The Y-chromosome F haplogroup contributes to the development of Barrett's esophagus-associated esophageal adenocarcinoma in a white male population.

Barrett's esophagus (BE) is a metaplastic condition of the distal esophagus, resulting from longstanding gastroesophageal reflux disease (GERD). BE predisposes for the highly malignant esophageal adenocarcinoma (EAC). Both BE and EAC have the highest frequencies in white males. Only a subset of patients with GERD develop BE, while <0.5% of BE will progress to EAC. Therefore, it is most likely that the development of BE and EAC is associated with underlying genetic factors. We hypothesized that in white males, Y-chromosomal haplogroups are associated with BE and EAC. To investigate this we conducted a multicenter study studying the frequencies of the Y-chromosomal haplogroups in GERD, BE, and EAC patients. We used genomic analysis by polymerase chain reaction and restriction fragment length polymorphism to determine the frequency of six Y-chromosomal haplogroups (DE, F(xJ,xK), K(xP), J, P(xR1a), and R1a) between GERD, BE, and EAC in a cohort of 1,365 white males, including 612 GERD, 753 BE patients, while 178 of the BE patients also had BE-associated EAC. Univariate logistic regression analysis was used to compare the outcomes. In this study, we found the R1a (6% vs. 9%, P = 0.04) and K (3% vs. 6%, P = 0.035) to be significantly underrepresented in BE patients as compared to GERD patients with an odds ratio (OR) of 0.63 (95% CI 0.42-0.95, P = 0.03) and of 0.56 (95% CI 0.33-0.96, P = 0.03), respectively, while the K haplogroup was protective against EAC (OR 0.30; 95% CI 0.07-0.86, P = 0.05). A significant overrepresentation of the F haplogroup was found in EAC compared to BE and GERD patients (34% vs. 27% and 23%, respectively). The F haplogroup was found to be a risk factor for EAC with an OR of 1.5 (95% CI 1.03-2.19, P = 0.03). We identified the R1a and K haplogroups as protective factors against development of BE. These haplogroups have low frequencies in white male populations. Of importance is that we could link the presence of the predominantly occurring F haplogroup in white males to EAC. It is possible that this F haplogroup is associated to genetic variants that predispose for the EAC development. In future, the haplogroups could be applied to improve stratification of BE and GERD patients with increased risk to develop BE and/or EAC.

Novel and recurrent variants of ATP2C1 identified in patients with Hailey-Hailey disease.

Hailey-Hailey disease (HHD) is a rare, late-onset autosomal dominant genodermatosis characterized by blisters, vesicular lesions, crusted erosions, and erythematous scaly plaques predominantly in intertriginous regions. HHD is caused by ATP2C1 mutations. About 180 distinct mutations have been identified so far; however, data of only few cases from Central Europe are available. The aim was to analyze the ATP2C1 gene in a cohort of Polish HHD patients. A group of 18 patients was enrolled in the study based on specific clinical symptoms. Mutations were detected using Sanger or next generation sequencing. In silico analysis was performed by prediction algorisms and dynamic structural modeling. In two cases, mRNA analysis was performed to confirm aberrant splicing. We detected 13 different mutations, including 8 novel, 2 recurrent (p.Gly850Ter and c.325-3 T > G), and 6 sporadic (c.423-1G > T, c.899 + 1G > A, p.Leu539Pro, p.Thr808TyrfsTer16, p.Gln855Arg and a complex allele: c.[1610C > G;1741 + 3A > G]). In silico analysis shows that all novel missense variants are pathogenic or likely pathogenic. We confirmed pathogenic status for two novel variants c.325-3 T > G and c.[1610C > G;1741 + 3A > G] by mRNA analysis. Our results broaden the knowledge about genetic heterogeneity in Central European patients with ATP2C1 mutations and also give further evidence that careful and multifactorial evaluation of variant pathogenicity status is essential.

Novel sporadic and recurrent mutations in KRT5 and KRT14 genes in Polish epidermolysis bullosa simplex patients: further insights into epidemiology and genotype-phenotype correlation.

Epidermolysis bullosa simplex (EBS) is a hereditary genodermatosis characterised by trauma-induced intraepidermal blistering of the skin. EBS is mostly caused by mutations in the KRT5 and KRT14 genes. Disease severity partially depends on the affected keratin type and may be modulated by mutation type and location. The aim of our study was to identify the molecular defects in KRT5 and KRT14 in a cohort of 46 Polish and one Belarusian probands with clinical suspicion of EBS and to determine the genotype-phenotype correlation. The group of 47 patients with clinical recognition of EBS was enrolled in the study. We analysed all coding exons of KRT5 and KRT14 using Sanger sequencing. The pathogenic status of novel variants was evaluated using bioinformatical tools, control group analysis (DNA from 100 healthy population-matched subjects) and probands' parents testing. We identified mutations in 80 % of patients and found 29 different mutations, 11 of which were novel and six were found in more than one family. All novel mutations were ascertained as pathogenic. In the majority of cases, the most severe genotype was associated with mutations in highly conserved regions. In some cases, different inheritance mode and clinical significance, than previously reported by others, was observed. We report 11 novel variants and show novel genotype-phenotype correlations. Our data give further insight into the natural history of EBS molecular pathology, epidemiology and mutation origin.

Expression pattern of immune suppressive cytokines and growth factors in oesophageal adenocarcinoma reveal a tumour immune escape-promoting microenvironment.

Immunotherapy for solid cancers, such as oesophageal adenocarcinoma (OAC), is generally hampered by an unfavourable immunological tumour microenvironment. This prompted us to investigate the nature of the OAC environment. Biopsies of tumour and normal control tissues were collected from 17 OAC patients, and investigated using fluorescent immunohistochemistry (IHC) for the expression of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), transforming growth factor-beta, indoleamine 2-3 dioxygenase, CXCL3 and CXCR1, and for measuring a panel of cytokines by cytometric bead array (CBA), and for Granzyme B (GrB), Perforin and PI9 detection by semi-quantitative PCR (QPCR). IHC showed that expression of all the above-mentioned factors is upregulated in 80-93% of the tumours. By QPCR, the cytokine interleukin (IL)-8 was significantly upregulated in tumour samples (P < 0.05). IL-6, IL-10, GrB and Perforin did not show any significant difference between normal and tumour samples, whereas PI9 levels were significantly higher in normal when compared with the tumour samples. CBA confirmed upregulation of IL-8 and show upregulation of IL-1beta in the tumours (P < 0.05). Regarding IL-6 and interferon (IFN)-gamma, no significant differences were observed between normal and tumour tissues. The OAC microenvironment is characterized by a lack of cytokines and factors that normally would enhance anti-cancer responses, such as IFN-gamma and GrB, and by a high expression of several immuno-suppressive factors, such as COX-2, VEGF and IL-8. For future improvement of treatment efficacy of OAC patients, it will be of importance to combine immunotherapy with immune-modulating agents.

Gene expression profile comparison of Barrett's esophagus epithelial cell cultures and biopsies.

Barrett's esophagus (BE) is a metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar lined epithelium. The aim was to gain more insight in the process of metaplasia and to identify which genes are specifically expressed by the epithelial cells and the surrounding tissues in BE. Hereto, the gene expression profile of a BE epithelial primary cell culture was compared to that of a BE biopsy. To specifically obtain the epithelial cell layer, epithelial cells from biopsies of BE were cultured using a Barrett specific culturing medium. Serial analysis of gene expression was applied to obtain a transcription library of the primary epithelial cell culture. The transcriptome was analyzed and compared to a previously described transcriptome of a BE biopsy. Validation of results by reverse transcriptase-polymerase chain reaction was performed using tissues of 16 BE patients and 16 primary cell cultures. Over 43,000 tags were sequenced. Genes specifically expressed by the Barrett epithelial cells were for instance Lipocalin 2 and Cyclin D1, whereas annexin A10, trefoil factor (TFF)1 and TFF2 were specifically expressed in the BE biopsies. The comparison of the gene expression profiles of BE primary cultured epithelial cells with BE biopsy defines a subset of genes that are specifically expressed by the epithelial cells and another subset that most likely is expressed by the underlying stromal tissues in the BE biopsy specimens.