- Background: Distichiasis, an ocular disorder in which aberrant cilia (eyelashes) grow from the opening of the Meibomian glands of the eyelid, has been reported in Friesian horses. - infection occurs, may lead to loss of the eye. - A 371 kb run of homozygosity (ROH) on ECA13 was found in 13 of the 14 cases, providing evidence for a recessive mode of inheritance. - This deletion was associated with distichiasis, as 18 of the 19 cases were homozygous ( p . - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article. - The growth cycle of the eyelash in humans is noted to be longer than body hairs, taking approximately 5 months to complete [3]. - It has also been found that eyelashes have a shorter anagen phase and a longer telogen phase than other body hairs, contributing to the shorter length of the eyelashes [3].. - Meibomian glands are considered modified sebaceous glands because of the unique combination of lipids they secrete to assist in the prevention of evaporation of the tear film [5, 6].. - In the two published reports of distichiasis in horses, 19 of the 20 cases were of the Friesian breed (Fig. - Utter and Wotman [11] describe distichiasis causing re- current corneal ulceration in two Friesian horses located in the United States. - Specifically, five of the 21 cases were identified as half-siblings (offspring of S3, Fig. - unequal relatedness in the GWAS cohorts, an EMMAX analysis was performed. - Haplotype analysis of the ECA13 associated locus identi- fied a 371 kb run of homozygosity (ROH) in 13 out of 14 cases, which was significantly associated with disease (chi- squared p Table 1). - To confirm genotyping results and replicate associations, SNPs that reached genome-wide significance in the GWAS (27 SNPs from ECA5 and ECA13) or haplotype analyses (2 SNPs from ECA12) were genotyped in all horses from the GWAS plus a replication sample set in- cluding five additional cases and 37 additional controls.. - In the replication sample set, the loci on ECA5 and ECA12 no longer reached significance ( p >. - However, the ECA13 locus was further supported as it continued to reach genome wide significance in the. - a Normal eyelashes developing from the outer surface of the lid. - b Aberrant eyelashes growing from the Meibomian gland orifices of the inferior eyelid margin. - Represented is the most informative portion of the pedigree. - Specifically, pedigree analysis showed that five of 21 affected horses shared a common ancestor within a single generation (S3, indicated with an orange border), which was not related to any of the controls. - rs but none of these were concordant with phenotype in the three cases sequenced and therefore they were not investi- gated further.. - Replication testing supported further investigation of the ECA13 locus, coding variants in the WGS data for the three ECA13 positional candidate genes, FAM20C , PRKAR1B and PDGFA , were investigated. - No variants concordant with phenotype were identified in the three cases and two controls sequenced. - These variants were either perfectly con- cordant with the phenotype in the WGS sample set ( n = 3 cases and n = 2 controls) or were predicted by SnpEff to impact protein function (Table S1). - These 32 variants were genotyped via Agena MassARRAY spectrometry in the cohort of Friesian horses. - A significant ROH was identified in the ECA13 associated locus (chi-squared test for independence, p . - Visual inspection of the WGS data from the associated ECA13 locus identified a deletion in this region. - No cover- age in a 16.42 kb region in this locus was detected in the cases. - This variant was later confirmed as the only structural variant in the ECA13 associated haplotype using LUMPY.. - Sanger sequencing of the deletion in two cases and two controls identified the boundaries to occur at ECA13:g del. - Further genotyping of the deletion using an allele specific PCR assay in our entire phenotyped sample ( n = 94) determined that the deletion was significantly associated with phenotype as all but one case was homozygous for this structural variant (chi-squared p Table 4). - mutations in the Friesian breed was also assessed in a non-phenotyped subset of the sample ( n = 73). - None of the other prioritized WGS variants in the associated locus on ECA13 were found to be as con- cordant with phenotype as the 16 kb deletion (Table 3).. - These included 279 samples banked in the Bellone la- boratory (Haflingers: n = 51, Belgian Draft horses: n = 47, Thoroughbreds: n = 95, Quarter Horses: n horses for which WGS data were publicly available, and WGS data contributed by the McCue laboratory ( n = 389). - The deletion was only identified in 11 non-Friesian individuals in the heterozygous state (1.15%) (Table 5).. - To investigate if there was tissue-specific variation in gene expression in clinically normal horses, the three genes near the deletion ( FAM20C , PRKAR1B and PDGFA ) were assessed in the RNA-seq data. - help to further elucidate the putative impact of the dele- tion on gene function. - However, in assessing the deletion in the publicly available equine ChIP-Seq data, tissue- specific histone modifications were identified in seven of the eight tissues for which data are available (Fig. - ECA13:g.340918C >. - ECA13:g.343850A >. - A pedigree analysis of Friesian horses affected with disti- chiasis identified an average inbreeding coefficient of 14.1% in the cohort under investigation, which is higher than that reported for other breeds with closed stud- books (Thoroughbreds = 12.5% and Standardbreds . - 2019 [16] found that the Friesian horse was the most inbred and had the smallest effective population size of the nine breeds they studied based on an analysis of the inbreed- ing coefficients, the length of ROHs, and measures of linkage disequilibrium. - They reported an even higher average inbreeding coefficient 25.5% in the Friesian population they investigated [16]. - Interrogation of the associated ECA13 locus using high throughput short-read WGS data did not identify any variants within the coding sequence of the three pos- itional candidate genes. - No reads were detected in three cases within a 16.42 kb region (ECA13:g del), but were found in the controls at approximately half the coverage of the flanking sequence. - Del refers to the ECA13 16 kb deletion and Ref signifies detection of the reference allele.. - Genotyping for the deletion was completed through an allele specific PCR assay in the phenotyped cohort ( n = 94) and in an additional sample set of horses that were not phenotyped for disease, which estimated the allele frequency to be 32.34%.. - 6 Visualization of Histone Mark Functional Annotation Data in the ECA13 Deletion. - The diversity of histone marks among these tissues supports the presence of tissue-specific regulatory elements in the ECA13 deletion. - Per- forming a longitudinal study, with multiple ocular examinations over time to evaluate different phases of the hair cycle in horses genotyped for the ECA13:. - Addition- ally, performing ocular exams and genotyping for the ECA13 deletion on sires and dams of the horses homo- zygous for the deletion should aid in segregation analysis needed to further evaluate the recessive mode of inherit- ance proposed and enable a precise estimate of penetrance.. - A single horse with distichia was not homozygous for the ROH identified as a part of the GWAS ana- lysis and was heterozygous for the deletion. - Therefore, horses with distichiasis may be bred if they are asymptomatic, have mild clinical signs, or if they have not yet developed the disease, thus propa- gating the disorder in the Friesian population. - It is also possible that this deletion impacts regulation of a gene associated with a favorable phenotype in the breed, and thus is undergoing positive selection.. - This suggests that the deletion is not a Friesian-specific muta- tion, but rather an old mutation that predates the formation of the Friesian breed. - Evaluating the frequency of this muta- tion and its presence in horses that have undergone ocular phenotyping is warranted in the other breeds in which the mutation was identified.. - According to the EquCab3.0 reference genome [26], the identified deletion lies in the intergenic region be- tween FAM20C and PDGFA , which are both included in the associated haplotype identified on ECA13. - As such, PDGFA has been shown to play a role in the for- mation of submandibular salivary glands in mice [33]. - Analysis of the equine FAANG RNA-seq data from two normal horses showed no tissue-specific variation in gene expression for FAM20C , PDGFA , and PRKAR1B , and thus did not provide any insight into potential puta- tive impact of the deletion on gene function. - This study represents the first utilization of the FAANG histone ChIP-seq data to de- velop hypotheses related to regulatory regions of the genome and illustrates the importance of these datasets as a reference for future investigations. - of repression in five other tissues with H3K27me3 marks in the deleted region, support this site as a tissue- specific regulatory region. - Of the tissues assessed, lamina is the most similar to the ocular lid margin as both tis- sues have an extensive extracellular matrix. - We, therefore, hypothesize that an enhan- cer located in the deleted region plays a role in regulating the expression of PDGFA and that deletion of this enhan- cer site could modify gene expression. - The discovery of a novel variant associated with disti- chiasis will enable genetic testing allowing for marker assisted selection, which provides the opportunity to lower the incidence of the disorder in the Friesian popu- lation. - It will also help in the identification of horses likely to be affected by distichiasis, thus allowing horse owners to screen horses for