Thermomechanical Material Characterization of Polyethylene Terephthalate Glycol with 30% Carbon Fiber for Large-Format Additive Manufacturing of Polymer Structures.

Large-format additive manufacturing (LFAM) is used to print large-scale polymer structures. Understanding the thermal and mechanical properties of polymers suitable for large-scale extrusion is needed for design and production capabilities. An in-house-built LFAM printer was used to print polyethylene terephthalate glycol with 30% carbon fiber (PETG CF30%) samples for thermomechanical characterization. Thermogravimetric analysis (TGA) shows that the samples were 30% carbon fiber by weight. X-ray microscopy (XRM) and porosity studies find 25% voids/volume for undried material and 1.63% voids/volume for dry material. Differential scanning calorimetry (DSC) shows a glass transition temperature (Tg) of 66 °C, while dynamic mechanical analysis (DMA) found Tg as 82 °C. The rheology indicated that PETG CF30% is a good printing material at 220-250 °C. Bending experiments show an average of 48.5 MPa for flexure strength, while tensile experiments found an average tensile strength of 25.0 MPa at room temperature. Comparison with 3D-printed PLA and PETG from the literature demonstrated that LFAM-printed PETG CF30% had a comparative high Young's modulus and had similar tensile strength. For design purposes, prints from LFAM should consider both material choice and print parameters, especially when considering large layer heights.

Spotting the Pattern: A Review on White Coat Color in the Domestic Horse.

Traits such as shape, size, and color often influence the economic and sentimental value of a horse. Around the world, horses are bred and prized for the colors and markings that make their unique coat patterns stand out from the crowd. The underlying genetic mechanisms determining the color of a horse's coat can vary greatly in their complexity. For example, only two genetic markers are used to determine a horse's base coat color, whereas over 50 genetic variations have been discovered to cause white patterning in horses. Some of these white-causing mutations are benign and beautiful, while others have a notable impact on horse health. Negative effects range from slightly more innocuous defects, like deafness, to more pernicious defects, such as the lethal developmental defect incurred when a horse inherits two copies of the Lethal White Overo allele. In this review, we explore, in detail, the etiology of white spotting and its overall effect on the domestic horse to Spot the Pattern of these beautiful (and sometimes dangerous) white mutations.

Population Analysis Identifies 15 Multi-Variant Dominant White Haplotypes in Horses.

The influence of a horse's appearance on health, sentimental and monetary value has driven the desire to understand the etiology of coat color. White markings on the coat define inclusion for multiple horse breeds, but they may disqualify a horse from registration in other breeds. In domesticated horses (Equus caballus), 35 KIT alleles are associated with or cause depigmentation and white spotting. It is a common misconception among the general public that a horse can possess only two KIT variants. To correct this misconception, we used BEAGLE 5.4-phased NGS data to identify 15 haplotypes possessing two or more KIT variants previously associated with depigmentation phenotypes. We sourced photos for 161 horses comprising 12 compound genotypes with three or more KIT variants and employed a standardized method to grade depigmentation, yielding average white scores for each unique compound genotype. We found that 7 of the 12 multi-variant haplotypes resulted in significantly more depigmentation relative to the single-variant haplotypes (ANOVA). It is clear horses can possess more than two KIT variants, and future work aims to document phenotypic variations for each compound genotype.

Prospective Evaluation of Venous Excess Ultrasound for Estimation of Venous Congestion.

