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Genomic Association of Chronic Idiopathic Anhidrosis to a Potassium Channel Subunit in a Large Animal Model

  • Laura Patterson Rosa
    Affiliations
    Department of Animal Sciences, College of Agriculture and Life Sciences, University of Florida, Gainesville, Florida, USA

    UF Genetics Institute, University of Florida, Gainesville, Florida, USA
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  • Neely Walker
    Affiliations
    School of Animal Sciences, Louisiana State University, Baton Rouge, Louisiana, USA
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  • Martha Mallicote
    Affiliations
    Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA
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  • Robert J. MacKay
    Affiliations
    Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA
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  • Samantha A. Brooks
    Correspondence
    Correspondence: Samantha A. Brooks, Department of Animal Sciences, College of Agricultural and Life Sciences, University of Florida, 2250 Shealy Drive, PO Box 110910, Gainesville, Florida 32611, USA.
    Affiliations
    Department of Animal Sciences, College of Agriculture and Life Sciences, University of Florida, Gainesville, Florida, USA

    UF Genetics Institute, University of Florida, Gainesville, Florida, USA
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Open AccessPublished:May 31, 2021DOI:https://doi.org/10.1016/j.jid.2021.05.014
      Similar to humans, the horse relies predominantly on the evaporation of sweat from the skin surface to dissipate excess body heat. Loss of the sweat response or anhidrosis can result in life-threatening hyperthermia. Anhidrosis occurs more frequently in some breeds as well as occurs at an increased frequency among individuals with a family history, suggesting a heritable component to the pathology. Given the natural occurrence and indications of genetic components in the etiology, we utilized genomics to better understand the molecular mechanisms involved in sweat response. We performed a case-control (n = 200) GWAS targeting cases of chronic idiopathic anhidrosis in a controlled genetic background to discover the contributing regions and interrogated gene function for roles in the sweating mechanism. A region containing the KCNE4 gene, which encodes the β-subunit of a potassium channel protein with a possible function in sweat gland outflow, was associated (P = 1.13 × 10-07) with chronic idiopathic anhidrosis through GWAS. A candidate mutation (NC_009149.3:g.11813731A > G, rs68643109) disrupting the KCNE4 protein structure could explain the disease but requires further investigation in larger populations. We show the potential role of ion channels and cellular damage in sweat response, correlating anhidrosis as a possible effect of congenital channelopathy.

      Abbreviation:

      CIA (chronic idiopathic anhidrosis), K+ (potassium ion)

      Introduction

      Anhidrosis is the loss of the ability to sweat in response to appropriate stimuli (
      • Breuhaus B.A.
      Thyroid function in anhidrotic horses.
      ). A poorly understood disease in horses, idiopathic anhidrosis affects 2% of the horse population in Florida (
      • Johnson E.B.
      • MacKay R.J.
      • Hernandez J.A.
      An epidemiologic study of anhidrosis in horses in Florida.
      ). Equine sweat production begins with the secretion of a mixture of water, proteins, and electrolytes into a coiled tubular fundus within the gland (
      • Evans C.L.
      • Ross K.A.
      • Smith D.F.G.
      • Weil-malherbe H.
      A physiological explanation of equine tropical anhidrosis.
      ;
      • Kerr M.G.
      • Snow D.H.
      Composition of sweat of the horse during prolonged epinephrine (adrenaline) infusion, heat exposure, and exercise.
      ). Within the equine sweat gland, serpentine excretory ducts associate closely with hair follicles, a characteristic of apocrine-type glands despite the eccrine-type function (
      • Evans C.L.
      • Ross K.A.
      • Smith D.F.G.
      • Weil-malherbe H.
      A physiological explanation of equine tropical anhidrosis.
      ;
      • Jenkinson D.M.
      • Elder H.Y.
      • Bovell D.L.
      Equine sweating and anhidrosis part 2: anhidrosis.
      ). To dissipate excess body heat, the horse relies predominantly on the evaporation of copious amounts of sweat from the skin, a strategy unique to just three mammals: equids, humans, and the Erythrocebus patas monkey (
      • Robertshaw D.
      Sweat and heat-exchange in man and other mammals.
      ). Thermoregulation in the horse utilizes hair erection and a large transpiration capacity through the sweat glands distributed across nearly the entire body surface (
      • Thomassian A.
      • Anidrose Termogenica.
      ). Despite these efficient heat dissipation mechanisms, the equine thermoregulatory system can be overwhelmed, resulting in critical hyperthermia (
      • McCutcheon L.J.
      • Geor R.J.
      Sweating. Fluid and ion losses and replacement.
      ).
      In horses, clinical signs of anhidrosis include dry skin, tachypnea, hyperthermia, decreased water intake, alopecia, dull hair coat, and depression (
      • Currie A.K.
      • Seager S.W.
      ;
      • Geor R.J.
      • McCutcheon L.J.
      Thermoregulation and clinical disorders associated with exercise and heat stress.
      ). Severe hyperthermia results in collapse, convulsions, and death (
      • Warner A.
      • Mayhew I.G.
      Equine anhidrosis: a review of pathophysiologic mechanisms.
      ). Anhidrotic horses suffer considerably during work and warm seasons, leading to decreased athletic performance and a reduced QOL. They require intensive management, restricted physical activity, and early retirement from breeding or competition to survive (
      • Breuhaus B.A.
      Thyroid function in anhidrotic horses.
      ;
      • McEwan Jenkinson D.M.
      • Elder H.Y.
      • Bovell D.L.
      Equine sweating and anhidrosis Part 1 — equine sweating.
      ). Environmental triggers such as overtraining, failure to acclimate after travel to a hotter climate, and electrolyte imbalance can elicit an acute anhidrotic episode (
      • MacKay R.J.
      • Mallicote M.
      • Hernandez J.A.
      • Craft W.F.
      • Conway J.A.
      A review of anhidrosis in horses.
      ). In contrast, chronic idiopathic anhidrosis (CIA) is characterized by repeated symptomatic phases and defies environmental factors. An epidemiological study revealed that at the individual level, the risk for CIA varies significantly by breed and family history (OR of 21.67), highlighting a strong underlying genetic component to this disease (
      • Johnson E.B.
      • MacKay R.J.
      • Hernandez J.A.
      An epidemiologic study of anhidrosis in horses in Florida.
      ). These findings support the hypothesis of a hereditary component contributing to CIA.
      We selected stock-type horses as target breeds for this study (American Quarter Horse, American Paint Horse, and Appaloosa) and the Thoroughbred (the genetic foundation of stock-type breeds as well as still bred to modern individuals) on the basis of the frequency and distribution of the disease (
      • Johnson E.B.
      • MacKay R.J.
      • Hernandez J.A.
      An epidemiologic study of anhidrosis in horses in Florida.
      ). In this study, we report the results of a national survey to characterize CIA as well as a GWAS designed to identify the genetic factors contributing to the disease.

