Legal Medicine 16 (2014) 135–138
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Age estimation via quantification of signal-joint T cell receptor excision circles in Koreans Sohee Cho a, Jianye Ge b, Seung Bum Seo b, Kiha Kim a, Hye Young Lee a, Soong Deok Lee a,c,⇑ a
Department of Forensic Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea Institute of Applied Genetics, Department of Forensics and Investigative Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, United States c Institute of Forensic Medicine, Seoul National University College of Medicine, 28, Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea b
a r t i c l e
i n f o
Article history: Received 20 August 2013 Received in revised form 22 January 2014 Accepted 22 January 2014 Available online 31 January 2014 Keywords: Age estimation sjTREC Korean Blood
a b s t r a c t The estimation of age from biological samples (i.e., remains) at crime scenes could provide useful information about both victims and other persons related to criminal activities. Signal-joint T cell receptor excision circle (sjTREC) levels in peripheral blood decline with age, and negative correlations between sjTREC levels and age have been demonstrated in several ethnic groups. To validate the utility of sjTREC for age estimation in Koreans, Taqman qPCR was used to quantify the sjTREC level in samples obtained from 172 individuals ranging from 16 to 65 years old. We modified the previously reported method by using a shorter amplicon and confirmed the efficiency and utility of this method in this report. Our results showed that the linear negative regression curve between sjTREC levels and age was characterized by r = 0.807 and a standard error of 8.49 years. These results indicate that sjTREC level is an effective age estimation method in Koreans. The value of the standard error of quantification was not different from previous reports for other population groups. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In forensic practice, DNA typing is mainly used for individual identification and paternity testing. In recent years, many efforts have been made to obtain other useful information from DNA samples, including ethnicity, individual phenotypic traits [1,2], and age [3,4]. Age estimation could aid investigators in the absence of known DNA samples for comparison. To date, the age-dependent accumulation of D-aspartic acid, the accumulation of a 4977-bp deletion in mitochondrial DNA, and telomere shortening have all been analyzed as potential age markers [5,6]. However, the use of these techniques in real cases has been limited for several reasons, including the role of environmental effects and disease effects, the low methodological accuracy, and tissue- and genderspecific variations [7,8]. For example, striking heterogeneity of mitochondrial deletions in different tissues and different telomere dynamics in each compartment of the body has been found [7,9]. Therefore, more stable, standardized and accurate methods for age estimation are needed. ⇑ Corresponding author at: Department of Forensic Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea. Tel.: +82 2 740 8359; fax: +82 2 764 8340. E-mail address: [email protected] (S.D. Lee). http://dx.doi.org/10.1016/j.legalmed.2014.01.009 1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
One of the consequences of aging is the deterioration of the immune system including the thymus. Important functions of the thymus include producing T cells, expressing T cell receptors (TCRs), and facilitating TCR gene rearrangement. During the rearrangement of TCR gene segments, some unused regions are spliced out as ringshaped DNA that may be detected in naïve T cells. These episomal DNA include signal-joint T cell receptor excision circles (sjTRECs), which are a by-product of an intermediate rearrangement in the TCRa/d locus in developing TCR ab+ lymphocytes. sjTRECs are stable, do not replicate during cellular proliferation in the periphery, and are diluted by each round of cell division. The quantification of recent thymic emigrants (RTEs) has been utilized to clinically estimate thymic function in the context of immunological diseases and genetic disorders [10]. RTEs, which form the youngest subset of naïve T cells, represent the ability of the thymus to produce T cells and are usually quantified by measurement of sjTRECs [11]. The content of sjTRECs has been reported to be lower in older people [12,13]. This may be related to thymic involution (i.e., the shrinking of the thymus), which begins shortly after birth and increases with age [14]. Therefore, the sjTREC content per total T cell content or with respect to the level of a constant gene is expected to decrease with age. In recent years, several groups have reported that sjTREC levels in peripheral blood decline with age, indicating the potential
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S. Cho et al. / Legal Medicine 16 (2014) 135–138
applicability of this method for age estimation [15,16], including through the use of bloodstain analysis [17]. Many biological or socio-economic factors, including ethnic differences, can be considered in light of this. Herein, we report data for the relationship between sjTREC level and age in Koreans ascertained with newly designed PCR assay with shorter amplicon length.
of serially diluted plasmids encoding sjTREC. For positive control, 0.1 pg of plasmid [corresponding to 22,727 copies, calculated as: copy = (ng copy/mole)/(bp ng/g g/mole of bp)] was run in parallel. The Ct value was used to assess between-experiment consistency. All samples were run in duplicate and the average from these results was used for statistical analysis.