BACKGROUND: Venous excess ultrasound (VExUS) is a novel ultrasound technique previously reported as a noninvasive measure of venous congestion and predictor of cardiorenal acute kidney injury. RESEARCH QUESTION: Are there associations between VExUS grade and cardiac pressures measured by right heart catheterization (RHC) and cardiac biomarkers and clinical outcomes in patients undergoing RHC? STUDY DESIGN AND METHODS: We conducted a prospective cohort study at the Denver Health Medical Center from December 20, 2022, to March 25, 2023. All patients undergoing RHC underwent a blinded VExUS assessment prior to their procedure. Multivariable regressions were conducted to assess relationships between VExUS grade and cardiac pressures, biomarkers, and changes in weight among patients with heart failure, a proxy for diuretic success. Receiver operating characteristic curve and area under the curve (AUC) were derived for VExUS, inferior vena cava (IVC) diameter, and IVC collapsibility index (ICI) to predict right atrial pressure (RAP) > 10 and < 7 mm Hg. RESULTS: Among 81 patients, 45 of whom were inpatients, after adjusting for age, sex, and Charlson Comorbidity Index, there were significant relationships between VexUS grade of 2 (ß = 4.8; 95% CI, 2.6-7.1; P < .01) and 3 (ß = 11; 95% CI, 8.9-14; P < .01) and RAP, VExUS grade of 2 (ß = 6.8; 95% CI, 0.16-13; P = .045) and 3 (ß = 15; 95% CI, 7.3-22; P < .01) and mean pulmonary artery pressure, and VExUS grade of 2 (ß = 7.0; 95% CI, 3.9-10; P < .01) and 3 (ß = 13; 95% CI, 9.5-17; P < .01) and pulmonary capillary wedge pressure. AUC values for VExUS, IVC diameter, and ICI as predictors of RAP > 10 mm Hg were 0.9 (95% CI, 0.83-0.97), 0.77 (95% CI, 0.68-0.88), and 0.65 (95% CI, 0.52-0.78), respectively. AUC values for VExUS, IVC diameter, and ICI as predictors of RAP < 7 mm Hg were 0.79 (95% CI, 0.70-0.87), 0.74 (95% CI, 0.64-0.84), and 0.62 (95% CI, 0.49-0.76), respectively. In a subset of 23 patients with heart failure undergoing diuresis, there was a significant association between VExUS grade 3 and change in weight between time of RHC and discharge (P = .025). INTERPRETATION: Although more research is required, VExUS has the potential to increase diagnostic and therapeutic capabilities of physicians at the bedside and increase our understanding of the underappreciated problem of venous congestion.

Quantitative Analysis of Autophagy in Single Cells: Differential Response to Amino Acid and Glucose Starvation.

Autophagy is a highly conserved, intracellular recycling process by which cytoplasmic contents are degraded in the lysosome. This process occurs at a low level constitutively; however, it is induced robustly in response to stressors, in particular, starvation of critical nutrients such as amino acids and glucose. That said, the relative contribution of these inputs is ambiguous and many starvation medias are poorly defined or devoid of multiple nutrients. Here, we sought to generate a quantitative catalog of autophagy across multiple stages and in single, living cells under normal growth conditions as well as in media starved specifically of amino acids or glucose. We found that autophagy is induced by starvation of amino acids, but not glucose, in U2OS cells, and that MTORC1-mediated ULK1 regulation and autophagy are tightly linked to amino acid levels. While autophagy is engaged immediately during amino acid starvation, a heightened response occurs during a period marked by transcriptional upregulation of autophagy genes during sustained starvation. Finally, we demonstrated that cells immediately return to their initial, low-autophagy state when nutrients are restored, highlighting the dynamic relationship between autophagy and environmental conditions. In addition to sharing our findings here, we provide our data as a high-quality resource for others interested in mathematical modeling or otherwise exploring autophagy in individual cells across a population.

Molecular dynamics simulations provide insights into ULK-101 potency and selectivity toward autophagic kinases ULK1/2.

Kinase domains are highly conserved within protein kinases in both sequence and structure. Many factors, including phosphorylation, amino acid substitutions or mutations, and small molecule inhibitor binding, influence conformations of the kinase domain and enzymatic activity. The serine/threonine kinases ULK1 and ULK2 are highly conserved with N- and C-terminal domains, phosphate-binding P-loops, αC-helix, regulatory and catalytic spines, and activation loop DFG and APE motifs. Here, we performed molecular dynamics (MD) simulations to understand better the potency and selectivity of the ULK1/2 small molecule inhibitor, ULK-101. We observed stable bound states for ULK-101 to the adenosine triphosphate (ATP)-binding site of ULK2, coordinated by hydrogen bonding with the hinge backbone and the catalytic lysine sidechain. Notably, ULK-101 occupies a hydrophobic pocket associated with the N-terminus of the αC-helix. Large movements in the P-loop are also associated with ULK-101 inhibitor binding and exit from ULK2. Our data further suggests that ULK-101 could induce a folded P-loop conformation and hydrophobic pocket reflected in its nanomolar potency and kinome selectivity.

Two Novel Variants in MITF and PAX3 Associated With Splashed White Phenotypes in Horses.

