Article

Multiple alleles of ACAN associated with chondrodysplastic dwarfism in Miniature horses

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Abstract

Chondrodysplastic dwarfism in Miniature horses appeared to be a recessive genetic trait based on the occurrence of affected offspring by normal parents. Dwarf phenotypes vary and range from abnormal abortuses to viable offspring with evidence of skeletal dysplasia. A genome‐wide association study implicated a region of ECA1 with dwarfism in Miniature horses. Aggrecan (ACAN) was a candidate gene in that region, and exons were sequenced to compare DNA sequences for dwarf and non‐dwarf horses. Sequencing led to the discovery of variants in exons 2, 6, 7 and 15 associated with dwarfism. The four variants are identified with reference to Ecab 3.0 (GCF_002863925.1) as g.95291270del (rs1095048841), g.95284530C>T (ERP107353), g.95282140C>G (rs1095048823) and g.95257480_95257500del (rs1095048839) and designated here as D1, D2, D3* and D4 respectively. A previous study at another laboratory reported dwarfism associated with homozygosity for D3*. Homozygotes for those variants and compound heterozygotes for any combination of those variants always expressed a dwarfism phenotype. However, eight additional horses with dwarfism were found, seven of which were heterozygotes for D2, D3* or D4, suggesting the existence of additional ACAN alleles causing dwarfism. Among Miniature horses, the combined frequency of D1, D2, D3* and D4 was 0.163, suggesting a carrier rate of 26.2% for alleles causing chondrodysplastic dwarfism.

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... All parents and the 2 affected foals were wild type (N/N) for the other 3 mutations (D1, D2, and D3*) in the ACAN gene that were described previously. 4 Two additional missense polymorphisms were found when sequencing exons of the ACAN gene (g.95271452C>T/p.A1875V and g.95270484A>G/p. R2198G, RefSeq 100033876). ...
... However, given that the stallions were also demonstrated to be homozygous for the same single-nucleotide polymorphisms and both mares were heterozygous for g.95271452C>T and homozygous for g.95270484A>G, these amino acid changes did not interfere with the functionality of the protein. Unfortunately, as reported in another study, 4 we were also unable to sequence the initial region of coding exon 11 (~1,000 bases). ...
... 6 Phenotypically, these two D4/D4 dwarfs were similar to the D2/D2, D2/D3*, D2/D4, D3*/D3*, and D3*/D4 genotypes described previously in dwarf foals. 4,9 All of the dwarfs had a disproportionately short stature, a malformed skull, a shortened nasal bone, and mandibular prognathism, 4,9 but the front and hind cannon length appeared to be unique to D4/D4 dwarfs. Compared with a control foal, these animals appeared to have shortened cannon bones and hindlimb deformity, with a plantarodorsal deviation in the region of the tarsometatarsal joint. ...
Article
Four causative mutations (D1, D2, D3*, and D4) of chondrodysplastic dwarfism have been described in the equine aggrecan ( ACAN) gene. Homozygotes for one of these mutations and heterozygotes for any combination of these mutations exhibit the disproportionate dwarfism phenotype. However, no case description of homozygotes for D4 (D4/D4) has been reported in the literature, to our knowledge. We report 2 Miniature horses with the genotype D4/D4 in the ACAN gene. Clinically, the 2 dwarfs had a domed head that was large compared to the rest of the body, mandibular prognathism, and short and bowed limbs, mainly in the proximal region of the metatarsal bones. Radiographic examination revealed contour irregularities of the subchondral bone in the long bones and confirmed mandibular prognathism; histopathology revealed irregular chondrocyte organization. To determine the genotypes of the horses, we performed DNA extraction from white blood cells, PCR, and Sanger sequencing. Genotyping demonstrated that these 2 animals had the D4/D4 genotype in the ACAN gene. The D4/D4 dwarfs were clinically similar to animals with the other ACAN genotypes reported for this disease. Identification of heterozygous animals makes mating selection possible and is the most important control measure to minimize economic losses and casualties.
