A genetic predictive model for canine hip dysplasia: integration of Genome Wide Association Study (GWAS) and candidate gene approaches.

Canine hip dysplasia is one of the most prevalent developmental orthopedic diseases in dogs worldwide. Unfortunately, the success of eradication programs against this disease based on radiographic diagnosis is low. Adding the use of diagnostic genetic tools to the current phenotype-based approach might be beneficial. The aim of this study was to develop a genetic prognostic test for early diagnosis of hip dysplasia in Labrador Retrievers. To develop our DNA test, 775 Labrador Retrievers were recruited. For each dog, a blood sample and a ventrodorsal hip radiograph were taken. Dogs were divided into two groups according to their FCI hip score: control (A/B) and case (D/E). C dogs were not included in the sample. Genetic characterization combining a GWAS and a candidate gene strategy using SNPs allowed a case-control population association study. A mathematical model which included 7 SNPs was developed using logistic regression. The model showed a good accuracy (Area under the ROC curve = 0.85) and was validated in an independent population of 114 dogs. This prognostic genetic test represents a useful tool for choosing the most appropriate therapeutic approach once genetic predisposition to hip dysplasia is known. Therefore, it allows a more individualized management of the disease. It is also applicable during genetic selection processes, since breeders can benefit from the information given by this test as soon as a blood sample can be collected, and act accordingly. In the authors' opinion, a shift towards genomic screening might importantly contribute to reducing canine hip dysplasia in the future. In conclusion, based on genetic and radiographic information from Labrador Retrievers with hip dysplasia, we developed an accurate predictive genetic test for early diagnosis of hip dysplasia in Labrador Retrievers. However, further research is warranted in order to evaluate the validity of this genetic test in other dog breeds.

Genetic and microbial factors modulating the ubiquitin proteasome system in inflammatory bowel disease.

OBJECTIVE: Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. DESIGN: We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. RESULTS: Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (p(FDR)=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. CONCLUSIONS: Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors.

Candidate's single-nucleotide polymorphism predictors of treatment nonresponse to the first anti-TNF inhibitor in ankylosing spondylitis.

The objective of this study is to identify single-nucleotide polymorphisms (SNPs) predictors of treatment nonresponse to the first anti-TNF-alpha agent in ankylosing spondylitis (AS). Patients were classified as "nonresponders" if they failed to achieve improvement ≥50 % of the initial BASDAI. We selected candidate SNPs previously reported, associated with susceptibility or pathogenesis of AS and with other spondylarthropaties (SpAs). The predictors of nonresponse were modeled with multiple logistic regression. The predictive power of the genetic model of nonresponse to treatment was tested with AUC-ROC. One hundred and twenty-one (121) AS patients fulfilled the inclusion criteria. Of the candidate SNPs tested for association with treatment effectiveness, five independent predictors were identified: rs917997, rs755622, rs1800896, rs3740691, and rs1061622. The genetic model of nonresponse to treatment had a predictive power of 0.77 (95 % CI 0.68-0.86). Our study identified several polymorphisms which could be the useful genetic biomarkers in predicting nonresponse to anti-TNF-alpha therapy.

