Association of Breast and Ovarian Cancers With Predisposition Genes Identified by Large-Scale Sequencing.

Importance: Since the discovery of BRCA1 and BRCA2, multiple high- and moderate-penetrance genes have been reported as risk factors for hereditary breast cancer, ovarian cancer, or both; however, it is unclear whether these findings represent the complete genetic landscape of these cancers. Systematic investigation of the genetic contributions to breast and ovarian cancers is needed to confirm these findings and explore potentially new associations. Objective: To confirm reported and identify additional predisposition genes for breast or ovarian cancer. Design, Setting, and Participants: In this sample of 11 416 patients with clinical features of breast cancer, ovarian cancer, or both who were referred for genetic testing from 1200 hospitals and clinics across the United States and of 3988 controls who were referred for genetic testing for noncancer conditions between 2014 and 2015, whole-exome sequencing was conducted and gene-phenotype associations were examined. Case-control analyses using the Genome Aggregation Database as a set of reference controls were also conducted. Main Outcomes and Measures: Breast cancer risk associated with pathogenic variants among 625 cancer predisposition genes; association of identified predisposition breast or ovarian cancer genes with the breast cancer subtypes invasive ductal, invasive lobular, hormone receptor-positive, hormone receptor-negative, and male, and with early-onset disease. Results: Of 9639 patients with breast cancer, 3960 (41.1%) were early-onset cases (≤45 years at diagnosis) and 123 (1.3%) were male, with men having an older age at diagnosis than women (mean [SD] age, 61.8 [12.8] vs 48.6 [11.4] years). Of 2051 women with ovarian cancer, 445 (21.7%) received a diagnosis at 45 years or younger. Enrichment of pathogenic variants were identified in 4 non-BRCA genes associated with breast cancer risk: ATM (odds ratio [OR], 2.97; 95% CI, 1.67-5.68), CHEK2 (OR, 2.19; 95% CI, 1.40-3.56), PALB2 (OR, 5.53; 95% CI, 2.24-17.65), and MSH6 (OR, 2.59; 95% CI, 1.35-5.44). Increased risk for ovarian cancer was associated with 4 genes: MSH6 (OR, 4.16; 95% CI, 1.95-9.47), RAD51C (OR, not estimable; false-discovery rate-corrected P = .004), TP53 (OR, 18.50; 95% CI, 2.56-808.10), and ATM (OR, 2.85; 95% CI, 1.30-6.32). Neither the MRN complex genes nor CDKN2A was associated with increased breast or ovarian cancer risk. The findings also do not support previously reported breast cancer associations with the ovarian cancer susceptibility genes BRIP1, RAD51C, and RAD51D, or mismatch repair genes MSH2 and PMS2. Conclusions and Relevance: The results of this large-scale exome sequencing of patients and controls shed light on both well-established and controversial non-BRCA predisposition gene associations with breast or ovarian cancer reported to date and may implicate additional breast or ovarian cancer susceptibility gene candidates involved in DNA repair and genomic maintenance.

Diurnal variation in nicotine sensitivity in mice: role of genetic background and melatonin.

Despite the evidence that there is a daily rhythm in smoking behavior and that the effects of drugs of abuse exhibit diurnal variations, very few studies have explored the extent to which sensitivity to the effects of nicotine vary over the course of the day. In the studies described in this report, the melatonin proficient mouse strain C3H/Ibg and the melatonin deficient mouse strains C57BL/6J and DBA/2J were assessed for diurnal variations in sensitivity to the effects of nicotine. Results indicated that there is significant variation in sensitivity to both activity and body temperature depressant effects of nicotine in the melatonin proficient C3H/Ibg strain with maximal sensitivity occurring during the latter third of the light period of the light cycle and minimal sensitivity taking place during the last third of the dark phase of the light cycle. The melatonin deficient strains did not exhibit diurnal differences in sensitivity to the effects of nicotine suggesting a potential role for melatonin in modulating the effects of nicotine. Experiments with knockout mice lacking both the Mtnr1a and Mtnr1b melatonin receptors confirmed that the reduced sensitivity observed during the dark phase is melatonin dependent. Diurnal variation in nicotinic receptor expression also was measured in cortex, hippocampus, hypothalamus and striatum using [(125)I]-α-bungarotoxin and [(125)I]-epibatidine. [(125)I]-α-bungarotoxin binding in hypothalamus of C3H mice exhibited a diurnal pattern with maximal binding observed in the latter third of the light portion of the light cycle. No other significant differences in binding were detected.

Differential regulation of alpha7 nicotinic receptor gene (CHRNA7) expression in schizophrenic smokers.

