Clinical and pathological impact of VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in clear cell renal cell carcinoma.

VHL is mutated in the majority of patients with clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent studies have identified recurrent mutations in histone modifying and chromatin remodeling genes, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c. Current evidence suggests that BAP1 mutations are associated with aggressive disease. The clinical significance of the remaining genes is unknown. In this study, targeted sequencing of VHL and JARID1c (entire genes) and coding regions of BAP1, PBRM1, SETD2, and KDM6A was performed on 132 ccRCCs and matched normal tissues. Associations between mutations and clinical and pathological outcomes were interrogated. Inactivation of VHL (coding mutation or promoter methylation) was seen in 75% of ccRCCs. Somatic noncoding VHL alterations were identified in 29% of ccRCCs and may be associated with improved overall survival. BAP1 (11%), PBRM1 (33%), SETD2 (16%), JARID1c (4%), and KDM6A (3%) mutations were identified. BAP1-mutated tumors were associated with metastatic disease at presentation (P = 0.023), advanced clinical stage (P = 0.042) and a trend towards shorter recurrence free survival (P = 0.059) when compared with tumors exclusively mutated for PBRM1. Our results support those of recent publications pointing towards a role for BAP1 and PBRM1 mutations in risk stratifying ccRCCs. Further investigation of noncoding alterations in VHL is warranted.

An extremely rare abnormality of a double tracheoesophageal fistula without atresia of the esophagus; a case report and review of the literature.

Tracheoesophageal fistulas without atresia of the esophagus are rare abnormalities of the upper gastrointestinal tract with an incidence rate of between 1% and 5%. Even more infrequent are 2 tracheoesophageal fistulas without atresia of the esophagus. This case report illustrates the history of an infant with 2 tracheoesophageal fistulas. The corresponding literature was reviewed, and a diagnostic algorithm was described.

Genetic variation of Aflatoxin B1 aldehyde reductase genes (AFAR) in human tumour cells.

AFAR genes play a key role in the detoxification of the carcinogen Aflatoxin B(1) (AFB(1)). In the rat, Afar1 induction can prevent AFB(1)-induced liver cancer. It has been proposed that AFAR enzymes can metabolise endogenous diketones and dialdehydes that may be cytotoxic and/or genotoxic. Furthermore, human AFAR1 catalyses the rate limiting step in the synthesis of the neuromodulator gamma-hydroxybutyrate (GHB) and was found elevated in neurodegenerative diseases such as Alzheimer's and dementia with Lewy bodies (DLB). The human AFAR gene family maps to a genomic region in 1p36 of frequent hemizygous deletions in various human cancers. To investigate, if genetic variation of AFAR1 and AFAR2 exists that may alter protein detoxification capabilities and confer susceptibility to cancer, we have analysed a spectrum of human tumours and tumour cell lines for genetic heterogeneity. From 110 DNA samples, we identified nine different amino acid changes; two were in AFAR1 and seven in AFAR2. In AFAR1, we found genetic variation in the proposed substrate-binding amino acid 113, encoding Ala(113) or Thr(113). An AFAR2 variant had a Glu(55) substituted by Lys(55) at a position that is conserved among many aldo-keto reductases. This polarity change may have an effect on the proposed substrate binding amino acids nearby (Met(47), Tyr(48), Asp(50)). Further population analyses and functional studies of the nine variants detected may show if these variants are disease-related.

Suppression of polyploidy by the BRCA2 protein.

Mounting evidence implicates BRCA2 not only in maintenance of genome integrity but also in cell-cycle checkpoints. However, the contribution of BRCA2 in the checkpoints is still far from being understood. Here, we demonstrate that breast cancer cells MX-1 are unable to maintain genome integrity, which results in gross polyploidization. We generated MX-1 clones, stably expressing BRCA2, and found that BRCA2 acts to suppress polyploidy. Compared with MX-1, the ectopically BRCA2-expressing cells had different intracellular levels of Aurora A, Aurora B, p21, E2F-1, and pRb, suggesting a BRCA2-mediated suppression of polyploidy via stabilization of the checkpoint proteins levels.

CEBPbeta, JunD and c-Jun contribute to the transcriptional activation of the metastasis-associated C4.4A gene.

The glycosylphosphatidylinositol-anchored molecule C4.4A, which shares structural features with uPAR, is frequently expressed on carcinomas with upregulated expression during tumor progression. Moreover, rare expression on nontransformed epithelial cells is strongly increased during tissue remodeling, e.g., during wound healing. This strictly regulated expression prompted us to define transcriptional activation of the C4.4A gene. C4.4A transcription was analyzed in 2 syngenic rat tumor cell lines with low or high metastatic potential, respectively. Though genomic C4.4A DNA was present in both lines, C4.4A mRNA and transcription of a reporter construct containing the C4.4A promoter was only observed in the metastasizing subline. Deletions and point mutations in the C4.4A promoter-driven reporter construct revealed that activation of the TATA-less, GC-rich core promoter (-1 to -50 bp) does not suffice to initiate transcription that requires coactivation of a proximal response element (-71 to -88 bp) and can be further increased by more distal response elements (-89 to -133 bp). Mobility-shift and cotransfection studies showed that Sp3 binding enhances C4.4A transcription, whereas potential Sp1 binding sites were ineffective. C4.4A transcription essentially requires C/EBPbeta binding to a TRE/CCAAT composite element (-71 to -88 bp) as measured by ChIP assay. C4.4A transcription is strikingly enhanced by cotransfection with JunD or c-Jun, such that C4.4A is most strongly transcribed even in the C4.4A-negative tumor cell line after cotransfection with C/EBPbeta plus JunD or c-Jun. Thus, upregulation of C/EBPbeta during tumor progression and wound repair may well provide a sufficient trigger for transcription of the C4.4A gene.

Allelic variants of CAMTA1 and FLJ10737 within a commonly deleted region at 1p36 in neuroblastoma.

Deletion of a distal portion of 1p is seen in a wide range of human malignancies, including neuroblastoma. Here, a 1p36.3 commonly deleted region of 216 kb has been defined encompassing two genes, CAMTA1 and FLJ10737. Low expression of CAMTA1 has been recently shown to be an independent predictor of poor outcome in neuroblastoma patients. The present study surveys CAMTA1 and FLJ10737 for genetic alterations by fluorescence-based single strand conformation polymorphism (SSCP) using a panel of DNAs from 88 neuroblastomas, their matching blood samples and 97 unaffected individuals. Nucleotide variants encoding amino acid substitutions were found in both genes. One CAMTA1 variant (T1336I) was not detected in 97 unaffected individuals, another (N1177K) resides in a conserved domain of the CAMTA1 protein and was found hemizygous in six neuroblastomas. We found no evidence for somatic mutations in FLJ10737 or CAMTA1. Further investigations are needed to address the functional impact of the identified variants and their possible significance for neuroblastoma.

Telomere maintenance requires the RAD51D recombination/repair protein.

The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are required in mammalian cells for normal levels of genetic recombination and resistance to DNA-damaging agents. We report here that RAD51D is also involved in telomere maintenance. Using immunofluorescence labeling, electron microscopy, and chromatin immunoprecipitation assays, RAD51D was shown to localize to the telomeres of both meiotic and somatic cells. Telomerase-positive Rad51d(-/-) Trp53(-/-) primary mouse embryonic fibroblasts (MEFs) exhibited telomeric DNA repeat shortening compared to Trp53(-/-) or wild-type MEFs. Moreover, elevated levels of chromosomal aberrations were detected, including telomeric end-to-end fusions, a signature of telomere dysfunction. Inhibition of RAD51D synthesis in telomerase-negative immortalized human cells by siRNA also resulted in telomere erosion and chromosome fusion. We conclude that RAD51D plays a dual cellular role in both the repair of DNA double-strand breaks and telomere protection against attrition and fusion.

BRCA2: a genetic risk factor for breast cancer.

The identification of the breast cancer susceptibility genes BRCA1 and BRCA2 a few years ago has been greeted with great excitement and has raised hopes that they might illuminate the common mechanisms of this disease. Today we have to recognize that these expectations remain unfulfilled. Mutations in BRCA1 and BRCA2 account only for a relatively small proportion of breast cancers, even within the group of familiar clusters, they seem to be virtually non-existing in sporadic breast cancers. A substantial proportion of familiar breast cancer clusters has failed to provide evidence for an association with mutations in either BRCA1 or BRCA2, thus we have to look forward to the identification of additional breast cancer susceptibility genes. What has been most disappointing is that the mutation status of BRCA1/2 can provide only limited information for cancer risk. Initial assessments had indicated a risk of close to 90% for mutation carriers to develop breast cancer until age 75 - a value that turned out to be restricted to high-risk families in which the BRCA1 and BRCA2 genes had been genomically mapped. In unselected clusters the risk appears much lower, some estimates suggest less than 40%. Both BRCA1 and BRCA2 large encode proteins that appear to have a plethora of functions, with a conspicuous association to DNA repair and DNA recombination, and probably transcription activation. Defects in DNA repair can result in cancer predisposition syndromes and are recognized as being instrumental in cancer progression. Central questions have remained unanswered: What is the function of damaged BRCA1 and BRCA2 genes in breast cancer risk? What is the basis of large variations of risk conferred to the patients by identical mutations? How can the predictive value of mutation surveys be increased?