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Background The objective of the current study was to compare the diagnostic accuracy of 2 new real-time polymerase chain reaction-based urinary markers with each other and with urinary cytology, cystoscopy, and/or histology in patients being followed for non-muscle-invasive bladder cancer. Methods A total of 487 patients were enrolled in the study. Patients were evaluated using voided urine cytology, the Xpert Bladder Cancer Monitor, the Bladder EpiCheck test, and white light cystoscopy. Results The overall sensitivity was 27.17% for cytology, 64.13% for the Bladder EpiCheck test, and 66.3% for the Xpert Bladder Cancer Monitor. The overall specificity was 98.82% for cytology, 82.06% for the Bladder EpiCheck test, and 76.47% for the Xpert Bladder Cancer Monitor. The negative predictive value was very similar for the 3 tests at 83.56% for cytology, 89.42% for the Bladder EpiCheck test, and 89.35% for the Xpert Bladder Cancer Monitor. When combined, the Bladder EpiCheck test and Xpert Bladder Cancer Monitor detected overall 79.35% of the tumors: 70.37% in low-grade and 92.11% in high-grade tumors. Conclusions The Xpert Bladder Cancer Monitor and Bladder EpiCheck test were found to perform very well in terms of sensitivity. Together, the 2 tests detected approximately 92.11% of high-grade tumors. Their specificity was high but could not reach the excellent value of cytology. The negative predictive value was the same for both tests and was higher than that for cytology, especially when the tests were used together (92.24%). These 2 new tests hold promise as urinary biomarkers. They may be used in combination to maximize sensitivity in a less invasive way, thereby reducing invasiveness in the follow-up of patients with non-muscle-invasive bladder cancer and decreasing discomfort for the patients as well as complications and costs.

Objectives: Upper urinary tract urothelial carcinoma (UTUC) represents about 5–10% of all urothelial malignancies with an increasing incidence. The standard diagnostic tools for the detection of UTUC are cytology, computed tomography (CT) urography, and ureterorenoscopy (URS). No biomarker to be included in the daily clinical practice has yet been identified. The aim of our study was to evaluate the potential role of Xpert® Bladder-Cancer (BC)-Detection in the diagnosis of UTUC. Methods: Eighty-two patients underwent 111 URS with Xpert® BC-Detection, cytology, or Urovysion® analysis of UT for suspicion of UTUC. Twenty-four cases were excluded from the analysis due to a non-diagnostic Xpert® BC-Detection, cytology, or Urovysion®. Samples were analyzed with upper tract (UT) urinary cytology, with Xpert® BC-Detection on UT urines, and with Urovysion® Fluorescence in situ hybridization (FISH) test. After urine collection, the patients underwent retrograde pyelography and/or URS, and if positive a UT biopsy. The Xpert® BC-Detection was reported by the software as negative or positive [cut-off total Linear Discriminant Analysis (LDA) = 0.45]. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of cytology, Xpert® BC-Detection and Urovysion-FISH were calculated using URS and/or histology results as reference. Results: In all, 27 (31%) of 87 URS resulted positive, with 20 low-grade (LG) and 7 high-grade (HG) tumors. Overall sensitivity was 51.9% for cytology, 100% for Xpert® BC-Detection, and 92.6% for Urovysion. The sensitivity of cytology increased from 26% in LG to 100% in HG tumors. For Xpert® BC-Detection, sensitivity was 100% both in LG and in HG, and for Urovysion-FISH, it increased from 90% in LG to 100% in HG tumors. PPV was 82.4% for cytology, 35% for Xpert® BC-Detection, and 73.5% for Urovysion. NPV was 81.4% for cytology, 100% for Xpert® BC-Detection, and 96.2% for Urovysion. Conclusion: The excellent NPV of Xpert® BC-Detection allows to avoid unnecessary endoscopic exploration of the UT, reducing invasiveness and URS complications in the follow-up of UTUC.

Levodopa is the standard long-term dopamine replacement therapy to treat Parkinson’s disease (PD) symptoms. With time, levodopa may induce debilitating dyskinesias (LID), the treatment of which represents a large clinically unmet need. However, time-to-LID onset varies between patients, reflecting a possible genetic component. We performed an hypothesis-free whole-exome sequencing (WES)-based screening of time-to-LID onset and attempted replication of previously published candidate gene studies. A WES association analysis was carried out in 134 PD patients in a meta-analytical framework. Replication was attempted in an independent study of 97 PD patients. Variants from previously reported candidate genes (OPRM1, COMT, BDNF) were also specifically examined. We significantly replicated, for the first time, an association of variant rs1799971 in the OPRM1 gene with time-to-LID onset. Furthermore, we identified two novel potentially functional variants, in the MAD2L2 (rs2233019) and MAP7 (rs35350783) genes, which were significantly associated at the discovery stage. In the replication study, the two variants showed direction-consistent effects but did not achieve the replication significance threshold. Our study provides the first WES results for time-to-LID onset, where we replicate association at OPRM1, and suggest new variants in MAD2L2 and MAP7 genes that are significant in discovery, but require larger datasets for replication. The results are being made publicly available to allow for independent external validation.

