Expansion and mechanistic insights into de novo DEAF1 variants in DEAF1-associated neurodevelopmental disorders.

De novo deleterious and heritable biallelic mutations in the DNA binding domain (DBD) of the transcription factor deformed epidermal autoregulatory factor 1 (DEAF1) result in a phenotypic spectrum of disorders termed DEAF1-associated neurodevelopmental disorders (DAND). RNA-sequencing using hippocampal RNA from mice with conditional deletion of Deaf1 in the central nervous system indicate that loss of Deaf1 activity results in the altered expression of genes involved in neuronal function, dendritic spine maintenance, development, and activity, with reduced dendritic spines in hippocampal regions. Since DEAF1 is not a dosage-sensitive gene, we assessed the dominant negative activity of previously identified de novo variants and a heritable recessive DEAF1 variant on selected DEAF1-regulated genes in 2 different cell models. While no altered gene expression was observed in cells over-expressing the recessive heritable variant, the gene expression profiles of cells over-expressing de novo variants resulted in similar gene expression changes as observed in CRISPR-Cas9-mediated DEAF1-deleted cells. Altered expression of DEAF1-regulated genes was rescued by exogenous expression of WT-DEAF1 but not by de novo variants in cells lacking endogenous DEAF1. De novo heterozygous variants within the DBD of DEAF1 were identified in 10 individuals with a phenotypic spectrum including autism spectrum disorder, developmental delays, sleep disturbance, high pain tolerance, and mild dysmorphic features. Functional assays demonstrate these variants alter DEAF1 transcriptional activity. Taken together, this study expands the clinical phenotypic spectrum of individuals with DAND, furthers our understanding of potential roles of DEAF1 on neuronal function, and demonstrates dominant negative activity of identified de novo variants.

Mutations affecting the SAND domain of DEAF1 cause intellectual disability with severe speech impairment and behavioral problems.

Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1.

APOE genotype influences P3b amplitude and response to smoking abstinence in young adults.

RATIONALE: There is strong evidence that nicotine can enhance cognitive functions and growing evidence that this effect may be larger in young healthy APOE ε4 carriers. However, the moderating effects of the APOE ε4 allele on cognitive impairments caused by nicotine deprivation in chronic smokers have not yet been studied with brain indices. OBJECTIVE: We sought to determine whether young female carriers of the APOE ε4 allele, relative to noncarriers, would exhibit larger abstinence-induced decreases in P3b amplitude during a two-stimulus auditory oddball task. METHODS: We compared parietal P3bs in female chronic smokers with either APOE ε3/ε3 (n = 54) or ε3/ε4 (n = 20) genotype under nicotine-sated conditions and after 12-17-h nicotine deprivation. RESULTS: Nicotine deprivation significantly reduced P3b amplitudes in APOE ε4 carriers, but not in APOE-ε3/ε3 individuals, such that the difference seen prior to nicotine deprivation was eliminated. CONCLUSIONS: The results suggest that subjects with the APOE ε4 allele are more sensitive to nicotine, which could influence smoking patterns, the risk for nicotine dependence, and the cognitive effects of nicotine use in these individuals.

DRD2-related TaqIA polymorphism modulates motivation to smoke.

INTRODUCTION: TaqIA polymorphism, a genetic variant associated with the expression level of dopamine D2 receptors in the brain, has been linked to various aspects of smoking behavior, including smoking prevalence, affective withdrawal symptoms, and smoking cessation outcome. However, its involvement in motivation to smoke cigarettes has not been elucidated. METHODS: The present study examined the possible differences in self-reported reasons to smoke and craving for smoking in 160 smokers participating in a clinical trial. RESULTS: Individuals with at least one A1 allele of the TaqIA polymorphism were more likely to report smoking for stimulating effects and to reduce negative affect compared with those lacking an A1 allele. The association of the A1 genotype with a higher probability and stronger motive to smoker to enhance cognitive functioning was evident in female but not in male smokers. Female A1 carriers also expected a greater likelihood of smoking for pleasure than those without an A1 allele. A1 subjects reported stronger craving for cigarettes during early days and the last phase of a 6-week abstinence period. DISCUSSION: These results support the idea that dopaminergic transmission plays an important role in the neurobiological basis of reasons for smoking and that the TaqIA variant is one of the genetic factors underlying individual differences in these aspects. These findings also have implications for improving treatment strategies to help individuals quit smoking by controlling their motivation to continue cigarette consumption.

Gene-environment interactions across development: Exploring DRD2 genotype and prenatal smoking effects on self-regulation.

