Molecular and immunohistochemical analyses of uveal melanoma patient cohort.

Uveal melanoma is a rare form of melanoma and the most frequent primary eye malignancy in adults. The major molecular alterations underlying uveal melanoma pathogenesis affect mainly the GNAQ, GNA11, SF3B1, and BAP1 genes. In this study, we somatically genotyped 31 Brazilian uveal melanomas for BRAF, GNA11, GNAQ, SF3B1, and BAP1 gene mutations and assessed BRCA2 and p53 protein expression. GNAQ and GNA11 mutations were detected in 60%, and SF3B1 mutation rate was 30%. p53 Immunostaining was markedly positive in 5/31, and 3/31 samples showed negative BRCA2 expression. This study supports the importance of these key genes in uveal melanoma tumorigenesis; p53 and BRCA pathways seem to play a role in a subset of patients, possibly heralding unfavorable prognosis.

Spectrum of germline mutations in smokers and non-smokers in Brazilian non-small-cell lung cancer (NSCLC) patients.

Lung cancer (LC) is a leading cause of cancer-related mortality. Although smoking is the major risk factor, ~15% of all cases occur in never-smokers, suggesting that genetic factors play a role in LC predisposition. Indeed, germline mutations in the TP53 gene predispose to multiple cancer types, including LC. To date, few studies compared the somatic and germline mutational profiles of LC cases by smoking status, and none was reported in Brazilians. Whole-exome sequencing (WES) was performed on two pools (seven smokers and six non-smokers) of tumor-derived DNA using the Illumina HiSeq2000 platform. Files from pools were analyzed separately using Ingenuity®Variant AnalysisTM and Mendel,MD. Validation of all candidate variants was performed by Sanger sequencing. Subsequently, validated mutations were analyzed in germline DNA from the same patients and in ethnically matched controls. In addition, a single recurring Brazilian TP53 germline mutation (R337H) was genotyped in 45 non-small-cell lung cancer patients.Four novel germline variants in the ATAD2, AURKA, PTPRD and THBS1 genes were identified exclusively in smoker patients, and four germline missense variants in PLCD1, RAD52, CP and CDC6 genes were identified solely in non-smokers. There were 4/45 (8.9%) germline carriers of the R337H TP53 mutation. In conclusion, the recurring Brazilian TP53 mutation should be genotyped in all non-small-cell lung cancer in Brazil, regardless of smoking status. Distinct pathogenic mutations and novel sequence variants are detected in Brazilian non-small-cell lung cancer patients, by smoking status. The contribution of these sequence variants to LC pathogenesis remains to be further explored.

Whole-exome identifies RXRG and TH germline variants in familial isolated prolactinoma.

Familial isolated pituitary adenoma (FIPA) is a rare genetic disorder. In a subset of FIPA families AIP germline mutations have been reported, but in most FIPA cases the exact genetic defect remains unknown. The present study aimed to determine the genetic basis of FIPA in a Brazilian family. Three siblings presented with isolated prolactin genes. Further mutation screening was performed using whole-exome sequencing and all likely causative mutations were validated by Sanger sequencing. In silico analysis and secreting pituitary adenoma diagnosed through clinical, biochemical and imaging testing. Sanger sequencing was used to genotype candidate prolactinoma-mutated additional predictive algorithms were applied to prioritize likely pathogenic variants. No mutations in the coding and flanking intronic regions in the MEN1, AIP and PRLR genes were detected. Whole-exome sequencing of three affected siblings revealed novel, predicted damaging, heterozygous variants in three different genes: RXRG, REXO4 and TH. In conclusion, the RXRG and TH possibly pathogenic variants may be associated with isolated prolactinoma in the studied family. The possible contribution of these genes to additional FIPA families should be explored.

Calcium signalling from the type I inositol 1,4,5-trisphosphate receptor is required at early phase of liver regeneration.

BACKGROUND & AIMS: Liver regeneration is a multistage process that unfolds gradually, with different mediators acting at different stages of regeneration. Calcium (Ca(2+) ) signalling is essential for liver regeneration. In hepatocytes, Ca(2+) signalling results from the activation of inositol 1,4,5-trisphosphate receptors (InsP3 R) of which two of the three known isoforms are expressed (InsP3 R-I and InsP3 R-II). Here, we investigated the role of the InsP3 R-I-dependent Ca(2+) signals in hepatic proliferation during liver regeneration. METHODS: Partial hepatectomy (HX) in combination with knockdown of InsP3 R-I (AdsiRNA-I) was used to evaluate the role of InsP3 R-I on liver regeneration and hepatocyte proliferation, as assessed by liver to body mass ratio, PCNA expression, immunoblots and measurements of intracellular Ca(2+) signalling. RESULTS: AdsiRNA-I efficiently infected the liver as demonstrated by the expression of ß-galactosidase throughout the liver lobules. Moreover, this construct selectively and efficiently reduced the expression of InsP3 R-I, as evaluated by immunoblots. Expression of AdsiRNA-I in liver decreased peak Ca(2+) amplitude induced by vasopressin in isolated hepatocytes 2 days after HX. Reduced InsP3 R-I expression prior to HX also delayed liver regeneration, as measured by liver to body weight ratio, and reduced hepatocyte proliferation, as evaluated by PCNA staining, at the same time point. At later stages of regeneration, control hepatocytes showed a decreased expression of InsP3 R, as well as reduced InsP3 R-mediated Ca(2+) signalling, events that did not affect liver growth. CONCLUSION: Together, these results show that InsP3 R-I-dependent Ca(2+) signalling is an early triggering pathway required for liver regeneration.

Mitochondrial calcium regulates rat liver regeneration through the modulation of apoptosis.

UNLABELLED: Subcellular Ca(2+) signals control a variety of responses in the liver. For example, mitochondrial Ca(2+) (Ca(mit)(2+)) regulates apoptosis, whereas Ca(2+) in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca(mit)(2+) also affects liver regeneration. The Ca(2+)-buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin-mitochondrial targeting sequence-green fluorescent protein (PV-MITO-GFP). This construct properly localized to and effectively buffered Ca(2+) signals in the mitochondrial matrix. Additionally, the expression of PV-MITO-GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl-2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2-associated X protein (bax), apoptotic peptidase activating factor 1, and caspase-6]. PV-MITO-GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca(mit)(2+) buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl-2 and the decreased expression of bax. CONCLUSION: Together, these results reveal an essential role for Ca(mit)(2+) in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis.