PLoS ONE
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The correlation of salivary telomere length and single nucleotide polymorphisms of the ADIPOQ, SIRT1 and FOXO3A genes with lifestyle-related diseases in a Japanese population
DOI 10.1371/journal.pone.0243745 , Volume: 16 , Issue: 1
Article Type: research-article, Article History
Abstract

Background

It has been reported that genetic factors are associated with risk factors and onset of lifestyle-related diseases, but this finding is still the subject of much debate.

Objective

The aim of the present study was to investigate the correlation of genetic factors, including salivary telomere length and three single nucleotide polymorphisms (SNPs) that may influence lifestyle-related diseases, with lifestyle-related diseases themselves.

Methods

In one year at a single facility, relative telomere length and SNPs were determined by using monochrome multiplex quantitative polymerase chain reaction and TaqMan SNP Genotyping Assays, respectively, and were compared with lifestyle-related diseases in 120 Japanese individuals near our university.

Results

In men and all participants, age was inversely correlated with relative telomere length with respective p values of 0.049 and 0.034. In men, the frequency of hypertension was significantly higher in the short relative telomere length group than in the long group with unadjusted p value of 0.039, and the difference in the frequency of hypertension between the two groups was of borderline statistical significance after adjustment for age (p = 0.057). Furthermore, in men and all participants, the sum of the number of affected lifestyle-related diseases, including hypertension, was significantly higher in the short relative telomere length group than in the long group, with p values of 0.004 and 0.029, respectively. For ADIPOQ rs1501299, men’s ankle brachial index was higher in the T/T genotype than in the G/G and G/T genotypes, with p values of 0.001 and 0.000, respectively. For SIRT1 rs7895833, men’s body mass index and waist circumference and all participants’ brachial-ankle pulse wave velocity were higher in the A/G genotype than in the G/G genotype, with respective p values of 0.048, 0.032 and 0.035. For FOXO3A rs2802292, women’s body temperature and all participants’ saturation of peripheral oxygen were lower in the G/T genotype than in the T/T genotype, with respective p values of 0.039 and 0.032. However, relative telomere length was not associated with physiological or anthropometric measurements except for height in men (p = 0.016). ADIPOQ rs1501299 in men, but not the other two SNPs, was significantly associated with the sum of the number of affected lifestyle-related diseases (p = 0.013), by genotype. For each SNPs, there was no significant difference in the frequency of hypertension or relative telomere length by genotype.

Conclusion

Relative telomere length and the three types of SNPs determined using saliva have been shown to be differentially associated with onset of and measured risk factors for lifestyle-related diseases consisting mainly of cardiovascular diseases and cancer.

Han, Kubota, Tanaka, Hayashi, Seki, Sakai, Kawaguchi-Ihara, Arakawa, Murohashi, and Boon-Peng: The correlation of salivary telomere length and single nucleotide polymorphisms of the ADIPOQ, SIRT1 and FOXO3A genes with lifestyle-related diseases in a Japanese population

Introduction

Telomeres are a repetitive nucleotide sequences of TTAGGG at the end of a chromatid, maintaining the stability of chromosomes to avoid deterioration and fusion with other chromosomes [1]. Telomere length (TL) is influenced by genetic factors, with previous studies reporting heritability estimates ranging from 34 to 82% [2, 3]. This parameter is also cumulatively shaped by nongenetic influences throughout human life. In contrast, single nucleotide polymorphisms (SNPs) are inherited from parents and transmit heritable events [4].

Quantitative trait locus studies have mapped putative loci that may be involved in regulating TL to human chromosomes 3q26.1, 10q26.13 and 12q12.22 [5]. Indeed, a number of recent genome-wide association studies (GWAS) identified common SNPs near the telomerase RNA component (TERC ) associated with TL in European and Chinese populations [5, 6]. The strongest associations with TL were reported for the SNPs rs12696304 and rs16847897 near TERC on 3q26 . Although their functions are unclear, these genes appear to be involved in the maintenance of chromosome structures [7].

Research on the genetics of human longevity has identified hundreds of genes associated with longevity, but polymorphisms in only two genes (APOE and FOXO3 ) have demonstrated strong and consistent replications across multiple, diverse human populations [8]. The correlation between genetic factors such as TL and SNPs and lifestyle-related diseases (LRDs) is also still controversial [815]. Furthermore, these studies [5, 6, 815] were conducted in different population cohorts, including Chinese, American and European cohorts, and in different types of studies, including original papers, literature reviews and meta-analyses.

To better understand the link between genetic factors and human disorders and diseases, we studied the correlation among TL, three SNPs that may influence the risk factors for LRDs [9, 1618] or the onset of LRDs [19], LRD-related physiological and anthropometric measurements, and the onset of LRDs themselves in a Japanese population from limited areas near our university in one year at a single facility.

Materials and methods

Subjects

Since March 2011, an annual university-sponsored health course for community members has been conducted [20]. In each course, which was held ten times per year, health seminars and point-of-care testing were performed. In 2015, a total of 223 attendants were enrolled, and 122 out of 143 attendants in the year (85.3%) agreed to participate in an analysis of salivary TL and SNPs of the ADIPOQ, SIRT1 and FOXO3A genes. The inclusion criteria were Japanese descent, clinical stability and sufficient salivary DNA extraction for genetic factor analysis. The exclusion criterion was any acute illness. In two participants, the DNA amount was not enough for analysis, and the remaining 120 subjects were included in the present study (S1 Table). They ranged in age from 41 to 84 years and consisted of 34 men and 86 women with a mean age of 73.3 and 67.8 years, respectively. In three other healthy volunteers, TL was determined by using DNA from saliva and peripheral blood-derived leukocytes.

DNA extraction from saliva and blood

Participants expectorated at least two ml of passive drool saliva into a sterile, 50 ml polyethylene conical tube (Corning Science, Corning, NY, USA) for five min. These collection tubes were maintained on ice until use. Immediately, one ml of saliva was mixed with an equal volume of nuclei lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 50 mM sucrose, 100 mM NaCl, 2.4% SDS, 550 μg/ml proteinase K, pH 8.0) [21]. After digestion of the cell lysate overnight at 53°C, 400 μl of 5 M NaCl was added to the mixture, shaken vigorously for 15 s and placed on ice for 30 min. The DNA in the supernatant was extracted by using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich Co., LLC., Tokyo, Japan). The concentration of the isolated DNA was determined using a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Heparinized venous blood was collected from three healthy adult volunteers. Every 4 ml of blood was mixed with 1 ml of 5% (w/v) dextran (MW 266,000, Sigma-Aldrich) in phosphate-buffered saline (PBS) (Gibco, Grand Island, NY, USA), incubated for 30 min at room temperature, and the upper layer containing whole leukocytes was harvested [22]. In one volunteer, the whole leukocyte fraction in PBS was further layered onto Ficoll-Hypaque (Ficoll-Paque Plus, 1.077 g/ml, GE Healthcare Life Sciences, Buckinghamshire, England) and centrifuged at 400×g for 30 min. The whole leukocytes resolved into two distinct bands, the upper containing mononuclear cells (MNCs) and the lower containing neutrophils, and either fraction was harvested. MNCs in PBS with 10% fetal calf serum (Gibco) were further placed in Falcon culture dishes (Becton Dickinson Labware, Oxnard, CA, USA) and incubated for 2 h at 37°C in 5% CO2. After incubation, the supernatant with the lymphocyte fraction was collected and the nonadherent cells were further removed by vigorous washing with PBS. After the addition of 3 ml fresh warm 0.05% Trypsin-EDTA (Gibco) to the flask, the cells were further incubated for five min at 37°C in 5% CO2 and the adherent cells (monocyte fraction) were collected by tapping the side of the flask. As in the case of saliva, cellular DNA was extracted, and the concentration of the isolated DNA was determined. After blood and saliva cells bound on a glass slide by CytospinTM 4 Cytocentrifuge (Thermo Fisher Scientific K.K., Tokyo, Japan), Wright-Giemsa staining (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) was performed, and 100 nucleated cells were counted and classified for each of the three slide glass specimens by using an inverted microscope (Eclipse E600, Nikon, Tokyo, Japan) at 1,000× magnification.

Telomere length determination by the monochrome multiplex quantitative polymerase chain reaction method

TL quantitative polymerase chain reaction (QPCR) was performed with a Chromo4TM Real-Time PCR Detection System (Bio-Rad, Tokyo, Japan) [23]. The telomere primers (telg and telc, final concentrations 900 nM each) were used for the telomere signal and a set of single-copy gene (scg) primers within β–globin genes (final concentrations 200 nM each) were used as a reference with Power SYBRTM Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA).

The PCR cycle for monochrome multiplex QPCR (MMQPCR) was as follows: Stage 1: 1 cycle of 2 min at 95°C; Stage 2: 2 cycles of 5 s at 94°C and 15 s at 49°C; and Stage 3: 40 cycles of 5 s at 94°C, 10 s at 62°C, 15 s at 74°C with signal acquisition (for the amplification of telomere template), 10 s at 84°C, and 15 s at 88°C with signal acquisition (for the amplification of the scg template). The same standard genomic DNA was used to establish two standard curve reactions in every plate in the study, one for the telomere signal and one for the scg signal, which were used for calculation of the T/S ratios (ratios of “telomere signals per scg signals”). As each experimental sample was assayed in triplicate, three T/S results were obtained for each sample; the final reported result for a sample in a given run is the average of the three T/S values and was named relative telomere length (RTL). The average T/S is expected to be proportional to the average TL per cell. Samples with an RTL > 1.0 have an average TL greater than that of the standard DNA; samples with an RTL < 1.0 have an average TL shorter than that of the standard DNA.

TaqMan® PCR assay

Genotyping for ADIPOQ rs1501299, Sirt-1 rs7895833 and FOXO3A rs2802292 was performed using Custom TaqMan® SNP Genotyping Assays (Applied Biosystems) in which a fluorogenic probe consisting of an oligonucleotide labeled with both a fluorescent reporter dye (FAM or VIC) and a quencher dye is included in a typical PCR [24]. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence [25]. Each primer and probe set was used in the TaqMan® SNP Genotyping Assays (ID: C___7497299_10, C__29163689_10 and C__16097219_10; Applied Biosystems) in accordance with the information on the Applied Biosystems website (http://www.appliedbiosystems.com).

PCR was performed according to the manufacturer’s instructions provided by Applied Biosystems. In brief, one to 20 ng of template DNA dissolved in 2.25 μl in each well was loaded into 96-well plates for PCR. The total reaction volume was 5 μl after adding 2.5 μl of TaqMan Universal PCR Master Mix (2×) and 0.25 μl of 10× working stock of SNP genotyping assay buffer (Applied Biosystems).

The PCR thermal cycling was as follows: Stage 1: 1 cycle of 2 min at 50°C; Stage 2: 1cycle of 10 min at 95°C for initial denaturing; Stage 3: 40 cycles of 15 s at 92°C and 1 min at 60°C. Thermal cycling was performed using a Chromo4TM Real-Time PCR Detection System (Bio-Rad). Each 96-well plate contained unknown genotype samples and three reaction mixtures containing the reagents but no DNA (no-template control). The no-DNA control samples were necessary for Sequence Detection System (SDS) 7700 signal processing, as outlined in the TaqMan Allelic Discrimination Guide. The genotypes were determined visually based on the dye component fluorescence emission data depicted in the X-Y scatter plot of the SDS software.

Physiological and anthropometric measurements

Body height (cm) was measured using a metal height meter (YS-OS, AS ONE Co., Ltd., Osaka, Japan). Weight (kg), body mass index [BMI (kg/m2)] and percentage of body fat (BF) (%) were measured by a dual-frequency body composition analyzer (DC 430A, Tanita Co., Ltd., Tokyo, Japan) using bioelectrical impedance analysis. Waist circumference (WC) was measured at the umbilicus to the nearest cm with the participant standing and breathing normally. Body temperature (BT) (°C) was measured using an electronic thermometer inserted into the armpit for 30 sec (ET-C205S, Terumo Co., Ltd., Tokyo, Japan).

Hypertension (HT) was defined as systolic blood pressure (SBP) ≥140 mm Hg, diastolic blood pressure (DBP) ≥90 mm Hg, or the use of antihypertensive medications [26]. After a more than 5 min rest, the SBP and DBP were measured by a trained physician or nurse using a standard mercury sphygmomanometer (SeaStar, 070108041, Tokyo, Japan) with the participant in the sitting position. Two consecutive blood pressure measurements were taken at 2-min intervals, and the lower one was recorded as the blood pressure.

Measurement of carotid maximum intima-medial thickness (max IMT) (mm) through B-mode ultrasonographic imaging was performed using C3cv (Aloka Medical, Ltd., Tokyo, Japan) with a 6-MHz transducer in the right common carotid artery 1.5 to 3.0 cm proximal to the bifurcation [27].

Measurements of the ankle brachial index (ABI) and brachial-ankle pulse wave velocity (PWV) (cm/sec) were performed as follows. Brachial-ankle arterial blood pressures were simultaneously measured using a noninvasive automatic device (model BP-203RPE-III; Nihon Colin, Tokyo, Japan) after a 5-min rest in the supine position [27]. ABI was defined as the ratio of systolic blood pressure in the ankle and the higher side of the two brachial arteries. The PWV on each side was calculated as the transmission distance divided by the transmission time. The transmission time between the right arm and both ankles was calculated using the waveform. The transmission distance between the right brachium and ankle was automatically calculated according to the height of the patient. PWV was evaluated on the higher side.

Saturation of peripheral oxygen (SpO2) was measured using a pulse oximeter device (ko-001, Kohken Medical Co., Ltd., Tokyo, Japan).

Questionnaire survey

With the help of our trained staff, self-reported questionnaires were completed during attendance, assessing attendants’ personal medical history (PMH) and antihypertension medication use. PMH consisted of HT, stroke, acute myocardial infarction (AMI), chronic kidney disease (CKD) with therapy for edema, hyperkalemia or anemia [28] and cancer (S2 Table). The score for PMH was defined as the sum of the number of affected LRDs in PMH.

Ethical considerations

This study was approved by the Ethics Committee of Saitama Prefectural University (No. 27503). Before study enrollment, participants and normal volunteers who provided both saliva and peripheral blood samples were asked to sign a consent and assent form that described the background and procedures of the study.

Statistical analyses

Nonparametric statistics were used. Continuous variables are described as the mean ± standard deviation (SD), and categorical variables are described as proportions. Unadjusted differences between groups were performed by one-way analysis of variance (ANOVA) for continuous variables and chi-square tests for categorical variables. Multivariable linear regression analyses were used to test for adjusted differences with adjustments for the confounding effects of age and sex. Student’s t-test was performed to compare the differences in means between the two groups. A two-tailed p value less than 0.05 was used to determine significance. Statistical analyses were performed by IBM SPSS Statistics version 23.

Results

Salivary and blood relative telomere length

In any of the three volunteers studied, there was no significant difference in RTL between the DNA extracted from saliva and blood-derived whole leukocytes (S1A Fig). Similarly, in one volunteer, there was no significant difference in RTL between the DNA extracted from blood-derived neutrophils, lymphocytes and monocytes (S1B Fig).

Relative telomere length and LRD-related physiological and anthropometric measurements

First, we examined the correlation between RTL and age. Age and RTL were inversely correlated in men (p = 0.049, r = − 0.340, y = −9.258x + 82.429) and all participants (p = 0.034, r = − 0.192, y = −7.711x + 76.857) but not in women (p = 0.073, r = − 0.194, y = −8.235x + 75.766) (Fig 1 and S1 Table).

Cross-sectional analysis of age-dependent shortening of salivary relative telomere length.
Fig 1
For each point, the age (yr) of the participant (x-axis) is plotted against salivary relative telomere length (RTL) (y-axis) for men (upper column), women (middle column), and all participants (lower column). The linear regression equation and correlation coefficient were determined using Microsoft Excel.Cross-sectional analysis of age-dependent shortening of salivary relative telomere length.

Next, we examined the association between RTL and LRD-related physiological and anthropometric measurements. In men, the long RTL group (n = 17) had a significantly greater height (167.5 ± 6.6 vs 162.1 ± 5.3 cm) and had a significantly lower pulse rate (65.3 ± 12.6 vs 73.7 ± 15.5/min) than the short RTL group (n = 17) before (p = 0.011) and after (p = 0.016) and before (p = 0.041) but not after (p = 0.092) age adjustment (S3 Table). RTL was not associated with other LRD-related physiological or anthropometric measurements.

