Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene‐doping detection considering the presence of pseudogenes

Tozaki, Teruaki; Ohnuma, Aoi; Kikuchi, Mio; Ishige, Taichiro; Kakoi, Hironaga; Hirota, Kei‐ichi; Kusano, Kanichi; Nagata, Shun‐ichi

doi:10.1111/age.13174

Cited by 8 publications

(9 citation statements)

References 29 publications

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“…High INDEL density was observed on chromosomes 12 and 20. Previous studies observed a similar trend on the same chromosomes for SNVs and nonsynonymous substitutions [ 7 , 16 ]. Although the exact reason for this is unknown, one reason is thought to be the existence of metabolic and sensory perception regions on chromosome 12, and immune response and antigen-processing regions on chromosome 20 [ 18 , 19 , 20 ].…”

Section: Discussionsupporting

confidence: 73%

“…It was confirmed that 779,457 loci (49.8%) were novel INDELs by comparing them with the Ensembl (Release 108). Our current and previous studies [ 7 , 16 ] identified 12 million SNVs, 1.56 million short INDELs, and 62 processed pseudogenes in 101 Thoroughbred horses. Although Thoroughbreds were bred as a closed population from a small founder population, it was demonstrated that current Thoroughbred populations have diverse genome structures.…”

Section: Discussionmentioning

confidence: 74%

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Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses

Tozaki

Ohnuma

Kikuchi

et al. 2023

Genes

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Thoroughbreds are some of the most famous racehorses worldwide and are currently animals of high economic value. To understand genomic variability in Thoroughbreds, we identified genome-wide insertions and deletions (INDELs) and obtained their allele frequencies in this study. INDELs were obtained from whole-genome sequencing data of 101 Thoroughbred racehorses by mapping sequence reads to the horse reference genome. By integrating individual data, 1,453,349 and 113,047 INDELs were identified in the autosomal (1–31) and X chromosomes, respectively, while 18 INDELs were identified on the mitochondrial genome, totaling 1,566,414 INDELs. Of those, 779,457 loci (49.8%) were novel INDELs, while 786,957 loci (50.2%) were already registered in Ensembl. The sizes of diallelic INDELs ranged from −286 to +476, and the majority, 717,736 (52.14%) and 220,672 (16.03%), were 1-bp and 2-bp variants, respectively. Numerous INDELs were found to have lower frequencies of alternative (Alt) alleles. Many rare variants with low Alt allele frequencies (<0.5%) were also detected. In addition, 5955 loci were genotyped as having a minor allele frequency of 0.5 and being heterogeneous genotypes in all the horses. While short-read sequencing and its mapping to reference genome is a simple way of detecting variants, fake variants may be detected. Therefore, our data help to identify true variants in Thoroughbred horses. The INDEL database we constructed will provide useful information for genetic studies and industrial applications in Thoroughbred horses, including a gene-editing test for gene-doping control and a parentage test using INDELs for horse registration and identification.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 74%

Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses

Tozaki

Ohnuma

Kikuchi

et al. 2023

Genes

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“…100 However, there may be a risk of false positives using these methodologies if there is an intron-less "pseudogene" in the horse's genome, which corresponds to the transgene of interest. 101 An alternative method for detection of transgenic horses produced via SCNT proposes using whole-mitochondrial sequencing via tiled long-range PCR and NGS to detect differences in mitochondrial DNA between a transgenic horse and the registered dam. 102 The aforementioned studies were performed in vitro or in silico and to date, no studies have been carried out using transgenic animals or embryos.…”

Section: In Vivo Raav Gene Therapy In Horsesmentioning

confidence: 99%

“…A database of rare and common genetic variants in Thoroughbred horses has been established for the purpose of non‐targeted detection of transgenic equids 100 . However, there may be a risk of false positives using these methodologies if there is an intron‐less “pseudogene” in the horse's genome, which corresponds to the transgene of interest 101 . An alternative method for detection of transgenic horses produced via SCNT proposes using whole‐mitochondrial sequencing via tiled long‐range PCR and NGS to detect differences in mitochondrial DNA between a transgenic horse and the registered dam 102 .…”

Section: Introductionmentioning

confidence: 99%

Administration and detection of gene therapy in horses: A systematic review

Haughan

Ortved

Robinson

2022

Drug Testing and Analysis

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Gene therapy uses genetic modification of cells to produce a therapeutic effect.Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression.Gene therapy is currently prohibited in both human and equine athletes because of the potential to induce production of performance-enhancing proteins in the athlete's body, also referred to as "gene doping." Detection of gene doping is challenging and necessitates development of creative, novel analytical methods for doping control. Methods for detection of gene doping must be specific to and will vary depending on the type of gene therapy. The purpose of this paper is to present the results of a systematic review of gene editing, gene therapy, and detection of gene doping in horses.Based on the published literature, gene therapy has been administered to horses in a large number of experimental studies and a smaller number of clinical cases. Detection of gene therapy is possible using a combination of PCR and sequencing technologies. This summary can provide a basis for discussion of appropriate and inappropriate uses for gene therapy in horses by the veterinary community and guide expansion of methods to detect inappropriate uses by the regulatory community.

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“…NGS technology has also enabled whole-genome resequencing of species with reference genome sequences [ 24 , 25 , 26 ]. Using NGS, transgenes can be detected as deletions of introns [ 27 , 28 , 29 ], enabling screening for nontargeted transgenes by comparing the detected intron deletions to annotated gene information (i.e., the intron location in reference sequences). Amplicon sequencing to detect targeted genes using NGS has also been developed for transgene detection [ 30 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control

Tozaki

Ohnuma

Nakamura

et al. 2022

Genes

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The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

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Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene‐doping detection considering the presence of pseudogenes

Cited by 8 publications

References 29 publications

Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses

Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses

Administration and detection of gene therapy in horses: A systematic review

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control

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