Prevalence of the RAPGEF5 c.2624C>A and PLOD1 c.2032G>A variants associated with equine familial isolated hypoparathyroidism and Fragile Foal Syndrome in the US Thoroughbred population (1988–2019)

Summary

Background:

Equine familial isolated hypoparathyroidism (EFIH) and Fragile Foal Syndrome (FFS) are both fatal recessive conditions reported in Thoroughbred foals. The causal variants for EFIH (RAPGEF5 c.2624C>A; EquCab3.0. chr4: g.54108297G>T) and FFS (PLOD1 c.2032G>A; EquCab3.0, chr2: g.39927817) were recently reported. Prevalence assessment for these variants in a large cohort of samples is needed to provide evidence-based recommendations for genetic testing.

Objectives:

To estimate the frequency of the EFIH and FFS variant alleles in the United States Thoroughbred population between 1988–2019 and determine whether these are recent mutations or are increasing in frequency due to current breeding practices.

Study design:

Population Allele Frequency Study.

Methods:

Genomic DNA from hair and serum samples were genotyped for the EFIH and FFS. Allele frequencies between cohorts, based on year of birth (1988–2000, n=728) and (2001–2019, n=1059), as well as across the seven geographical regions of the United States were compared by Fischer’s Exact tests.

Results:

EFIH and FFS allele frequencies were not significantly different between the two time points studied (0.008 and 0.004 respectively in the older cohorts and 0.008 and 0.009 in most recent years). No EFIH or FFS homozygotes were detected. A sample from 1992 was identified as a carrier for EFIH and one from 1993 a carrier for FFS. Non-significant changes in geographical distribution of carriers for both traits were observed.

Main limitations:

The earliest samples available for study were from foals born in 1988.

Conclusions:

The EFIH and FFS variants are present at low frequency in the United States Thoroughbred population but are not recent mutations. There is no evidence to support changes in allele frequency over time. However, given the closed studbook and breeding practices, continued monitoring of breed allele frequencies and genetic testing is recommended to avoid the mating of carriers and production of affected foals.

Introduction

Equine familial isolated hypoparathyroidism

Equine familial isolated hypoparathyroidism (EFIH; EquCab 3.0. chr4: g.54108297G>T; OMIA-002458–9796) is a fatal condition in Thoroughbred foals characterised by muscle contractions and seizures due to hypocalcemia^1,2. This condition was first characterised as idiopathic hypocalcemia by Beyer et al. in 1997¹. This initial report described five Thoroughbred foals that presented with severe neonatal hypocalcemia, which included a combination of increased phosphorus, decreased ionised calcium, decreased to normal parathyroid hormone, and unidentifiable parathyroid tissue at necropsy. Ages of presenting foals ranged from 4 to 35 days old with clinical signs including seizure activity and tetany¹. Subsequent clinical investigations have described an additional four affected foals².

Calcium supplementation effectively prevents seizures in these foals, indicating that the seizure activity is due to the hypocalcemia and not a primary neurologic insult². This condition is invariably fatal, with foals dying due to the severe hypocalcemia or from euthanasia due to poor prognosis^1,2. In 2019, researchers at UC Davis identified a nonsense variant in the Rap Guanine Nucleotide Exchange Factor 5 (RAPGEF5 c.2624C>A p.Ser875*; EquCab 3.0. chr4: g.54108297G>T) as the genetic basis for idiopathic hypocalcemia and renamed the disease EFIH based on the comparison to familial isolated hypoparathyroidism in humans². This study determined that all four affected foals were homozygous for this variant and thus the mode of inheritance was recessive. A preliminary prevalence estimate was determined in a sample population of 82 Thoroughbreds and allele frequency was 1.8%², estimating 707 out of 20,000 foals each year as carriers. To date, this variant has been identified only in the Thoroughbred breed.

Fragile Foal Syndrome

Fragile Foal Syndrome Type 1 (FFS; EquCab 3.0, chr2: g.39927817; OMIA-001982–9796 (FFS)) is an Ehlers-Danlos-like syndrome caused by a missense mutation in the PLOD1 (c.2032G>A, p.Gly678Arg) gene, which plays an important role in posttranslational modification during collagen fibril formation³. This condition has been documented in 19 foals and/or late term aborted fetuses who are homozygous for the variant^3–8. This recessive condition is characterised by collagen dysplasia, including severe cutaneous lacerations and lesions, thin and friable skin, loose connective tissue attachments, open abdominal cavities, oedema, and haematomas^3,7. Fragile Foal Syndrome Type 1 was initially identified in Warmbloods³ but subsequent research found the presence of the allele in the Thoroughbred⁹ and other breeds⁵. Most recently, the first clinical case was confirmed in the Thoroughbred breed in 2021⁴. Preliminary prevalence estimates in a sample population of 716 US Thoroughbreds determined the FFS allele frequency to be 1.2%⁹, estimating 480 out of 20,000 foals each year as carriers.

