Lower Prevalence of Alzheimer’s Disease among Tibetans: Association with Religious and Genetic Factors

Abstract

Background:

The prevalence of dementia differs among racial groups, the highest prevalence being in Latin America (8.5%) compared to sub-Saharan African regions (2–4%). The most common type of dementia is Alzheimer’s disease (AD).

Objective:

To estimate the prevalence of AD in the Qinghai-Tibet plateau and to investigate the related factors.

Methods:

This was a cross-sectional, multistage cluster sampling design survey. Data was collected from May 2014 to September 2014 from 4,060 Tibetan aged >60 years. Participants underwent clinical examinations and neuropsychological evaluations. MALDI-TOF was used to test the genotypes of CLU, TFAM, TP53INP1, IGHV1-67, CR1, ApoE, and BIN1. Logistic regression models were used to ascertain the associations with AD.

Results:

The prevalence of AD among Tibetan individuals aged >60 years was 1.33% (95% CI: 0.98–1.69). The CLU haplotypes AA+GA (odds ratio (OR) = 4.483; 95% CI: 1.069–18.792) of rs2279590 was correlated with AD. The CLU haplotypes GG+GC (OR = 0.184; 95% CI: 0.038–0.888) of rs9331888 and kowtow (OR = 0.203; 95% CI 0.046–0.896) were negatively correlated with AD.

Conclusion:

A low prevalence of AD was found in Tibetans from the Qinghai-Tibet plateau. Multivariate analysis might suggest that regular “mind-body” religious meditative activities may be negatively associated with AD in this population, as well as the CLU genotype at rs9331888.

Keywords

Alzheimer’s disease gene polymorphisms Qinghai-Tibet Plateau religion Tibetan

INTRODUCTION

Alzheimer’s disease (AD) is the most common type of dementia and is characterized by a decline in memory and other cognitive skills that affects a person’s ability to perform everyday activities [1]. A severe cognitive deficit is observed in patients with AD and prejudices the quality of life of family members. However, at present, there is no efficient measure to prevent AD and it cannot be reversed by available treatments.

Recently, a systematic meta-analysis revealed that the age-standardized prevalence of dementia for people aged ≥60 years varies from 5% to 7% in most world regions [2]. The highest prevalence is observed in Latin America (8.5%) and the lowest prevalence is observed in sub-Saharan Africa (2–4%) [2]. This meta-analysis also indicated that the prevalence of dementia differs among racial groups [2]. The total prevalence of dementia is about 3.0% in China, as reported in a meta-analysis of 48 epidemiologic studies carried out from 1980 to 2010 [3].

Tibetans have been living at high altitude longer than any other ethnic groups. They have deep religious faith, particular living habits, greater hypoxic and hypercapnic ventilatory response, larger lungs, greater lung diffusion capacity, and better lung function compared to people living in low altitude regions [4]. In a recent report, Tibetans had a higher prevalence of hyperlipidemia than other Chinese populations, which might be derived from their living environment, living habits, customs, gene polymorphisms, and religious behaviors [5]. Tibetans have a deep religious culture that is characterized by daily prayers and meditative activities. Kowtow is the act of deep respect shown by prostration. Other meditative actions performed by Tibetans include reciting sutras, turning prayer wheels, and turning prayer beads. Previous studies have shown that meditative processes improve cognitive functions [6, 7].

Along with ApoE polymorphisms, 20 common susceptibility loci have been identified to be associated with AD [8 –10]. However, no report specifically examined Tibetans. Therefore, the aim of this survey was to estimate the prevalence of AD in elderly Tibetans as well as to identify associations between AD and gene polymorphisms potentially involved.

MATERIALS AND METHODS

Subjects

From May 2014 to September 2014, a population-based cross-sectional survey with a multistage cluster sampling design was carried out. Subjects were recruited from the Qinghai province of China. First, four representative cities were selected in Qinghai. Then, six counties within the selected cities were chosen randomly. Lastly, 65 rural villages within the selected counties were sampled randomly.

Then, to be selected, participants had to be unrelated Tibetans aged >60 years who had been living for at least 10 years in Qinghai and whose ancestors had been living in Qinghai. Participants were excluded in the presence of: 1) dementia caused by poisoning or systemic diseases; 2) pseudo-dementia caused by depression; 3) cerebrovascular or nervous system diseases (including vascular dementia); 4) severe acute disease or with malignant tumor; or 5) recent history of suffering from cold, chronic inflammation, acute disease, metabolic disease, or infectious disease.

This study was approved by the Ethics Committee of our hospital. Written informed consent was obtained from each participant or from the legal guardians in case of cognitive impairment. The study was done in accord with the ethical standards of the Committee on Human Experimentation, and in accord with the Helsinki Declaration of 1975.

A total of 4,060 subjects participated in the survey. Among them, eight were diagnosed with vascular dementia. Due to missing data, 3,974 participants were finally included in the analyses.

Interviews

The investigation group consisted of experts and interviewers. The evaluation panel included three neurologists and three neuropsychologists with special expertise in cognitive impairments. There were three groups of interviewers, each including one senior neurology graduate student, one junior neurologist, and one interpreter. All interviewers and experts received the same training on neuropsychological diagnosis and assessment. The inter-rater reliability for videotapes of cognitive tests and diagnostic procedures was required to exceed 0.90 [11].

Each interviewing group conducted individual semi-structured interviews with participants and their close caregivers at the residence. Interviews usually lasted 1.5 h. Detailed data on socio-demographic characteristics, medical history, lifestyle, current medications,and family history were collected. One of the inter-viewers tested the participant with a series of neuropsychological tests. First, the cognitive function was assessed with the Mini-Mental State Examination [12, 13]. Second, functional autonomy was evaluated with the Activity of Daily Living Scale [14]. Third, the differentiation between degenerative dementia and vascular dementia was assessed by the Hachinski Ischemic Scale [15]. The other interviewer took a detailed history of all cognitive impairments including time and mode of onset, possible triggers, affected domains, the course of the condition, impact on daily activities, changes in mood or behavior, results of computed tomographic scans or magnetic resonance imaging, and treatment and its effects. Last, standardized general and neurological examinations were performed.

All interviewers involved in diagnosing AD were blinded to lifestyle and genetic variables.

Diagnostic criteria of AD

All information was recorded on the questionnaire of basic information, reviewed by the panel and interviewers, and diagnoses were made each workday. If consensus was not reached, the expert would return to the residence to reevaluate the participant for a final diagnosis the following day.

Diagnostic criteria for dementia were based on the fourth edition of the DSM-IV [16]. Diagnostic criteria for AD was based on the criteria issued by the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association [17]. Diagnostic criteria for vascular dementia was based on the criteria of the National Institute of Neurological Disorders and Stroke-Association International pour la Recherche et l’Enseignement en Neurosciences [18]. Other types of dementia (including mixed dementia, frontotemporal dementia, dementia with Lewy bodies, Parkinson’s disease with dementia, etc.) were not analyzed in the present survey.

Dietary habits, smoking, and alcohol

Consumption of beef, mutton, dairy products, bean products, chicken, fish, and vegetables were defined as eating more than 1.25 kilograms per week. Eggs intake habits were defined as eating more than three eggs per week [19 –21]. Smoking was defined as smoking ≥100 cigarettes a year. Alcohol consumption was defined as drinking more than 1 time a week for at least 1 year [22]. Kowtow was defined as performing this religious action more than three times per day. Reciting sutra, turning prayer beads, and turning a prayer wheel were defined as doing so for more than 3 hours per day.

Laboratory measurements

Venous blood samples (5 ml) were obtained after an overnight fast, stored in EDTA-K₂ tubes, and stored at –20°C. A DNA extraction kit (Promega, Madison, WI, USA) was used to extract DNA. Finally, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) was used to test the different genotypes (Complete PLEX Gold Genotyping Reagent Ser 384; Sequenom, Inc., San Diego, CA, USA). Single-nucleotide polymorphisms (SNPs) were selected by reviewing previous studies [1 , 23]. Thirteen SNPs were selected (Supplementary Table 1).

PCR amplification primers and single-base extension primers of SNP loci were designed using the Genotyping Tools of the Sequenom Company and the MassARRAY Assay Design software (Supplementary Table 1). PCR amplification was carried out using multiple PCR technology in 384-well plates. The reaction volume was 5μl. PCR reaction conditions were: 94°C for 4 min; 45 cycles of 94°C for 20 s, 56°C for 30 s, 72°C for 1 min; and final extension at 72°C for 3 min. PCR products were treated with shrimp alkaline phosphatase (SAP) to remove the free dNTPs. The single base extension reaction was carried out after SAP treatment. Conditions were: 94°C for 30 s; followed by 43 cycles at 94°C for 5 s, 52°C for 5 s, and 80°C for 5 s; and final extension at 72°C for 3 min. PCR products were purified using resin. Dried resin was added with the extension product. The plate was sealed and rotated at low speed for 30 min. The resin was centrifuged and removed.

A Mass ARRAY Nanodispenser RS1000 spotting instrument was used to spot the purified extension products on a 384-well SpectroCHIP (Sequenom, Inc., San Diego, CA, USA) chips. The chips were analyzed by MALDI-TOF. The results were analyzed using the TYPER 4.0 software (Sequenom, Inc., San Diego, CA, USA).

Statistical analysis

Validity and reliability of the questionnaire of basic information were tested using the Cronbach’s alpha. Participants were divided into the AD and non-AD groups. To identify associations, subjects with cognitive impairment were matched 1:10 with subjects without impairment. For genotypes, subjects with cognitive impairment were matched 1:1.5 with subjects without impairment. Estimates of the prevalence of AD were calculated for the overall population and for subgroups stratified by age and gender. Age-standardized and gender-standardized prevalence was calculated based on the population distribution of China in 2010 [24]. The 95% confidence intervals (CIs) were calculated for prevalence, adjusted for age and gender. Analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA). The analysis of gene polymorphisms data was performed using SHEsis [25]. Two-sided p-values <0.05 were considered significant.

RESULTS

Characteristics of the participants

A total of 3,974 participants completed this survey. The rate of participation was 88.37% . Tables 1–3 present the characteristics of the participants. Compared to non-AD participants, patients with AD were older (p < 0.001), fewer were married (p = 0.026), and more of them lived with their children (p = 0.015) (Table 1). Dietary habits are presented in Table 2. Compared to non-AD participants, less patients with AD performed religious activities (all p < 0.05) (Table 2). Compared to non-AD participants, more patients with AD suffered from chronic obstructive pulmonary disease (p < 0.001) and had a history of head trauma (p < 0.001). Compared to subjects with AD, subjects without AD were eating less dairy products (p = 0.026), more bean products (p < 0.001), more chicken (p < 0.001), more fish (p = 0.002), and more eggs (p = 0.002).

Prevalence of AD

The prevalence of AD among residents aged 60 years and older was 1.33% (95% CI, 0.98–1.69). AD was more prevalent in females (prevalence ratio = 1.59; 95% CI, 1.10–2.09) compared to males (prevalence ratio = 0.92; 95% CI, 0.44–1.39). Meanwhile, the age group ≥85 years old (prevalence ratio = 12.05; 95% CI, 7.10–17.00) had the highest prevalence of AD (Table 4).

Association with non-hereditary factors

Variables with p-values <0.05 in univariate analyses (Supplementary Table 2) were entered in a multivariate logistic regression analysis (Table 5). Age of 75–79, 80–84, and ≥85, eating beef, and head trauma were positively associated with AD. Kowtow, turning prayer beads, and eating chicken were negatively associated with AD.

Gene polymorphisms

Among all patients, 39 specimens in the AD group and 56 specimens in the no AD group were available for SNP analyses. Age (p = 0.306) and gender (p = 0.524) were comparable between the two groups.