      Results

      Survey analysis

      A total of 385 likely cases were submitted through the survey: concomitant diagnosis for diseases with similar and confounding signs excluded 160 individuals; 75 had experienced only one anhidrotic episode and did not meet the criteria for the chronic form of the disease. A total of 172 survey responses were analyzed. Horses from the Thoroughbred breed comprised a large proportion (32.7%) of the CIA case responses (Figure 1). Males (115 geldings, 5 stallions) represented most CIA cases (69.1%). CIA cases ranged in age from 5 to 33 years, and those affected year round (mean = 18.66, SD = 7.48) were significantly older than seasonally affected individuals (mean = 14.7, SD = 6.02; χ2 [1, n = 165] = 13.58, P = 0.0002 [Bonferroni correction P ≤ 0.0029]).
      Figure thumbnail gr1
      Figure 1Ponies, Thoroughbreds, and Warmbloods are the most frequently reported among anhidrosis cases, after stock-type horses (n = 172). TWH, Tennessee walking horse.
      Horse caretakers most frequently noted a lack of sweat on the hindquarters (n = 167), followed by dyspnea/tachypnea (n = 154) and lack of sweat on the neck (n = 153) or on the back (n = 107). Rectal temperatures >40 °C (104 °F) were observed only in 30.8% of survey cases. Survey respondents also reported polydipsia (n = 48) and anorexia (n = 14), signs not previously described for this condition (Table 1). Mapping of the postal code of where affected horses reside illustrated a distribution across the United States and including diverse climate zones/regions (Figure 2).
      Table 1Affected Horse’s Clinical Signs Reported by Survey Respondents
      ConditionNumber of ReportsPercentage of Reports
      Lack of sweating on the hindquarter16797.1%
      Shortness of breath/rapid breathing15489.5%
      Lack of sweating on the neck15389.0%
      Lack of sweating under the saddle/on the back10762.2%
      Elevated heart rate (tachycardia)8650.0%
      Lethargy (reluctant to work or move)7141.3%
      Higher body temperature (>40 °C/104 °F)5330.8%
      Increased or excessive water consumption (polydipsia)4827.9%
      Thinning or loss of hair (alopecia)3419.8%
      Dry flaky skin2816.3%
      Decreased water consumption2514.5%
      Poor appetite (anorexia)148.1%
      The survey had the option to report more than one signal per horse.
      Figure thumbnail gr2
      Figure 2Anhidrosis cases are not isolated to the hot climates of the southern United States (n = 172; map generated by maptive.com, geographic map generator). Concentration in the states of Florida and Louisiana is due to the regional advertisement of the study. CT, Connecticut; DE, Delaware; INEGI, Instituto Nacional de Estadistica y Geografia (Mexico), NJ, New Jersey; RI, Rhode Island.

      Equine CIA genome-wide association analysis

      Genotypes were generated through this work for 115 cases originated from the survey and 78 stock-type controls from the University of Florida (Gainesville, FL) equine herd. A collaborator from the University of Kentucky provided supplementary control genotypes of 153 Thoroughbreds. Using an identity by state-matched case to control (1:1) totaling 200 individuals, the GWAS resulted in four markers exceeding the significance threshold post-Bonferroni correction (raw P < 6.03 × 10–7) (Table 2 and Figure 3), well-supported by neighboring SNPs (r2 > 0.04). Five genes were annotated within the chromosome 6 region, whereas the chromosome 7 markers revealed a region rich in zinc-finger transcription factors with high paralogy. The two remaining loci were not assembled in EquCab2 but were found on chromosome 7 and chromosome X in EquCab3.
      Table 2GWAS Significant Markers and Respective Locations
      Candidate RegionGenes
      Marker IDP-ValueChrEquCab2 BPEquCab 2EquCab3
      AX-1038221511.13 × 10–7611896273chr6:11443300-12249279chr6:11212563-12024244SGPP2
      FARSB
      MOGAT
      ACSL3
      KCNE4
      AX-1036035662.35 × 10–7Unk27305043chrUn:27228677-27348533chrX:43239168-43244703∗ASB1
      AX-1034848323.63 × 10–07Unk19421320chrUn:19131513-19421320chr7:49335006-49454862‬Zinc-finger rich region
      AX-1029664815.35 × 10–7746963254chr7:46013490-47961836chr7:47298766-49253548Zinc-finger rich region
      Abbreviation: Chr, chromosome; ID, identification.
      Figure thumbnail gr3
      Figure 3Genome-wide association of CIA case/control status (n = 200). (a) GWAS identified 12 significant markers (P = 6.03 × 10–7, Bonferroni = solid black line) (b) Quantile‒quantile plot demonstrating a genomic inflation factor (λ) of 1.22951. (c, d) Markers from the candidate region and respective genes from the Equus caballus annotation release 102 (NCBI), color coded by r2 value. CIA, chronic idiopathic anhidrosis; NCBI, National Center for Biotechnology Information.

      Candidate variant analysis

      We inspected two stock-type resequenced genomes (one chronic case, one control) for causative variants. Four of the five genes had no changes in the coding sequence or were related to anhidrosis phenotypes in other species. However, we identified three variants in the case genome within the KCNE4 gene: one synonymous (NC_009149.3:g.11813106G > A, rs68643107) and two missense (NC_009149.3:g.11813731A > G, rs68643109 and NC_009149.3:g.11814204A > C, p. Asn156His, rs1151561929).
      Equine KCNE4 likely has a human-like gene structure, with two total exons (National Center for Biotechnology Information Equus caballus annotation release 103) (
      • Abbott G.W.
      Novel exon 1 protein-coding regions N-terminally extend human KCNE3 and KCNE4.
      ). Most other mammalian species possess only one exon, and therefore, there is little phylogenetic data with which to infer conservation of residues for the human/horse upstream exon, a key piece of evidence for functional prediction tools such as Polymorphism Phenotyping, version 2 (
      • Adzhubei I.A.
      • Schmidt S.
      • Peshkin L.
      • Ramensky V.E.
      • Gerasimova A.
      • Bork P.
      • et al.
      A method and server for predicting damaging missense mutations.
      ) (Supplementary Figure S1). Therefore, to investigate the possible functional impacts of the missense changes, rs68643109 and rs1151561929, we performed a protein structure analysis on Iterative Threading ASSEmbly Refinement (
      • Yang J.
      • Yan R.
      • Roy A.
      • Xu D.
      • Poisson J.
      • Zhang Y.
      The I-TASSER Suite: protein structure and function prediction.
      ) on the basis of the translated KCNE4 protein model from the National Center for Biotechnology Information annotation release 103. The A allele at rs68643109 is predicted to support an α-helix structure instead of a random coil (G allele) in the 10th amino acid, altering the expected protein tertiary structure (Figure 4) (
      • Yang J.
      • Yan R.
      • Roy A.
      • Xu D.
      • Poisson J.
      • Zhang Y.
      The I-TASSER Suite: protein structure and function prediction.
      ). The rs1151561929 missense change had no impact on the predicted protein structure.
      Figure thumbnail gr4
      Figure 4Top-ranked I-TASSER structural predictions for KCNE4 modeled after the NCBI gene prediction mRNA (XM_023642424.1). (a) The predicted structure for the reference (CIA unaffected) sequence resembles other KCNE superfamily protein structures, (b) whereas the protein bearing the rs68643109 variant is folded over itself and with an extra coil structure (orange arrows). CIA, chronic idiopathic anhidrosis; I-TASSER, Iterative Threading ASSEmbly Refinement; NCBI, National Center for Biotechnology Information.