Mutations causing depigmentation are relatively common in Equus caballus (horse). Over 40 alleles in multiple genes are associated with increased white spotting (as of February 2023). The splashed white phenotype, a coat spotting pattern described as appearing like the horse has been splashed with white paint, was previously associated with variants in the PAX3 and MITF genes. Both genes encode transcription factors known to control melanocyte migration and pigmentation. We report two novel mutations, a stop-gain mutation in PAX3 (XM_005610643.3:c.927C>T, ECA6:11,196,181, EquCab3.0) and a missense mutation in a binding domain of MITF (NM_001163874.1:c.993A>T, ECA16:21,559,940, EquCab3.0), each with a strong association with increased depigmentation in Pura Raza Española horses (P = 1.144E-11, N = 30, P = 4.441E-16, N = 39 respectively). Using a quantitative method to score depigmentation, the PAX3 and MITF mutations were found to have average white scores of 25.50 and 24.45, respectively, compared to the average white coat spotting score of 1.89 in the control set. The functional impact for each mutation was predicted to be moderate to extreme (I-TASSER, SMART, Variant Effect Predictor, SIFT). We propose to designate the MITF mutant allele as Splashed White 9 and the PAX3 mutant allele as Splashed White 10 per convention.

5'UTR Variant in KIT Associated With White Spotting in Horses.

Mutations in KIT, a gene that influences melanoblast migration and pigmentation, often result in mammalian white spotting. As of February 2023, over 30 KIT variants associated with white spotting were documented in Equus caballus (horse). Here we report an association of increased white spotting on the skin and coat with a variant in the 5'UTR of KIT (rs1149701677: g.79,618,649A>C). Horses possessing at least one alternate allele demonstrate phenotypic characteristics similar to other KIT mutations: clear borders around unpigmented regions on the body, face, and limbs. Using a quantitative measure of depigmentation, we observed an average white score of 10.70 among individuals with rs1149701677, while the average score of the control, homozygous reference sample was 2.23 (P = 1.892e-11, n = 109, t-test). The rs1149701677 site has a cross-species conservation score of 3.4, one of the highest scores across the KIT 5'UTR, implying regulatory importance for this site. Ensembl also predicted a "moderately impactful" functional effect for the rs1149701677 variant. We propose that this single nucleotide variant likely alters the regulation of KIT, which in turn may disrupt melanoblast migration causing an increase in white spotting on the coat. Alternatively, the rs1149701677 variant may be in linkage with another nearby variant with an as-yet-undiscovered functional impact. We propose to term this new allele "Holiday White" or W35 based on conventional nomenclature.

Validating a Nutrition Ranking System for Food Pantries Using the Healthy Eating Index-2015.

In 2020, charitable food organizations began adopting Healthy Eating Research (HER) nutrition guidelines, which rank individual foods in tiers (e.g., green, yellow, or red) based on each food's nutrient profile. This study aimed to validate this HER tier-ranked system against the Healthy Eating Index-2015 (HEI) and develop a formula to summarize the percentages of tier-ranked foods in an overall nutritional quality index that correlated with HEI. Using secondary data of foods selected by clients in 16 Minnesota food pantries (n = 503 "client carts"), we generated an HEI score and percentages of green, yellow, and red foods for each cart. As validation, we tested the association between HEI scores and the percentages of tier-ranked foods and compared the means of the tier-ranked variables using quintiles of the HEI scores. HEI scores were positively associated with percentages of green foods and negatively associated with percentages of red foods. Next, we used statistical learning to generate weights to maximize the correlation of the tier-ranked variables and the HEI scores and used these weights to create an index. The index demonstrated a moderate-to-strong correlation with HEI and can be used as a single measure to summarize the overall nutritional quality for sets of tier-ranked foods.

Waistline to gumline: a retrospective analysis of body mass index in treated periodontitis patients undergoing periodontal maintenance therapy.