Background The objective of this study was to evaluate the diagnostic accuracy of the Bladder EpiCheck test in the follow-up of patients with non-muscle-invasive bladder cancer (NMIBC) and to compare it with the accuracy of urinary cytology, cystoscopy, and/or histology. Methods In total, 243 patients were enrolled in the current study. Patients were evaluated by voided urine cytology, by the Bladder EpiCheck test, and by white-light cystoscopy. Results Overall sensitivity was 33.3% for cytology, 62.3% for Bladder EpiCheck, and 66.7% for the 2 tests combined. The sensitivity of cytology increased from 7.7% in low-grade (LG) tumors to 66.6% in high-grade (HG) tumors; whereas, for the Bladder EpiCheck test, the sensitivity was 46.1% in LG tumors and 83.3% in HG tumors. Combined cytology and Bladder EpiCheck testing yielded an overall sensitivity of 56.4% for LG tumors and 90% for HG tumors. Overall specificity was 98.6% for cytology, 86.3% for Bladder EpiCheck, and 85.6% for the 2 tests combined. The positive predictive value was 92% for cytology and 68.2% for Bladder EpiCheck. For the 2 tests combined, it was 68.6%. The negative predictive value was similar for the 2 tests: 75.8% for cytology, 82.9% for Bladder EpiCheck, and 84.5% for the 2 tests combined. Conclusions The sensitivity of the Bladder EpiCheck test was significantly higher than that of cytology. The test performed very well in terms of specificity but could not reach the high value of cytology. The positive predictive value was higher for Bladder EpiCheck, whereas the negative predictive value was approximately the same for both tests.

AimsCystoscopy and urine cytology represent the gold standard for monitoring superficial bladder cancer (BC). Xpert BC Monitor is a new urinary marker based on the evaluation of five target mRNAs overexpressed in patients with bladder cancer. The aim of our study was to evaluate the diagnostic accuracy of Xpert BC Monitor in follow-up of patients with non-muscle invasive bladder cancer (NMIBC).Methods230 patients were included in this prospective study. Xpert BC Monitor cut-off was set to 0.5. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of cytology, Xpert BC Monitor and their combination were calculated and compared with cystoscopy/histology.Results52/230 patients showed a NMIBC recurrence, 45 low grade (LG) and 7 high grade (HG). Overall sensitivity was 11.5% for cytology, 46.2% for Xpert BC Monitor and 48.1% for the two tests combined. Sensitivity of cytology increased from 4.4% in LG to 57.1% in HG tumours whereas for the Xpert BC Monitor it was 40% in LG and 85.7% in HG tumours. Combined cytology and Xpert BC Monitor yielded an overall sensitivity of 42% for LG and 85.7% for HG. Overall specificity was 97.2% for cytology, 77% for Xpert BC Monitor and 75.8% for the two tests.ConclusionsSensitivity for the Xpert BC Monitor Test was significantly higher than for cytology. The test performed very well in terms of specificity but could not reach the value of cytology, while PPV and NPV performed approximately the same for both tests.

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson’s disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Purpose Mutations in SZT2 have been previously reported in several cases of early onset epilepsy and intellectual disability. In this study we investigate potential causal mutations in two male siblings affected by early onset epilepsy, intellectual disability and macrocephaly. Methods We use family-based whole-exome sequencing to identify candidate variants. Results We report the identification of two potential causal SZT2 mutations in compound heterozygous state. We observe considerable differences in the clinical phenotype severity of the two affected individuals. The cerebral MRI revealed no abnormalities in the older affected brother, while in the youngest one it revealed a right frontal polymicrogiria. Moreover, while good seizure control was achieved in the older affected individual the younger brother is affected by pharmacoresistant epilepsy, progressive spastic paraplegia, cortical myoclonus and a more severe intellectual disability. We also analyzed the relative location of the reported pathogenic mutations in the SZT2 protein. Conclusion Variable phenotypic expressivity is observed for this condition, while the location and type of mutations in SZT2 also has a potential impact on epilepsy severity. These findings extend our knowledge of epileptogenic conditions related to SZT2 and mTOR signaling.

