Search

Since entering into force in 2014, the Human Research Act (HRA) and its ordinances have provided the regulatory framework in Switzerland for accessing health-related personal data and biological material for the purposes of research related to human diseases and to the structure and functioning of the human body. This legislation aims to protect participants’ privacy. At the same time, the scientific community wants to generate knowledge. Aside from the HRA, data privacy in Switzerland is more widely covered by the Federal Act on Data Protection (FADP) and in some cases by the EU’s General Data Protection Regulation (GDPR). When preparing and conducting a clinical study, researchers have to comply with a number of requirements and guidelines in order to respect the rights of patients (i.e. data privacy) and fulfil their duties to the scientific community (i.e. data sharing). This article discusses how these statutory requirements apply to specific clinical study documents, processes, and tools.

AIMS We aimed to identify familial hypercholesterolaemia mutation carriers among participants to the Lausanne Institutional Biobank (BIL). Our experimental workflow was designed as a proof-of-concept demonstration of the resources and services provided by our integrated institutional clinical research support platform. METHODS Familial hypercholesterolaemia was used as a model of a relatively common yet often underdiagnosed and inadequately treated Mendelian disease. Clinical and laboratory information was extracted from electronic hospital records. Patients were selected using elevated plasma cholesterol levels (total cholesterol ≥7.5 mM or low-density lipoprotein cholesterol ≥5 mM), premature coronary artery disease status and age (18-60 yr) as main inclusion criteria. LDLR, APOB and PCSK9 were analysed by high-throughput DNA sequencing. The most relevant mutations were confirmed by Sanger sequencing. RESULTS Of 23 737 patients contacted by the BIL, 17 760 individuals consented to participate and 13 094 wished to be recontacted if there were findings requiring clinical action. Plasma cholesterol records were available for 5111 participants, of whom 94 were selected for genetic screening. Twenty-five of the tested patients presented with premature coronary artery disease while 69 had no such diagnosis. Seven heterozygous carriers of eight rare coding missense variants were identified. Three mutations were pathogenic (APOB p.R3527Q) or likely pathogenic (LDLR p.C27W, LDLR p.P526S) for hypercholesterolaemia, while the others were either benign or of unknown significance. One patient was a double heterozygote for variants APOB p.R3527Q and LDLR p.P526S. CONCLUSION This work illustrates how clinical and translational research can benefit from a dedicated platform integrating both a hospital-based biobank and a data support team.

Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples.

Polycystic diseases and left-right (LR) axis malformations are frequently linked to cilia defects. Renal cysts also arise in mice and frogs lacking Bicaudal C (BicC), a conserved RNA-binding protein containing K-homology (KH)domains and a sterile alpha motif (SAM). However, a role for BicC in cilia function has not been demonstrated. Here, we report that targeted inactivation of BicC randomizes left-right (LR) asymmetry by disrupting the planar alignment of motile cilia required for cilia-driven fluid flow. Furthermore,depending on its SAM domain, BicC can uncouple Dvl2 signaling from the canonical Wnt pathway, which has been implicated in antagonizing planar cell polarity (PCP). The SAM domain concentrates BicC in cytoplasmic structures harboring RNA-processing bodies (P-bodies) and Dvl2. These results suggest a model whereby BicC links the orientation of cilia with PCP, possibly by regulating RNA silencing in P-bodies.

Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

Telomerase is the ribonucleoproteic complex involved in maintaining telomere size. It is expressed in germ and stem cells but not in normal somatic cells. In most tumors, telomerase is reactivated. In humans, telomerase activity is tightly regulated by expression of the hTERT gene. In a previous study, we found a direct correlation between methylation of the hTERT promoter and hTERT gene expression. In order to demonstrate this correlation, demethylation experiments were performed with the demethylating agent 5aza-2'-deoxycytidine (5azadC). Three telomerase-positive tumor cell lines (Lan-1, HeLa, and Co115), presenting a hypermethylated hTERT promoter, were treated with different doses and types of treatment for a long period. Analysis of methylation revealed a final hTERT promoter demethylation up to 95%. Quantification of hTERT mRNA showed that transcription was strongly repressed during drug exposure. In contrast, expression of c-Myc, an activator of hTERT promoter, was barely down-regulated or increased by the treatment. Using a TRAP assay, telomerase activity was semiquantified in all experiments. It strongly decreased or was suppressed after two to four passages. Finally, telomere length was measured by Southern blot. Their averages were not modified, but ranges concentrated around the mean. Thus, it is likely that hTERT promoter hypermethylation would be necessary for its expression.