Genetic predisposition to early recurrence in clinically localized prostate cancer.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Currently available nomograms to predict preoperative risk of early biochemical recurrence (EBCR) after radical prostatectomy are solely based on classic clinicopathological variables. Despite providing useful predictions, these models are not perfect. Indeed, most researchers agree that nomograms can be improved by incorporating novel biomarkers. In the last few years, several single nucleotide polymorphisms (SNPs) have been associated with prostate cancer, but little is known about their impact on disease recurrence. We have identified four SNPs associated with EBCR. The addition of SNPs to classic nomograms resulted in a significant improvement in terms of discrimination and calibration. The new nomogram, which combines clinicopathological and genetic variables, will help to improve prediction of prostate cancer recurrence. OBJECTIVES: To evaluate genetic susceptibility to early biochemical recurrence (EBCR) after radical prostatectomy (RP), as a prognostic factor for early systemic dissemination. To build a preoperative nomogram to predict EBCR combining genetic and clinicopathological factors. PATIENTS AND METHODS: We evaluated 670 patients from six University Hospitals who underwent RP for clinically localized prostate cancer (PCa), and were followed-up for at least 5 years or until biochemical recurrence. EBCR was defined as a level prostate-specific antigen >0.4 ng/mL within 1 year of RP; preoperative variables studied were: age, prostate-specific antigen, clinical stage, biopsy Gleason score, and the genotype of 83 PCa-related single nucleotide polymorphisms (SNPs). Univariate allele association tests and multivariate logistic regression were used to generate predictive models for EBCR, with clinicopathological factors and adding SNPs. We internally validated the models by bootstrapping and compared their accuracy using the area under the curve (AUC), net reclassification improvement, integrated discrimination improvement, calibration plots and Vickers' decision curves. RESULTS: Four common SNPs at KLK3, KLK2, SULT1A1 and BGLAP genes were independently associated with EBCR. A significant increase in AUC was observed when SNPs were added to the model: AUC (95% confidence interval) 0.728 (0.674-0.784) vs 0.763 (0.708-0.817). Net reclassification improvement showed a significant increase in probability for events of 60.7% and a decrease for non-events of 63.5%. Integrated discrimination improvement and decision curves confirmed the superiority of the new model. CONCLUSIONS: Four SNPs associated with EBCR significantly improved the accuracy of clinicopathological factors. We present a nomogram for preoperative prediction of EBCR after RP.

Genetic factors conferring an increased susceptibility to develop Crohn's disease also influence disease phenotype: results from the IBDchip European Project.

OBJECTIVE: Through genome-wide association scans and meta-analyses thereof, over 70 genetic loci (Crohn's disease (CD) single nucleotide polymorphisms (SNPs)) are significantly associated with CD. We aimed to investigate the influence of CD-SNPs and basic patient characteristics on CD clinical course, and develop statistical models to predict CD clinical course. DESIGN: This retrospective study included 1528 patients with CD with more than 10 years of follow-up from eight European referral hospitals. CD outcomes of interest were ileal (L1), colonic (L2) and ileocolonic disease location (L3); stenosing (B2) or penetrating behaviour (B3); perianal disease; extraintestinal manifestations; and bowel resection. A complicated disease course was defined as stenosing or penetrating behaviour, perianal disease and/or bowel resection. Association between CD-SNPs or patient characteristics and specified outcomes was studied. RESULTS: Several CD-SNPs and clinical characteristics were statistically associated with outcomes of interest. The NOD2 gene was the most important genetic factor, being an independent predictive factor for ileal location (p=2.02 × 10(-06), OR=1.90), stenosing (p=3.16 × 10(-06), OR=1.82) and penetrating (p=1.26 × 10(-02), OR=1.25) CD behaviours, and need for surgery (p=2.28 × e-05, OR=1.73), and as such was also the strongest factor associated with a complicated disease course (p=6.86 × 10(-06), OR=2.96). Immunomodulator (azathioprine/6-mercaptopurine and methotrexate) use within 3 years after diagnosis led to a reduction in bowel stenoses (p=1.48 × 10(-06), OR=0.35) and surgical rate (p=1.71 × 10(-07), OR=0.34). Association between each outcome and genetic scores, created using significant SNPs in the univariate analysis, revealed large differences in the probability of developing fistulising disease (IL23R, LOC441108, PRDM1, NOD2; p=9.64e-4, HR=1.43), need for surgery (IRGM, TNFSF15, C13ORF31, NOD2; p=7.12 × 10(-03), HR=1.35), and stenosing disease (NOD2, JAK2, ATG16L1; p=3.01 × 10(-02), HR=1.29) among patients with low and high score. CONCLUSIONS: This large multicentre cohort study has found several genetic and clinical factors influencing the clinical course of CD. NOD2 and early immunomodulator use are the clinically most meaningful predictors for its clinical course.