Urachal carcinoma (UrC) is an exceedingly rare neoplasm that develops from the urachus, an embryologic remnant of the urogenital sinus and allantois. The most commonly encountered histologic subtype is adenocarcinoma. The aim of this study is to characterize a series of UrC by morphology, immunohistochemistry, and molecular analysis. We retrospectively investigated seven cases of UrCs and assessed patient symptoms, imaging, histologic features, immunohistochemical profile, molecular characteristics, pathologic stages, and type of treatment. Immunostaining for CK7, CK20, Muc-2, CDX2, GATA3, β-catenin, and CK34βE12 was carried out on each neoplasm and on seven non-neoplastic urachal remnants as the control group. Additionally, a mutational analysis was performed using the QIAact Actionable Insights Tumor Panel Kit, which analyzes KRAS, NRAS, KIT, BRAF, PDGFRA, ALK, EGFR, ERBB2, PIK3CA, ERBB3, ESR1, and RAF1. Our cohort comprised five females and two males with a mean age of 64 years. UrCs consisted of two mucinous cystadenocarcinomas and five invasive, non-cystic adenocarcinomas. Carcinoma antigen expression profile was positive for CK20 and negative for CK34βE12 and GATA3 in all cases. Five of seven cases stained positively for Muc-2 and CDX2. On the contrary, non-neoplastic urachal remnants were immunoreactive for CK34βE12, CK7, and GATA3. Mutational analysis gave a positive result in four out of seven (57.1%) cases. All four positive tumors showed RAS mutation and one an additional mutation in PIK3CA. Urachal tumors exhibit peculiar morphologic, immunohistochemical, and molecular features. Due to the advanced stage at presentation, individualized treatment should be undertaken.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PGC-1α, encoded by PPARGC1A, is a transcriptional co-activator that has been implicated in the pathogenesis of neurodegenerative disorders. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located far upstream of the reference gene promoter, are CNS-specific and are more abundant than reference gene transcripts in whole brain. These CNS-specific transcripts encode two main full-length and several truncated isoforms via alternative splicing. Truncated CNS-isoforms include 17 kDa proteins that lack the second LXXLL motif serving as an interaction site for several nuclear receptors. We now determined expression levels of CNS- and reference gene transcripts in 5 brain regions of 21, 8, and 13 deceased subjects with idiopathic PD, Lewy body dementia and controls without neurodegenerative disorders, respectively. We observed reductions of CNS-specific transcripts (encoding full-length isoforms) only in the substantia nigra pars compacta of PD and Lewy body dementia. However, in the substantia nigra and globus pallidus of PD cases we found an up-regulation of transcripts encoding the 17 kDa proteins that inhibited the co-activation of several transcription factors by full-length PGC-1α proteins in transfection assays. In two established animal models of PD, the PPARGC1A expression profiles differed from the profile in human PD in that the levels of CNS- and reference gene transcripts were decreased in several brain regions. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that appeared protective for PD in a clinical cohort and a post-mortem sample (P = .0002). Thus, functional and genetic studies support a role of the CNS-specific PPARGC1A locus in PD.

Objective: The aims of the present study were to profile the expression of several candidate microRNAs (miRNAs) in blood from l-dopa-treated and drug-naive patients with Parkinson disease (PD) vs unaffected controls and to interpret the miRNA expression data in a biological context. Methods: We analyzed RNAs from peripheral blood of 36 l-dopa–treated, 10 drug-naive patients with PD and unaffected controls matched 1:1 by sex and age. We evaluated expression by reverse transcription–quantitative real-time PCR, and we analyzed data using a 2-tailed paired t test. To detect miRNA targets, several miRNA resources were combined to generate an overall score for each candidate gene using weighted rank aggregation. Results: Significant overexpression of miR-103a-3p ( p p = 0.002), and miR-29a-3p ( p = 0.005) in treated patients with PD was observed, and promising candidate target genes for these were revealed by an integrated in silico analysis. Conclusions: We revealed 3 candidate biomarkers for PD. miRNAs 30b-5p and 29a-3p replicated a documented deregulation in PD albeit opposite to published data, while for miR-103a-3p, we demonstrated for the first time an overexpression in treated patients with PD. Expression studies in patients and/or in isolated peripheral blood mononuclear cells before and after l-dopa administration are necessary to define the involvement of l-dopa treatment in the observed overexpression. Our in silico analysis to prioritize targets of deregulated miRNAs identified candidate target genes, including genes related to neurodegeneration and PD. Despite the preliminary character of our study, the results provide a rationale for further clarifying the role of the identified miRNAs in the pathogenesis of PD and for validating their diagnostic potential.

