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

Huiyuan Qi,Takashi Mori,Kodzue Kinoshita,Yukiko Matsui,Miho Inoue-Murayama,Kaori Matsumoto

doi:10.1038/s41598-021-99424-4

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.

Highlights

Age is an important ecological tool for wildlife conservation
If the sequences were highly conserved, it was assumed that the markers would have high applicability to other felines. To further verify this by experiment, we used the blood samples of the snow leopard (Panthera uncia) available to our team[25,26], an endangered large cat species that shares long genetic distance with the domestic cat[27], to test the markers and models developed with domestic cat samples
If the estimation accuracy was influenced by chronic kidney disease (CKD), it would imply that the markers should be used with a combination of disease biomarkers or pre-knowledge of the health condition of target individuals

Summary

Introduction

Age is an important ecological tool for wildlife conservation. As it is related to reproduction[1,2] and mortality rates[3], knowing the accurate age of an animal is helpful in estimating the structure of populations[4] and, in the prediction of the present and future extinction risk for a wildlife population. The model and markers designed for domestic cats are expected to be useful for other wild felines, as related species share similar age-related DNA methylation changes as implied by previous studies on humans and chimpanzees[12], and on dogs and wolves[19]. To verify this hypothesis, we compared the sequences of age-estimation markers used in our models among all felines whose genome data were published. Our study presents the first age-estimation model applied to non-human species, including diseased individuals, by which we could devise specific ways to apply our model in the future. We analysed the methylation rate of two epigenetic aging marker candidates (Table 1): (1) ELOVL230–32 (elongation of very long chain fatty acid protein) and (2) RALYL20 (RALY RNA binding protein-like) via methylation-sensitive high-resolution melting (MS-HRM)[30,31,33,34,35]

Methods

Results

Conclusion