2. Materials and methods
2.5. Statistical analysis
2.1. Human blood samples and DNA extraction
Statistical analysis was performed using IBM SPSS Statistics 20 and Microsoft Excel, and plots were generated from analysis using SPSS. The normality of the data was examined using the Kolmogorov–Smirnov test, and correlation was assessed using Pearson’s correlation coefficient. The significance of mean difference among age groups was performed using One-way ANOVA test.
Samples were collected from 172 Korean volunteers aged 16–65 years old. Informed consent was obtained from individuals, and the study was approved by the ethical committee of the Institutional Review Board of the Seoul National University Hospital Biomedical Research Institute. Peripheral blood samples were collected into sodium heparin green top vacutainers (BD Bioscience, San Jose, CA, USA). Genomic DNA was immediately extracted from 300 lL of whole blood using a MaxwellÒ 16 Blood DNA Purification Kit (Promega, Madison, WI, USA) and a MaxwellÒ 16 instrument (Promega), and stored at 20 °C until use. The obtained DNA was quantified using the QuantifilerÒ Human DNA Quantification Kit (Life Technologies, CA, USA). 2.2. sjTREC primers and probes The forward and reverse sjTREC (accession No. NT_026437) PCR primers were designed to allow amplification of the target sequence only when the TCRd gene was deleted from chromosomal DNA and the sjTREC fragment was formed. The modified primer (S) sequences were as follows, 50 -TGCTGACACCTCTGGTTTTTGTAA-30 (F; forward) and 50 -GTGCCAGCTGCAGGGTTTAG-30 (R; reverse) [18]. The sequence for the Taqman probe was 50 -FAM-CACGGTGA TGCATAGGCACCTGC-TAMRA-30 . The previously reported primers (L), 50 -CCATGCTGACACCTCTGGTT-30 (F; forward) and 50 -TCGTGAG AACGGTGAATGAAG-30 (R; reverse) [14] which were slightly longer than our primers, were also tried. 2.3. PCR amplification Plasmid containing sjTREC (accession No. NT_026437) sequences was constructed in pCR2.1-TOPO plasmid (Life Technologies) and used as a positive control. Extracted genomic DNA and plasmids were amplified in 20-lL reaction mixtures containing 1 Gold buffer (Life Technologies), 2 mM MgCl2, 0.25 mM dNTP, 800 nM of each primer and 1.6 unit of Gold Taq polymerase (Life Technologies). The cycling conditions were 94 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and a final soak at 72 °C for 7 min. The amplified products were separated by 2% agarose gel electrophoresis. 2.4. Real-time PCR using TaqMan probes The sjTREC content of each sample was quantified using an ABI Prism 7000 machine (Life Technologies) and the results were analyzed with SDS v2.1 software from the same manufacturer. Real-time PCR was performed in 20-lL reaction mixtures containing 50 ng of genomic DNA, 800 nM of each primer, 250 nM of Taqman probe and 2 TaqManÒ Universal PCR Master Mix II (Life Technologies). The thermal cycling conditions were 50 °C for 2 min and 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. To determine the optimal amount of DNA for detection, dilutions of 10 DNA samples representing men and women ranging from 23 to 63 years of age at intervals of 10 years were tested. A standard curve was generated by PCR amplification
3. Results 3.1. Modification of the sjTREC quantification method and its efficiency When using the previously reported primers, the Ct value was uncheckable in some old aged samples over 60 years of age. Furthermore the slight non-specific amplification, which seems to be a result of primer–dimer formation, was found (data not shown). In light of this, we designed new primer set (S) that targeted a shorter amplicon length (Fig. 1, adapted from [11]). This primer set yielded 93 bp amplicon (Fig. 2a), and exact sequences and size were verified by sequencing analysis (data not shown). The PCR efficiency using sjTREC(S) primer was obtained with diluted sjTREC plasmid ranging from 107 to 101 copies in Taqman qPCR reactions, and it showed that PCR efficiencies of both old and new assays are high enough. (Fig. 2b and c). 3.2. Distribution of sjTREC levels in a Korean population Ct values for all the samples including aged sample over 60 years of age were obtained, and the Ct value tended to increase with age as it has been known. A negative correlation between sjTREC levels and age (r = 0.807) and a regression line with a fit of R2 = 0.648 were obtained (Fig. 3a). Standard error of the estimate was 8.49 years and a total decline of approximately 1.5 Log was confirmed. 3.3. The variation of sjTREC levels in groups The tested samples were divided into 5-years groups (Fig. 3b and Table 1) to compare variations in small age ranges. A distinct drop was not observed between the adjacent groups, but only the levels declined slightly between the 15–19 and 30–34 years old groups, declined more sharply at 35–45 years, and showed increased variation thereafter. The range of variation was slightly wider in groups of older. 4. Discussion Several investigators have reported differences in sjTREC contents by age among variously aged individuals, with forensic application in mind [15,16]. In these reports, negative correlation values between sjTREC contents and age in the tested samples showed a slight difference (R2 = 0.835 in [15] and R2 = 0.668 in [16], respectively). Some authors have also reported on the difference in sjTREC contents between healthy donors and patients [10,19]. Since sjTREC level is dependent on several factors including genetic and environmental variations, we considered here a difference between ethnic populations, with the Korean population
