Genetic variability of the ABCC2 gene and clinical outcomes in pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, caused by various factors, such as the aggressiveness of the disease, the limited therapeutic options and the lack of early detection and risk markers. The ATP binding cassette subfamily C member 2 (ABCC2) protein plays a critical role in response to various drugs and is differentially expressed in gemcitabine sensitive and resistant cells. Moreover, single nucleotide polymorphisms (SNPs) in the gene have been associated with differential outcomes and prognosis in several tumour types. The aim of this study was to investigate the possible association between SNPs in the ABCC2 gene and overall survival (OS) in PDAC patients. We analysed 12 polymorphisms, including tagging-SNPs covering all the genetic variability of the ABCC2 gene and genotyped them in 1415 PDAC patients collected within the Pancreatic Disease ReseArch (PANDoRA) consortium. We tested the association between ABCC2 SNPs and PDAC OS using Cox proportional hazard models. We analysed PDAC patients dividing them by stage and observed that the minor alleles of three SNPs showed an association with worse OS [rs3740067: hazard ratio (HR) = 3.29, 95% confidence interval (CI) = 1.56-6.97, P = 0.002; rs3740073: HR = 3.11, 95% CI = 1.52-6.38, P = 0.002 and rs717620: HR = 2.90, 95% CI = 1.41-5.95, P = 0.004, respectively] in stage I patients. In patients with more advanced PDAC, we did not observe any statistically significant association. Our results suggest that rs3740067, rs3740073 and rs717620 could be promising prognostic markers in stage I PDAC patients

Lack of association of CD44-rs353630 and CHI3L2-rs684559 with pancreatic ductal adenocarcinoma survival.

Although pancreatic ductal adenocarcinoma (PDAC) survival is poor, there are differences in patients' response to the treatments. Detection of predictive biomarkers explaining these differences is of the utmost importance. In a recent study two genetic markers (CD44-rs353630 and CHI3L2-rs684559) were reported to be associated with survival after PDAC resection. We attempted to replicate the associations in 1856 PDAC patients (685 resected with stage I/II) from the PANcreatic Disease ReseArch (PANDoRA) consortium. We also analysed the combined effect of the two genotypes in order to compare our results with what was previously reported. Additional stratified analyses considering TNM stage of the disease and whether the patients received surgery were also performed. We observed no statistically significant associations, except for the heterozygous carriers of CD44-rs353630, who were associated with worse OS (HR = 5.01; 95% CI 1.58-15.88; p = 0.006) among patients with stage I disease. This association is in the opposite direction of those reported previously, suggesting that data obtained in such small subgroups are hardly replicable and should be considered cautiously. The two polymorphisms combined did not show any statistically significant association. Our results suggest that the effect of CD44-rs353630 and CHI3L2-rs684559 cannot be generalized to all PDAC patients

Common variability in oestrogen-related genes and pancreatic ductal adenocarcinoma risk in women

The incidence of pancreatic ductal adenocarcinoma (PDAC) is different among males and females. This disparity cannot be fully explained by the difference in terms of exposure to known risk factors; therefore, the lower incidence in women could be attributed to sex-specific hormones. A two-phase association study was conducted in 12,387 female subjects (5436 PDAC cases and 6951 controls) to assess the effect on risk of developing PDAC of single nucleotide polymorphisms (SNPs) in 208 genes involved in oestrogen and pregnenolone biosynthesis and oestrogen-mediated signalling. In the discovery phase 14 polymorphisms showed a statistically significant association (P < 0.05). In the replication none of the findings were validated. In addition, a gene-based analysis was performed on the 208 selected genes. Four genes (NR5A2, MED1, NCOA2 and RUNX1) were associated with PDAC risk, but only NR5A2 showed an association (P = 4.08 × 10−5) below the Bonferroni-corrected threshold of statistical significance. In conclusion, despite differences in incidence between males and females, our study did not identify an effect of common polymorphisms in the oestrogen and pregnenolone pathways in relation to PDAC susceptibility. However, we validated the previously reported association between NR5A2 gene variants and PDAC risk

Polygenic and multifactorial scores for pancreatic ductal adenocarcinoma risk prediction

Most cases of pancreatic ductal adenocarcinoma (PDAC) are asymptomatic in early stages, and the disease is typically diagnosed in advanced phases, resulting in very high mortality. Tools to identify individuals at high risk of developing PDAC would be useful to improve chances of early detection

