Imputation of canine genotype array data using 365 whole-genome sequences improves power of genome-wide association studies.

Genomic resources for the domestic dog have improved with the widespread adoption of a 173k SNP array platform and updated reference genome. SNP arrays of this density are sufficient for detecting genetic associations within breeds but are underpowered for finding associations across multiple breeds or in mixed-breed dogs, where linkage disequilibrium rapidly decays between markers, even though such studies would hold particular promise for mapping complex diseases and traits. Here we introduce an imputation reference panel, consisting of 365 diverse, whole-genome sequenced dogs and wolves, which increases the number of markers that can be queried in genome-wide association studies approximately 130-fold. Using previously genotyped dogs, we show the utility of this reference panel in identifying potentially novel associations, including a locus on CFA20 significantly associated with cranial cruciate ligament disease, and fine-mapping for canine body size and blood phenotypes, even when causal loci are not in strong linkage disequilibrium with any single array marker. This reference panel resource will improve future genome-wide association studies for canine complex diseases and other phenotypes.

Banking on the Last Gift: Cornell's Signature Program of Postmortem Tissue Procurement.

High-quality, well-annotated, healthy tissue specimens are crucial to the success of basic and translational research, but often difficult to procure. Postmortem (PM) tissue collections provide the opportunity to collect these healthy biospecimens. PM procurement programs led by biobanks can further contribute by providing researchers with rare biospecimens collected with short postmortem intervals (PMI) in controlled environments. To support biomedical and translational research, the Cornell Veterinary Biobank (CVB), an ISO 20387 accredited core resource at the Cornell University College of Veterinary Medicine, has performed PM tissue collections from research and privately owned animals since 2013. The CVB PM collection team, consisting of a board-certified veterinary pathologist, a licensed veterinary technician collection specialist, and a data capture specialist, performs rapid tissue collections during controlled warm necropsies, with an accepted PMI of ≤2 hours and a target PMI of ≤1 hour. A retrospective analysis of PM collections between 2013 and 2020 was completed, consisting of 4077 aliquots of 1582 biospecimens from 69 donors (48 canine, 16 feline, and 5 equine). An average of 22.93 biospecimens per donor were collected (range: 1-49). The average PMI for standard collections was 43.48 ± 2.30 minutes, starting on average 20.81 ± 1.61 minutes after time of death. Thus far, the CVB has a favorable utilization rate, with 414 aliquots (10.15%) from 350 specimens (20.12%) and 45 animals (65.22%) distributed to researchers. The success of the CVB PM tissue biobanking program, collecting high-quality biospecimens with short PMIs, was due to support from veterinary pathologists, the competence of CVB personnel, and the continuous evolution of methods within a quality management system. Improvement of PM tissue collection programs in biobanks, with standardized practices for all processes and specialized personnel, can enhance the quality and increase utilization of its biospecimens and associated data.

Purpose, Partnership, and Possibilities: The Implementation of the Dog Aging Project Biobank.

Background: Biobanks have been supporting longitudinal prospective and retrospective studies by providing standardized services for the acquisition, transport, processing, storage, and distribution of high-quality biological material and associated data. Here, we describe how the Dog Aging Project (DAP), a large-scale longitudinal study of the domestic dog (Canis familiaris) with translational applications for humans, developed a biobank of canine biospecimens and associated data. Design and methods: This was accomplished by working with the Cornell Veterinary Biobank, the first biobank in the world to receive accreditation to ISO 20387:2018-General Requirements for Biobanking. The biobank research team was involved in the early collection stages of the DAP, contributing to the development of appropriate workflows and processing fit-for-purpose biospecimens. In support of a dynamic strategy for real-time adjustment of processes, a pilot phase was implemented to develop, test, and optimize the biospecimen workflows, followed by an early phase of collection, processing, and banking of specimens from DAP participants. Results: During the pilot and early phases of collection, the DAP Biobank stored 164 aliquots of whole blood, 273 aliquots of peripheral blood mononuclear cells, 130 aliquots of plasma, and 70 aliquots of serum, and extracted high molecular weight genomic DNA suitable for whole-genome sequencing from 109 whole blood specimens. These specimens, along with their associated preanalytical data, have been made available for distribution to researchers. Conclusion: We discuss the challenges and opportunities encountered during the implementation of the DAP Biobank, along with novel strategies for promoting biobanking sustainability such as partnering with a DAP quality assurance manager and a DAP marketing and communication specialist and developing a pilot grant structure to fund small innovative research projects.

