•Dog leucocyte antigen (DLA) class II genes were examined in laboratory Beagles.•Laboratory Beagles represent a different immunogenetic pool compared with pet Beagles.•Beagles do not express the full range of DLA haplotypes seen in the canine population.•Beagles could be suitable for vaccine studies if genetic diversity is maintained.
Beagles are commonly used in vaccine trials as part of the regulatory approval process. Genetic restriction within this breed and the impact this might have on vaccine responses are rarely considered. This study was designed to characterise diversity of dog leucocyte antigen (DLA) class II genes in a breeding colony of laboratory Beagles, whose offspring are used in vaccine studies. DLA haplotypes were determined by PCR and sequence-based typing from genomic DNA extracted from blood. Breeding colony Beagles had significantly different DLA haplotype frequencies in comparison with pet Beagles and both groups showed limited DLA diversity. Restricted DLA class II genetic variability within Beagles might result in selective antigen presentation and vaccine responses that are not necessarily representative of those seen in other dog breeds.
Beagle; Dog leucocyte antigen; Major histocompatibility complex
To identify new genetic associations with juvenile and adult dermatomyositis (DM).
We performed a genome-wide association study (GWAS) of adult and juvenile DM patients of European ancestry (n = 1178) and controls (n = 4724). To assess genetic overlap with other autoimmune disorders, we examined whether 141 single nucleotide polymorphisms (SNPs) outside the major histocompatibility complex (MHC) locus, and previously associated with autoimmune diseases, predispose to DM.
Compared to controls, patients with DM had a strong signal in the MHC region consisting of GWAS-level significance (P < 5x10−8) at 80 genotyped SNPs. An analysis of 141 non-MHC SNPs previously associated with autoimmune diseases showed that three SNPs linked with three genes were associated with DM, with a false discovery rate (FDR) < 0.05. These genes were phospholipase C like 1 (PLCL1, rs6738825, FDR=0.00089), B lymphoid tyrosine kinase (BLK, rs2736340, FDR=0.00031), and chemokine (C-C motif) ligand 21 (CCL21, rs951005, FDR=0.0076). None of these genes was previously reported to be associated with DM.
Our findings confirm the MHC as the major genetic region associated with DM and indicate that DM shares non-MHC genetic features with other autoimmune diseases, suggesting the presence of additional novel risk loci. This first identification of autoimmune disease genetic predispositions shared with DM may lead to enhanced understanding of pathogenesis and novel diagnostic and therapeutic approaches.
dermatomyositis; adult; juvenile; shared autoimmunity genes
There is a known association between myositis and cancer. The risk is greater in dermatomyositis (DM) than polymyositis (PM), although reliable methods to predict cancer risk in specific patients with myositis are not presently available. This study was undertaken to determine whether risk of developing cancer in myositis can be predicted by antibody profiling.
A cross‐sectional study of UK Caucasian adults with PM (n = 109), DM (n = 103) and connective tissue disease overlap (myositis/CTD‐overlap, n = 70). Patients were tested for a comprehensive range of myositis‐specific/associated autoantibodies. Sensitivity and specificity analyses were performed for the optimal identification of cancer risk.
Sixteen patients had cancer‐associated myositis (CAM) (15 DM, 1 myositis/CTD‐overlap). CAM patients were older at disease onset, and patients without myositis‐specific/associated autoantibodies on “routine” laboratory testing (negative for anti‐Jo‐1, anti‐PM‐Scl, anti‐U1‐RNP, anti‐U3‐RNP, anti‐Ku antibodies) had a significantly increased risk of CAM. Possession of the antibody against 155 kDa and 140 kDa protein specificities (anti‐155/140 antibody) represented a significant risk factor for CAM, and was found exclusively in DM. A positive anti‐155/140 antibody result proved highly specific, moderately sensitive, with high negative predictive value for CAM. A “negative routine myositis antibody panel” result was highly sensitive, with high negative predictive value for CAM. The combination of these two approaches was 94% sensitive, detecting 15 of 16 CAM, with 100% sensitivity and negative predictive value in DM.
These results may help clinicians predict which patients with myositis are at greater risk of developing cancer, thus identifying those requiring aggressive diagnostic evaluation and intensive cancer surveillance at myositis onset and follow‐up.
To determine whether interferon‐gamma (IFN‐γ) and interleukin‐4 (IL‐4) genes confer susceptibility for the idiopathic inflammatory myopathies (IIMs).
