Assessment of collagen genes involved in fragmented medial coronoid process development in Labrador Retrievers as determined by affected sibling-pair analysis

OBJECTIVE: To evaluate the involvement of various collagen genes in the development of fragmented medial coronoid process (FCP) in Labrador Retrievers. SAMPLE POPULATION: 93 dogs originating from 13 litters were used in the study; FCP was diagnosed in 35 dogs, and each affected dog had at least 1 sibling that was also affected. Twelve dams and sires were included in the analysis. All dogs were purebred Labrador Retrievers except for 2 litters (offspring of a female Golden Retriever-Labrador Retriever mixed-breed dog). PROCEDURES: For each dog, DNA was isolated from blood samples. Polymorphic microsatellite markers adjacent to 14 candidate genes (ie, COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL6A3, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, and COL24A1) were analyzed by use of PCR assays; genotypes were determined via automated detection of DNA products. The level of allele sharing between pairs of affected siblings was assessed. RESULTS: Among the 93 dogs, allele sharing of the 14 collagen genes was determined as follows: COL1A1, 45%; COL1A2, 47%; COL2A1, 37%; COL3A1, 32%; COL5A1, 43%; COL5A2, 32%; COL6A3, 36%; COL9A1, 45%; COL9A2, 49%; COL9A3, 38%; COL10A1, 46%; COL11A1, 52%; COL11A2, 47%; and COL24A1, 47%. CONCLUSIONS AND CLINICAL RELEVANCE: Because siblings share 50% of their genome at random, the fact that the percentages of allele sharing among the analyzed collagen genes were not significantly > 50% indicates that these genes are not determinant candidates for FCP in Labrador Retrievers. The gene for the vitamin D receptor could also be excluded because of its proximity to COL2A1.     

Evaluation of candidate genes as a cause of chondrodysplasia in Labrador retrievers

Chondrodysplasia (CD) is a disabling, hereditary disease in Labradors with short limbs, warranting genetic screening in families at risk. Segregation analysis of eight litters with 13 affected dogs showed that autosomal recessive inheritance was consistent with the observed incidence of CD in the litters. Possible involvement of eight candidate collagen genes (COL9A1, COL9A2, COL9A3, COMP, MATN3, COL2A1, COL11A1 and COL11A2) and of a sulfate transporter glycoprotein (SLC26A2) gene in eight affected dogs and in 14 related control Labradors was investigated. Assuming recessive inheritance, the candidate genes could not be implicated in CD.     

Dilated cardiomyopathy (DCM) in dogs--pathological, clinical, diagnosis and genetic aspects

Dilated cardiomyopathy (DCM) is a heart disease which is often found in humans and animals. The age of onset of this progressive disease varies between 3 and 7 years of age. A juvenile form of DCM has been found in Portuguese Water Dogs and Doberman Pinscher Dogs. Some breeds such as Doberman pinscher, Newfoundland, Portuguese Water dog, Boxer, Great Dane, Cocker Spaniel and Irish Wolfhound exhibit a higher prevalence to DCM. There also seems to be a sex predisposition as male dogs are affected more often than female dogs and in Great Danes an X-linked recessive inheritance is likely. In Newfoundland and Boxer an autosomal dominant inheritance was found whereas an autosomal recessive inheritance was described in Portuguese Water Dogs. Atrial fibrillation as a cause or consequence of DCM is assumed for certain breeds. The causes of DCM are widely unknown in dogs. A genetic basis for this heart disease seems to exist. Apart from a few exceptions the mode of inheritance and the possible underlying gene mutations are not known for DCM in dogs. In humans mutations in several genes responsible for DCM have been identified. Comparative genetic analyses in dogs using genes causing DCM in men and a genome-wide scan with anonymus markers were not able to detect causative mutations or genomic regions harboring gene loci linked to DCM. The investigation of the genetic basis of canine DCM may lead to new insights into the pathogenesis of DCM and may result in new therapeutic approaches and breeding strategies.     

Expression of fibrosis-related genes in canine chronic hepatitis

Molecular regulation of fibrosis in chronic canine hepatitis is poorly understood. The authors employed quantitative polymerase chain reaction (PCR) to determine the expression levels of genes reported to be related to fibrosis in other species (human, mouse, and rat) and to elucidate the relationship of these genes with the degree of fibrosis and the presence or absence of ascites and/or jaundice in dogs with hepatitis. Nine fibrosis-related genes were assayed: PDGFB, PDGFD, MMP2, TIMP1, THBS1, COL1A1, COL3A1, TGFB1, and TGFB2. Liver samples of 15 dogs with chronic hepatitis and 4 healthy control dogs were obtained via laparoscopic biopsy and subjected to histologic and quantitative PCR analyses. The expression of all 9 genes showed significant positive correlation (P<.01, r>.70) with the degree of fibrosis. Furthermore, the expression levels of all genes except TGFB1 were significantly higher (P<.05) in dogs with hepatic failure-related symptoms (ascites/jaundice). Results suggest that these 9 genes are integral to the development of fibrosis in canine chronic hepatitis.     

An association study of 20 candidate genes with cryptorchidism in Siberian Husky dogs

Cryptorchidism (CO) as a common developmental defect in purebred dogs causes health concerns of reduced fertility and increases risk of testicular malignancies. A total of 49 single nucleotide polymorphisms (SNPs) discovered from 20 candidate genes was investigated to analyse their associations with CO in Siberian Huskies. The sibling‐transmission disequilibrium test on 38 discordant full‐sibs revealed seven SNPs in the collagen type II α 1 (COL2A1) gene were significantly (p < 0.05) or suggestively (p < 0.10) associated with CO. Further analyses showed that only one SNP (rs23358342) in this gene remained suggestively significant (p < 0.1) on a data set of full‐sibs with additional related dogs, but not significant on all 156 Siberian Huskies. Based on the statistical results and the involvement of COL2A1 in the testicle development and descent, we could not exclude COL2A1 as a potential candidate gene for CO in Siberian Huskies. Further studies are necessary to clarify these results from our relatively small sample size.     

Absent expression of collagen XVII (BPAG2, BP180) in canine familial localized junctional epidermolysis bullosa

Hereditary junctional epidermolysis bullosa (JEB) represents a subset of mechanobullous diseases associated with defective hemidesmosome/anchoring filament proteins leading to cleavage in the lamina lucida of the epidermal basement membrane. In humans, most cases of JEB have been related to a deficiency of either laminin-5, collagen XVII (BPAG2, BP180) or integrin β4.
We describe the existence of a previously unreported form of familial localized non-lethal JEB in German Shorthaired Pointer littermates. Acral, auricular and oral erosions and ulcers were observed. Severe ulceration of the footpads was present.
Skin biopsy specimens of non-lesional and lesional skin of affected dogs were screened for a defect in basement membrane proteins using indirect immunofluorescence and immunoperoxidase testing. Epidermal staining for laminin-5 and integrin α6β4 was similar in affected and normal control dogs. Lack of expression of collagen XVII was uniquely identified in all sections of JEB probands compared with normal control dogs.
The defective expression of collagen XVII is likely to be caused by mutation(s) of the COL17A1 gene, as previously reported in humans. This is, to date, the first report of a deficient basement membrane protein in canine JEB.     

New form of X-linked dominant hereditary nephritis in dogs

OBJECTIVE: To determine features of a new form of hereditary nephritis (HN) in dogs. ANIMALS: Parents and 16 first-generation offspring (8 males, 8 females). PROCEDURE: Adolescent dogs that developed renal failure were euthanatized and necropsied. Unaffected dogs were monitored until they were at least 2 years old. Studies included light and electron microscopy of kidneys obtained from affected and unaffected dogs and immunolabeling for collagen-IV chains in renal and epidermal basement membranes (BM). The nucleotide sequence of a portion of exon 35 of the COL4A5 gene was determined in genomic DNA isolated from affected and unaffected males. RESULTS: 7 of 8 male and 2 of 8 female offspring had proteinuria and juvenile-onset chronic renal failure, which progressed more rapidly in the males. Labeling for alpha3-alpha6(IV) chains was completely absent in renal BM of affected males and segmentally absent in affected females. Expression of alpha1-alpha2(IV) chains in glomerular BM (GBM) of affected dogs was increased. Labeling for alpha5-alpha6(IV) chains in epidermal BM was absent in affected males and segmental in affected females. Ultrastructural changes characteristic of HN were observed in GBM of affected dogs. The sequence of exon 35 of COL4A5 was normal in affected dogs. CONCLUSIONS: This renal disease is an example of X-linked dominant HN, with typical abnormalities of GBM ultrastructure and alpha(IV) chain expression. CLINICAL RELEVANCE AND IMPLICATIONS FOR HUMAN MEDICINE: Dogs with this naturally acquired progressive renal disease can be used to investigate the pathogenesis and treatment of similar disorders in human beings and dogs.     

