Monogenic diabetes in children and young adults: Challenges for researcher, clinician and patient

Monogenic diabetes results from one or more mutations in a single gene which might hence be rare but has great impact leading to diabetes at a very young age. It has resulted in great challenges for researchers elucidating the aetiology of diabetes and related features in other organ systems, for clinicians specifying a diagnosis that leads to improved genetic counselling, predicting of clinical course and changes in treatment, and for patients to altered treatment that has lead to coming off insulin and injections with no alternative (Glucokinase mutations), insulin injections being replaced by tablets (e.g. low dose in HNFα or high dose in potassium channel defects -Kir6.2 and SUR1) or with tablets in addition to insulin (e.g. metformin in insulin resistant syndromes). Genetic testing requires guidance to test for what gene especially given limited resources. Monogenic diabetes should be considered in any diabetic patient who has features inconsistent with their current diagnosis (unspecified neonatal diabetes, type 1 or type 2 diabetes) and clinical features of a specific subtype of monogenic diabetes (neonatal diabetes, familial diabetes, mild hyperglycaemia, syndromes). Guidance is given by clinical and physiological features in patient and family and the likelihood of the proposed mutation altering clinical care. In this article, I aimed to provide insight in the genes and mutations involved in insulin synthesis, secretion, and resistance, and to provide guidance for genetic testing by showing the clinical and physiological features and tests for each specified diagnosis as well as the opportunities for treatment

A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs

The quiescent cell population of tumours poses a barrier to the success of many cancer therapies. Most chemotherapeutic drugs target proliferating cells, but the growth fraction of many tumours is low. Based on the multicellular tumour spheroid model, a system was developed using human colon adenocarcinoma (DLD-1) cells to mimic the microenvironment of quiescent microregions of solid tumours. The quiescent tumour spheroids (TSQ) showed decreased expression of the proliferation marker Ki-67 and increased expression of the quiescence marker p27kip1 compared to proliferating spheroids (TSP). The quiescent status of the TSQ was confirmed by long-term growth assessment. The quiescence was completely reversible demonstrating that the TSQ retained the ability to proliferate and morphological assessment by light microscopy confirmed the absence of significant apoptosis. When the efficacy of widely used chemotherapeutic drugs was determined, vinblastine, doxorubicin, cisplatin and 5-fluorouracil (5-FU) all produced significant cell death in the TSP. However, while still effective, the potencies of doxorubicin and cisplatin were significantly reduced in TSQ. In contrast, 5-FU and vinblastine did not produce cell death in the TSQ. In summary, TSQ show considerable resistance to a panel of established chemotherapeutic agents and represent a useful model for evaluating the efficacy of drugs and other cancer therapies in quiescent tumours

Cks1 Is Required for Tumor Cell Proliferation but Not Sufficient to Induce Hematopoietic Malignancies

The Cks1 component of the SCFSkp2 complex is necessary for p27Kip1 ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27Kip1. To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo

Mechanisms of relapse in acute leukaemia: involvement of p53 mutated subclones in disease progression in acute lymphoblastic leukaemia

Mutations of the p53 tumour suppressor gene are infrequent at presentation of both acute myeloblastic leukaemia (AML) and acute lymphoblastic leukaemia (ALL), being found in between 5–10% of AML and 2–3% of ALL. Here we have studied the frequency of detection of p53 mutations at relapse of both AML and B-precursor ALL. In those patients with detectable mutations at relapse we investigated whether the mutation was detectable at presentation and was thus an early initiating event or whether it had arisen as a late event associated with relapse. Bone marrow samples from 55 adults and children with relapsed AML (n = 41) or ALL (n = 14) were analysed for p53 gene alterations by direct sequencing of exons 5–9. For samples where a p53 mutation was found at relapse, analysis of presentation samples was carried out by direct sequencing of the exon involved, or by allele-specific polymerase chain reaction (PCR) if the mutation could not be detected using direct sequencing. A p53 mutated gene was found at relapse in seven out of 55 cases. The frequency was higher in relapsed ALL (four out of 14 cases; 28.6%) compared to AML (three out of 41 cases; 7.3%). In five out of the seven cases presentation samples were available to study for the presence of the mutation. In two out of two AML patients the p53 mutation was detectable in the presentation sample by direct sequencing. In three ALL patients analysis of presentation material by direct sequencing showed a small mutant peak in one case, the other two being negative despite the sample analysed containing > 90% blast cells. However in both of these patients, the presence of p53 mutation was confirmed in the presentation sample using allele-specific PCR. In one of these patients the emergence of a subclone at relapse was confirmed by clonality analysis using IgH fingerprinting. Our results confirm that in ALL p53 mutations are present in a proportion of patients at relapse. Furthermore cells carrying the mutation are detectable at presentation in a minor clone suggesting that p53 mutations in ALL may be a mechanism contributing to disease relapse. © 1999 Cancer Research Campaig

Facial-muscle weakness, speech disorders and dysphagia are common in patients with classic infantile Pompe disease treated with enzyme therapy

Classic infantile Pompe disease is an inherited generalized glycogen storage disorder caused by deficiency of lysosomal acid α-glucosidase. If left untreated, patients die before one year of age. Although enzyme-replacement therapy (ERT) has significantly prolonged lifespan, it has also revealed new aspects of the disease. For up to 11 years, we investigated the frequency and consequences of facial-muscle weakness, speech disorders and dysphagia in long-term survivors. Sequential photographs were used to determine the timing and severity of facial-muscle weakness. Using standardized articulation tests and fibreoptic endoscopic evaluation of swallowing, we investigated speech and swallowing function in a subset of patients. This study included 11 patients with classic infantile Pompe disease. Median age at the start of ERT was 2.4 months (range 0.1-8.3 months), and median age at the end of the study was 4.3 years (range 7.7 months −12.2 years). All patients developed facial-muscle weakness before the age of 15 months. Speech was studied in four patients. Articulation was disordered, with hypernasal resonance and reduced speech intelligibility in all four. Swallowing function was studied in six patients, the most important findings being ineffective swallowing with residues of food (5/6), penetration or aspiration (3/6), and reduced pharyngeal and/or laryngeal sensibility (2/6). We conclude that facial-muscle weakness, speech disorders and dysphagia are common in long-term survivors receiving ERT for classic infantile Pompe disease. To improve speech and reduce the risk for aspiration, early treatment by a speech therapist and regular swallowing assessments are recommended

Chemical genetics approach to restoring p27Kip1 reveals novel compounds with antiproliferative activity in prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>The cyclin-dependent kinase (CDK) inhibitor p27^Kip1is downregulated in a majority of human cancers due to ectopic proteolysis by the ubiquitin-proteasome pathway. The expression of p27 is subject to multiple mechanisms of control involving several transcription factors, kinase pathways and at least three different ubiquitin ligases (SCF^SKP2, KPC, Pirh2), which regulate p27 transcription, translation, protein stability and subcellular localization. Using a chemical genetics approach, we have asked whether this control network can be modulated by small molecules such that p27 protein expression is restored in cancer cells.</p> <p>Results</p> <p>We developed a cell-based assay for measuring the levels of endogenous nuclear p27 in a high throughput screening format employing LNCaP prostate cancer cells engineered to overexpress SKP2. The assay platform was optimized to Z' factors of 0.48 - 0.6 and piloted by screening a total of 7368 chemical compounds. During the course of this work, we discovered two small molecules of previously unknown biological activity, SMIP001 and SMIP004, which increase the nuclear level of p27 at low micromolar concentrations. SMIPs (small molecule inhibitors of p27 depletion) also upregulate p21^Cip1, inhibit cellular CDK2 activity, induce G1 delay, inhibit colony formation in soft agar and exhibit preferential cytotoxicity in LNCaP cells relative to normal human fibroblasts. Unlike SMIP001, SMIP004 was found to downregulate SKP2 and to stabilize p27, although neither SMIP is a proteasome inhibitor. Whereas the screening endpoint - nuclear p27 - was robustly modulated by the compounds, SMIP-mediated cell cycle arrest and apoptosis were not strictly dependent on p27 and p21 - a finding that is explained by parallel inhibitory effects of SMIPs on positive cell cycle regulators, including cyclins E and A, and CDK4.</p> <p>Conclusions</p> <p>Our data provide proof-of-principle that the screening platform we developed, using endogenous nuclear p27 as an endpoint, presents an effective means of identifying bioactive molecules with cancer selective antiproliferative activity. This approach, when applied to larger and more diverse sets of compounds with refined drug-like properties, bears the potential of revealing both unknown cellular pathways globally impinging on p27 and novel leads for chemotherapeutics targeting a prominent molecular defect of human cancers.</p

The theory of expanded, extended, and enhanced opportunities for youth physical activity promotion

Background Physical activity interventions targeting children and adolescents (≤18 years) often focus on complex intra- and inter-personal behavioral constructs, social-ecological frameworks, or some combination of both. Recently published meta-analytical reviews and large-scale randomized controlled trials have demonstrated that these intervention approaches have largely produced minimal or no improvements in young people\u27s physical activity levels. Discussion In this paper, we propose that the main reason for previous studies\u27 limited effects is that fundamental mechanisms that lead to change in youth physical activity have often been overlooked or misunderstood. Evidence from observational and experimental studies is presented to support the development of a new theory positing that the primary mechanisms of change in many youth physical activity interventions are approaches that fall into one of the following three categories: (a) the expansion of opportunities for youth to be active by the inclusion of a new occasion to be active, (b) the extension of an existing physical activity opportunity by increasing the amount of time allocated for that opportunity, and/or (c) the enhancement of existing physical activity opportunities through strategies designed to increase physical activity above routine practice. Their application and considerations for intervention design and interpretation are presented. Summary The utility of these mechanisms, referred to as the Theory of Expanded, Extended, and Enhanced Opportunities (TEO), is demonstrated in their parsimony, logical appeal, support with empirical evidence, and the direct and immediate application to numerous settings and contexts. The TEO offers a new way to understand youth physical activity behaviors and provides a common taxonomy by which interventionists can identify appropriate targets for interventions across different settings and contexts. We believe the formalization of the TEO concepts will propel them to the forefront in the design of future intervention studies and through their use, lead to a greater impact on youth activity behaviors than what has been demonstrated in previous studies

Screening for neonatal diabetes at day 5 of life using dried blood spot glucose measurement.

The majority of infants with neonatal diabetes mellitus present with severe ketoacidosis at a median of 6 weeks. The treatment is very challenging and can result in severe neurological sequelae or death. The genetic defects that cause neonatal diabetes are present from birth. We aimed to assess if neonatal diabetes could be diagnosed earlier by measuring glucose in a dried blood spot collected on day 5 of life.This article is freely available via Open Access. Click on the Additional Link above to access the full-text via the publisher's site.Published