Comparison of clinical features between patients with anti-synthetase syndrome and dermatomyositis: Results from the MYONET registry.

OBJECTIVES: To compare clinical characteristics, including the frequency of cutaneous, extramuscular manifestations, and malignancy, between adults with anti-synthetase syndrome (ASyS) and dermatomyositis (DM). METHODS: Using data regarding adults from the MYONET registry, a cohort of DM patients with anti-Mi2/-TIF1É£/-NXP2/-SAE/-MDA5 autoantibodies, and a cohort of ASyS patients with anti-tRNA synthetase autoantibodies (anti-Jo1/-PL7/-PL12/-OJ/-EJ/-Zo/-KS) were identified. Patients with DM sine dermatitis or with discordant dual autoantibody specificities were excluded. Sub-cohorts of patients with ASyS with or without skin involvement were defined based on presence of DM-type rashes (heliotrope rash, Gottron's papules/sign, violaceous rash, shawl sign, V sign, erythroderma, and/or periorbital rash). RESULTS: In total 1,054 patients were included (DM, n = 405; ASyS, n = 649). In ASyS cohort, 31% (n = 203) had DM-type skin involvement (ASyS-DMskin). A higher frequency of extramuscular manifestations, including Mechanic's hands, Raynaud's phenomenon, arthritis, interstitial lung disease, and cardiac involvement differentiated ASyS-DMskin from DM (all p< 0.001), whereas higher frequency of any of four DM-type rashes: heliotrope rash (n = 248, 61% vs n = 90, 44%), violaceous rash (n = 166, 41% vs n = 57, 9%), V sign (n = 124, 31% vs n = 28, 4%), and shawl sign (n = 133, 33% vs n = 18, 3%) differentiated DM from ASyS-DMskin (all p< 0.005). Cancer-associated myositis (CAM) was more frequent in DM (n = 67, 17%) compared with ASyS (n = 21, 3%) and ASyS-DMskin (n = 7, 3%) cohorts (both p< 0.001). CONCLUSION: DM-type rashes are frequent in patients with ASyS; however, distinct clinical manifestations differentiate these patients from classical DM. Skin involvement in ASyS does not necessitate increased malignancy surveillance. These findings will inform future ASyS classification criteria and patient management.

The temporal relationship between cancer and adult onset anti-transcriptional intermediary factor 1 antibody-positive dermatomyositis.

OBJECTIVES: To characterize the 10 year relationship between anti-transcriptional intermediary factor 1 antibody (anti-TIF1-Ab) positivity and cancer onset in a large UK-based adult DM cohort. METHODS: Data from anti-TIF1-Ab-positive/-negative adults with verified diagnoses of DM from the UK Myositis Network register were analysed. Each patient was followed up until they developed cancer. Kaplan-Meier methods and Cox proportional hazard modelling were employed to estimate the cumulative cancer incidence. RESULTS: Data from 263 DM cases were analysed, with a total of 3252 person-years and a median 11 years of follow-up; 55 (21%) DM cases were anti-TIF1-Ab positive. After 10 years of follow-up, a higher proportion of anti-TIF1-Ab-positive cases developed cancer compared with anti-TIF1-Ab-negative cases: 38% vs 15% [hazard ratio 3.4 (95% CI 2.2, 5.4)]. All the detected malignancy cases in the anti-TIF1-Ab-positive cohort occurred between 3 years prior to and 2.5 years after DM onset. No cancer cases were detected within the following 7.5 years in this group, whereas cancers were detected during this period in the anti-TIF1-Ab-negative cases. Ovarian cancer was more common in the anti-TIF1-Ab-positive vs -negative cohort: 19% vs 2%, respectively (P < 0.05). No anti-TIF1-Ab-positive case <39 years of age developed cancer, compared with 21 (53%) of those ≥39 years of age. CONCLUSION: Anti-TIF1-Ab-positive-associated malignancy occurs exclusively within the 3 year period on either side of DM onset, the risk being highest in those ≥39 years of age. Cancer types differ according to anti-TIF1-Ab status, and this may warrant specific cancer screening approaches.

High erythrocyte levels of the n-6 polyunsaturated fatty acid linoleic acid are associated with lower risk of subsequent rheumatoid arthritis in a southern European nested case-control study.