the disorder and potentially provide intervention prior to the development of clinical signs and irreversible corneal damage leading to better welfare for these horses.. - Further exploration of the func- tional consequences of this deletion may help to explain the underlying etiology of distichiasis and penetrance in Friesian horses, humans, dogs and other species.. - Ninety-nine privately owned, registered Friesian horses were phenotyped for inclusion in this study and remained in their owners’ care for the duration of the study. - A complete ocular examination of the adnexa (ad- joining surfaces), anterior and posterior segments of both eyes was performed by a diplomate of the European or American College of Veterinary Ophthalmologists.. - In the replication sample set, horses with other ocu- lar pathologies were excluded as additional controls.. - Quality control consisted of the exclusion of samples with call rates ≤0.95 and SNPs with call rates ≤0.95 or minor allele frequencies of <. - A visual inspection of the haplotype from the genome-wide associated regions was performed and a chi-squared test for independence comparing the frequency of the identified haplotype in cases and controls was completed.. - The coding re- gions of the genes present in the 371 kb associated haplotype region on ECA13 plus 1 Mb flanking both the 5′ and 3′ sides of the haplotype were also assessed. - Vari- ants were prioritized for further analysis based on: (1) perfect concordance with phenotype in the identified ROH in the samples used for the WGS analysis. - and (2) SnpEff predicted functional effect of high, moderate or low regardless of concordance with phenotype in the WGS sample set. - Sanger sequencing of two cases and two controls was performed to identify the boundaries of the ECA13 deletion. - The addition of 1 μL BSA was needed to amplify the 3′ end of the deletion. - The se- quences were compared to the EquCab3.0 reference genome using the NCBI BLAST tool to determine the precise 5′ and 3′ boundaries of the deletion [52]. - A chi-square test for independence was performed to compare the preva- lence of the deletion between the cases and the con- trols. - Once the discovery of the deletion was made by our team, a DNA diagnostic test was developed and it is now commercially available at the UC Davis Veter- inary Genetics Laboratory. - Additional tests available at the UC Davis Veter- inary Genetics Laboratory were used to genotype a subset of the Friesian samples ( n = 73) for the hydro- cephalus and dwarfism mutations. - DNA samples from other horse breeds, banked in the Bellone laboratory, namely Haflingers ( n = 51), Belgian Draft horses ( n = 47), Thoroughbreds ( n = 95) and Quarter Horses ( n = 86) were also genotyped for the ECA13 deletion as de- scribed above. - Genome STRiP [54] was utilized to evaluate an additional 95 unmapped paired-end equine WGS BAM files from the SRA, as well as 389 horse genomes banked in the McCue laboratory.. - Novel variants logged in the European Variant Archive (project PRJEB34362). - The mission of the Morris Animal Foun- dation is to bridge science and resources to advance the health of animals.. - 26296: Tools to Link Phenotype to Genotype in the Horse, The American Quarter Horse Association, and a University of Minnesota Multistate grant.. - A portion of the datasets generated and/or analyzed during the current study are not publicly available as they are still being investigated for another related project but are available from the corresponding author on reasonable request. - The remaining datasets generated and/or analyzed during the current study are available in the European Variation Archive (PRJEB34362: https://www.ebi.ac.uk/ena/browser/view/PRJEB34362, and PRJEB36380: https://www.ebi.ac.uk/ena/browser/view/PRJEB36380).. - Ocular exams and sampling was performed with approval of the Institutional Animal Care and Use Committee of the University of California, Davis (#20450) and the Animal Ethics Committee of the Utrecht University (DEC file 2013.III.01.012).. - Carcinoma of sebaceous glands of the eyelid. - Utility of meibography in the evaluation of Meibomian glands morphology in normal and diseased eyelids. - The genomic makeup of nine horse populations sampled in the Netherlands.. - Aortic rupture and aorto-pulmonary fistulation in the Friesian horse:. - characterisation of the clinical and gross post mortem findings in 24 cases.. - Functionally annotating regulatory elements in the equine genome using histone mark ChIP-Seq. - Characterization of noncoding regulatory DNA in the human genome. - Inbreeding and the inheritance of fertility in the thoroughbred mare. - A microsatellite analysis of five colonial Spanish horse populations of the southeastern United States. - polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w 1118
Xem thử không khả dụng, vui lòng xem tại trang nguồn hoặc xem
Tóm tắt