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      Cohort Profile: The Healthy Aging Longitudinal Study in Taiwan (HALST)

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          Why was the cohort set up? During the past century, there have been dramatic improvements in life expectancy in Taiwan, with the average life span increasing from 30.0 and 32.1 years for men and women in 1908 to 77 and 83.5 years in 2016, respectively. 1 As a consequence of this demographic transition, the population in Taiwan has rapidly been ageing. Currently, persons aged 65 and older comprise about 12.5% of the Taiwanese population; this proportion is projected to reach 14% in 2018 and 20% in 2025. 2 With the ageing of a population comes an increase in prevalence of chronic diseases and geriatric syndromes, and an expansion in healthcare costs that impose a huge burden on the whole society. 3 For example, cancer, coronary heart disease, stroke, diabetes, hypertension and chronic kidney disease have been listed among the 10 leading causes of death among the elderly in Taiwan for the past decade. 4 The high prevalence of chronic kidney disease in the elderly (estimated to be over 37% 5 ) is another example indicating that an ageing population is one of the most important factors behind the high incidence and prevalence of end-stage renal disease (ESRD) in Taiwan. 6 Thus, it is essential to understand more about risk factors attributable to the ethnicity-specific ageing process so that effective prevention programmes can be developed for the elderly. To address different age-related health issues, several longitudinal Chinese ageing studies have previously been conducted in Taiwan or other Asian countries, such as the Chinese Longitudinal Health Longevity Survey (CLHLS), 7 the China Health & Retirement Longitudinal Study (CHARLS), 8 the Beijing Longitudinal Study of Aging (BLSA), 9 the Taiwan Longitudinal Study of Aging (TLSA) 10 and the Singapore Chinese Longitudinal Aging Study. 11 However, most of these established senescent cohorts, which were mainly followed up by collecting self-reported information, had not acquired comprehensive biomedical profiles for their participants. To further understand determinants of healthy longevity and geriatric issues, additional functional status measurements and biochemical data collections have been appended to some sub-studies 7 , 8 , 12 but their sample sizes were far smaller compared with their original cohorts. For example, lack of statistical power (n = 639) is one of major limitations of the Social Environment and Biomarkers of Aging Study (SEBAS), 12 a sub-study of the TLSA, in spite of its enrichment with biochemical, genetic and functional measurements. To overcome the common barriers in geriatric cohort research, the Healthy Aging Longitudinal Study in Taiwan (HALST) was therefore established and funded by the National Health Research Institutes in Taiwan to address issues related to healthy ageing (ClinicalTrials.gov: NCT02677831). The main study objectives of the HALST are to investigate: (i) factors that may influence trajectories of physical functioning; (ii) impacts of healthy lifestyles on incidence of chronic diseases, quality of life and mortality; (iii) individual, social and environmental determinants of cardiovascular diseases; (iv) association of neuropsychiatric risk factors and well-being; and (v) interaction between genetic traits and environmental risk factors in frailty versus successful ageing processes in older adults. The project also involves a variety of ancillary sub-studies focusing on important health-related issues that are unique to local people (such as betel quid chewing, dietary pattern, hepatitis due to viral infection and chronic renal disease). These issues are frequently overlooked but are crucial for the development of health-promotion programmes in older populations. Who is in the cohort? The HALST is designed as a longitudinal study recruiting community-dwellers aged 55 and above in seven selected areas in Taiwan: two in the north (Shilin District and Yangmei Township), two in the central region (Miaoli City and Changhua City), two in the south (Puzi Township and Lingya District) and one in the east (Hualien). These seven locations (Figure 1) cover both urban and rural areas, as well as different ethnic groups speaking different dialects, representing the diverse socio-demographic characteristics of the Taiwanese population. In each catchment area, a regional hospital was selected to be the medical facility for clinical examinations, and all eligible residents living within about a 2-km radius of this local hospital were ascertained from the household registry archives. By using a systemic sampling method, beginning with around 3000–3500 residents aged 55 and above in each catchment area, we created a recruitment roster within the target population. To ensure that our study sample covered a sufficient number of the elderly with different socio-demographic backgrounds, the older adults (≥ 65 years of age) were over-sampled (70% for those ≥ 65 years and 30% for those in 55–64 years); on the other hand, the sampling distributions of gender and educational level (none, primary school, high school and above) are based on the demographic distribution within a li (village), a basic house registration unit defined by the Taiwan government. Individuals with any of the following conditions at the recruiting interview were excluded: highly contagious infectious diseases (including scabies and open tuberculosis), severe illnesses (including cancer under treatment), physician-diagnosed dementia, bedridden and/or too frail to stand and walk, severe mental disorder or cognitive impairment with a Mini-Mental Status Score < 16, 13 mental retardation or severe hearing loss. Individuals who were then hospitalized or institutionalized were also excluded. Figure 2 presents a flow chart with details of the subject selection process and the number of participants who completed the first annual follow-up telephone interview. Compared with the recruited subjects, the non-participants were more likely to be women, older and illiterate. In the first-wave survey (2009–13), we enrolled 5664 community-dwellers aged 55 or above. Figure 1. Map of participating sites in the HALST. Figure 2. Selection flow chart for the HALST participants. How often have they been followed up? The longitudinal assessments conducted in the HALST consist of home interviews and hospital-based clinical examinations every 5 years. The first-wave survey (recruitment and baseline survey) was carried out in 2009–13. The field study team took about 6–8 months to finish the work in one catchment area, starting in Miaoli City and then moving to the next (Shilin District was the last) for the processes of recruitment, interview and examination. The second-wave follow-up (2014–19) of home interviews and examinations is currently under way. After enrolment, those who have completed both home interviews and hospital-based examinations (n = 5349) are to be followed up by telephone contact every year for updates on vital status and health-related conditions. What has been measured? In the sampling area of each site, community residents who met the inclusion criteria were invited to participate in the HALST study. A home interview was arranged for those who completed the consent form. Within 2 weeks after the home visit, study participants received a physical examination and provided morning spot urine and up to 30 ml of fasting blood specimens in one of the local hospitals. The home interview took about 90 to 120 min to complete; and the clinical examination required about 120 min. All