OBJECTIVES: Findings relating to dietary intake of n-3 polyunsaturated fatty acids (PUFA) and risk of rheumatoid arthritis (RA) are mixed. Erythrocyte membrane PUFA is an accurate objective biomarker of PUFA status; however, there are little data on erythrocyte membrane PUFA and risk of RA. The objective was therefore to compare erythrocyte membrane PUFA between pre-RA individuals and matched controls from a population-based sample, and specifically to test the hypothesis that higher levels of longer chain n-3 PUFA are associated with lower risk of RA. METHODS: The European Prospective Investigation into Cancer and Nutrition (EPIC) is a large European prospective cohort study of apparently healthy populations. We undertook a nested case-control study by identifying RA cases with onset after enrolment (pre-RA) in four EPIC cohorts in Italy and Spain. Confirmed pre-RA cases were matched with controls by age, sex, centre, and date, time and fasting status at blood collection. Conditional logistic regression analysis was used to estimate associations of PUFA with the development of RA, adjusting for potential confounders including body mass index, waist circumference, education level, physical activity, smoking status and alcohol intake. RESULTS: The study analysed samples from 96 pre-RA subjects and 258 matched controls. In this analysis, the median time to diagnosis (defined as time between date of blood sample and date of diagnosis) was 6.71 years (range 0.8-15). A significant inverse association was observed with n-6 PUFA linoleic acid (LA) levels and pre-RA in the fully adjusted model (highest tertile: OR 0.29; 95% CI 0.12 to 0.75; P for trend 0.01). No association was observed with any individual n-3 PUFA, total n-3 PUFA or total n-3:n-6 ratio. CONCLUSIONS: Erythrocyte levels of the n-6 PUFA LA were inversely associated with risk of RA, whereas no associations were observed for other n-6 or n-3 PUFA. Further work is warranted to replicate these findings and to investigate if lower LA levels are a bystander or contributor to the process of RA development.

The EuroMyositis registry: an international collaborative tool to facilitate myositis research.

AIMS: The EuroMyositis Registry facilitates collaboration across the idiopathic inflammatory myopathy (IIM) research community. This inaugural report examines pooled Registry data. METHODS: Cross-sectional analysis of IIM cases from 11 countries was performed. Associations between clinical subtypes, extramuscular involvement, environmental exposures and medications were investigated. RESULTS: Of 3067 IIM cases, 69% were female. The most common IIM subtype was dermatomyositis (DM) (31%). Smoking was more frequent in connective tissue disease overlap cases (45%, OR 1.44, 95% CI 1.09 to 1.90, p=0.012). Smoking was associated with interstitial lung disease (ILD) (OR 1.32, 95% CI 1.06 to 1.65, p=0.013), dysphagia (OR 1.43, 95% CI 1.16 to 1.77, p=0.001), malignancy ever (OR 1.78, 95% CI 1.36 to 2.33, p<0.001) and cardiac involvement (OR 2.40, 95% CI 1.60 to 3.60, p<0.001).Dysphagia occurred in 39% and cardiac involvement in 9%; either occurrence was associated with higher Health Assessment Questionnaire (HAQ) scores (adjusted OR 1.79, 95% CI 1.43 to 2.23, p<0.001). HAQ scores were also higher in inclusion body myositis cases (adjusted OR 3.85, 95% CI 2.52 to 5.90, p<0.001). Malignancy (ever) occurred in 13%, most commonly in DM (20%, OR 2.06, 95% CI 1.65 to 2.57, p<0.001).ILD occurred in 30%, most frequently in antisynthetase syndrome (71%, OR 10.7, 95% CI 8.6 to 13.4, p<0.001). Rash characteristics differed between adult-onset and juvenile-onset DM cases ('V' sign: 56% DM vs 16% juvenile-DM, OR 0.16, 95% CI 0.07 to 0.36, p<0.001). Glucocorticoids were used in 98% of cases, methotrexate in 71% and azathioprine in 51%. CONCLUSION: This large multicentre cohort demonstrates the importance of extramuscular involvement in patients with IIM, its association with smoking and its influence on disease severity. Our findings emphasise that IIM is a multisystem inflammatory disease and will help inform prognosis and clinical management of patients.

Systematic protein-protein interaction and pathway analyses in the idiopathic inflammatory myopathies.

BACKGROUND: The idiopathic inflammatory myopathies (IIM) are autoimmune diseases characterised by acquired proximal muscle weakness, inflammatory cell infiltrates in muscle and myositis-specific/associated autoantibodies. It is unclear which pathways are involved in IIM, and the functional relationship between autoantibody targets has not been systematically explored. Protein-protein interaction and pathway analyses were conducted to identify pathways relevant to disease, using autoantibody targets and gene products of IIM-associated single nucleotide polymorphism (SNP) loci. METHODS: Protein-protein interactions were analysed using Disease Association Protein-Protein Link Evaluator (DAPPLE). Gene ontology and pathway analyses were conducted using Database for Annotation Visualisation and Integrated Discovery (DAVID) and Gene Relationships Across Implicated Loci (GRAIL). Analyses were undertaken including the targets of published autoantibodies, significant and suggestive SNPs from an IIM association study and autoantibody targets plus SNPs combined. RESULTS: The protein-protein interaction networks formed by autoantibody targets and associated SNPs showed significant direct and/or indirect connectivity (p < 0.05). Autoantibody targets plus associated SNPs combined resulted in more significant indirect and common interactor connectivity, suggesting autoantibody targets and proteins encoded by IIM-associated loci may be involved in common pathways. Tumour necrosis factor receptor-associated factor 6 (TRAF6) was identified as a hub protein, and UBE3B, HSPA1A, HSPA1B and PSMD3 also were identified as genes with significant connectivity. Pathway analysis identified that autoantibody targets and associated SNP regions are significantly interconnected (p < 0.01), and confirmed autoantibody target involvement in translational and post-translational processes. 'Ubiquitin' was the only keyword strongly linking significant genes across regions in all three GRAIL analyses of autoantibody targets and IIM-associated SNPs. CONCLUSIONS: Autoantibody targets and IIM-associated loci show significant connectivity and inter-relatedness, and identify several key genes and pathways in IIM pathogenesis, possibly mediated via the ubiquitination pathway.

Assessment of osteoarthritis candidate genes in a meta-analysis of nine genome-wide association studies.

OBJECTIVE: To assess candidate genes for association with osteoarthritis (OA) and identify promising genetic factors and, secondarily, to assess the candidate gene approach in OA. METHODS: A total of 199 candidate genes for association with OA were identified using Human Genome Epidemiology (HuGE) Navigator. All of their single-nucleotide polymorphisms (SNPs) with an allele frequency of >5% were assessed by fixed-effects meta-analysis of 9 genome-wide association studies (GWAS) that included 5,636 patients with knee OA and 16,972 control subjects and 4,349 patients with hip OA and 17,836 control subjects of European ancestry. An additional 5,921 individuals were genotyped for significantly associated SNPs in the meta-analysis. After correction for the number of independent tests, P values less than 1.58 × 10(-5) were considered significant. RESULTS: SNPs at only 2 of the 199 candidate genes (COL11A1 and VEGF) were associated with OA in the meta-analysis. Two SNPs in COL11A1 showed association with hip OA in the combined analysis: rs4907986 (P = 1.29 × 10(-5) , odds ratio [OR] 1.12, 95% confidence interval [95% CI] 1.06-1.17) and rs1241164 (P = 1.47 × 10(-5) , OR 0.82, 95% CI 0.74-0.89). The sex-stratified analysis also showed association of COL11A1 SNP rs4908291 in women (P = 1.29 × 10(-5) , OR 0.87, 95% CI 0.82-0.92); this SNP showed linkage disequilibrium with rs4907986. A single SNP of VEGF, rs833058, showed association with hip OA in men (P = 1.35 × 10(-5) , OR 0.85, 95% CI 0.79-0.91). After additional samples were genotyped, association at one of the COL11A1 signals was reinforced, whereas association at VEGF was slightly weakened. CONCLUSION: Two candidate genes, COL11A1 and VEGF, were significantly associated with OA in this focused meta-analysis. The remaining candidate genes were not associated.

The role of ECE1 variants in cognitive ability in old age and Alzheimer's disease risk.