A genome-wide association study to investigate genetic loci associated with primary glaucoma in American Cocker Spaniels.

OBJECTIVE: To identify genetic associations with primary glaucoma (PG) in American Cocker Spaniels using a genome-wide association study (GWAS). ANIMALS: A nationwide ambidirectional case-control cohort study was performed in American Cocker Spaniels that had an ophthalmic examination performed by a veterinarian. Ninety-four dogs with PG (cases) and 111 dogs without glaucoma (controls) met phenotypic criteria and had a blood sample collected after receiving informed owner consent. PROCEDURES: Genomic DNA was extracted from whole blood samples and genotyped (CanineHD BeadChip, Illumina Inc). A case-control GWAS using a linear mixed model was performed, and 3 significance thresholds were calculated (1) using a Bonferroni correction on all single nucleotide polymorphisms (SNPs) included in the GWAS, (2) using a Bonferroni correction on only the unlinked SNPs from a pruned data set, and (3) using 10,000 random phenotype permutations. RESULTS: Following genotype data quality control, 89 cases and 93 controls were included in the GWAS. We identified an association on canine chromosome (CFA10); however, it did not reach statistical significance. Potential candidate genes within the surrounding linkage disequilibrium interval include coiled-coil domain containing 85A (CCDC85A) and extracellular growth factor containing fibulin extracellular matrix protein 1 (EFEMP1). CLINICAL RELEVANCE: Primary glaucoma in the American Cocker Spaniel is a complex heterogeneous disease that may be influenced by a locus on CFA10. The candidate genes CCDC85A and EFEMP1 within the identified linkage disequilibrium interval have been shown to be involved in human open-angle glaucoma.

Complex Feline Disease Mapping Using a Dense Genotyping Array.

The current feline genotyping array of 63 k single nucleotide polymorphisms has proven its utility for mapping within breeds, and its use has led to the identification of variants associated with Mendelian traits in purebred cats. However, compared to single gene disorders, association studies of complex diseases, especially with the inclusion of random bred cats with relatively low linkage disequilibrium, require a denser genotyping array and an increased sample size to provide statistically significant associations. Here, we undertook a multi-breed study of 1,122 cats, most of which were admitted and phenotyped for nine common complex feline diseases at the Cornell University Hospital for Animals. Using a proprietary 340 k single nucleotide polymorphism mapping array, we identified significant genome-wide associations with hyperthyroidism, diabetes mellitus, and eosinophilic keratoconjunctivitis. These results provide genomic locations for variant discovery and candidate gene screening for these important complex feline diseases, which are relevant not only to feline health, but also to the development of disease models for comparative studies.

A Biobank's Journey: Implementation of a Quality Management System and Accreditation to ISO 20387.