The alpha7 neuronal nicotinic receptor gene (CHRNA7) has been implicated in the pathophysiology of schizophrenia by genetic and pharmacological studies. Expression of the alpha7* receptor, as measured by [(125)I]alpha-bungarotoxin autoradiography, is decreased in postmortem brain of schizophrenic subjects compared to non-mentally ill controls. Most schizophrenic patients are heavy smokers, with high levels of serum cotinine. Smoking changes the expression of multiple genes and differentially regulates gene expression in schizophrenic hippocampus. We examined the effects of smoking on CHRNA7 expression in the same tissue and find that smoking differentially regulates expression of both mRNA and protein for this gene. CHRNA7 mRNA and protein levels are significantly lower in schizophrenic nonsmokers compared to control nonsmokers and are brought to control levels in schizophrenic smokers. Sufficient protein but low surface expression of the alpha7* receptor, seen in the autoradiographic studies, suggests aberrant assembly or trafficking of the receptor.

Chrna7 genotype is linked with alpha7 nicotinic receptor expression but not alpha7 RNA levels.

Studies using the radio-labeled nicotinic receptor antagonist [(125)I]-alpha-bungarotoxin, which binds to alpha7 subunit containing nicotinic receptors, have demonstrated that mouse strains vary considerably in the number of alpha7-containing nicotinic receptors in brain. In addition, brain region specific differences in alpha-bungarotoxin binding between the mouse strains C3H/Ibg and DBA/2 have been linked to polymorphisms in Chrna7, the gene that encodes the alpha7 subunit. In the studies described here, we evaluated whether the relationship between Chrna7 genotype and individual differences in alpha-bungarotoxin binding levels in adult brain might be due to an effect of Chrna7 genotype on alpha7 RNA levels. Quantitative autoradiography of coronal brain slices from F2 mice derived from the parental strains C3H/Ibg and DBA/2 demonstrate that Chrna7 genotype is not linked to alpha7 RNA levels. In contrast, quantitative autoradiography confirmed the linkage of Chrna7 genotype with alpha-bungarotoxin binding levels in hippocampus, striatum, and more precisely defined areas within these brain regions where Chrna7 genotype is associated with the level of alpha-bungarotoxin binding. The fact that Chrna7 genotype is linked to individual differences in alpha-bungarotoxin binding, but not alpha7 RNA levels, suggests that the observed linkage between Chrna7 genotype and alpha-bungarotoxin levels may be due to genetic influences on the post-transcriptional regulation of alpha7 nicotinic receptor expression.

Regulation of a novel alphaN-catenin splice variant in schizophrenic smokers.

The alphaN-catenin (CTNNA2) gene represents a promising candidate gene for schizophrenia based upon previous genetic linkage, expression, and mouse knockout studies. CTNNA2 is differentially regulated by smoking in schizophrenic patients. In this report, the genomic structure of a primate-specific alphaN-catenin splice variant (alphaN-catenin III) is described. A comparison of alphaN-catenin III mRNA expression across postmortem hippocampi from schizophrenic and non-mentally ill smokers and non-smokers revealed a significant decrease in expression among patient non-smokers compared to all other groups. The recent evolutionary divergence of this gene, as well as the differences in gene expression in postmortem brain of schizophrenic non-smokers, supports the role of alphaN-catenin III as a novel disease susceptibility gene.

Permutation-based adjustments for the significance of partial regression coefficients in microarray data analysis.

The aim of this paper is to generalize permutation methods for multiple testing adjustment of significant partial regression coefficients in a linear regression model used for microarray data. Using a permutation method outlined by Anderson and Legendre [1999] and the permutation P-value adjustment from Simon et al. [2004], the significance of disease related gene expression will be determined and adjusted after accounting for the effects of covariates, which are not restricted to be categorical. We apply these methods to a microarray dataset containing confounders and illustrate the comparisons between the permutation-based adjustments and the normal theory adjustments. The application of a linear model is emphasized for data containing confounders and the permutation-based approaches are shown to be better suited for microarray data.

alpha7 nicotinic receptor gene promoter polymorphisms in inbred mice affect expression in a cell type-specific fashion.

Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.

Smoking, Genetics and Schizophrenia: Evidence for Self Medication.

Schizophrenia is a common mental illness with a high prevalence of smoking. More than 80% of schizophrenics smoke compared to 25% of the general population. Both schizophrenia and tobacco use have strong genetic components, which may overlap. It has been suggested that smoking in schizophrenia may be a form of self-medication in an attempt to treat an underlying biological pathology. Smoking normalizes auditory evoked potential and eye tracking deficits in schizophrenia, as well as improving cognitive function. Nicotine acts through a family of nicotinic receptors with either high or low affinity for nicotine. The loci for several of these receptors have been genetically linked to both smoking and to schizophrenia. Smoking changes gene expression for more than 200 genes in human hippocampus, and differentially normalizes aberrant gene expression in schizophrenia. The α7* nicotinic receptor, linked to schizophrenia and smoking, has been implicated in sensory processing deficits and is important for cognition and protection from neurotoxicity. Nicotine, however, has multiple health risks and desensitizes the receptor. A Phase I trial of DMXB-A, an α7* agonist, shows improvement in both P50 gating and in cognition, suggesting that further development of nicotinic cholinergic drugs is a promising direction in schizophrenia research.