... 2,5,11 . Clinical diagnosis of chondrodysplastic dwarfism in Miniature horses has already been described in Brazil 12 , and the D4/D4 genotype was recently characterized in the same country 11 . ...
... The clinical signs observed in the Miniature horses with dwarfism described here were similar to those previously reported 2,5,11,12 . As ACAN-D1 was not observed in the dwarf Miniature horses of the present study, the clinical signs that this variant can cause (cleft palate with protruding tongue, large abdominal hernia and embryonic or late term loss) 2 were not reported in the affected animals of the present study. Therefore, our results also suggest that the genotype of an animal cannot be assumed only from the phenotypic characteristics, except for genotypes associated with ACAN-D1 2 . ...
Article
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Chondrodysplastic dwarfism in Miniature horses is an autosomal recessive disorder previously associated with four mutations (D1, D2, D3*, and D4) in the aggrecan (ACAN) gene. The aim of this study was to identify additional variants in the candidate ACAN gene associated with chondrodysplastic dwarfism in Miniature horses. Fifteen dwarf Miniature horses were found to possess only one of the dwarfism-causing variants, and two possessed none of the variants. The ACAN exons (EquCab3.0) of seven dwarf Miniature horses were sequenced. A missense SNP in coding exon 11 (g.95271115A > T, c.6465A > T-RefSeq XM_005602799.2), which resulted in the amino acid substitution p.Leu2155Phe (RefSeq XP_005602856.2), was initially associated with the dwarf phenotype. The variant was tested and found present in 14 dwarf foals as well as one parent of each, and both parents of a dwarf possessing two copies. Genetic testing of 347 phenotypically normal Miniature horses demonstrated that none had more than one of the dwarf alleles or c.6465A > T. However, a study of large breeds revealed the presence of c.6465A > T, which was present in homozygosis in two Mangalarga Marchador horses. We suggest that c.6465A > T as a marker of disequilibrium or complex interactions in the Miniature horse genome could contribute to the associated dwarfism.
... Dwarfism has been reported in many species and is a consequence of mutations that disrupt normal gene function related to growth, often with negative health consequences. For horses, variants in three genes have been associated with dwarfism: SHOX (Rafati et al. 2016), B4GALT7(Leegwater et al. 2016) and ACANEberth et al. 2018). The most common form of dwarfism in Miniature horses is a consequence of mutations in the gene ACANEberth et al. 2018). ...
... For horses, variants in three genes have been associated with dwarfism: SHOX (Rafati et al. 2016), B4GALT7(Leegwater et al. 2016) and ACANEberth et al. 2018). The most common form of dwarfism in Miniature horses is a consequence of mutations in the gene ACANEberth et al. 2018). ...
Article
Homozygous and compound heterozygous Miniature horses for ACAN alleles D1, D2, D3* and D4 exhibit chondrodysplastic dwarfism (OMIA 001271‐9796). In a previous study, the carrier rate for these four alleles, combined, was 26.2%. The purpose of this study was to investigate whether carriers of these dwarfism‐causing alleles had a shorter withers height than non‐carriers. A total of 245 Miniature horses were tested for these four ACAN alleles and also were measured for withers height. Of these horses, 98 were carriers and 147 were non‐carriers. A statistically significant difference of 1.43 inches was observed with the carriers being shorter (P = 1.72E − 11). The range of heights for the two groups overlapped, indicating that other factors, including genes, have an impact on withers height. However, the high carrier rate of these dwarfism‐causing variants may be due to selection for decreased height.
... In the ROHet of 39.2-43.05 Mb on SSC16, the Aggrecan (ACAN) gene is known to affect cartilage development, and homozygous and compound heterozygous of the four ACAN alleles cause chondrodysplastic dwarfism in Miniature horses (Eberth et al., 2018). In this study, we found that heterozygosity for this genomic region actually led to a reduced phenotype for ADG trait (614.50 g/day for heterozygosity vs. 619.86 ...