Biological plausibility and other prior information could help select genome-wide association (GWA) findings for further follow-up, but there is no consensus on which types of knowledge should be considered or how to weight them. We used experts' opinions and empirical evidence to estimate the relative importance of 15 types of information at the single-nucleotide polymorphism (SNP) and gene levels. Opinions were elicited from 10 experts using a two-round Delphi survey. Empirical evidence was obtained by comparing the frequency of each type of characteristic in SNPs established as being associated with seven disease traits through GWA meta-analysis and independent replication, with the corresponding frequency in a randomly selected set of SNPs. SNP and gene characteristics were retrieved using a specially developed bioinformatics tool. Both the expert and the empirical evidence rated previous association in a meta-analysis or more than one study as conferring the highest relative probability of true association, whereas previous association in a single study ranked much lower. High relative probabilities were also observed for location in a functional protein domain, although location in a region evolutionarily conserved in vertebrates was ranked high by the data but not by the experts. Our empirical evidence did not support the importance attributed by the experts to whether the gene encodes a protein in a pathway or shows interactions relevant to the trait. Our findings provide insight into the selection and weighting of different types of knowledge in SNP or gene prioritization, and point to areas requiring further research.

Restless legs syndrome (RLS) is a sleep-related movement disorder that affects up to 15 % of the population. Linkage studies have identified several genomic loci in single families (12q, 14q, 9p, 2q, 20p and 16p, respectively). However, confirmation of these loci has not always been achieved, and causative mutations have not yet been identified. The locus on chromosome 2q33 (RLS4) was identified in two South Tyrolean families who shared a haplotype of microsatellite marker alleles across an 8.2-cM region. To pinpoint the gene localisation within RLS4, additional families from the same geographic region were evaluated, and linkage was replicated in one family. Within the candidate region, we initially found a haplotype of 23 single nucleotide polymorphism markers spanning 131.6 Kb shared by all affected members of the three linked families. Using a next generation sequencing approach, we further restricted the shared candidate region to 46.9 Kb over the potassium channel-related gene KCTD18 and exons 10-13 of SPATS2L.

Activated partial thromboplastin time (aPTT) and prothrombin time (PT) are clinical tests commonly used to screen for coagulation-factor deficiencies. One genome-wide association study (GWAS) has been reported previously for aPTT, but no GWAS has been reported for PT. We conducted a GWAS and meta-analysis to identify genetic loci for aPTT and PT. The GWAS for aPTT was conducted in 9,240 individuals of European ancestry from the Atherosclerosis Risk in Communities (ARIC) study, and the GWAS for PT was conducted in 2,583 participants from the Genetic Study of Three Population Microisolates in South Tyrol (MICROS) and the Lothian Birth Cohorts (LBC) of 1921 and 1936. Replication was assessed in 1,041 to 3,467 individuals. For aPTT, previously reported associations with KNG1, HRG, F11, F12, and ABO were confirmed. A second independent association in ABO was identified and replicated (rs8176704, p = 4.26 × 10(-24)). Pooling the ARIC and replication data yielded two additional loci in F5 (rs6028, p = 3.22 × 10(-9)) and AGBL1 (rs2469184, p = 3.61 × 10(-8)). For PT, significant associations were identified and confirmed in F7 (rs561241, p = 3.71 × 10(-56)) and PROCR/EDEM2 (rs2295888, p = 5.25 × 10(-13)). Assessment of existing gene expression and coronary artery disease (CAD) databases identified associations of five of the GWAS loci with altered gene expression and two with CAD. In summary, eight genetic loci that account for ∼29% of the variance in aPTT and two loci that account for ∼14% of the variance in PT were detected and supported by functional data.

The genetic determinants of variation in iron status are actively sought, but remain incompletely understood. Meta-analysis of two genome-wide association (GWA) studies and replication in three independent cohorts was performed to identify genetic loci associated in the general population with serum levels of iron and markers of iron status, including transferrin, ferritin, soluble transferrin receptor (sTfR) and sTfR-ferritin index. We identified and replicated a novel association of a common variant in the type-2 transferrin receptor (TFR2) gene with iron levels, with effect sizes highly consistent across samples. In addition, we identified and replicated an association between the HFE locus and ferritin and confirmed previously reported associations with the TF, TMPRSS6 and HFE genes. The five replicated variants were tested for association with expression levels of the corresponding genes in a publicly available data set of human liver samples, and nominally statistically significant expression differences by genotype were observed for all genes, although only rs3811647 in the TF gene survived the Bonferroni correction for multiple testing. In addition, we measured for the first time the effects of the common variant in TMPRSS6, rs4820268, on hepcidin mRNA in peripheral blood (n = 83 individuals) and on hepcidin levels in urine (n = 529) and observed an association in the same direction, though only borderline significant. These functional findings require confirmation in further studies with larger sample sizes, but they suggest that common variants in TMPRSS6 could modify the hepcidin-iron feedback loop in clinically unaffected individuals, thus making them more susceptible to imbalances of iron homeostasis.

