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Estimation of Human Age According to Telomere Shortening in

Peripheral Blood Leukocytes of Tibetan

Article  in  The American journal of forensic medicine and pathology: official publication of the National Association of Medical Examiners · September 2009
DOI: 10.1097/PAF.0b013e318187df8e · Source: PubMed

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 ORIGINAL ARTICLE

Estimation of Human Age According to Telomere Shortening in

Peripheral Blood Leukocytes of Tibetan
Fu Ren, PhD, Changyong Li, PhD, Huanjiu Xi, MSc, Youfeng Wen, PhD, and Keqiang Huang, MSc

provide an estimate of age when the forensic sample carried no

Abstract: In most normal somatic cells, the terminal restriction fragments
morphologic information.
(TRF) length and age are inversely correlated, which can be used to
determine individual age. However, very little is known about the quantita-
tive relationship between human telomeres and age. The aim of the present MATERIALS AND METHODS
study was to investigate age-, gender-, and ethnicity-related changes in Whole blood samples were obtained from 105 unrelated
telomere length in human peripheral blood leukocytes (PBLs). Changes with healthy individuals ranging in age from 0 (newborn) to 81 years old
age in telomere lengths were assessed by Southern blotting. The results of Chinese Tibetans, an ethnic group living in the Tibet Autonomous
shown that telomeres shorten in human PBLs in an age-dependent manner Region (Table 1). Donors were randomly chosen among individuals
(r ⫽ ⫺0.913, P ⬍ 0.01). The formula for age estimation according to whose ancestors had lived in the region for at least 3 generations.
telomere shortening was Y ⫽ ⫺16.539X ⫹ 236.287 (Y: age, year; X: mean Moreover, 28 healthy Han population individuals aged 5 to 80 years
TRF length, kb). We analyzed the mean TRF length in males and females (Table 2) were contrasted against Tibetan population. Individuals in
and found that males had shorter telomeres than females. Moreover, we the sample were selected to have a balanced sex and age distribution.
compared the TRF length of Tibetan and Han population in China and found The research protocol was approved by the Human Subjects Com-
that telomere length did not differ between 2 populations. We conclude that mittee at the Department Anatomy of Liaoning Medical University.
estimation of human age according to telomere shortening in PBLs is a novel All subjects provided a written informed consent.
method especially when there is no morphologic information, furthermore, Genomic DNA was extracted from the peripheral blood using
the gender must be considered when age estimation is carried out based on the phenol/chloroform method. We determined the TRF length as
telomere shortening. telomere length using the Telo TTAGGG telomere length assay
(Roche, Germany). Two microgram of genomic DNA was digested
Key Words: age estimation, telomere, Southern blotting, Tibetan with RsaI and HinfI restriction enzymes. After DNA digestion, the
(Am J Forensic Med Pathol 2009;30: 252–255) DNA fragments were separated by 0.8% agarose gel electrophoresis
and transferred by Southern blotting to a positively charged nylon
membrane (Roche Diagnostics). The blotted DNA fragments were
hybridized to a telomeric probe (digoxigenin 3⬘-end labeled 5⬘-
H uman telomeres, located at the ends of eukaryotic chromo-
somes, are composed of TTAGGG repeats, which can protect
chromosomes from degradation, fusion, and recombination.1 In
关CCCTAA兴3) at 42°C and incubated with a DIG-specific antibody
covalently coupled to alkaline phosphatase. The telomere probe was
visualized by alkaline phosphatase metabolising CDP-Star, a highly
normal human somatic cells, because of inherent limitations in the sensitive chemiluminescence substrate. The mean TRF length was
mechanics of DNA replication, telomeres shorten at each cell determined by comparing signals relative to a molecular weight
division. In the absence of telomerase, when telomere shortening marker on x-ray film. The TRF length was estimated according to
reaches a critical limit, cells cease to divide and reach replicative the manufacturers’ instructions (Telo TTAGGG telomere length
senescence. Thus, telomere length in a given cell may serve as a assay) using the Telomeric 1.2 (Available at: http://bioinformatics.
marker of its replicative history and of the residual capacity for fccc.edu). After completion of all TRF measurements in all samples,
further cell division. It is reported that human telomeres shorten statistical analysis was performed to obtain linear regression equa-
gradually with age,2–5 and this information can be used to determine tions for estimating the age of humans from peripheral blood
individual age. However, very little is known about the quantitative samples by telomere shortening. Gender and ethnicity differences
relationship between human telomere DNA and donor age. As a were assessed using the unpaired Student t test or one-way analysis
result, it is difficult to determine age by existing data.6 In addition, of variance.
there is no research report of systemic population about telomere
shortening. In the present study, we show an inverse correlation
between terminal restriction fragments (TRF) length and age in RESULTS
peripheral blood leukocytes (PBLs) of Tibetan. This correlation may Because there is no reliable method to directly measure
be applied in forensic medicine where telomere shortening would telomere length in human cells, we used mean TRF length to detect
the changes in the length of the terminal TTAGGG arrays.2,7 We
assessed the mean TRF length of PBLs in 105 Tibetan individuals
Manuscript received August 2, 2007; accepted December 29, 2007.
(Figs. 1, 2) and found that telomeres shorten with age. The rate
From the Institute of Anthropology, Department of Anatomy, Liaoning Medical of telomere attrition across the age range was approximately 51
University, Jinzhou, Liaoning Province, People’s Republic of China. bp per year.
Supported by the National Natural Science Foundation of China (No. 30270696), When the sample was divided into age groups (0 – 4 years,
the Natural Science Foundation of Liaoning Province (No. 20052206), and the
Basic Science and Technology Research Program Foundation of Ministry of
5–14 years, 15– 64 years, and ⬎65 years) (Fig. 3), the results
Education (No. 206031). showed differences of TRF among different age groups were highly
Reprints: Fu Ren, PhD, Institute of Anthropology, Department of Anatomy, Liaoning significant (P ⬍ 0.001). These data indicated that changes with age
Medical University, No. 40, Section 3, Songpo Rd, Jinzhou, Liaoning Province, in telomere lengths were significant.
People’s Republic of China 121001. E-mail: [email protected].
Copyright © 2009 by Lippincott Williams & Wilkins
A straight line was obtained by regression analysis between
ISSN: 0195-7910/09/3003-0252 the mean TRF length and age, the correlation coefficient being r ⫽
DOI: 10.1097/PAF.0b013e318187df8e ⫺0.913 (Fig. 4). The detected mean TRF length of each sample can