Biobanks play an integral role in research and precision medicine by acquiring, processing, storing, and distributing high-quality, clinically annotated biological material. Compliance with biobanking standards and the implementation of quality management systems (QMS) can improve the quality of the biological material and associated data (BMaD). By undergoing third-party assessments, biobanks can demonstrate compliance to these standards and instill confidence in their users. In the 8 months following the publication of the International Organization for Standardization (ISO) 20387:2018 General Requirements for Biobanking standard, the Cornell Veterinary Biobank (CVB) became compliant with the standard requirements, including developing and implementing a QMS. This was achieved through the documentation of all biobanking processes, demonstration of personnel competence, the stringent control of documents and records, and ongoing evaluation of processes and the QMS. Procedures describing the control of documents and records were implemented first to provide a foundation on which to build the QMS, followed by procedures for documenting the identification of risks and opportunities, improvements, and corrective actions following nonconforming outputs. Internal audit and management review programs were developed to verify QMS performance and to monitor quality objectives. Procedures for the governance and management of the biobank were developed, including the following: organizational structure; confidentiality and impartiality policies; facility and equipment maintenance, calibration, and monitoring; personnel training and competency; and evaluation of external providers. All processes on scope were described, along with the validation and verification of methods, to ensure the fitness-for-purpose of the BMaD and the reproducibility of biobanking processes. Training sessions were held during implementation of the QMS to ensure all personnel would conform to the procedures. In April 2019, the CVB underwent third-party assessment by the American Association of Laboratory Accreditation (A2LA) and became the first biobank in the world to receive accreditation to ISO 20387:2018.

Biobanking in the COVID-19 Era and Beyond: Part 2. A Set of Tool Implementation Case Studies.

The COVID-19 era has brought about a number of novel challenges for the global biobanking community. An array of diverse tools (e.g., standards, best practices, and plans) exists to support quality and fitness-for-purpose in biobank operations. The International Society for Biological and Environmental Repositories (ISBER) COVID-19 Response Task Force has set out to identify needs and gaps in these tools and make recommendations for the next generation of available tools, having closely examined the COVID-19-related challenges. While conducting this work to examine the relationships between tools and biobank adaptability, a subgroup of the task force conducted a parallel effort to develop and describe individual COVID-19 era case studies based on a number of operating biobanks. Each case study presents a different combination of implemented tools. Observations and lessons learned from these case studies are provided, and experiences with tool implementation are discussed. This information is supplemented by data relating to tool usefulness that was obtained through an ISBER survey discussed in a companion article. The knowledge gained from this study will be combined with other task force efforts to make recommendations to better position the biobanking community in their response to future emergencies.

Biobanking in the COVID-19 Era and Beyond: Part 1. How Early Experiences Can Translate into Actionable Wisdom.

The era of COVID-19 has brought about a number of novel challenges for the global biobanking community. To better position the biobanking community to cope with current and future challenges, the International Society for Biological and Environmental Repositories (ISBER) COVID-19 Response Task Force was convened to identify needs and gaps in biobanking tools (existing resources that support good practice), for example, standards, best practices, business, etc. and to make recommendations to benefit the community. Toward these goals, the Task Force assembled a set of questions to explore individual biobanks' experiences, with emphasis on identification of key challenges and approaches, including tools employed. A survey was designed with the use of these questions and administered by ISBER. This article presents a summary of the aggregated data obtained from the survey responses, illustrating some of the major issues encountered and identifying which tools the survey respondents found most useful. In particular, this article focuses on the challenges identified during the early months of the COVID-19 era. Recommendations are provided to support biobank emergency preparedness for the future, address lessons learned, and propose solutions to bridge identified gaps. The analysis and the complete survey dataset will also inform the larger Task Force goal to develop specific tool recommendations.

Development and use of DNA archives at veterinary teaching hospitals to investigate the genetic basis of disease in dogs.

The DNA archives developed at veterinary medical teaching hospitals will be important resources for mapping disease loci and identifying underlying genes. The most important feature of a DNA archive is accurate identification or exclusion of diseases in each animal. Such archives will be complimentary resources to tissue banks that are currently available.

Gene expression in hip soft tissues in incipient canine hip dysplasia and osteoarthritis.

