Clinical Multigene Panel Testing Identifies Racial and Ethnic Differences in Germline Pathogenic Variants Among Patients With Early-Onset Colorectal Cancer.

PURPOSE: The early-onset colorectal cancer (EOCRC) burden differs across racial/ethnic groups, yet the role of germline genetic predisposition in EOCRC disparities remains uncharacterized. We defined the prevalence and spectrum of inherited colorectal cancer (CRC) susceptibility gene variations among patients with EOCRC by race and ethnicity. PATIENTS AND METHODS: We included individuals diagnosed with a first primary CRC between age 15 and 49 years who identified as Ashkenazi Jewish, Asian, Black, Hispanic, or White and underwent germline genetic testing of 14 CRC susceptibility genes performed by a clinical testing laboratory. Variant comparisons by racial and ethnic groups were evaluated using chi-square tests and multivariable logistic regression adjusted for sex, age, CRC site, and number of primary colorectal tumors. RESULTS: Among 3,980 patients with EOCRC, a total of 530 germline pathogenic or likely pathogenic variants were identified in 485 individuals (12.2%). By race/ethnicity, 12.7% of Ashkenazim patients, 9.5% of Asian patients, 10.3% of Black patients, 14.0% of Hispanic patients, and 12.4% of White patients carried a germline variant. The prevalence of Lynch syndrome (P = .037), as well as APC, CHEK2, MLH1, monoallelic MUTYH, and PTEN variants, varied by race/ethnicity among patients with EOCRC (all P < .026). Ashkenazim and Hispanic patients had significantly higher odds of presenting with a pathogenic APC variant, which included p.I1307K (odds ratio [OR], 2.67; 95% CI, 1.30 to 5.49; P = .007) and MLH1 variant (OR, 8.69; 95% CI, 2.68 to 28.20; P = .0003), respectively, versus White patients in adjusted models. CONCLUSION: Germline genetic features differed by race/ethnicity in young patients with CRC, suggesting that current multigene panel tests may not be representative of EOCRC risk in diverse populations. Further study is needed to optimize genes selected for genetic testing in EOCRC via ancestry-specific gene and variant discovery to yield equitable clinical benefits for all patients and to mitigate inequities in disease burden.

Racial/Ethnic and Sex Differences in Somatic Cancer Gene Mutations among Patients with Early-Onset Colorectal Cancer.

Molecular features underlying colorectal cancer disparities remain uncharacterized. Here, we investigated somatic mutation patterns by race/ethnicity and sex among 5,856 non-Hispanic white (NHW), 535 non-Hispanic Black (NHB), and 512 Asian/Pacific Islander (API) patients with colorectal cancer (2,016 early-onset colorectal cancer patients: sequencing age <50 years). NHB patients with early-onset nonhypermutated colorectal cancer, but not API patients, had higher adjusted tumor mutation rates than NHW patients. There were significant differences for LRP1B, FLT4, FBXW7, RNF43, ATRX, APC, and PIK3CA mutation frequencies in early-onset nonhypermutated colorectal cancers between racial/ethnic groups. Heterogeneities by race/ethnicity were observed for the effect of APC, FLT4, and FAT1 between early-onset and late-onset nonhypermutated colorectal cancer. By sex, heterogeneity was observed for the effect of EP300, BRAF, WRN, KRAS, AXIN2, and SMAD2. Males and females with nonhypermutated colorectal cancer had different trends in EP300 mutations by age group. These findings define genomic patterns of early-onset nonhypermutated colorectal cancer by race/ethnicity and sex, which yields novel biological clues into early-onset colorectal cancer disparities. SIGNIFICANCE: NHBs, but not APIs, with early-onset nonhypermutated colorectal cancer had higher adjusted tumor mutation rates versus NHWs. Differences for FLT4, FBXW7, RNF43, LRP1B, APC, PIK3CA, and ATRX mutation rates between racial/ethnic groups and EP300, KRAS, AXIN2, WRN, BRAF, and LRP1B mutation rates by sex were observed in tumors of young patients. See related commentary by Shen et al., p. 530 . This article is highlighted in the In This Issue feature, p. 517.

Pharmacological manipulation of DNA methylation normalizes maternal behavior, DNA methylation, and gene expression in dams with a history of maltreatment.

The quality of parental care received during development profoundly influences an individual's phenotype, including that of maternal behavior. We previously found that female rats with a history of maltreatment during infancy mistreat their own offspring. One proposed mechanism through which early-life experiences influence behavior is via epigenetic modifications. Indeed, our lab has identified a number of brain epigenetic alterations in female rats with a history of maltreatment. Here we sought to investigate the role of DNA methylation in aberrant maternal behavior. We administered zebularine, a drug known to alter DNA methylation, to dams exposed during infancy to the scarcity-adversity model of low nesting resources, and then characterized the quality of their care towards their offspring. First, we replicate that dams with a history of maltreatment mistreat their own offspring. Second, we show that maltreated-dams treated with zebularine exhibit lower levels of adverse care toward their offspring. Third, we show that administration of zebularine in control dams (history of nurturing care) enhances levels of adverse care. Lastly, we show altered methylation and gene expression in maltreated dams normalized by zebularine. These findings lend support to the hypothesis that epigenetic alterations resulting from maltreatment causally relate to behavioral outcomes.

Female pups receive more maltreatment from stressed dams.

The effects of exposure to developmental stress often diverge for males and females. Using the scarcity-adversity model of low nesting resources outside the home cage, our lab has discovered sex differences in both behavioral and epigenetic consequences of repeated exposure to caregiver maltreatment. For the measures we have performed to date, we have found more consequences for females. The reasons underlying this sex disparity are unknown. In the current experiment, we aimed to discern the quality of maternal care received by male and female pups in our model. As we have previously found more behavioral and epigenetic consequences in females, we hypothesized that females receive more adverse care compared to their male littermates. Our hypothesis was supported; in our maltreatment condition, we found that female pups received more adverse care than males. This sex difference in adverse care was not present in our two control conditions (cross-foster and normal maternal care). These data lend support to the notion that one reason females in our model incur more behavioral and epigenetic consequences is a result of greater mistreatment by the dam.

Pharmacological Manipulation of DNA Methylation in Adult Female Rats Normalizes Behavioral Consequences of Early-Life Maltreatment.

Exposure to adversity early in development alters brain and behavioral trajectories. Data continue to accumulate that epigenetic mechanisms are a mediating factor between early-life adversity and adult behavioral phenotypes. Previous work from our laboratory has shown that female Long-Evans rats exposed to maltreatment during infancy display both aberrant forced swim behavior and patterns of brain DNA methylation in adulthood. Therefore, we examined the possibility of rescuing the aberrant forced swim behavior in maltreated-adult females by administering an epigenome-modifying drug (zebularine) at a dose previously shown to normalize DNA methylation. We found that zebularine normalized behavior in the forced swim test in maltreated females such that they performed at the levels of controls (females that had been exposed to only nurturing care during infancy). These data help link DNA methylation to an adult phenotype in our maltreatment model, and more broadly provide additional evidence that non-targeted epigenetic manipulations can change behavior associated with early-life adversity.

Using c-Jun to identify fear extinction learning-specific patterns of neural activity that are affected by single prolonged stress.

Neural circuits via which stress leads to disruptions in fear extinction is often explored in animal stress models. Using the single prolonged stress (SPS) model of post traumatic stress disorder and the immediate early gene (IEG) c-Fos as a measure of neural activity, we previously identified patterns of neural activity through which SPS disrupts extinction retention. However, none of these stress effects were specific to fear or extinction learning and memory. C-Jun is another IEG that is sometimes regulated in a different manner to c-Fos and could be used to identify emotional learning/memory specific patterns of neural activity that are sensitive to SPS. Animals were either fear conditioned (CS-fear) or presented with CSs only (CS-only) then subjected to extinction training and testing. C-Jun was then assayed within neural substrates critical for extinction memory. Inhibited c-Jun levels in the hippocampus (Hipp) and enhanced functional connectivity between the ventromedial prefrontal cortex (vmPFC) and basolateral amygdala (BLA) during extinction training was disrupted by SPS in the CS-fear group only. As a result, these effects were specific to emotional learning/memory. SPS also disrupted inhibited Hipp c-Jun levels, enhanced BLA c-Jun levels, and altered functional connectivity among the vmPFC, BLA, and Hipp during extinction testing in SPS rats in the CS-fear and CS-only groups. As a result, these effects were not specific to emotional learning/memory. Our findings suggest that SPS disrupts neural activity specific to extinction memory, but may also disrupt the retention of fear extinction by mechanisms that do not involve emotional learning/memory.

Phenotypic outcomes in adolescence and adulthood in the scarcity-adversity model of low nesting resources outside the home cage.

Early life adversity is known to disrupt behavioral trajectories and many rodent models have been developed to characterize these stress-induced outcomes. One example is the scarcity-adversity model of low nesting resources. This model employs resource scarcity (i.e., low nesting materials) to elicit adverse caregiving conditions (including maltreatment) toward rodent neonates. Our lab utilizes a version of this model wherein caregiving exposures occur outside the home cage during the first postnatal week. The aim of this study was to determine adolescent and adult phenotypic outcomes associated with this model, including assessment of depressive- and anxiety-like behaviors and performance in different cognitive domains. Exposure to adverse caregiving had no effect on adolescent behavioral performance whereas exposure significantly impaired adult behavioral performance. Further, adult behavioral assays revealed substantial differences between sexes. Overall, data demonstrate the ability of repeated exposure to brief bouts of maltreatment outside the home cage in infancy to impact the development of several behavioral domains later in life.

Do social functioning and symptoms improve with continuation antidepressant treatment of persistent depressive disorder? An observational study.

OBJECTIVE: To determine efficacy of continued treatment with the serotonin norepinephrine reuptake inhibitor duloxetine on symptom reduction and functional improvement in outpatients with dysthymia. METHOD: Fifty outpatients with DSM-IV-TR diagnosed dysthymia who had participated in a 10 week double-blind, placebo-controlled study of duloxetine received open treatment for three months. Nineteen duloxetine responders continued duloxetine, 24 patients initially treated with placebo started open duloxetine treatment, and 7 duloxetine non-responders were treated with desvenlafaxine or bupropion, selected by clinician choice. RESULTS: Patients continuing duloxetine maintained symptom improvement, 84% meeting response and 63% remission criteria at week 22. Patients initially treated with placebo showed similarly high levels of response (83%) and remission (62%) at week 22, and most duloxetine non-responders subsequently responded to other antidepressants. Duloxetine-continuation patients improved modestly between weeks 10 and 22 on measures of social and cognitive functioning and temperament. Despite this improvement concurrently across several functional domains, 66.7% of patients continuing duloxetine remained in the impaired range of functioning according to the Social Adjustment Scale (SAS). CONCLUSIONS: Continued duloxetine treatment appears to be effective in maintaining symptom response in dysthymic disorder, and has positive effects on social functioning. However, the majority of patients do not show normalization of functioning, even when controlling for remission status. Additional treatments should be considered to target residual impairments in social functioning in mood remitted patients with persistent depressive disorder.

Neural circuits via which single prolonged stress exposure leads to fear extinction retention deficits.

Single prolonged stress (SPS) has been used to examine mechanisms via which stress exposure leads to post-traumatic stress disorder symptoms. SPS induces fear extinction retention deficits, but neural circuits critical for mediating these deficits are unknown. To address this gap, we examined the effect of SPS on neural activity in brain regions critical for extinction retention (i.e., fear extinction circuit). These were the ventral hippocampus (vHipp), dorsal hippocampus (dHipp), basolateral amygdala (BLA), prelimbic cortex (PL), and infralimbic cortex (IL). SPS or control rats were fear conditioned then subjected to extinction training and testing. Subsets of rats were euthanized after extinction training, extinction testing, or immediate removal from the housing colony (baseline condition) to assay c-Fos levels (measure of neural activity) in respective brain region. SPS induced extinction retention deficits. During extinction training SPS disrupted enhanced IL neural activity and inhibited BLA neural activity. SPS also disrupted inhibited BLA and vHipp neural activity during extinction testing. Statistical analyses suggested that SPS disrupted functional connectivity within the dHipp during extinction training and increased functional connectivity between the BLA and vHipp during extinction testing. Our findings suggest that SPS induces extinction retention deficits by disrupting both excitatory and inhibitory changes in neural activity within the fear extinction circuit and inducing changes in functional connectivity within the Hipp and BLA.

Environmental influences on the female epigenome and behavior.

Environmental factors have long-lasting effects on brain development and behavior. One way experiences are propagated is via epigenetic modifications to the genome. Environmentally-driven epigenetic modifications show incredible brain region- and sex-specificity, and many brain regions affected are ones involved in maternal behavior. In rodent models, females are typically the primary caregiver and thus, any environmental factors that modulate the epigenotype of the mother could have consequences for her current and future offspring. Here we review evidence of the susceptibility of the female epigenome to environmental factors, with a focus on brain regions involved in maternal behavior. Accordingly, implications for interventions that target the mother's epigenome and parenting behavior are discussed.

Cholinergic neuronal lesions in the medial septum and vertical limb of the diagonal bands of Broca induce contextual fear memory generalization and impair acquisition of fear extinction.

Previous research has shown that the ventral medial prefrontal cortex (vmPFC) and hippocampus (Hipp) are critical for extinction memory. Basal forebrain (BF) cholinergic input to the vmPFC and Hipp is critical for neural function in these substrates, which suggests BF cholinergic neurons may be critical for extinction memory. In order to test this hypothesis, we applied cholinergic lesions to different regions of the BF and observed the effects these lesions had on extinction memory. Complete BF cholinergic lesions induced contextual fear memory generalization, and this generalized fear was resistant to extinction. Animals with complete BF cholinergic lesions could not acquire cued fear extinction. Restricted cholinergic lesions in the medial septum and vertical diagonal bands of Broca (MS/vDBB) mimicked the effects that BF cholinergic lesions had on contextual fear memory generalization and acquisition of fear extinction. Cholinergic lesions in the horizontal diagonal band of Broca and nucleus basalis (hDBB/NBM) induced a small deficit in extinction of generalized contextual fear memory with no accompanying deficits in cued fear extinction. The results of this study reveal that MS/vDBB cholinergic neurons are critical for inhibition and extinction of generalized contextual fear memory, and via this process, may be critical for acquisition of cued fear extinction. Further studies delineating neural circuits and mechanisms through which MS/vDBB cholinergic neurons facilitate these emotional memory processes are needed. © 2015 Wiley Periodicals, Inc.

Pi-Extended Ethynyl 21,23-Dithiaporphyrins: A Synthesis and Comparative Study of Electrochemical, Optical, and Self-Assembling Properties.

21,23-Dithiaporphyrins were synthesized containing pi-extending ethynyl substituents at the meso positions. These porphyrins displayed highly bathochromic and broadened absorbance profiles spanning 400-900 nm with molar absorptivities ranging from 2500 to 300,000 M(-1) cm(-1). Electrochemically, these ethynyl dithiaporphyrins undergo a single oxidation at 0.44 or 0.57 V and reduction at -1.17 or -1.08 V versus a ferrocene/ferrocenium internal standard depending on the type of functionalization appended to the ethynyl group. DFT calculations predict that the delocalization of the frontier molecular orbitals should expand onto the meso positions of the ethynyl 21,23-dithiaporphyrins; shrinking the HOMO-LUMO energy gap by destabilizing the HOMO energy. Indeed, the DFT results agree with our optical and electrochemical assessments. Finally, differential scanning calorimetry combined with cross-polarized optical microscopy and powder X-ray diffraction was used to assess the ability of these porphyrins for long-range order. For the ethynylphenyl alkoxy 21,23-dithiaporphyin, birefringent, soft-crystalline-like domains were observed by polarized microscopy, which are marginally sustained by a low-level of crystallinity detected in the XRD, suggesting that long-range ordering is possible. Overall, ethynyl 21,23-dithiaporphyrins are able to harvest much lower energy light and possess lower oxidation and reduction potentials compared to their pyrrolic analogues, which are desirable properties for applications in organic electronics.

Inhibiting corticosterone synthesis during fear memory formation exacerbates cued fear extinction memory deficits within the single prolonged stress model.

Using the single prolonged stress (SPS) animal model of post-traumatic stress disorder (PTSD), previous studies suggest that enhanced glucocorticoid receptor (GR) expression leads to cued fear extinction retention deficits. However, it is unknown how the endogenous ligand of GRs, corticosterone (CORT), may contribute to extinction retention deficits in the SPS model. Given that CORT synthesis during fear learning is critical for fear memory consolidation and SPS enhances GR expression, CORT synthesis during fear memory formation could strengthen fear memory in SPS rats by enhancing GR activation during fear learning. In turn, this could lead to cued fear extinction retention deficits. We tested the hypothesis that CORT synthesis during fear learning leads to cued fear extinction retention deficits in SPS rats by administering the CORT synthesis inhibitor metyrapone to SPS and control rats prior to fear conditioning, and observed the effect this had on extinction memory. Inhibiting CORT synthesis during fear memory formation in control rats tended to decrease cued freezing, though this effect never reached statistical significance. Contrary to our hypothesis, inhibiting CORT synthesis during fear memory formation disrupted extinction retention in SPS rats. This finding suggests that even though SPS exposure leads to cued fear extinction memory deficits, CORT synthesis during fear memory formation enhances extinction retention in SPS rats. This suggests that stress-induced CORT synthesis in previously stressed rats can be beneficial.

Sex differences in the single prolonged stress model.

Post traumatic stress disorder (PTSD) is a debilitating anxiety disorder resulting from traumatic stress exposure. Females are more likely to develop PTSD than males, but neurobiological mechanisms underlying female susceptibility are lacking. This can be addressed by using nonhuman animal models. Single prolonged stress (SPS), a nonhuman animal model of PTSD, results in cued fear extinction retention deficits and hippocampal glucocorticoid receptor (GR) upregulation in male rats. These effects appear linked in the SPS model, as well as in PTSD. However, the effects of SPS on cued fear extinction retention and hippocampal GRs in female rats remain unknown. Thus, we examined sex differences in SPS-induced cued fear extinction retention deficits and hippocampal GR upregulation. SPS induced cued fear extinction retention deficits in male rats but not female rats. SPS enhanced GR levels in the dorsal hippocampus of female rats, but not male rats. SPS had no effects on ventral hippocampal GR levels, but ventral hippocampal GR levels were attenuated in female rats relative to males. These results suggest that female rats are more resilient to the effects of SPS. The results also suggest that GR upregulation and cued fear extinction retention deficits can be dissociated in the SPS model.

Forcing aggregation of cyanine dyes with salts: a fine line between dimers and higher ordered aggregates.

It is uncommon to read about cyanine dyes in the literature and not have their aggregation discussed. They are of high interest considering their propensity to undergo self-organization in aqueous solution, leading to interesting photophysical properties resulting from the formation of their dimers and higher ordered aggregates. Currently, the study of their aggregation is in high demand due to their diverse application range including dye-sensitized solar cells. However, their aggregation in high salt solutions is under studied, and the effect on aggregation in congruence with high ionic strength is often overlooked. In a previous study, our group established the role of specific ion effects and in particular the necessity of matching water affinity to induce aggregation of a cationic cyanine dye, thiazole orange. In order to advance the understanding of this topic, we present in this article the diverse aggregation of cyanine dyes, as a single monovalent salt can cause different aggregation responses in a variety of these dyes. We established via absorption spectroscopy combined with chemometric analyses that the inherent monomer-dimer equilibrium of a dye depends on its geometry. More interestingly, experimental data coupled with DFT calculations reveal that not only the geometry of a dye but also its charge location plays a role in the aggregate morphology formed by the interaction of a cationic cyanine dye and an anion. It is thought that contact ion pair formation and effective charge screening generated within that ion pair are responsible for aggregates with a greater order.

Tuning light absorption in pyrene: synthesis and substitution effects of regioisomeric donor-acceptor chromophores.

Three isomeric donor-acceptor (DA) chromophores based on pyrene were synthesized to study the effects of substitution pattern on intramolecular charge-transfer absorption through pyrene. These chromophores are nonfluorescent and absorb light in the long-wavelength region approaching 700 nm, making them promising light-harvesters. Their optical properties depend greatly on the substitution pattern of the donor, but their electrochemical properties are relatively unaffected.

The Baeyer-Villiger reaction: solvent effects on reaction mechanisms.

This study focuses on the Baeyer-Villiger reaction of propanone and performic acid, with formic acid as catalyst. Continuum solvation methods (EIF-PCM and CPCM) and two density functionals (B3LYP and MPWB1K) are used to study solvent effects on two types of reaction mechanisms: concerted non-ionic and stepwise ionic. The ionic mechanism is the one found in most organic chemistry textbooks; it begins with the protonation of the ketone by the acid catalyst, even though this reaction normally takes place in non-polar solvents such as dichloromethane. Our calculations show that the concerted non-ionic pathway, which is the least energetic in non-polar solvents such as dichloromethane, becomes more energetic the more polar the solvent. After investigating a variety of non-ionic and ionic pathways in water, it is found that the addition step seems to be ionic but the migration step, which is rate-determining, is uncatalyzed, non-ionic and fully concerted. These results confirm the experimental findings in solvents of low to medium polarity that the rate constant of the reaction decreases as the solvent polarity increases. Moreover, we find that contrary to what is commonly accepted, in the addition and migration ionic steps the deprotonation of the ionic species occurs in a concerted manner with the other chemical events taking place.