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Heterozygosity can effectively reflect the diverse models of population structure and demographic history. However, the genomic distribution of heterozygotes and the correlation between regions of heterozygosity (runs of heterozygosity, ROHet) and phenotypes are largely understudied in livestock. The objective of this study was to identify ROHet in the Duroc pig genome, and investigate the relationships between ROHet and eight important economic traits. Here, we genotyped 3,770 American Duroc (S21) and 2,096 Canadian Duroc (S22) pigs using 50 K single nucleotide polymorphism array to analyze heterozygosity. A total of 145,010 and 84,396 ROHets were characterized for S21 and S22 populations, respectively. ROHet segments were mostly enriched in 1–2 Mb length classification (75.48% in S21 and 72.25% in S22). The average genome length covered by ROHet was 66.53 ± 12.20 Mb in S21 and 73.32 ± 13.77 Mb in S22 pigs. Additionally, we detected 20 and 13 ROHet islands in S21 and S22 pigs. Genes in these genomic regions were mainly involved in the biological processes of immunity and reproduction. Finally, the genome-wide ROHet-phenotypes association analysis revealed that 130 ROHets of S21 and 84 ROHets of S22 were significantly associated with eight economic traits. Among the candidate genes in the significant ROHet regions, 16 genes related to growth, metabolism, and meat quality were considered as candidate genes for important economic traits of pigs. This work preliminarily explores the effect of heterozygosity-rich regions in the pig genome on production performance and provides new insights for subsequent research on pig genetic improvement.
... Peripheral blood (from live animals) or hair bulb tissue (from dead animals) was collected from all horses for ACAN genotyping tests. PCR and Sanger sequencing to detect the previously described ACAN gene mutations [10] were performed according to previously described methods [11] (Table S1). ...
Article
Dwarfism is a skeletal disorder that causes abnormal growth. In Miniature horses, dwarfism can occur as chondrodysplastic dwarfism, an autosomal recessive disorder associated with five mutations (D1, D2, D3*, D4 and c.6465A>T variant) in the aggrecan (ACAN) gene. The aim of this study was to evaluate the expression of aggrecan (at the gene and protein level) and specific cytokines (IL-1β, IL-6, and TNF-α) in the articular cartilage of Miniature horses with chondrodysplastic dwarfism (D4/c.6465A>T genotype). Metatarsal bone samples from eight dwarf Miniature horses were collected for histopathological analysis, and articular cartilage was collected to detect and quantify aggrecan levels through Western blotting and determine the relative expression levels of ACAN, IL-1β, IL-6, and TNF-α through qPCR. All affected animals presented chondrodysplasia-like lesions with disorganization of the chondrocyte layers and reduced the amount of an extracellular matrix. No significant difference in aggrecan expression levels in uncleaved samples from the dwarf and control groups (composed of phenotypically normal animals of similar age and breed (P = 0.7143)) was found using Western blotting. qPCR revealed that ACAN gene expression was higher in the affected animals than in normal animals (P = 0.0119). No significant difference in cytokine levels was detected between the groups. Mutant aggrecan may interfere with normal cellular function, leading to chondrodysplasia and the observed phenotypic findings.
... As a core transcription factor in embryonic stem cells, ZNF281 (zinc finger protein 281) was found to be related to spontaneous osteochondrogenic differentiation [38]. Other studies showed the strong association of LCORL/NCAPG, HMGA2, PROP1, LASP [39], ZFAT, DIAPH3 [40], ACTN2, ADAMTS17, GH1, ANKRD1 [40], and ACAN [41,42] with miniature size and dwarfism in a variety of pony breeds, including Shetland [41,43], miniature [44], Welsh ponies [45], German warmblood horses [46], American miniature horses, Brazilian ponies [47], and Jeju ponies [48], as well as B4GALT7 [49] and PROP1 [50] in Friesian horses. These genes were not found in this study, which may be due to the expression specificity of lncRNAs in different horse breeds [27] and different omics levels, since TBX3, under strong selection in Chinese ponies, was also identified in this study. ...