interviewing and examination processes are based on the standardized manual of operation; the field sites are periodically inspected by the responsible investigators every season; and a routine call-back interview for quality and reliability control is performed for around 8% of the enrolled subjects by random selection. As seen in Table 1, information obtained through the measurements and analyses employed in the HALST can be organized into four parts: home visit, clinical examination, laboratory analysis and follow-up telephone survey. ‘Home visit’ and ‘clinical examination’, including blood and urine samples collection, constitute the formal investigation conducted every 5 years; ‘follow-up telephone survey’ is the annual survey of vital status and new health events occurring between formal home visit and clinical examination. The main measures are aimed at collecting information on physical function and geriatric conditions (e.g. lifestyle profiles, cardiovascular diseases, cognitive and mental health and longevity-related genetic factors) necessary for our research interests. In addition, the design of measures and instruments is mainly based on three practical considerations: (i) our results could be compared with those from well-recognized ageing-related studies, such as the Chicago Healthy Aging Study and Baltimore Longitudinal Study of Aging; (ii) the instruments have been used in similar population-based studies in ethnic Chinese communities; and (iii) the Chinese-language version questionnaires were chosen from those validated and widely recognized for use in community-based studies such as assessments of leisure time physical activity, 14 the Chinese version of Lawton and Brody’s measure 15 for instrumental activities of daily living (IADLs), 16 the Mini-Mental State Exam (MMSE), 17 the Center for Epidemiologic Studies Depression Scale (CES-D) 18 and the Short-Form 12 Health Survey (SF-12). 19 , 20 The food intakes were measured from a food frequency questionnaire (FFQ) containing more than 80 items of Chinese food. The validation of this FFQ has been reported elsewhere. 21 Specifically, we show participants wooden blocks representing the volume of the food to approximately measure for each food item; then the frequency and the volume are converted accordingly. The final results are the amount consumed per day. Table 1. Measurements in the Healthy Aging Longitudinal Study in Taiwan (HALST) Type Measures Instruments Interviewer-administered home visit Questionnaires Physical functioning Barthel Index, Lawton-Brody IADL Scale Frailty CHS frailty phenotype, the Edmonton frail scale, CSHA-CFA, SOF Cognitive function MMSE Mental health 20-item CES-D Health-related quality of life SF-12 Diet assessment FFQ Others: evaluation of general conditions, social demography, health conditions, geriatric conditions (fall, pain), chronic disease risk factor, sleep, use of healthcare, lifestyle (smoking, alcohol, betel, physical activity, nutritional supplements) and family history of chronic diseases Physical assessment Performance-based measures Peak flow test, grip strength, SPPB Clinical examination Examinations Anthropometry Height and weight, and hip circumference Brief physical examination Lower extremity function Single-leg stand test, timed up-and-go test, 6-min walk Cardiovascular function Blood pressure, heart rate, electrocardiogram, ankle-brachial index measurement, heart rate variability Cognitive function Digit symbol substitution test, clock drawing test Vision Visual acuity Mental health PRIME-MD Questionnaires Clinical assessment of cardiovascular symptoms Rose questionnaire, TIA questionnaire Other: vision, hearing, incontinence Laboratory analysis Blood tests Routine biochemistry Cholesterol, triglyceride, HDLC, LDLC, globulin, albumin, total protein, AST, ALT, GGT, insulin, glucose, creatinine, BUN, uric acid Haematology HbA1c, complete blood count (RBC, WBC, platelet), haemoglobin, HCT, MCV, MCH, MCHC, differential count of WBC Inflammation-related High-sensitivity CRP (hsCRP), intact PTH, ionized calcium, vitamin B12, folic acid Hepatitis virus titre HBsAg, Anti-HCVAb Coagulation factor D-dimer, fibrinogen DNA Genes associated with ageing (SNPs) Other IL-6, TNF-R1, IGF-1, sIL-6r, vitamin D Urine test Routine urinalysis Colour, clarity, specific gravity, pH, glucose, protein, occult blood, urobilinogen, bilirubin, nitrite, ketone body, RBC, WBC, epithelial cells, casts, crystals, bacteria, parasites, urinary albumin, urine creatinine, leukocyte esterase Follow-up telephone questionnaire Questionnaires Self-rated health status, physical functioning, pain, weight changes, smoking, physical activity, vision, falls and fractures, depressive symptoms, new events and health conditions, specific examinations and surgeries BSRS-5 IADL, Instrumental Activity of Daily Living; CHS, Cardiovascular Health Study; CSHA-CFA, Chinese-Canadian Study of Health and Ageing Clinical Frailty scale physical version; SOF, Study of Osteoporotic Fractures; MMSE, Mini-Mental State Examination; CES-D, Center for the Epidemiologic Studies Depression Scale; SF-12, Short Form 12; FFQ, Food Frequency Questionnaire; SPPB, Short Physical Performance Battery; PRIME-MD, Primary Care Evaluation of Mental Disorders; TIA, transient ischaemic attack; HDLC, high-density lipoprotein cholesterol; LDLC, low-density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transpeptidase; BUN, blood urea nitrogen; HbA1c, glycated haemoglobin; RBC, red blood cells; WBC, white blood cells; HCT, haematocrit; MCV, mean cell volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; CRP, C-reactive protein; PTH, parathyroid hormone; HBsAg, hepatitis B surface antigen; Anti-HCVAb, anti-hepatitis C virus antibody; IL-6, interleukin-6; TNF-R1, tumour necrosis factor-R1; IGF-1, insulin-like growth factor-1; SNPs, single nucleotide polymorphisms; sIL-6r, soluble interleukin-6 receptor; BSRS-5, Brief Symptom Rating Scale-5. For the laboratory analysis, routine fasting blood and morning urine tests are analysed at a certified clinical laboratory. In addition to the routine standardization and calibration tests performed by the laboratory, duplicate samples for about 5% of the specimens blinded to the laboratory are submitted together with other control samples to test reliability. We created three different levels (high, medium and low) of serum pools from control samples to assess accuracy of the assays operated by the central laboratory. All the remaining blood is centrifuged, aliquoted and stored in a -80°C freezer at the National Health Research Institutes, where other blood tests—including inflammatory markers, blood clotting markers, hepatitis B and hepatitis C markers and genetic assays—are undertaken in the principal investigators’ laboratories. The results of annual validity and reliability tests regarding between-run and within-run quality control of some major laboratory items are acceptable. In addition to the measures set in the routine investigations, a number of measures in relation to our main research interests are also conducted through ancillary sub-studies by collaborative researchers. For example, to better understand the relationship between bone/muscle mass and older adults’ health, examinations of bone mineral density and whole body scan using dual-energy X-ray absorptiometry were carried out at the Puzi, Changhua and Hualien sites. In addition to the home interviews and hospital-based examinations which are conducted every 5 years, we also perform annual telephone contact to update participants’ health conditions such as changes in body weight, lifestyle behaviours, newly diagnosed diseases and conditions and new events of fall and fall-induced fractures and hospitalizations. To ascertain mortality and cause of death, we link the identification number of the HALST participants to the National Death Registry Database on a yearly basis. Similarly, medical records are requested for the ascertainment of any hospitalized events. In addition, we also assess health outcomes, healthcare utilization and medical costs from the National Health Insurance database (NHID) for those who have signed an informed consent (n = 5152, 91%) for the data linkage. What has been found? Key findings and publications Table 2 shows some selected baseline socio-demographic characteristics by different age groups. Women outnumbered men for those younger than 75. The prevalence of widowhood increased with older ages. The percentages of self-identified ‘mainlanders’ (immigrants from China) and no education were the highest (20.0% and 24.6%, respectively) in the oldest group. As regards lifestyle characteristics, about 13% of the study participants were current smokers and 3% were betel-quid chewers. The prevalence of these risky behaviours declined as the participants became older. The percentage in each age group that engaged in leisure-time physical activity was about the same (around 71%). With regard to the self-reported major cardiovascular diseases (such as heart disease and stroke) and some age-related conditions (such as cataract, arthritis and prostatic disorders in males), the prevalence generally increased with age. Table 2. Numbers of study subjects in the HALST cohort presenting with selected socio-demographic characteristics (and percentages in parentheses) and reported health conditions Total (n = 5664) 55–64 years (n = 1686) 65–74 years (n = 2497) ≥ 75 years (n = 1481) P Sex 0.001  Male 2676 (47.2) 800 (47.4) 1121 (44.9) 755 (51.0)  Female 2988 (52.8) 886 (52.6) 1376 (55.1) 726 (49.0) Marital status < 0.001  Married 4140 (73.1) 1428 (84.7) 1823 (73.0) 889 (60.0)  Widowed 1235 (21.8) 121 (7.2) 570 (22.8) 544 (36.7)  Other a 289 (5.1) 137 (8.1) 104 (4.2) 48 (3.2) Ethnicity b < 0.001  Fukien 3326 (58.8) 1017 (60.4) 1573 (63.0) 736 (49.8)  Hakka 1638 (28.9) 475 (28.2) 746 (29.9) 417 (28.2)  Mainlander 574 (10.1) 157 (9.3) 121 (4.8) 296 (20.0)  Aborigine 122 (2.2) 36 (2.1) 56 (2.2) 30 (2.0) Level of education < 0.001  No education 799 (14.1) 49 (2.9) 386 (15.5) 364 (24.6)  Primary school 2322 (41.0) 570 (33.8) 1135 (45.5) 617 (41.7)  High school 1628 (28.8) 643 (38.2) 648 (26.0) 337 (22.8)  University 911 (16.1) 422 (25.1) 328 (13.1) 161 (10.9) Smoking status < 0.001  Current smoker 723 (12.8) 290 (17.2) 293 (11.7) 140 (9.5)  Past smoker 894 (15.8) 208 (12.3) 350 (14.0) 336 (22.7)  Non-smoker 4047 (71.5) 1188 (70.5) 1854 (74.2) 1005 (67.9) Betel quid < 0.001  Current chewer 180 (3.2) 86 (5.1) 76 (3.0) 18 (1.2)  Past chewer 512 (9.0) 194 (11.5) 218 (8.7) 100 (6.8)  Non-chewer 4972 (87.8) 1406 (83.4) 2203 (88.2) 1363 (92.0) Engage in physical activity c 4039 (71.3) 1196 (70.9) 1799 (72.0) 1044 (70.5) 0.533 Falls in previous year 1103 (19.5) 247 (14.7) 488 (19.5) 368 (24.8) < 0.001 Chronic disease d  Heart disease 1215 (21.5) 248 (14.7) 532 (21.3) 435 (29.4) < 0.001  Stroke 303 (5.3) 44 (2.6) 147 (5.9) 112 (7.6) < 0.001  Cataract 2214 (39.1) 236 (14.0) 1045 (41.9) 933 (63.0) < 0.001  Arthritis 986 (17.4) 207 (12.3) 441 (17.7) 338 (22.8) < 0.001  Osteoporosis 1118 (19.7) 300 (17.8) 538 (21.5) 280 (18.9) 0.007  Prostatic disorders e 875 (32.7) 143 (17.9) 384 (34.3) 348 (46.1) < 0.001 aIncludes divorced, separated, and single. bThe ethnicity of participants was classified based on the origin of the participants’ fathers. cEngage in physical activity: having any leisure-time physical activity in the past year. dPhysician-diagnosed chronic disease. eThe percentage was calculated for males. Table 3 reveals baseline biomarker profiles of the HALST participants. Systolic blood pressure increased but diastolic blood pressure decreased along with age. In addition, the levels of haemoglobin, albumin, glomerular filtration rate, cholesterol and triglyceride, and gait speed also all decreased with age, whereas the prevalence of under-weight [body mass index (BMI) < 20 kg/m2] increased with age. For those older than 75 years, > 11% and > 6% had BMI < 20 kg/m2 and serum albumin < 4 g/dl, respectively, indicating a risk of malnutrition that medical personnel should be on the alert for. Some common chronic diseases—such as hypertension, diabetes, chronic kidney disease and anaemia—also increased with age. In general, the intake of food and nutrients decreases in older participants, except for beans and dairy intake in males (data not shown). Regarding the physical function performance, the mean gait speed of those older than 75 (0.7 m/s) was slower than the cutoff point suggested in the European consensus, indicating the need for refining the definition of sarcopenia for the Asian population. 22 Similar situations can also be found with gender-specific handgrip strength (about 5 kg lower than that of Caucasian counterparts) 22 and distance in 6-min walking test (> 15% of those older than 75 years could walk no farther than 250 m). Table 3. Baseline biomarker profiles for the study subjects in the HALST cohort Total (n = 5664) 55–64 years (n = 1686) 65–74 years (n = 2497) ≥ 75 years (n = 1481) P BMI (kg/m2) 24.6 (3.5) 24.7 (3.5) 24.7 (3.5) 24.2 (3.5) < 0.001 SBP(mmHg) 128.6 (18.8) 122.6 (17.3) 129.4 (18.0) 134.4 (19.7) < 0.001 DBP(mmHg) 70.6 (10.8) 72.2 (11.1) 70.7 (10.6) 68.4 (10.6) < 0.001 Fasting glucose (mg/dl) 111.9 (31.4) 110.0 (29.8) 113.2 (33.8) 111.6 (28.7) 0.007 HbA1c (%) 6.2 (1.1) 6.1 (1.0) 6.3 (1.1) 6.3 (1.1) < 0.001 Haemoglobin (g/dl) 13.6 (1.5) 14.0 (1.5) 13.7 (1.4) 13.2 (1.5) < 0.001 Albumin (g/dl) 4.4 (0.2) 4.4 (0.2) 4.4 (0.2) 4.3 (0.2) < 0.001 GFR (ml/min/1.73 m 2 ) 83.2 (22.0) 91.2 (20.4) 83.6 (21.1) 72.8 (21.2) < 0.001 ALT (U/l) 27.0 (19.0) 29.3 (21.6) 27.3 (19.2) 23.7 (14.2) < 0.001 AST (U/l) 28.9 (14.6) 28.8 (13.8) 29.2 (16.8) 28.4 (11.1) 0.228 Uric acid (mg/dl) 6.0 (1.6) 5.8 (1.5) 5.9 (1.5) 6.3 (1.6) < 0.001 LDL-C (mg/dl) 118.1 (33.1) 121.4 (33.9) 118.6 (32.7) 113.0 (32.3) < 0.001 HDL-C (mg/dl) 52.5 (13.7) 52.8 (13.9) 52.6 (13.4) 52.0 (13.8) 0.288 TG (mg/dl) 124.1 (87.6) 131.5 (102.6) 123.2 (86.6) 116.7 (66.4) < 0.001 Gait speed (m/s) a 0.9 (0.3) 1.0 (0.3) 0.9 (0.3) 0.7 (0.3) < 0.001 Handgrip strength (kg) b 28.4 (10.2) 32.5 (10.3) 28.3 (9.6) 23.8 (9.0) < 0.001 SPPB c 10.0 (2.0) 11.0 (1.0) 11.0 (2.0) 9.0 (3.0) < 0.001 6-min walk (m) d 382.2 (82.0) 421.5 (69.6) 381.2 (74.9) 328.0 (80.8) < 0.001 BMI (kg/m2) < 0.001  < 20 432 (8.1) 107 (6.6) 174 (7.3) 151 (11.3)  20–24.9 2672 (50.0) 824 (50.6) 1175 (49.5) 673 (50.3)  25–29.9 1876 (35.1) 574 (35.3) 870 (36.6) 432 (32.3)  ≥ 30 360 (6.7) 123 (7.6) 156 (6.6) 81 (6.1) Hypertension e 3017 (53.3) 657 (39.0) 1370 (54.9) 990 (66.9) < 0.001 Diabetes f 1365 (25.5) 343 (21.1) 652 (27.4) 370 (27.5) < 0.001 Haemoglobin < 12 g/dl 609 (11.4) 118 (7.3) 242 (10.2) 249 (18.5) < 0.001 Albumin < 4 g/dl 186 (3.5) 31 (1.9) 67 (2.8) 88 (6.6) < 0.001 GFR < 60 ml/min/1.73 m 2 696 (13.0) 91 (5.6) 255 (10.7) 350 (26.0) < 0.001 ACR ≥ 30 mg/g 1278 (24.0) 252 (15.6) 535 (22.6) 491 (36.7) < 0.001 LDL-C ≥ 200 mg/dl 74 (1.4) 30 (1.8) 34 (1.4) 10 (0.7) 0.037 TG ≥ 200 mg/dl 594 (11.1) 223 (13.7) 252 (10.6) 119 (8.9) < 0.001 Uric acid ≥ 8 mg/dl 591 (11.1) 138 (8.5) 246 (10.4) 207 (15.4) < 0.001 Hepatitis B carrier 498 (9.4) 210 (12.9) 202 (8.5) 86 (6.5) < 0.001 Hepatitis C carrier 273 (5.1) 73 (4.5) 119 (5.0) 81 (6.1) 0.141 Slow gait speed  < 0.8 m/s 1994 (35.7) 281 (16.8) 848 (34.3) 865 (60.3) < 0.001  < 0.7 m/s 1298 (23.3) 131 (7.8) 523 (21.1) 644 (44.9) < 0.001 Low handgrip strength (kg)  male < 30, female < 20 1520 (27.1) 155 (9.3) 594 (23.9) 771 (52.8) < 0.001  male < 25, female < 15 616 (11.0) 49 (2.9) 184 (7.4) 383 (26.2) < 0.001 SPPB ≤ 9 1199 (21.8) 105 (6.3) 452 (18.5) 642 (45.9) < 0.001 6-min walk < 250 m 293 (5.9) 18 (1.1) 103 (4.6) 172 (15.4) < 0.001 Data are n (%) unless indicated otherwise. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; GFR, glomerular filtration rate; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; TG, triglyceride; ACR, urine albumin-to-creatinine ratio. a5580 study subjects had received measurement of gait speed. b5612 study subjects had received measurement of handgrip strength. c5490 study subjects had received measurement of SPPB. d4954 study subjects had received measurement of 6-min walk. eHypertension: SBP ≥ 140 mmHg, or DBP ≥ 90 mmHg, or taking anti-hypertensive drugs. fDiabetes: fasting glucose ≥ 126 mg/dl, or HbA1c ≥ 6.5%, or taking anti-diabetic drugs. Among those who had completed the first annual telephone interview (n = 4930), 123 (2.49%) had a new diagnosis of hypertension, 99 (2.01%) developed diabetes, 34 (0.69%) had stroke and 38 (0.77%) had cancer diagnosed in the previous year of the first telephone survey. We also found, in the previous year, 752 (15.26%) had falls, 589 (11.95%) had been admitted to hospitals, 267 (5.53%) had body weight loss of more than 3 kg and 52 (1.09%) had various degrees of depression syndrome [BSRS-5 score 10–14: 45 (0.94%), score ≥ 15: 7 (0.15%)]. In addition to the unique characteristics described above, several interesting results have been found in the HALST study. For example, we found a strong relationship between dietary fibre intake and physical performance in the elderly, providing potential practical preventive strategies for frail older adults. 23 Those who had higher education, higher BMI and lower fish and milk intake were found to be more likely to have vitamin D insufficiency. 24 The results of gait speed and handgrip strength performed by the HALST participants were adopted to refine cutoffs and prevalence of sarcopenia in Taiwan. 25 We also illustrated a synergistic impact of sarcopenia and obesity on elders’ physical performance. 26 The HALST study is a member of the TaiChi consortium, joining international efforts to identify genetic determinants of atherosclerosis and metabolic-related traits in multi-ethnic populations. For the past 3 years, collaboration within the TaiChi consortium has been fruitful. For example, four new genetic loci have been found related to obesity; 27 some novel genetic variants associated with HbA1c, plasma triglycerides and risk of coronary artery disease were identified; 28 , 29 a novel independent type 2 diabetes locus was found in the Chinese population; 30 and some other important findings were also published in renowned journals. 31–37 By linking with the NHID, we have recently conducted a prospective study and found that the older adults performing a healthy lifestyle (higher diet score, physical activity and psychosocial score) would be less likely to develop diabetes (manuscript under revision). More findings based on the follow-up data will be realized when we finish the second wave which started in 2014. The HALST, because of its prospective nature and extended data linkage, is a good epidemiological research platform to better understand multidimensional health risks in the elderly. What are the main strengths and weaknesses? Strengths The HALST study has several strengths. First, composed of comprehensive geriatric assessment and extended biochemical and genetic measurements, the HALST is more feasible, compared with other Chinese longitudinal ageing studies, to investigate factors related to healthy ageing. We have established some international collaboration to conduct genetic and biomarker studies for ageing-related genetic traits. Results from the HALST study will provide information unique to Asian societies and allow a direct comparison with those from Western countries which differ in lifestyle and in genetic and environmental characteristics. Second, the study design includes data linkage with National Health Insurance databases, the mortality registry, the cancer registry, and medical records, which allows a tracking of participant incidence of health-related events and use of healthcare. Third, the HALST has a close link with the Chicago Healthy Aging Study. 38 Most methods of procedures (MOP) between these two studies are similar. This provides opportunities to make ethnicity and cross-country comparisons in various geriatric research issues. For example, researchers on both sides have recently been working on developing and cross-validating a sensitive but important questionnaire about filial piety which is unique to Chinese culture. Finally, all measurements in the HALST have been conducted by a well-trained team containing 15 fieldworkers who are trained to strictly follow the study protocols. Data collection, management, validation and processing are also being carried out to an exceptionally high standard. Weaknesses First, the HALST cohort is not a completely random sample from the elderly population in Taiwan; instead, the study is targeted at recruiting enough people with different socio-demographic backgrounds. This incomplete representation of Taiwan’s general population limits the data applicability for estimation of disease prevalence. However, our study focuses on searching for the risk factors of ageing-related diseases and conditions, so the sampling effects would be minimal. Second, as with other longitudinal studies for ageing, response rate, sample attrition and missing data are always great concerns in the interpretation of the results. Third, although our study has a relatively large sample size, several years of longitudinal observations are necessary before it can obtain statistical power for new outcomes. Can I get hold of the data? Where can I find out more? The HALST study group encourages domestic and international research collaboration. To learn more about the HALST study, access the data and explore potential collaboration, please contact the principal investigator, Dr. Chao A. Hsiung [hsiung@nhri.org.tw]. HALST in a nutshell HALST is a prospective cohort study aiming at investigating multidimensional determinants of healthy aging—including lifestyle behaviours and genetic, metabolic and inflammatory factors—in an older Asian population. A total of 5664 community-dwellers aged 55 and over, recruited from seven selected cities/counties to represent the socio-demographic diversity of the Taiwanese population, participated in home interviews and hospital-based clinical examinations in the first-wave survey (2009–13). Participants have annual telephone contact to update health-related conditions and hospitalized events. The HALST dataset has been linked to Taiwan’s National Health Insurance database, the national mortality registry, the national cancer registry and medical records. The HALST dataset comprises a broad scope of measurements, including socio-demographic information, lifestyle pattern, dietary habit, metabolic profile, inflammatory biomarkers, cognitive function, depression assessment, physical function, medication and genetic components. The first-wave HALST data (2009–13) have been compiled and are available for analysis; the second-wave survey (2014–19) is ongoing. Further enquiries about research collaboration should be addressed to the principal investigator, Dr. Chao A. Hsiung [hsiung@nhri.org.tw]. Funding This study was supported by the National Health Research Institutes in Taiwan [project no. BS-097-SP-04, PH-098-SP-02, PH-099-SP-01, PH-100-SP-01, PH-101-SP-01, PH-102-SP-01, PH-103-SP-01, PH-104-SP-01].

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          Trans-Ethnic Fine-Mapping of Lipid Loci Identifies Population-Specific Signals and Allelic Heterogeneity That Increases the Trait Variance Explained

          Introduction Genome-wide association studies (GWAS) have identified many common genetic variants associated with human diseases and complex traits (www.genome.gov/gwastudies), including ∼100 loci associated with triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), or total cholesterol [1]–[5]. A majority of the lead SNPs at these loci have shown small effect sizes, leaving much of the trait heritability unexplained. Some of this missing heritability may be due to the incomplete coverage of functional common or rare variants and the poor representation of appropriate proxies on commercial genotyping arrays [6], [7]. Other missing heritability may result from a failure to detect the full spectrum of causative variants present at GWAS-identified loci. Fine-mapping of GWAS signals should increase the power to detect variants that influence trait variability. Genotyping of additional variants at GWAS loci can identify SNPs with stronger evidence of association than the reported GWAS index SNPs and may help detect or further localize the underlying causal variants [7], [8]. The Metabochip is a high-density custom genotyping array designed to replicate and fine-map known GWAS signals for metabolic and atherosclerotic/cardiovascular endpoints, and more extensively, to identify all signals around the index SNPs [9], [10]. The fine-mapping SNPs spanned a wide range of allele frequencies including rare (minor allele frequency (MAF) 10−4 and was not annotated as a nonsense or nonsynonymous substitution. We also investigated whether association signals were population-specific, which we defined as association signals with variants that are not variable in the samples from the other two ancestry groups in this study or in the 1000 Genomes Project populations that represent those groups among total European ancestry (EUR), total East Asian ancestry (ASN), or total west African ancestry (AFR). In African Americans, sequential conditional analyses revealed that 10 of the 22 loci with evidence of association exhibited two or more signals at P 10−4 and had no annotation suggesting potential function. d Variance explained was estimated based on PAGE samples (n = 5,593). e P values of initial association in East Asians and Europeans. f Conditional analyses at LDL-C locus PCSK9 were restricted to 5,593 PAGE samples because SNPs rs67608943 (Y142X), rs72646508 (L253F) and rs11591147 (R46L) were not polymorphic in HyperGEN samples. The seven signals at PCSK9 in African Americans included six nonsense or nonsynonymous variants previously shown to associate with LDL-C levels and to affect PCSK9 expression or function [20]–[22], along with an unreported intronic variant (Table 1). The strongest signals were a nonsense variant rs28362286 (C679X, Figure 1A) and a nonsynonymous variant rs28362263 (A443T, Figure 1B), which showed no reduction of association evidence when conditioned on C679X. Conditional analysis on both C679X and A443T yielded a third signal at rs28362261 (N425S, Figure 1C); and further conditional analyses successively implicated rs67608943 (Y142X, Figure 1D), rs72646508 (L253F, Figure 1E), and an intronic variant rs11800243 (Figure 1F). The seventh signal, which did not reach the Pconditional 10−4 and had no annotation suggesting potential function. d Variance explained by SNPs at each locus was estimated based on European samples. e P values of initial association in African Americans and East Asians. At the TOMM40-APOE-APOC4 cluster, the seven signals in African Americans explained 6.6% of the LDL-C phenotypic variance compared to 4.1% explained by the strongest signal R176C, which had reported functional effects [23] (Table 1, Figure S1). These seven signals were not entirely independent of one another. The fourth signal, rs157588, showed association with LDL-C (P = 2.0×10−7) only after conditioning on the top three signals, but not in the original unconditioned association analysis (P = 0.72). The trait-decreasing allele (G allele: freq = 0.176) of rs157588 was present on haplotypes containing the trait-increasing allele of the third signal rs1038026 (A allele: freq = 0.351), thus the association of the fourth signal increased in significance after accounting for linkage disequilibrium (r2/D′ = 0.35/0.92) with the third signal at the same locus. Haplotype analysis revealed that compared to the reference A-A (increasing-increasing) haplotype, the G-G (decreasing-decreasing) haplotype only displayed modest association with LDL-C (P = 7.5×10−3), but the A–G (rs1038026 increasing- rs157588 decreasing) haplotype showed significant association with decreased level of LDL-C (P = 1.5×10−10) (Table S5). In Europeans (Table 2) and East Asians (Table 3), three and two signals were identified at TOMM40-APOE-APOC4, respectively. The known functional variant R176C exhibited the strongest evidence of association across the three ancestry groups, with effect sizes of −0.536, −0.505, and −0.411 mmol/L in individuals of African American, European, and East Asian ancestry, respectively (Table 1). However, another APOE variant rs429358 (C130R), that together with R176C, defines the three major isoforms of APOE (ε2, ε3, and ε4) [7], [24], was not successfully genotyped, therefore the LDL-C association with either C130R or the APOE haplotype was unavailable in this study. 10.1371/journal.pgen.1003379.t003 Table 3 Lipid loci with multiple signals in East Asians. SNP Annotation Effect/non-effect allele East Asian (n = 9,449) Variance explained by the strongest signald Variance explained by all signalsd African American (n = 6,832) European (n = 10,829) EAF LD (r 2/D′)a βb P initial βb P conditional c EAF βb P e EAF βb P e APOA5 for TG rs651821 APOA5: -3A>G T/C 0.725 ---- −0.145 7.2E-68 ---- ---- 2.6% 3.4% 0.851 −0.037 1.4E-03 0.921 −0.151 8.5E-36 rs2075291 APOA5-G185C A/C 0.064 0.09/1.00 0.201 3.7E-37 0.106 7.2E-10 0.002 0.204 0.028 0.0003 0.269 0.23 rs11604424 ZNF259-intron T/C 0.650 0.39/1.00 −0.075 2.0E-21 −0.045 4.8E-05 0.725 −0.020 0.032 0.765 −0.101 6.5E-40 TOMM40-APOE-APOC4 for LDL-C rs7412 APOE-R176C T/C 0.086 ---- −0.411 1.1E-64 ---- ---- 8.0% 9.0% 0.110 −0.536 6.7E-75 0.056 −0.505 5.4E-76 rs769449 APOE-intron A/G 0.086 0.00/1.00 0.173 2.8E-12 0.191 3.8E-06 0.024 0.302 1.1E-06 0.160 0.121 1.7E-12 CETP for HDL-C rs17231506 CETP-5′UTR T/C 0.168 ---- 0.073 3.6E-28 ---- ---- 1.0% 2.3% 0.146 0.071 2.9E-12 0.284 0.090 2.2E-58 rs7499892 CETP-intron T/C 0.164 0.00/1.00 −0.052 2.8E-15 −0.065 2.0E-07 0.372 −0.066 1.4E-16 0.173 −0.097 4.6E-48 ABO for LDL-C rs9411476 ABO-downstream A/G 0.162 ---- 0.106 1.1E-08 ---- ---- 0.8% 1.8% 0.121 0.043 0.14 0.005 −0.190 0.037 rs191637055 ADAMTSL-intron A/C 0.998 0.00/1.00 −0.688 1.1E-03 −1.055 4.0E-05 0.977 −0.014 0.83 0 ---- ---- a LD (r 2/D′) with SNP showing the strongest evidence of association at each locus. b β: effect size from an additive model and corresponding to the effect allele, in the unit of mmol/L for HDL-C, LDL-C and natural log transformed TG. c P values of sequential conditional analyses, in which we added the SNP with the strongest evidence of association into the regression model as a covariate and tested for the next strongest SNP until the strongest SNP showed a conditional P value>10−4 and had no annotation suggesting potential function. d Variance explained by SNPs at each locus was estimated based on CLHNS samples (n = 1,716). e P values of initial association in African Americans and Europeans. In Europeans, 21 signals at nine of the 31 loci exhibited multiple signals for at least one of the three lipid traits at P 9,000 East Asian individuals in this study. In East Asians, we observed three signals at the TG locus APOA5, and two signals at three loci including TOMM40-APOE-APOC4 cluster for LDL-C, CETP for HDL-C, and ABO for LDL-C (Table 3). At the four loci that exhibited multiple signals, all the association signals increased the explained phenotypic variance by an average of 1.3-fold compared to the strongest signal across loci. The second signal at APOA5 was the nonsynonymous variant G185C previously reported to affect the protein function [25]. Although G185C was not unique to East Asians, the frequency was very low in African Americans (MAF = 0.002, P = 0.028) and Europeans (MAF = 0.0003, P = 0.23), and the low allele frequency meant that this study had less than 5% statistical power to detect the association in these groups. At APOA5, which exhibited multiple signals in all three populations (Table 1, Table 2, Table 3), the strongest TG-associated SNPs differed and were not in high LD (r2 10−3, Table 3). The SNP LD r2 values between the African American and East Asian signals were less than 0.02 in both populations, suggesting that they represent distinct APOA5 signals in the two ancestry groups. In addition, the APOA5 signal rs3741298 (P = 9.7×10−44, MAF = 0.222) in Europeans exhibited evidence of association with TG in African Americans (P = 9.8×10−5, MAF = 0.327) and East Asians (P = 1.2×10−20, MAF = 0.357), but