Improved prediction of biochemical recurrence after radical prostatectomy by genetic polymorphisms.

PURPOSE: Single nucleotide polymorphisms are inherited genetic variations that can predispose or protect individuals against clinical events. We hypothesized that single nucleotide polymorphism profiling may improve the prediction of biochemical recurrence after radical prostatectomy. MATERIALS AND METHODS: We performed a retrospective, multi-institutional study of 703 patients treated with radical prostatectomy for clinically localized prostate cancer who had at least 5 years of followup after surgery. All patients were genotyped for 83 prostate cancer related single nucleotide polymorphisms using a low density oligonucleotide microarray. Baseline clinicopathological variables and single nucleotide polymorphisms were analyzed to predict biochemical recurrence within 5 years using stepwise logistic regression. Discrimination was measured by ROC curve AUC, specificity, sensitivity, predictive values, net reclassification improvement and integrated discrimination index. RESULTS: The overall biochemical recurrence rate was 35%. The model with the best fit combined 8 covariates, including the 5 clinicopathological variables prostate specific antigen, Gleason score, pathological stage, lymph node involvement and margin status, and 3 single nucleotide polymorphisms at the KLK2, SULT1A1 and TLR4 genes. Model predictive power was defined by 80% positive predictive value, 74% negative predictive value and an AUC of 0.78. The model based on clinicopathological variables plus single nucleotide polymorphisms showed significant improvement over the model without single nucleotide polymorphisms, as indicated by 23.3% net reclassification improvement (p = 0.003), integrated discrimination index (p <0.001) and likelihood ratio test (p <0.001). Internal validation proved model robustness (bootstrap corrected AUC 0.78, range 0.74 to 0.82). The calibration plot showed close agreement between biochemical recurrence observed and predicted probabilities. CONCLUSIONS: Predicting biochemical recurrence after radical prostatectomy based on clinicopathological data can be significantly improved by including patient genetic information.

Can we identify a power-oriented polygenic profile?

Using the model originally developed by Williams and Folland (J Physiol 586: 113-121, 2008), we determined 1) a "total genotype score" (TGS, from the accumulated combination of the 6 polymorphisms, with a maximum value of "100" for the theoretically optimal polygenic score) in a group of elite power athletes, endurance athletes, and nonathletic controls, and 2) the probability for the occurrence of Spanish individuals with the "perfect" power-oriented profile (i.e., TGS = 100). We analyzed six polymorphism that are candidates to explain individual variations in elite power athletic status or power phenotypes (ACE I/D, ACTN3 R577X, AGT Met235Thr, GDF-8 K153R, IL6 -174 G/C, and NOS3 -786T>C) in 53 elite track and field power athletes (jumpers, sprinters), 100 nonathletic controls, and 100 elite endurance athletes (distance runners and road cyclists) (all Spanish Caucasian males). The mean TGS was significantly higher in power athletes (70.8 +/- 17.3) compared with endurance athletes (60.4 +/- 15.9; P < 0.001) and controls (63.3 +/- 13.2; P = 0.012), whereas it did not differ between the latter two groups (P = 0.366). A total of five power athletes (9.4%, all sprinters) had a theoretically "optimal" TGS of 100 vs. 0 subjects in the other two groups. The probability of a Spanish individual possessing a theoretically optimal polygenic profile for up to the six candidate polymorphisms we studied was very small, i.e., approximately 0.2% (or 1 in 500 Spanish individuals). We have identified a polygenic profile that allows us, at least partly, to distinguish elite power athletes from both endurance athletes and nonathletic population.

The -174 G/C polymorphism of the IL6 gene is associated with elite power performance.

The -174 G/C polymorphism [rs1800795] of the IL6 gene is a candidate to explain individual variations in health and exercise related phenotypes. We compared -174 G/C genotypic and allelic frequencies in three groups of men of the same Caucasian (Spanish) descent: elite endurance athletes (cyclists, runners; n=100); elite power athletes (jumpers, throwers, sprinters; n=53) and non-athletic controls (n=100). The frequency of the GG genotype (P=0.030) and G allele (P=0.026) was higher in the power athletes group compared with the control group. The frequency of the GG genotype (P=0.033) and G allele (P=0.013) was also higher in the power athletes group compared with the endurance athletes group. The odds ratio of being a power athlete if the subject had the GG genotype (dominant model) was 2.471 (95% confidence interval: 1.242-4.915) compared to the control group or the endurance athlete group. We did not find differences between the control and endurance athlete groups. In summary, our findings suggest that the G allele of the IL6 -174 G/C polymorphism might favour sprint/power sports performance.

Are elite endurance athletes genetically predisposed to lower disease risk?

We compared a polygenic profile that combined 33 disease risk-related mutations and polymorphisms among nonathletic healthy control subjects and elite endurance athletes. The study sample comprised 100 healthy Spanish male nonathletic (sedentary) control subjects and 100 male elite endurance athletes. We analyzed 33 disease risk-related mutations and polymorphisms. We computed a health-related total genotype score (TGS, 0-100) from the accumulated combination of the 33 variants. We did not observe significant differences in genotype or allele distributions among groups, except for the rs4994 polymorphism (P < 0.001). The computed health-related TGS was similar among groups (23.8 +/- 1.0 vs. 24.2 +/- 0.8 in control subjects and athletes, respectively; P = 0.553). Similar results were obtained when computing specific TGSs for each main disease category (cardiovascular disease and cancer). We observed no evidence that male elite endurance athletes are genetically predisposed to have lower disease risk than matched nonathletic control subjects.

Serum chitotriosidase activity, a marker of activated macrophages, predicts new cardiovascular events independently of C-reactive protein.

BACKGROUND: C-reactive protein (CRP) is a well-established inflammation marker associated with cardiovascular risk. However, its relationship with chitotriosidase activity, a novel marker of activated macrophages highly expressed in human atherosclerotic plaques, is unknown. Therefore, we sought to determine if serum chitotriosidase activity predicts the risk of new coronary events, and to analyze its relationship with CRP. METHODS: Chitotriosidase activity and genotype, and high-sensitivity CRP were measured at baseline in 133 middle-aged men with stable coronary heart disease, who were followed for the occurrence of cardiovascular morbidity and mortality for a mean of 4 years. We studied the value of these proteins in predicting the risk of new cardiovascular events. RESULTS: Serum chitotriosidase activity was higher in the group of subjects with a prespecified major event (nonfatal myocardial infarction, nonfatal ischemic stroke, coronary revascularization procedures and death from cardiovascular causes) than in the group of subjects without event, 116 +/- 30.9 nmol/ml x h versus 74.2 +/- 5.69 nmol/ml x h, respectively (p = 0.042). The baseline values of chitotriosidase activity and CRP did not correlate (R = 0.104, p = 0.266), but both parameters were related to a reduction of event-free survival in the Cox regression analysis, with relative risks of 2.61 (p = 0.060) and 2.56 (p = 0.019), respectively. Chitotriosidase activity seems to be a better marker for new events occurring after 2 years of follow-up than in the first 2 years. Both markers had similar predictive values, and their sensitivity (64%) and negative predictive value (84%) were improved when combined. CONCLUSIONS: Our results suggest that serum chitotriosidase activity predicts the risk of new cardiovascular events in the following 4 years. This new cardiovascular risk marker is independent of CRP and, when combined, the prediction of the risk of new cardiovascular events and the identification of a lower risk group seem to improve.

Tendon xanthomas in familial hypercholesterolemia are associated with a differential inflammatory response of macrophages to oxidized LDL.

Tendon xanthomas (TX) are pathognomonic lipid deposits commonly found in familial hypercholesterolemia (FH) patients. The aim of this study was to determine whether macrophages from FH patients with TX (TX+) have higher predisposition to foam cells formation after oxidized LDL (oxLDL) overload than those from FH patients without TX (TX-), and if their differential gene expression profile could explain these different phenotypes. Total RNA pools from macrophages from FH patients TX+ and TX- were analyzed using Affymetrix oligonucleotide arrays to evaluate the gene expression profile in presence and absence of oxLDL. Also, the intracellular lipid content was measured by fluorescence flow cytometry. Results of these studies suggest that macrophages from FH subjects TX+ compared to those TX- have a differential response to oxLDL, since they show higher intracellular cholesterol ester accumulation and a differential gene expression profile. The gene array data were validated by relative quantitative real-time RT-PCR and quantitative ELISA in culture media and plasma samples. FH subjects TX+ showed increased plasma tryptase, TNF-alpha, IL-8 and IL-6 concentrations. We propose that TX formation are associated with higher intracellular lipid content, and higher inflammatory response of macrophages in response to oxLDL.

Reliable low-density DNA array based on allele-specific probes for detection of 118 mutations causing familial hypercholesterolemia.

BACKGROUND: Patients with familial hypercholesterolemia (FH) have a high risk of premature cardiovascular disease (PCVD). Mutations in the LDL receptor (LDLR) gene and the R3500Q mutation in the apolipoprotein B (APOB) gene are known to cause FH, but lack of high-throughput methods makes routine genetic diagnosis difficult. The objective of this work was to develop a DNA array for large-scale identification of mutant LDLR alleles. METHODS: We developed a low-density oligonucleotide microarray to identify 118 DNA sequence variations (117 for the LDLR gene and 1 for the APOB gene). We verified specificity and sensitivity by analyzing 1180 previously sequenced DNA samples, and conducted a blind study screening 407 Spanish patients with a clinical diagnosis of FH. RESULTS: The DNA array confirmed the previous genotyping results in almost all cases. In the blind study, the microarray detected at least 1 mutation in 51% of the patients for whom clinical diagnosis was classified as certain according to Dutch FH-MEDPED criteria; it also identified mutations in 37% of those with a diagnosis of probable/possible FH, thus giving a definite diagnosis. Patients harboring null mutations had shorter PCVD-free survival times and higher relative risk of PCVD than patients with a missense mutation. CONCLUSIONS: The proposed DNA array allows large-scale population screening and provides molecular information regarding mutation type and its correlation with clinical severity of FH, which can be used to develop therapeutic strategies.

[Differences in clinical presentation between subjects with a phenotype of familial hypercholesterolemia determined by defects in the LDL-receptor and defects in Apo B-100]. / Diferencias en la presentación clínica en sujetos con fenotipo de hipercolesterolemia familiar por defectos en el receptor LDL y por defectos de la apo B-100.

INTRODUCTION AND OBJECTIVES: Familial hypercholesterolemia and familial defective Apo B-100 are phenotypically indistinguishable. At present they can be distinguished by genetic analysis. PATIENTS AND METHODç We compared the clinical features of 13 subjects with familial defective Apo B-100 and 39 subjects with familial hypercholesterolemia. We used data from first degree relatives to compare morbidity and mortality between the two groups. RESULTS: We found statistically significant differences in total cholesterol and LDL cholesterol, which were lower in the familial defective Apo B-100 group (TC = 357 37.3 mg/dl vs 415 79.7 mg/dl and LDLc = 270 34.2 mg/dl vs 355 72.4 mg/dl). We found no differences in xanthomas, corneal arcus, smoking status, vascular events, blood pressure, BMI or waist/hip ratio. There were no differences between the two groups in the proportions of patients with cardiovascular disease or patients who died. We found statistically significant differences between the groups (p = 0.023) in the mean age at first vascular event (familial hypercholesterolemia and first degree relatives: 45.3 19.9 years; familial defective Apo B-100 and first degree relatives: 51.5 20.8 years). CONCLUSIONS: We conclude that familial defective Apo B-100 results in clinically milder hypercholesterolemia than familial hypercholesterolemia, and that discerning between them could be helpful to stratify the risk in persons with hereditary hypercholesterolemia.