S. Cho et al. / Legal Medicine 16 (2014) 135–138
137
Fig. 1. Schematic diagram of the Taqman qPCR assay strategy. The destiny of a T-cell lineage as either ab+ or cd+ is determined by which TCR chain set is expressed via rearrangement of TCR gene segments during T-cell development. For production of ab+ T cells, which comprise 70–75% of the total T-cell pool, d gene segments are excised from a region of the chromosome where a gene segments coexist. When d segments remain as part of the chromosome, the utilized primers do not amplify the target sequence. However, the formation of sjTREC (i.e., deletion of the d segments) allows amplification. The target includes the recombination region of recombination signal sequences (RSS) flanking the dRec and wJa genes.
Fig. 2. Specificity and efficiency of primers. Amplified PCR products using sjTREC(L) and sjTREC(S) primer with standard plasmid on agarose gel (a). Standard curves generated by quantitative real-time PCR using sjTREC(L) (b) and sjTREC(S) (c) primer. Amplification was performed with 10-fold serial diluted sjTREC-containing plasmids ranging from 107 to 101 copies. X-axis and Y-axis indicate log10[copy number of plasmid] and its Ct value, respectively, and R2 and the slope value presents the correlation between the logged number of plasmid and measured Ct value.
as our focus. In this study, we did not compare the difference between populations directly, but it was expected that our assumed hypothesis would not be significant. Direct comparison needs to be performed in future studies in this area. Some slight differences found in this work may have been affected by the method of quantifying sjTREC used, the analytic method applied to our data or variation in sample size [16]. The method of normalization is worth elaborating upon in this kind of quantification work. For example, the measured sjTREC level can be normalized with a single-copy gene genomic DNA [15,16,18] or the equivalent microgram of DNA is applied to measurement [20]. However, there seems no golden rule for setting such standards. In our reports, the number of TCRa chain constant region gene, which is reflected in a ab+ T-cell proliferation-dependent manner, were first considered for normalization. However, a slight negative correlation between age and the Ct
value of the TCRa gene (R2 = 0.274; data not shown) was observed. It has been suggested that factors such as the T-cell proliferation rate or T-cell death rate could influence the sjTREC level [21], which could potentially offset additional age-related differences in sjTREC levels. Therefore it was thought that using the measurement of sjTREC molecules per lg of genomic DNA was more suitable for this purpose. Furthermore, DNA quantity is usually considered to be a standard in forensics, and it is thought that applying another target together is less convenient for real application. The error of the estimate was 8.49 years in this report. This is not significantly different from the 8.9 years in Zubakov et al. [15]. Even with this age range, this method seems to be the most accurate age estimation method available when considering former methods such as measuring telomere length or accumulated mtDNA deletions. A dramatic drop of sjTREC contents in puberty
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The concentration of samples was set to 50 ng as in previous reports [15], and the optimal amount of sample needed for this test was not examined precisely in this study. We found that we could obtain appropriate results using a lower amount, less than 12.5 ng in younger people (data not shown). For older groups higher amount of DNA is required. This kind of sample concentration dependent difference for different age group represents another source of information for age estimation methodology. Alongside efforts to improve the overall efficiency of the test, more research in greater or a variety of samples related to immune system is expect to expand the application in forensics. Acknowledgment This work was supported by Grant No. 1315000438 from the National Forensic Service (NFS) in Korea. References
Fig. 3. The distribution of sjTREC levels in our sample of 172 Koreans. The levels of sjTREC in the investigated Korean individuals aged 16–65 years were plotted and the linear regression curve was found (a). Samples were divided into 5 years groups (b), and the result was illustrated as box plots. The top and bottom of each box denote the 75th and 25th percentiles, respectively, with 95% confidence intervals (whiskers) extending from each box. The bar in the box denotes the median, and outliers are plotted as closed circles ().
