| 1. IntroductionDementia is an overall term for diseases and conditions characterized by a decline in memory or other thinking skills that affects a person’s ability to perform everyday activities. Alzheimer’s disease (AD) is the most common type of dementia. A severe cognitive deficit is displayed in AD patient that prejudices the quality of life of family members and healthcare system as well. However, no efficient measure is performed in preventing AD at present. Similarly, AD and other dementias cannot be reversed with current treatments.Tibetans have been living at high altitude longer than any other ethnic group, they have deep religious faith, particular living habits, greater hypoxic and hypercapnic ventilatory response, larger lungs, greater lung diffusing capacity and better lung function than lowlanders. As for genetic adaptation to high altitude, they have been concerned for more than 10 years by scientists. In recent report, Tibetans had higher prevalence of hyperlipidemia than other populations in China, which might be derived from their living environment, living habits, special customs, gene polymorphism and religious behaviors.Recently, a systematic meta-analysis by Prince etc. of the prevalence of dementia demonstrated that age-standardized prevalence for those aged≥60 years varied in a narrow band,5%-7% in most world regions, and the highest existed in Latin America (8.5%), a distinctively lower prevalence in the four sub-Saharan African regions (2%-4%). This review indicates that the prevalence of dementia differs among racial groups. Meanwhile, Zhang etc. reported that the pooled prevalence of the population over 60 years of AD was 1.9% and the total prevalence of dementia was 3.0% in China, via a meta-analysis of 48 epidemiologic studies from 1980 to 2010.Along with the APOE polymorphism,20 common susceptibility loci have been identified associated with AD. However, there had not been reported any of them were tested in Tibetan. Therefore, as few extant epidemiologic investigations have focused on Tibetans in Qinghai-Tibet plateau, we conducted this survey to estimate the prevalence of dementia and AD in elderly Tibetans as well as the risk factors and gene polymorphism.2. Methods2.1. Study design and samplesFrom May 2014 to September 2014, a population-based cross-sectional survey with a multistage cluster sampling design was performed. Separate subjects were recruited from Qinghai province of China. First,4 representative cities were selected in Qinghai. Second,6 counties within the selected cities were chosen randomly. Last,65 rural villages within the selected counties were sampled at random.In the investigation, all eligible individuals were Tibetan Chinese in ethnicity aged 60 years and older which were drawn from 65 rural villages (n=4506). According to preceding survey data, every resident was listed for at least 10 years in the census of the community. Those listed in the census but lived in nursing rooms were also included. In order to guarantee a maximum participation rate, we conducted a door-to-door survey in some villages where transportation was difficult. Subjects who refused to participate, or were deceased, or were untraceable, or had a life-threatening illness were excluded. Finally,4060 residents participated in the survey. In the process of measuring data, beef, mutton, dairy products, bean products, chicken,fish and vegetables intake habits were defined as eating more than 1.25 kilograms per week. Eggs intake habits were defined as eating more than 3 eggs per week. Kowtow was defined as doing more than 3 times per day. Recite sutras, turn prayer beads, turn prayer-wheel were defined as doing more than 3 hours per day.In data analysis, incomplete data and incredible data had been eliminated. As just 8 residents were diagnosed with vascular dementia (VD), we did not analyze the statistic about VD in this research. At last, the data of 3974 residents were used to analyze the characteristics of samples.To find risk factors, we screened a control group in the ratio of 10:1 with the case group from people without cognitive impairments randomly. As well, to analyze gene polymorphism, we screened a control group in the ratio of 1.5:1 with the case group from people without cognitive impairments. Also, they did not have biological kin and difference in age and gender with cases.5ml venous blood samples harvested from the morning fasting venous blood, stored in EDTA-K2 tubes, cryopreserved in -20℃, use the steps of DNA distilling kit to extract DNA. Finally, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) was used to test genotypes of DNA.Single-nucleotide polymorphisms (SNPs) were selected by consult previous studies and the results of genome-wide association studies. A total of 13 SNPs were selected.Informed consent of each participate was obtained either from their guardian or themselves. The protocol for this study was approved and reviewed by the ethics committee.2.2. Assessment and diagnosis2.2.1. TrainingThe investigation group consisted of both experts and interviewers. A panel comprised of 3 neurologists and 3 neuropsychologists with special expertise in cognitive