Genetic factors dynamically interact with both pre- and postnatal environmental influences to shape development. Considerable attention has been devoted to gene-environment interactions (G x E) on important outcomes (A. Caspi & T. E. Moffitt, 2006). It is also important to consider the possibility that these G x E effects may vary across development, particularly for constructs like self-regulation that emerge slowly, depend on brain regions that change qualitatively in different developmental periods, and thus may be manifested differently. To illustrate one approach to exploring such developmental patterns, the relation between variation in the TaqIA polymorphism, related to D2 dopamine receptor expression and availability, and prenatal exposure to tobacco was examined in two exploratory studies. First, in 4-week-old neonates, genotype-exposure interactions were observed for attention and irritable reactivity, but not for stress dysregulation. Second, in preschool children, genotype was related to Preschool Trail Making Test (K. A. Espy and M. F. Cwik, 2004) task performance on conditions requiring executive control; children with both the A1+ genotype and a history of prenatal tobacco exposure displayed disproportionately poor performance. Despite study limitations, these results illustrate the importance of examining the interplay between genetic and prenatal environmental factors across development.

FAS expression inversely correlates with PTEN level in prostate cancer and a PI 3-kinase inhibitor synergizes with FAS siRNA to induce apoptosis.

Fatty acid synthase (FAS), a key enzyme of the fatty acid biosynthetic pathway, has been shown to be overexpressed in various types of human cancer and is, therefore, considered to be an attractive target for anticancer therapy. However, the exact mechanism of overexpression of the FAS gene in tumor cells is not well understood. In this report, we demonstrate that the expression of the tumor suppressor gene PTEN has a significant inverse correlation with FAS expression in the case of prostate cancer in the clinical setting, and inhibition of the PTEN gene leads to the overexpression of FAS in vitro. We also found that the combination of the expression status of these two genes is a better prognostic marker than either gene alone. Furthermore, our results indicate that the specific inhibition of FAS gene by siRNA leads to apoptosis of prostate tumor cells, and inhibition of PI 3-kinase pathway synergizes with FAS siRNA to enhance tumor cell death. These results provide a strong rationale for exploring the therapeutic use of an inhibitor of the PTEN signaling pathway in conjunction with the FAS siRNA to inhibit prostate tumor growth.

PTEN up-regulates the tumor metastasis suppressor gene Drg-1 in prostate and breast cancer.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) has been shown to be inactivated in a wide variety of cancers, and the role of this gene as a tumor suppressor has been well established. On the other hand, results of recent animal studies as well as clinical evidence indicate that PTEN is also involved in tumor metastasis suppression. Although PTEN is known to play a key role in controlling cell growth and apoptosis, how PTEN exerts the metastasis suppressor function remains largely unknown. Recently, a microarray analysis identified the Drg-1 gene (differentiation related gene 1) as one of the potential targets of PTEN. The Drg-1 gene has been shown to suppress tumor metastasis in animal models of prostate and colon cancer, and the expression of this gene is significantly reduced with advancement of prostate and breast cancers in clinical setting. In this study, we explored the possibility that PTEN controls tumor metastasis by regulating the expression of the Drg-1 gene. Our results indicate that overexpression of PTEN significantly augments the endogenous expression of Drg-1 protein, whereas inhibition of PTEN by small interfering RNA decreases Drg-1 in a dose- and time-dependent manner. We also found that the control of the Drg-1 gene by PTEN seems to be at the transcriptional level, and that a phospho-Akt inhibitor restores the Drg-1 expression, indicating that PTEN controls Drg-1 by an Akt-dependent pathway. Consistent with these results, our immunohistochemical analysis revealed that PTEN expression correlates significantly with Drg-1 in both prostate and breast cancer cases. Furthermore, combination of the two markers, PTEN and Drg-1, emerged as a significantly better predictor of prostate and breast cancer patient survival than either marker alone.

DEAF1 binds unmethylated and variably spaced CpG dinucleotide motifs.

DEAF1 is a transcriptional regulator associated with autoimmune and neurological disorders and is known to bind TTCG motifs. To further ascertain preferred DEAF1 DNA ligands, we screened a random oligonucleotide library containing an "anchored" CpG motif. We identified a binding consensus that generally conformed to a repeated TTCGGG motif, with the two invariant CpG dinucleotides separated by 6-11 nucleotides. Alteration of the consensus surrounding the dual CpG dinucleotides, or cytosine methylation of a single CpG half-site, eliminated DEAF1 binding. A sequence within the Htr1a promoter that resembles the binding consensus but contains a single CpG motif was confirmed to have low affinity binding with DEAF1. A DEAF1 binding consensus was identified in the EIF4G3 promoter and ChIP assay showed endogenous DEAF1 was bound to the region. We conclude that DEAF1 preferentially binds variably spaced and unmethylated CpG-containing half-sites when they occur within an appropriate consensus.

Effects of nicotine on emotional distraction of attentional orienting: evidence of possible moderation by dopamine type 2 receptor genotype.