Relative telomere length and score for personal medical history

Frequencies of HT for men, women and all participants according to RTL quartiles are shown in Fig 2A. The frequency of HT decreased in the order of 1st, 2nd, 3rd and 4th RTL quartiles except for 4th RTL quartiles for women and all participants. Longer RTL quartiles had an increasingly lower frequency of HT, especially for men. The p values for men, women and all participants after adjustments for both sex and age were 0.130, 0.723 and 0.270, respectively. In men, the frequency of HT was significantly lower in the long RTL group (3rd and 4th RTL quartiles) than in the short group (1st and 2nd RTL quartiles) with unadjusted p value of 0.039, and the difference in the frequency of HT between the two groups was of borderline statistical significance after adjustment for age (p = 0.057) (Fig 2B). The p values for women and all participants after adjustments for both sex and age were 0.597 and 0.146, respectively.

Frequencies of hypertension by quartiles of salivary relative telomere length.
Fig 2
(A) Frequencies of hypertension (HT) for men, women and all participants in the 1st [66.7% (6/9), 52.4% (11/21) and 56.7% (17/30), respectively], 2nd [62.6% (5/8), 45.5% (10/22) and 50.0% (15/30), respectively], 3rd [30.0% (3/10), 40.0% (8/20) and 36.7% (11/30), respectively] and 4th [28.6% (2/7), 43.5% (10/23) and 40.0% (12/30), respectively] quartiles of relative telomere length (RTL) are shown. The frequency of HT was determined by using data in S1 Table. The RTL levels increase in the order of 1st, 2nd, 3rd and 4th RTL quartiles. For men, women and all participants, the p value was computed using multivariable linear regression analyses to investigate the association between frequency of HT and RTL, and was expressed after adjustments for both sex and age. (B) Frequencies of HT for men, women and all participants are also shown in two groups, one with a combination of 1st and 2nd RTL quartiles [64.7% (11/17), 48.8% (21/43) and 53.3% (32/60), respectively], and the other with a combination of 3rd and 4th RTL quartiles [29.4% (5/17), 41.9% (18/43) and 38.3% (23/60), respectively]. The p value was determined and expressed as shown in (A).Frequencies of hypertension by quartiles of salivary relative telomere length.

In both men and all participants, the PMH score was also significantly higher in the short RTL group than in the long RTL group after adjustments for both age and sex with respective p values of 0.004 and 0.029 (Table 1).

Table 1
The association of score for personal medical history with relative telomere length.
SexRelative telomere lengthp value
shortlong 
Men0.941±0.556 (17)0.353±0.493 (17)0.004
Women0.692±0.694 (39)0.524±0.632 (41)0.33
All0.768±0.660 (56)0.483±0.599 (58)0.029

For men, women and all participants, the score for personal medical history (PMH) was shown by relative telomere length (RTL) as shown in the materials and methods. The score for PMH was determined and is expressed as the mean ± standard deviation of the sum of the number of affected lifestyle-related diseases in PMH as shown in the materials and methods. The number of participants whose PMH score was completed was shown in parentheses. The p value was computed using multivariable linear regression analysis to investigate the association between PMH score and RTL, and was expressed after adjustments for both sex and age.

SNPs, LRD-related physiological and anthropometric measurements and scores for personal medical history

For ADIPOQ rs1501299, the ABI of men was significantly higher in carriers of the T/T genotype than in those with the G/G and G/T genotypes, with p values of 0.001 and 0.000, respectively (Table 2). For SIRT1 rs7895833, the BMI and WC of men were significantly higher in carriers of the A/G genotype than in those with the G/G genotype, with respective p values of 0.048 and 0.032. The PWV of all participants was also significantly higher in carriers of the A/G genotype than in those with the G/G genotype, with a p value of 0.035. For FOXO3A rs2802292, the BT of women and SpO2 of all participants were significantly lower in carriers of the G/T genotype than in those with the T/T genotype, with respective p values of 0.039 and 0.032. ADIPOQ rs1501299 in men, but not the other two SNPs, was significantly associated with the PMH score by genotype (p = 0.013).