impairment. There were 3 groups of interviewers, including 1 senior neurology graduate student,1 junior neurologist and 1 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 were required to exceed 0.90.2.2.2. Diagnostic proceduresEach interviewer group conducted individual, semi-structured interviews with participants and their close informants at the residence of respondents. Interviews usually lasted 0.5 to 1 hour. Detailed data on socio-demographic characteristics, medical history, lifestyle, current medications and family history were collected. One of the interviewers tested the participant with a series of neuropsychological tests. First, the cognitive function was assessed with Mini-Mental State Examination. Second, the social function was evaluated with the Activity of Daily Living Scale. Third, the differentiation of dementia was assessed by the Hachinski Ischemic Scale. The other interviewer took a detailed history of all cognitive impairments, including time and mode of onset, possible triggers, affected domains, course of condition, impact on daily activities, changes in mood or behavior, results of computed tomographic scans or magnetic resonance images, and treatment along with its effects. Last, standardized general and neurological examinations were performed.All information collected was recorded on the questionnaire of basic information, reviewed by the panel and interviewers, and diagnose 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.2.2.3. Diagnostic criteriaDiagnostic criteria for dementia were based on the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders. After a diagnosis of dementia was made, participants were divided into 3 subtypes:AD, VaD, and other types of dementia. Diagnostic criteria for AD based on the criteria issued by the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association. Diagnostic criteria for VaD was based on the criteria of the National Institute of Neurological Disorders and Stroke-Association International pour la Recherche et l’Enseignement en Neurosciences. According to various globally accepted criteria, other types of dementia (OD) included mixed dementia, fronto-temporal dementia, dementia with Lewy-bodies, Parkinson’s disease with dementia etc. was not analyzed in the present survey.2.3. Statistical analysis2.3.1. Standardized prevalenceEstimates of the prevalence of dementia and AD were calculated for the overall population and for subgroups stratified by age and gender. Age-standardized and gender-standardized prevalence rates were calculated based on the population distribution of China in 2010. The prevalence ratios (PRs) with 95% confidence intervals (CIs) were calculated adjusted for age and gender.2.3.2. Characteristics of samplesValidity and reliability of the questionnaire of basic information were tested by Cronbach’s alpha. The data of participants were divided into AD group and non-AD group, analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA) and differences between the groups were considered statistically significant when the P value was less than 0.05.2.3.3. Logistic regression of non-hereditary risk factors.Logistic regression models were used to ascertain the risk factors of AD considering the main effects of socio-demographic characteristics, lifestyle and comorbidity. All analyses were performed using SPSS 20.0(SPSS Inc., Chicago, IL, USA) and differences were considered statistically significant when the P value was less than 0.05.2.3.4 Gene polymorphisms detection.The analysis of gene polymorphisms data was performed using SHEsis. Logistic regression models were used to ascertain the risk factors of AD considering the main effects of non-hereditary factors and genetic factors. Differences were considered statistically significant when the P value was less than 0.05.3. Results3.1. Demographic characteristics.A total of 3982 residents were identified as completed the survey. The rate of participation was 88.37%. The total number of dementia, AD and VD were 61,53 and 8, respectively. In participants with AD, the gender ratios were skewed towards female (73.6%), the age ratios were inclined to age 85 and older (37.7%), which had the lowest proportion of population in all age groups.3.2. Prevalence of dementia and ADThe prevalence ratios (PRs) of dementia and AD among residents aged 60 years and older was 1.53%(95% CI,1.15-1.92) and 1.33%(95% CI,0.98-1.69), respectively. The data presented a higher prevalence of female (PR,1.59; 95% CI, 1.10-2.09) compared to male (PR,0.92; 95% CI,0.44-1.39) in AD. Meanwhile, the age group of over 85 years old (PR,12.05; 95%CI,7.10-17.00) had the highest prevalence of dementia and AD, apparently. Simultaneously, the data were standardized according to age and gender distribution of the Chinese population in 2010.3.3. Validity and reliability of the questionnaire of basic information.Cronbach’s alpha of the 2 reexamining subjects were 0.717 and 0.689, the data of questionnaire was credible.3.4. Retrospective study of difference between AD and non-AD group.Between AD and non-AD group, there were no significant differences in gender, literacy, habitual residence, vegetables intake habits and a history