Age estimation using methylation-sensitive high-resolution melting (MS-HRM) in both healthy felines and those with chronic kidney disease

Abstract

Age is an important ecological tool in wildlife conservation. However, it is difficult to estimate in most animals, including felines—most of whom are endangered. Here, we developed the first DNA methylation-based age-estimation technique—as an alternative to current age-estimation methods—for two feline species that share a relatively long genetic distance with each other: domestic cat (Felis catus; 79 blood samples) and an endangered Panthera, the snow leopard (Panthera uncia; 11 blood samples). We measured the methylation rates of two gene regions using methylation-sensitive high-resolution melting (MS-HRM). Domestic cat age was estimated with a mean absolute deviation (MAD) of 3.83 years. Health conditions influenced accuracy of the model. Specifically, the models built on cats with chronic kidney disease (CKD) had lower accuracy than those built on healthy cats. The snow leopard-specific model (i.e. the model that resets the model settings for snow leopards) had a better accuracy (MAD = 2.10 years) than that obtained on using the domestic cat model directly. This implies that our markers could be utilised across species, although changing the model settings when targeting different species could lead to better estimation accuracy. The snow leopard-specific model also successfully distinguished between sexually immature and mature individuals.

Age estimation based on blood DNA methylation levels in brown bears

“…We selected target genomic locations adjacent to 12 genes, namelyGSE1 , VGF , SLC12A5 , SCGN , KCNK12 ,OTUD7A , BCL6B , POU4F2 , ELOVL2 , RALYL ,KISS1R , and CAPS2 (Table 1). The methylation levels of CpG sites adjacent to these genes change with age in humans (Day et al 2013;Florath et al 2014;Bekaert et al 2015;Lowe et al 2018), dogs (Ito et al 2017;Yamazaki et al 2021), and cats (Qi et al 2021). We identified homologous sequences in the genomic regions of brown bears containing the target CpG sites using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information.…”

“…To evaluate whether age, sex, or growth environment affects the deviation of the age estimation model, linear regressions were generated using Δage (predicted age -chronological age) and |Δage| (absolute difference between predicted age and chronological age) as dependent variables and three factors, as well as the interactions among each factor pair, as explanatory variables (Qi et al 2021). Model construction and selection were conducted using the "lm" command and "MnMIn" package in the R software.…”

“…In early epigenetic research efforts for age estimation, the focus was on forensic research in humans using various biological samples, including blood, muscle, saliva, buccal swabs, and semen (Horvath 2013;Lee et al 2015;Bekaert et al 2015). Subsequently, similar techniques have been established for laboratory animals such as mice (Wang et al, 2017;Petkovich et al, 2017;Stubbs et al, 2017) and naked mole rats (Heterocephalus glaber ; Lowe et al 2020), companion animals such as dogs and cats (Thompson et al 2017;Qi et al 2021;Raj et al 2021) (Biggar & Storey 2014;Alvarado et al 2015;Yamazaki et al 2021).…”

Infection by a helminth parasite is associated with changes in DNA methylation in the house sparrow

“…$$$$ \frac{a^{\ast }M}{100-M}=\frac{\mathrm{Df}}{{\mathrm{Df}}_{\mathrm{max}}-\mathrm{Df}} $$$$where “ a ” is a coefficient, M is the methylation level of each standard sample, Df is the Df value for each standard sample, and Df max is the maximum Df value for the 100% methylated standard. The “nls” function in R 4.2.1 was used to implement the regression model and calculate the estimated value of “ a ” as in Qi et al (2021). Typical standard curves for NR1D1 and CLDN22 are shown in Figure A5 in Appendix .…”

Age estimation of Asian elephants (Elephas maximus) using methylation-sensitive high-resolution melting (MS-HRM)

“…Most importantly, many studies have reported correlations between DNA methylation levels at speci c CpG islands in certain genes and age, mainly in humans [19,[28][29][30]. However, recently, increasing numbers of studies on other species have been carried out, such as in dogs (Canis familiaris) [31], cats (Felis catus) [32], chimpanzees (Pan troglodytes) [33], bats (Myotis bechsteinii) [34,35], seabirds (Ardenna tenuirostris) [36], green turtles (Chelonia mydas) [37], sh [38,39], and several cetacean species [40][41][42][43].…”