A large cross‐sectional study of UK caucasian adults with polymyositis (PM, n = 101), dermatomyositis (DM, n = 94) and myositis overlapping with a connective tissue disease (myositis/CTD‐overlap, n = 70) was completed. 177 ethnically matched controls were available for comparison. Single‐nucleotide polymorphisms (SNPs) within intronic regions coding for IL‐4, IFN‐γ and a microsatellite marker within intron 1 of the IFN‐γ gene were typed.
Strong linkage disequilibrium was present between SNPs in each gene. In the IFN‐γ gene, a weak allelic association was observed in PM versus controls at rs1861493 (odds ratio (OR) 1.6, 95% confidence interval (CI) 1.03 to 2.4). The microsatellite IFN‐γ CA(14) allele was associated with risk for IIMs overall (OR 3.3, 95% CI 1.4 to 7.8), the strongest association being observed within the anti‐U1‐ribonucleoprotein (RNP) group (OR 6.0, 95% CI 1.5 to 23.1), and persisting after adjustment for known myositis human leucocyte antigen (HLA) class II associations.
Genetic markers in the IFN‐γ gene demonstrate significant allelic associations with the IIMs in a UK Caucasian population. The SNPs tested in this study within the region coding for IL‐4 fail to show significant associations with susceptibility to IIM disease.
Objective. Treatment-resistant muscle wasting is an increasingly recognized problem in idiopathic inflammatory myopathy (IIM). TNF-α is thought to induce muscle catabolism via activation of nuclear factor-kappa B (NF-κB). Several genes share homology with the NF-κB family of proteins. This study investigated the role of NF-κB-related genes in disease susceptibility in UK Caucasian IIM.
Methods. Data from 362 IIM cases [274 adults, 49 (±14.0) years, 72% female; 88 juveniles, 6 (±3.6) years, 73% female) were compared with 307 randomly selected Caucasian controls. DNA was genotyped for 63 single nucleotide polymorphisms (SNPs) from NF-κB-related genes. Data were stratified by IIM subgroup/serotype.
Results. A significant allele association was observed in the overall IIM group vs controls for the IKBL-62T allele (rs2071592, odds ratio 1.5, 95% CI 1.21, 1.89, corrected P = 0.0086), which strengthened after stratification by anti-Jo-1 or -PM-Scl antibodies. Genotype analysis revealed an increase for the AT genotype in cases under a dominant model. No other SNP was associated in the overall IIM group. Strong pairwise linkage disequilibrium was noted between IKBL-62T, TNF-308A and HLA-B*08 (D′ = 1). Using multivariate regression, the IKBL-62T IIM association was lost after adjustment for TNF-308A or HLA-B*08.
Conclusion. An association was noted between IKBL-62T and IIM, with increased risk noted in anti-Jo-1- and -PM-Scl antibody-positive patients. However, the IKBL-62T association is dependent on TNF-308A and HLA-B*08, due to strong shared linkage disequilibrium between these alleles. After adjustment of the 8.1 HLA haplotype, NF-κB genes therefore do not independently confer susceptibility in IIM.
polymyositis; dermatomyositis; single nucleotide polymorphisms; immunogenetics; autoantibodies; NF-κB; TNF
Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in the elderly. It is characterized by changes in joint structure including degeneration of the articular cartilage and its etiology is multifactorial with a strong postulated genetic component. We performed a meta-analysis of four genome-wide association (GWA) studies of 2,371 knee OA cases and 35,909 controls in Caucasian populations. Replication of the top hits was attempted with data from additional ten replication datasets. With a cumulative sample size of 6,709 cases and 44,439 controls, we identified one genome-wide significant locus on chromosome 7q22 for knee OA (rs4730250, p-value=9.2×10−9), thereby confirming its role as a susceptibility locus for OA. The associated signal is located within a large (500kb) linkage disequilibrium (LD) block that contains six genes; PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, beta), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like), and BCAP29 (the B-cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.
A breed-specific polymyositis is frequently observed in the Hungarian Vizsla. Beneficial clinical response to immunosuppressive therapies has been demonstrated which points to an immune-mediated aetiology. Canine inflammatory myopathies share clinical and histological similarities with the human immune-mediated myopathies. As MHC class II associations have been reported in the human conditions we investigated whether an MHC class II association was present in the canine myopathy seen in this breed. 212 Hungarian Vizsla pedigree dogs were stratified both on disease status and degree of relatedness to an affected dog. This generated a group of 29 cases and 183 “graded” controls: 93 unaffected dogs with a first degree affected relative, 44 unaffected dogs with a second degree affected relative, and 46 unaffected dogs with no known affected relatives. Eleven DLA class II haplotypes were identified, of which, DLA-DRB1*02001/DQA1*00401/DQB1*01303, was at significantly raised frequency in cases compared to controls (OR = 1.92, p = 0.032). When only control dogs with no family history of the disease were compared to cases, the association was further strengthened (OR = 4.08, p = 0.00011). Additionally, a single copy of the risk haplotype was sufficient to increase disease risk, with the risk substantially increasing for homozygotes. There was a trend of increasing frequency of this haplotype with degree of relatedness, indicating low disease penetrance. These findings support the hypothesis of an immune-mediated aetiology for this canine myopathy and give credibility to potentially using the Hungarian Vizsla as a genetic model for comparative studies with human myositis.