the significance levels of the association with rs3741298 were substantially attenuated by conditioning on the strongest signals S19W in African Americans (P = 0.10) and rs651821 in East Asians (P = 0.88). In Europeans, the associations with rs3741298 were partially removed when conditioning on S19W and rs651821 (Pconditional  = 1.7×10−28 and 3.1×10−17, respectively). The European signal rs3741298 was moderately correlated with the African American signal S19W (LD r2  = 0.21 and 0.10 in the 1000 Genomes Project EUR samples (European ancestry) and in PAGE African American samples, respectively), and with the East Asian signal rs651821 (LD r2  = 0.31 and 0.28 in 1000 Genomes Project EUR and ASN samples, respectively). Notably, the effect sizes of the two reported functional variants S19W [26] and G185C [25] at APOA5 were similar across the three groups (S19W, African American: 0.136; East Asian: 0.136; European: 0.121 and G185C, African American: 0.204; East Asian: 0.201; European: 0.269 mmol/L in loge scale) despite the limited power to detect significant evidence of association at low allele frequencies. These findings support the hypothesis that causative variants may have a similar genetic impact on trait variation across populations if not influenced by hidden gene-gene or gene-environment interactions [27]. We also observed that the second European signal rs75919952 exhibited nominal evidence of association (P initial = 0.018, MAF = 0.041), but was not associated with TG in the other two groups (Table 2). The lack of association may be due to insufficient power (15% and 55% in African Americans and East Asians, respectively; assuming α = 0.05) corresponding to the lower allele frequency (MAF = 0.012) in African Americans, the smaller sample sizes in both populations, or underlying interactions. Trans-ethnic high-density genotyping narrowed the region of association signals We next examined whether trans-ethnic meta-analysis or comparison across ancestries would refine the association signals by narrowing the genomic regions where functional variants might be expected to reside. The trans-ethnic analysis allowed the refinement of association signals at loci of GCKR, PPP1R3B, ABO, LCAT, and ABCA1 (Table 4, Table S3A–S3C). The signal at GCKR was localized to the reported functional variant P446L [28] due to the limited LD in African Americans (Figure S2A–S2D). Notably, there were seven and six variants in high LD (r2 >0.8) with P446L in the 1000 Genomes Project ASN and EUR samples, but no SNP with LD r2 >0.8 in African American individuals. At the signal ∼200 kb from the PPP1R3B gene for which no functional regulatory variant(s) have been reported, the association signal was narrowed from 4 SNPs spanning 36 kb (P 0.94) (Figure 2). The lead SNP rs6601299 was in high LD with 11 variants in the 1000 Genomes Project EUR samples but only highly correlated with two and one variant in the 1000 Genomes Project AFR samples (West African ancestry) and PAGE African American individuals, respectively. At the ABO locus, trans-ethnic meta-analysis revealed six SNPs exhibiting stronger evidence of association (P 2.3×10−7) (Figure S3A–S3D). At the locus LCAT for HDL-C, the association signals spanned ∼800 kb, ∼360 kb, and ∼360 kb in Europeans, East Asians, and African Americans, with a ∼50 kb overlapping region. Trans-ethnic meta-analysis of all samples localized the signal to four variants spanning this 50 kb region (Figure S4A–S4D). At HDL-C locus ABCA1, the reported GWAS index SNP rs1883025 consistently showed the strongest association within each of the three ancestry groups that we examined, but the significance level of the association was similar to those of the nearby SNPs. Trans-ethnic meta-analysis refined the signal by revealing that rs1883025 (P = 4.3×10−17) and rs2575876 (P = 1.8×10−15) displayed much stronger association than the neighboring SNPs (P>8.4×10−10) (Figure S5A–S5D). 10.1371/journal.pgen.1003379.g002 Figure 2 Trans-ethnic high-density genotyping narrows the association signal at the HDL-C locus PPP1R3B. Association in Europeans (A), East Asians (B), African Americans (C) and in a combined trans-ethnic meta-analysis (D). Index SNP rs6601299 colored in purple is the variant showing strongest evidence of association in the combined trans-ethnic meta-analysis. 10.1371/journal.pgen.1003379.t004 Table 4 Trans-ethnic fine-mapping narrowed the association signals. Locus/Trait SNP Meta-analysis African American East Asian European P metaa Directionb P hetc I 2 d MAF β P MAF β P MAF β P GCKR/TG rs1260326 (P446L) 1.6E-42 +++++++++++ 0.72 0 0.149 0.07 2.2E-08 0.484 0.06 1.5E-13 0.350 0.07 4.4E-24 PPP1R3B/HDL rs6601299 8.8E-10 −−−−−−−−−+− 0.16 30.6 0.121 −0.06 8.0E-08 0.052 −0.02 0.29 0.160 −0.03 1.1E-04 ABO/LDL rs2519093 2.2E-13 +++++++++++ 0.91 0 0.105 0.10 6.6E-04 0.187 0.09 5.4E-07 0.196 0.07 1.7E-05 LCAT/HDL rs3785100 (SLC12A4-E4G) 9.0E-12 −−−−−+−−−−− 0.55 0 0.217 −0.04 6.5E-07 0.096 −0.03 1.7E-03 0.155 −0.03 1.6E-04 ABCA1/HDL rs1883025 4.3E-17 −+−−−−−−−−− 0.63 0 0.336 −0.02 0.018 0.271 −0.04 3.7E-11 0.209 −0.03 4.5E-07 a P meta: P values from meta-analysis combining samples of African American, East Asian and European ancestries. b Direction: effect direction of each individual studies in the order of ARIC, MEC, WHI batch1, WHI batch2, HyperGEN, CLHNS, TAICHI, Finnish T2D, Finnish unaffected, Norwegian T2D and Norwegian unaffected. c P het: P values for heterogeneity, indicating whether observed effect sizes are homogeneous across ancestry samples. d I 2: index of the degree of heterogeneity. Reported functional variants were frequently the most strongly associated ones at a signal Among loci associated with at least one lipid trait (P 0.95) with the most strongly associated variants and showed similar evidence of association (APOB-rs934198, P = 3.7×10−17; LPL-rs1803924, P = 1.1×10−11). If we include these two variants, then 16 of the 19 (84%) reported functional variants displayed the strongest association P-value at the primary, secondary, or successive signals. The remaining three reported functional variants: LDLR-rs688 (N591N), LPL-rs1801177 (D9N), and HMGCR-rs3761740 (911C>A), were poorly tagged (LD r2 G) [32] Yes rs651821 1st ASN 0.275 Same variant APOA5: rs2075291 (G185C) [25] Yes rs2075291 2nd ASN 0.064 Same variant GCKR: rs1260326 (L446P) [28] Yes rs1260326 1st AA, EUR 0.149–0.350 Same variant SORT1: rs12740374 [18] Yes rs12740374 1st AA 0.247 Same variant CETP: rs17231520 [33] Yes rs17231520 3rd AA 0.069 Same variant LIPC: rs2070895 [34] Proxy: rs1077834 (LD r2  = 1.00) rs1077834 1st, 2nd AA, EUR 0.481 LD r2  = 1.00 APOB: rs7575840 [35] Yes rs934198 1st EUR 0.298 LD r2  = 0.98 LPL: rs328 (S447X) [36] Yes rs1803924 1st ASN 0.095 LD r2  = 0.96 LDLR: rs688 (N591N) [37] Yes rs73015011, rs112898275 1st AA, EUR ---- LD r2 A) [39] Proxy: rs17238330 (LD r2  = 1.00) rs12916 1st EUR ---- LD r2 G [40] No ---- ---- ---- ---- ---- LPL: rs268 (N291S) [41] No ---- ---- ---- ---- ---- ABCA1: rs9282541 (R230C) [42] No ---- ---- ---- ---- ---- ABCA1: rs2066715 (V825I) [43] No ---- ---- ---- ---- ---- LCAT: rs28940887(R159W) [44] No ---- ---- ---- ---- ---- PLTP: R235W [45] No ---- ---- ---- ---- ---- LIPG: rs77960347 (A396S) [46] No ---- ---- ---- ---- ---- LIPG: rs34474737 [47] No ---- ---- ---- ---- ---- * AA, African American; EUR, European; ASN, East Asian. Among the 16 reported functional variants and proxies that exhibited the strongest association P-value at a signal (Table 5), R176C at APOE was strongest in all three populations and GCKR L446P was identified in both African Americans and Europeans. The remaining 14 variants showed the strongest associations in only one of the populations, including 10 in African Americans, three in East Asians, and one in Europeans. Five of the 10 variants in African Americans were at the PCSK9 locus. Furthermore, nine of the 16 variants represented the strongest signal at a given locus, three for a 2nd signal, and four for the 3rd or additional signals. These functional variants covered a wide allele frequency spectrum (MAF: 0.003–0.481), including five less common or rare variants observed only in African Americans. Discussion This study evaluated densely spaced SNPs at 58 lipid loci across three ancestrally diverse populations. The results support evidence that allelic heterogeneity is a frequent feature of polygenic traits [5], [49] and extend the findings to non-European populations, especially to African ancestry populations that have high levels of haplotype diversity. The results also provide strong evidence that fine mapping at GWAS loci can identify population-specific signals. Despite comparable sample sizes, we identified more signals per locus and more signals overall in African Americans (34 signals at 10 loci) compared to Europeans (21 signals at nine loci) and East Asians (nine signals at four loci), and 15 of the 34 signals identified in African Americans were population-specific (Table 1, Table 2, Table 3). These observations may reflect the larger number of SNPs genotyped in African Americans (Table S2), variation across populations subject to natural selection during human evolution [14], or genetic drift [50]. Due