252 | www.amjforensicmedicine.com Am J Forensic Med Pathol • Volume 30, Number 3, September 2009
Am J Forensic Med Pathol • Volume 30, Number 3, September 2009 Estimation of Human Age in PBLs

TABLE 1. Number and Age Distribution of the Subjects

From Tibetan
Age Group (Yr) Male Female Total
0–4 5 4 9
5–14 7 5 12
15–24 7 8 15
25–34 6 6 12
35–44 5 6 11
45–54 5 5 10
55–64 7 5 12
65–74 5 7 12
75–81 6 6 12
FIGURE 2. The mean TRF length of different age groups in
Total 53 52 105 human PBLs.

TABLE 2. Number and Age Distribution of the Subjects

From Han Population
Age Group (Yr) Male Female Total
5–14 4 2 6
15–64 7 6 13
65–80 5 4 9
Total 16 12 28

FIGURE 3. Telomere shortening with age. The sample was

divided into age groups (0 – 4 years, 5–14 years, 15– 64
years, and ⬎65 years).

FIGURE 1. A representative Southern blot analysis demon-

strating length of TRF in PBLs. Lane 1: 17 years; lane 2: 12
years; lane 3: 0 year; M: Molecular weight marker.

be included in the following formula to roughly calculate the age of FIGURE 4. Regression of telomere length measurements in
the subject: Y ⫽ ⫺16.539X ⫹ 236.287 ⫾ 9.832 (Y: age, year; X: PBLs by Southern blotting against age.
mean TRF length, kb; 9.832: standard error). To evaluate the
accuracy of this method, the actual ages of the subjects were
compared with estimated ages using the mean prediction error (ME). age groups and data were reanalyzed. Estimated ages were within
The result showed that ME was 9.213 years and a standard error of ⫾10 years of actual ages in 56.4% of male subjects, and 62.0% of
estimate was 9.832 years. In addition, estimated ages were within female subjects.
⫾10 years of actual ages in 49.5% of all subjects. The accuracy of To investigate gender-specific changes in telomere lengths in
age estimation was increased when the subjects were divided into 2 human PBLs, we analyzed the mean TRF length in male and female