Canine hip dysplasia and developmental dysplasia of the human hip share demographic, phenotypic, and clinical features including the predisposition to develop osteoarthritis in affected joints. To support the results of genetic mapping studies for CHD and its concomitant osteoarthritis with functional information, we performed RNA-seq on hip capsule and teres ligament of affected and unaffected dogs. RNA seq showed that expressed genes segregated according age, capsule or ligament, and hip phenotype. Expression of HHIP, DACT2, and WIF1 was significantly higher in capsule from control hips than dysplastic hips indicating a disruption of the hedgehog signaling pathway. Expression of SPON 1, a key component of the WNT pathway, was increased significantly in both dysplastic capsule and ligament while FBN2 and EMILIN3 were significantly increased in dysplastic capsule. Of genes associated with human hip osteoarthritis, expression of ACAN, IGF1, CILP2, COL11A1, COL8A1, and HAPLN was increased significantly in dysplastic capsule. The significant increase in expression of PLA2F, TNFRSF, TMEM, and IGFBP in dysplastic capsule indicated an injury response. Gene set enrichment analysis revealed that genes involved in extracellular matrix structure, epithelial to mesenchymal transition, myogenesis, growth factor signaling, cancer and immune pathways were enriched in dysplastic capsule. For teres ligament from dysplastic joints, genes in retinoic signaling pathways and those encoding extracellular matrix molecules, but not proteoglycans, were enriched. Hip tissues respond to abnormal mechanics early in dysplastic hip development and these pathways present targets for intervention in the early synovitis and capsulitis secondary to canine and human hip dysplasia. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:313-324, 2019.

Complex disease and phenotype mapping in the domestic dog.

The domestic dog is becoming an increasingly valuable model species in medical genetics, showing particular promise to advance our understanding of cancer and orthopaedic disease. Here we undertake the largest canine genome-wide association study to date, with a panel of over 4,200 dogs genotyped at 180,000 markers, to accelerate mapping efforts. For complex diseases, we identify loci significantly associated with hip dysplasia, elbow dysplasia, idiopathic epilepsy, lymphoma, mast cell tumour and granulomatous colitis; for morphological traits, we report three novel quantitative trait loci that influence body size and one that influences fur length and shedding. Using simulation studies, we show that modestly larger sample sizes and denser marker sets will be sufficient to identify most moderate- to large-effect complex disease loci. This proposed design will enable efficient mapping of canine complex diseases, most of which have human homologues, using far fewer samples than required in human studies.

The associations between serum adiponectin, leptin, C-reactive protein, insulin, and serum long-chain omega-3 fatty acids in Labrador Retrievers.

Obesity has been associated with an increased inflammatory response and insulin resistance due to adipose tissue-derived adipokines and increases in C-reactive protein (CRP). Dogs appear to be similar to other species with the exception of adiponectin, which might not be affected by obesity status. Serum long-chain polyunsaturated fatty acid concentrations have been positively and negatively associated with serum adipokines. The aim of the study was to examine the relationship between leptin, CRP, adiponectin, and insulin to body condition score (BCS) and to the long-chain omega-3 fatty acids in serum lipoproteins, including alpha-linolenic acid, eicosapentaenoic acid (EPA), docosapentanenoic acid (DPA), and docosahexaenoic acid (DHA) as a reflection of dietary omega-3 status in the Labrador Retriever. Seventy-seven Labrador Retrievers were evaluated for BCS, percent fasting serum lipoprotein fatty acid concentrations, as well as serum leptin, adiponectin, insulin, and CRP. A multivariable general linear regression model was constructed to examine the association between the dependent variables leptin, CRP, adiponectin, and insulin and the predictor variables of BCS, age, and sex, as well as concentrations of alpha-linolenic acid, EPA, DHA, and DPA. Adiponectin concentration was positively associated with age (P<0.0008), EPA (P=0.027) and negatively associated with DHA (P=0.008). Leptin concentration was positively associated with an increased DHA (P=0.009), BCS (P<0.0001), age (P=0.02), and decreased DPA (P=0.06). Insulin concentration was only associated with BCS (P<0.0001), and no meaningful associations were found for CRP. Longer chain omega-3 fatty acids may play a role in regulating adiponectin concentrations in dogs. However, because insulin concentrations were associated only with BCSs, further examination of the role of adiponectin in canine obesity is warranted. EPA and DPA may reduce the overall inflammatory state in dogs as these omega-3 fatty acids reflect increased adiponectin (increased EPA and decreased DHA) and decreased leptin (decreased DHA and increased DPA).