to the varied number of signals per locus, different associated markers, and different effect sizes, the phenotypic variance explained differs across populations [51]–[53]. Sampling variability, epistasis, and gene-environment interactions may cause over- or under-estimation of the proportion of explained phenotypic variance. In this study, we also observed that many population-specific signals, including those at PCSK9 and APOA5, are largely confirmatory [20], [22], [54]; however, the association evidence at other signals, in particular the additional signals at APOE, LDLR, and APOC1 identified by the conditional analyses, requires replication in future studies. At PCSK9, the strongest signal C679X identified in African Americans is population-specific and showed substantially stronger evidence of association with LDL-C (P = 4.1×10−22) compared to the GWAS index SNP rs2479409 [5] (P = 0.12) and the most strongly associated SNP R46L identified via fine-mapping [7] (P = 2.3×10−3), both of which were previously reported in Europeans. The proportion of phenotypic variance explained in African Americans increased from 0.16% by the GWAS index SNP to 1.3% by the Metabochip signal C679X, and all variants at the locus together explained 3.6% of the total variation in LDL-C, providing evidence that heritability at identified loci may be underestimated by GWAS [7]. A limitation of these variance estimates is that calculations included the SNPs based simply on their significant association P values rather than the variants with biological function, which could over-estimate effects due to the winner's curse. Results across the genotyped loci demonstrated that the majority of signals were represented by common variants, yet high-density genotyping also identified less common and rare variants associated with lipid traits. At PCSK9, the MAFs of six out of the seven signals were 43 kb away from the narrowed association signals observed in this study (Table 4). Refining signals by trans-ethnic meta-analysis largely relies not only on the existence of distinct LD patterns across ancestry groups but also on shared functional variants. If functional variants are shared across populations, as observed with GCKR-P446L, performing trans-ethnic meta-analysis and integrating LD information across different populations may refine the signal. On the contrary, if trait variation is influenced by distinct functional variants across populations, as our data suggest for APOA5 (Figure S6A–S6D), the lead SNPs produced by meta-analysis would be influenced by the sample size, magnitude of genetic effects, and allele frequencies. Similarly, in the case of population-specific functional variants, such as those at PCSK9, the results from meta-analysis would reflect the association in one particular population rather than the combined effect across populations if signals unique to this population drive the results. Therefore, accurate assessment of allelic variability is needed on a population-by-population and locus-by-locus basis. Although genotype imputation has become a standard practice to increase genome coverage in GWAS by predicting the genotypes at SNPs that are not directly genotyped, imputation accuracy tends to be lower for rare variants owing to the lower degree of LD and the more challenging haplotype reconstruction [60]. In addition, African American samples pose a challenge for imputation due to their varying degree of admixture [61]. A major strength of our study is that all variants we tested for association were directly genotyped using the Metabochip, which was designed to provide a high-density coverage for both overall SNPs and low frequency variants concentrated around GWAS-identified loci and/or signals [9], [10]. This approach increases the reliability of our association results overall, but in particular the variants with low allele frequencies. In conclusion, we performed a large-scale trans-ethnic fine-mapping study to investigate the established lipid loci using the Metabochip high-density genotyping array and focusing on diverse groups including African Americans, East Asians, and Europeans. Our results highlight the value of high-density genotyping in diverse populations to identify a wider spectrum of susceptibility variants at established loci, both in terms of additional signals and in terms of population-specific and/or potentially functional variants. The additional signals revealed through the sequential conditional analyses lead to a 1.3- to 1.8-fold increase in the explained phenotypic variance across the different populations. In addition, integrating diverse LD patterns across diverse ancestry groups allows for the refinement of association signals. Lastly, our findings that 74% of the reported functional variants exhibited the strongest association at these densely typed signals suggest that at loci and signals where functional variants are unknown, the variants with strongest association may be good candidates for functional assessment. Materials and Methods Study populations and phenotypes The 6,832 African Americans studied are comprised of individuals from the Atherosclerosis Risk in Communities Study (ARIC) [62], the Multiethnic Cohort Study (MEC) [63], and the Women's Health Initiative (WHI) [64], [65] that are part of Population Architecture using Genomics and Epidemiology (PAGE) consortium [66] and from Hypertensive Genetic Epidemiology Network (HyperGEN) [67]. The 9,449 East Asian samples are comprised of 1,716 Filipinos from the Cebu Longitudinal Health and Nutrition Survey (CLHNS) [68] and 7,733 Chinese from Taiwan-Metabochip Study for Cardiovascular Disease (TAICHI). The 10,829 European samples are comprised of Finnish and Norwegian individuals; the Finns are from the Finland-United States Investigation of NIDDM Genetics (FUSION), Dehko 2D 2007 (D2D2007), Diabetes Prevention Study (DPS), Dose-Responses to Exercise Training (DR's EXTRA), and Metabolic Syndrome in Men (METSIM) [69], [70], and the Norwegians were from the cohorts of Nord-Trøndelag Health Study (HUNT 2) and the Tromsø Study (TROMSO) [71], [72]. All study protocols were approved by Institutional Review Boards at their respective sites. Brief descriptions of the studies are provided in the Text S1. General characteristics and measurements of TG, HDL-C, and LDL-C in each cohort are summarized in Table S1. Values of triglycerides were natural log transformed to approximate normality in each study sample separately. Genotyping We genotyped all study samples with the Metabochip according to the manufacturer's protocol (Illumina, San Diego, CA, USA). Table S1 summarizes the quality control criteria of genotyping, including call rate, sample success rate, Hardy-Weinberg equilibrium, and MAF that varied across studies. Statistical analyses We applied multiple linear regression models and assumed an additive mode of inheritance to test for association between genotypes and HDL-C, LDL-C, or log-transformed triglycerides. We performed each test of association separately in each of the 11 groups (Table S1) prior to meta-analysis. We constructed principal components (PCs) using the software EIGENSOFT. We used age and sex as covariates in each individual cohort; other cohort-specific covariates including age2, enrollment site, socioeconomic status, and principal components varied across studies (Table S1). The European samples include type 2 diabetes (T2D) cases and unaffected controls; to avoid confounding due to T2D status, samples were analyzed separately as Finnish T2D patients, Finnish unaffected individuals, Norwegian T2D patients, and Norwegian unaffected individuals. We first conducted the meta-analysis within the African Americans, East Asians, and Europeans separately. We then performed combined trans-ethnic meta-analyses by combining the statistics of each the 11 participating groups to assess the association with the SNPs at the 58 lipids loci. At loci that exhibited evidence of association at P 10−4 and had no annotation or literature evidence that suggested a functional role. For single SNP analyses, we applied PLINK (http://pngu.mgh.harvard.edu/~purcell/plink/) [73] for population-based studies. We used the R package GWAF [74] for the family-based study of HyperGEN. We applied an inverse variance-weighted fixed-effect meta-analysis implemented in METAL [75]. Unless otherwise noted, linkage disequilibrium estimates were obtained from the 1000 Genomes Project November 2010 release. SNP positions correspond to hg18. We performed haplotype analysis at LDL-C locus TOMM40-APOE-APOC4 in 5,593 unrelated African Americans from the PAGE consortium, using the ‘haplo.stat’ R package. Haplotypes and haplotype frequencies were estimated using the R function ‘haplo.em’. The association between haplotypes and LDL-C was assessed using the R function ‘haplo.glm’. An additive model was assumed, in which the regression coefficient β represents the expected change in LDL-C level with each additional copy of the specific haplotype compared with the reference haplotype, which was set as the A-A (trait increasing-increasing) haplotype. We created the regional association plots using LocusZoom [76]. To plot the association