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As a nutrient sensor, the placenta plays a key role in regulating fetus growth and development. Long non-coding RNAs (lncRNAs) have been shown to regulate growth-related traits. However, the biological function of lncRNAs in horse placentas remains unclear. To compare the expression patterns of lncRNAs in the placentas of the Chinese Ningqiang (NQ) and Yili (YL) breeds, we performed a transcriptome analysis using RNA sequencing (RNA-seq) technology. NQ is a pony breed with an average adult height at the withers of less than 106 cm, whereas that of YL is around 148 cm. Based on 813 million high-quality reads and stringent quality control procedures, 3011 transcripts coding for 1464 placental lncRNAs were identified and mapped to the horse reference genome. We found 107 differentially expressed lncRNAs (DELs) between NQ and YL, including 68 up-regulated and 39 down-regulated DELs in YL. Six (TBX3, CACNA1F, EDN3, KAT5, ZNF281, TMED2, and TGFB1) out of the 233 genes targeted by DELs were identified as being involved in limb development, skeletal myoblast differentiation, and embryo development. Two DELs were predicted to target the TBX3 gene, which was found to be under strong selection and associated with small body size in the Chinese Debao pony breed. This finding suggests the potential functional significance of placental lncRNAs in regulating horse body size.
... 2014b; Frischknecht et al. 2016;Metzger et al. 2018). It is important that height is treated distinctly from dwarfism, which is simply inherited in several populations including the Miniature Horse ( Metzger et al. 2017;Eberth et al. 2018) and Friesian ( Leegwater et al. 2016; Table 3). ...
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The horse reference genome from the Thoroughbred mare Twilight has been available for a decade and, together with advances in genomics technologies, has led to unparalleled developments in equine genomics. At the core of this progress is the continuing improvement of the quality, contiguity and completeness of the reference genome, and its functional annotation. Recent achievements include the release of the next version of the reference genome (EquCab3.0) and generation of a reference sequence for the Y chromosome. Horse satellite‐free centromeres provide unique models for mammalian centromere research. Despite extremely low genetic diversity of the Y chromosome, it has been possible to trace patrilines of breeds and pedigrees and show that Y variation was lost in the past approximately 2300 years owing to selective breeding. The high‐quality reference genome has led to the development of three different SNP arrays and WGSs of almost 2000 modern individual horses. The collection of WGS of hundreds of ancient horses is unique and not available for any other domestic species. These tools and resources have led to global population studies dissecting the natural history of the species and genetic makeup and ancestry of modern breeds. Most importantly, the available tools and resources, together with the discovery of functional elements, are dissecting molecular causes of a growing number of Mendelian and complex traits. The improved understanding of molecular underpinnings of various traits continues to benefit the health and performance of the horse whereas also serving as a model for complex disease across species.
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Short stature is a common pediatric disorder affecting 3% of the population. However, the clinical variability and genetic heterogeneity prevents the identification of the underlying cause in about 80% of the patients. Recently, heterozygous mutations in the ACAN gene coding for the proteoglycan aggrecan, a main component of the cartilage matrix, were associated with idiopathic short stature. To ascertain the prevalence of ACAN mutations and broaden the phenotypic spectrum in patients with idiopathic short stature we performed sequence analyses in 428 families. We identified heterozygous nonsense mutations in four and potentially disease-causing missense variants in two families (1.4%). These patients presented with a mean of -3.2 SDS and some suggestive clinical characteristics. The results suggest heterozygous mutations in ACAN as a common cause of isolated as well as inherited idiopathic short stature.
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Aggrecan, encoded by ACAN, is a major proteoglycan component of the extracellular matrix in the growth plate and articular cartilage. Aggrecan provides the hydrated gel structure important for the load-bearing properties of joints and plays a key role in cartilage and bone morphogenesis. At least 25 pathological ACAN mutations have been identified in patients with highly variable phenotypes of syndromic or non-syndromic short stature. This review provides an overview of the current understanding of ACAN and the clinical and genetic findings concerning aggrecan-associated diseases.
