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Research Department of Genetics
University Medical Center Groningen

Methods for estimating biological age

Kees Nedeveen, Sept. 2012 - Jan. 2013


The LifeLines investigation is a project that started in 2006 in order to investigate healthy aging. It will ultimately cover a total of 165,000 individuals from the provinces of Groningen, Friesland and Drenthe. These individuals will be observed for a period of 30 years during which information will be gathered using, for instance, questionnaires, blood samples, and stool samples. During my HBO graduation project at the Department of Genetics, Universitary Medical Centre Groningen (UMCG), I have tested and optimized multiple biological age estimation methods in order to enable their use on the ‘LifeLines Deep’ samples (a subsample of about 1500 LifeLines participants).

The age estimation methods that have been tested can be divided into two groups, with each group taking advantage of a different biological process. The first group of methods makes use of the fact that telomere length reduces as you age. Telomere length is mainly reduced through the end replication problem during cell division, meaning that, at each cell division, around 20 basepairs are lost due to imperfect replication. Other reasons for reduced telomere length are disease and oxidative stress. Only an estimation of the aging process can be made using methods based on telomere length, since the speed at which the telomere repeats are lost varies from person to person. The second group of methods uses single joint T-cell receptor excision circles (sjTRECS), these are a by-product of T-cell formation by the thymus. Since the thymus slowly degrades over the years, the sjTREC concentration will slowly decrease, allowing the sjTREC concentration to be used as an age estimation method. A problem with the sjTREC concentration is that it can be altered by sickness, which will lead to an increase in T-cells and therefore their by-product, the sjTRECS.

The Cawthon (2002, 2009) and O’Callaghan (2008) methods were used to determine the correlation between biological age and telomere length in an individual. In addition, the correlation between sjTREC concentration and biological age was also determined using the method of Zubakov et al. (2010). Besides setting up the sjTREC age estimation with healthy volunteers, the method was also tested on patients with rheumatoid arthritis or celiac disease in order to determine the effects of disease on sjTREC concentration. Since disease should increase the number of T-cells and thus the number of sjTRECs present, patients are expected to have a lower biological age compared to the biological age of sameage healthy individuals.

After testing and optimizing the telomere length methods, we determined the reproducible results. A correlation of 0.411 between age and telomere length was found for the Cawthon (2002) method and a correlation of 0.17 for the O’Callaghan (2008) method. The Cawthon (2009) multiplex method could not be properly optimized on the VIIA 7 PCR because of the technical limitations of this specific PCR, leading to irreproducible results. The Cawthon (2009) method was therefore abandoned. After testing and optimizing the sjTREC concentration method, we also obtained reproducible results for this method, resulting in a correlation of 0.70 between age and sjTREC concentration in healthy volunteers, and correlations of 0.61 and 0.51 in patients suffering from celiac disease and rheumatoid arthritis, respectively.

Except for the Cawthon (2009) multiplex method, all the methods could be used for analysing the LifeLines Deep samples. Which method should be used depends on the goal of any subsequent experiments. In experiments where variation between chronological age and biological age is being investigated, a method with a lower correlation should be used, and therefore the use of one of the telomere length estimations would be preferable.

In cases like forensic research, where a high correlation between chronological and biological age is desirable, the use of the sjTREC age estimation method is preferred. This is because the correlation between sjTREC concentration and age is higher than the correlation between telomere length and age. Furthermore, no primer-dimer-sensitive primer pairs are needed when measuring the sjTREC concentration, the lack of these pairs leads to more accurate PCR results. This is because primer-dimer formation leads to the presence of excess fluorescent signaling by a product formed of the forward and the reverse primers. The use of telomere length or sjTREC concentration in order to determine age should only be used to make a rough estimate, since age estimations that are off by 20 years or more have occasionally been measured.

No definitive conclusions can be drawn when comparing the sjTREC concentration and age correlation in samples from healthy volunteers against patients with rheumatoid arthritis or celiac disease. This inability to draw a definitive conclusion was mainly due to a lack of age-related samples in each group, since each of the groups was composed of participants only in the age range of 31–62 years old.


Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002;30(10):e47.

Cawthon RM. Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic Acids Res. 2009;37(3):e21. doi: 10.1093/nar/gkn1027.

O’Callaghan N, Dhillon V, Thomas P, Fenech M. A quantitative real-time PCR method for absolute telomere length. Biotechniques. 2008; 44(6):807-9. doi: 10.2144/000112761.

Zubakov D, Liu F, van Zelm MC, Vermeulen J, Oostra BA, van Duijn CM, Driessen GJ, van Dongen JJ, Kayser M, Langerak AW. Estimating human age from T-cell DNA rearrangements. Curr Biol. 2010;20(22):R970-1. doi: 10.1016/j.cub.2010.10.022.
Last modified:20 June 2016 2.40 p.m.