results in Europeans and East Asians, we used the LocusZoom-implemented LD estimates from the 1000 Genomes Project (June 2010) CEU and CHB+JPT samples, whose LD structures are similar to our samples with European and East Asian ancestries. We applied the user-supplied LD calculated from the genotype data of the PAGE African American samples to plot the regional association in African Americans [9], because the LD patterns may vary from any pre-computed LD sources implemented in LocusZoom. We evaluated the proportion of variance explained by a single SNP or any given locus by including the SNP or a set of SNPs into a linear regression model with all covariates used in association analysis and calculating the R2 for the full model. We subtracted the variance explained by a basic model in which only covariates were included from the variance we obtained from the full model. We performed these analyses using SAS version 9.2 (SAS Institute, Cary, NC, USA). Supporting Information Figure S1 LDL-C locus TOMM40-APOE-APOC4 exhibited seven signals in African Americans. Each SNP was colored according to its LD (r2 ) in PAGE consortium with the strongest SNP rs7412 (R176C) colored in purple. (PDF) Click here for additional data file. Figure S2 Association at TG locus GCKR in Europeans (A), East Asians (B), African Americans (C), and trans-ethnic meta-analysis (D). Index SNP rs1260326 (P446L) is the variant showing the strongest evidence of association in trans-ethnic meta-analysis. (PDF) Click here for additional data file. Figure S3 Association at LDL-C locus ABO in Europeans (A), East Asians (B), African Americans (C), and trans-ethnic meta-analysis (D). Index SNP rs2519093 is the variant showing the strongest evidence of association in trans-ethnic meta-analysis. (PDF) Click here for additional data file. Figure S4 Association at HDL-C locus LCAT in Europeans (A), East Asians (B), African Americans (C), and trans-ethnic meta-analysis (D). Index SNP rs3785100 (SLC12A4-E4G) is the variant showing the strongest evidence of association in trans-ethnic meta-analysis. (PDF) Click here for additional data file. Figure S5 Association at HDL-C locus ABCA1 in Europeans (A), East Asians (B), African Americans (C), and trans-ethnic meta-analysis (D). Index SNP rs1883025 is the variant showing the strongest evidence of association in trans-ethnic meta-analysis. (PDF) Click here for additional data file. Figure S6 Association at TG locus APOA5 in Europeans (A), East Asians (B), African Americans (C), and trans-ethnic meta-analysis (D). The SNPs rs3741298, rs651821 (-3A>G), rs3135506 (S19W), and rs662799 that exhibited the smallest P values in Europeans, East Asians, African Americans, and the trans-ethnic meta-analysis are indicated. (PDF) Click here for additional data file. Table S1 Characteristics of the study samples. (PDF) Click here for additional data file. Table S2 Number of SNPs at each locus for analysis in each of the three ancestry groups. (PDF) Click here for additional data file. Table S3 Lead SNP at TG (A), HDL-C (B), and LDL-C (C) loci within each ancestry group and their relative significance compared to reported GWAS index SNPs. (PDF) Click here for additional data file. Table S4 SNPs with the strongest association at TG (A), HDL-C (B) and LDL-C (C) loci in combined trans-ethnic meta-analysis and their associations within ancestry groups. (PDF) Click here for additional data file. Table S5 LDL-C association with haplotypes consisting of the third (rs1038026) and the fourth (rs157588) signals at TOMM40-APOE-APOC4 cluster. (PDF) Click here for additional data file. Text S1 Study description. (DOCX) Click here for additional data file.
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            Epidemiology of sarcopenia among community-dwelling older adults in Taiwan: a pooled analysis for a broader adoption of sarcopenia assessments.

            To develop cut-off points of muscle mass, gait speed and handgrip strength; and to examine the prevalence of sarcopenia, and the relationship between sarcopenia stages and functional limitations and disability by using these cut-off points. We pooled individual participant data of 2867 community-dwelling older adults from five cohort studies. We defined the cut-off point of a muscle mass index (ASM/ht(2)) as the values of two standard deviations below the sex-specific means of a young population or as the 20th percentile of the sex-specific distribution in our study population. The gait speed and handgrip strength cut-off points were defined as the 20th percentile of their population distributions. We also measured functional limitations, using the Short Physical Performance Battery, and the number of activities of daily living and instrumental activities of daily living difficulties. We identified the cut-off points of ASM/ht(2), gait speed and handgrip strength. By applying these cut-off points to our study population, the prevalence of sarcopenia varied from 3.9% (2.5% in women and 5.4% in men) to 7.3% (6.5% in women and 8.2% in men). A higher sarcopenia stage was independently associated with a lower summary performance score, as well as more activities of daily living and instrumental activities of daily living difficulties (P < 0.05 for all). The prevalence of sarcopenia in community-dwelling older adults is comparable with those in other populations. A dose-response relationship exists between sarcopenia stages and functional limitations/disability. The European Working Group on Sarcopenia in Older People consensus definition using these cut-off points is suitable for determining sarcopenia cases in the elderly population of Taiwan. © 2014 Japan Geriatrics Society.
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              • Record: found
              • Abstract: found
              • Article: not found

              Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene.

              Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.
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                Author and article information

                Journal
                Int J Epidemiol
                Int J Epidemiol
                ije
                International Journal of Epidemiology
                Oxford University Press
                0300-5771
                1464-3685
                August 2017
                27 February 2017
                27 February 2017
                : 46
                : 4
                : 1106-1106j
                Affiliations
                [1 ]Institute of Population Health Sciences, National Health Research Institutes, Zhunan, Taiwan
                [2 ]Department of Health Services Administration, China Medical University, Taichung, Taiwan
                [3 ]Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
                [4 ]Department of Cardiology, Hope Doctors Hospital, Miaoli, Taiwan
                [5 ]Puzi Hospital, Ministry of Health and Welfare, Chiayi, Taiwan
                [6 ]Department of Family Medicine, Yee Zen General Hospital, Taoyuan, Taiwan
                [7 ]Department of Family Medicine, Changhua Christian Hospital, Changhua, Taiwan
                [8 ]School of Medicine, Chung Shan Medical University, Taichung, Taiwan
                [9 ]Department of Community Health, Mennonite Christian Hospital, Hualien, Taiwan
                [10 ]Department of Surgery, Yuan’s General Hospital, Kaohsiung, Taiwan
                [11 ]Department of Neurology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
                [12 ]School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
                [13 ]Department of Epidemiology and Biostatistics, University of California, San Francisco, CA, USA
                [14 ]Molecular Biochemistry and Expression Laboratories, Cedars-Sinai Medical Center, Los Angeles, CA, USA
                [15 ]Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
                Author notes
                [* ]Corresponding author. Institute of Population Health Sciences, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli 350, Taiwan. E-mail: hsiung@ 123456nhri.org.tw
                Article
                dyw331
                10.1093/ije/dyw331
                5837206
                28369534
                3bde1bb9-1ac5-458c-87cc-7ab6c16ba820
                © The Author 2017. Published by Oxford University Press on behalf of the International Epidemiological Association

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence ( http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work properly cited. For commercial re-use, please contact journals.permissions@oup.com

                History
                : 20 October 2016
                Page count
                Pages: 11
                Funding
                Funded by: National Health Research Institutes 10.13039/501100004737
                Award ID: BS-097-SP-04, PH-098-SP-02, PH-099-SP-01, PH-100-SP-01, PH-101-SP-01, PH-102-SP-01, PH-103-SP-01, PH-104-SP-01
                Categories
                Cohort Profiles

                Public health
                Public health

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