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Nanomelia is a recessively inherited connective tissue disorder of chicken affecting cartilage development. Other investigators have demonstrated that it involves low aggrecan production and diminished aggrecan mRNA levels. Based on genetic linkage studies showing a high likelihood that the mutation responsible for the nanomelic phenotype lay within the aggrecan gene, a series of experiments was performed to define the molecular basis of the trait. Aggrecan mRNA was present in the nucleus of the nanomelic chondrocyte but greatly reduced in the cytoplasmic compartment, a finding suggestive of a premature stop codon within the aggrecan transcript. Since no defect in mRNA splicing could be demonstrated by ribonucleasease protection studies, direct DNA sequencing was initiated by polymerase chain reaction of the mRNA and of genomic DNA. A stop codon was demonstrated at codon 1513, which is located in the eighth repeat of the chondroitin sulfate 2 domain of the large tenth exon. The mutation creates a unique BasBI restriction site which readily distinguishes the mutant and wild-type alleles.
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We previously reported the cloning and sequencing of a 1.5-kilobase cDNA which encoded a portion of the chondroitin sulfate domain from the chick cartilage proteoglycan core protein and the localization of a species-specific monoclonal antibody epitope. Using polymerase chain reaction amplification and primer extension, cDNA clones which code for the entire proteoglycan core protein have now been obtained from a 10-day chick embryo cDNA library. The composite sequence is 6464 nucleotides long, coding for a protein of 2109 amino acid residues with a calculated M(r) = 223,500. The overall arrangement of globular and carbohydrate-attachment domains is similar to human and rat chondrosarcoma aggrecan, but there are significant differences in detailed homology between chick and mammalian core proteins. Most significantly a highly repetitive region (19 repeat units of 20 residues each), not found in either human or rat, enlarges one of the characteristic serine-glycine containing regions (designated CS-2) while the other serine-glycine containing domain (designated CS-1) is approximately one-fourth the length of the mammalian CS-1. Analysis of a polymerase chain reaction-amplified fragment encoding the chick-specific repeat region revealed a single base mutation at position 4553 (G to T transversion) that converted the codon GAA for glutamate at amino acid 1513 to TAA, a stop codon, in nanomelic chondrocytes. Genomic DNA from nanomelic liver was also digested with restriction enzyme BsaBI to verify the G to T transversion. This single mutation leads to a shortened core protein precursor with a calculated M(r) = 158,300. The resulting phenotype, nanomelia, arises because the truncated core protein is neither processed to a mature proteoglycan, nor secreted from the chondrocyte.
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Cartilage extracellular matrix (ECM) is composed primarily of type II collagen and large, link stabilized aggregates of hyaluronic acid and chondroitin sulfate proteoglycan (aggrecan). Maturation and function of these complex macromolecules are dependent upon sequential processing events which occur during their movements through specific subcellular compartments in the constitutive secretory pathway. Failure to complete these events successfully results in assembly of a defective ECM and may produce skeletal abnormalities. Nanomelia is a lethal genetic mutation of chickens characterized by shortened and malformed limbs. Previous biochemical studies have shown that cultured nanomelic chondrocytes synthesize a truncated aggrecan core protein precursor that disappears with time; however, the protein does not appear to be processed by the Golgi or secreted. The present study investigates the intracellular trafficking of the defective aggrecan precursor using immunofluorescence, immunoelectron microscopy and several inhibitors. Results indicate that nanomelic chondrocytes assemble an ECM that contains type II collagen, but lacks aggrecan. Instead, aggrecan precursor was localized intracellularly, within small cytoplasmic structures corresponding to extensions of the endoplasmic reticulum (ER). At no time were precursor molecules observed in the Golgi. In contrast, normal and nanomelic chondrocytes exhibited no difference in the intracellular or extracellular distribution of type II procollagen. Therefore, retention of the aggrecan precursor appears to be selective. Incubation of chondrocytes at 15 degrees C resulted in the retention and accumulation of product in the ER. After a return to 37 degrees C, translocation of the product to the Golgi was observed for normal, but not for nanomelic, chondrocytes, although the precursors disappeared with time. Ammonium chloride, an inhibitor of lysosomal function, had no effect on protein loss, suggesting that the precursor was removed by a non-lysosomal mechanism, possibly by ER-associated degradation. Based on these studies, we suggest that nanomelic chondrocytes are a useful model for examining cellular trafficking and sorting events and the processes by which abnormal products are targeted for retention or degradation. Further investigations should provide insight into the mechanisms underlying chondrodystrophies and other related diseases.