The ß-amyloid peptide may play a central role in Alzheimer's disease (AD) pathogenesis. We have evaluated variants in seven Aß-degrading genes (ACE, ECE1, ECE2, IDE, MME, PLAU, and TF) for association with AD risk in the Genetic and Environmental Risk in Alzheimer's Disease Consortium 1 (GERAD1) cohort, and with three cognitive phenotypes in the Lothian Birth Cohort 1936 (LBC1936), using 128 and 121 SNPs, respectively. In GERAD1, we identified a significant association between a four-SNP intragenic ECE1 haplotype and risk of AD in individuals that carried at least one APOE ε4 allele (P = 0.00035, odds ratio = 1.61). In LBC1936, we identified a significant association between a different two-SNP ECE1 intragenic haplotype and non-verbal reasoning in individuals lacking the APOE ε4 allele (P = 0.00036, ß = -0.19). Both results showed a trend towards significance after permutation (0.05 < P < 0.10). A follow-up cognitive genetic study evaluated the association of ECE1 SNPs in three additional cohorts of non-demented older people. Meta-analysis of the four cohorts identified the significant association (Z < 0.05) of SNPs in the ECE-1b promoter with non-verbal reasoning scores, particularly in individuals lacking the APOE ε4 allele. Our genetic findings are not wholly consistent. Nonetheless, the AD associated intronic haplotype is linked to the 338A variant of known ECE1b promoter variant, 338C>A (rs213045). We observed significantly less expression from the 338A variant in two human neuroblastoma cell lines and speculate that this promoter may be subject to tissue-specific regulation.

Genetic association study of NF-κB genes in UK Caucasian adult and juvenile onset idiopathic inflammatory myopathy.

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.

Does inflammation predispose to recurrent vascular events after recent transient ischaemic attack and minor stroke? The North West of England transient ischaemic attack and minor stroke (NORTHSTAR) study.

BACKGROUND AND HYPOTHESIS: Inflammation is implicated in the pathogenesis and outcome of ischaemic injury. Poststroke inflammation is associated with outcome but it remains unclear whether such inflammation precedes or results from ischaemic injury. We hypothesised that inflammatory markers are associated with an increased risk of recurrent vascular events soon after transient ischaemic attack and minor stroke. METHODS: This was a multicentre, prospective, nested case-control study. Plasma concentrations of C-reactive protein, interleukin-6, interleukin-1-receptor antagonist and fibrinogen, leucocyte counts, erythrocyte sedimentation rate and inflammatory gene allele frequencies were analysed in 711 patients with recent transient ischaemic attack or minor stroke. Cases were defined by the incidence of one or more recurrent vascular events during the three-month follow-up. Association of inflammatory markers with case-status was determined using conditional logistic regression. RESULTS: Plasma concentrations of C-reactive protein, interleukin-1-receptor antagonist and interleukin-6 were not associated with case-status. In secondary analyses, only erythrocyte sedimentation rate was significantly associated with case-status (odds ratio 1·39, 95% confidence interval 1·03-1·85; P=0·03), but this effect did not persist after adjustment for smoking and past history of transient ischaemic attack or stroke. Single nucleotide polymorphisms in four inflammatory genes (interleukin-6, fibrinogen, P-selectin and vascular cell adhesion molecule-1) were nominally associated with case-status. CONCLUSIONS: Circulating inflammatory markers were not associated with recurrent vascular events. Nominally significant associations between genetic markers and case-status will require replication. These data provide little evidence for an inflammatory state predisposing to stroke and other vascular events in a susceptible population.

A candidate gene study of canine joint diseases.

Canine osteoarthritis (OA) commonly occurs in association with articular diseases, such as hip dysplasia (HD), elbow dysplasia (ED), or cranial cruciate ligament rupture (CCLR). We hypothesized that a common genomic risk for the development of canine joint disease and canine OA would be identified by evaluating the allele frequencies of candidate gene single nucleotide polymorphisms (SNPs) in dogs with OA associated with different articular diseases when compared with a general population of breed-matched dogs. DNA was extracted from blood samples obtained from Labrador Retrievers and Golden Retrievers surgically treated for ED, HD, and CCLR and confirmed to have radiographic evidence of OA. One hundred and thirteen SNPs in 20 candidate genes were genotyped. No significant associations were identified for SNPs or haplotypes in the candidate genes for the diseases evaluated. The candidate gene approach for the study of genetic association is unlikely to be successful for complex canine diseases such as OA without prior trait mapping evaluation.

Vascular endothelial growth factor production is regulated by gene polymorphisms.

BACKGROUND: Vascular endothelial growth factor (VEGF) has a key role in angiogenesis and in transplantation. The level of VEGF is related to the differences in the DNA sequence of its promoter region. OBJECTIVES: In this study, the association between the combination of VEGF -1154 G and -2578 C alleles and VEGF production by LPS-stimulated PBMCs was investigated. In addition; the relationship between VEGF polymorphisms and the influence of TNF- and IL-4 on VEGF production was studied. METHODS: VEGF -1154 G/A and -2578 C/A were detected using ARMS-PCR. To determine the impact of combinations of these two polymorphisms on VEGF production; PBMCs were stimulated by LPS and VEGF production was measured by ELISA. RESULTS: The combinations of -1154 GG/-2578 CC and -1154 GG/-2578 CA were significantly associated with higher VEGF production (p<0.0001). Production of VEGF was significantly influenced by TNF- in individuals who had certain VEGF genotype combinations. Although VEGF production was dramatically suppressed by IL-4, it was not dependent on VEGF genotype. CONCLUSIONS: Since TNF- has influence on the graft outcome, to avoid allocation of grafts from high TNF- producer donors to recipients, it might be useful to predict and minimize graft rejection by having prior knowledge of TNF- and also VEGF genotypes especially -1154 G/A and -2578 C/A VEGF.

