for publication.
Funding
The present study was supported by the Department of Science and Technology, Ministry of Science and Technology, Government of India (DST/CSRI/2018/162G) and Indian Council of Medical Research (45/01/2022-ANTHRO/BMS).
Data Availabilty Statement
The data supporting the findings of this study will be made available by the authors on reasonable request.
Declarations
Conflict of interests
The authors declare no competing interests.
Human Ethics
The present study was conducted following the principles of the Declaration of Helsinki for biomedical studies involving human subjects. Moreover, the present study was approved by the Institutional Ethics Committee, Department of Anthropology, University of Delhi (Ref No. Anth/2018/2890/1/28-12-2018). Prior to recruitment and data collection, pre-informed voluntary written consent, transcribed in local language was obtained from each participant.
Consent for publication
All the authors have provided consent for the publication of this manuscript.
Footnotes
Publisher's Note
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data supporting the findings of this study will be made available by the authors on reasonable request.