INTRODUCTION: Growing evidence suggests that attentional bias to, and distraction by, emotional stimuli may moderate affective states and motivation for nicotine and other drug use. METHODS: The present study assessed the effects of nicotine and dopamine receptor genotype on distraction by emotional pictures, during a modified spatial attention task, in 46 overnight-deprived smokers. RESULTS: Relative to placebo, 14mg nicotine patch produced shorter overall reaction times (RTs) and individuals with two dopamine type 2 receptor (DRD2) A2 alleles exhibited the greatest RT benefit from nicotine following emotionally negative pictures after the longest cue-target delay (800ms), but benefitted least from nicotine following positive pictures after the shortest delay (400ms). In contrast, at the shortest delay, A1 carriers did not benefit from nicotine following emotionally negative pictures but did following positive ones. CONCLUSIONS: These genetic differences in the effects of nicotine on attention immediately following emotionally positive versus negative stimuli may reflect differential excitatory and inhibitory transmitter processes related to approach (reward) and avoidance (punishment) sensitivities of dopamine-related neural networks that support positive and negative affect.

pH-Sensitive MR Responses Induced by Dendron-Functionalized SPIONs.

We report a series of investigations of the pH-sensitive magnetic resonance (MR) responses of various surface-functionalized SPIONs (superparamagnetic iron oxide nanoparticles). First, functionalization of ~12 nm highly monocrystalline SPION cores with three different generations of melamine-dendrons was optimized to give agents with high molar relaxivities (e.g. R2m ~300 mM-1·s-1 at 7 T and R1m ~20-30 mM-1·s-1 at 0.5 T) and excellent aqueous stabilities. Molar relaxivities were found to exhibit great sensitivity to pH at physiologically-relevant ionic strengths, with sharp inflections observed at pH values near the pKa of the melamine monomer. The strength of the effect was observed to grow with increasing dendron generation (with concomitant shift in the position of the main pH inflection). Opposing behavior in R2m and R2m * trends may be exploited to provide a ratiometric MR response to pH. Combined with TEM and corresponding MR measurements from solutions of varying ionic strengths, these results are consistent with the pH-sensitive behavior originating from transient, reversible SPION clustering modulated by an interplay between SPION surface charge density and solution ionic strength. Studies of SPION cellular uptake and MR response in HeLa cell cultures are also presented. Finally, comparisons with the MR responses of SPIONs with alternative functionalities-derivatives of nitrilotriacetic acid or poly(1-vinylimidazole)-indicate that these types of pH-sensitive MR responses can be highly dependent upon the chemical composition of the surface species (and thus amenable to modulation through rational design).

Deformed epidermal autoregulatory factor-1 (DEAF1) interacts with the Ku70 subunit of the DNA-dependent protein kinase complex.

Deformed Epidermal Autoregulatory Factor 1 (DEAF1) is a transcription factor linked to suicide, cancer, autoimmune disorders and neural tube defects. To better understand the role of DEAF1 in protein interaction networks, a GST-DEAF1 fusion protein was used to isolate interacting proteins in mammalian cell lysates, and the XRCC6 (Ku70) and the XRCC5 (Ku80) subunits of DNA dependent protein kinase (DNA-PK) complex were identified by mass spectrometry, and the DNA-PK catalytic subunit was identified by immunoblotting. Interaction of DEAF1 with Ku70 and Ku80 was confirmed to occur within cells by co-immunoprecipitation of epitope-tagged proteins, and was mediated through interaction with the Ku70 subunit. Using in vitro GST-pulldowns, interaction between DEAF1 and the Ku70 subunit was mapped to the DEAF1 DNA binding domain and the C-terminal Bax-binding region of Ku70. In transfected cells, DEAF1 and Ku70 colocalized to the nucleus, but Ku70 could not relocalize a mutant cytoplasmic form of DEAF1 to the nucleus. Using an in vitro kinase assay, DEAF1 was phosphorylated by DNA-PK in a DNA-independent manner. Electrophoretic mobility shift assays showed that DEAF1 or Ku70/Ku80 did not interfere with the DNA binding of each other, but DNA containing DEAF1 binding sites inhibited the DEAF1-Ku70 interaction. The data demonstrates that DEAF1 can interact with the DNA-PK complex through interactions of its DNA binding domain with the carboxy-terminal region of Ku70 that contains the Bax binding domain, and that DEAF1 is a potential substrate for DNA-PK.

Neurotransmission-related genetic polymorphisms, negative affectivity traits, and gender predict tobacco abstinence symptoms across 44 days with and without nicotine patch.

Genetic and personality trait moderators of tobacco abstinence-symptom trajectories were assessed in a highly controlled study. Based on evidence suggesting their importance in stress reactivity and smoking, moderators studied were serotonin transporter gene (5-HTTLPR) and dopamine D2 receptor gene (DRD2) polymorphisms and personality traits related to negative affect (NA). Smokers were randomly assigned to quit smoking with nicotine or placebo patches. Financial incentives resulted in 80% verified abstinence across the 44-day study. Individuals with 1 or 2 short alleles of 5-HTTLPR (S carriers) experienced larger increases in NA symptoms than did those without a short allele. Nicotine replacement therapy (NRT) alleviated anxiety only in S carriers. NRT reduced NA to a greater extent in DRD2 A1 carriers than in A2A2 individuals during the 1st 2 weeks of treatment (when on the 21-mg patch); however, A1 carriers experienced a renewal of NA symptoms when switched to the 7-mg patch and when off the patch, while A2A2 individuals continued to benefit from NRT. The results suggest that the effects of genotype and treatment may vary across different durations of abstinence, treatment doses, and genotypes.

Dopamine receptor (DRD2) genotype-dependent effects of nicotine on attention and distraction during rapid visual information processing.

The effects of nicotine, distractor type, and dopamine type-2 receptor (DRD2) genotype on rapid visual information processing (RVIP) task performance were assessed in habitual smokers. Four RVIP tasks differed in terms of distractor location (central vs. peripheral) and distractor type (numeric vs. emotional). Each participant performed each of the tasks on two different days, once while wearing an active nicotine patch and once while wearing a placebo patch. Overall, the nicotine patch produced more accurate detection of and faster reaction times to target sequences; however, these effects varied with distractor type and genotype. Nicotine speeded reaction time more with left-visual-field (LVF) than right-visual-field (RVF) emotional distractors but speeded reaction time more with RVF than LVF numeric distractors, especially when the distractor digit matched the target sequence in terms of numeric oddness or evenness. Nicotine tended to facilitate performance more in individuals with at least one A1 allele than in homozygous A2A2 individuals, especially with numeric distractors presented to the left hemisphere. Nicotine tended to reduce distraction by negative stimuli more than other types of stimuli. Few gender differences were observed. The overall pattern of results was consistent with the view that nicotine modulates selective attention or subsequent information processing in a manner that depends partly on the emotional versus numeric nature of task distractors, DRD2 genotype, and the brain hemisphere that initially processes the distractors (visual field of distractor).

Identification of a nuclear export signal and protein interaction domains in deformed epidermal autoregulatory factor-1 (DEAF-1).

Deformed epidermal autoregulatory factor-1 (DEAF-1) is a DNA-binding protein required for embryonic development and linked to clinical depression and suicidal behavior in humans. Although primarily nuclear, cytoplasmic localization of DEAF-1 has been observed, and this suggests the presence of a nuclear export signal (NES). Using a series of fluorescent fusion proteins, an NES with a novel spacing of leucines (LXLX(6)LLX(5)LX(2)L) was identified near the COOH-terminal MYND domain at amino acids 454-476. The NES was leptomycin B-sensitive and mutation of the leucine residues decreased or eliminated nuclear export activity. In vitro pull downs and an in vivo fluorescent protein interaction assay identified a DEAF-1/DEAF-1 protein interaction domain within the NES region. DNA binding had been previously mapped to a positively charged surface patch in the novel DNA binding fold called the "SAND" domain. A second protein-protein interaction domain was identified at amino acids 243-306 that contains the DNA-binding SAND domain and also an adjacent zinc binding motif and a monopartite nuclear localization signal (NLS). Deletion of these adjacent sequences or mutation of the conserved cysteines or histidine in the zinc binding motif not only inhibits protein interaction but also eliminates DNA binding, demonstrating that DEAF-1 protein-protein interaction is required for DNA recognition. The identification of an NES and NLS provides a basis for the control of DEAF-1 subcellular localization and function, whereas the requirement of protein-protein interaction by the SAND domain appears to be unique among this class of transcription factors.

Effects of quitting smoking on EEG activation and attention last for more than 31 days and are more severe with stress, dependence, DRD2 A1 allele, and depressive traits.

Changes in physiology and attentional performance associated with smoking abstinence were characterized in 67 female smokers during low-stress and high-stress conditions. Abstinence was associated with decreases in cognitive performance, heart rate, and electroencephalographic (EEG) activation but with no change in serum estradiol or progesterone. Effects of quitting showed no tendency to resolve across the 31 days of abstinence. EEG deactivation and heart rate slowing were greater during a math task (high stress) than during relaxation (low stress). Individuals high in trait depression or nicotine dependence or with at least one dopamine D(2) receptor A1 allele experienced greater EEG deactivation following abstinence, especially in the right hemisphere during the stressful task. Thus, findings support the situation x trait adaptive response model of abstinence effects and emphasize the value of multiple dependent measures when characterizing abstinence responses.