Telomere regulation: lessons learnt from mice and men,potential opportunities in horses

Telomeres are genetically conserved nucleoprotein complexes located at the ends of chromosomes that preserve genomic stability. In large mammals, somatic cell telomeres shorten with age, owing to the end replication problem and lack of telomere-lengthening events (e.g. telomerase and ALT activity). Therefore, telomere length reflects cellular replicative reserve and mitotic potential. Environmental insults can accelerate telomere attrition in response to cell division and DNA damage. As such, telomere shortening is considered one of the major hallmarks of ageing. Much effort has been dedicated to understanding the environmental perturbations that accelerate telomere attrition and therapeutic strategies to preserve or extend telomeres. As telomere dynamics seem to reflect cumulative cellular stress, telomere length could serve as a biomarker of animal welfare. The assessment of telomere dynamics (i.e. rate of shortening) in conjunction with telomere-regulating genes and telomerase activity in racehorses could monitor long-term animal health, yet it could also provide some unique opportunities to address particular limitations with the use of other animal models in telomere research. Considering the ongoing efforts to optimise the health and welfare of equine athletes, the purpose of this review is to discuss the potential utility of assessing telomere length in Thoroughbred racehorses. A brief review of telomere biology in large and small mammals will be provided, followed by discussion on the biological implications of telomere length and environmental (e.g. lifestyle) factors that accelerate or attenuate telomere attrition. Finally, the utility of quantifying telomere dynamics in horses will be offered with directions for future research.     

Genetic contributions to precocity traits in racing Thoroughbreds

Adaptation to early training and racing (i.e. precocity), which is highly variable in racing Thoroughbreds, has implications for the selection and training of horses. We hypothesised that precocity in Thoroughbred racehorses is heritable. Age at first sprint training session (work day), age at first race and age at best race were used as phenotypes to quantify precocity. Using high‐density SNP array data, additive SNP heritability () was estimated to be 0.17, 0.14 and 0.17 for the three traits respectively. In genome‐wide association studies (GWAS) for age at first race and age at best race, a 1.98‐Mb region on equine chromosome 18 (ECA18) was identified. The most significant association was with the myostatin (MSTN) g.66493737C>T SNP (P = 5.46 × 10^?12 and P = 1.89 × 10^?14 respectively). In addition, two SNPs on ECA1 (g.37770220G>A and g.37770305T>C) within the first intron of the serotonin receptor gene HTR7 were significantly associated with age at first race and age at best race. Although no significant associations were identified for age at first work day, the MSTN:g.66493737C>T SNP was among the top 20 SNPs in the GWAS (P = 3.98 × 10^?5). Here we have identified variants with potential roles in early adaptation to training. Although there was an overlap in genes associated with precocity and distance aptitude (i.e. MSTN), the HTR7 variants were more strongly associated with precocity than with distance. Because HTR7 is closely related to the HTR1A gene, previously implicated in tractability in young Thoroughbreds, this suggests that behavioural traits may influence precocity.     

Genome‐wide association study of osteochondrosis in the tarsocrural joint of Dutch Warmblood horses identifies susceptibility loci on chromosomes 3 and 10

Equine osteochondrosis is a developmental joint disease that is a significant source of morbidity affecting multiple breeds of horse. The genetic variants underlying osteochondrosis susceptibility have not been established. Here, we describe the results of a genome‐wide association study of osteochondrosis using 90 cases and 111 controls from a population of Dutch Warmblood horses. We report putative associations between osteochondrosis and loci on chromosome 3 (BIEC2‐808543; P = 5.03 × 10^?7) and chromosome 10 (BIEC2‐121323; P = 2.62 × 10^?7).     

Leukocyte telomere length in major depression: correlations with chronicity, inflammation and oxidative stress--preliminary findings

Background

Depression is associated with an unusually high rate of aging-related illnesses and early mortality. One aspect of “accelerated aging” in depression may be shortened leukocyte telomeres. When telomeres critically shorten, as often occurs with repeated mitoses or in response to oxidation and inflammation, cells may die. Indeed, leukocyte telomere shortening predicts early mortality and medical illnesses in non-depressed populations. We sought to determine if leukocyte telomeres are shortened in Major Depressive Disorder (MDD), whether this is a function of lifetime depression exposure and whether this is related to putative mediators, oxidation and inflammation.

