Progress 05/01/24 to 04/30/25
Outputs
Target Audience:International equine and animal research community; undergraduate and graduate students; the Equine Industry; horse breed associations; veterinarians/theriogenologists; horse breeders and horse owners. Changes/Problems:The use of thesingle, containerized pipeline (WAGS) to standardize variant calling when aligning short read data to a reference genome and call variants across more than 1100 individual horse genomes to identify novel candidate genes and variants is critical for the progress of this project. However, running this pipeline by our collaborators at the University of Minnesota has taken longer time than initially planned. Therefore, we realistically anticipate that we will request a non-cost extension of this project for 12 months. What opportunities for training and professional development has the project provided?During this reporting period, the PI and co-PIs of the project have trained one postdoctoral fellow, one graduate student and five undergraduate students working on parts of the project. The findings have been presented at several international and national meetings, symposia and conferences. How have the results been disseminated to communities of interest?Communities of interest for this research include but are not limited to veterinarians, horse breeders and owners. Through our Molecular Cytogenetics and Animal Genetics services which provide clinical karyotyping and parentage and disease testing, respectively, we stay in touch via emails and phone calls with the communities of interest. The fact that research on the genomics of equine disorders of sex development is ongoing, already encourages collaboration through which new cases have been identified, and additional samples procured. Ongoing research and the preliminary findings have also been disseminated through invited online video presentations at British Equine Veterinary Association (BEVA) Advanced Reproduction Discussion Forum. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will accomplish the following: Short-read whole genome data analysis continues with a hypothesis-free genome-wide variant discovery to identify additional potential variants not captured by the candidate gene investigation. We will use an expanded equine genome variant database to identify genome-wide candidate variants in cases. We will use a single, containerized pipeline (WAGS) to standardize variant calling when aligning short read data to a reference genome. This pipeline drastically reduces resource usage and run time and is amenable to very large datasets. Variant calling will be done across more than 1100 individual horse genomes including the 91 cases and 200 in-house controls already enrolled in this project. This will potentially identify new genes involved in the observed phenotypes. Long-read WGS data is analyzed for the discovery of impactful structural variants through the ongoing horse pangenome project which includes 84 haplotypes of which 36 (18 long-read genomes) have been generated in course of this project. Briefly, all horse genomes will be aligned to each other to create a pangenome graph using minigraph-cactus. Identified unique sequence variation and differences in genome organization between the haplotypes will then be analyzed for potential phenotypic impact, especially in cases where variation overlaps with coding sequence and regulatory regions. A previous graph version identified over 32,000 structural variants meet these criteria. Short-read whole genome data will then be aligned to the pangenome graph for combined analysis using the variant graph (vg) toolkit to increase the likelihood for identification of novel variation and understanding of genome organization. Continue evaluation of the identified candidate variants/mutations for effects on protein function and structure using predictive modeling. Present the findings at Plant and Animal Genome, PAG (annual, San Diego, CA); biennial International Havemeyer Foundation Horse Genome workshop; annual meetings of Texas Genetics Society and Texas Forum for Reproductive Sciences.
Impacts
What was accomplished under these goals?