Telomerase, the ribonucleoprotein complex involved in telomere maintenance, is composed of two main components: hTERT and hTERC. hTERT seems to be the rate-limiting factor for telomerase activity, although hTERC expression was also shown to correlate to a certain extent with telomerase reactivation. To determine whether the absence of hTERC expression could be the consequence of DNA methylation, we quantified hTERC RNA in 60 human samples (19 telomerase-negative normal tissues, nine telomerase-positive and 22 telomerase-negative tumor tissues, eight telomerase-positive and two telomerase-negative cell lines) using a quantitative dot blot on RT-PCR products. Most of the normal tissues did not express hTERC whereas, in telomerase-positive cell lines and in telomerase-positive tumor tissues, a strong up-regulation was observed, suggesting that hTERC transcription is up-regulated during tumorigenesis. The two telomerase-negative cell lines did not express hTERC. In a series of 22 telomerase-negative soft tissue sarcomas (STS), half did not express hTERC at all, or only weakly, whereas a wide range of expression was observed in the other half. As methylation might be involved in hTERC silencing, we examined the methylation pattern in all samples by direct sequencing and methylation-specific single stand conformation analysis after bisulfite modification. hTERC methylation was never observed, neither in normal nor in tumor tissues. Furthermore, there was no correlation between hTERC expression and proliferation, telomere length or hTERT expression in telomerase-negative STS. In contrast, three of eight telomerase-positive cell lines and the two telomerase negative cell lines were found to be hypermethylated, suggesting that the methylation observed may occur during cell line establishment. In conclusion, this study shows that hTERC expression is indeed regulated during carcinogenesis, but this regulation is unlikely to depend on hTERC methylation, cell proliferation rate, telomere length or hTERT expression.

Telomerase has been found to be reactivated in a majority of cancers but is inactive in most somatic cells. Our principal goal was to determine the potential use of the telomeric repeat amplification protocol (TRAP) assay as marker for malignancy in cytological effusions. The simple selection criterion was the cytological diagnosis, and routine samples were classified into malignant (58 samples) and nonmalignant (233 samples). Of the malignant samples, 44/58 (76%) were positive by TRAP assay. Of the 14 telomerase-negative cytology-positive samples, RNA integrity was poor in 9, indicating suboptimal sample conservation for molecular analysis. In 3 of the remaining 5 samples with a negative TRAP assay, a high number of malignant cells was observed, and these cells might have been telomerase-negative. Thus, the sensitivity of TRAP assay for the presence of malignant cells was about 76%. In the cytologically nonmalignant effusions, the presence of telomerase activity was observed in 24% (55/233). Of these, 6% were highly suspicious for malignancy, 9% were doubtful, and 9% were cytologically nonmalignant effusions confirmed by a follow-up of 12 mo or more. According to these data, the specificity of the TRAP assay to detect tumor cells in effusions ranged only between 82-91%. Our results indicate that, although the TRAP assay is positive in 6-15% of putative malignant effusions, the relatively high number of TRAP false-negative and false-positive cases renders this test unsuitable for routine diagnostic purposes.