Table 1 The levels of Log [sjTREC per lg DNA] among groups divided into 5-years groups. Group (age)
n
Mean
SD
SE
Min
Max
15–19 20–24 25–29 30–34 35–39 40–44 45–49 50–54 55–59 60–65
36 21 14 18 19 13 13 16 14 8
4.448 4.352 4.210 4.181 3.852 3.836 3.563 3.487 3.459 3.023
0.221 0.222 0.251 0.264 0.396 0.345 0.335 0.413 0.349 0.199
0.037 0.048 0.067 0.062 0.091 0.096 0.093 0.103 0.093 0.070
3.997 3.909 3.779 3.616 3.199 2.955 2.945 2.923 2.864 2.750
5.008 4.927 4.617 4.536 4.539 4.257 4.022 4.261 4.012 3.337
[19] was not observed, but a small change of the level between 15–35 years of age and a decrease with wider variation thereafter among divided groups [22] were confirmed similarly in our age range, 15–65 years old (Fig. 3b). If we could collect more information on which factors having an effect on immunological age estimation methods, the usability of this method in forensic case work samples could be increased.
[1] Kayser M, de Knijff P. Improving human forensics through advances in genetics, genomics and molecular biology. Nat Rev Genet 2011;12(3):179–92. [2] Kayser M, Schneider PM. DNA-based prediction of human externally visible characteristics in forensics: motivations, scientific challenges, and ethical considerations. Forensic Sci Int Genet 2009;3(3):154–61. [3] Ritz-Timme S, Cattaneo C, Collins MJ, Waite ER, Schütz HW, Kaatsch HJ, et al. Age estimation: the state of the art in relation to the specific demands of forensic practise. Int J Legal Med 2000;113(3):129–36. [4] Meissner C, Ritz-Timme S. Molecular pathology and age estimation. Forensic Sci Int 2010;203(1–3):34–43. [5] Hewakapuge S, van Oorschot RA, Lewandowski P, Baindur-Hudson S. Investigation of telomere lengths measurement by quantitative real-time PCR to predict age. Leg Med (Tokyo) 2008;10(5):236–42. [6] von Wurmb-Schwark N, Higuchi R, Fenech AP, Elfstroem C, Meissner C, Oehmichen M, et al. Quantification of human mitochondrial DNA in a real time PCR. Forensic Sci Int 2002;126(1):34–9. [7] Butler MG, Tilburt J, DeVries A, Muralidhar B, Aue G, Hedges L, et al. Comparison of chromosome telomere integrity in multiple tissues from subjects at different ages. Cancer Genet Cytogenet 1998;105(2):138–44. [8] Ritz-Timme S, Collins MJ. Racemization of aspartic acid in human proteins. Ageing Res Rev 2002;1(1):43–59. [9] Friedrich U, Griese E, Schwab M, Fritz P, Thon K, Klotz U. Telomere length in different tissues of elderly patients. Mech Ageing Dev 2000;119(3):89–99. [10] Geenen V, Poulin JF, Dion ML, Martens H, Castermans E, Hansenne I, Moutschen M, Sékaly RP, Cheynier R. Quantification of T cell receptor rearrangement excision circles to estimate thymic function: an important new tool for endocrine-immune physiology. J Endocrinol 2003;176(3):305–11. [11] Hazenberg MD, Verschuren MC, Hamann D, Miedema F, van Dongen JJ. T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation. J Mol Med (Berl) 2001;79(11):631–40. [12] Zhang SL, Bhandoola A. Losing TREC with age. Immunity 2012;36(2):163–5. [13] Naylor K, Li G, Vallejo AN, Lee WW, Koetz K, Bryl E, et al. The influence of age on T cell generation and TCR diversity. J Immunol 2005;174(11):7446–52. [14] Aspinall R, Andrew D. Thymic involution in aging. J Clin Immunol 2000;20(4):250–6. [15] Zubakov D, Liu F, van Zelm MC, Vermeulen J, Oostra BA, van Duijn CM, et al. Estimating human age from T-cell DNA rearrangements. Curr Biol 2010;20(22):R970–1. [16] Ou X, Zhao H, Sun H, Yang Z, Xie B, Shi Y, et al. Detection and quantification of the age-related sjTREC decline in human peripheral blood. Int J Legal Med 2011;125(4):603–8. [17] Lang PO, Govind S, Dramé M, Aspinall R. Measuring the TREC ratio in dried blood spot samples: intra-and inter-filter paper cards reproducibility. J Immunol Methods 2013;389(1–2):1–8. [18] Gerstel-Thompson JL, Wilkey JF, Baptiste JC, Navas JS, Pai SY, Pass KA, et al. Highthroughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunode ficiency in population-based newborn screening. Clin Chem 2010;56(9): 1466–74. [19] Zhang L, Lewin SR, Markowitz M, Lin HH, Skulsky E, Karanicolas R, et al. Measuring recent thymic emigrants in blood of normal and HIV-1-infected individuals before and after effective therapy. J Exp Med 1999;190(5):725–32. [20] Douek DC, McFarland RD, Keiser PH, Gage EA, Massey JM, Haynes BF, et al. Changes in thymic function with age and during the treatment of HIV infection. Nature 1998;396(6712):690–5. [21] Ye P, Kirschner DE. Reevaluation of T cell receptor excision circles as a measure of human recent thymic emigrants. J Immunol 2002;168(10):4968–79. [22] Lorenzi AR, Patterson AM, Pratt A, Jefferson M, Chapman CE, Ponchel F, et al. Determination of thymic function directly from peripheral blood: a validated modification to an established method. J Immunol Methods 2008;339(2): 185–94.
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Age estimation via quantification of signal-joint T cell receptor excision circles in Koreans Sohee Cho a, Jianye Ge b, Seung Bum Seo b, Kiha Kim a, Hye Young Lee a, Soong Deok Lee a,c,⇑ a
Department of Forensic Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea Institute of Applied Genetics, Department of Forensics and Investigative Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, United States c Institute of Forensic Medicine, Seoul National University College of Medicine, 28, Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea b
a r t i c l e
i n f o
Article history: Received 20 August 2013 Received in revised form 22 January 2014 Accepted 22 January 2014 Available online 31 January 2014 Keywords: Age estimation sjTREC Korean Blood
a b s t r a c t The estimation of age from biological samples (i.e., remains) at crime scenes could provide useful information about both victims and other persons related to criminal activities. Signal-joint T cell receptor excision circle (sjTREC) levels in peripheral blood decline with age, and negative correlations between sjTREC levels and age have been demonstrated in several ethnic groups. To validate the utility of sjTREC for age estimation in Koreans, Taqman qPCR was used to quantify the sjTREC level in samples obtained from 172 individuals ranging from 16 to 65 years old. We modified the previously reported method by using a shorter amplicon and confirmed the efficiency and utility of this method in this report. Our results showed that the linear negative regression curve between sjTREC levels and age was characterized by r = 0.807 and a standard error of 8.49 years. These results indicate that sjTREC level is an effective age estimation method in Koreans. The value of the standard error of quantification was not different from previous reports for other population groups. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In forensic practice, DNA typing is mainly used for individual identification and paternity testing. In recent years, many efforts have been made to obtain other useful information from DNA samples, including ethnicity, individual phenotypic traits [1,2], and age [3,4]. Age estimation could aid investigators in the absence of known DNA samples for comparison. To date, the age-dependent accumulation of D-aspartic acid, the accumulation of a 4977-bp deletion in mitochondrial DNA, and telomere shortening have all been analyzed as potential age markers [5,6]. However, the use of these techniques in real cases has been limited for several reasons, including the role of environmental effects and disease effects, the low methodological accuracy, and tissue- and genderspecific variations [7,8]. For example, striking heterogeneity of mitochondrial deletions in different tissues and different telomere dynamics in each compartment of the body has been found [7,9]. Therefore, more stable, standardized and accurate methods for age estimation are needed. ⇑ Corresponding author at: Department of Forensic Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea. Tel.: +82 2 740 8359; fax: +82 2 764 8340. E-mail address: [email protected] (S.D. Lee). http://dx.doi.org/10.1016/j.legalmed.2014.01.009 1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
One of the consequences of aging is the deterioration of the immune system including the thymus. Important functions of the thymus include producing T cells, expressing T cell receptors (TCRs), and facilitating TCR gene rearrangement. During the rearrangement of TCR gene segments, some unused regions are spliced out as ringshaped DNA that may be detected in naïve T cells. These episomal DNA include signal-joint T cell receptor excision circles (sjTRECs), which are a by-product of an intermediate rearrangement in the TCRa/d locus in developing TCR ab+ lymphocytes. sjTRECs are stable, do not replicate during cellular proliferation in the periphery, and are diluted by each round of cell division. The quantification of recent thymic emigrants (RTEs) has been utilized to clinically estimate thymic function in the context of immunological diseases and genetic disorders [10]. RTEs, which form the youngest subset of naïve T cells, represent the ability of the thymus to produce T cells and are usually quantified by measurement of sjTRECs [11]. The content of sjTRECs has been reported to be lower in older people [12,13]. This may be related to thymic involution (i.e., the shrinking of the thymus), which begins shortly after birth and increases with age [14]. Therefore, the sjTREC content per total T cell content or with respect to the level of a constant gene is expected to decrease with age. In recent years, several groups have reported that sjTREC levels in peripheral blood decline with age, indicating the potential
136
S. Cho et al. / Legal Medicine 16 (2014) 135–138
applicability of this method for age estimation [15,16], including through the use of bloodstain analysis [17]. Many biological or socio-economic factors, including ethnic differences, can be considered in light of this. Herein, we report data for the relationship between sjTREC level and age in Koreans ascertained with newly designed PCR assay with shorter amplicon length.
of serially diluted plasmids encoding sjTREC. For positive control, 0.1 pg of plasmid [corresponding to 22,727 copies, calculated as: copy = (ng copy/mole)/(bp ng/g g/mole of bp)] was run in parallel. The Ct value was used to assess between-experiment consistency. All samples were run in duplicate and the average from these results was used for statistical analysis.
2. Materials and methods
2.5. Statistical analysis
2.1. Human blood samples and DNA extraction
Statistical analysis was performed using IBM SPSS Statistics 20 and Microsoft Excel, and plots were generated from analysis using SPSS. The normality of the data was examined using the Kolmogorov–Smirnov test, and correlation was assessed using Pearson’s correlation coefficient. The significance of mean difference among age groups was performed using One-way ANOVA test.
Samples were collected from 172 Korean volunteers aged 16–65 years old. Informed consent was obtained from individuals, and the study was approved by the ethical committee of the Institutional Review Board of the Seoul National University Hospital Biomedical Research Institute. Peripheral blood samples were collected into sodium heparin green top vacutainers (BD Bioscience, San Jose, CA, USA). Genomic DNA was immediately extracted from 300 lL of whole blood using a MaxwellÒ 16 Blood DNA Purification Kit (Promega, Madison, WI, USA) and a MaxwellÒ 16 instrument (Promega), and stored at 20 °C until use. The obtained DNA was quantified using the QuantifilerÒ Human DNA Quantification Kit (Life Technologies, CA, USA). 2.2. sjTREC primers and probes The forward and reverse sjTREC (accession No. NT_026437) PCR primers were designed to allow amplification of the target sequence only when the TCRd gene was deleted from chromosomal DNA and the sjTREC fragment was formed. The modified primer (S) sequences were as follows, 50 -TGCTGACACCTCTGGTTTTTGTAA-30 (F; forward) and 50 -GTGCCAGCTGCAGGGTTTAG-30 (R; reverse) [18]. The sequence for the Taqman probe was 50 -FAM-CACGGTGA TGCATAGGCACCTGC-TAMRA-30 . The previously reported primers (L), 50 -CCATGCTGACACCTCTGGTT-30 (F; forward) and 50 -TCGTGAG AACGGTGAATGAAG-30 (R; reverse) [14] which were slightly longer than our primers, were also tried. 2.3. PCR amplification Plasmid containing sjTREC (accession No. NT_026437) sequences was constructed in pCR2.1-TOPO plasmid (Life Technologies) and used as a positive control. Extracted genomic DNA and plasmids were amplified in 20-lL reaction mixtures containing 1 Gold buffer (Life Technologies), 2 mM MgCl2, 0.25 mM dNTP, 800 nM of each primer and 1.6 unit of Gold Taq polymerase (Life Technologies). The cycling conditions were 94 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and a final soak at 72 °C for 7 min. The amplified products were separated by 2% agarose gel electrophoresis. 2.4. Real-time PCR using TaqMan probes The sjTREC content of each sample was quantified using an ABI Prism 7000 machine (Life Technologies) and the results were analyzed with SDS v2.1 software from the same manufacturer. Real-time PCR was performed in 20-lL reaction mixtures containing 50 ng of genomic DNA, 800 nM of each primer, 250 nM of Taqman probe and 2 TaqManÒ Universal PCR Master Mix II (Life Technologies). The thermal cycling conditions were 50 °C for 2 min and 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. To determine the optimal amount of DNA for detection, dilutions of 10 DNA samples representing men and women ranging from 23 to 63 years of age at intervals of 10 years were tested. A standard curve was generated by PCR amplification
3. Results 3.1. Modification of the sjTREC quantification method and its efficiency When using the previously reported primers, the Ct value was uncheckable in some old aged samples over 60 years of age. Furthermore the slight non-specific amplification, which seems to be a result of primer–dimer formation, was found (data not shown). In light of this, we designed new primer set (S) that targeted a shorter amplicon length (Fig. 1, adapted from [11]). This primer set yielded 93 bp amplicon (Fig. 2a), and exact sequences and size were verified by sequencing analysis (data not shown). The PCR efficiency using sjTREC(S) primer was obtained with diluted sjTREC plasmid ranging from 107 to 101 copies in Taqman qPCR reactions, and it showed that PCR efficiencies of both old and new assays are high enough. (Fig. 2b and c). 3.2. Distribution of sjTREC levels in a Korean population Ct values for all the samples including aged sample over 60 years of age were obtained, and the Ct value tended to increase with age as it has been known. A negative correlation between sjTREC levels and age (r = 0.807) and a regression line with a fit of R2 = 0.648 were obtained (Fig. 3a). Standard error of the estimate was 8.49 years and a total decline of approximately 1.5 Log was confirmed. 3.3. The variation of sjTREC levels in groups The tested samples were divided into 5-years groups (Fig. 3b and Table 1) to compare variations in small age ranges. A distinct drop was not observed between the adjacent groups, but only the levels declined slightly between the 15–19 and 30–34 years old groups, declined more sharply at 35–45 years, and showed increased variation thereafter. The range of variation was slightly wider in groups of older. 4. Discussion Several investigators have reported differences in sjTREC contents by age among variously aged individuals, with forensic application in mind [15,16]. In these reports, negative correlation values between sjTREC contents and age in the tested samples showed a slight difference (R2 = 0.835 in [15] and R2 = 0.668 in [16], respectively). Some authors have also reported on the difference in sjTREC contents between healthy donors and patients [10,19]. Since sjTREC level is dependent on several factors including genetic and environmental variations, we considered here a difference between ethnic populations, with the Korean population
S. Cho et al. / Legal Medicine 16 (2014) 135–138
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Fig. 1. Schematic diagram of the Taqman qPCR assay strategy. The destiny of a T-cell lineage as either ab+ or cd+ is determined by which TCR chain set is expressed via rearrangement of TCR gene segments during T-cell development. For production of ab+ T cells, which comprise 70–75% of the total T-cell pool, d gene segments are excised from a region of the chromosome where a gene segments coexist. When d segments remain as part of the chromosome, the utilized primers do not amplify the target sequence. However, the formation of sjTREC (i.e., deletion of the d segments) allows amplification. The target includes the recombination region of recombination signal sequences (RSS) flanking the dRec and wJa genes.
