Bioinformatic evaluation of transcriptional regulation of WNT pathway genes with reference to diabetic nephropathy

Objective: Diabetic nephropathy (DN) is a microvascular complication of diabetes. Members of the WNT/ β-catenin pathways have been implicated in interstitial fibrosis and glomerular sclerosis, characteristic hallmarks of DN. These processes are controlled, in part, by transcription factors (TFs), proteins which bind to gene promoter regions attenuating their regulation. We sought to identify predicted cis-acting transcription factor binding sites (TFBS) over-represented within the promoter regions of WNT pathway members compared to genes across the genome.Methods: We assessed the frequency of 62 TFBS motifs from the JASPAR databases on 65 WNT pathway genes. P-values were estimated on the hypergeometric distribution for each TF. Gene expression profiles of enriched motifs were examined from DN-related datasets to assess clinical significance.Results: TFBS motifs transcription factor AP-2 alpha (TFAP2A), myeloid zinc finger 1 (MZF1), and specificity protein 1 (SP1) were significantly enriched within WNT pathway genes (P-values<6.83x10-29, 1.34x10-11 and 3.01x10-6 respectively). MZF1 gene expression was significantly increased in DN in a whole kidney dataset (fold change = 1.16; 16% increase; P = 0.03). TFAP2A gene expression was decreased in an independent dataset (fold change = -1.02; P = 0.03). SP1 was not differentially expressed in any datasets examined.Conclusions: Three TFBS profiles are significantly enriched within the WNT pathway genes examined highlighting the use of in silico analyses for identifying key regulators of this pathway. Modification of TF binding to gene promoter regions involved in DN pathology may limit progression, making refinement of targeted therapeutic strategies possible through clearer delineation of their role.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma:analysis of a clinic-based population

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

Time-dependent tegumental surface changes in juvenile Fasciola gigantica in response to triclabendazole treatment in goat

Triclabendazole (TCBZ), the anthelmintic drug active against both mature and immature liver flukes, was used to investigate the effect of in vivo treatment on the tegumental surface of juvenile Fasciola gigantica. Five goats were infected with 150 F. gigantica metacercariae each by oral gavage. Four of them were treated with single dose of TCBZ at 10mg/kg at four weeks post-infection. They were euthanized at 0 (untreated), 24, 48, 72 and 96h post treatment. Juvenile flukes were manually retrieved from the goat livers and processed for scanning electron microscopy. In control flukes, the anterior region was adorned with sharply pointed spines projecting away from the surface, while in the posterior region, spines become shorter and narrower, loosing serration and with the appearance of distinct furrows and papillae. The dorsal surface retained the same pattern of surface architecture similar to that of ventral surface. Flukes obtained from 24h post-treatment did not show any apparent change and were still very active. However, there were limited movements and some blebbing, swelling, deposition of tegumental secretions and some flattening displayed by the flukes of 48h post-treatment. All the worms were found dead 72h post-treatment and showed advanced level of tegumental disruptions, consisting of severe distortion of spines, sloughing off the tegument to expose the basal lamina, formation of pores and isolated patches of lesions. By 96h post-treatment, the disruption was extremely severe and the tegument was completely sheared off causing deeper lesions that exposed the underlying musculature. The disruption was more severe at posterior than anterior region and on ventral than dorsal surface. The present study further establishes the time-course of TCBZ action in vivo with 100% efficacy against the juvenile tropical liver fluke.

Successful second unrelated donor BMT in a child with juvenile chronic myeloid leukaemia:documentation of chimaerism using the polymerase chain reaction

A 3-year old child with juvenile chronic myeloid leukaemia received a T cell-depleted BMT from a male unrelated donor. There was early graft failure associated with increasing splenomegaly and hypersplenism. Splenectomy was performed 53 days post-transplant and was followed by autologous marrow recovery with return of leukaemia. A second unrelated donor BMT was performed 9 months later using T cell-replete marrow from a similarly matched female donor. Grade 2 GVHD involving the skin and gut responded to treatment with steroids. Chimaerism was assessed using Y-specific polymerase chain reaction (PCR) and microsatellites. Samples taken at the time of splenectomy showed no donor marrow engraftment but there was significant engraftment in the spleen. Following the second transplant, donor-type haematopoiesis was documented using a panel of microsatellite probes. The patient remains well 6 months after transplant. Splenectomy should be considered prior to transplant in patients with significant splenomegaly and hypersplenism. Partial chimaerism in the spleen, but not bone marrow, post-BMT, has not previously been documented. PCR technology is a useful and highly sensitive way to assess chimaerism post-BMT and is informative in sex-matched cases, whilst the small amount of material required is advantageous in paediatric patients.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2sigma;2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2sigma;2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca<inf>o</inf>²⁺) homeostasis. To elucidate the role of AP2sigma;2 in Ca<inf>o</inf>²⁺ regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2sigma;2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2sigma;2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2sigma;2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2sigma;2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2sigma;2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Methotrexate polyglutamates as a potential marker of adherence to long-term therapy in children with juvenile idiopathic arthritis and juvenile dermatomyositis: an observational, cross-sectional study

Introduction: Methotrexate (MTX) is a cornerstone of treatment in a wide variety of inflammatory conditions, including juvenile idiopathic arthritis (JIA) and juvenile dermatomyositis (JDM). However, owing to its narrow therapeutic index and the considerable interpatient variability in clinical response, monitoring of adherence to MTX is important. The present study demonstrates the feasibility of using methotrexate polyglutamates (MTXPGs) as a biomarker to measure adherence to MTX treatment in children with JIA and JDM.
Methods: Data were collected prospectively from a cohort of 48 children (median age 11.5 years) who received oral or subcutaneous (SC) MTX therapy for JIA or JDM. Dried blood spot samples were obtained from children by finger pick at the clinic or via self- or parent-led sampling at home, and they were analysed to determine the variability in MTXPG concentrations and assess adherence to MTX therapy.
Results: Wide fluctuations in MTXPG total concentrations (>2.0-fold variations) were found in 17 patients receiving stable weekly doses of MTX, which is indicative of nonadherence or partial adherence to MTX therapy. Age (P = 0.026) and route of administration (P = 0.005) were the most important predictors of nonadherence to MTX treatment. In addition, the study showed that MTX dose and route of administration were significantly associated with variations in the distribution of MTXPG subtypes. Higher doses and SC administration of MTX produced higher levels of total MTXPGs and selective accumulation of longer-chain MTXPGs (P < 0.001 and P < 0.0001, respectively).
Conclusions: Nonadherence to MTX therapy is a significant problem in children with JIA and JDM. The present study suggests that patients with inadequate adherence and/or intolerance to oral MTX may benefit from SC administration of the drug. The clinical utility of MTXPG levels to monitor and optimise adherence to MTX in children has been demonstrated.Trial Registration: ISRCTN Registry identifier: ISRCTN93945409 . Registered 2 December 2011.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.

Nuclear cataract shows significant familial aggregation in an older population after adjustment for possible shared environmental factors.

PURPOSE: To quantify the association between siblings in age-related nuclear cataract, after adjusting for known environmental and personal risk factors. METHODS: All participants (probands) in the Salisbury Eye Evaluation (SEE) project and their locally resident siblings underwent digital slit lamp photography and were administered a questionnaire to assess risk factors for cataract including: age, gender, lifetime sun exposure, smoking and diabetes history, and use of alcohol and medications such as estrogens and steroids. In addition, blood pressure, body mass index, and serum antioxidants were measured in all participants. Lens photographs were graded by trained observers masked to the subjects' identity, using the Wilmer Cataract Grading System. The odds ratio for siblings for affectedness with nuclear cataract and the sibling correlation of nuclear cataract grade, after adjusting for covariates, were estimated with generalized estimating equations. RESULTS: Among 307 probands (mean age, 77.6 +/- 4.5 years) and 434 full siblings (mean age, 72.4 +/- 7.4 years), the average sibship size was 2.7 per family. After adjustment for covariates, the probability of development of nuclear cataract was significantly increased (odds ratio [OR] = 2.07, 95% confidence interval [CI], 1.30-3.30) among individuals with a sibling with nuclear cataract (nuclear grade > or = 3.0). The final fitted model indicated a magnitude of heritability for nuclear cataract of 35.6% (95% CI: 21.0%-50.3%) after adjustment for the covariates. CONCLUSIONS: Findings in this study are consistent with a genetic effect for age-related nuclear cataract, a common and clinically significant form of lens opacity.

Cortical, but not posterior subcapsular, cataract shows significant familial aggregation in an older population after adjustment for possible shared environmental factors.

PURPOSE:

To quantify the risk for age-related cortical cataract and posterior subcapsular cataract (PSC) associated with having an affected sibling after adjusting for known environmental and personal risk factors.

DESIGN:

Sibling cohort study.

PARTICIPANTS:

Participants in the ongoing Salisbury Eye Evaluation (SEE) study (n = 321; mean age, 78.1+/-4.2 years) and their locally resident siblings (n = 453; mean age, 72.6+/-7.4 years) were recruited at the time of Rounds 3 and 4 of the SEE study. INTERVENTION/TESTING METHODS: Retroillumination photographs of the lens were graded for the presence of cortical cataract and PSC with the Wilmer grading system. The residual correlation between siblings' cataract grades was estimated after adjustment for a number of factors (age; gender; race; lifetime exposure to ultraviolet-B light; cigarette, alcohol, estrogen, and steroid use; serum antioxidants; history of diabetes; blood pressure; and body mass index) suspected to be associated with the presence of cataract.

RESULTS:

The average sibship size was 2.7 per family. Multivariate analysis revealed the magnitude of heritability (h(2)) for cortical cataract to be 24% (95% CI, 6%-42%), whereas that for PSC was not statistically significant (h(2) 4%; 95% CI, 0%-11%) after adjustment for the covariates. The model revealed that increasing age, female gender, a history of diabetes, and black race increased the odds of cortical cataract, whereas higher levels of provitamin A were protective. A history of diabetes and steroid use increased the odds for PSC.

CONCLUSIONS:

This study is consistent with a significant genetic effect for age-related cortical cataract but not PSC.

Familial aggregation of hyperopia in an elderly population of siblings in Salisbury, Maryland.

PURPOSE: To determine whether hyperopia aggregates in families in an older mixed-race population. DESIGN: Cross-sectional familial aggregation study using sibships. METHODS: We recruited 759 subjects (mean age, 73.4 years) in 241 families through the population-based Salisbury Eye Evaluation study. Subjects underwent noncycloplegic refraction if best-corrected visual acuity (BCVA) was <or=20/40, had lensometry to measure their currently worn spectacles if BCVA was >20/40 with spectacles, or were considered to be plano (refraction of zero) if the BCVA was >20/40 without spectacles. Preoperative refraction from medical records was used for bilaterally pseudophakic subjects. RESULTS: Utilizing hyperopia cutoffs from 1.00 to 2.50 diopters, age-, race-, and gender-adjusted odds ratios for hyperopia with an affected sibling ranged from 2.72 (95% confidence interval [CI], 1.84-4.01) to 4.87 (95% CI, 2.54-9.30). The odds of hyperopia increased with age until 75 years, after which they remained relatively constant. Black men were significantly less likely to be hyperopic than white men, white women, or black women. CONCLUSIONS: Hyperopia appears to be under strong genetic control in this older population.

Heritability of refractive error and familial aggregation of myopia in an elderly American population.

PURPOSE: To determine the heritability of refractive error and the familial aggregation of myopia in an older population. METHODS: Seven hundred fifty-nine siblings (mean age, 73.4 years) in 241 families were recruited from the Salisbury Eye Evaluation (SEE) Study in eastern Maryland. Refractive error was determined by noncycloplegic subjective refraction (if presenting distance visual acuity was < or =20/40) or lensometry (if best corrected visual acuity was >20/40 with spectacles). Participants were considered plano (refractive error of zero) if uncorrected visual acuity was >20/40. Preoperative refraction from medical records was used for pseudophakic subjects. Heritability of refractive error was calculated with multivariate linear regression and was estimated as twice the residual between-sibling correlation after adjusting for age, gender, and race. Logistic regression models were used to estimate the odds ratio (OR) of myopia, given a myopic sibling relative to having a nonmyopic sibling. RESULTS: The estimated heritability of refractive error was 61% (95% confidence interval [CI]: 34%-88%) in this population. The age-, race-, and sex-adjusted ORs of myopia were 2.65 (95% CI: 1.67-4.19), 2.25 (95% CI: 1.31-3.87), 3.00 (95% CI: 1.56-5.79), and 2.98 (95% CI: 1.51-5.87) for myopia thresholds of -0.50, -1.00, -1.50, and -2.00 D, respectively. Neither race nor gender was significantly associated with an increased risk of myopia. CONCLUSIONS: Refractive error and myopia are highly heritable in this elderly population.

Glycosylation of Vitamin D Binding Protein Reduced in Juvenile Idiopathic Arthritis Patients At Risk of Disease Extension.

Background/Purpose:Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish the subset of patients in whom inflammation extends to affect a large number of joints, early in the disease process. The post-translational modifications to candidate protein markers were verified by a novel deglycosylation strategy.Methods:SF samples from 57 patients were obtained around time of initial diagnosis of JIA. At 1 year from inclusion patients were categorized according to ILAR criteria as oligoarticular arthritis (n=26), extended oligoarticular (n=8) and polyarticular disease (n=18). SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with vitamin D binding protein (VDBP) expression and siaylation further verified by immunohistochemistry, ELISA test and immunoprecipitation. Candidate biomarkers were compared to conventional inflammation measure C-reactive protein (CRP). Sialic acid residues were enzymatically cleaved from immunopurified SF VDBP, enriched by hydrophilic interaction liquid chromatography (HILIC) and analysed by mass spectrometry.Results:Hierarchical clustering based on the expression levels of a set of 23 proteins segregated the extended-to-be oligoarticular from the oligoarticular patients. A cleaved isoform of VDBP, spot 873, is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Conversely total levels of vitamin D binding protein are elevated in plasma and ROC curves indicate an improved diagnostic sensitivity to detect patients at risk of disease extension, over both spot 873 and CRP levels. Sialysed forms of intact immunopurified VDBP were more prevalent in persistent oligoarticular patient synovial fluids.Conclusion:The data indicate that a subset of the synovial fluid proteome may be used to stratify patients to determine risk of disease extension. Reduced conversion of VDBP to a macrophage activation factor may represent a novel pathway contributing to increased risk of disease extension in JIA patients.