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Mouse cartilage matrix deficiency (cmd) is an autosomal recessive disorder caused by a genetic defect of aggrecan, a large chondroitin sulfate proteoglycan in cartilage. The homozygotes (-/-) are characterized by cleft palate and short limbs, tail, and snout. They die just after birth because of respiratory failure, and the heterozygotes (+/-) appear normal at birth. Here we report that the heterozygotes show dwarfism and develop spinal misalignment with age. Within 19 months of age, they exhibit spastic gait caused by misalignment of the cervical spine and die because of starvation. Histological examination revealed a high incidence of herniation and degeneration of vertebral discs. Electron microscopy showed a degeneration of disc chondrocytes in the heterozygotes. These findings may facilitate the identification of mutations in humans predisposed to spinal degeneration.
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The colour locus historically referred to as C in the horse is linked to microsatellites markers on horse chromosome 21. Preliminary results demonstrated linkage of Ccr, thought to be the cream dilution variant of the C locus, to HTG10. An analysis of horse chromosome 21 using additional families confirmed and established a group of markers linked to Ccr. This work also improved the resolution of previously reported linkage maps for this chromosome. Linkage analysis unambiguously produced the map order: SGCV16-(19.1 cM)-HTG10-(3.8 cM)-LEX60/COR73-(1.3 cM)-COR68-(4.5 cM)- Ccr-(11.9 cM)-LEX31. Comparative and synteny data suggested that the horse C locus is not tyrosinase (TYR).
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Spondyloepiphyseal dysplasia (SED) encompasses a heterogeneous group of disorders characterized by shortening of the trunk and limbs. The autosomal dominant SED type Kimberley (SEDK) is associated with premature degenerative arthropathy and has been previously mapped in a multigenerational family to a novel locus on 15q26.1. This locus contains the gene AGC1, which encodes aggrecan, the core protein of the most abundant proteoglycan of cartilage. We screened AGC1 for mutations and identified a single-base-pair insertion, within the variable repeat region of exon 12 in affected individuals from the family with SEDK, that introduces a frameshift of 212 amino acids, including 22 cysteine residues, followed by a premature stop codon. This is the first identification of an AGC1 mutation causing a human disorder. This finding extends the spectrum of mutated genes that may cause SED and thus will aid in the molecular delineation of this complex group of conditions.
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Whole-genome association studies (WGAS) bring new computational, as well as analytic, challenges to researchers. Many existing genetic-analysis tools are not designed to handle such large data sets in a convenient manner and do not necessarily exploit the new opportunities that whole-genome data bring. To address these issues, we developed PLINK, an open-source C/C++ WGAS tool set. With PLINK, large data sets comprising hundreds of thousands of markers genotyped for thousands of individuals can be rapidly manipulated and analyzed in their entirety. As well as providing tools to make the basic analytic steps computationally efficient, PLINK also supports some novel approaches to whole-genome data that take advantage of whole-genome coverage. We introduce PLINK and describe the five main domains of function: data management, summary statistics, population stratification, association analysis, and identity-by-descent estimation. In particular, we focus on the estimation and use of identity-by-state and identity-by-descent information in the context of population-based whole-genome studies. This information can be used to detect and correct for population stratification and to identify extended chromosomal segments that are shared identical by descent between very distantly related individuals. Analysis of the patterns of segmental sharing has the potential to map disease loci that contain multiple rare variants in a population-based linkage analysis.
A method and server for predicting damaging missense mutations
  • I.A. Adzhubei
  • S. Schmidt
  • L. Peshkin
  • V.E. Ramensky
  • A. Gerasimova
  • P. Bork
  • A.S. Kondrashov
  • S.R. Sunyaev
A method and server for predicting damaging missense mutations
  • Adzhubei