Methodology

Leukocyte telomere length was compared between 18 unmedicated MDD subjects and 17 controls and was correlated with lifetime depression chronicity and peripheral markers of oxidation (F2-isoprostane/Vitamin C ratio) and inflammation (IL-6). Analyses were controlled for age and sex.

Principal Findings

The depressed group, as a whole, did not differ from the controls in telomere length. However, telomere length was significantly inversely correlated with lifetime depression exposure, even after controlling for age (p<0.05). Average telomere length in the depressed subjects who were above the median of lifetime depression exposure (≥9.2 years'' cumulative duration) was 281 base pairs shorter than that in controls (p<0.05), corresponding to approximately seven years of “accelerated cell aging.” Telomere length was inversely correlated with oxidative stress in the depressed subjects (p<0.01) and in the controls (p<0.05) and with inflammation in the depressed subjects (p<0.05).

Conclusions

These preliminary data indicate that accelerated aging at the level of leukocyte telomeres is proportional to lifetime exposure to MDD. This might be related to cumulative exposure to oxidative stress and inflammation in MDD. This suggest that telomere shortening does not antedate depression and is not an intrinsic feature. Rather, telomere shortening may progress in proportion to lifetime depression exposure.     

Age‐associated microRNA expression in human peripheral blood is associated with all‐cause mortality and age‐related traits

Recent studies provide evidence of correlations of DNA methylation and expression of protein‐coding genes with human aging. The relations of microRNA expression with age and age‐related clinical outcomes have not been characterized thoroughly. We explored associations of age with whole‐blood microRNA expression in 5221 adults and identified 127 microRNAs that were differentially expressed by age at P < 3.3 × 10^?4 (Bonferroni‐corrected). Most microRNAs were underexpressed in older individuals. Integrative analysis of microRNA and mRNA expression revealed changes in age‐associated mRNA expression possibly driven by age‐associated microRNAs in pathways that involve RNA processing, translation, and immune function. We fitted a linear model to predict ‘microRNA age’ that incorporated expression levels of 80 microRNAs. MicroRNA age correlated modestly with predicted age from DNA methylation (r = 0.3) and mRNA expression (r = 0.2), suggesting that microRNA age may complement mRNA and epigenetic age prediction models. We used the difference between microRNA age and chronological age as a biomarker of accelerated aging (Δage) and found that Δage was associated with all‐cause mortality (hazards ratio 1.1 per year difference, P = 4.2 × 10^?5 adjusted for sex and chronological age). Additionally, Δage was associated with coronary heart disease, hypertension, blood pressure, and glucose levels. In conclusion, we constructed a microRNA age prediction model based on whole‐blood microRNA expression profiling. Age‐associated microRNAs and their targets have potential utility to detect accelerated aging and to predict risks for age‐related diseases.     

A candidate-SNP retrospective cohort study for fracture risk in Japanese Thoroughbred racehorses

Fractures are medical conditions that compromise the athletic potential of horses and/or the safety of jockeys. Therefore, the reduction of fracture risk is an important horse and human welfare issue. The present study used molecular genetic approaches to determine the effect of genetic risk for fracture at four candidate SNPs spanning the myostatin (MSTN) gene on horse chromosome 18. Among the 3706 Japanese Thoroughbred racehorses, 1089 (29.4%) had experienced fractures in their athletic life, indicating the common occurrence of this injury in Thoroughbreds. In the case/control association study, fractures of the carpus (carpal bones and distal radius) were statistically associated with g.65809482T/C (P = 1.17 x 10^-8), g.65868604G/T (P = 2.66 x 10^-9), and g.66493737C/T (P = 6.41 x 10^-8). In the retrospective cohort study using 1710 racehorses born in 2000, the relative risk (RR) was highest for male horses at g.65868604G/T, based on the dominant allele risk model (RR = 2.251, 95% confidence interval 1.407–3.604, P = 0.00041), and for female horses at g.65868604G/T, based on the recessive allele risk model (RR = 2.313, 95% confidence interval 1.380–3.877, P = 0.00163). Considering the association of these SNPs with racing performance traits such as speed, these genotypes may affect the occurrence of carpus fractures in Japanese Thoroughbred racehorses as a consequence of the non-genetic influence of the genotype on the distance and/or intensity of racing and training. The genetic information presented here may contribute to the development of strategic training programs and racing plans for racehorses that improve their health and welfare.     

