American Journal of Forensic Medicine & Pathology:
The Study on Telomere Length for Age Estimation in a Thai Population
Srettabunjong, Supawon MD, LLB, MSc, MTox*; Satitsri, Saravut BSc*; Thongnoppakhun, Wanna PhD†; Tirawanchai, Nednapis PhD‡
From the Departments of *Forensic Medicine, †Research and Development, and ‡Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Manuscript received October 10, 2013; accepted March 7, 2014.
This study was supported in part by the Siriraj Graduate Thesis Scholarship. The sponsor had no involvement in the development of the article or decisions related to the article.
The authors report no conflicts of interest.
Reprints: Supawon Srettabunjong, MD, LLB, MSc, MTox, Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. E-mail: firstname.lastname@example.org.
Age is one of the key parameters in establishing a physical characteristic profile of an individual. For biological evidence left in crime scenes such as blood, saliva, hair, etc, the evidence owner’s age can be determined only by DNA extracted from these materials. Previous researches have found that there are certain DNA regions with specialized characteristic and function called telomere being able to predict age. The present study was to determine the correlation between telomere length and age as well as the effect of sex on the correlation and to create linear regression equation for age estimation in Thai population for forensic purposes. Blood samples obtained from unrelated healthy Thai fresh cadavers without anatomical organ abnormalities were used in this study. All cadaver subjects underwent the postmortem examination in jurisdiction of the Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, and Institute of Forensic Medicine, Police General Hospital. Fifty blood samples from both sexes of all ages divided into 6 groups for equal age distribution (0–11, 12–23, 24–35, 36–47, 48–59, and 60 years old and older) were collected for a total of 100 samples. The extracted genomic DNA samples were then subjected to telomere length estimation by terminal restriction fragment (TRF) assay. The results showed that the mean TRF length was inversely correlated with age (r = −0.625), and sex did not have a statistically significant influence on the association between age and mean TRF length (P > 0.05). The obtained linear regression equation was y = 113.538 ± 9.604 − 0.012 × (R2 = 0.391; P < 0.001). However, the correlation was too low to be used for age estimation with high certainty and a possible reason for this in part would be the postmortem DNA degradation at some level, even using fresh cadaver blood, and other biological factors such as ethnicity and DNA sources. Roughly, those individuals who had a mean TRF length longer than 6.3 kilobase (kb), between 5.5 and 6.3 kb, and shorter than 5.5 kb aged younger than 28 years, 30 to 44 years, and older than 46 years, respectively (P < 0.01). As a preliminary study, this study highlighted that telomere length could act as a useful biomarker of aging in human population and might be used for rough age estimation in a Thai population. However, further studies with a larger sample size and/or in living human bloods as well as other cell types are recommended to support the results of this study.
Age is one of the key parameters in establishing a physical characteristic profile of an individual. For trace biological evidence left in crime scenes, particularly violent crimes, such as blood, saliva, hair, etc, the evidence owner’s age can be determined only by DNA extracted from these materials. Previous researches have shown that there are certain DNA regions with specialized characteristic and function called telomere being associated with age and thus having potential to predict age.1–6 Therefore, there has been an attempt to validate such notion and apply this information to forensic cases.
Telomeres are short and highly conserved hexanucleotide repeats located at the end of each eukaryotic chromosome. Telomeres play an important role in cell division in the last stage of replication process of S phase, preserving chromosome stability and integrity. In human chromosomes, telomeres are sets of tandem repeats of sequence such as (TTAGGG)n, 10 to 15 kilobase (kb) long.7 Telomere repeats are generated next to subtelomeric region by telomerase, of which enzyme content is telomerase reverse transcriptase (Tert). The enzyme can recognize 3′-OH at the end of chromosomes and adds nucleic acid into telomere repeats de novo by using telomerase RNA component (Terc), which has RNA sequences used for templates for making new telomere repeats. Telomere repeats then template for RNA primer to bind and synthesize complementary DNA strand from 5′ to 3′ in the complementary strand. Telomere end, therefore, has a 3′ overhang of G-rich strand generated from postreplicative process of the C-rich.5 The 3′ overhang of G-rich can form telomere loop, which folds back and binds with double-strand DNA to prevent DNA repair and protect telomere and chromosome. Telomeres can bind to protein complexes called shelterin, which function as chromosome protection and telomere length regulation.5 Unlike unicellular organisms, human cells have a limited amount of telomerase and the inability of DNA polymerase to copy at the very end of a chromosome results in telomere shortening, thus called end-replication problem.8 Telomere shortening increases directly to increasing age in most human somatic cells. It has been estimated that 50 to 150 base pairs (bp) of telomeric DNA are lost with each proliferation cycle during mitosis.6,9 In the absence of telomere, when telomere shortening reaches a critical limit, the cells cease to divide and reach replicative senescence.