© 2009 Lippincott Williams & Wilkins www.amjforensicmedicine.com | 253

Ren et al Am J Forensic Med Pathol • Volume 30, Number 3, September 2009

adolescence, morphologic methods based on the radiologic exami-

nation of dental and skeletal development are to be recommended.
However, these methods are often qualitative, and the accuracy of
most morphologic methods is much reduced in adulthood.14,15 In
addition, these methods cannot be practically applied to subjects
who carried no morphologic information such as bloodstain and
parenchyma. To estimate the individual age using techniques of
molecular biology, will be of importance in forensic medicine.16
Michikawa et al, reported that estimation of age at death can be
based on quantitation of the 4977 bp deletion of mitochondrial DNA
in human skeletal muscle.17 However, this method cannot be applied
to subjects of an advanced age and who have heart disease or other
disorders.18 Excitedly, a biochemical method based on aspartic acid
racemization in dentine provides the most accurate estimates of age,
followed by special morphologic dental and skeletal methods.14,19,20
FIGURE 5. Telomere lengths in human PBLs from male and This method is recommended for use particularly in adult age
female. estimation.14 Currently, based on accuracy of estimated age, sim-
plicity of the method, time required, and reproducibility, tooth
dentin is considered one of the best target tissues.21
We took a different approach to estimate age based on
telomere shortening in PBLs. It was a continuation of a previously
published pilot study to examine the possible application of the TRF
length as an indicator of age. Notably, Tibetan nationality is an
ethnic group that lives in such areas of China as Tibet, Qinghai,
Sichuan, Yunnan and Gansu province, on Qingzang plateau. We
detected the mean TRF length of PBLs of Tibetan live in the Tibet
Autonomous Region and compared the difference between Tibetan
and Han populations in this study. We demonstrated that telomeres
shorten in an age-dependent manner in PBLs. Moreover, changes of
TRF length among 4 age groups (0 – 4 years, 5–14 years, 15– 64
years, and ⬎65 years) were highly significant. These findings
suggest that the loss of telomere DNA in hematopoietic cells is a
dynamic process and telomere length can be an index of individual
identification of different age.
It is well known that Tibet Autonomous Region is on Qing-
zang plateau where UV irradiation is very intense. Exposure to UV
and/or to oxidative damage would be expected to introduce exten-
sive damage into telomeres. Therefore, telomere shortening can
FIGURE 6. Telomere lengths in Tibetan and Han populations. occur with effects of activated oxygen in human cells.22 In this
study, we analyzed ethnicity differences by comparing Tibetan and
Han population in China and found telomere length did not differ
PBLs (Fig. 5). In all age groups, age-adjusted telomere length was between 2 populations. This may be due to the fact that our model
longer in females than in males. Moreover, the results showed did not accommodate other unmeasured factors, such as shared
gender differences in 2 age groups (5–14 years old and 55– 64 years diets, living conditions, and so forth. Moreover, a limitation of this
old) were significant (12.64 ⫾ 0.30 vs. 12.20 ⫾ 0.65 kb for females study is the small sample size, especially for Han population.
and males, P ⫽ 0.001; 11.32 ⫾ 0.03 vs. 10.67 ⫾ 0.58 kb for females Unfortunately, ethnicity differences about telomere shortening with
and males, P ⫽ 0.025; respectively). age are not published in the literature. Therefore, large-scale inves-
To determine whether telomere lengths were of ethnicity tigations should be undertaken to explore further ethnicity differ-
differences, we compared the TRF length of Tibetan and Han ences at a wide age range.
population in China (Fig. 6). The results showed telomere length did We reported in this study that telomere length was signifi-
not differ between Tibetan and Han population (P ⬎ 0.05). At 5 to cantly longer in PBLs from females than in those from males,
14 years old, the TRF length were 12.92 ⫾ 0.89 kb and 13.27 ⫾ 0.42 specially, in 2 age groups (5–14 years old and 55– 64 years old).
kb for Tibetan and Han population, respectively. At 15 to 64 years However, telomere length did not differ between male and female
old, the TRF length were 11.61 ⫾ 0.84 kb and 11.81 ⫾ 0.45 kb, newborns. These findings were accordant with published data.23 It is
respectively. After the age of 65, the TRF length were 10.36 ⫾ 0.41 well known that telomerase, consists of 2 main components, telo-
kb and 10.51 ⫾ 0.42 kb, respectively. mere RNA which acts as a template for telomere synthesis, and
telomere reverse transcription which catalyzes the elongation, seems
DISCUSSION to serve as a factor of stabilization telomere length.24 Kyo et al
Age estimation in cadavers, human remains and living indi- reported that telomere reverse transcription is both a direct and
viduals has received considerable attention in forensic medicine, in indirect target of estrogen, implying the existence of hormone-
which it is a widely used method for individual identification. So far, dependent mechanisms of control of telomerase activity.25 There-
most common macroscopic methods are based on dental wear and fore, the gender must be considered when an individual age is
histologic evaluation of skeletal remodeling.8 –13 In childhood and estimated based on telomere shortening in PBLs. We still need