WGS data generation. By now, short-read WGS data has been generated for 91 cases, of which 18 cases have also long-read WGS data. With this, sequence data generation is complete and exceeds initial plans. WGS data analysis and candidate variant discovery. We are investigating chromosomally normal DSD cases of three clinical phenotypes: (i) females with X-monosomy-like gonadal dysgenesis (n=31); (ii) 64,XY SRY-positive female-like (n=19), and (iii) 64,XX intersex (n=41). All 91 case genomes have been aligned to the recently available T2T Thoroughbred horse reference genome assembly and genotyped with a cohort of 200 in-house horse genomes as controls. The genomic variants were evaluated for potential impact on 194 candidate genes involved in sex determination, sex differentiation, and ovarian dysgenesis. The variants were filtered for high predicted impact on a gene known to cause similar phenotypes in other mammals, rare (< 2%) in the overall population, and homozygous in the case. The findings are: 48 missense, nonsense, frameshift, or indel mutations were identified in 34 genes across 31/91 (34%) DSD cases. Four genes (AR, FREM2, FRAS1, STAG3) had mutations in two or more cases. 10 cases had multiple mutations, either across different genes or within the same gene but at different positions. Notably, the NR0B1 gene had the same mutation in two cases from different phenotype groups - intersex and X-monosomy-like. Mutations in the Androgen Receptor (AR) gene were exclusively found in 64,XY SRY-positive female-like horses. Altogether, we identified 7 different AR mutations in 7/19 (37%) cases of this phenotype group. All mutations affected AR function resulting in androgen insensitivity syndrome. 13 mutations were identified in genes associated with human ovarian dysgenesis in 7/31 (23%) mares with X-monosomy-like phenotype. Identified variants are currently being evaluated for effects on protein function and structure using predictive modeling. Endocrine profiles for anti-Müllerian hormone (AMH), inhibin B, and testosterone have been obtained for 64 horses: 22 intersex, eight 64,XY SRY-positive female-like, and 30 X-monosomy-like. With this, the goals of Objective #2 are accomplished. The endocrine data are combined with variant discovery to better understand the effect of candidate genes and mutations. Current findings suggest that: Equine DSDs are implicated by many genes. Most cases have mutations in two or more genes suggesting additive effect. Different mutations in the same gene (AR) can affect protein function and phenotype in a similar way (androgen insensitivity).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
4. Hailey Anderson, Sam Stroupe, Alan J. Conley, Casey Caruso, Rebecca Cotterman, Rytis Juras, Brian W. Davis, Terje Raudsepp. 2025. Equine Disorders of Sex Development Involve Mutations in Several Sex Development Key Genes. Plant and Animal Genome 32, January 10-15, 2025, San Diego, USA. Platform and Poster presentations.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
5. Hailey Anderson, Sam Stroupe, Rytis Juras, Brian W. Davis, Terje Raudsepp. 2025. Mutations in Key Genes Involved in Equine Disorders of Sex Development: A Focus on the Androgen Receptor Gene. VMBS Trainee Research Symposium, February 6, 2025, College Station, USA. Poster and Flash Talk presentations.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
6. Hailey Anderson, Sam Stroupe, Rytis Juras, Brian W. Davis, Terje Raudsepp. 2025. Mutations in the Androgen Receptor Gene and Other Sex Development Key Genes Are Associated with Equine Disorders of Sex Development. Texas Genetics Society, March 20-22, 2025, College Station, USA. Platform presentation.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
7. Hailey Anderson, Sam Stroupe, Rytis Juras, Brian W. Davis, Terje Raudsepp. 2025. Mutations in the Androgen Receptor Gene and Other Key Genes Associated with Equine Disorders of Sex Development. Equine Science Society Symposium 2025, June 3-6, Fort Collins, Colorado, USA. Platform presentation.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
8. Hailey Anderson, Sam Stroupe, Rytis Juras, Brian W. Davis, Terje Raudsepp. 2025. Candidate Gene Investigation for Equine Disorders of Sex Development. 40th International Society for Animal Genetics (ISAG) Conference, July 20-25, 2025, Daejeon, South Korea.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
9. Sam Stroupe, Jonah N. Cullen, Sian A Durward-Akhurst, Matteo Paini, Massimo Delledonne, Jessica Petersen, Terje Raudsepp, Ted Kalbfleisch, Molly McCue, Brian W. Davis. 2025. Moving Towards Personalized Pangenomic Veterinary Medicine in Equids. 40th International Society for Animal Genetics (ISAG) Conference, July 20-25, 2025, Daejeon, South Korea.