      Discussion

      Insights from the survey

      Previous work examining the epidemiology of anhidrosis successfully relied solely on owner- and veterinarian-reported diagnoses; therefore, we applied a similar approach (
      • Johnson E.B.
      • MacKay R.J.
      • Hernandez J.A.
      An epidemiologic study of anhidrosis in horses in Florida.
      ;
      • Mayhew I.G.
      • Ferguson 2nd, H.O.
      Clinical, clinicopathologic, and epidemiologic features of anhidrosis in Central Florida thoroughbred horses.
      ;
      • Warner A.E.
      • Mayhew I.G.
      Equine anhidrosis: a survey of affected horses in Florida.
      ). Although regional advertising strategies resulted in over-representation of responses for horses in Florida and Louisiana, we received reports of CIA-affected horses from diverse climates. CIA diagnosis could be more frequent in the southern United States as a result of more days of hot and humid weather exacerbating CIA signs (

      NOAA. National Climatic Data Center. Annual Report of Climate of 2005-2012, https://www.ncdc.noaa.gov/about-ncdc/annual-reports.html, 2020 (accessed 19 June 2020)

      ). Tropical climates contribute to more frequent reporting of equine anhidrosis (
      • Warner A.
      • Mayhew I.G.
      Equine anhidrosis: a review of pathophysiologic mechanisms.
      ); yet, both chronic and acute anhidrosis are frequently recognized in many nontropical regions (
      • Hodgson D.R.
      • Davis R.E.
      • McConaghy F.F.
      Thermoregulation in the horse in response to exercise.
      ;
      • Warner A.
      • Mayhew I.G.
      Equine anhidrosis: a review of pathophysiologic mechanisms.
      ,
      • Warner A.E.
      • Mayhew I.G.
      Equine anhidrosis: a survey of affected horses in Florida.
      ).

      Possible role of potassium in CIA

      KCNE4 is a potassium channel β subunit with an inhibitory effect on the KCNQ1 channel (α subunit) (
      • Grunnet M.
      • Jespersen T.
      • Rasmussen H.B.
      • Ljungstrøm T.
      • Jorgensen N.K.
      • Olesen S.P.
      • et al.
      KCNE4 is an inhibitory subunit to the KCNQ1 channel.
      ). The KCNQ1 gene encodes a six transmembrane domain voltage-gated potassium channel that controls the Kv7.1 potassium current (
      • Abbott G.W.
      Biology of the KCNQ1 potassium channel.
      ) by interacting with multiple possible β subunits, including KCNE4 (
      • Abbott G.W.
      Biology of the KCNQ1 potassium channel.
      ). KCNE4 contributes to myocardial repolarization and vascular tone in mice (
      • Abbott G.W.
      • Jepps T.A.
      Kcne4 deletion sex-dependently alters vascular reactivity.
      ). The role of the KCNQ1 channel in sweat secretion is not well-documented; however, the gene product is seven-fold more expressed in wild-type mice footpads than in the footpads from the sweat gland‒absent mouse model EdaTa Tabby (
      • Cui C.Y.
      • Sima J.
      • Yin M.
      • Michel M.
      • Kunisada M.
      • Schlessinger D.
      Identification of potassium and chloride channels in eccrine sweat glands.
      ). Both KCNE4 and KCNQ1 are expressed in horse skin (
      • Mansour T.A.
      • Scott E.Y.
      • Finno C.J.
      • Bellone R.R.
      • Mienaltowski M.J.
      • Penedo M.C.
      • et al.
      Tissue resolved, gene structure refined equine transcriptome.
      ).
      When bound to KCNE1, KCNQ1 activation is accelerated in higher (32 °C) temperatures (20 °C) compared with that in cooler temperatures, resulting in increased potassium ion (K+) excretion (
      • Abbott G.W.
      Biology of the KCNQ1 potassium channel.
      ). Normal equine sweat glands may have an adaptive mechanism limiting the K+ excretion response to thermal stress (
      • Jenkinson D.M.
      • Loney C.
      • Elder H.Y.
      • Montgomery I.
      • Mason D.K.
      Effects of season and lower ambient temperature on the structure of the sweat glands in anhidrotic horses.
      ). Skin cells from anhidrotic horses have decreased short circuit responses and fail to respond to β-adrenergic agonist stimulation, indicating an impairment of epithelial ion transport during anhidrosis (
      • Wilson D.C.
      • Corbett A.D.
      • Steel C.
      • Pannirselvam R.
      • Bovell D.L.
      A preliminary study of the short circuit current (Isc) responses of sweat gland cells from normal and anhidrotic horses to purinergic and adrenergic agonists.
      ). Furthermore, the little sweat that can be gathered from anhidrotic horses has a significantly higher concentration of K+ and chloride ion (2.7 and 1.8 fold, respectively) (
      • Marlin D.J.
      • Schroter R.C.
      • Scott C.M.
      • White S.
      • Nyrop K.A.
      • Maykuth P.L.
      • et al.
      Sweating and skin temperature responses of normal and anhidrotic horses to intravenous adrenaline.
      ). In sweat glands from normally sweating horses, a temporary flattened cellular configuration with fewer cytoplasmic vesicles postmaximal secretion is suggestive of prolonged K+ channel activity during sweat production (
      • Jenkinson D.M.
      • Montgomery I.
      • Elder H.Y.
      • Mason D.K.
      • Collins E.A.
      • Snow D.H.
      Ultrastructural variations in the sweat glands of anhidrotic horses.
      ;
      • McEwan Jenkinson D.M.
      • Elder H.Y.
      • Bovell D.L.
      Equine sweating and anhidrosis Part 1 — equine sweating.
      ;
      • Pollock A.S.
      • Arieff A.I.
      Abnormalities of cell volume regulation and their functional consequences.
      ). In contrast, immunohistochemistry of CIA cases shows that cells within the sweat gland permanently lose their normally cuboidal morphology, becoming flattened with diversely shaped nuclei, extremely vacuolated cytoplasm (
      • Bovell D.L.
      • Lindsay S.L.
      • Corbett A.D.
      • Steel C.
      Immunolocalization of aquaporin-5 expression in sweat gland cells from normal and anhidrotic horses.
      ), and thickened basal lamina, leading to irreversible cellular damage (
      • Jenkinson D.M.
      • Elder H.Y.
      • Bovell D.L.
      Equine sweating and anhidrosis part 2: anhidrosis.
      ,
      • Jenkinson D.M.
      • Loney C.
      • Elder H.Y.
      • Montgomery I.
      • Mason D.K.
      Effects of season and lower ambient temperature on the structure of the sweat glands in anhidrotic horses.
      ,
      • Jenkinson D.M.
      • Montgomery I.
      • Elder H.Y.
      • Mason D.K.
      • Collins E.A.
      • Snow D.H.
      Ultrastructural variations in the sweat glands of anhidrotic horses.
      ). In contrast, the sweat glands of acutely anhidrotic horses appear histologically normal, suggesting a transient or functional defect (
      • Evans C.L.
      • Nisbet A.M.
      • Ross K.A.
      A histological study of the sweat glands of normal and dry-coated horses.
      ;
      • Peter J.E.
      • Boge P.
      • Morris P.G.
      • Gordon B.J.
      Anhidrosis in a thoroughbred.
      ). Therefore, the chronic inability to resume proper sweating may be due to irreversible structural damage of the gland, possibly due to excess K+ secretion (
      • Jenkinson D.M.
      • Elder H.Y.
      • Bovell D.L.
      Equine sweating and anhidrosis part 2: anhidrosis.
      ,
      • Jenkinson D.M.
      • Montgomery I.
      • Elder H.Y.
      • Mason D.K.
      • Collins E.A.
      • Snow D.H.
      Ultrastructural variations in the sweat glands of anhidrotic horses.
      ).
      Kcne4-knockout mice have a significantly inhibited response to the β-adrenoreceptor agonist isoprenaline (
      • Abbott G.W.
      • Jepps T.A.
      Kcne4 deletion sex-dependently alters vascular reactivity.
      ), suggesting that Kcne4 mediates the intracellular β-adrenoreceptor response. Given the importance of β-adrenergic signaling in the control of equine sweat glands (
      • Bijman J.
      • Quinton P.M.
      Predominantly beta-adrenergic control of equine sweating.
      ;
      • Wilson D.C.
      • Corbett A.D.
      • Steel C.
      • Pannirselvam R.
      • Bovell D.L.
      A preliminary study of the short circuit current (Isc) responses of sweat gland cells from normal and anhidrotic horses to purinergic and adrenergic agonists.
      ), this pathway is frequently proposed as a causative mechanism for equine anhidrosis (
      • Evans C.L.
      • Smith D.F.
      • Ross K.A.
      • Weil-Malherbe H.
      Physiological factors in the condition of “dry coat” in horses.
      ,
      • Evans C.L.
      • Smith D.F.G.
      • Weil-Malherbe H.
      The relation between sweating and the catechol content of the blood in the horse.
      ;
      • McEwan Jenkinson D.M.
      • Elder H.Y.
      • Bovell D.L.
      Equine sweating and anhidrosis Part 1 — equine sweating.