254 | www.amjforensicmedicine.com © 2009 Lippincott Williams & Wilkins

Am J Forensic Med Pathol • Volume 30, Number 3, September 2009 Estimation of Human Age in PBLs

sufficient power to detect significant gender-related differences in 6. Takasaki T, Tsuji A, Ikeda N, et al. Age estimation in dental pulp DNA based
the age-dependent telomere shortening. on human telomere shortening. Int J Legal Med. 2003;117:232–234.
Several previous studies were devoted to assess the accuracy 7. Allsopp RC, Vaziri H, Patterson C, et al. Telomere length predicts replicative
capacity of human fibroblasts. Proc Natl Acad Sci USA. 1992;89:10114 –
of age estimation using the morphologic methods and proposed 10118.
some applicable techniques. However, in the forensic practice, there 8. Aicardi G, Vignolo M, Milani S, et al. Assessment of skeletal maturity of the
is often no morphologic information. Thus, it is difficult to obtain hand-wrist and knee: a comparison among methods. Am J Hum Biol. 2000;
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tor. Unfortunately, in the present study, we found that the ME, was age at death of adult males. J Forensic Sci. 2006;51:213–229.
9.213 years and a standard error of estimate was 9.832 years, which 10. Martrille L, Ubelaker DH, Cattaneo C, et al. Comparison of four skeletal
of the variation was gross. Therefore, our method can only give a methods for the estimation of age at death on White and Black adults.
J Forensic Sci. 2007;52:302–307.
rough estimation of age of a subject in forensic examination.
11. Lamendin H, Baccino E, Humbert JF, et al. A simple technique for age
Possible future developments along the results presented in this estimation in adult corpses: the two criteria dental method. J Forensic Sci.
study relate to big sample size would lead to better age estimation. 1992;37:1373–1379.
It might be worthwhile to collect blood from subjects of known age, 12. Kvaal SI, Kolltveit KM, Thomsen IO, et al. Age estimation of adults from
then have the investigator who is estimating age be blinded to the dental radiographs. Forensic Sci Int. 1995;74:175–185.
age of the decedent, thus mimicking the conditions in forensic 13. Cameriere R, Ferrante L, Belcastro MG, et al. Age estimation by pulp/tooth
pathology. ratio in canines by peri-apical x-rays. J Forensic Sci. 2007;52:166 –170.
Although a practical estimation of age depends on many 14. Ritz-Timme S, Cattaneo C, Collins MJ, et al. Age estimation: the state of the
factors such as environmental and genetic factors, one can obtain the art in relation to the specific demands of forensic practice. Int J Legal Med.
2000;113:129 –136.
effective information related to age using this method in forensic
15. Cameriere R, Brogi G, Ferrante LM, et al. Reliability in age determination by
samples such as blood that carry no morphologic information which Pulp/Tooth ratio in upper canines in skeletal remains. J Forensic Sci.
can be used to estimate the rough age of the subject.26 This method 2006;51:861– 864.
that ourselves and other authors reported will solve the problem of 16. Lahnert P. An improved method for determining telomere length and its use
age estimation according to micro amount of tissue or bloodstain, in assessing age in blood and saliva. Gerontology. 2005;51:352–356.
using various material evidence roundly. Moreover, use of this 17. Michikawa Y, Mazzucchelli F, Bresolin N, et al. Aging-dependent large
method is feasible regardless of age of the subject. However, it accumulation of point mutations in the human mtDNA control region for
requires a large-scale anthropological investigation and statistical replication. Science. 1999;286:774 –779.
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ACKNOWLEDGMENTS racemization of root dentin by internal standard method. Forensic Sci Int.
The authors thank Dr. Rongjian Su for providing technical 2004;141:127–130.
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