Given the previously mentioned information, there would be some correlations between telomere lengths and individual age, leading to formulation of a linear regression equation for predicting age from unknown human biological evidences to narrow suspects and to identify victims either of the crime or of the disaster for forensic purposes. However, several studies have shown that there are some factors affecting shortened telomere such as oxidative stress,10 smoking,11–13 and obesity.12 Telomere lengths are also significantly shorter in certain cancers such as bladder cancer,11 lung cancer,14 colorectal carcinoma,15 and in some diseases such as ulcerative colitis,16 liver cirrhosis,17 chronic heart failure,18 atherosclerosis,19 myocardial infarction,20 vascular dementia,21 and diabetes mellitus.22 There have been some studies observing a significant correlation between telomere length and age in humans and certain kinds of animals, with a variety of methods used to quantify telomere length.2–6,23–27
Because aging is a complex procedure and involves multiple factors and mechanisms at different levels and stages in life, ethnicity also has a tremendous influence on aging. Previous researches have shown that shorter telomere length is associated with older age,2–6,28–30 male sex,28–31 and white race.28,30 Furthermore, the strength of the association with age is highly dependent on the age range of the population.2–6,28–31 Therefore, this study aimed to determine the possible correlation between leukocyte telomere lengths and age using terminal restriction fragment (TRF) assay in fresh healthy Thai cadavers as well as the effect of sex on this association and to create a linear regression equation to predict individual age in a Thai population that could be able to apply it into real forensic cases.
MATERIALS AND METHODS
This study was a cross-sectional study using blood samples obtained from 100 unrelated Thai fresh cadavers with no anatomical organ abnormalities as to the relevant diseases mentioned previously and no previous history of bone marrow transplantation. All cadaver subjects underwent the postmortem examination under the jurisdiction of the Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, and Institute of Forensic Medicine, Police General Hospital within 18 hours after death. The subjects were divided into 2 groups by sex. Each group consisted of 50 cadavers of all age divided into 6 groups for equal age distribution as follows: 0–11, 12–23, 24–35, 36–47, 48–59, and 60 years old and older. All blood samples were preserved in EDTA tube and stored at 4°C shortly after being drawn from the femoral vein of cadavers.
Extraction of Genomic DNA
Extraction of high molecular weight genomic DNA of peripheral blood leukocytes was performed using High Pure PCR Template Preparation Kit (Roche Diagnostics Inc, Basel, Switzerland) according to the manufacturer’s instructions within 24 hours of blood drawing. The quality and quantity of the obtained genomic DNA were measured spectrophotometrically using NanoPhotometer (Implen, München, Germany). All the DNA samples were immediately stored at −20°C until use for TRF assay.
Terminal Restriction Fragment Assay
Terminal restriction fragment assay was performed using the Southern blot technique with the TeloTAGGG Telomere Length Assay kit (Roche Diagnostics Inc, Mannheim, Germany) according to the manufacturer’s instructions with minor modifications. Briefly, genomic DNA was subjected to enzyme restriction, HinfI and RsaI mixture for 2 hours at 37°C. Telomere restriction fragments were then separated in 0.8% agarose gel (SeaKem LE Agarose; Cambrex Bio Science Rockland Inc, East Rutherford, NJ) with 1× tris-acetate EDTA buffer by electrophoretic separation. The DNA fragments were then denatured, neutralized, and transferred to a positively charged nylon membrane (Roche Diagnostics Inc, Mannheim, Germany) by Southern blotting using VacuGene base (LKB Bromma 2016 Vacugene, Pharmacia, Stockholm, Sweden) at 5 kPa with 20× saline sodium citrate buffer for 3 hours at room temperature. The DNA fragments were then cross-linked to the membrane by UV light using a UV transilluminator (FOTO/UV 26; Fotodyne Inc, Wis), washed with 2× saline sodium citrate buffer, and air dried. The blotted DNA fragments were incubated with DIG Easy Hyp at 42°C for 1 hour, hybridized with telomere-specific digoxigenin–labeled hybridization probe at 42°C for 3 hours, incubated with anti–telomere-specific digoxigenin–alkaline phosphatase at room temperature for 30 minutes, and visualized by CDP-Star chemiluminescent substrate. Finally, the chemiluminescent signals from the membrane were then exposed to x-ray film (Fuji Film Corporation, Tokyo, Japan).
