analysing dna methylation levels of the age predictor gene...
TRANSCRIPT
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