Ehlers-Danlos-like syndrome in 2 dogs: clinical,histologic, and ultrastructural findings

Background: Ehlers-Danlos syndrome comprises a group of rare inherited connective tissue diseases characterized by skin hyperextensibility, joint laxity, skin and vessel fragility, and poor wound healing.
Objective: The purpose of this report was to describe the clinical, histologic, and ultrastructural findings in 2 dogs with collagenopathies consistent with Ehlers-Danlos syndrome.
Methods: Two dogs were examined clinically; skin extensibility index was calculated. Skin biopsies obtained from the dorsum were examined by light and electron microscopy.
Results: Both dogs had clinical signs of skin hyperextensibility and fragility, lower skin elasticity, vessel fragility, and poor wound healing. One dog had a hip dislocation, and the other had bilateral medial patellar luxation (grade II), subcutaneous hematomas produced by minimal trauma, and generalized periodontitis. Histologic and ultrastructural examination confirmed abnormalities in the structure and arrangement of collagen fibrils. Fibroblasts were characterized by variable dilatation of the rough endoplasmic reticulum, and anomalous elastic fibers (elaunin fibers) were present in the dermis.
Conclusion: Although the primary defects underlying collagenopathies in animals are still unknown, analysis of the ultrastructural changes in collagen fibrils and clinical findings could facilitate better characterization of these disorders in dogs.     

Remission of Histiocytic Ulcerative Colitis in Boxer Dogs Correlates with Eradication of Invasive Intramucosal Escherichia coli

Background: Historically, histiocytic ulcerative (HUC) (or granulomatous) colitis of Boxer dogs was considered an idiopathic immune-mediated disease with a poor prognosis. Recent reports of dramatic responses to enrofloxacin and the discovery of invasive Escherichia coli within the colonic mucosa of affected Boxer dogs support an infectious etiology.
Hypothesis: Invasive E. coli is associated with colonic inflammation in Boxer dogs with HUC, and eradication of intramucosal E. coli correlates with clinical and histologic remission.
Animals: Seven Boxer dogs with HUC.
Methods: Prospective case series. Colonic biopsies were obtained at initial evaluation in 7 dogs, and in 5 dogs after treatment with enrofloxacin. Biopsies were evaluated by standardized histopathology, and fluorescence in situ hybridization (FISH) with probes to eubacteria and E. coli .
Results: Intramucosal E. coli was present in colonic biopsies of 7/7 Boxers with HUC. Clinical response was noted in all dogs within 2 weeks of enrofloxacin (7±3.06 mg/kg q24 h, for 9.5±3.98 weeks) and was sustained in 6 dogs (median disease-free interval to date of 47 months, range 17–62). FISH was negative for E. coli in 4/5 dogs after enrofloxacin. E. coli resistant to enrofloxacin were present in the FISH-positive dog that relapsed.
Conclusions and Clinical Relevance: The correlation between clinical remission and the eradication of mucosally invasive E. coli during treatment with enrofloxacin supports the causal involvement of E. coli in the development of HUC in susceptible Boxer dogs. A poor response to enrofloxacin treatment might be due to colonization with enrofloxacin-resistant E. coli .     

Desmosomal gene evaluation in Boxers with arrhythmogenic right ventricular cardiomyopathy

OBJECTIVE: To sequence the exonic and splice site regions of the 4 desmosomal genes associated with the human form of familial arrhythmogenic right ventricular cardiomyopathy (ARVC) in Boxers with ARVC and identify a causative mutation. ANIMALS: 10 unrelated Boxers with ARVC and 2 unaffected Labrador Retrievers (control dogs). PROCEDURES: Exonic and splice site regions of the 4 genes encoding the desmosomal proteins plakophilin-2, plakoglobin, desmoplakin, and desmoglein-2 were sequenced. Sequences were compared for nucleotide sequence changes between affected dogs and the published sequences for clinically normal dogs and between affected dogs and the control dogs. Base-pair changes were considered to be causative for ARVC if they were detected in an affected dog but not in unaffected dogs, and if they involved a conserved amino acid and changed that amino acid to one of a different polarity, acid-base status, or structure. RESULTS: A causative mutation for ARVC in Boxers was not identified, although single nucleotide polymorphisms were detected in some affected dogs within exon 3 of the plakophilin-2 gene; exon 3 of the plakoglobin gene; exons 3 and 7 of the desmoglein-2 gene; and exons 6, 14, 15, and 24 of the desmoplakin gene. None of these changed the amino acid of the respective protein. CONCLUSIONS AND CLINICAL RELEVANCE: Mutations within the desmosomal genes associated with the development of ARVC in humans do not appear to be causative for ARVC in Boxers. Genomewide scanning for genetic loci of interest in dogs should be pursued.     