Determination of Telomere Length
The exposed x-ray films were captured using a high-definition scanner and uploaded into analytical software Gene Tools (Syngene Inc, Md) for analysis of the signals. Telomere length is given as the average TRF length and the signal intensity is determined by comparing the telomere smear of each sample to a molecular weight standard. The mean TRF length of each sample was calculated by integrating the signal intensity of the TRF bands on the exposed x-ray film as a function of its mean molecular weight. An example of an autoradiograph obtained from a set of samples used in the study was shown in Figure 1, the telomere smear being visible in the top half of each lane.
All the statistical analyses were performed using SPSS software program version 17.0 (IBM Corporation, Chicago, Ill). Continuous variables were shown as means with SDs. The Pearson correlation was used for analyzing a simple linear regression equation and correlation coefficient. The unpaired Student t test or one-way analysis of variance was used to assess the difference between age groups and sex. P values of less than 0.05 (2 sided) were considered statistically significant.
The Effect of Age on Telomere Length
Because there is no reliable method to measure absolute telomere length directly in human cells, this study used the mean TRF lengths as the mean telomere lengths of each individual subject. The mean age of subjects in this study was 38.58 ± 21.65 years with the range of 30 months and 95 years (median, 38 years). The mean telomere length was 6007.73 ± 1084.96 bp with the range of 4184.74 and 9917.57 bp (median, 5741.65 bp). This study obtained the negative correlation between the mean TRF length and the age of the subjects by calculating regression analysis (r = −0.625; P < 0.001) as shown in Figure 2. The regression constant was accurate to within 95% confidence limits.
The Linear Regression Equation for Age Estimation
As shown in Figure 2 and Table 1, the derived formula for age estimation was x = −0.012 y + 113.538 ± 9.604 (R2 = 0.391; P < 0.001), where x represents the age in years, y represents the mean TRF length or telomere length in kilobase, and 9.604 represents the SE.
The relationship between telomere length and age could be divided into 3 groups according to telomere length, that is, greater than 6.3 kb, 5.5 to 6.3 kb, and less than 5.5 kb. Individuals having a mean TRF length of more than 6.3 kb aged younger than 28 years, between 5.5 and 6.3 kb aged 28 to 44 years, and lesser than 5.5 kb aged older than 44 years. These results documented that the decline in mean TRF length differed with age group with statistical significance (P < 0.01) as shown in Table 2.
The Effect of Sex on the Association of Telomere Length and Age
Regression analysis was performed to study the association of telomere length and age in men (n = 50) and women (n = 50) separately as shown in Figure 3 and Table 3. The straight solid and dotted lines showed the inversely relationship between telomere length and age in men and women (r = −0.645 [R2 = 0.416; P < 0.0001] and r = −0.617 [R2 = 0.381; P < 0.0001], respectively). However, no significant difference between the age-telomere length relationship and sex was obtained (P = 0.132).
Telomere shortening in an age-dependent manner and in some age-related diseases observed in certain cells has been the research of interest for past decades.2–6,10–31 Because age is one of the characteristics that determines individual identification, age estimation in biological evidence, especially with no morphologic information such as trace evidence, is very important and has received considerable attention in forensic medicine. Therefore, estimating the trace evidence owner’s age using techniques of molecular biology will be of importance in forensic cases.
Hewakapuge et al24 investigated age-related changes in telomere length from buccal cells in white population with various ethnic origins using quantitative polymerase chain reaction (Q-PCR) assay. They delineated the low correlation between telomere lengths and age because of a large interindividual variation in telomere length and ethnically mixed samples and thus were not able to develop a formula for predicting age from telomere length. The Q-PCR assay is a high-throughput method and requires a very small DNA sample of variable quality. However, the method cannot measure telomere length directly but relatively by determining telomere length in T/S ratio (telomere repeat copy number/known single copy gene copy number) in experimental samples relative to a reference sample.24,25 Also, its overall coefficient of variance is quite large (often quoted as 5%–10%), thus easily obscuring the observed differences in mean telomere length between samples and strengthening the differences between individuals of the same age.4 Furthermore, the T/S ratio was not highly correlated with TRF length and the stability of the reference gene is not guaranteed. With these reasons, Q-PCR has been questioned as an accurate technique for measuring telomere length for forensic applications.24,25
Baird et al26 introduced another method for measuring telomere length called single-telomere length analysis (STELA) in 2003. The STELA method can accurately measure the full spectrum of the telomere length of specific chromosomes, especially those with shorter telomeres, but there have not been enough primers to reveal all chromosomes. In addition, because of its low throughput, inconsistent amplification among the samples, and inconsistent correlation with the resulting amplified telomere lengths, STELA is more amenable to basic studies of telomere dynamics and has not been used in forensic studies.4,23
Baerlocher and Lansdorp27 used flow cytometry and quantitative fluorescent in situ hybridization (Q-FISH) to measure the average telomere length in 2004. The method is automated and sensitive for various cells present in the human blood. However, the fluorescence is more variable and the results are not very accurate or reproducible when analyzing small amounts of sample. In addition, calibration of the controls to fit the fluorescence signal on a linear scale is constantly needed, making it more time-consuming and technically demanding.23