Fig. 2. Specificity and efficiency of primers. Amplified PCR products using sjTREC(L) and sjTREC(S) primer with standard plasmid on agarose gel (a). Standard curves generated by quantitative real-time PCR using sjTREC(L) (b) and sjTREC(S) (c) primer. Amplification was performed with 10-fold serial diluted sjTREC-containing plasmids ranging from 107 to 101 copies. X-axis and Y-axis indicate log10[copy number of plasmid] and its Ct value, respectively, and R2 and the slope value presents the correlation between the logged number of plasmid and measured Ct value.
as our focus. In this study, we did not compare the difference between populations directly, but it was expected that our assumed hypothesis would not be significant. Direct comparison needs to be performed in future studies in this area. Some slight differences found in this work may have been affected by the method of quantifying sjTREC used, the analytic method applied to our data or variation in sample size [16]. The method of normalization is worth elaborating upon in this kind of quantification work. For example, the measured sjTREC level can be normalized with a single-copy gene genomic DNA [15,16,18] or the equivalent microgram of DNA is applied to measurement [20]. However, there seems no golden rule for setting such standards. In our reports, the number of TCRa chain constant region gene, which is reflected in a ab+ T-cell proliferation-dependent manner, were first considered for normalization. However, a slight negative correlation between age and the Ct
value of the TCRa gene (R2 = 0.274; data not shown) was observed. It has been suggested that factors such as the T-cell proliferation rate or T-cell death rate could influence the sjTREC level [21], which could potentially offset additional age-related differences in sjTREC levels. Therefore it was thought that using the measurement of sjTREC molecules per lg of genomic DNA was more suitable for this purpose. Furthermore, DNA quantity is usually considered to be a standard in forensics, and it is thought that applying another target together is less convenient for real application. The error of the estimate was 8.49 years in this report. This is not significantly different from the 8.9 years in Zubakov et al. [15]. Even with this age range, this method seems to be the most accurate age estimation method available when considering former methods such as measuring telomere length or accumulated mtDNA deletions. A dramatic drop of sjTREC contents in puberty
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The concentration of samples was set to 50 ng as in previous reports [15], and the optimal amount of sample needed for this test was not examined precisely in this study. We found that we could obtain appropriate results using a lower amount, less than 12.5 ng in younger people (data not shown). For older groups higher amount of DNA is required. This kind of sample concentration dependent difference for different age group represents another source of information for age estimation methodology. Alongside efforts to improve the overall efficiency of the test, more research in greater or a variety of samples related to immune system is expect to expand the application in forensics. Acknowledgment This work was supported by Grant No. 1315000438 from the National Forensic Service (NFS) in Korea. References
Fig. 3. The distribution of sjTREC levels in our sample of 172 Koreans. The levels of sjTREC in the investigated Korean individuals aged 16–65 years were plotted and the linear regression curve was found (a). Samples were divided into 5 years groups (b), and the result was illustrated as box plots. The top and bottom of each box denote the 75th and 25th percentiles, respectively, with 95% confidence intervals (whiskers) extending from each box. The bar in the box denotes the median, and outliers are plotted as closed circles ().
Table 1 The levels of Log [sjTREC per lg DNA] among groups divided into 5-years groups. Group (age)
n
Mean
SD
SE
Min
Max
15–19 20–24 25–29 30–34 35–39 40–44 45–49 50–54 55–59 60–65
36 21 14 18 19 13 13 16 14 8
4.448 4.352 4.210 4.181 3.852 3.836 3.563 3.487 3.459 3.023
0.221 0.222 0.251 0.264 0.396 0.345 0.335 0.413 0.349 0.199
0.037 0.048 0.067 0.062 0.091 0.096 0.093 0.103 0.093 0.070
3.997 3.909 3.779 3.616 3.199 2.955 2.945 2.923 2.864 2.750
5.008 4.927 4.617 4.536 4.539 4.257 4.022 4.261 4.012 3.337
[19] was not observed, but a small change of the level between 15–35 years of age and a decrease with wider variation thereafter among divided groups [22] were confirmed similarly in our age range, 15–65 years old (Fig. 3b). If we could collect more information on which factors having an effect on immunological age estimation methods, the usability of this method in forensic case work samples could be increased.
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