Telomere Length Trajectory and Its Determinants in Persons with Coronary Artery Disease: Longitudinal Findings from the Heart and Soul Study

Background

Leukocyte telomere length, an emerging marker of biological age, has been shown to predict cardiovascular morbidity and mortality. However, the natural history of telomere length in patients with coronary artery disease has not been studied. We sought to investigate the longitudinal trajectory of telomere length, and to identify the independent predictors of telomere shortening, in persons with coronary artery disease.

Methodology/Principal Findings

In a prospective cohort study of 608 individuals with stable coronary artery disease, we measured leukocyte telomere length at baseline, and again after five years of follow-up. We used multivariable linear and logistic regression models to identify the independent predictors of leukocyte telomere trajectory. Baseline and follow-up telomere lengths were normally distributed. Mean telomere length decreased by 42 base pairs per year (p<0.001). Three distinct telomere trajectories were observed: shortening in 45%, maintenance in 32%, and lengthening in 23% of participants. The most powerful predictor of telomere shortening was baseline telomere length (OR per SD increase = 7.6; 95% CI 5.5, 10.6). Other independent predictors of telomere shortening were age (OR per 10 years = 1.6; 95% CI 1.3, 2.1), male sex (OR = 2.4; 95% CI 1.3, 4.7), and waist-to-hip ratio (OR per 0.1 increase = 1.4; 95% CI 1.0, 2.0).

Conclusions/Significance

Leukocyte telomere length may increase as well as decrease in persons with coronary artery disease. Telomere length trajectory is powerfully influenced by baseline telomere length, possibly suggesting negative feedback regulation. Age, male sex, and abdominal obesity independently predict telomere shortening. The mechanisms and reversibility of telomeric aging in cardiovascular disease deserve further study.     

Genetic risk for squamous cell carcinoma of the nictitating membrane parallels that of the limbus in Haflinger horses

Squamous cell carcinoma (SCC) is the most common cancer affecting the equine eye, with a higher incidence documented in Haflinger horses. Recently, a missense variant in the gene damage specific DNA binding protein 2 (DDB2, p.Thr338Met) on ECA12 was identified as a risk factor for the development of limbal SCC in Haflinger horses. SCC also occurs on the nictitating membrane; therefore, investigating the role of this missense variant in nictitating membrane SCC is warranted. In this study, a common ancestor was identified among Haflinger horses affected with limbal SCC or with nictitating membrane SCC, thus supporting a recessive risk factor for the development of cancer at both ocular locations. Analysis of genotype data from Haflinger horses with and without nictitating membrane SCC revealed that the same region on ECA12 associated with limbal SCC was also associated with nictitating membrane SCC (P < 2.04 × 10^?5). Fine mapping of this locus using 25 cases and 49 controls supported the hypothesis that DDB2:c.1013C>T, p.Thr338Met, is a risk factor for nictitating membrane SCC, as 88% of our cases were homozygous for this variant and no other polymorphism was more strongly associated (P = 4.13 × 10^?14). These data indicate that the genetic risk is the same for the development of both limbal and nictitating membrane SCC in Haflinger horses and validates utilization of genetic testing of the DDB2 variant for both clinical management and the guidance of mating decisions.     

A cohort study of racing performance in Japanese Thoroughbred racehorses using genome information on ECA18

Using 1710 Thoroughbred racehorses in Japan, a cohort study was performed to evaluate the influence of genotypes at four single nucleotide polymorphisms (SNPs) on equine chromosome 18 (ECA18), which were associated in a previous genome‐wide association study for racing performance with lifetime earnings and performance rank. In males, both g.65809482T>C and g.65868604G>T were related to performance rank (P = 0.005). In females, g.65809482T>C (P = 1.76E‐6), g.65868604G>T (P = 6.81E‐6) and g.66493737C>T (P = 4.42E‐5) were strongly related to performance rank and also to lifetime earnings (P < 0.05). When win‐race distance (WRD) among all winning racehorses and best race distance (BRD) among elite racehorses were considered as the phenotypes, significant associations (P < 0.001) were observed for all four SNPs. The favourable race distance of both elite (BRD) and novice racehorses (WRD) was also associated with genotypes in the ECA18 region, indicating the presence of a gene in this region influencing optimum race distance in Thoroughbred racehorses. Therefore, the association with performance rank is likely due to the bias in the race distances. The location of the SNPs within and proximal to the gene encoding myostatin (MSTN) strongly suggests that regulation of the MSTN gene affects racing performance. In particular, the g.65809482T>C, g.65868604G>T and g.66493737C>T SNPs, or their combinations, may be genetic diagnostic markers for racing performance indicators such as WRD and BRD.     

Telomere length and dynamics predict mortality in a wild longitudinal study

Explaining variation in life expectancy between individuals of the same age is fundamental to our understanding of population ecology and life history evolution. Variation in the length and rate of loss of the protective telomere chromosome caps has been linked to cellular lifespan. Yet, the extent to which telomere length and dynamics predict organismal lifespan in nature is still contentious. Using longitudinal samples taken from a closed population of Acrocephalus sechellensis (Seychelles warblers) studied for over 20 years, we describe the first study into life‐long adult telomere dynamics (1–17 years) and their relationship to mortality under natural conditions (n = 204 individuals). We show that telomeres shorten with increasing age and body mass, and that shorter telomeres and greater rates of telomere shortening predicted future mortality. Our results provide the first clear and unambiguous evidence of a relationship between telomere length and mortality in the wild, and substantiate the prediction that telomere length and shortening rate can act as an indicator of biological age further to chronological age when exploring life history questions in natural conditions.     

Comparison of Thoroughbred and Arabian horses using RAPD markers

We compared pools of DNA from 10 Thoroughbred horses and 10 Arabian horses for the presence of randomly amplified polymorphic DNA (RAPD) markers which might be useful in distinguishing between the breeds. Using 212 decamer oligonucleotides and our polymerase chain reaction (PCR) conditions, 173 of the primers produced scoreable bands. The number of bands ranged from 0 to 9 with an average of 3·6. In family studies using 11 arbitrarily selected primers, five of the 11 primers produced polymorphic bands which exhibited Mendelian inheritance as dominant markers. When comparing the pooled DNA from Thoroughbred and Arabian horses we found 10 primers which identified markers present in the pooled DNA from one breed but absent in the pool from the other breed. Testing individual horses revealed that only two markers were wholly absent for one group while being present among members of the other. Primer UBC-85 (5′-GTGCTCGTGC-3′) detected a pair of markers absent in Thoroughbred horses but present among 11 of 31 Arabian horses. These markers were 1500 and 1700 base pairs (bp) long and designated UBC-85^C and UBC-85^D, respectively. Primer UBC-126 (5′-CTTTCGTGCT-3′) detected a 1000 bp marker (designated UBC-126^C) absent in 20 of 20 Thoroughbred horses but present in 31 of 31 Arabian horses. UBC-126^C would be particularly effective for breed comparisons, especially if the DNA band were cloned, sequenced and an allelic marker present in Thoroughbred horses but rare or absent among Arabian horses was identified. The distribution of such markers among other horse breeds might be useful to infer relationships among breeds. These kinds of markers may also be useful in detecting unwanted crossbreeding between two horse breeds.     

Genetic mapping of recurrent exertional rhabdomyolysis in a population of North American Thoroughbreds

Recurrent exertional rhabdomyolysis is a heritable disorder that results in painful skeletal muscle cramping with exercise in up to 10% of all Thoroughbred racehorses. Here, we report a genome‐wide association study with 48 282 SNPs analyzed among 48 case and 37 control Thoroughbreds. The most significant SNPs spanned approximately 13 Mb on ECA16, and the P‐value of the most significant SNP after correcting for population structure was 8.0 × 10^?6. This region on ECA16 was further evaluated by genotyping 247 SNPs in both the initial population and a second population of 34 case and 98 control Thoroughbreds. Several SNPs across the 13‐Mb region on ECA16 showed significance when each population was analyzed separately; however, the exact positions of the most significant SNPs within this region on ECA16 varied between populations. This variability in location may be attributed to lack of power owing to insufficient sample sizes within each population individually, or to the relative distribution of long, conserved haplotypes, characteristic of the Thoroughbred breed. Future genome‐wide association studies with additional horses would likely improve the power to resolve casual loci located on ECA16 and increase the likelihood of detecting any additional loci on other chromosomes contributing to disease susceptibility.     

The cosmopolitan maternal heritage of the Thoroughbred racehorse breed shows a significant contribution from British and Irish native mares

The paternal origins of Thoroughbred racehorses trace back to a handful of Middle Eastern stallions, imported to the British Isles during the seventeenth century. Yet, few details of the foundation mares were recorded, in many cases not even their names (several different maternal lineages trace back to 'A Royal Mare'). This has fuelled intense speculation over their origins. We examined mitochondrial DNA from 1929 horses to determine the origin of Thoroughbred foundation mares. There is no evidence to support exclusive Arab maternal origins as some historical records have suggested, or a significant importation of Oriental mares (the term used in historic records to refer to Middle East and western Asian breeds including Arab, Akhal-Teke, Barb and Caspian). Instead, we show that Thoroughbred foundation mares had a cosmopolitan European heritage with a far greater contribution from British and Irish Native mares than previously recognized.     

Short fetal leukocyte telomere length and preterm prelabor rupture of the membranes

Background

Rupture of the fetal membranes is a common harbinger of imminent labor and delivery. Telomere shortening is a surrogate for oxidative stress (OS) and senescence. Fetal leukocyte and placental membrane DNA telomere lengths were evaluated to determine their association with preterm prelabor rupture of the membranes (pPROM) or spontaneous preterm births with intact membranes (PTB), compared to term birth.

Methods

Telomere lengths were quantified in cord blood leukocytes (n = 133) from three major groups: 1) pPROM (n = 28), 2) PTB (n = 69) and 3) uncomplicated full term births (controls, n = 35), using real-time quantitative PCR. Placental membrane specimens (n = 18) were used to correlate fetal leukocyte and placental telomere lengths. Telomere length differences among the groups were analyzed by ANOVA. Pearson correlation coefficients determined relationships between leukocyte and placental membrane telomere lengths.

Results

In pregnancies with intact membranes, fetal leukocyte telomere length was inversely proportional to gestational age. The mean telomere length decreased as gestation progressed, with the shortest at term. pPROM had telomere lengths (9962±3124 bp) that were significantly shorter than gestational age-matched PTB (11546±4348 bp, p = 0.04), but comparable to term births (9011±2497 bp, p = 0.31). Secondary analyses revealed no effects of race (African American vs. Caucasian) or intraamniotic infection on telomere length. A strong Pearson''s correlation was noted between fetal leukocyte and placental membrane telomere lengths (ρ = 0.77; p<0.01).

Conclusions

Fetal leukocyte telomere length is reduced in pPROM compared to PTB but is similar to term births. pPROM represents a placental membrane disease likely mediated by OS-induced senescence.     

A Sequence Polymorphism in MSTN Predicts Sprinting Ability and Racing Stamina in Thoroughbred Horses

Variants of the MSTN gene encoding myostatin are associated with muscle hypertrophy phenotypes in a range of mammalian species, most notably cattle, dogs, mice, and humans. Using a sample of registered Thoroughbred horses (n = 148), we have identified a novel MSTN sequence polymorphism that is strongly associated (g.66493737C>T, P = 4.85×10⁻⁸) with best race distance among elite racehorses (n = 79). This observation was independently validated (P = 1.91×10⁻⁶) in a resampled group of Thoroughbreds (n = 62) and in a cohort of Thoroughbreds (n = 37, P = 0.0047) produced by the same trainer. We observed that C/C horses are suited to fast, short-distance races; C/T horses compete favorably in middle-distance races; and T/T horses have greater stamina. Evaluation of retrospective racecourse performance (n = 142) and stallion progeny performance predict that C/C and C/T horses are more likely to be successful two-year-old racehorses than T/T animals. Here we describe for the first time the identification of a gene variant in Thoroughbred racehorses that is predictive of genetic potential for an athletic phenotype.     

PCR-RFLP analysis of the cytochrome b gene in horse mitochondrial DNA

The mitochondrial DNA sequence of cytochrome b gene in a Thoroughbred horse was determined. By comparing DNA sequences between the Thoroughbred and published sequence data (two horses and one Grevyi zebra), polymerase chain reaction (PCR) primers were designed for amplification of a 590 bp DNA fragment in the cytochrome b gene, and PCR-restriction fragment length polymorphism (RFLP) analysis was studied in 140 horses of six breeds using three restriction enzymes ( AciI, BamHI, RsaI ). Two morphs were found using each of the three enzymes. By combining three enzymes morphs, the 140 horses examined were classified into four types. Type 2 was most frequent in all breeds.     

Age-related telomere attrition in the human putamen

Age is a major risk factor for neurodegenerative diseases. Shortening of leucocyte telomeres with advancing age, arguably a measure of “biological” age, is a known phenomenon and epidemiologically correlated with age-related disease. The main mechanism of telomere shortening is cell division, rendering telomere length in post-mitotic cells presumably stable. Longitudinal measurement of human brain telomere length is not feasible, and cross-sectional cortical brain samples so far indicated no attrition with age. Hence, age-related changes in telomere length in the brain and the association between telomere length and neurodegenerative diseases remain unknown. Here, we demonstrate that mean telomere length in the putamen, a part of the basal ganglia, physiologically shortens with age, like leukocyte telomeres. This was achieved by using matched brain and leukocyte-rich spleen samples from 98 post-mortem healthy human donors. Using spleen telomeres as a reference, we further found that mean telomere length was brain region-specific, as telomeres in the putamen were significantly shorter than in the cerebellum. Expression analyses of genes involved in telomere length regulation and oxidative phosphorylation revealed that both region- and age-dependent expression pattern corresponded with region-dependent telomere length dynamics. Collectively, our results indicate that mean telomere length in the human putamen physiologically shortens with advancing age and that both local and temporal gene expression dynamics correlate with this, pointing at a potential mechanism for the selective, age-related vulnerability of the nigro-striatal network.     

Haplotype diversity in the equine myostatin gene with focus on variants associated with race distance propensity and muscle fiber type proportions

Two variants in the equine myostatin gene (MSTN), including a T/C SNP in the first intron and a 227‐bp SINE insertion in the promoter, are associated with muscle fiber type proportions in the Quarter Horse (QH) and with the prediction of race distance propensity in the Thoroughbred (TB). Genotypes from these loci, along with 18 additional variants surrounding MSTN, were examined in 301 horses of 14 breeds to evaluate haplotype relationships and diversity. The C allele of intron 1 was found in 12 of 14 breeds at a frequency of 0.27; the SINE was observed in five breeds, but common in only the TB and QH (0.73 and 0.48 respectively). Haplotype data suggest the SINE insertion is contemporary to and arose upon a haplotype containing the intron 1 C allele. Gluteal muscle biopsies of TBs showed a significant association of the intron 1 C allele and SINE with a higher proportion of Type 2B and lower proportion of Type 1 fibers. However, in the Belgian horse, in which the SINE is not present, the intron 1 SNP was not associated with fiber type proportions, and evaluation of fiber type proportions across the Belgian, TB and QH breeds shows the significant effect of breed on fiber type proportions is negated when evaluating horses without the SINE variant. These data suggest the SINE, rather than the intron 1 SNP, is driving the observed muscle fiber type characteristics and is the variant targeted by selection for short‐distance racing.