Four novel genes associated with longevity found in Cane Corso purebred dogs

doi:10.21203/rs.3.rs-1293017/v1

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Four novel genes associated with longevity found in Cane Corso purebred dogs

https://doi.org/10.21203/rs.3.rs-1293017/v1

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Background: Longevity-related genes have been found in several animal species as well as in humans. The goal of this study was to perform genetic analysis of long-lived Cane Corso dogs with the aim to find genes that are associated with longevity.

Results: SNPs with particular nucleotides were significantly overrepresented in long-lived dogs in four genes, TDRP, MC2R, FBXO25 and FBXL21. In FBXL21, the longevity-associated SNP localises to the exon. In the FBXL21 protein, tryptophan in long-lived dogs replaced arginine present in reference dogs.

Conclusions: Four SNPs associated with longevity in dogs were identified using GWAS. We conclude that genes TDRP, MC2R, FBXO25 and FBXL21 are associated with longevity in Cane Corso dogs.

Longevity-associated genes

GWAS

Cane Corso Italiano dog

extending lifespan

longevity testing

Longevity and lifespan extension represent a subject of interest and research in various organisms from yeasts to vertebrates including humans [1–3]. According to recent studies, extended lifespan is a polygenic trait influenced not only genetically, but also by epigenetic, environmental, and behavioural factors [4–6]. Heritability of extended lifespan varies from 20–35% [5]. So far, 33 genes have been associated with longevity in mice and thanks to genetic mapping, next generation sequencing and genome-wide association studies (GWAS), dozens of candidate genes and hundreds of associated single nucleotide polymorphisms (SNPs) were described in humans [2, 5, 6]. Nevertheless, the recent knowledge does not allow any significant extension of the lifespan in humans and animals.

In the last years, GWAS have been done in dogs with the aim to map genetic loci associated with general or breed-specific diseases [7–9] and eliminate the risk alleles. In addition to that, understanding the genetic component of longevity in dogs is also important for selective breeding. Extended lifespan, especially in large dog breeds with a generally short lifespan, is a desirable goal. Interestingly, long co-evolution of dogs and humans led to sharing not just their lifestyle, but also common diseases such as diabetes [10, 11]. In combination with artificial selection, genetic diversity among breeds and health care comparable to humans, dogs represent an interesting model organism for longevity research.

So far, only a few studies have investigated the genetic background of the dog longevity. A whole-genome sequencing study of two extremely old dogs showed multiple potential loci that could be closely associated with longevity [12]. Another study focusing on genes associated with cancer mortality and longevity also suggests the need for further research direction in this field [13]. Such results are challenging for further research of genes and loci associated with longevity in dogs.

This study focuses on finding genes associated with longevity in purebred Cane Corso dogs. Cane Corso is a large Molossian breed with a median lifespan of merely 9.29 years [14], which exemplifies the fact that the lifespan of large dog breeds is significantly shorter compared to small breeds. Earlier death in larger breeds is often caused by gastrointestinal and musculoskeletal diseases, such as canine hip dysplasia (CHD) [15–18]. The inheritance of CHD [19, 20] and coat colour [21] have been described in the breed Cane Corso. This study identifies four candidate genes associated with longevity in the Cane Corso dogs using the GWAS method and subsequent confirmative sequencing.

Buccal swab samples from purebred Cane Corso dogs were collected during the years 2016–2020. The health status of the dogs was not evaluated. For the purpose of this research, samples were divided into two groups defined by the age of the dogs examined. The group of old dogs contained samples from individuals older than 12 years, which were considered as long-lived dogs according to a previous study of the Cane Corso breed longevity [14]. For the reference group, we sampled dogs in the age between 2–9 years. Overall, 20 samples of long-lived dogs and 20 samples of reference dogs were used for this study. Since dogs from the reference group could possibly be long-living, monitoring of these dogs will continue to evaluate our results.

DNA was isolated from buccal swab samples using a Qiagen DNeasy Blood & Tissue Kit and standard phenol-chloroform DNA isolation protocol. DNA was eluted in 15 to 50 µl elution solution. Concentration and purity of isolated DNA was checked using a spectrophotometer. The required length of 5000 base pairs (bp) for the SNP genotyping was checked in 2% agarose gel. Suitable samples were diluted or concentrated to the required concentration of DNA for 20–30 ng/µl. Out of all the isolated samples, nine samples from the old group and 15 reference samples had sufficient concentration and bp length and were suitable for SNP genotyping. Samples were genotyped using Illumina CanineHD Beadchip at Neogen laboratory, 4131 N. 48th St. Lincoln, NE 68504, USA. This chip allows analysis of 172,115 SNPs.

Statistical analysis and necessary steps that preceded the association analysis were performed using PLINK v1.90b6.16 [22]. Received data were checked according to commonly used quality parameters. Firstly, SNPs that were missing in more than 10% (geno 0.1) of the samples were excluded from further analysis. Of the rest of the markers, those that were missing in more than 1% (geno 0.01) of all samples were also excluded. All samples kept for further analysis have more than 95% of SNP markers genotyped (mind 0.05). After this first step of data cleaning, 71,660 variants and 24 dogs passed the data clean up. SNPs with minor allele frequencies lower than 5% (maf 0.05) were also excluded from the association analysis. Although the number of samples was low and the model organism was a highly inbred domestic animal, markers that did not fit in Hardy Weinberger Equilibrium in threshold 0.0001 were excluded. 47,915 SNP variants and 24 dogs passed for further genome-wide association analysis. A Manhattan plot for visualization of association analysis was constructed in R Studio [23].

According to the result of GWAS, genomic position of all candidate SNPs was checked in the CanFam 3.1 reference genome. Seven candidate SNPs and their close surroundings, located in seven genes, were PCR amplified and sequenced in 40 samples including those used for GWAS. Samples were sequenced in SEQme s.r.o., 26301 Dobris, Czech Republic.

Statistical significance of the distribution of candidate SNPs within the long-lived and reference groups was tested in R Studio [23] using Fisher’s exact test [24].

GWAS

Overall, 24 SNPs passed through the set significance threshold (1.0e-04) (Fig. 1). Eleven SNPs that passed the significance threshold were located within the described genes in the CanFam 3.1 reference genome. Four SNPs located in the intron region of the genes and one SNP located in the 3'UTR part of one gene were selected for sequencing in the entire sample panel. Next, two SNPs located in the exon region, which passed through the significance threshold of (1.0e-03) and had the highest P-value, were also sequenced in the entire sample panel. Results from the association analysis for the seven selected SNPs and their position in particular genes are shown in Table 1.

Table 1

Table of GWAS results for 7 candidate SNPs sorted by the highest P-value.
P-value	Gene	Position	CHR	SNP	BP	A1	F_A	F_U	A2	CHISQ	OR
3.705e-07	TDRP	3'UTR	25	BICF2P365740	37486799	T	0.7222	0.03333	C	25.84	75.4
2.772e-05	MC2R	intron	1	BICF2P1140956	24364734	G	0.7778	0.1667	A	17.57	17.5
3.969e-05	NGDN	intron	8	BICF2P384545	3705090	C	0.6111	0.06667	T	16.89	22
3.969e-05	RARB	intron	23	BICF2G630382633	18647529	C	0.6111	0.06667	T	16.89	22
6.334e-05	FBXO25	intron	25	BICF2P999174	37425075	A	0.4444	0	G	16	NA
1.478e-04	FBXL21	exon	11	chr11_23852542	23852542	T	0.6667	0.1333	C	14.4	13
3.413e-04	PARP9	exon	33	chr33_25671811	25671811	A	0.7222	0.2	C	12.83	10.4

Sequencing

In all the selected SNPs, differences were found between old and reference samples (Fig. 2).

In the most significant SNP BICF2P365740 (CHR 25, BP position 37486799), located in the 3'UTR part of the TDRP gene, nucleotide T was considered as an allele associated with longevity. Twenty-five percent of the old group were homozygous with genotype TT compared to 0% in the reference group. Fifty percent of the old group were heterozygous with genotype TC compared to 30% of the reference group. Only 25% of the old group were homozygous with genotype CC compared to 70% of the reference group (Fig. 2). Association of allele T with longevity was significant in Fisher’s exact test (p = 0.04712). Genotype TT was proved to be associated with longevity, as it was found exclusively in long-lived dogs.

The second most significant SNP BICF2P1140956 (CHR 1, BP 24364734) is located in the first intron of the MC2R gene. For this position, nucleotide G was considered as an allele associated with longevity (Fig. 2). Thirty-seven percent of the old group were homozygous with genotype GG compared to 5% in the reference group. Thirty-two percent of the old group were heterozygous with genotype GA compared to 37% of the reference group. Thirty-two percent of the old group were homozygous with genotype AA compared to 58% of the reference group. Association of allele G with longevity was significant in Fisher’s exact test (p = 0.01966).

For SNP BICF2P999174 (CHR 25, BP 37425075), located in the second intron of the FBXO25 gene, nucleotide A was considered as an allele associated with longevity (Fig. 2). Five percent of the old group were homozygous with genotype AA compared to 0% in the reference group. Forty-seven percent of the old group were heterozygous with genotype GA compared to 15% of the reference group. Forty-seven percent of the old group were homozygous with genotype GG compared to 85% of the reference group. Association of allele A with longevity was significant in Fisher’s exact test (p = 0.04074). Genotype AA was proved to be associated with longevity, as we have found it exclusively in long-lived dogs.

For SNP chr11_23852542 (CHR 11, BP 23852542), located in the third exon of the FBXL21 gene, nucleotide T was considered as an allele associated with longevity (Fig. 2). Thirty-one percent of the old group were homozygous with genotype TT compared to 0% in the reference group. Thirty-eight percent of the old group were heterozygous with genotype TC compared to 30% of the reference group. Thirty-one percent of the old group were homozygous with genotype CC compared to 70% of the reference group. Association of allele T with longevity was significant in Fisher’s exact test (p = 0.0111). With this nucleotide substitution from C to T in the SNP position, amino acid arginine is replaced by tryptophan. Genotype TT was proved to be associated with longevity and we observed it exclusively in long-lived dogs.

For SNP chr33_25671811 (CHR 33, BP 25671811), located in the second exon of the PARP9 gene, nucleotide A was considered as an allele associated with longevity (Fig. 2). Twenty-eight percent of the old group were homozygous with genotype AA compared to 5% in the reference group. None of the samples either from the old group or from the reference group were heterozygous with genotype AC. Seventy-two percent of the old group were homozygous with genotype CC compared to 95% of the reference group. Association of allele A with longevity was not significant in Fisher’s exact test (p = 0.08968). No statistically significant association with longevity was demonstrated for this gene. Therefore, we do not consider this gene to be associated with longevity.

For SNP BICF2P384545 (CHR 8, BP 3705090), located in the first intron of the NGDN gene, nucleotide C was considered as an allele associated with longevity (Fig. 2). Nineteen percent of the old group were homozygous with genotype CC compared to 10% in the reference group. Thirty-one percent of the old group were heterozygous with genotype TC compared to 16% of the reference group. Fifty percent of the old group were homozygous with genotype TT compared to 74% of the reference group. Association of allele C with longevity was not significant in Fisher’s exact test (p = 0.3044). No statistically significant association with longevity was demonstrated for this gene. Therefore, we do not consider this gene to be associated with longevity.

Results for SNP BICF2G630382633 (CHR 23, BP 18647529), located in the third intron of the RARB gene, suggested that nucleotide C was considered as an allele associated with longevity (Fig. 2). Twenty-seven percent of the old group were homozygous with genotype CC compared to 10% in the reference group. Twenty-seven percent of the old group were heterozygous with genotype TC compared to 10% of the reference group. Forty-six percent of the old group were homozygous with genotype TT compared to 80% of the reference group. Association of allele C with longevity was not significant in Fisher’s exact test (p = 0.1523). No statistically significant association with longevity was demonstrated for this gene. Therefore, we do not consider this gene to be associated with longevity.

Longevity-associated genes have been found in several animal species as well as in humans [1–3]. Such genes have not yet been described in dogs. In this study, we describe four SNPs that are associated with longevity in the Cane Corso breed. According to GWAS, the most significant SNP associated with longevity was located in the 3'UTR of the TDRP gene. TDRP was not previously described as associated with longevity, but it is associated with spermatogenesis and sperm motility [25]. Untranslated regions at the 3' end can play an important role in the regulation of translation and mRNA stability [26]. We found two SNPs located in introns of two genes with a statistically significant association with longevity. MC2R (adrenocorticotropic hormone receptor) was previously described as associated with longevity in humans \([\)6], which suggests that the association of this gene with longevity is more general. FBXO25 plays a role in promoting tumour growth [27, 28].

Determining the causal relationship between a particular nucleotide substitution and longevity can be crucial in identifying the predisposition of longevity at the molecular genetic level. From this point of view, SNPs located in gene exons are of greatest importance. We detected one gene where the SNP associated with longevity is located in an exon. In the FBXL21 gene, a nucleotide substitution leads to an amino acid change resulting in tryptophan presence in long-lived dogs, while arginine is produced in reference dogs. FBXL21 has an important role in the oscillation of the circadian clock [29]. Optimal circadian rhythms seem to have an influence on aging [30]. Circadian rhythms have most often been described in terms of their phases and amplitudes, and how these respond, in both health and disease, to a single exposure to synchronizers. Daily fluctuation of several physiological functions, which has been studied in dogs, demonstrates a daily rhythmicity [31, 32]. Substitution of one amino acid can lead to a change in the structure of the protein produced, which can then cause a change in its function. Analysis of the structure of such proteins will be the subject of our further research.

All the described genes could have a direct influence on extending lifespan. Three of the investigated genes, PARP9, NGDN and RARB, were not significantly associated with longevity in the Cane Corso breed.

Our findings also have a specific practical significance. Testing the SNPs in genes that are associated with longevity determined in this study will allow prediction of the possible longevity of the tested dog. Crossing dogs with longevity potential could allow breeding of long-lived dog lineages. This is very important for every breeder and dog owner. The results found in this study can only be used for longevity testing in the Cane Corso Italiano breed.

Finding out whether the genes we have found are associated with longevity in other dog breeds will be the subject of our further research. This study has its limits due to the low number of sampled dogs. It will be useful to confirm these results in a study with a larger number of samples. Also, selective breeding of the carriers of the longevity allele is a way to test the validity of our results.

Four SNPs shown to be associated with longevity in Cane Corso dogs were identified using GWAS and DNA sequencing. Genes TDRP, MC2R, FBXO25 and FBXL21 are associated with longevity in Cane Corso dogs.

Testing of SNPs in genes that are associated with longevity would allow prediction of possible longevity of the tested dogs. The results found in this study can only be used for longevity testing in the Cane Corso Italiano dog breed. The possibility of using SNPs to determine longevity in other dog breeds is the subject of our future research.

CHD

Canine hip dysplasia

GWAS

Genome-wide association study

SNP

single nucleotide polymorphism

We would like to thank all the donors of buccal swab samples from Cane Corso dogs. We would like to also thank Gabriela Suchanová for proofreading and English correction.

Author Contributions

Conceptualization, Evžen Korec; Methodology, Lenka Ungrová, Jiří Hejnar and Adéla Grieblová; Project administration, Evžen Korec; Supervision, Evžen Korec and Jiří Hejnar; Validation, Evžen Korec and Jiří Hejnar; Visualization, Lenka Ungrová; Writing – original draft, Evžen Korec and Lenka Ungrová; Writing – review & editing, Jiří Hejnar. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Availability of data and materials

The datasets used in this study are available from https://www.ebi.ac.uk/eva/?Study-Browser&browserType=sgv , project accession: PRJEB51024.

Declarations

Ethics approval and consent to participate

All samples were obtained non-invasively. Owners of the dogs collected and provided all samples.

Consent for publication

Not applicable.

Competing Interest

The authors declare no competing interests.

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You are reading this latest preprint version

Four novel genes associated with longevity found in Cane Corso purebred dogs

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Abstract

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Background

Methods

Results

GWAS

Sequencing

Discussion

Conclusions

Abbreviations

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References

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