While previous prevalence estimates suggest that both EFIH and FFS are relatively rare conditions, these are two of the only three conditions for which a genetic mutation has been identified in the Thoroughbred^2,4. Given that approximately 20,000 Thoroughbreds are registered annually as a part of the breed registry in the US¹⁰, genotyping a larger and representative cohort of samples provides for a more robust estimate of allele frequency for both the EFIH and FFS variants. With the average price of a yearling Thoroughbred across yearling sales in 2021 standing at $80,145¹⁰ and the subsequent costs of breeding fees and veterinary care, even a single loss can result in substantial economic impact. This larger randomised sample set, compared to the initial studies of Thoroughbreds^2,4, will also allow us to determine whether or not this is a recent mutation. Further, comparing the frequency of older samples to new samples will determine if this variant is undergoing selection in the population, and provide a more precise estimate of allele frequency across generations and within the current breeding stock. Results will provide for evidence-based recommendations on genetic testing, which can guide breeding decisions and clinical diagnosis confirmation, therefore minimising economic loss in the industry.

Due to the lack of previous genetic testing, as well as recent identification of these variants in the Thoroughbred breed, we hypothesise that the EFIH (RAPGEF5 c.2624C>A; EquCab 3.0. chr4: g.54108297G>T) and FFS (PLOD1 c.2032G>A; EquCab 3.0, chr2: g.39927817) genetic variants are present at low allele frequency in the Thoroughbred population and date back to the first archived DNA sample (1988). Objectives of the study were to (1) determine the allele frequency of the RAPGEF5 (EquCab 3.0. chr4: g.54108297G>T) and PLOD1 (EquCab 3.0, chr2: g.39927817) variants in a large random cohort of Thoroughbred horses across the 7 geographical regions of the US between 1988–2000 and 2001–2019 and (2) determine if these are recent variants in the Thoroughbred population and whether these variants are undergoing selection by comparing the allele frequencies of these two time points.

Materials and Methods

Sample Selection

The sample population was randomly selected from hair and serum samples, banked at the UC Davis Veterinary Genetics Laboratory, that have been submitted annually from the foal crop for parentage testing at the time of registration. Lists of samples registered per year of birth were subset by the seven geographical regions of the United States (Figure 1) and the last two generations (2001–2019), representing the current breeding stock, were compared to the oldest samples available since the beginning of DNA typing in horses. Four males and four females from each geographical region per year were randomly selected for even geographical distribution of samples for this study, totaling 56 samples per year for 1988–2000 (n=728) and 2001–2019 (n=1059). Based on previously published allele frequency calculations⁵, an overall sample size of n=400–500 samples is adequate to detect significant differences in allele frequencies between these two time periods should they exist. The Jockey Club granted express permission to access Thoroughbred hair and serum samples banked at the UC Davis Veterinary Genetics Laboratory as part of the Jockey Club parentage testing program.

Figure 1:

The seven geographical regions of the United States: Pacific Costal, Rocky Mountain, South-West, Southern, Mid-West, Mid-Atlantic, and New England.

Created with mapchart.net

Genetic Testing

DNA was isolated from hair follicles as previously described¹¹. A modified technique was used for DNA extraction from serum samples. Fifty microlitres of serum per sample was placed in a 0.5 ml PCR Clean Microcentrifuge Tube (Eppendorf Inc). Tubes were centrifuged to concentrate cellular content and excess serum was removed. Fifty microlitres of hair lysis buffer (5 μl Roche Fast start Taq [Roche Applied Science] PCR buffer, 5 μl 25 mM MgCl₂, 0.25 μl Tween 20, and 40 μl water) and 3 μl of Proteinase K (2 mg/mL) were added to each tube. Tubes were incubated at 60°F for 45 minutes for enzymatic digestion, then at 95°F for an additional 45 minutes for denaturing of Proteinase K. One microlitre of hair or serum extract was used for PCR amplification. DNA was amplified for both the RAPGEF5 c.2624C>A (EquCab 3.0. chr4: g.54108297G>T) and PLOD1 c.2032G>A (EquCab 3.0, chr2: g.39927817) variants using the commercially available PCR assay at the UC Davis Veterinary Genetics Laboratory. To ensure accurate genotyping, assays were run with three positive controls (unaffected for both variants, EFIH carrier, FFS carrier) and one negative control (water, no DNA). Positive controls genotyped as expected and negative controls did not yield detectable PCR product. For EFIH, samples were genotyped as (1) N/N genotype does not have the variant that causes EFIH (unaffected), (2) N/EFIH genotype, indicating they carry one copy of the variant (carriers), or (3) EFIH/EFIH genotype with two copies of the variant (affected). For FFS, samples were genotyped as (1) N/N genotype does not have the variant that causes FFS (unaffected), (2) N/FFS genotype, indicating they carry one copy of the variant (carriers), or (3) FFS/FFS genotype with two copies of the variant (affected).

Allele frequencies for EFIH and FSS were calculated by year of birth and by geographical location as well as for each of the time frames using Microsoft Excel. Additionally, 95% confidence intervals for the two time-period populations were calculated using the modified Wald Method¹². Statistical differences in allele frequencies between time periods were evaluated using a two-tailed Fisher’s Exact Test with significant differences established at p<0.05.

