DNA methylation profiling of esophageal adenocarcinoma using Methylation Ligation-dependent Macroarray (MLM).

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.

Bicaudal C, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and leftward flow.

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.

SARS-CoV-2 seroprevalence in healthcare workers of a Swiss tertiary care centre at the end of the first wave: a cross-sectional study.

OBJECTIVE: To assess the SARS-CoV-2 transmission in healthcare workers (HCWs) using seroprevalence as a surrogate marker of infection in our tertiary care centre according to exposure. DESIGN: Seroprevalence cross-sectional study. SETTING: Single centre at the end of the first COVID-19 wave in Lausanne, Switzerland. PARTICIPANTS: 1874 of 4074 responders randomly selected (46% response rate), stratified by work category among the 13 474 (13.9%) HCWs. MAIN OUTCOME MEASURES: Evaluation of SARS-CoV-2 serostatus paired with a questionnaire of SARS-CoV-2 acquisition risk factors internal and external to the workplace. RESULTS: The overall SARS-CoV-2 seroprevalence rate among HCWs was 10.0% (95% CI 8.7% to 11.5%). HCWs with daily patient contact did not experience increased rates of seropositivity relative to those without (10.3% vs 9.6%, respectively, p=0.64). HCWs with direct contact with patients with COVID-19 or working in COVID-19 units did not experience increased seropositivity rates relative to their counterparts (10.4% vs 9.8%, p=0.69 and 10.6% vs 9.9%, p=0.69, respectively). However, specific locations of contact with patients irrespective of COVID-19 status-in patient rooms or reception areas-did correlate with increased rates of seropositivity (11.9% vs 7.5%, p=0.019 and 14.3% vs 9.2%, p=0.025, respectively). In contrast, HCWs with a suspected or proven SARS-CoV-2-infected household contact had significantly higher seropositivity rates than those without such contacts (19.0% vs 8.7%, p<0.001 and 42.1% vs 9.4%, p<0.001, respectively). Finally, consistent use of a mask on public transportation correlated with decreased seroprevalence (5.3% for mask users vs 11.2% for intermittent or no mask use, p=0.030). CONCLUSIONS: The overall seroprevalence was 10% without significant differences in seroprevalence between HCWs exposed to patients with COVID-19 and HCWs not exposed. This suggests that, once fully in place, protective measures limited SARS-CoV-2 occupational acquisition within the hospital environment. SARS-CoV-2 seroconversion among HCWs was associated primarily with community risk factors, particularly household transmission.

Epigenetic alteration of the Wnt inhibitory factor-1 promoter occurs early in the carcinogenesis of Barrett's esophagus.

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.

Identification and molecular characterisation of Lausanne Institutional Biobank participants with familial hypercholesterolaemia - a proof-of-concept study.

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.

Bicc1 links the regulation of cAMP signaling in polycystic kidneys to microRNA-induced gene silencing.

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.

Imprinting of tumor-suppressor genes in human placenta.

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.

Unusual distribution of DNA methylation within the hTERT CpG island in tissues and cell lines.

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.

Demethylation of the human telomerase catalytic subunit (hTERT) gene promoter reduced hTERT expression and telomerase activity and shortened telomeres.

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.

Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity.

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.

The human telomerase RNA gene (hTERC) is regulated during carcinogenesis but is not dependent on DNA methylation.

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.