      ). In culture, nonanhidrotic horse sweat glands exposed to increased agonist stimulation develop desensitization and downregulation of the β-adrenoceptors through a refractory effect (
      • Freedman N.J.
      • Lefkowitz R.J.
      Desensitization of G protein-coupled receptors.
      ;
      • Rakhit S.
      • Murdoch R.
      • Wilson S.M.
      Persistent desensitisation of the beta 2 adrenoceptors expressed by cultured equine sweat gland epithelial cells.
      ). A defect in β-adrenergic signaling, possibly mediated through altered KCNE4 function, could explain why anhidrotic horses have a lowered response to β-agonists such as terbutaline (
      • Hubert J.D.
      • Beadle R.E.
      • Norwood G.
      Equine anhidrosis.
      ;
      • MacKay R.J.
      Quantitative intradermal terbutaline sweat test in horses.
      ).
      The KCNE4 rs68643109 variant protein structural prediction suggests tertiary structure changes. If these changes were to result in loss of KCNE4 β-subunit binding affinity, relaxing the regulatory inhibition of the KCNQ1 channel (
      • Grunnet M.
      • Jespersen T.
      • Rasmussen H.B.
      • Ljungstrøm T.
      • Jorgensen N.K.
      • Olesen S.P.
      • et al.
      KCNE4 is an inhibitory subunit to the KCNQ1 channel.
      ), they would also lead to prolonged exposure of the β2 receptor to agonists and a lack of current activation inhibition, ultimately causing desensitization (
      • Jenkinson D.M.
      • Elder H.Y.
      • Bovell D.L.
      Equine sweating and anhidrosis part 2: anhidrosis.
      ). This would be a progressive process dependent on thermoregulatory sweat as the trigger, leading to a chronic degenerative decrease in the sweat response capacity.
      In the mouse, the activity of KCNE4 varies significantly with age, resulting in a decrease in the inhibitory effect of Kv7.1 channels in older mice (
      • Abbott G.W.
      • Jepps T.A.
      Kcne4 deletion sex-dependently alters vascular reactivity.
      ;
      • Crump S.M.
      • Hu Z.
      • Kant R.
      • Levy D.I.
      • Goldstein S.A.
      • Abbott G.W.
      Kcne4 deletion sex- and age-specifically impairs cardiac repolarization in mice.
      ). Equine CIA may also change with age because older horses are over-represented among cases suffering from year-round CIA, compared with those with milder seasonal signs (χ2 [1, n = 165] = 13.58, P = 0.0002, Bonferroni threshold P ≤ 0.0029). We also observed a significant effect of sex within our GWAS cohort, with males being over-represented among CIA cases (χ2 [1, n = 200] = 25.672, P < 0.0001). This could in part be due to sampling bias because geldings are more commonly kept as working horses where an inability to sweat would be more readily observed by caretakers and because relatively few stallions were available for sampling. However, Kcne4 gene expression does vary by sex and correlates with cardiac pathology severity in mice (
      • Abbott G.W.
      • Jepps T.A.
      Kcne4 deletion sex-dependently alters vascular reactivity.
      ;
      • Crump S.M.
      • Hu Z.
      • Kant R.
      • Levy D.I.
      • Goldstein S.A.
      • Abbott G.W.
      Kcne4 deletion sex- and age-specifically impairs cardiac repolarization in mice.
      ). In this model, intact males and postmenopausal females possess higher expression levels, whereas castrated males have lower expression levels of Kcne4, comparable with premenopausal female ranges (
      • Crump S.M.
      • Hu Z.
      • Kant R.
      • Levy D.I.
      • Goldstein S.A.
      • Abbott G.W.
      Kcne4 deletion sex- and age-specifically impairs cardiac repolarization in mice.
      ). Given the cardiac and vascular pathologies associated with this murine allele (
      • Abbott G.W.
      • Jepps T.A.
      Kcne4 deletion sex-dependently alters vascular reactivity.
      ;
      • Crump S.M.
      • Hu Z.
      • Kant R.
      • Levy D.I.
      • Goldstein S.A.
      • Abbott G.W.
      Kcne4 deletion sex- and age-specifically impairs cardiac repolarization in mice.
      ) and the likely impact of the rs68643109 equine variant on protein structure, similar cardiovascular phenotypes should be investigated in horses carrying this allele.
      The Kcne4 rs68643109 missense mutation, should its association with CIA disease hold true in additional populations, establishes a noteworthy hypothesis for CIA treatment. In humans, ion channelopathies result in diverse conditions ranging from epilepsies to cystic fibrosis (
      • Imbrici P.
      • Liantonio A.
      • Camerino G.M.
      • De Bellis M.
      • Camerino C.
      • Mele A.
      • et al.
      Therapeutic approaches to genetic ion channelopathies and perspectives in drug discovery.
      ). In the latter, defects in the CFTR gene, a chloride channel, cause an increased concentration of chloride observed in sweat, along with proinflammatory and fibrogenic responses, especially in the lung (
      • Bernstein M.L.
      • McCusker M.M.
      • Grant-Kels J.M.
      Cutaneous manifestations of cystic fibrosis.
      ;
      • Mall M.A.
      • Galietta L.J.V.
      Targeting ion channels in cystic fibrosis.
      ). Naturally occurring channelopathies in the horse could be an advantageous treatment/research model for therapeutics (
      • Balfour-Lynn I.M.
      • King J.A.
      CFTR modulator therapies–effect on life expectancy in people with cystic fibrosis [e-pub ahead of print].
      ;
      • Imbrici P.
      • Liantonio A.
      • Camerino G.M.
      • De Bellis M.
      • Camerino C.
      • Mele A.
      • et al.
      Therapeutic approaches to genetic ion channelopathies and perspectives in drug discovery.
      ). Given the postulated protein structural changes and the potentially degenerative outcomes of loss of regulation of KCNQ1, the KCNE4 variant is a promising candidate for CIA and a strong prospect for further study.
      Equine CIA is not limited to specific climatic regions and affects diverse horse breeds. GWAS within one breed type revealed a strongly supported candidate region containing the KCNE4 gene, and sequence analysis revealed an SNP likely to alter KCNE4 protein function. These findings suggest a genetic factor in CIA etiology, yet require further exploration, including the validation of the association between this sequence variant and CIA in a new cohort as well as expanded phenotyping and sex-matching of controls. Investigation of possible concomitant pathologies as well as longitudinal and epidemiological studies evaluating age and sex correlations would also improve our understanding of the mechanisms underlying sweat response. Given that CIA is exacerbated by warmer weather, global warming will likely increase the prevalence of this condition in the future (
      • Körner C.
      • Basler D.
      Phenology under global warming.
      ). We anticipate that this work will be followed by the development of genetic diagnostic tools for equine CIA as well as innovative treatments for channelopathies causing anhidrosis.

      Materials and Methods

      Ethics statement

      All samples were collected voluntarily under the University of Florida Institutional Animal Care and Use Committees 201408411.

      Recruitment and survey analysis

      Sampling utilized a survey distributed from 2016 to 2018 (Qualtrics, Provo, UT) (

      Qualtrics [computer program]. Seattle, UT: Qualtrics: online survey software & insight platform; 2014.

      ) and/or written veterinary health history describing clinical signs and a hair sample from each horse for DNA extraction. We mapped the location of each CIA-affected horse by the reported postal code utilizing Maptive.com (https://www.maptive.com/). For the reported 390 horses, 5 were volunteered controls, leaving 385 likely cases. We removed 160 horses from further analyses owing to concomitant diagnosis for diseases with similar and confounding signs (i.e., Equine Asthma). A total of 75 horses had experienced only one anhidrotic episode and did not meet the criteria for the chronic form of the disease. This left 172 horses, where 115 were reported as registered stock-type breeds (American Quarter Horse, American Paint Horse, and Appaloosa, n = 58) and Thoroughbred (n = 57) (Supplementary Figure S1). A total of 78 stock-type horses previously banked at the University of Florida, plus genotypes of 153 Thoroughbreds previously banked at the University of Kentucky (Lexington, KY), both groups with no known clinical history for CIA, were included as controls. Statistical analysis of survey responses was conducted in JMP, Version 14.1 (SAS Institute Inc., Cary, NC) (

      SAS Institute [computer program]. Cary, NC: JMP. Stat Methods 2018.

      ), utilizing Pearson’s chi-square test and a Bonferroni adjusted α level of 0.025 (0.05/2).

      DNA extraction and genotyping

      DNA extracted as previously described (
      • Cook D.
      • Brooks S.
      • Bellone R.
      • Bailey E.
      Missense mutation in exon 2 of SLC36A1 responsible for champagne dilution in horses.
      ) was genotyped utilizing the Affymetrix 670k Axiom Equine Genotyping Array, giving an average genotyping rate of 0.9956 (GeneSeek, Lincoln, NE; Affymetrix/Thermo Fisher Scientific, Waltham, MA) for the 178 newly collected samples (100 cases and 78 controls). We retained only PolyHighResolution markers (n = 389,105) in the Axiom Analysis Suite Software (Affymetrix/Thermo Fisher Scientific) and filtered for quality using PLINK, version 1.90b3.39 (
      • Purcell S.
      • Neale B.
      • Todd-Brown K.
      • Thomas L.
      • Ferreira M.A.R.
      • Bender D.
      • et al.
      PLINK: a tool set for whole-genome association and population-based linkage analyses.
      ) (--maf 0.05 --geno 0.05; 589 and 118,018 SNPs removed). We assessed the association with genotyping batch using PLINK (178 samples batch #1 and 153 samples batch #2). SNP loci differing significantly in genotype by batch (force discovery rate < 0.05; 7,846 total markers) were excluded (
      • Benjamini Y.
      • Hochberg Y.
      Controlling the false discovery rate: a practical and powerful approach to multiple testing.
      ;

      Holl HM, Brooks SA. Transitioning from Illumina to Affymetrix: platform concordance and lessons learned. Paper presented at: International Plant and Animal Genome Conference XXVI. January 13–17, 2018; San Diego, CA.

      ). After filtering for quality, 262,652 informative markers remained.

      GWAS

      To reduce population stratification, clustering in PLINK (
      • Purcell S.
      • Neale B.
      • Todd-Brown K.
      • Thomas L.
      • Ferreira M.A.R.
      • Bender D.
      • et al.
      PLINK: a tool set for whole-genome association and population-based linkage analyses.
      ) matched cases to controls using identity by state at a 1:1 ratio (--cluster --mcc 1 1). A principal component analysis implemented in Genome-wide complex trait analysis (
      • Yang J.
      • Lee S.H.
      • Goddard M.E.
      • Visscher P.M.
      GCTA: a tool for genome-wide complex trait analysis.
      ) assessed relatedness in the matched population (n = 200) (Supplementary Figure S2).
      We performed the GWAS in Genome-wide complex trait analysis utilizing a Genetic Relationship Matrix‒corrected Mixed Linear Model Association—Restricted Maximum Likelihood approach on the identity by state matched population (n = 200). Markers above the Bonferroni corrected (0.05 ÷ 262,652 markers) P = 6.03 × 10–7 were inspected for supporting linkage (r2) in PLINK (--chr [ChromosomeNumber] --r2 --ld-snp [MarkerID] --ld-window-r2 0.00--ld-window 1000). Loci with an r2 ≥ 0.4 to the lowest P-value SNP defined the boundaries of candidate regions considered for further analysis (
      • Patterson Rosa L.
      • Mallicote M.F.
      • Long M.T.
      • Brooks S.A.
      Metabogenomics reveals four candidate regions involved in the pathophysiology of Equine Metabolic Syndrome.
      ;
      • Weich K.
      • Affolter V.
      • York D.
      • Rebhun R.
      • Grahn R.
      • Kallenberg A.
      • et al.
      Pigment intensity in dogs is associated with a copy number variant upstream of KITLG [published correction appears in Genes (Basel) 2021;12:75].
      ).