Linkage disequilibrium (D’ > 0.5, r² < 0.8) was found in the loci for CLU, TP53INP1, and TFAM, indicating that haplotypes could be analyzed for these three genes. In the AD and no AD groups, the frequency of all other SNPs loci conformed to the Hardy-Weinberg equilibrium (p > 0.05), insuring the reliability of their application to evaluate larger groups.

When not considering the impact of the APOE ɛ4 allele, allele A of rs2279590 and allele C of rs2306604 were more frequent in the AD group, while allele C of rs11136000 had a lower frequency in the AD group (Table 6). The genotypes AA+GA of rs2279590 were positively associated with AD, while the genotypes GC of rs9331888 and GG+GC of rs9331888 were negatively associated with AD (Supplementary Table 3).

There was no difference in the distribution of ApoE alleles between the two groups (p = 0.876) (Supplementary Table 4). Within the ApoE ɛ4 carriers, there was no difference in any locus between the two groups (Supplementary Table 5; all p > 0.05). Within the ApoE ɛ4 non-carriers, allele A of rs2279590 and allele C of rs2306604 were more frequent in the AD group, while allele C of rs11136000 was less frequent in the AD group (Supplementary Table 6).

In the analysis of haplotypes, the CLU haplotype AG of rs2279590 and rs9331888, and the CLU haplotype TG of rs11136000 and rs9331888 were more frequent in the AD group. The CLU haplotype GG of rs2279590 and rs9331888, and the CLU haplotype CG of rs11136000 and rs9331888 had a lower frequency in the AD group.

Within the ApoE ɛ4 carriers, there were no differences in haplotypes between the two groups. Within the ApoE ɛ4 non-carriers, the CLU haplotype AG of rs2279590 and rs9331888, and the CLU haplotype TG of rs11136000 and rs9331888 showed a higher frequency in the AD group. The CLU haplotype GG of rs2279590 and rs9331888, the CLU haplotype CG of rs11136000 and rs9331888, and the TFAM haplotype GT of rs1937 and rs2306604 showed a lower frequency in the AD group (Supplementary Table 7).

Multivariate analyses for the association with non-hereditary and genetic factors

History of head trauma was not included in the model because of the too small number of subjects. The model showed that AA+GA of rs2279590 was positively associated with AD, while GG+GC of rs9331888 and kowtow were negatively associated with AD (Table 7).

DISCUSSION

The “World Alzheimer Report 2014: the key points” [23] reports that more than 5 million Americans are living with AD, and that 1 in 3 elderly die with AD or other dementia. Approximately 500,000 people die each year because of AD. Tibetans have been living on the plateau for generations, and genetically adapted to the harsh life conditions [26]. In addition, they have special living habits, customs, and religious faith that may influence the occurrence of AD [27]. Therefore, the aim of this study was to estimate whether the prevalence of AD in this population and to investigate the related factors. Results showed that the prevalence of AD among Tibetan individuals was low. CLU haplotypes AA+GA (OR = 4.483; 95% CI: 1.069–18.792) of rs2279590 was associated with AD. CLU haplotypes GG+GC (OR = 0.184; 95% CI: 0.038–0.888) of rs9331888 and kowtow (OR = 0.203; 95% CI 0.046–0.896) were negatively associated with AD.

In Jamaica, the age-standardized prevalence of dementia in people >60 years old is about 5.32% [28], while it is 5.4% in Europe and 6.5% in North America [29]. The prevalence of AD reported in a meta-analysis was 1.9% in Chinese people >60 years old [3]. In this survey, the prevalence of AD in Tibetans was 1.33% . The age-standardized prevalence of AD was 0.18% , which was lower than in previous reports [30]. A previous study has shown that the results of the mini-mental status examination in Tibetans were similar to those of mainland China, but that the distributions of age, education level, and gender in relation to the score distribution were different between Tibetans and mainland Chinese [31].