An update on the immunogenetics of idiopathic inflammatory myopathies: major histocompatibility complex and beyond.

PURPOSE OF REVIEW: To update the reader on immunogenetic advances in idiopathic inflammatory myopathy (IIM) over the past 18 months. RECENT FINDINGS: In Caucasian IIM, despite a shared association with the human leukocyte antigen (HLA) 8.1 ancestral haplotype (HLA-DRB1*03-DQA1*05-DQB1*02), anti-Jo-1 and anti-PM-Scl antibody-positive cases have differing IIM clinical phenotypes. A study of the HLA-DPB1 region has shown that DPB1*0101 is associated with anti-Jo-1 positivity but not with anti-PM-Scl. IIM single nucleotide polymorphism studies have demonstrated associations in the protein tyrosine phosphatase, nonreceptor type 22, tumour necrosis factor alpha and interleukin-1 genes. The GM 13 allotype has been confirmed as a risk factor in Caucasian IIM. In inclusion body myositis, the HLA 8.1 ancestral haplotype may not only influence disease susceptibility but also disease expression. A follow-up study including a meta-analysis of the apolipoprotein E gene in inclusion body myositis suggests that this gene does not confer risk of disease. SUMMARY: Although a substantial part of the genetic risk for developing adult and juvenile IIM lies within the major histocompatibility complex, recent research suggests that genetic regions outside of the major histocompatibility complex are also potentially involved in conferring IIM disease susceptibility, although with more modest effect sizes. An ongoing and internationally coordinated IIM genome-wide association scan may provide further insights into IIM immunogenetics.

TNF, LTA, HSPA1L and HLA-DR gene polymorphisms in HIV-positive patients with hypersensitivity to cotrimoxazole.

AIMS: Sulfamethoxazole in combination with trimethoprim (cotrimoxazole) is used for prophylaxis and treatment of several opportunistic infections in HIV-infected patients. It is associated with a high incidence of hypersensitivity reactions, which is thought to have an immune basis. Genetic polymorphisms in MHC are known to predispose to hypersensitivity reactions to a structurally diverse group of drugs in HIV-positive patients. The aim of the study was to determine whether functional polymorphisms in TNF, LTA, HSPA1L and HLA-DRB1 genes influence the risk of cotrimoxazole hypersensitivity in HIV-infected patients. METHODS: We genotyped 136 HIV-positive patients with (n = 53) and without (n = 83) cotrimoxazole hypersensitivity using a combination of PCR-based techniques, including PCR-restriction fragment length polymorphisms, PCR-sequence specific oligonucleotides and real-time PCR. Genotypes and the haplotype frequencies were analyzed using the chi(2) test in the Haploview and CLUMP programs. RESULTS: No statistically significant difference in SNP or haplotype frequencies were found in HIV-infected sulfamethoxazole hypersensitive patients compared with controls. CONCLUSION: Our data show that MHC polymorphisms are not major predisposing factors for cotrimoxazole hypersensitivity, although we cannot exclude a minor contribution. An environmental factor (i.e., HIV infection) seems to predominate over any of the genetic factors so far investigated in increasing the risk of cotrimoxazole hypersensitivity.

Molecular phenotypic descriptors of Dupuytren's disease defined using informatics analysis of the transcriptome.