Investigation of TLR1-9 genes and miR-155 expression in dogs infected with canine distemper

This study aimed to determine the relationship of toll-like receptor (TLR) 1-9 genes and microRNA (miR) -155 expression levels with hematologic parameters in dogs diagnosed with canine distemper. In the study, two groups were used pre-treatment and post-treatment. Infected dogs were diagnosed with canine distemper with the help of a rapid test kit and Real Time-Polymerase Chain Reaction (RT-PCR). Based on the correlation coefficients between the expression levels of the genes examined within the scope of the study and hematologic values, a positive correlation was found between the TLR2 gene and the monocyte (MON) value and between the TLR4 gene and the platelet (PLT) value in the pre-treatment group. A strong positive correlation was identified between TLR3 and TLR9 genes and erythrocyte (RBC) and hemoglobin (HGB) values; between TLR5 gene and RBC, HGB and hematocrit (HCT) values and between TLR9 gene and RBC and HGB values in the post-treatment group, on the other hand, a positive correlation was found between TLR1 gene and MON and neutrophil (GRAN) values; between TLR3 gene and HCT value and between TLR9 gene and MON and HCT values. The study concluded that miR-155 and TLR8 gene were upregulated at a statistically significant level (P < 0.05) Post-treatment in dogs infected with canine distemper and there was a positive correlation between the upregulation of miR-155 and the upregulation of TLR8 in the same period. This result suggests that the upregulated miR-155 expression post-treatment increased TLR8 gene expression. In the light of these findings, it miR-155 may have the potential to be used in clinical practice in the treatment or prognosis of dogs infected with canine distemper.     

基于单倍型肉牛屠宰性状全基因组关联分析研究

单倍型标记与数量性状基因座(quantitative trait loci，QTL)之间具有较强的连锁不平衡(linkage disequilibrium，LD)关系，在基因定位和因果突变鉴定方面具有较高的应用价值。为了评估单倍型标记在基因组研究中的作用，本研究在华西牛资源群体中，选取该群体于2008—2021年间屠宰的共计1 478头平均月龄为24个月的个体进行研究，其中公牛1 333头，母牛145头。利用770K高密度芯片数据，基于LD阈值(r²>0.3)及固定单核苷酸多态(single nucleotide polymorphism,SNP)个数(5个连续SNP)两种方法进行单倍型构建，分别采用单位点SNP标记和两种单倍型标记共3种标记，基于GCTA的混合线性模型(mixed linear model,MLM)，开展宰前活重(LW)和屠宰率(DP)等屠宰性状的全基因组关联分析(genome-wide association study，GWAS)，定位影响屠宰性状的显著(P<0.05) SNPs、单倍型块和候选基因，同时比较3种标记的GWAS结果，评估3种标记的优劣。结果显示，3种标记在全基因组范围内共找到16个的显著SNPs及单倍型区域，主要分布于1、5、6、14、16、17和28号染色体上，同时鉴定到FAM184B、PPM1K、LCORL、RIMS2等10个与屠宰性状相关的候选基因，其中，基于SNP标记方法鉴定到的3个候选基因，在利用基于单倍型标记的方法中也鉴定到，且单倍型鉴定到的显著性位点或区域大多位于基因内部。在两种单倍型构建方法中，与基于固定SNP个数构建单倍型进行GWAS相比，基于LD阈值的构建方法鉴定到了更多候选基因。本研究结果表明，以单倍型开展GWAS可以综合考虑SNP位点间连锁关系，能较好地揭示复杂性状的遗传结构。     

Echocardiographic assessment of right ventricular systolic function in Boxers with arrhythmogenic right ventricular cardiomyopathy

Objectives

To determine whether there are differences in measures of longitudinal right ventricular (RV) systolic function among Boxers with arrhythmogenic right ventricular cardiomyopathy (ARVC) compared with healthy control Boxer dogs. To explore relationships between markers of RV systolic function and age, body weight, gender, arrhythmia frequency, and markers of left ventricular (LV) systolic function in Boxer dogs.

麦洼牦牛初乳期乳腺中2个特异表达EST片段的克隆与分析

为研究牦牛初乳期乳腺特异表达的基因,采用抑制消减杂交和斑点杂交法对麦洼牦牛初乳期(产犊后2 d)和常乳期(产犊后18 d)特异表达的基因进行了分离鉴定。结果发现2个在初乳期间特异表达的EST片段,并命名为LAO和COL1α1。测序结果LAO片段长698 bp,GenBank比对,其核苷酸序列与西藏黄牛(Bos taurus)、小家鼠(Mus musculus)、家犬(Canis familiaris)等的L-氨基酸氧化酶基因有较高的序列相似性,其序列一致性分别达到96%、73%、72%;COL1α1片段长337 bp,比对结果,其序列与Bos taurus、马鹿(Cervus elaphus)、家犬(Canis lupus fa-miliaris)等的Ⅰ型胶原蛋白α1有较高的序列相似性,其序列一致性分别为100%、100%、99%。2个序列与牛基因组的Blast分析表明,LAO片段定位于3号染色体LOC782545区,该区域编码1个L-氨基酸氧化酶基因;另一片段定位于19号染色体LOC615867区,该区域编码1个Ⅰ型胶原α1基因。研究表明,L-氨基酸氧化酶基因和Ⅰ型胶原蛋白α1基因是牦牛初乳期乳腺特异表达蛋白,这2个基因在牦牛初乳期乳腺组织中较高的表达水平可能与初乳期乳腺特殊的生理状态有关。