The TRF assay is another method used for measuring telomere length and is suitable for species with limited genome resources available to estimate telomere length from individuals. This method needs a considerable amount of good quality DNA and has its strength for its reproducibility with coefficient of variance of less than 2% and expression of values in absolute base pair units.4 However, it measures both the telomeric and subtelomeric regions, thus possibly resulting in artificial inflation of the mean telomere length. Previous studies relevant to predicting age from telomere lengths in forensic perspective using TRF assay have shown a linear regression equation correlated between age and telomere shortening.2,3,6 However, they observed a wide variability of TRF length in the individuals of the same age. Tsuji et al2 studied such a correlation in Japanese population and attributed variability among the data sets to environmental and genetic factors (such as diseases). Ren et al6 also studied the effect of age on telomere length in two ethnic populations in China, Tibetan and Han. They found that telomere lengths did not differ between the two populations, possibly because of a small sample size with narrow age range, and that the differences in the data were likely due to sex, that is, men having telomere lengths shorter than women, significant in 2 age groups (5–14 years old and 55–64 years old).
Consistent with previous studies,2,3,6 this study found significant progressive reduction of TRF length with increased age in healthy peripheral blood and the regression coefficient was −0.625, indicating that older cadavers have less telomeric DNA. A summary of these observations including the present study is shown in Table 4. The findings suggest that the loss of telomeric DNA in hematopoietic cells is a dynamic process and telomere length could be an index of individual age. Although the relationship of telomere length with age was significant, the effect was not as strong as that of previous reports.2,3,6 The observed differences in the effects of the correlation could be mainly due to differences in ethnicity, DNA source, and laboratory technique used in the studies. Also, a possible reason for this in part would be the postmortem DNA degradation at some level, even using fresh cadaver blood. However, a large variation among people of the same age group was also observed in these studies including the present study (Fig. 2), which could be due to multiple factors involving individual genetics and the environment affecting the telomere dynamics,2,3,6,31,32 suggesting that telomere length might have a limited application as an age estimation for personal identification. Changes of TRF length among 3 age groups (<28, 28–44, and >44 years) were highly significant (P < 0.01), indicating that this result would serve as a rough age estimation in Thai population for forensic purposes.
This study did not find an effect of sex on the age-telomere length relationship as shown in Figure 3, consistent with previous reports.6,33 However, Ren et al6 observed the significant sex-related differences in certain age groups. In addition, some studies29,34,35 demonstrated that mean telomere length seemed to be shorter in male sex than in female sex, possibly because of the telomere length at birth, the faster rate of telomere attrition in male sex, and the effect of estrogen on telomerase36 and oxidative stress.37 Also, telomerase acts differently on chromosomes with varying telomere lengths. Although telomere length in humans is heritable, positively linked to paternal age, and highly tissue specific, telomere shortening can be influenced by unknown risk factors with unclear sensitive periods and can affect aging differently over the life of an individual.33 In this study, stronger correlation between telomere length and age was also observed in men (R2 = 0.416) than in women (R2 = 0.381), but no statistical significance was obtained.
In conclusion, this would be the first study to observe the age-dependent telomere length in healthy cadaver blood confirmed by autopsy findings. As a pilot study, it highlighted a strong correlation between age and leukocyte telomere length, indicating the significance of telomere length as a biomarker of aging and rough age estimation. However, there are some limitations for a general biomarker because the variability of the telomere length between individuals, especially in the same age group, is high. To reduce the overall variability in the data, a study exploring the correlation between single specific chromosome telomere length and age may be needed to better determine the exact strength of the correlation to better serve as a powerful tool to predict individual age in trace biological evidence. The result of this study may require corroboration because of its small sample size. Further researches with the same strict criterion population, greater sample sizes, and wider age ranges may tighten the relationship between telomere length and age in Thai population before applying it to forensic practice. In addition, the correlation obtained was too low to be used for age estimation with high certainty and a possible reason for this in part could be the postmortem DNA degradation at some level, even using fresh cadaver blood. Further studies in living human bloods or other tissues such as hair roots are recommended to support the results of this study. Although TRF assay allowed acceptable estimates of telomere lengths to be made, more precise estimates of telomere length could have been obtained to measure the exact telomere length for accurate and precise telomere measurement.
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age estimation; blood; telomere length; terminal restriction fragment assay
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