As aging is the highest risk factors of AD [1], the shorter life expectancy of people living in the Qinghai province might be responsible for the low prevalence of AD. According to the “China Statistical Yearbook, 2014” [32], the life expectancy in the Qinghai province is 69.96 years, compared to 74.92 in the Shanxi province. A recent study [33] reported that the prevalence of AD is 3.7% in people >60 years old in the Shanxi province. Therefore, it may be hypothesized that the low prevalence cold be associated with shorter life expectancy in the Qinghai province. Nevertheless, aging is not the only risk factor for AD [1]. In addition, the prevalence of AD in people aged >80 years was 5.07% in this present study while a recent survey indicated a prevalence of 13.6% [33]. Furthermore, the Shanxi province is a plain mainly inhabited by Han people, whose customs, living habits, environment, and religious behaviors are very different from that of Tibetans.

In this study, there were no differences in literacy, which contradicts a previous report [1] and could be due to the low literacy of Tibetans in the entire Qinghai region [31, 32]. In multivariate analyses of non-hereditary associations, being aged ≥75 years was associated with AD. Beef intake habits and head trauma were also associated with AD. As red meat intake increases the risk of cardiovascular and cerebrovascular diseases, healthy blood vessels help ensure that the brain is supplied with oxygen and nutrients [1]. Meanwhile, chicken intake, kowtow, and turning prayer beads showed protective effects against AD. Chicken contains more unsaturated fatty acids than red meat, which could reduce blood low-density lipoprotein cholesterol levels. Kowtow is a “mind-body” process, and Tibetans believe that kowtow express their respect for Buddha and their religious faith to a maximum extent. In addition, kowtow is a meditative process, which has been shown to improve cognitive functions [6, 7].

The CLU gene encodes apolipoprotein J/clusterin, and was significantly associated with AD [34, 35]. TP53INP1 is a genetic locus that was only recently associated with AD [36]. TP53INP1 has mainly been shown to be involved in cancers [37] and its exact role in AD remains to be elucidated. A number of previous studies have associated genetic variants of the mitochondrial transcription factor A (TFAM) with AD [38, 39]. When considering the genetic factors, ApoE ɛ4 polymorphism was not different between the two groups, which contradicts a previous study. This might be due to the small sample size or a possible under-representation of this allele since Tibetans hardly intermarried with other ethnic groups for generations. The CLU haplotype AA+GA of rs2279590 was associated with AD. The CLU rs2279590 polymorphism has been reported to be significantly associated with hemoglobin A (1c) level in the Japanese population [14]. The CLU haplotype GG+GC of rs9331888 is relevant to AD protection; rs9331888 is located at a non-coding region and might affect the expression of protein, which affects the susceptibility to AD. Interestingly, Kowtow still showed an independent positive impact for AD protection, but other factors such as diet and environment were not verified in the last regression model.

This study is not without limitations. Even if the whole population study was large, the sub-sample for genetic analyses was small. The AD diagnoses were made by discussion between the experts, which may introduce some bias. In addition, the generalizability of the present study is limited to people living in high altitude. The comparison of the prevalence between Tibetans and the Shanxi province is limited by the difference of life expectancy (70 versus 75 years) and its impact on the development of AD. Larger studies are required to get the exact picture of the whole population.

In conclusion, the present study suggests that the prevalence of AD in Tibetans was lower than in the general Chinese population. Multivariate analysis might suggest that regular “mind-body” religious meditative activities may be negatively associated with AD in this population, as well as the CLU genotype at rs9331888. These results may provide some probable new directions for research on AD prevention.

Footnotes

ACKNOWLEDGMENTS

We thank our colleagues from the Southern Medical University, the civil authorities in Qinghai, the Qinghai Centers for Disease Control and Prevention, the civil authorities in Hainan of Qinghai, and the Qinghai Red Cross Hospital for their help. This survey was supported by the State Key Scientific Research Programs (1981020421) and The Central Government Support Social Organizations to Participate In Social Service in 2014 Programs (B033).

Authors’ disclosures available online ().

Appendix

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