Insulin-like growth factors are implicated in the development of diabetic nephropathy. IGF-binding protein 2 (IGFBP2) and IGF2 are expressed in the kidney, but their associations with diabetic nephropathy are unclear. We therefore tested the hypothesis that circulating levels of IGF2 and IGFBP2 predict longitudinal renal function in individuals with type 2 diabetes.
Design and methods
IGFBP2 and IGF2 measurements were performed in 436 individuals (263 males) with type 2 diabetes. Linear mixed-effect regression analysis was used to model the relationship between plasma IGFBP2 concentration and longitudinal changes in estimated glomerular filtration rate (eGFR) over an 8-year period. Analyses were also performed for IGF1, IGF2, IGFBP1 and IGFBP3 concentrations as predictors of longitudinal renal outcomes.
High IGFBP2 concentration at baseline was associated with a decreased eGFR over an 8-year period (β=−0.02, (95% confidence interval −0.03 to −0.01), P<0.001). High IGFBP1, IGFBP2 and IGFBP3 were also associated with low baseline eGFR concentration.
This study demonstrates that IGFBP2 is a predictor of longitudinal deterioration of renal function in type 2 diabetes.
IGFBP2; longitudinal trends; renal function; real-world data
The current disease model for leishmaniasis suggests that only a proportion of infected individuals develop clinical disease, while others are asymptomatically infected due to immune control of infection. The factors that determine whether individuals progress to clinical disease following Leishmania infection are unclear, although previous studies suggest a role for host genetics. Our hypothesis was that canine leishmaniasis is a complex disease with multiple loci responsible for the progression of the disease from Leishmania infection.
Genome-wide association and genomic selection approaches were applied to a population-based case-control dataset of 219 dogs from a single breed (Boxer) genotyped for ∼170,000 SNPs. Firstly, we aimed to identify individual disease loci; secondly, we quantified the genetic component of the observed phenotypic variance; and thirdly, we tested whether genome-wide SNP data could accurately predict the disease.
We estimated that a substantial proportion of the genome is affecting the trait and that its heritability could be as high as 60%. Using the genome-wide association approach, the strongest associations were on chromosomes 1, 4 and 20, although none of these were statistically significant at a genome-wide level and after correcting for genetic stratification and lifestyle. Amongst these associations, chromosome 4: 61.2–76.9 Mb maps to a locus that has previously been associated with host susceptibility to human and murine leishmaniasis, and genomic selection estimated markers in this region to have the greatest effect on the phenotype. We therefore propose these regions as candidates for replication studies. An important finding of this study was the significant predictive value from using the genomic information. We found that the phenotype could be predicted with an accuracy of ∼0.29 in new samples and that the affection status was correctly predicted in 60% of dogs, significantly higher than expected by chance, and with satisfactory sensitivity-specificity values (AUC = 0.63).
Imputation is an extremely valuable tool in conducting and synthesising genome-wide association studies (GWASs). Directly typed SNP quality control (QC) is thought to affect imputation quality. It is, therefore, common practise to use quality-controlled (QCed) data as an input for imputing genotypes. This study aims to determine the effect of commonly applied QC steps on imputation outcomes. We performed several iterations of imputing SNPs across chromosome 22 in a dataset consisting of 3177 samples with Illumina 610k (Illumina, San Diego, CA, USA) GWAS data, applying different QC steps each time. The imputed genotypes were compared with the directly typed genotypes. In addition, we investigated the correlation between alternatively QCed data. We also applied a series of post-imputation QC steps balancing elimination of poorly imputed SNPs and information loss. We found that the difference between the unQCed data and the fully QCed data on imputation outcome was minimal. Our study shows that imputation of common variants is generally very accurate and robust to GWAS QC, which is not a major factor affecting imputation outcome. A minority of common-frequency SNPs with particular properties cannot be accurately imputed regardless of QC stringency. These findings may not generalise to the imputation of low frequency and rare variants.