of hypertension, ischemia stroke, hemorrhagic stroke and cancer. However, age, marital status, birthplace, life partners, alcohol, smoking, religious behaviors, a history of chronic obstructive pulmonary disease, head trauma and diet habits had obvious differences between 2 group.3.5. Logistic regression of non-hereditary risk factors.To verify the potential risk factors, univariate logistic regression and multivariate logistic regression analysis were running between AD group and control group. In univariate logistic regression, the data suggested that age of 75-79 (OR, 4.497; 95%CI,1.691-12.487),80-84 (OR,4.583; 95%CI,1.427-14.719) and ≥85 (OR,38.500; 95%CI,12.630-117.356), beef (OR,2.162; 95%CI, 1.099-4.252), mutton (OR,2.464; 95%CI,1.165-5.211), dairy products (OR, 2.212; 95%CI,1.075-4.549), chronic obstructive pulmonary disease (OR,11.070; 95%CI,1.805-67.874) and head trauma (OR,14.510; 95%CI,1.293-162.889) had a positive correlation with AD. Religious behaviors like kowtow (OR,0.095; 95%CI,0.029-0.309), recite sutra (OR,0.279; 95%CI,0.155-0.503), turn prayer beads (OR,0.331; 95%CI,0.184-0.595), turn prayer-wheel (OR,0.511; 95%CI,0.275-0.951), chicken (OR,0.079; 95%CI,0.011-0.580) and egg (OR, 0.231; 95%CI,0.081-0.658) intake habits and smoking (OR,0.195; 95%CI, 0.046-0.824) had a negative correlation with AD.In multivariate logistic regression analysis, the model showed that age of 75-79 (OR,4.497; 95%CI,1.691-12.487),80-84 (OR,4.583; 95%CI,1.427-14.719), ≥85(OR,35.728; 95% CI,9.640-132.418), beef intake habit (OR,6.174; 95% CI,2.460-15.492) and head trauma (OR,44.622; 95% CI,2.546-782.170) had a positive correlation with AD. While Kowtow (OR,0.109; 95%CI,0.027-0.439), turn prayer beads (OR,0.198; 95%CI,0.084-0.467) and chicken intake habit (OR, 0.050; 95%CI,0.005-0.520) had a negative correlation with AD.3.6. Gene polymorphisms detection and analysis.There were 39 specimens in AD group and 56 specimens in control group. The geometric mean of age (p=0.306) in AD and control were 80.69±9.00 and 79.11±11.04, the geometric mean of gender (p=0.524) in AD and control were 1.74±0.439 and 1.70±0.461, without difference in age and gender between 2 group. The time of flight mass spectra (TOF MS) products of 13 locus’ gene polymorphisms detection were attached to appendix according to the limited space of the article.In AD and control group, the frequency of rs11136000 etc. locus conformed to Hardy-Weinberg equilibrium (x2<3.84, p>0.05), insuring the reliability of their application to evaluate larger groups. The linkage disequilibrium (D’>0.5, r2<0.8) were found in locus of CLU, TP53INP1 and TFAM, which proved haplotype could be analyzed within these 3 genes.Without the impact of APOE ε 4, allele A (OR,3.567; 95%CI,1.397-9.111) of rs2279590, allele C (OR,2.005; 95%CI,1.009-3.984) of rs2306604 had higher frequency in AD group. Allele C (OR,0.350; 95%CI,0.140-0.878) of rs11136000 had lower frequency in AD group. The model AA+GA (OR,3.264; 95%CI, 1.248-8.538) of rs2279590 had a positive correlation with AD. The model GC (OR, 0.422; 95%CI,0.182-0.980) of rs9331888, GG+GC (OR,0.373; 95%CI, 0.144-0.963) of rs9331888 had a negative correlation with AD.The difference of APOE ε 4 (p=0.876) between AD and control had not been found, however, we still separated subjects with APOE ε 4 (+) and APOE ε 4 (-). Within APOE ε 4 (+), no locus showed diversity between 2 group. Within APOE ε 4 (-), allele A (OR,3.567; 95%CI,1.397-9.111) of rs2279590, allele C (OR, 2.005; 95%CI,1.009-3.984) of rs2306604 had higher frequency in AD group. Allele C (OR,0.350; 95%CI,0.140-0.878) of rs11136000 had lower frequency in AD group.In analysis of haplotypes, CLU haplotype AG (OR,2.721; 95%CI, 1.113-6.651) of rs2279590 and rs9331888, CLU haplotype TG (OR,2.815; 95%CI,1.199-6.608) of rs11136000 and rs9331888 had higher frequency in AD group. CLU haplotype GG (OR,0.438; 95%CI,0.230-0.832) of rs2279590 and rs9331888, CLU haplotype CG (OR,0.398; 95%CI,0.206-0.768) of rs11136000 and rs9331888 had lower frequency in AD group. Within APOE ε 4 (+), no haplotypes showed diversity between 2 group. Within APOE ε 4 (-), CLU haplotype AG (OR,2.918;95%CI,1.111-7.659) of rs2279590 and rs9331888, CLU haplotype TG (OR,2.996;95%CI,1.193-7.521) of rs11136000 and rs9331888 had higher frequency in AD group. CLU haplotype GG (OR,0.338;95%CI,0.152-0.750) of rs2279590 and rs9331888, CLU haplotype CG (OR,0.291;95%CI,0.126-0.672) of rs11136000 and rs9331888, TFAM haplotype GT(OR,0.499;95%CI,0.251-0.991) of rs1937 and rs2306604 had lower frequency in AD group.3.7. Non-hereditary factors and genetic factors in logistic regression models for AD.As there were just 2 participants had a history of head trauma in AD group, and 0 in control group, we did not put it into logistic regression. Considering kowtow, turn prayer beads, beef, chicken and discrepant genotypes, in multivariate logistic regression analysis, the model showed that model AA+GA (OR,4.483;95%CI, 1.069-18.792) of rs2279590 had positive correlation with AD. Model GG+GC (OR, 0.184; 95%CI,0.038-0.888) of rs9331888 and kowtow (OR,0.203; 95%CI, 0.046-0.896) had negative correlation with AD.4. Discussion.