Telomerase is inactive in most somatic cells, but has been found to be reactivated in a majority of cancers. Our principal goal was to test whether the presence of telomerase activity concurred with positive cytology, and was thus of potential use in detecting cancer cells in effusions. The telomeric repeat amplification protocol (TRAP) assay and cytological examination were performed in a blinded fashion on 91 unselected effusions, for which laboratory processing was done according to standard procedures. In our series, 30% (27/91) of samples were found to be malignant by cytology. Of these, 19 (70%) were also positive in the TRAP assay. Of the 8 telomerase-negative cytology-positive samples, RNA integrity was generally poor, indicating suboptimal sample conservation for molecular analysis. Negative cytology in the presence of telomerase activity was observed in 17 effusions. Of these, 11 were from patients with advanced cancer, and thus a diagnosis of malignant effusion should be suspected. The TRAP assay for telomerase activity holds promise in the analysis of effusions, but its routine use as an adjunct to cytology awaits further confirmation of its positive predictive value. Diagn. Cytopathol. 2001;24:174–180. © 2001 Wiley-Liss, Inc.

Transcriptional deregulation in cancer has been shown to be associated with epigenetic alterations, in particular to tumor-suppressor- gene (TSG) promoters. In contrast, DNA methylation of TSGs is not considered to be present in normal differentiated cells. Nevertheless, we previously showed that the promoter of the tumor-suppressor gene APC is methylated, for one allele only, in normal gastric cells. Recently, RASSF1A has been shown to be imprinted in normal human placenta. To clarify putative TSG methylation in the placenta, 23 normal placental tissues from the first trimester, both decidua and villi, and four normal non-gestational endometrium were screened for DNA methylation by methylation-sensitive single-strand conformation analysis (MS-SSCA) and sequencing after bisulfite modification, on a panel of 12 genes known to be implicated in carcinogenesis. In all placental villi, four TSG promoters-APC, SFRP2, RASSF1A and WIF1-were hypermethylated, whereas all decidua and normal endometrium did not show any methylation. Allele-specific methylation analysis revealed that this methylation was monoallelic. Furthermore, comparison with maternal DNA indicated that APC and WIF1 were methylated on the maternal allele, whereas SFRP2 was methylated on the paternal allele. Sequence analysis of WIF1 mRNA revealed that only the unmethylated paternal allele was transcribed. The imprinting status of these TSGs is conserved during pregnancy. These results indicate that TSG imprinting is pre-existent in normal human placenta and should not be confused with carcinogenesis or pathology-induced methylation.

The promoter region of the gene encoding the human telomerase reverse transcriptase (hTERT) is located in a CpG island and was shown to be regulated, at least in part, by DNA methylation. However, the observed correlation between hTERT methylation and gene expression was opposite to the general model of regulation by DNA methylation. We established a detailed mapping of methylcytosines at the CpG island (-1539 to +1732) surrounding the hTERT promoter in tissues and cell lines. In telomerase-positive samples, a methylation of all the CpG sites was observed for the hTERT promoter region (-500 to +1), whereas the exonic part (+1 to +450) revealed an unstable methylation pattern. Incomplete methylation of the proximal exon region could be necessary for, at least, a low level of hTERT transcription. In conclusion, hypermethylation of the CpG island plays a complex but essential role in the expression of hTERT in telomerase-positive cells.

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation-sensitive single-strand conformation analysis (MS-SSCA) and direct sequencing using bisulfite-modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p < 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT-expressing telomerase-positive samples and was absent in telomerase-negative samples (p < 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression. © 2002 Wiley-Liss, Inc.