Periodontal disease affects 42% of adults in the United States. Both the periodontal microbiome and the host immune inflammatory response may be influenced by overweight/obesity status. This retrospective analysis sought to determine the associations of periodontal disease parameters with body mass index (BMI) and obesity status in patients undergoing periodontal maintenance therapy. The records of 418 patients who were undergoing periodontal maintenance after periodontitis treatment were examined, and the patients' demographic characteristics (sex, age, and race/ethnicity), self-reported BMI, periodontal disease condition, number of sites with probing depth ≥ 4 mm, missing teeth, and sites with bleeding on probing (BOP) were recorded. Patients were determined to have active moderate to severe periodontitis if they presented with 2 or more sites in 2 different quadrants with clinical attachment loss ≥ 5 mm and probing depth ≥ 5 mm. Individuals were also categorized into 3 groups: underweight/normoweight, BMI < 25; overweight, BMI 25 ≤ 30; or obese, BMI ≥ 30. In the study population, BMI ranged from 16.827 to 51.389. The periodontitis status was not significantly associated with a BMI status of overweight (odds ratio [OR] = 1.388 [95% CI, 0.961- 2.006]) or obese (OR = 1.168 [95% CI, 0.77-1.757]). Female sex (OR = 0.561 [95% CI, 0.343-0.918]) and age (OR = 0.983 [95% CI, 0.967-0.999]) were negatively associated with active periodontitis status. Obese patients demonstrated significantly more sites with BOP than either underweight/normoweight or overweight patients, and a BMI indicating obesity was associated with increasing age (P < 0.001) and higher number of missing teeth (P = 0.0064). In a population of patients undergoing periodontal maintenance therapy, BMI was associated with age and missing teeth, and obese status was associated with a significantly higher number of sites with BOP.

A KIT Variant Associated with Increased White Spotting Epistatic to MC1R Genotype in Horses (Equus caballus).

Over 40 identified genetic variants contribute to white spotting in the horse. White markings and spotting are under selection for their impact on the economic value of an equine, yet many phenotypes have an unknown genetic basis. Previous studies also demonstrate an interaction between MC1R and ASIP pigmentation loci and white spotting associated with KIT and MITF. We investigated two stallions presenting with a white spotting phenotype of unknown cause. Exon sequencing of the KIT and MITF candidate genes identified a missense variant in KIT (rs1140732842, NC_009146.3:g.79566881T>C, p.T391A) predicted by SIFT and PROVEAN as not tolerated/deleterious. Three independent observers generated an Average Grade of White (AGW) phenotype score for 147 individuals based on photographs. The KIT variant demonstrates a significant QTL association to AGW (p = 3.3 × 10−12). Association with the MC1R Extension locus demonstrated that, although not in LD, MC1R e/e (chestnut) individuals had higher AGW scores than MC1R E/- individuals (p = 3.09 × 10−17). We also report complete linkage of the previously reported KIT W19 allele to this missense variant. We propose to term this variant W34, following the standardized nomenclature for white spotting variants within the equine KIT gene, and report its epistatic interaction with MC1R.

Chemical Biology Screening Identifies a Vulnerability to Checkpoint Kinase Inhibitors in TSC2-Deficient Renal Angiomyolipomas.

The tuberous sclerosis complex (TSC) is a rare genetic syndrome and multisystem disease resulting in tumor formation in major organs. A molecular hallmark of TSC is a dysregulation of the mammalian target of rapamycin (mTOR) through loss-of-function mutations in either tumor suppressor TSC1 or TSC2. Here, we sought to identify drug vulnerabilities conferred by TSC2 tumor-suppressor loss through cell-based chemical biology screening. Our small-molecule chemical screens reveal a sensitivity to inhibitors of checkpoint kinase 1/2 (CHK1/2), regulators of cell cycle, and DNA damage response, in both in vitro and in vivo models of TSC2-deficient renal angiomyolipoma (RA) tumors. Further, we performed transcriptional profiling on TSC2-deficient RA cell models and discovered that these recapitulate some of the features from TSC patient kidney tumors compared to normal kidneys. Taken together, our study provides a connection between mTOR-dependent tumor growth and CHK1/2, highlighting the importance of CHK1/2 inhibition as a potential antitumor strategy in TSC2-deficient tumors.

Nutrition standards for the charitable food system: challenges and opportunities.

Food insecurity is associated with increased risk of chronic disease and poor dietary intake. The United States charitable food system, a complex network of food banks, pantries and congregate meal sites, provides food for millions of low-income households each year. Food banks and pantries play a critical role in supporting food security and are an important contributor to dietary intake for its clients. In recent years, there has been an increased focus on sourcing and supplying more nutritious foods within the charitable food system. Despite this, there is a lack of alignment in how the charitable food system defines and tracks the nutritional quality of food.In 2019, Healthy Eating Research convened a panel of nutrition, charitable food system and food policy experts to create a set of evidence-based nutrition standards. Standards were developed based on a review of the literature and existing nutrition ranking systems, while also considering the operational needs and capacity of the charitable food system. The panel provided recommendations for eleven distinct food categories: fruits and vegetables, grains, protein, dairy, non-dairy alternatives, beverages, mixed dishes, processed and packaged snacks, desserts, condiments and cooking staples, and other miscellaneous items. Products are ranked into three tiers, choose often (green), choose sometimes (yellow) or choose rarely (red), based on designated saturated fat, sodium, and added sugar thresholds. This paper outlines the expert panel's approach and summarizes the barriers and opportunities for implementing these standards across the charitable food system.

Demystifying the Genetic Origins of the Mangalarga Horse Through the Influential Stallion Turbante J.O.

Pedigrees and horse written ancestry contain numerous inconsistencies and divergence between farm histories, owner accounts and registration records. In particular, the origins of the Brazilian Mangalarga, or "Mangalarga Paulista'' horse breed is controversial, and the breed's popular history claims that one of its most famous individuals, Turbante J.O., may have been sired by an unknown Hanoverian stallion. Turbante J.O. sired over 1678 offspring and is present in about 71% of the male pedigrees. We genotyped Turbante J.O. and 29 registered Mangalarga individuals using a commercially available ancestry service and compared genomic to pedigree-based estimates. DNA was extracted for this sire from frozen semen samples. Other breed-average genomic ancestries for the Arabian, Thoroughbred, Saddlebred, and Hanoverian were utilized for comparison. Pedigree-based inbreeding coefficient (Fped) of Turbante J.O. and the 17 other Mangalargas were analyzed, and while Turbante J.O.'s Fped is estimated to be 18.5%, the genomic-based inbreeding coefficient is 33%. Pedigree-based co-ancestry coefficients estimate that about 3% of his ancestry should reflect Thoroughbred and Arabian heritage, however, the genomic analysis of Turbante J.O. identified 100% Iberian ancestry, and 99% in common with other Mangalarga individuals followed by other autochthonous Brazilian breeds, with no evidence of Hanoverian parentage. We demonstrate higher pedigree-estimated inbreeding coefficient errors than previously reported, perhaps a result of the pedigree depth, and the ability of genomic ancestral analysis to answer questions that pedigree analyses cannot. Due to the genomic relatedness, these results may provide more detailed guidelines in maintaining genetic diversity in this breed through selective outbreeding.

Spectrum of germline and somatic mitochondrial DNA variants in Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is caused by loss of function variants in either TSC1 or TSC2 and is characterized by broad phenotypic heterogeneity. Currently, there is limited knowledge regarding the role of the mitochondrial genome (mtDNA) in TSC pathogenesis. In this study, we aimed to determine the prevalence and spectrum of germline and somatic mtDNA variants in TSC and identify potential disease modifiers. Analysis of mtDNA amplicon massively parallel sequencing (aMPS) data, off-target mtDNA from whole-exome sequencing (WES), and/or qPCR, revealed mtDNA alterations in 270 diverse tissues (139 TSC-associated tumors and 131 normal tissue samples) from 199 patients and six healthy individuals. Correlation of clinical features to mtDNA variants and haplogroup analysis was done in 102 buccal swabs (age: 20-71 years). No correlation was found between clinical features and either mtDNA variants or haplogroups. No pathogenic variants were identified in the buccal swab samples. Using in silico analysis, we identified three predicted pathogenic variants in tumor samples: MT-ND4 (m.11742G>A, p. Cys328Tyr, VAF: 43%, kidney angiomyolipoma), MT-CYB (m.14775T>C, p. Leu10Pro, VAF: 43%, LAM abdominal tumor) and MT-CYB (m.15555C>T, p. Pro270Leu, VAF: 7%, renal cell carcinoma). Large deletions of the mitochondrial genome were not detected. Analysis of tumors from 23 patients with corresponding normal tissue did not reveal any recurrent tumor-associated somatic variants. The mtDNA/gDNA ratio between tumors and corresponding normal tissue was also unchanged. Overall, our findings demonstrate that the mitochondrial genome is highly stable across tissues and within TSC-associated tumors.

