Utilization of multigene panels in hereditary cancer predisposition testing: analysis of more than 2,000 patients.

PURPOSE: The aim of this study was to determine the clinical and molecular characteristics of 2,079 patients who underwent hereditary cancer multigene panel testing. METHODS: Panels included comprehensive analysis of 14-22 cancer susceptibility genes (BRCA1 and BRCA2 not included), depending on the panel ordered (BreastNext, OvaNext, ColoNext, or CancerNext). Next-generation sequencing and deletion/duplication analyses were performed for all genes except EPCAM (deletion/duplication analysis only). Clinical histories of ColoNext patients harboring mutations in genes with well-established diagnostic criteria were assessed to determine whether diagnostic/testing criteria were met. RESULTS: Positive rates were defined as the proportion of patients with a pathogenic mutation/likely pathogenic variant(s) and were as follows: 7.4% for BreastNext, 7.2% for OvaNext, 9.2% for ColoNext, and 9.6% for CancerNext. Inconclusive results were found in 19.8% of BreastNext, 25.6% of OvaNext, 15.1% of ColoNext, and 23.5% of CancerNext tests. Based on information submitted by clinicians, 30% of ColoNext patients with mutations in genes with well-established diagnostic criteria did not meet corresponding criteria. CONCLUSION: Our data point to an important role for targeted multigene panels in diagnosing hereditary cancer predisposition, particularly for patients with clinical histories spanning several possible diagnoses and for patients with suspicious clinical histories not meeting diagnostic criteria for a specific hereditary cancer syndrome.

Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL) facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS). Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.

The role of mitochondrial uncoupling in 3,4-methylenedioxymethamphetamine-mediated skeletal muscle hyperthermia and rhabdomyolysis.

Use of the popular club drug ecstasy (3,4-methylenedioxymethamphetamine, MDMA) can result in life-threatening hyperthermia and rhabdomyolysis. Recent studies show a link between skeletal muscle uncoupling proteins in MDMA-mediated hyperthermia. The mechanisms by which MDMA interacts with skeletal muscle mitochondria are largely unknown. The present study was designed to comprehensively evaluate the effects of MDMA on bioenergetics and toxicity of skeletal muscle. Using (31)P nuclear magnetic resonance (NMR) and serum creatine kinase levels, we demonstrate evidence for uncoupling of oxidative phosphorylation in the skeletal muscle of MDMA (40 mg/kg)-treated rats. In vivo, rats treated with MDMA had significantly elevated serum creatine kinase levels, a marker of rhabdomyolysis, 4 h post-MDMA treatment (955 +/- 132 IU/l) compared with saline-treated controls (373.2 +/- 59 IU/l). beta-ATP signal areas after MDMA treatment showed significant reductions (15%) from the baseline values with corresponding increases in inorganic phosphate (88% increases) and decreases in intracellular pH. Clark electrode experiments on isolated skeletal muscle mitochondria in vitro (1-5 mM MDMA) and ex vivo in MDMA-treated animals demonstrated no evidence of uncoupling of oxidative phosphorylation. In vitro experiments using L6 myotubules cocultured with primary hepatocytes demonstrated the presence of uncoupling protein-3 in the L6 myotubules, but no evidence of a direct effect of MDMA or its potential metabolites on cellular creatine kinase concentrations. These findings suggest that MDMA uncouples skeletal muscle mitochondria in vivo but that this uncoupling is the result of indirect mechanisms.

The effects of ecstasy (MDMA) on rat liver bioenergetics.

OBJECTIVES: Use of the drug ecstasy (3,4-methylenedioxymethamphetamine [MDMA]) can result in life-threatening hyperthermia. Agents that uncouple mitochondrial oxidative phosphorylation are known to cause severe hyperthermia. In the present study, the authors tested the hypothesis that MDMA directly uncouples oxidative phosphorylation in rat liver mitochondria. METHODS: Effects on mitochondrial bioenergetics were assessed both in vitro and ex vivo. In vitro studies consisted of measuring the effects of MDMA (0.1-5.0 mmol/L) on states of respiration in isolated rat liver mitochondria and on mitochondrial membrane potential in a rat liver cell line. In ex vivo studies, mitochondrial rates of respiration were measured in the livers of rats one hour after treatment with MDMA (40 mg/kg subcutaneously). RESULTS: With the in vitro mitochondrial preparations, only concentrations of 5 mmol/L MDMA showed evidence of uncoupling with a slight increase in state 4 respiration and a corresponding decrease in the respiratory control index. MDMA (0.1-5.0 mmol/L) failed to decrease the mitochondrial membrane potential in 3,3-dihexyloxacarbocyanide iodide-stained WB-344 cells after either one or 24 hours of incubation. Ex vivo rates of respiration obtained from the livers of rats one hour after treatment with MDMA (40 mg/kg subcutaneously) showed no evidence of mitochondrial uncoupling. CONCLUSIONS: These data suggest that while high concentrations of MDMA have some mild uncoupling effects in isolated mitochondria, these effects do not translate to cell culture or ex vivo studies in treated animals. These data do not support the view that the hyperthermia induced by MDMA is from a direct effect on mitochondrial oxidative phosphorylation.