Table 2
The association of SNPs with LRD-related physiological and anthropometric measurements and scores for personal medical history.
SNPsSexGenotypeAgeBMIWCBTABIPWVSpO2PMH
no. (%)y.o.score
n = 120n = 120n = 110n = 120n = 106n = 120n = 120n = 119n = 114
ADIPOQMenGG 18 (53)75±423.3±1.588±536.1±0.81.15±0.06a1656±28896.3±1.40.89±0.58c
GT 14 (41)72±722.2±2.683±836.1±0.61.14±0.06b1545±22496.5±1.40.356±0.50c
TT 2 (6)73±320.6±6.479±1636.1±0.11.38±0.20a, b1472±3396.5±0.70.50±0.71c
WomenGG 56 (65)68±721.8±3.083±936.0±0.51.12±0.071574±33896.2±1.30.61±0.64
GT 21 (24)65±922.1±3.185±1036.1±0.51.11±0.061471±30096.8±1.00.45±0.61
TT 9 (11)70±521.8±2.280±835.7±0.71.09±0.091813±35796.4±1.61.00±0.87
AllGG 74 (62)70±722.2±2.784±936.0±0.61.13±0.071594±32696.3±1.30.68±0.63
GT 35 (29)68±722.1±2.984±1036.1±0.61.12±0.061500±27196.7±1.20.41±0.56
TT 11 (9)70±521.5±3.080±935.6±0.61.14±0.151751±34896.4±1.40.91±0.83
SIRT1MenAA 3 (9)71±421.0±2.082±635.5±2.01.16±0.081619±15696.0±1.00.67±0.58
AG 12 (35)75±424.1±2.1d90±5e36.2±0.71.18±0.121703±20196.1±1.60.75±0.45
GG 19 (56)72±622.0±2.3d82±8e36.1±0.41.15±0.061531±28696.7±1.30.58±0.69
WomenAA 7 (8)73±223.4±1.585±535.8±0.81.11±0.071590±28496.0±2.20.86±0.90
AG 39 (45)68±621.5±2.783±936.1±0.51.12±0.061651±35596.4±1.30.66±0.68
GG 40 (47)66±822.0±3.283±1036.0±0.51.11±0.071496±32396.4±1.10.53±0.60
AllAA 10 (8)73±322.6±2.084±535.7±1.21.12±0.071599±24496.0±1.80.80±0.79
AG 51 (43)70±722.2±2.884±936.1±0.51.13±0.081663±325f96.3±1.40.68±0.63
GG 59 (49)68±822.0±2.983±1036.0±0.51.13±0.081507±310f96.5±1.20.54±0.63
FOXO3AMenTT 19 (56)73±622.6±2.385±836.0±0.51.18±0.101534±16596.6±1.20.63±0.68
GT 15 (44)74±422.7±2.685±836.1±0.91.14±0.061682±32996.1±1.60.67±0.49
WomenTT 50 (58)69±721.9±3.084±1036.1±0.5g1.12±0.061565±33696.6±1.20.59±0.68
GT 36 (42)67±721.9±2.981±835.8±0.5g1.11±0.071586±35096.1±1.30.65±0.66
AllTT 69 (58)70±722.1±2.884±1036.1±0.51.14±0.081556±29896.6±1.2h0.60±0.67
GT 51 (42)69±722.1±2.883±835.9±0.71.12±0.081614±34496.1±1.4h0.65±0.61
For men, women and all participants, and for any of the three types of SNPs by genotype, data are expressed as the mean ± standard deviation. The p value was computed using multivariable linear regression analysis to investigate the association of genotype with lifestyle-related disease-related physiological and anthropometric measurements and scores for personal medical history, and was expressed after adjustments for both sex and age.
Abbreviations: SNPs, single nucleotide polymorphisms; Age, age (yr); RTL, relative telomere length; BMI, body mass index (kg/m2); WC, waist circumference (cm); BT, body temperature (°C); ABI, ankle brachial index; PWV, brachial-ankle pulse wave velocity (cm/sec); SpO2, saturation of peripheral oxygen (%); PMH, personal medical history.
a p = 0.001
b p = 0.000
c p = 0.013
d p = 0.048
e p = 0.032
f p = 0.03
g p = 0.039
h p = 0.032

In men, women and all participants, the results for LRD-related physiological and anthropometric measurements, RTL and frequency of HT not associated with any of the three SNPs by genotype are shown in S4 Table.

Reproducibility of salivary relative telomere length measurements

Finally, to examine inter-assay reproducibility, we repeated the measurements of salivary RTL in the same 120 DNA samples as in Fig 1, again in triplicate, on another day. Fig 3 shows the strong correlation between the salivary RTL determined by the first and second runs (R2 = 0.9061).

Reproducibility of salivary relative telomere length in independent runs.
Fig 3
The same 120 salivary DNA samples assayed in Fig 1 were assayed again on different days. The linear regression equation and correlation coefficient were determined using Microsoft Excel.Reproducibility of salivary relative telomere length in independent runs.

Discussion

We successfully determined RTL and genotypes of the three SNPs by using DNA from passive drool saliva in any of the 120 participants. However, caution is warranted when comparing TL measured from saliva obtained through passive drool and saliva obtained using swabs or sponges as the percentage of buccal cells in the latter is significantly higher than that of the former [29]. We confirmed in three volunteers that there was no significant difference in RTL measured using passive drool saliva and blood-derived whole leukocytes. In one volunteer, the proportion of neutrophils in passive drool saliva was as high as 78.27 ± 6.03% (mean ± SD of triplicate measurements). To date, a growing body of evidence has shown that TL measurement using passive drool saliva instead of blood-derived whole leukocytes is quite useful [3032].

As previously reported [33], the long RTL group (3rd plus 4th) had a significantly lower frequency of HT than the short group (1st plus 2nd) only in men with unadjusted p value of 0.039, and the difference was of borderline statistical significance after age adjustment (p = 0.057). Furthermore, compatible with a previous report [34], our present study found a significant association between the PMH score and RTL in men and all participants. In the present study, LRDs consisted mainly of cardiovascular diseases and cancer. Inconsistent with previous reports [35, 36], however, RTL did not correlate with SBP, DBP or pulse pressure because of the frequent use of antihypertension medication in the present study (40/120; 33.3%).

The overall effect of individually unique environmental factors during adult life, such as energy intake, lifestyle, socioeconomic status and mental stress, is relatively small compared with the joint effect on TL of heritability and shared environmental factors, which is estimated at ~87% [37]. In addition, in recent years, medical treatment and preventive medicine have made remarkable progress [38]. Interestingly, the Mendelian randomization approach, which usually avoids the potential confounding effect of environmental exposure on the relation between phenotypically measured TL and risk of ischemic stroke, has shown that genetically predicted TL is not associated with ischemic stroke [8]. The effect of heritability on TL is presumed to be weaker in the middle-aged to elderly subjects in the present study than in the younger subjects. For these reasons, in the present study as well, it seems likely that RTL appears to be more strongly associated with the combination of heritability and shared environmental factors than heritability or individual-specific environmental factors alone, in terms of predisposition to the development of LRDs.