Caratterizzazione dell'espressione e della funzione delle Emiline in topo ed in zebrafish

Emilins are a family of glycoproteins of the extracellular matrix with common structural organization and containing a characteristic N-terminal cysteine-rich domain. During my PhD, I studied expression and function in zebrafish and in mice of the corresponding genes, and I focused my attention on Emilin3, coding for the only component of the family lacking the C-terminal gC1q domain. As concerns my work on zebrafish, by database searching we identified and cloned eight members of this gene family. Their expression pattern during embryonic development, analyzed by whole-mount in situ hybridization, is characteristic and partially overlapping with expression of Emilins in mice. The most interesting pattern is the one of Emilin-3: while other Emilin genes reveal a mesenchymal or cardiovascular expression profile, the two zebrafish genes coding for Emilin-3 are abundantly expressed at early stages in the notochord and in the floor plate and at later stages in the branchial arches. This suggests that Emilin-3 may play a distinct role in the extracellular matrix, in comparison with other members of Emilin family. To gain insight into the function of these genes in vivo, we started functional studies by in situ hybridization of embryos in which relevant molecular pathways are blocked by treatment with specific drugs and by microinjection of mRNA and morfolino oligonucleotides in fertilized oocytes. Concerning the studies in mice, these experiments represented a large part of my PhD work and they were concentrated on the gene coding for Emilin-3. In the past years, targeted Emilin3 gene inactivation in murine embryonic stem (ES) cells was already undertaken in the laboratory where I performed my PhD work. However, generation of Emilin3 knockout mice was unsuccesful and these previous attempts revealed that, unlike other Emilins, targeted inactivation of the Emilin3 gene is particularly difficult. The frequency of homologous recombination of this gene is extremely low and transmission of the inactivated allele to the F1 generation was not reached, probably also due to karyotypic instability of targeted ES cell clones. Therefore, I carried out a new Emilin3 gene targeting approach, performed by means of a large scale experiment with a new ES cell line. I transfected euploid R1 ES cells with a Emilin3 targeting construct and, after double positive-negative selection, 1505 clones were isolated; 995 of these individual cell clones were further expandend and investigated for identifying clones in which a correct homologous recombination event had occurred. With such large number of clones, molecular analysis by Southern blotting, a standard but time- and money-consuming procedure, was not an easily affordable screening method for detecting rare homologus recombinant clones. Therefore, I set up an optimal screening procedure by PCR. Six positive ES clones were identified, and after additional characterization four of them were used for generation of chimeric mice by microinjection of ES cells into host blastocysts and implant in foster females. By crossing these chimeric mice, I obtained for two independent clones heterozygous and finally homozygous Emilin3 knockout mice, and I started to undertake the phenotypic analysis of Emilin-3 null mice. Finally, I also performed a detailed karyotypic analysis of targeted Emilin3 ES clones introduced in vivo, in comparison to the original ES cell line and other mutant ES cell clones, with the aim of correlating the karyotype of ES cells with their ability to transmit the inactivated allele to the germ-line

Expression pattern and functional studies of Emilin genes in zebrafish

Expression pattern and functional studies of Emilin genes in zebrafis

Expression pattern and function analysis of Emilin genes in zebrafish

Expression pattern and function analysis of Emilin genes in zebrafis

Emilin genes are duplicated and dynamically expressed during zebrafish embryonic development

Emilins are a family of extracellular matrix proteins with common structural organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other emilins were recently isolated in mammals. To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure with the human ortholog. In situ hybridization revealed that expression of zebrafish emilin genes is regulated in a spatiotemporal manner during embryonic development, with overlapping and site-specific patterns mostly including mesenchymal structures. Expression of certain emilin genes in peculiar areas, such as the central nervous system or the posterior notochord, suggests that they may play a role in key morphogenetic processes

EMILIN3, an extracellular matrix molecule with restricted distribution in skin

EMILIN3 is an extracellular matrix glycoprotein that displays a dynamic and restricted expression pattern in connective tissues during post-natal life. In this study, we report the characterization of EMILIN3 deposition in the skin. In addition, to unravel the functions of this protein in skin homeostasis, we generated Emilin3 null mice and provide evidence that EMILIN3 is dispensable for hair follicle growth and maintenance throughout adult life