A Multi-Site Analysis of the Prevalence of Food Insecurity in the United States, before and during the COVID-19 Pandemic.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic profoundly affected food systems including food security. Understanding how the COVID-19 pandemic impacted food security is important to provide support and identify long-term impacts and needs. OBJECTIVE: The National Food Access and COVID research Team (NFACT) was formed to assess food security over different US study sites throughout the pandemic, using common instruments and measurements. This study presents results from 18 study sites across 15 states and nationally over the first year of the COVID-19 pandemic. METHODS: A validated survey instrument was developed and implemented in whole or part through an online survey of adults across the sites throughout the first year of the pandemic, representing 22 separate surveys. Sampling methods for each study site were convenience, representative, or high-risk targeted. Food security was measured using the USDA 6-item module. Food security prevalence was analyzed using ANOVA by sampling method to assess statistically significant differences. RESULTS: Respondents (n = 27,168) indicate higher prevalence of food insecurity (low or very low food security) since the COVID-19 pandemic, compared with before the pandemic. In nearly all study sites, there is a higher prevalence of food insecurity among Black, Indigenous, and People of Color (BIPOC), households with children, and those with job disruptions. The findings demonstrate lingering food insecurity, with high prevalence over time in sites with repeat cross-sectional surveys. There are no statistically significant differences between convenience and representative surveys, but a statistically higher prevalence of food insecurity among high-risk compared with convenience surveys. CONCLUSIONS: This comprehensive study demonstrates a higher prevalence of food insecurity in the first year of the COVID-19 pandemic. These impacts were prevalent for certain demographic groups, and most pronounced for surveys targeting high-risk populations. Results especially document the continued high levels of food insecurity, as well as the variability in estimates due to the survey implementation method.

Hereditary Equine Regional Dermal Asthenia homozygote adult working horse with mild signs - A Case Report.

Hereditary Equine Regional Dermal Asthenia (HERDA) is an autosomal recessive condition present in the American Quarter Horse and other related breeds. Resulting from a mutation in the peptidyl-prolyl cis-trans isomerase B (PPIB) gene, HERDA is homologous to Ehlers-Danlos syndrome in humans. Characterized by fragile, hyperelastic, skin, HERDA affected horses often present first with slow-healing wounds usually on the dorsum, and resulting in atrophic scars, seromas, and ulcers. As there is no treatment for the condition affected horses are typically reported to be unrideable, and if persistent wounds are sufficiently severe, may require euthanasia. This case report describes clinical presentation and genetic diagnostics of HERDA in an 8-year-old horse with notably mild clinical signs, previously undiagnosed. On recommendation from the referring veterinarian, the horse owners pursued genetic diagnostics for HERDA following development of painful dorsal skin lesions under the saddle area during a riding clinic. The individual was confirmed homozygous for HERDA c.115G>A missense mutation in the PPIB gene by commercial testing service (Etalon Diagnostics Inc.). Further objective studies on the severity and clinical presentation of HERDA are necessary to evaluate complex elements of this disease. Furthermore, mildly affected individuals may be underdiagnosed as a result of not demonstrating the clinical signs that commonly encourage genetic testing.

Two Variants of KIT Causing White Patterning in Stock-Type Horses.

Over 30 polymorphisms in the KIT Proto-Oncogene Receptor Tyrosine Kinase (KIT) gene have been implicated in white spotting patterns ranging from small areas to full dermal depigmentation in the horse. We performed a candidate-gene exon sequencing approach on KIT and MITF, 2 known causatives of white spotting patterns, within 2 families of horses of unknown white spotting. Family 1 (Fam1, N = 5) consisted of a Quarter Horse stallion and 4 offspring with white spotting pattern ranging from legs, lower ventral, and head regions with jagged borders, to almost complete white. The second family (Fam2, N = 7) consisted of 6 half-sibling American Paint Horse/Quarter Horse and their dam, demonstrating unpigmented limbs with belly spots and an extensive white patterning on the face. This approach resulted in 2 variants significantly associated with familial phenotypes, where Fam1 variant is an indel leading to a frameshift mutation, and Fam2 a non-synonymous SNP. We validated the variants within an unrelated population of horses (Fam2 variant, P = 0.00271944) as well as for protein functional impact with ExPASy, Protter, Phyre2, SMART, PROVEAN, SIFT, and I-TASSER, confirming the reported associations. Fam1 associated variant, deemed W31, alters the protein sequence, leading to an early stop codon truncating the normal amino acid sequence from 972 to just 115 amino acids. Fam2 associated variant, deemed W32, may have a subtle impact on receptor function or could be in linkage with a non-coding or regulatory change creating the mild spotting pattern observed in this family.