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:International equine and animal research community; undergraduate and graduate students; the Equine Industry; horse breed associations; veterinarians/theriogenologists; horse breeders and horse owners. Changes/Problems:No problems to report. The project is progressing as planned. What opportunities for training and professional development has the project provided?During this reporting period, the PI and co-PIs of the project have trained two undergraduate students and one graduate student working on parts of the project. The findings will be presented at 14th International Havemeyer Foundation Horse Genome Workshop, May 12-15, 2024, Caen, France. How have the results been disseminated to communities of interest?Communities of interest for this research include but are not limited to veterinarians, horse breeders and owners. Through our Molecular Cytogenetics and Animal Genetics services which provide clinical karyotyping and parentage and disease testing, respectively, we stay in touch via emails and phone calls with the communities of interest. Even though, we do not have, yet, candidate genomic regions or variants identified, the fact that research on the genomics of equine disorders of sex development is ongoing, already encourages collaboration through which new cases will be identified, and additional samples procured. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will accomplish the following: Short-read whole genome data analysis continues. We will use an expandedequine genome variant database to identify candidate variants in cases. The second step of sequence analysis uses a single, containerized pipeline (WAGS) developed by researchers in Minnesota (authored by Dr. Jonah Cullen) to standardize variant calling when aligning short read data to a reference genome. This pipeline drastically reduces resource usage and run time and is amenable to very large datasets. In summer of 2023 we began rolling out the pipeline at Texas A&M to adapt it for the use in horses. Dr. Cullen has implemented more than 25 fixes to WAGS to facilitate its use on the Texas A&M High Performance Computing Cluster, requisite to variant calling across more than 1100 horses that will be included in this variant database. Currently, variant calling is proceeding with datasets from approximately 1,100 horses (including our ~200 horse genomes). The results should provide unprecedented resolution regarding the DNA sequence variation across more than 100 horse breeds. Long-read whole genome sequence data analysis continues and will be combined with the analysis using the above-mentioned WAGS pipeline. Upon the completion of variant calling through the WAGS pipeline across 1100 horses of 100 breeds, we will explore the frequency of each tagging variants in the context of all sampled breeds. Present the findings at Plant and Animal Genome, PAG (annual, San Diego, CA), annual meetings of Texas Genetics Society and Texas Forum for Reproductive Sciences and invite collaborators from UC Davis (Prof. Alan Conley) to present the findings of endocrine profiling on a seminar of the Interdisciplinary Faculty of Reproductive Biology.
Impacts
What was accomplished under these goals?
RESEARCH SAMPLE COLLECTION: Through our Karyotyping service and communication with veterinarians, breeders, and owners, we procured samples from 11 additional cases. These included four 64,XX intersex horses, one 64,XY SRY-positive female-like horse, and six 64,XX female horses with X-monosomy-like gonadal dysplasia. Objective #1: Genomic DNA has been isolated from 88 case horses: 31 64,XX females with X-monosomy-like phenotype, 41 64,XX intersex horses, and 16 64,XY SRY-positive female-like horses. Because short-read sequencing at Texas A&M core facilities became cheaper, Illumina short-read sequence data was generated for all 88 horses (initially, we proposed to sequence 30 horses, 10 per each phenotype group). All raw sequence data passed QC and was initially aligned to the current horse reference genome EquCab3. In the meanwhile, through collaborations in equine genomics community (Dr. Ted Kalbfleisch), we gained access to the most recent and most comprehensive, near-gapless telomere-to-telomere (T2T) horse reference genome (unpublished and not yet publicly available) and are currently aligning the 88 case genomes to the T2T genome together with 108 in-house generated non-case horse genomes of 21 horse breeds. We have a variant database (vcf file) for these 196 samples ready to be used for candidate variant discovery in cases during the next stage of the project. Also, we will expand the current equine variant database with over 600 whole genome short-read equine sequences available from NCBI. High Molecular Weight (HMW) genomic DNA has been isolated for 18 cases: six for each of the three phenotype groups. Long-read High-Fidelity PacBio sequences have been obtained for 16 cases and passed QC. We are waiting for the last two case long-read genomes. Processing long-read data is in progress and will be used for the discovery of CNVs and complex structural variants. Objective #2: Endocrine profiles have been obtained from blood serum of 64 horses: 22 intersex horses, eight 64,XY SRY-positive female-like horses, thirty 64,XX females with X-monosomy-like phenotype, and four horses with cytogenetically confirmed X-monosomy (as controls). We measured levels of the Anti-Müllerian hormone (AMH), Inhibin B, and Testosterone. The most informative data was obtained for AMH and testosterone and we have identified several individuals in the three phenotype groups with highly abnormal endocrine profiles in relation to their genetic sex and gonadal phenotype. With this, the goals of Objective #2 are accomplished. The endocrine data will be combined with genomics analysis to pinpoint candidate genes and genomic regions.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Brian W. Davis, Rytis Juras, Melanie Barbee, Austin Byrom, Terje Raudsepp. Exploring the genomic component of equine sex development and reproduction. 14th International Havemeyer Foundation Horse Genome Workshop, May 12-15, 2024, Caen, France (platform presentation).
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