The level of body iron storage and the erythropoietic need for iron are indicated by the serum levels of ferritin and soluble transferrin receptor (sTfR), respectively. A meta-analysis of five genome-wide association studies on sTfR and ferritin revealed novel association to the PCSK7 and TMPRSS6 loci for sTfR and the HFE locus for both parameters. The PCSK7 association was the most significant (rs236918, P = 1.1 x 10E-27) suggesting that proprotein convertase 7, the gene product of PCSK7, may be involved in sTfR generation and/or iron homeostasis. Conditioning the sTfR analyses on transferrin saturation abolished the HFE signal and substantially diminished the TMPRSS6 signal while the PCSK7 association was unaffected, suggesting that the former may be mediated by transferrin saturation whereas the PCSK7-associated effect on sTfR generation appears to be more direct.

The QRS interval, from the beginning of the Q wave to the end of the S wave on an electrocardiogram, reflects ventricular depolarization and conduction time and is a risk factor for mortality, sudden death and heart failure. We performed a genomewide association meta-analysis in 40,407 individuals of European descent from 14 studies, with further genotyping in 7,170 additional Europeans, and we identified 22 loci associated with QRS duration ( P < 5 x 10(-8)). These loci map in or near genes in pathways with established roles in ventricular conduction such as sodium channels, transcription factors and calcium-handling proteins, but also point to previously unidentified biologic processes, such as kinase inhibitors and genes related to tumorigenesis. We demonstrate that SCN10A, a candidate gene at the most significantly associated locus in this study, is expressed in the mouse ventricular conduction system, and treatment with a selective SCN10A blocker prolongs QRS duration. These findings extend our current knowledge of ventricular depolarization and conduction.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1-2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.

Vinculin is found in all adherens junctions, while metavinculin, a larger splice variant, is coexpressed with vinculin only in smooth and cardiac muscle. To understand the significance of metavinculin expression, we compared ligand binding between turkey vinculin and metavinculin. Residues 1–258 were found essential for head-tail interactions in both proteins. The tail domains (VT and MVT, respectively) both bind to F-actin. However, while VT bundles F-actin, MVT generates highly viscous F-actin webs. In transfected PtK2 cells, VT causes F-actin needles or coils, while MVT-expressing cells display a diffuse F-actin distribution. Thus, the MVT-specific insert induces an F-actin supraorganization different from the VT-based form, suggesting that metavinculin has a specific role in muscle.

Twenty-one expressed sequence tags (ESTs) belonging to unidentified transcripts were mapped to 9 intervals at 11p 15.5-p15.3. Thirteen were mapped to a region of an estimated size of 7 Mb with the help of a YAC contig. The remaining eight were mapped outside this region, and the centromeric or telomeric position of the ESTs relative to the YAC contig was defined with the help of a cellular hybrid containing a derivative chromosome 11 with a break-point within the region covered by the contig. The average size of the intervals, where the ESTs were mapped, was estimated to be 0.3-0.4 Mb. The refinement of the chromosomal location of these ESTs could be of great help in the identification of potential candidate genes for disease locus mapping at 11p15.

Prioritization is the process whereby a set of possible candidate genes or SNPs is ranked so that the most promising can be taken forward into further studies. In a genome-wide association study, prioritization is usually based on the p-values alone, but researchers sometimes take account of external annotation information about the SNPs such as whether the SNP lies close to a good candidate gene. Using external information in this way is inherently subjective and is often not formalized, making the analysis difficult to reproduce. Building on previous work that has identified fourteen important types of external information, we present an approximate Bayesian analysis that produces an estimate of the probability of association. The calculation combines four sources of information: the genome-wide data, SNP information derived from bioinformatics databases, empirical SNP weights, and the researchers’ subjective prior opinions. The calculation is fast enough that it can be applied to millions of SNPS and although it does rely on subjective judgments, those judgments are made explicit so that the final SNP selection can be reproduced. We show that the resulting probability of association is intuitively more appealing than the p-value because it is easier to interpret and it makes allowance for the power of the study. We illustrate the use of the probability of association for SNP prioritization by applying it to a meta-analysis of kidney function genome-wide association studies and demonstrate that SNP selection performs better using the probability of association compared with p-values alone.

Using protein kinase C, we have studied the influence of intramolecular interactions on phosphorylation in vinculin. We show that vinculin and its 90 kDa head and 29/27 kDa tail fragments, generated by V8 proteolytic cleavage, are differentially phosphorylated. While intact vinculin and the isolated head domain are only weakly labelled, the isolated tail fragment is much more strongly phosphorylated. In the presence of the tail, the head is fully protected from the kinase. These data are consistent with our observation that native vinculin is primarily phosphorylated within the tail domain and suggest a function of vinculin phosphorylation in the regulation of the vinculin conformation.

The osmotic behavior of myosin subfragment 1 was studied at 22 degrees C and pH 7.45 in 0.1 m KCl, 2 mm MgCl2, and 10 mm triethanolamine or in 25 mm phosphate, 2 mm MgCl2, and 2 mm MgADP. It was found that, in 0.1 m KCl, myosin subfragment 1 behaved as a spheroidal particle, with an average diameter of 8.09 nm, composed of two myosin subfragment 1 molecules. The lower limit of the thermodynamic dimerization constant was estimated to be 3.5 x 10(4) M-1. Above 5 mm as monomer, myosin subfragment 1 departed from the behavior expected of a dimeric spheroidal model because of the onset of a "hydration force." This force measured at the contact distance between particles equals 2.18 x 10(7) dynes/cm2 and falls off exponentially with a decay distance of 0.27 nm. In 25 mm orthophosphate, myosin subfragment 1, with an increase in the protein osmotic pressure, shifted from the behavior of a sphere to that of a cylinder. Between 1 x 10(5) and 4 x 10(5) dynes/cm2, the behavior of myosin subfragment 1 was different in the presence and in the absence of MgADP. In particular, at 1.8 x 10(5) dynes/cm2, the protein osmotic pressure in frog muscle, myosin subfragment 1 behaved as a sphere of 3.21-nm radius in the presence of MgADP and as a cylinder with a length to diameter ratio of 2.07 in the absence of MgADP. Under the solution conditions used in this work, S1 never behaved as a fully extended particle.

At the physiological protein osmotic pressure, the angle formed between the long axis of the actin monomer and the pointed end of the filament axis is roughly 61 degrees in F-actin and about 90 degrees in the myosin subfragment 1--decorated F-actin. This implies that, in the course of the contractile cycle, actin itself contributes, by about 4 nm, to the displacement of the actin filament toward the center of the sarcomer.

The diameter of the actin filament is influenced by osmotic stress, being 9.0 nm at 1×10 5 dynes/cm 2 and 6.8 nm at 9.00×10 6 dynes/cm 2 . At 1.81×10 5 dynes/cm 2 , the protein osmotic pressure in frog muscle, the diameter is 7.95 nm. The diameter of the tropomyosin-decorated actin filament is also influenced by osmotic stress even though, at the low pressures (up to 2×10 5 dynes/cm 2 ), the decorated filament is significantly more resistant to compression than the undecorated actin filament.

Genomic copy number variants (CNVs) are a common, heritable source of inter-individual differences in genomic sequence. Their influence on phenotypic variability and their involvement in the pathogenesis of several common diseases is well established and the object of many current studies. In the course of examining CNV association to various quantitative traits in a general population, we have detected a strong association of CNVs over the four TCR genes to lymphocyte and neutrophil numbers in blood. In a small replication series, we have further characterized the nature of these CNVs and found them not to be germline, but dependent on the origin of analysed DNA. Germline deletion and rearrangement around the T-cell receptor (TCR) genes naturally occurs in white blood cells. Blood DNA derived from persons with high lymphocyte counts generates variable intensity signals which behave like germline CNVs over these genes. As DNA containing a relative high proportion of these CNV-like events involving the TCR genes has the ability to influence genotype counts of SNPs in the regions of these genes, care should be taken in interpreting and replicating association signals on variants within these genes when blood-derived DNA is the only source of data.

The imprinted expression of the IGF2 and H19 genes is controlled by the Imprinting Centre 1 (IC1) at chromosome 11p15.5. This is a methylation-sensitive chromatin insulator that works by binding the zinc-finger protein CTCF in a parent-specific manner. Microdeletions abolishing some of the CTCF target sites (CTSs) of IC1 have been associated with the Beckwith-Wiedemann syndrome (BWS). However, the link between these mutations and the molecular and clinical phenotypes was debated. We have identified two novel families with IC1 deletions, in which individuals with the clinical features of the BWS are present in multiple generations. By analysing the methylation pattern at the IGF2 - H19 locus together with the clinical phenotypes in the individuals with maternal and those with paternal transmission of five different deletions, we demonstrate that maternal transmission of 1.4-1.8 kb deletions in the IC1 region co-segregates with the hypermethylation of the residual CTSs and BWS phenotype with complete penetrance, whereas normal phenotype is observed upon paternal transmission. Although gene expression could not be assayed in all cases, the methylation detected at the IGF2 DMR2 and H19 promoter suggests that IC1 hypermethylation is consistently associated with biallelic activation of IGF2 and biallelic silencing of H19. Comparison of these deletions with a 2.2 kb one previously reported by another group indicates that the spacing of the CTSs on the deleted allele is critical for the gain of the abnormal methylation and penetrance of the clinical phenotype. Furthermore, we observe that the hypermethylation resulting from the deletions is always mosaic, suggesting that the epigenetic defect at the IGF2-H19 locus is established post-zygotically and may cause body asymmetry and heterogeneity of the clinical phenotype. Finally, the IC1 microdeletions are associated with a high incidence of Wilms' tumour, making their molecular diagnosis particularly important for genetic counselling and tumour surveillance at follow-up. © 2007 Oxford University Press.

Corrigendum: Novel loci affecting iron homeostasis and their effects in individuals at risk for hemochromatosis

Fusion between the NUP98 and NSD3genes in a patient with acute myeloid leukemia associated with t(8;11)(p11.2;p15), is reported for the first time. The t(8;11)(p11.2;p15) was identified by classical cytogenetics. Fluorescence in situ hybridization (FISH) analysis revealed a split signal with a mix of BAC 118H17 and 290A12, indicating the translocation disrupted NUP98. FISH restriction at 8p11-12 showed a split of BAC 350N15. Molecular investigations into candidate genes in this BAC showed the NUP98 fusion partner at 8p11.2 was the NSD3 gene. To date the NSD3 gene has never been implicated in hematologic malignancies.

Chromosome 11p15 deletion is frequent in human tumors, suggesting the presence of at least one tumor suppressor gene within this region. While mutation analyses of local genes revealed only rare mutations, we have previously described a mechanism, gain of imprinting, that leads to loss of expression of genes located on the maternal 11p15 chromosome in human hepatocarcinomas. Loss of expression was often associated with loss of maternal-specific methylation at the KvDMR1 locus. Here, we show that loss of the maternal KvDMR1 methylation is common, ranging from 30 to 50%, to a variety of adult neoplasms, including liver, breast, cervical and gastric carcinomas. We found that other 11p15.5 loci were concomitantly hypomethylated, indicating that loss of KvDMR1 methylation occurred in the context of a common mechanism affecting the methylation of a large 11p15 subchromosomal domain. These epigenetic abnormalities were not detected in any normal somatic tissue. Therefore, it seems possible that, contrary to the repression of promoter activity caused by hypermethylation, loss of gene expression at 11p15.5 may result from the activation, by hypomethylation, of one or more negative regulatory elements.

Genomic imprinting is a reversible condition that causes parental-specific silencing of maternally or paternally inherited genes. Analysis of DNA and RNA from 52 human hepatocarcinoma samples revealed abnormal imprinting of genes located at chromosome 11p15 in 51% of 37 informative samples. The most frequently detected abnormality was gain of imprinting, which led to loss of expression of genes present on the maternal chromosome. As compared with matched normal liver tissue, hepatocellular carcinomas showed extinction or significant reduction of expression of one of the alleles of the CDKN1C , SLC22A1L , and IGF2 genes. Loss of maternal-specific methylation at the KvDMR1 locus in hepatocarcinoma correlated with abnormal expression of CDKN1C and IGF2 , suggesting a function for KvDMR1 as a long-range imprinting center active in adult tissues. These results point to the role of epigenetic mechanisms leading to loss of expression of imprinted genes at chromosome region 11p15 in human tumors.

Chromosome region 11p15.5 harbors unidentified genes involved in neoplasms and in the genetic disease Beckwith–Wiedemann syndrome. The genetic analysis of a 170-kb region at 11p15.5 between loci D11S601 and D11S679 resulted in the identification of six transcriptional units. Three genes, hNAP2 , CDKN1C , and KVLQT1 , are well characterized, whereas three genes are novel. The three additional genes were designated BWR1A , BWR1B , and BWR1C . Full-length cDNAs for these three genes were cloned and nucleotide sequences were determined. While our work was in progress, BWR1C cDNA was described as IPL [Qian, N., Franck, D., O’Keefe, D., Dao, D., Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. & Tycko, B. (1997) Hum. Mol. Genet. 6, 2021–2029]. The cloning and mapping of these genes together with the fine mapping of the three known genes indicates that the transcriptional map of this region is likely to be complete. Because this region frequently is altered in neoplasms and in the genetic disease Beckwith–Wiedemann syndrome, we carried out a mutational analysis in tumor cell lines and Beckwith–Wiedemann syndrome samples that resulted in the identification of genetic alterations in the BWR1A gene: an insertion that introduced a stop codon in the breast cancer cell line BT549 and a point mutation in the rhabdomyosarcoma cell line TE125-T. These results indicate that BWR1A may play a role in tumorigenesis.

We have modeled the effect of protein osmotic pressure on the orientation of the monomer in F-actin, in tropomyosin-F-actin, in the myosin subfragment-1 decorated F-actin and in the myosin subfragment-1 decorated tropomyosin-F-actin. According to the model, at the physiological protein osmotic pressure (18 kPa), the elastic moduli by bending of the monomer in F-actin and in tropomyosin-F-actin are calculated to be 4.74 MPa and 5.8 MPa, respectively. The elastic moduli by bending of the monomer in the myosin subfragment-1 decorated F-actin and in the myosin subfragment-1 decorated tropomyosin-F-actin are calculated to be 22MPa and 22.3MPa, respectively. These latter values are in excellent agreement with the values of the elastic moduli by stretching found for the fibres of frog and rabbit muscle. We have also calculated that, at the physiological protein osmotic pressure, the myosin subfragment-1 decorated F-actin rigor complex can develop a force of 3.96 pN, a force correctly oriented to promote the sliding of the actin filament toward the center of the sarcomere. The magnitude of this force is comparable to that reported for intact skeletal muscle. In contrast, the myosin subfragment-1 decorated tropomyosin-F-actin rigor complex develops a much smaller driving force, that favours relaxation. Apparently tropomyosin uncouples the osmotic and the mechanical event. It is proposed that the elastic energy for muscle contraction is provided by protein osmotic pressure.

The diameter of the actin filament decreases with an increase of the protein osmotic pressure. This phenomenon is accompanied by a decrease of the angle (alpha) formed between the long axis of the actin monomer and the pointed end of the filament axis. At 1.8 x 10(5) dyn/cm2 (the protein osmotic pressure in frog muscle) the diameter is 8.34 nm and the angle (alpha) is 61.5 degrees. The interfilament distance of tropomyosin-decorated actin filaments, at a set of different osmotic pressures, is larger than that of F-actin filaments. This suggests that the two tropomyosin helices project out of the contour of the actin filament. The tropomyosin-decorated actin filament is more rigid than F-actin. At 1.8 x 10(5) dyn/cm2, the angle (alpha) is 76.4 degrees, as compared to the value of 61.5 degrees for F-actin. The interfilament distance of troponin-tropomyosin-decorated actin filaments is sensitive to Ca2+: in the physiological range of protein osmotic pressure it decreases from 13.3 nm, in the presence of 2 mM EGTA, to 12.2 nm in the presence of 0.2 mM CaCl2. Two alternative models are proposed to explain the decrease in interfilament distance. (a) Calcium shifts tropomyosin along the actin monomer, toward the filament axis (the classical model). (b) Calcium releases the rigidity of the tropomyosin-decorated filament and restores the original plasticity of F-actin. The consequent decrease of the angle (alpha) brings the tropomyosin helices nearer to the filament axis, without any real movement of tropomyosin along the actin monomer.

The effect of alpha-actinin from chicken gizzard on the properties of F-actin solutions at 37 degrees C and at 4 degrees C was investigated. Beside the well-known increase of the gelling activity of alpha-actinin, it was found that lowering temperature to 4 degrees C: (a) modifies the shape of the alpha-actinin-F-actin binding isotherm; (b) increases the light scattering of the alpha-actinin-F-actin mixtures; (c) induces the formation of ribbons and bundles of F-actin. It was also observed that, by warming to 37 degrees C, the bundles of F-actin formed at 4 degrees C were dissociated into quasi-parallel actin filaments running at a distance of 25-42 nm from each other. On subsequent cooling to 4 degrees C, these parallel filaments were rapidly assembled into bundles. As at 37 degrees C, alpha-actinin displays a potent gelling activity on bundles but not on filaments of actin, and as the gelling activity at 4 degrees C is accompanied by the formation of actin bundles, it is concluded that actin bundling is a necessary condition to promote the actin-gelling activity.