Results

EFIH allele frequencies were not significantly different (p=0.85) between the older population (0.008; 95% CI, 0.005–0.015) and the more recent population (0.008; 95% CI, 0.005–0.012, Table 1). The oldest sample with the EFIH variant detected was born in 1992 (Figure 2). Of the 1,789 horses genotyped in this study, 28 were identified as EFIH carriers and no EFIH homozygotes were detected (combined carrier frequency of 0.016).

Table 1:

Summary of EFIH and FFS allele and carrier frequencies between 1988–2000 (n=728) and 2001–2019 (n=1059) detected using the commercially available PCR assay at the UC Davis Veterinary Genetics Laboratory.

		1988–2000 (n=728)	2001–2019 (n=1059)
EFIH	Allele Frequency	0.008	0.008
	95% CI	0.005 to 0.015	0.005 to 0.012
	Carrier Frequency	0.016	0.015
	95% CI	0.009 to 0.029	0.009 to 0.025
FFS	Allele Frequency	0.004	0.009
	95% CI	0.0017 to 0.0092	0.006 to 0.014
	Carrier Frequency	0.008	0.018
	95% CI	0.003 to 0.018	0.011 to 0.028

Figure 2:

The allele frequencies of EFIH and FFS per year from 1988–2019 detected using the commercially available PCR assay at the UC Davis Veterinary Genetics Laboratory.

Similarly, the FFS allele was not significantly different (p=0.10) between the older population (0.004; 95% CI, 0.002–0.009) when compared to the more recent population (0.009, 95% CI, 0.006–0.014, Table 1). The oldest sample with the FFS variant detected was born in 1993 (Figure 2). Of the 1,789 horses genotyped in this study, a carrier frequency of 0.014 (i.e. 25 carriers identified in the total sample set) was identified. Also similar to EFIH, no FFS homozygotes were detected in these data, and no horses genotyped as carriers for both the EFIH and FFS variant alleles. When geographical distribution of carriers for both EFIH and FFS between time periods was assessed (Figure 3), association testing showed no significant differences across the seven geographical regions between the two-time points evaluated (p>0.05).

Figure 3:

The geographical distribution of Thoroughbred EFIH and FFS carriers between 1988–2000 and 2001–2019 across the seven geographical regions of the United States.

Discussion

The objectives of this study were to determine the allele frequency of the RAPGEF5 (c.2624C>A p.Ser875*; EquCab 3.0. chr4: g.54108297G>T) and PLOD1 (c.2032G>A, p.Gly678Arg; EquCab 3.0, chr2: g.39927817) variants in a large random cohort of Thoroughbred horses across the 7 geographical regions of the US between 1988–2000 and 2001–2019 and to determine if these are recent variants in the Thoroughbred population. We confirmed low allele frequencies for RAPGEF5 (EquCab 3.0. chr4: g.54108297G>T) and PLOD1 in both the older and more recent populations. The first sample detected with the EFIH variant was from a Thoroughbred that was born in 1992 and the FFS variant was identified in a Thoroughbred that was born in 1993, indicating their existence in the population for at least 30 years. Statistical significance between populations would indicate differences in the prevalence of the variant by selection through breeding. Given that there was no statistical difference in either allele or carrier frequency between the two time points evaluated, these data do not support that these variants have undergone selection in the Thoroughbred population. However, for EFIH, while allele frequencies had a relative increase in 1997 and 2014 (Figure 2), they were not significantly different. Further research is warranted to analyse correlation of popular sires carrying these variants during specific years. Carrier frequencies of 1.51% for EFIH and 1.79% for FFS from 2001–2019 (Table 1) suggest that about 658 out of 20,000 foals born annually are carriers for EFIH or FFS. This demonstrates the need to continue to monitor allele frequencies within the population to avoid increases in carrier frequencies that could occur from changes in breeding practices.

There were no homozygous horses for either the EFIH or FFS, which is most likely explained by the fatality of both of these recessive conditions. The samples utilised in this study were collected at the time of registration for parentage testing, which is generally 3–4 months following the foaling date¹³. Only one FFS case has been reported in the literature and that foal was euthanised immediately following delivery⁵. The oldest EFIH Thoroughbred foal reported to date was euthanised at 35 days old², which would have been prior to registration and submission of a DNA sample. Additionally, observed shifts in the geographical distribution of both EFIH and FFS carriers among the time periods (Figure 3) were evaluated and were not significant (p>0.05). Further research would be necessary to thoroughly evaluate geographical regions and correlating factors such as sire popularity.

This study demonstrated that both the EFIH (RAPGEF5 c.2624C>A; EquCab 3.0. chr4: g.54108297G>T) and FFS (PLOD1 c.2032G>A; EquCab 3.0, chr2: g.39927817) variants are present at very low frequency in the US Thoroughbred population but are not recent mutations. Continued monitoring of these alleles in the Thoroughbred breed is recommended to ensure breeding practices are not inadvertently increasing the frequency of these diseases. Further, despite low carrier frequencies in the current breeding population, genetic testing is advisable to avoid the mating of carriers and production of EFIH and FFS affected foals given the economic impact of a single loss.

Data availability statement

The data that support the findings of this study are available at the request from the corresponding authors.