PURPOSE: Dupuytren's disease (DD) is a fibroproliferative disorder of unknown etiopathogenesis, which may cause progressive, permanent contracture of digits. Previous studies provide compelling evidence that genetic alterations play an important role. Macroscopically affected areas demonstrate phenotypic differences between the two structurally distinct fibrotic elements in DD (ie, the nodule and the cord). In this study, we set out to (1) compare gene expression profiles between DD and transverse carpal fascia of control subjects (external control); (2) profile DD cords and nodules from the palm against the unaffected transverse palmar fascia (internal control); and (3) identify biologically important candidate genes from the transcriptome profiles. METHODS: RNA samples from DD nodules (n = 4), cords (n = 4), and internal control (n = 4) as well as external control (n = 4) from unaffected individuals were subjected to differential gene expression profile analysis. Changes of more than 2-fold in DD groups and controls were recorded. Quantitative reverse transcriptase-polymerase chain reactions were performed to validate 16 implicated genes, which included developmental control genes, matrix metalloproteinases, and apoptotic genes. RESULTS: Several genes associated with DD formation were common across all 6 pairwise analyses. Genes markedly upregulated shared common expression levels across all pairwise analysis studies. Pairs involving the DD nodule arrays were notably distinguishable from all other permutations. The majority of genes dysregulated in the DD cords demonstrated an increase in fold change when compared with the DD nodule tissues. Key collagens, collagenases, metalloproteinases, and inhibitors were identified. Genes involved in cytoskeleton development and lipid metabolism were markedly dysregulated. Confirmations of these alterations were obtained in quantitative reverse transcriptase-polymerase chain reaction. CONCLUSIONS: These data demonstrate a gradation in expression of certain genes in DD tissue phenotypes compared with control fascia. Transcriptome profiling is predictive not only of disease but also of disease phenotype. These results indicate a number of important candidate genes associated with DD formation, which may provide clues for molecular mechanisms involved in DD pathogenesis.

Evaluation of 15 candidate genes for dilated cardiomyopathy in the Newfoundland dog.

Dilated cardiomyopathy (DCM) is a disease of the myocardium, which causes heart failure and premature death. It has been described in humans and several domestic animals. In the Newfoundland dog, DCM is an autosomal dominant disease with late onset and reduced penetrance. We analyzed 15 candidate genes for their involvement in DCM in the Newfoundland dog. Polymorphic microsatellite markers and single Nucleotide Polymorphisms were genotyped in 4 families of Newfoundland dogs segregating dilated cardiomyopathy for the genes encoding alpha-cardiac actin (ACTC), caveolin (CAVI), cysteine-rich protein 3 (CSRP3), LIM-domain binding factor 3 (LDB3), desmin (DES), lamin A/C (LMNA), myosin heavy polypeptide 7 (MYH7), delta-sarcoglycan (SGCD), troponin I (TNNTI3), troponin T (TNNT2), alpha-tropomyosin (TPMI), titin (TTN) and vinculin (VCL). A Logarithm of the odds (LOD) score of less than -2.0 in 2-point linkage analysis indicated exclusion of all but 2 genes, encoding CSRP3 and DES. A (LOD) score between -1.5 and -2.0 for CSRP3 and DES makes these genes unlikely causes of DCM in this dog breed. For the phospholamban (PLN) and titin cap (TTN) genes, a direct mutation screening approach was used. DNA sequence analysis of all exons showed no evidence that these genes are involved in DCM in the Newfoundland dog.

Canine candidate genes for dilated cardiomyopathy: annotation of and polymorphic markers for 14 genes.

BACKGROUND: Dilated cardiomyopathy is a myocardial disease occurring in humans and domestic animals and is characterized by dilatation of the left ventricle, reduced systolic function and increased sphericity of the left ventricle. Dilated cardiomyopathy has been observed in several, mostly large and giant, dog breeds, such as the Dobermann and the Great Dane. A number of genes have been identified, which are associated with dilated cardiomyopathy in the human, mouse and hamster. These genes mainly encode structural proteins of the cardiac myocyte. RESULTS: We present the annotation of, and marker development for, 14 of these genes of the dog genome, i.e. alpha-cardiac actin, caveolin 1, cysteine-rich protein 3, desmin, lamin A/C, LIM-domain binding factor 3, myosin heavy polypeptide 7, phospholamban, sarcoglycan delta, titin cap, alpha-tropomyosin, troponin I, troponin T and vinculin. A total of 33 Single Nucleotide Polymorphisms were identified for these canine genes and 11 polymorphic microsatellite repeats were developed. CONCLUSION: The presented polymorphisms provide a tool to investigate the role of the corresponding genes in canine Dilated Cardiomyopathy by linkage analysis or association studies.

Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis.

BACKGROUND: The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use. The aim of this study was to modify the PAXgene Blood RNA System kit protocol for application to small, sick children, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression.Aliquots of 0.86 mL PAXgene reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease. RESULTS: Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02). CONCLUSION: We have successfully modified the PAXgene blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis.