AZD4320, A Dual Inhibitor of Bcl-2 and Bcl-xL, Induces Tumor Regression in Hematologic Cancer Models without Dose-limiting Thrombocytopenia.

PURPOSE: Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2-selective inhibitor, has had success in the clinic, another family member, Bcl-xL, has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-xL inhibitor that broadens the therapeutic activity while minimizing Bcl-xL-mediated thrombocytopenia. EXPERIMENTAL DESIGN: We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-xL and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time. RESULTS: We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-xL, and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2- and Bcl-xL-dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2-selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-xL-dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models. CONCLUSIONS: AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-xL inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.

Discovery and pharmacological characterization of AZD3229, a potent KIT/PDGFRα inhibitor for treatment of gastrointestinal stromal tumors.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma driven by mutations in KIT or platelet-derived growth factor α (PDGFRα). Although first-line treatment, imatinib, has revolutionized GIST treatment, drug resistance due to acquisition of secondary KIT/PDGFRα mutations develops in a majority of patients. Second- and third-line treatments, sunitinib and regorafenib, lack activity against a plethora of mutations in KIT/PDGFRα in GIST, with median time to disease progression of 4 to 6 months and inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) causing high-grade hypertension. Patients with GIST have an unmet need for a well-tolerated drug that robustly inhibits a range of KIT/PDGFRα mutations. Here, we report the discovery and pharmacological characterization of AZD3229, a potent and selective small-molecule inhibitor of KIT and PDGFRα designed to inhibit a broad range of primary and imatinib-resistant secondary mutations seen in GIST. In engineered and GIST-derived cell lines, AZD3229 is 15 to 60 times more potent than imatinib in inhibiting KIT primary mutations and has low nanomolar activity against a wide spectrum of secondary mutations. AZD3229 causes durable inhibition of KIT signaling in patient-derived xenograft (PDX) models of GIST, leading to tumor regressions at doses that showed no changes in arterial blood pressure (BP) in rat telemetry studies. AZD3229 has a superior potency and selectivity profile to standard of care (SoC) agents-imatinib, sunitinib, and regorafenib, as well as investigational agents, avapritinib (BLU-285) and ripretinib (DCC-2618). AZD3229 has the potential to be a best-in-class inhibitor for clinically relevant KIT/PDGFRα mutations in GIST.

Differential Activity of ATR and WEE1 Inhibitors in a Highly Sensitive Subpopulation of DLBCL Linked to Replication Stress.

DNA damage checkpoint kinases ATR and WEE1 are among key regulators of DNA damage response pathways protecting cells from replication stress, a hallmark of cancer that has potential to be exploited for therapeutic use. ATR and WEE1 inhibitors are in early clinical trials and success will require greater understanding of both their mechanism of action and biomarkers for patient selection. Here, we report selective antitumor activity of ATR and WEE1 inhibitors in a subset of non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) cell lines, characterized by high MYC protein expression and CDKN2A/B deletion. Activity correlated with the induction of replication stress, indicated by increased origin firing and retardation of replication fork progression. However, ATR and WEE1 inhibitors caused different amounts of DNA damage and cell death in distinct phases of the cell cycle, underlying the increased potency observed with WEE1 inhibition. ATR inhibition caused DNA damage to manifest as 53BP1 nuclear bodies in daughter G1 cells leading to G1 arrest, whereas WEE1 inhibition caused DNA damage and arrest in S phase, leading to earlier onset apoptosis. In vivo xenograft DLBCL models confirmed differences in single-agent antitumor activity, but also showed potential for effective ATR inhibitor combinations. Importantly, insights into the different inhibitor mechanisms may guide differentiated clinical development strategies aimed at exploiting specific vulnerabilities of tumor cells while maximizing therapeutic index. Our data therefore highlight clinical development opportunities for both ATR and WEE1 inhibitors in non-GCB DLBCL subtypes that represent an area of unmet clinical need. SIGNIFICANCE: ATR and WEE1 inhibitors demonstrate effective antitumor activity in preclinical models of DLBCL associated with replication stress, but new mechanistic insights and biomarkers of response support a differentiated clinical development strategy.

Prevalence of advance care directives in the community: a telephone survey of three Australian States.

BACKGROUND: The community prevalence of advance care directives (ACD) is low despite known benefits of advance care planning for patients, families and health professionals. AIM: To determine the community prevalence of instructional and appointing ACD in New South Wales, Victoria and Queensland and factors associated with completion of these documents. METHODS: A telephone survey of adults living in New South Wales, Victoria and Queensland (n = 1175) about completion of instructional ACD (making their own decisions about future healthcare) and appointing ACD (appointing another to decide). Quota sampling occurred based on population size by state, gender and age, with oversampling in smaller jurisdictions (Victoria and Queensland). RESULTS: Overall response rate was 33%. Six per cent of respondents reported completing an instructional ACD while 12% reported completing an appointing ACD. Female gender, higher educational level, personal experience of a major health scare and being widowed were significant predictors of completing an instructional ACD. Older age, higher educational level and being widowed were significant predictors of completing an appointing ACD. CONCLUSIONS: Despite long-standing efforts to increase advance care planning, community prevalence of ACD remains low, particularly for instructional ACD. This study found some different predictors for instructional ACD compared with appointing ACD, and also a potential role for experiential factors in triggering uptake. These findings suggest supplementing general community awareness campaigns with more nuanced and targeted efforts to improve ACD completion.

Community Knowledge of Law on End-of-life Decision-making: An Australian Telephone Survey.

The law has a clear role to play in supporting patients and their substitute decision-makers (SDMs) to be involved in end-of-life (EOL) decision-making. Although existing literature suggests that knowledge of EOL law is variable among health professionals, there is little information about the extent and sources of such knowledge within the general community. A telephone survey of a representative sample of adults in three Australian States used six case scenarios to examine the extent to which adults know their legal duties, rights and powers as patients or SDMs; the sources from which people derive relevant legal knowledge; experiences of EOL decision-making; and individual characteristics associated with levels of knowledge. The results show considerable variation in levels of legal knowledge dependent primarily of the area of decision-making presented, some sizeable gaps in people's knowledge of EOL law, and varied awareness of how to access appropriate information on this subject. This study points to the need to increase community legal literacy around EOL decision-making, enhance awareness of the role of law in these circumstances and promote the availability of reliable and accessible information on the law at the time when it is needed.

Pharmacological Inhibition of PARP6 Triggers Multipolar Spindle Formation and Elicits Therapeutic Effects in Breast Cancer.

: PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity. SIGNIFICANCE: These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.

Does implementation matter if comprehension is lacking? A qualitative investigation into perceptions of advance care planning in people with cancer.

PURPOSE: While advance care planning holds promise, uptake is variable and it is unclear how well people engage with or comprehend advance care planning. The objective of this study was to explore how people with cancer comprehended advance care plans and examine how accurately advance care planning documentation represented patient wishes. METHODS: This study used a qualitative descriptive design. Data collection comprised interviews and an examination of participants' existing advance care planning documentation. Participants included those who had any diagnosis of cancer with an advance care plan recorded: Refusal of Treatment Certificate, Statement of Choices, and/or Enduring Power of Attorney (Medical Treatment) at one cancer treatment centre. RESULTS: Fourteen participants were involved in the study. Twelve participants were female (86%). The mean age was 77 (range: 61-91), and participants had completed their advance care planning documentation between 8 and 72 weeks prior to the interview (mean 33 weeks). Three themes were evident from the data: incomplete advance care planning understanding and confidence, limited congruence for attitude and documentation, advance care planning can enable peace of mind. Complete advance care planning understanding was unusual; most participants demonstrated partial comprehension of their own advance care plan, and some indicated very limited understanding. Participants' attitudes and their written document congruence were limited, but advance care planning was seen as helpful. CONCLUSIONS: This study highlighted advance care planning was not a completely accurate representation of patient wishes. There is opportunity to improve how patients comprehend their own advance care planning documentation.

Using Webinars for the Education of Health Professionals and People Affected by Cancer: Processes and Evaluation.

Technology provides an opportunity to engage with a variety of audiences to provide cancer education, information and support. Webinars are one such format that allow live presentations by experts that can be accessed online, from people's homes or other convenient locations. In 2015, Cancer Council Victoria (CCV) undertook a program of work to design and evaluate the effectiveness of a suite of webinars: four designed for people affected by cancer and two for health professionals. Webinars included a series of expert presentations, a panel discussion and an interactive component where participants posed questions to the panel. Evaluation included analysis of online metrics and a post-event survey covering experience and satisfaction with the webinar, self-reported changes in knowledge of key webinar concepts and confidence to discuss concepts with health professionals or patients. A total of 438 people participated in the webinars (41.5% of 1056 registrations), and 207 post-event surveys were completed by participants (47.3%). Overall, 90.1% indicated that webinar content was relevant to their interests and needs. Self-ratings of knowledge, awareness of resources and confidence to discuss webinar topics increased after the webinar. The majority (63.9%) had not participated in a webinar before, and 92.6% were interested in participating in future webinars. Over half of respondents (52.8%) had not accessed CCV resources before. This work provided a new opportunity to consolidate consistency of delivery and evaluation of webinars, demonstrating they are an effective, acceptable, accessible and sustainable vehicle for delivering information and support to health professionals and people affected by cancer.

Community knowledge of law at the end of life: availability and accessibility of web-based resources.

Objective The aim of the present study was to identify online resources community members may access to inform themselves about their legal duties and rights in end-of-life decision making. Methods Resource mapping identified online resources that members of the public in New South Wales, Victoria and Queensland are likely to identify, and assessed the ease or difficulty in locating them. Resources were then critically analysed for accessibility of language and format using the Patient Education Materials Assessment Tool (PEMAT). Results Identified resources differed considerably based on whether search terms identified by community members or experts were used. Most resources focused on advance directives, enduring powers of attorney and substitute decision making. Relatively few provided information about legal duties (e.g. powers and responsibilities of substitute decision makers) or resolving conflict with health practitioners. Accessibility (understandability and actionability) of resource content varied. Conclusions Although numerous resources on end-of-life law are available online, community members may not be able to identify relevant resources or find resource content accessible. What is known about the topic? Research on participation by patients in decision making about their treatment has focused primarily on medical rather than legal knowledge. What does this paper add? The present study investigated which online resources community members may access to inform themselves about the law on end-of-life decision making. The resources identified were analysed for ease of location and content accessibility. What are the implications for practitioners? Authors of online resources on end-of-life decision making should consider whether their resources can be: (1) identified by search terms used by the public; (2) understood by a general audience; and (3) readily used to promote reader action.

Generation of stable PDX derived cell lines using conditional reprogramming.

Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.

AZD5153: A Novel Bivalent BET Bromodomain Inhibitor Highly Active against Hematologic Malignancies.

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563-74. ©2016 AACR.

AZD9150, a next-generation antisense oligonucleotide inhibitor of STAT3 with early evidence of clinical activity in lymphoma and lung cancer.

Next-generation sequencing technologies have greatly expanded our understanding of cancer genetics. Antisense technology is an attractive platform with the potential to translate these advances into improved cancer therapeutics, because antisense oligonucleotide (ASO) inhibitors can be designed on the basis of gene sequence information alone. Recent human clinical data have demonstrated the potent activity of systemically administered ASOs targeted to genes expressed in the liver. We describe the preclinical activity and initial clinical evaluation of a class of ASOs containing constrained ethyl modifications for targeting the gene encoding the transcription factor STAT3, a notoriously difficult protein to inhibit therapeutically. Systemic delivery of the unformulated ASO, AZD9150, decreased STAT3 expression in a broad range of preclinical cancer models and showed antitumor activity in lymphoma and lung cancer models. AZD9150 preclinical activity translated into single-agent antitumor activity in patients with highly treatment-refractory lymphoma and non-small cell lung cancer in a phase 1 dose-escalation study.

Inhibition of platelet-derived growth factor receptor α by MEDI-575 reduces tumor growth and stromal fibroblast content in a model of non-small cell lung cancer.

Platelet-derived growth factor receptor α (PDGFRα) is a receptor tyrosine kinase that promotes cell survival and is expressed in both the tumor and the stromal components of human cancers. We have developed a fully human monoclonal antibody, MEDI-575, that selectively binds to human PDGFRα with high affinity, with no observable affinity for murine PDGFRα. To more fully characterize the role of PDGFRα in the regulation of tumor stroma, we evaluated the in vivo antitumor effects of MEDI-575 in tumor-bearing severe combined immunodeficient (SCID) mice and in genetically altered SCID mice expressing human PDGFRα in place of murine PDGFRα. We used the Calu-6 non-small cell lung cancer model because it lacks an in vitro proliferative response to PDGFRα activation. Antitumor activity was observed when the study was performed in mice expressing the human receptor, but no activity was observed in the mice expressing the murine receptor. Immunohistologic analysis of the tumors from mice expressing human PDGFRα showed a highly significant reduction in stromal fibroblast content and only minor changes in tumor proliferative index in tumors exposed to MEDI-575 compared with the results seen in vehicle-treated tumors or in tumors from mice expressing murine PDGFRα. Additional in vitro studies indicated that exposure of primary cancer-associated fibroblasts to MEDI-575 can directly affect proliferation and key signaling pathways in these cells. These results highlight the potential for observing antitumor activity with MEDI-575 through modulation of the stromal component of tumors and confirm that the PDGFRα pathway can play a role in maintaining a tumor microenvironment conducive to tumor growth.

Comparisons of the efficacy of a Jak1/2 inhibitor (AZD1480) with a VEGF signaling inhibitor (cediranib) and sham treatments in mouse tumors using DCE-MRI, DW-MRI, and histology.

Jak1/2 inhibition suppresses STAT3 phosphorylation that is characteristic of many cancers. Activated STAT3 promotes the transcription of factors that enhance tumor growth, survival, and angiogenesis. AZD1480 is a novel small molecule inhibitor of Jak1/2, which is a key mediator of STAT3 activation. This study examined the use of diffusion-weighted (DW) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) biomarkers in assessing early tumor response to AZD1480. Cediranib (AZD2171), a vascular endothelial growth factor signaling inhibitor, was used as a comparator. Thirty mice were injected with Calu-6 lung cancer cells and randomized into the three treatment groups: AZD1480, cediranib, and sham. DW-MRI and DCE-MRI protocols were performed at baseline and at days 3 and 5 after treatment. The percent change from baseline measurements for K(trans), ADC, and v(e) were calculated and compared with hematoxylin and eosin (H&E), CD31, cParp, and Ki-67 histology data. Decreases in K(trans) of 29% (P < .05) and 53% (P < .05) were observed at days 3 and 5, respectively, for the cediranib group. No significant changes in K(trans) occurred for the AZD1480 group, but a significant increase in ADC was demonstrated at days 3 (63%, P < .05) and 5 (49%, P < .05). CD31 staining indicated diminished vasculature in the cediranib group, whereas significantly increased cParp staining for apoptotic activity and extracellular space by image analysis of H&E were present in the AZD1480 group. These imaging biomarker changes, and corresponding histopathology, support the use of ADC, but not K(trans), as a pharmacodynamic biomarker of response to AZD1480 at these time points.

Discovery of (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent.

Structure-activity relationship analysis identified (+)-N-(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), from a series of novel kinesin spindle protein (KSP) inhibitors, as exhibiting both excellent biochemical potency and pharmaceutical properties suitable for clinical development. The selected compound arrested cells in mitosis leading to the formation of the monopolar spindle phenotype characteristic of KSP inhibition and induction of cellular death. A favorable pharmacokinetic profile and notable in vivo efficacy supported the selection of this compound as a clinical candidate for the treatment of cancer.

The Child Attention-Deficit Hyperactivity Disorder Teacher Telephone Interview (CHATTI): reliability and validity.

BACKGROUND: The ICD-10 and DSM-IV diagnostic criteria for hyperkinetic disorder and attention-deficit hyperactivity disorder (ADHD) require symptoms or impairment in two or more settings. Thus, information on children's symptoms in school is usually required. This paper presents the Child ADHD Teacher Telephone Interview (CHATTI), an instrument aimed at systematically obtaining this information. AIMS: To examine the stability, test-retest reliability and criterion validity of the CHATTI for children referred with a suspected diagnosis of ADHD. METHOD: Data were obtained from 79 teachers, of whom 36 were interviewed on two occasions. RESULTS: Overall, the CHATTI shows good stability, test-retest reliability and criterion validity for symptom scores. Test-retest reliability for some individual items was low. Reliability for the operationalised criteria of 'pervasiveness' (i.e. symptoms at school and home) and 'school impairment' was excellent (kappa=1). CONCLUSIONS: The CHATTI appears to be a promising tool for assessing ADHD symptoms in a school setting and could be useful in clinical as well as research settings.

No evidence of association between HLA-DRB1 and attention deficit hyperactivity disorder.

OBJECTIVES: There has been a recent resurgence in interest in the role of autoimmunity in childhood neuropsychiatric disorders. Significant association between HLA-DRB1 and attention deficit hyperactivity disorder (ADHD) in a case-control study of 31 subjects has been reported but there have been no other published studies following up these results. We attempted to replicate these findings. METHODS: In a well-characterized sample of 173 children with ADHD, using a fully automated sequence-specific oligonucleotide method for HLA genotyping, association between ADHD and HLA-DRB1 was tested for using the Transmission Disequilibrium Test and case-control analysis. RESULTS: Transmission Disequilibrium Test analysis yielded a chi-square of 10.694 with a simulated global P value of 0.1641 for the full sample, and a chi-square value of 11.307 with a simulated global P value of 0.1323 for the complete trios only. CONCLUSION: There was no evidence of association of HLA-DRB1 and ADHD.

Sodium channel beta4, a new disulfide-linked auxiliary subunit with similarity to beta2.

The principal alpha subunit of voltage-gated sodium channels is associated with auxiliary beta subunits that modify channel function and mediate protein-protein interactions. We have identified a new beta subunit termed beta4. Like the beta1-beta3 subunits, beta4 contains a cleaved signal sequence, an extracellular Ig-like fold, a transmembrane segment, and a short intracellular C-terminal tail. Using TaqMan reverse transcription-PCR analysis, in situ hybridization, and immunocytochemistry, we show that beta4 is widely distributed in neurons in the brain, spinal cord, and some sensory neurons.beta4 is most similar to the beta2 subunit (35% identity), and, like the beta2 subunit, the Ig-like fold of beta4 contains an unpaired cysteine that may interact with the alpha subunit. Under nonreducing conditions, beta4 has a molecular mass exceeding 250 kDa because of its covalent linkage to Nav1.2a, whereas on reduction, it migrates with a molecular mass of 38 kDa, similar to the mature glycosylated forms of the other beta subunits. Coexpression of beta4 with brain Nav1.2a and skeletal muscle Nav1.4 alpha subunits in tsA-201 cells resulted in a negative shift in the voltage dependence of channel activation, which overrode the opposite effects of beta1 and beta3 subunits when they were present. This novel, disulfide-linked beta subunit is likely to affect both protein-protein interactions and physiological function of multiple sodium channel alpha subunits.

No evidence of association of two 5HT transporter gene polymorphisms and attention deficit hyperactivity disorder.

OBJECTIVES: Three studies to date have found evidence (or a trend for evidence) of linkage and association between the long allele of the 44 base pair repeat insertion/deletion 5-HTT functional polymorphism (5-HTTLPR) and attention deficit hyperactivity disorder (ADHD). In an attempt to replicate these findings, we examined this polymorphism and a variable number tandem repeat in the second intron of 5-HTT for association with ADHD. METHODS: One hundred and fifty children who met diagnostic criteria for ADHD and their parents (where available) were genotyped for these polymorphisms. Analysis was undertaken using the transmission disequilibrium test and haplotype analysis, as well as case-control comparisons using a control group of 121 individuals. RESULTS: No association between either the 5-HTTLPR or the variable number tandem repeat (VNTR) in ADHD was found (extended transmission disequilibrium test; P=0.37 and P=0.62, respectively). Haplotype analysis was also non-significant. Further analysis revealed no evidence of association in the subgroups of those without conduct disorder and in medication non-responders. CONCLUSIONS: Failure to replicate findings from previous studies may be due to a lack of statistical power. However, given recent findings by Kent et al. (2002) of association with another polymorphism in the 5HTT gene, we hypothesise that previous positive findings may have arisen by the LPR and VNTR being in linkage disequilibrium with the true susceptibility polymorphism.

Association analysis of monoamine oxidase A and attention deficit hyperactivity disorder.

Attention deficit hyperactivity disorder (ADHD) is a highly heritable disorder. Although the causes of ADHD are unknown, dopaminergic, serotonergic and nor-adrenergic pathways have been strongly implicated. Monoamine Oxidase A (MAOA) is involved in the degradation of all three of these neurotransmitters and therefore has been suggested as a strong candidate gene for ADHD. Animal and human studies have implicated MAOA and 5-HT in impulsive and aggressive behavior. We therefore additionally postulated that MAOA might be associated with a subtype of ADHD where aggressive and impulsive features are especially prominent. We have tested this hypothesis by genotyping two polymorphisms (the 30-bp VNTR in the promoter and the Fnu4HI 941T-->G) in MAOA that are associated with altered MAOA function. Our sample consisted of 171 British Caucasian children 6-16 years of age fulfilling DSM-III R, DSM-IV or ICD-10 criteria for ADHD/Hyperkinetic Disorder. Using case control analysis and then the TDT, no association was found between these two MAOA polymorphisms and ADHD. Case control analysis of the VNTR showed an association with a subgroup of children with comorbid conduct problems (OR = 2.0, 95% CI = 1.09, 3.5), and TDT analysis indicated a statistical trend toward association. Our findings highlight the importance of phenotype definition and the need for the MAOA VNTR to be further examined.