In the present study, men in the long RTL group were shown to be significantly taller than those in the short RTL group. Although it is undeniable that bone mineral density may be maintained with long TL [39], it would be more correct to say that height is not a risk factor for LRDs.

Associations between the SNPs and LRD-related physiological or anthropometric measurements in the present study were as follows: for ADIPOQ rs1501299 , in accordance with previous papers [16, 19], men’s ABI was higher in the T/T genotype than in the G/G or G/T genotype; for SIRT1 rs7895833 , consistent with previous papers [17, 18], men’s BMI and waist circumference and all participants’ PWV were higher in the A/G genotype than in the G/G genotype; for FOXO3A rs2802292, women’s BT and all participants’ SpO2 were lower in the G/T genotype than in the T/T genotype. We confirmed again in the peresent study that the Japanese allele frequencies for SIRT1 rs7895833 [17], which are different from those of Caucasians [18], might explain why Japanese individuals show less marked obesity than Caucasians. Consistent with a role for FOXO3 in suppression of insulin/insulin-like growth factor and thus growth to extend lifespan, the longevity-associated G allele variant of FOXO3A rs2802292 seems to be associated with lower BT and SpO2 [9, 40].

Compatible with previous reports [16, 19], only ADIPOQ rs1501299, but not the other two types of SNPs showed a significant correlation with both risk factors for and PMH score of LRDs. In any of the three SNPs, there was no significant difference in the frequency of HT or RTL by genotype.

Many studies have shown that TL is a heritable trait, and SNPs in several candidate genes including TERT (telomerase reverse transcriptase), TERC (telomerase RNA component), OBFC1 (oligonucleotide/oligosaccharide-binding folds containing one), CTC1 (conserved telomere maintenance component 1) and ZNF676 (zinc fingerprotein676) have been identified to be significantly associated with TL [2, 41]. These genes have limited genetic variations and encode proteins that are thought to be involved either in TL maintenance or with telomere binding proteins necessary for telomere stability and structure. Exceptionally, fat mass- and obesity-associated (FTO) genes have been shown to be associated with obesity and TL [7], and the FOXO3 G allele of SNP rs2802292 significantly protected against aged-related TL loss relative to that of carriers of the common TT genotype [42]. However, we could not find any association between RTL and genotype in any of the three SNPs examined in the present study.

SNPs are the most common type of human genetic variation and have been associated with disease development and phenotypic forecasting [43]. Although GWAS identified genetic variants involved in complex phenotypes, the fraction of heritability of common traits and diseases explained by the identified loci is small [44]. The reason for this small proportion of the heritability of these complex traits is still unclear, but the causes can involve epistatic effects, genetic interactions inside undiscovered pathway or underestimated genotype-environment interactions [45, 46].

SIRT1 also regulates a stress-response transcription factor, FOXO3 , thereby modulating cellular senescence/aging, skeletal muscle function, cardiovascular homeostasis, and human longevity [9, 40, 47]. Furthermore, telomeres have been shown to be closely associated with sirtuin [48] and FOXO3 [42]. Therefore, further studies are required to clarify the exact correlation between the two SNPs and TL.

DNA methylation is capable of controlling the gene expression of common traits and influencing the development of aberrant health outcomes under external exposure [49]. Recently, an epigenetic ‘mortality risk score’ based on whole blood DNA methylation at 10 mortality-related CpG sites has been shown to be strongly associated with TL and even more strongly associated with all-cause mortality [50]. However, TL failed to correlate with all-cause mortality. DNA methylation studies of the SNPs may further clarify the relationship among SNPs, TL and LRDs [51].

In the present study, the frequency of the significant association of genetic factors with LRDs was higher in men than in women. It is presumed that the reason is, in part, that men have a higher prevalence of LRDs, especially cardiovascular disease, and a shorter life expectancy than women in specific age groups, 50–70 [52]. The number of subjects in this study was small, 34 men and 86 women, with a total of 120, so the results of the study may be limited; however, none of the 34 men were excluded (S1 Table). In any case, further studies are required to confirm our present results.

Conclusion

Over the course of a year and in a single facility, we showed that RTL and the three types of SNPs determined using saliva are differentially associated with onset of and measured risk factors for LRDs consisting mainly of cardiovascular diseases and cancer in Japanese individuals living in a small area near our university.

Acknowledgements

We thank our lab members for supporting the annual university-sponsored course for community members. We also thank Dr. Yukio Kamezawa (Saitama Prefectural University) for generously providing cell-separation reagents and for helpful discussions on this study.

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13 Sep 2020

PONE-D-20-22106

The correlation of salivary telomere length and single nucleotide polymorphisms of

ADIPOQ ,SIRT1 and FOXO3A genes with lifestyle-related diseases in Japanese

PLOS ONE

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Reviewer #1: This is an interesting and well conducted study on the correlation of genetic factors (3SNPs) and lifestyle-related diseases in a Japanese cohort. Several physiological parameters have been measured for that. Language is in general acceptable but has to be corrected by a native speaker since articles are often missing or misplaced and there are other language and grammar issues as well.

Although it is very good that you confirmed the correspondence of TL in saliva and blood cells I would suggest to move figure 1 to the supplementary since it is just a methodological detail and only very few samples ( 3 or 1) have been included for blood related markers.

For table 1 please include n-numbers for each group and better describe in the text how exactly you determined the prevalence for LRDs.

Importantly, since your study cohort included much more women than men: is it possible that several significances in men are due to the much smaller sample size?

For table 2 please make the letters the p-values at the end refer to larger since it is very difficult to spot them, perhaps use bold font as well. When you discuss heritability of TL, please make sure to emphasise that the % of heritability decreases with age-it is higher in young people than older and you have a middle-age/older population in your study.

Presentation of figure 3 needs to be improved. For 3A, what is % if you want to present the prevalence? Please clarify! 3B needs its own axis labelling! For both graphs please show error bars and statistical significances.

Reviewer #2: 1) The abstract requires profound revision, dividing sections of introductions, methods, results and conclusions. In this form, the rationale of the study is not reported also.

2) Tables: authors should fix the tables that are very not easy-to-get in these forms.

3) Sample size analysis is lacking in statistical sections. This is relevant for such type of genetic data (and numbers are not so convincing).

4) Major issue: authors aims to address a correlation between a modifiable marker (telomere length) with modifiable exposure to environmental factors, “blocking” for a genetic trait. However, there is a lack of causality which is given by the non-causal effect of polymorphisms. How often are expected these variants to be frequent in your populations? Again (sample size is lacking)? Are the variants in accordance with Hardy-Weinberg equilibrium?

Based on these lacks conclusions in the abstact as well as in the text are not supported by data. Telomeres cannot be defined “independently associated”. Please, I strongly suggest to revise this point.

5) How much authors are confident on the third number decimal as for telomere length? Since data are coming from an “estimated” fold-change of a pcr-based method, generally it would be hard to be so precise. How many triplicates intra- and inter-day have been repeated for telomere length ?

6) The heading of table 1 is not clear: it is reporting a 2X2 association between gender and telomere length, so how do authors comment on the heading of the table?

**********

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Reviewer #1: Yes: Gabriele Saretzki

Reviewer #2: No

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24 Oct 2020

‘Response to Reviewers’

Hoh Boon-Peng, PhD

Academic Editor

PLOS ONE, and

Two reviewers

Thank you for your kind and accurate guidance. Responses to academic editor and reviewers are as follows.

Hoh Boon-Peng, PhD

Academic Editor

PLOS ONE

First of all, we apologize for making two mistakes and correcting them. The first is the mistake for statistical processing of quantification of personal medical history. Then, we showed all 120 subjects’ data including age, sex, relative telomere length (RTL) and personal medical history in S1 Table. Due to the correction, the contents of Table 1 have been slightly changed. The second is that we noticed the significant correlation between SNPs ADIPOQ rs1501299 and personal medical history in men. Due to the correction, the contents of Table 2 have been slightly changed.

Next, we will answer your questions in order below.

1. We asked American Journal Expert (AJE) for language editing and proofreading. Certificate of the completion of calibration by AJE is attached.

2. We confirmed by using G*Power software (HHU) that 120 samples are appropriate to provide sufficient power of study. In addition, this study was performed in a single institute in one year in Japanese population. This is described in the revised manuscript.

3. In our paper, a total of 120 subjects were analyzed, 34 men and 86 women. The characteristics of the 120 participants were shown in S1 Table. The description of relative telomere length, three SNPs, hypertension was completed in all 120 subjects. The personal medical history was complete in all 34 men and 80 women, a total of 114. The total of five diseases checked by our trained staff during attendance consisted of hypertension, acute myocardial infarction, stroke and chronic kidney disease, which mainly develop based on vascular lesions, and the rest is cancer (any malignant neoplasms). The sum of the number of affected diseases was compared with the relative telomere length.

Answer to Journal requirements

1. We corrected manuscript to meet PLOS ONE’s style requirements, as far as possible.

2. Our manuscript has been corrected by American Journal Experts, and the certificate of the completion of calibration is attached.

3. “Request for personal medical history” in both Japanese and English was added and shown in S2 Table.

4. I will soon ensure ORCiD ID.

5. We deleted all “data not shown”. All relevant data were shown in S1, S3 and S4 Tables.

Answers to the reviewers’ comments.

1. We have rewritten our revised manuscript extensively according to your guidance.

2. In the Materials and Methods, we rewritted statistical analyses properly.

3. All data were added and shown in S1, S3 and S4 tables.

4. We asked American Journal Expert (AJE) for language editing and proofreading. Certificate of the completion of calibration by AJE is attached. In addition, we read the manuscript many times and corrected the mistakes.

5. Answers to review comments.

Reviewer #1

Thank you for your important advice. We think that our manuscript has been greatly improved owing to the proofreading by American Journal Experts. The certificate of the completion of calibration is attached.

Fig 1 has been changed to S1 Fig, as instructed.

For Table 1, the number of subjects is shown for each group, as instructed.

The number of 34 male subjects is not large, but none were excluded. In addition, relative telomere length determination and personal medical history hearing have been completed for all, as shown in S1 Table. We discussed in depth in the Discussion why the frequency of significant differences are higher in men in the present study.

According to your suggestion for Table 2, we changed the letters the p-values at the end refer to bold font

As suggested, “The effect of heritability on TL is presumed to be weaker in the middle-aged to elderly subjects in the present study than in the younger subjects.“ has been added to the Discussion.

We are very sorry for making an inappropriate description in Fig 3. We replaced prevalence with frequency (percentage). We added axis labelling to both A and B. In the frequency [for example 66.7% (6/9)] only one number comes out, and it is impossible to show as mean ± SD. Multivariable linear regression analyses were used to identify statistical significance. As the number of statistical comparisons is too large to describe, only one (p = 0.057) is shown.

Reviewer #2

1) The abstract has been significantly revised, and was divided to introduction, objectives, methods, results and conclusions.

2) Tables 1 and 2 have been improved to the maximum to make them easier to read.