genome-wide association study; imputation; quality control; single nucleotide polymorphism
Animals might be able to use highly polymorphic genetic markers to recognize very close relatives and avoid inbreeding [1, 2]. The major histocompatibility complex (MHC) is thought to provide such a marker [1, 3–6] because it influences individual scent in a broad range of vertebrates [6–10]. However, direct evidence is very limited [1, 6, 10, 11]. In house mice (Mus musculus domesticus), the major urinary protein (MUP) gene cluster provides another highly polymorphic scent signal of genetic identity [8, 12–15] that could underlie kin recognition. We demonstrate that wild mice breeding freely in seminatural enclosures show no avoidance of mates with the same MHC genotype when genome-wide similarity is controlled. Instead, inbreeding avoidance is fully explained by a strong deficit in successful matings between mice sharing both MUP haplotypes. Single haplotype sharing is not a good guide to the identification of full sibs, and there was no evidence of behavioral imprinting on maternal MHC or MUP haplotypes. This study, the first to examine wild animals with normal variation in MHC, MUP, and genetic background, demonstrates that mice use self-referent matching of a species-specific [16, 17] polymorphic signal to avoid inbreeding. Recognition of close kin as unsuitable mates might be more variable across species than a generic vertebrate-wide ability to avoid inbreeding based on MHC.
Increased expression of epithelial cell-derived neutrophil-activating peptide (ENA-78) has been reported in several immune and inflammatory conditions suggesting its role in inflammatory response. We have identified two single nucleotide polymorphisms in the promoter and exon 2 of the ENA-78 gene by scanning the full length gene using DHPLC DNA fragment analysis and DNA sequencing.
The polymorphism at position +398 (A/G from the first ATG codon) in exon 2 results in a synonymous substitution not resulting in an amino acid change. The promoter polymorphism was found at position −156 (C/G from the first ATG codon). An assay was designed for the detection of the polymorphisms using SNapshot ddNTP primer extension, followed by capillary electrophoresis (ABI 3100).
Allele and genotype frequencies for the promoter −156 polymorphism are presented for 107 healthy Spanish and 54 UK Caucasians. Frequencies for the exon 2 polymorphism are also presented for 63 UK Caucasians.
ENA-78; polymorphism; biallelic
Osteoarthritis (OA) has a complex aetiology with a strong genetic component. Genome-wide association studies implicate several nuclear genes in the aetiology, but a major component of the heritability has yet to be defined at the molecular level. Initial studies implicate maternally inherited variants of mitochondrial DNA (mtDNA) in subgroups of patients with OA based on gender and specific joint involvement, but these findings have not been replicated.
The authors studied 138 maternally inherited mtDNA variants genotyped in a two cohort genetic association study across a total of 7393 OA cases from the arcOGEN consortium and 5122 controls genotyped in the Wellcome Trust Case Control consortium 2 study.
Following data quality control we examined 48 mtDNA variants that were common in cohort 1 and cohort 2, and found no association with OA. None of the phenotypic subgroups previously associated with mtDNA haplogroups were associated in this study.
We were not able to replicate previously published findings in the largest mtDNA association study to date. The evidence linking OA to mtDNA is not compelling at present.
Gene Polymorphism; Osteoarthritis; Pharmacogenetics
Obesity as measured by body mass index (BMI) is one of the major risk factors for osteoarthritis. In addition, genetic overlap has been reported between osteoarthritis and normal adult height variation. We investigated whether this relationship is due to a shared genetic aetiology on a genome-wide scale.
We compared genetic association summary statistics (effect size, p value) for BMI and height from the GIANT consortium genome-wide association study (GWAS) with genetic association summary statistics from the arcOGEN consortium osteoarthritis GWAS. Significance was evaluated by permutation. Replication of osteoarthritis association of the highlighted signals was investigated in an independent dataset. Phenotypic information of height and BMI was accounted for in a separate analysis using osteoarthritis-free controls.
We found significant overlap between osteoarthritis and height (p=3.3×10−5 for signals with p≤0.05) when the GIANT and arcOGEN GWAS were compared. For signals with p≤0.001 we found 17 shared signals between osteoarthritis and height and four between osteoarthritis and BMI. However, only one of the height or BMI signals that had shown evidence of association with osteoarthritis in the arcOGEN GWAS was also associated with osteoarthritis in the independent dataset: rs12149832, within the FTO gene (combined p=2.3×10−5). As expected, this signal was attenuated when we adjusted for BMI.
We found a significant excess of shared signals between both osteoarthritis and height and osteoarthritis and BMI, suggestive of a common genetic aetiology. However, only one signal showed association with osteoarthritis when followed up in a new dataset.
Osteoarthritis; Gene Polymorphism; Epidemiology