      Whole genome resequencing and variant analysis

      We selected one chronic case and one control (both stock type) on the basis of health history, accessibility to high-quality samples (leucocytes), same breed, and similar management practices/location for genomic DNA sequencing utilizing the 10x Chromium library technology and Illumina HiSeq 3000 (10x Genomics, San Francisco, CA; Novogene, Sacramento, CA). We mapped the resulting raw reads to the EquCab2 and EquCab3 (GenBank assembly accession: GCA_000002305.1 and GCA_002863925.1) reference genomes utilizing LongRanger following the standard protocols (10x Genomics) (Supplementary Table S1).
      Gene annotation was examined utilizing the National Center for Biotechnology Information E. caballus Annotation Release 103 (accession number GCF_002863925.1). We conducted a comprehensive web search of scientific literature on the genes from candidate regions using Google Scholar and PubMed (
      • Shultz M.
      Comparing test searches in PubMed and Google Scholar.
      ). Visual inspection of genomic regions for polymorphisms within coding regions was performed on Loupe (10x Genomics). We searched for previously discovered polymorphisms within the candidate regions on ENSEMBL release 94, EquCab2 (INSDC Assembly GCA_000002305.1, Sep 2007) database. For regions within unassembled contigs in EquCab2 (chromosome Unk), localization within EquCab3 was performed on UCSC Genome Browser, utilizing the lift-over and Blat tools (
      • Casper J.
      • Zweig A.S.
      • Villarreal C.
      • Tyner C.
      • Speir M.L.
      • Rosenbloom K.R.
      • et al.
      The UCSC genome browser database: 2018 update.
      ;
      • Hinrichs A.S.
      • Raney B.J.
      • Speir M.L.
      • Rhead B.
      • Casper J.
      • Karolchik D.
      • et al.
      UCSC data integrator and variant annotation integrator.
      ). Protein structure was predicted on Iterative Threading ASSEmbly Refinement (
      • Yang J.
      • Yan R.
      • Roy A.
      • Xu D.
      • Poisson J.
      • Zhang Y.
      The I-TASSER Suite: protein structure and function prediction.
      ), retaining top-ranked/scored predictions to evaluate the structural differences triggered by novel variants (
      • Yang J.
      • Yan R.
      • Roy A.
      • Xu D.
      • Poisson J.
      • Zhang Y.
      The I-TASSER Suite: protein structure and function prediction.
      ).

      Data availability statement

      Genotyping data are hosted at Mendeley Data (https://data.mendeley.com/datasets/h7wk6bk86h/1) with the project https://doi.org/10.17632/h7wk6bk86h.1. Whole-genome sequences are available at the Sequence Read Archive repository, Bioproject: PRJNA641747.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      Our many thanks to the horse owners who made this study possible and our utmost appreciation to Ernie Bailey, University of Kentucky (Lexington, KY), for sharing genomic data used as controls, for his expertise and guidance throughout our study, as well as for his help in reviewing the manuscript. This work was supported by the American Quarter Horse Foundation Funding Grant (2016–2017).

      Author Contributions

      Conceptualization: LPR, NW, MM, RJM, SAB; Data Curation: LPR; Formal Analysis: LPR; Funding Acquisition: LPR, SAB; Investigation: LPR; Methodology: LPR, MM, RJM, SAB; Project Administration: LPR; Resources: SAB, NW, MM, RJM; Supervision: SAB, NW, MM, RJM; Visualization: LPR; Writing - Original Draft Preparation: LPR; Writing - Review and Editing: LPR, NW, MM, RJM, SAB

      Supplementary Materials

      Figure thumbnail fx1
      Supplementary Figure S1Flowchart of the number of anhidrosis-affected subjects through recruitment, eligibility, and selection for genotyping steps.
      Figure thumbnail fx2
      Supplementary Figure S2Tridimensional scatterplot of PC1, PC2, and PC3. On the basis of the genotype data, this shows the diversity of study horses registered as stock-type (squares) or Thoroughbreds (filled circles). Individual data points are colored by CIA case (red) or control (blue) phenotype. CIA, chronic idiopathic anhidrosis; PC, principal component.
      Supplementary Table S1Summary of Whole-Genome Sequencing Data
      ControlCIA Case
      Read length (bp)2 × 1502 × 150
      Total reads749,105,480759,099,216
      Mapped reads92.0%92.0%
      Phased SNPs98.2%92.3%
      Mean depth36.9×37.4×
      Longest phase block (bp)13,518,62712,798,901
      N50 phase block (bp)2,797,8762,417,368
      Large structural variant calls499387
      Short deletion calls16,94315,828
      Abbreviations: bp, base pair; CIA, chronic idiopathic anhidrosis.

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