3) We confirmed by using G*Power software (HHU) that 120 samples are appropriate to provide sufficient power of study. In addition, this study was performed at a single facility in one year in a Japanese population. This is described in the revised manuscript. In our paper, a total of 120 subjects were analyzed, 34 men and 86 women. The characteristics of the 120 participants were shown in S1 Table. The description of relative telomere length, three SNPs, hypertension was completed in all 120 subjects. The personal medical history was complete in all 34 men and 80 women, a total of 114. The number of subjects was indicated in Tables 1 and 2, and Tables S3 and S4. We added the description of “The number of subjects in this study was small, 34 men and 86 women, with a total of 120, so the results of the study may be limited.” to the Discussion.

4) We are very grateful for pointing out a very important issue. We deeply apologize for making an inappropriate statement of “independently associated”. This statement in both Abstract and Discussion has been deleted.

5) We measured RTL according to the method by Cawthon (Nucleic Acids Res 2009; 37:e21, reference 23. In our paper). He showed that the correlation of RTL with terminal restriction fragment length measured by Southern blot was strong (R2 = 0.844). In some samples, we repeated the measurements of RTL, again in triplicate, on another day. For analysis, however, the results of the first measurement were used for any of the subjects.

6) In our present study, in men, women and men and women in total, clinical characteristics of the study subjects including score for personal medical history were compared across the median or quartiles of RTL by using the methods shown in statistical analyses. Then, we consider that the heading of the Table 1 of “The association of score for personal medical history with relative telomere length” is correct.

6. Do you want your identity to be public for this peer?

Yes

Submitted filename: PO,Rev,22Oct,Response to Reviewers,IM.docx

11 Nov 2020

PONE-D-20-22106R1

The correlation of salivary telomere length and single nucleotide polymorphisms of the

ADIPOQ ,SIRT1 and FOXO3A genes with lifestyle-related diseases in a Japanese population

PLOS ONE

Dear Dr. Murohashi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The authors addressed the majority of the comments, however a proper description and interpretation of the results is missing. Most of the results consist of the 2 figures and tables, without a a clear explanation of those rather complex data. This has to be improved and amended to make it easier for the reader to follow the content of the study which is pretty complex.

==============================

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PLOS ONE

Additional Editor Comments (if provided):

The authors addressed the majority of the comments, however a proper description and interpretation of the results is missing. Most of the results consist of the 2 figures and tables, without a a clear explanation of those rather complex data. This has to be improved and amended to make it easier for the reader to follow the content of the study which is pretty complex.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Although the authors addressed the majority of my initial comments, I find that a proper description and interpretation of the results is really missing. Most of the results consist of the 2 figures and tables, but I a missing an explanation of those rather complex data. For example, for fig. 2 the authors state that they have performed various complex statistical methods while just giving 1 non-significant p-value (a, p=0.057). This has to be much better described and understandably interpreted for the reader, Normally, the results text mentions every single figure and table while here the authors write something in the text which I find difficult to relate to particular graphs, tables or statistical comparisons. Please try to give your results in better way as it is now.

Also, the heading for fig. 2 reads as "hypertensive frequency" which is my vie should be rather "Frequencies of hypertension" since the frequency isn't really suffering from hypertension.

**********

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Reviewer #1: Yes: Gabriele Saretzki

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24 Nov 2020

We believe that we have responded properly to any of the questions and requests, and now the submitting paper has been markedly improved owing to the help by academic editor and rewieers.

Submitted filename: 2nd Rev, 24 Nov, Response to Reviewers.docx

26 Nov 2020

The correlation of salivary telomere length and single nucleotide polymorphisms of the

ADIPOQ ,SIRT1 and FOXO3A genes with lifestyle-related diseases in a Japanese population

PONE-D-20-22106R2

Dear Dr. Murohashi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Hoh Boon-Peng, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

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Reviewer #1: Yes: Dr. Gabriele Saretzki


11 Jan 2021

PONE-D-20-22106R2

The correlation of salivary telomere length and single nucleotide polymorphisms of the ADIPOQ, SIRT1 and FOXO3A genes with lifestyle-related diseases in a Japanese population

Dear Dr. Murohashi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Academic Editor

PLOS ONE

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

https://www.researchpad.co/tools/openurl?pubtype=article&doi=10.1371/journal.pone.0243745&title=The correlation of salivary telomere length and single nucleotide polymorphisms of the <i>ADIPOQ</i>, <i>SIRT1</i> and <i>FOXO3A</i> genes with lifestyle-related diseases in a Japanese population&author=&keyword=&subject=Research Article,Biology and Life Sciences,Cell Biology,Chromosome Biology,Chromosomes,Chromosome Structure and Function,Telomeres,Telomere Length,Biology and Life Sciences,Genetics,Single Nucleotide Polymorphisms,Biology and Life Sciences,Genetics,Heredity,Genetic Mapping,Variant Genotypes,Biology and Life Sciences,Anatomy,Body Fluids,Saliva,Medicine and Health Sciences,Anatomy,Body Fluids,Saliva,Biology and Life Sciences,Physiology,Body Fluids,Saliva,Biology and Life Sciences,Anatomy,Anthropometry,Medicine and Health Sciences,Anatomy,Anthropometry,Biology and Life Sciences,Genetics,Heredity,Biology and Life Sciences,Computational Biology,Genome Analysis,Genome-Wide Association Studies,Biology and Life Sciences,Genetics,Genomics,Genome Analysis,Genome-Wide Association Studies,Biology and Life Sciences,Genetics,Human Genetics,Genome-Wide Association Studies,Biology and Life Sciences,Cell Biology,Chromosome Biology,Chromosomes,Chromosome Structure and Function,Telomeres,