Helena Correia Dias 1,2,3, Eugénia Cunha 2, Francisco Corte Real 3, Licínio Manco 1

1 Research Centre for Anthropology and Health (CIAS), Department of Life Sciences, University of Coimbra, Portugal.

2 Centre for Functional Ecology (CEF), Laboratory of Forensic Anthropology, Department of Life Sciences, University of Coimbra, Portugal.

3 National Institute of Legal Medicine and Forensic Sciences, Portugal.

Adult’s age-at-death estimation is one of the main concerns in the forensic

field. Recently, DNA methylation of some genes emerges as a powerful tool

in age-at-death estimation [1]. A correlation between DNA methylation status

of the EDARADD (EDAR associated death domain) gene (on chromosome

1q42.3) and chronological age of individuals was previously shown [2, 3]. In

this study, we investigated the correlation between DNA methylation patterns

of four CpGs from EDARADD gene and chronological age in a Portuguese

sample.

Bisulfite PCR-sequencing showed to be a simple, efficient and

economic method to investigate DNA methylation levels at

EDARADD gene. C3 showed to be an accurate age predictor site and

C2 a site that should be considered in future age estimation models.

Blood samples of 47 healthy individuals (32 females, 15 males; aged 1-95

years old) were collected after informed consent and according institutional

and ethical guidelines. Genomic DNA was extracted and a method based on

the bisulfite conversion using EZ DNA Methylation-GoldTM Kit (Zymo

Research, CA, USA), followed by PCR and Sanger sequencing was used to

evaluate the DNA methylation patterns in four CpGs of EDARADD gene.

The methylation status of cytosines in each CpG dinucleotides was estimated

by measuring the ratio of the cytosine peak height to the sum of cytosine and

thymine peak heights, according to [4]. Simple linear regressions were used

to analyze relationships between the CpGs methylation levels and

chronological age. Statistical analysis was performed using SPSS, version

24.0.

A negative correlation between EDARADD DNA methylation levels and

chronological age was observed (Figure 1). A strong correlation between

methylation levels and age was observed for C3 (R = 0.912; p = 4.75×10-19),

explaining 83% of variation in age, followed by C2 (R = 0.797; p = 2.05×10-

11) (adjusted R2 = 0.627). The C4 site showed a lower correlation (R = 0.610;

p = 0.000005) (adjusted R2 = 0.358) and C1 revealed no significant

correlation with age (R = 0.046; p = 0.759). Data showed in table 1.

[1] F. Kader, M. Ghai, Forensic Sci Int 249 (2015) 255–265. [2] B. Bekaert, A. Kamalandua, S.C. Zapico, W. Van de Voorde, R. Decorte,Forensic Sci Int Genet Supplement Series 5 (2015) e144-e145. [3] B. Bekaert, A. Kamalandua, S.C. Zapico, W. Van de Voorde, R. Decorte,Epigenetics 10 (10) (2015) 922-930. [4] M. Jiang, Y. Zhang, J. Fei, X. Chang, W. Fan, X. Qian, T. Zhang, D. Lu, Laboratory Investigation90 (2010) 282–290.

CpG R Corrected

R2

Standard

error

P value

C1 0.046 -0.020 31,066 0.759

C2 0.797 0.627 18,783 2.05×10-11

C3* 0.912 0,828 12,745 4.75×10-19

C4* 0.610 0.358 24,649 0.000005

Table 1: Univariate regression analysis of 4 CpGs of EDARADD

gene.

*Investigated according to the literature.

Analysing DNA methylation levels of the age

predictor gene EDARADD using the bisulfite

PCR-sequencing method

Figure 2: Predicted age versus chronological age of the 47

individuals based in C3 methylation levels.

Predicted age of the sampled individuals based on the strongest

correlation site C3, showed a mean absolute deviation from

chronological age of 10 years (Figure 2). For two individuals aged 1

years old, negative prediction values were obtained and were set at 0.

As in previous studies [2, 3], C3 showed a strong correlation with age.

However, higher significant values were obtained for C2, a CpG site

that was not considered in previous studies, in relation with C4 that

was previously shown highly correlated with age [3].

Figure 1: Negative correlation between DNA methylation levels from CpG3

of EDARADD gene and chronological age (years).

Introduction

Results and discussion

Methods

Results and discussion

Conclusion

Sponsors

References