《World Alzheimer Report 2014:the key points》 report that more than 5 million Americans are living with AD, lin 3 seniors die with AD or another dementia, approximately 500,000 people dying each year because they have AD. Tibetans live on the plateau for generations, with genetic adaption for plateau, special live habits and customs and religious faith for buddle, which may influence the prevalence of AD or dementia. We conduct this survey to estimate the prevalence in elderly Tibetans. It should be noted that this study used a standardized procedure to diagnose dementia and AD. The results of the survey may provide new sights to prevent the onset of AD.In West Europe, age-standardized prevalence rate of dementia in persons over 60 years old was 5.32%. The prevalence of dementia and AD reported in a meta-analysis were 3.0% and 1.9% aged 60 years and older in China. In this survey, the prevalence of dementia and AD in Tibetans were 1.53% and 1.33%, respectively. And the age-standardized prevalence of dementia and AD were 0.20% and 0.18% which were standardized on the Chinese population distribution in 2010, which were significantly lower than previous reports about Chinese and proves AD to be the most common type of dementia in Tibetan.As aging is the highest risk factors of AD, we conjecture whether the low prevalence correlate with the short life expectancy. According to 《China Statistical Yearbook,2014》, life expectancy of Qinghai province is 69.96 while Shanxi province is 74.92. A recent study report that the prevalence of AD is 3.7% among people over 60 years and older in Shanxi province, therefore, we might imagine that the low prevalence has some correlation with the short life expectancy. However,《2014 Alzheimer’s Disease Facts and Figures》 mentions that aging is not the unique risk factor for AD. In addition, the prevalence of AD aged 80 years and older is 5.07% in our survey while that in the recent survey is 13.6%. Also, Shanxi province is a plain of Han settlement, whose customs, live habits, environment, religious behaviors are different with Tibetan. Therefore, we analyze the data to seek for potential impact factors of AD. Also to point out, just 8 VD has been diagnosed, we hypothesized this might be attributed to the higher mortality after stroke because of the inconvenient medical condition in rural villages and pastoral areas.Retrospective study indicate that the literacy show no difference between AD and control group, which is opposite with the previous report. Reason for this might be the low literacy of Tibetan in the entire region. In multivariate logistic regression of non-hereditary risk factors, aged 75 years and older is risk factor for AD, the older age, the higher risk. Also, beef intake habits and head trauma are risk factors. As take in more red meet increase the risk for cardiovascular and cerebrovascular diseases, a healthy heart helps ensure that enough blood is pumped through blood vessels, and healthy blood vessels help ensure that the brain is supplied with the oxygen and nutrient-rich blood it needs to function normally. These results are keep track of the previous reports. Meanwhile, chicken intake habits, kowtow and turn prayer beads show protective effects for AD. This might be reason for chicken contain more unsaturated fatty acid compared with red meet, which could reduce the low density lipoprotein cholesterol content in blood. Kowtow is mind-body workout, Tibetan believes that kowtow could express their respect for buddle and religious faith to maximum extent. Meanwhile, they are meditating at the same time. Therefore, we believe that kowtow not only effectively increase daily exercise, but also comfort their spirit and stabilize their mood, and then improve cognitive function comprehensively.When put together with genetic factors, APOE ε 4 polymorphisms has no difference in 2 groups, this is inconsistent with previous research. We hypothesized the reasons for this might be small sample size or Tibetan hardly intermarried with other ethnic group for generations. However, CLU model AA+GA of rs2279590 show more relevant to AD risk. CLU gene codes for Apolipoprotein J/Clusterin, as a significant risk variant for Alzheimer’s disease AD. CLU rs2279590 polymorphism has been reported to be significantly associated with hemoglobin A (1c) level in Japanese population. CLU model GG+GC of rs9331888 show more relevant to AD protection. Rs9331888 locate at non-coding region and might affect the expression of protein which affects the susceptibility to AD. Interestingly, Kowtow still show positive impact for AD protection, but other factors such as diet and environment are not verified in the last regression model.5. ConclusionThis is the first study to systematically investigate the prevalence and gene polymorphisms of AD in Tibetan. Our study presented that the prevalence of dementia and AD in Tibetan were lower than the average level of general population in China, Qinghai-Tibet Plateau is the most typically high altitude environment worldwide, the results of this research might suggest new sights and therapeutic consideration for researchers of AD prevention. |
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