Background: Aberrant methylation of multiple promoter CpG islands is a common event in human cancers. Methylation profile of cancer-related genes has extraordinary potential in basic research as well as translational medicine. The aim of this study was to replace the tedious and inefficient bisulfite-based methods with a simple technology which could permit a rapid and robust analysis of large numbers of genes in large numbers of tumor samples simultaneously. Materials and methods: Genomic DNA was first sonicated, then couples of primers for each specific gene promoter were annealed to this DNA. In a one-step reaction, the CfoI methylation-sensitive enzyme digested unmethylated DNA, whereas the Taq ligase joined primers annealed to the methylated sequence. Tagged ligation products were amplified by PCR and finally hybridized to a nylon membrane macroarray. Methylation Ligation-dependent Macroarray (MLM) allows the establishment of DNA methylation profiles on many samples (up to 21) and on many gene promoters (up to 45) in a simple reaction in 2-3 days. To validate this novel method, we used it to detect aberrant methylation in 14 cell lines and in DNA samples of 40 patients with a colorectal cancer. Results: In tumor cell lines, among the 42 genes investigated 23.7 (11-32) were found methylated whereas only 9 have been observed methylated in a normal fibroblast cell line (BJ). In normal colorectal tissues, about 12/40 gene promoters were found methylated; in colorectal cancers 16 to 36 genes were methylated. Methylated gene promoters identified in colorectal cancers were ADAMTS18 (78%), APC (28%), CDKN2A (20%), DKK2 (50%), GPx3 (48%), Mal (72%), MLH1 (30%), NELL1 (72%), SFRP4 (35%), SFRP5 (52%) and WRN (20%): Conclusion: The MLM is a fast, non expensive and reliable technology that profiles the DNA methylation status of >40 promoter CpG islands in various human samples. This technology can be used in research for rapid methylation profiling of numerous samples and for identification of new epigenetic biomarkers, but also in clinic for rapid cancer diagnosis, for predictive response to cancer therapy and for distinction between progressive and non-progressive diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2011-3023

The role of Wnt antagonists in the carcinogenesis of esophageal adenocarcinoma (EAC) remains unclear. We hypothesized that downregulation of the Wnt inhibitory factor-1 (WIF-1) might be involved in the neoplastic progression of Barrett's esophagus (BE). We analyzed the DNA methylation status of the WIF-1 promoter in normal, preneoplastic, and neoplastic samples from BE patients and in EAC cell lines. We investigated the role of WIF-1 on EAC cell growth and the chemosensitization of the cells to cisplatin. We found that silencing of WIF-1 correlated with promoter hypermethylation. EAC tissue samples showed higher levels of WIF-1 methylation compared to the matched normal epithelium. In addition, we found that WIF-1 hypermethylation was more frequent in BE samples from patients with EAC than in BE samples from patients who had not progressed to EAC. Restoration of WIF-1 in cell lines where WIF-1 was methylation-silenced resulted in growth suppression. Restoration of WIF-1 could sensitize the EAC cells to the chemotherapy drug cisplatin. Our results suggest that silencing of WIF-1 through promoter hypermethylation is an early and common event in the carcinogenesis of BE. Restoring functional WIF-1 might be used as a new targeted therapy for the treatment of this malignancy.

In lower and higher eukaryotes, a family of tightly related proteins designated eRF1 (for eukaryotic release factor 1) catalyses termination of protein synthesis at all three stop codons. The human genome contains four eRF1 homologous sequences localised on chromosomes 5, 6, 7 and X. We report here the cloning and the structural analysis of the human eRF1 gene family. It appears that the gene located on chromosome 5 alone is potentially functional, whereas the other three sequences resemble processed pseudogenes. This is the first description of the structural organisation of the human eRF1 gene, which has been remarkably conserved during evolution and which is essential in the translation termination process.

Genetic defects in autosomal-dominant polycystic kidney disease (ADPKD) promote cystic growth of renal tubules, at least in part by stimulating the accumulation of cAMP. How renal cAMP levels are regulated is incompletely understood. We show that cAMP and the expression of its synthetic enzyme adenylate cyclase-6 (AC6) are up-regulated in cystic kidneys of Bicc1(-)(/-) knockout mice. Bicc1, a protein comprising three K homology (KH) domains and a sterile alpha motif (SAM), is expressed in proximal tubules. The KH domains independently bind AC6 mRNA and recruit the miR-125a from Dicer, whereas the SAM domain enables silencing by Argonaute and TNRC6A/GW182. Bicc1 similarly induces silencing of the protein kinase inhibitor PKIα by miR-27a. Thus, Bicc1 is needed on these target mRNAs for silencing by specific miRNAs. The repression of AC6 by Bicc1 might explain why cysts in ADPKD patients preferentially arise from distal tubules.