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SummaryAccurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next‐generation‐sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical‐grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically‐reliable diagnostic test and minimize false‐negative results we developed an open‐source tool (CoverMi) to accurately determine base‐coverage and the ‘discoverability’ of known mutations for every sample. We validated our 33‐gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS‐based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.

Acute myeloid leukemia (AML) is characterized by the accumulation of immature blood cell precursors in the bone marrow. Pharmacologically overcoming the differentiation block in this condition is an attractive therapeutic avenue, which has only achieved success in a subtype of AML, acute promyelocytic leukemia (APL). Attempts to emulate this success in other AML subtypes have thus far been unsuccessful. Autophagy is a conserved protein degradation pathway with important roles in mammalian cell differentiation, particularly within the hematopoietic system. In this study we demonstrate the functional importance of autophagy in APL cell differentiation. We show that autophagy is increased during ATRA-induced granulocytic differentiation of the APL cell line NB4, and that this is associated with increased expression of LC3-II and GATE-16 proteins involved in autophagosome formation. Autophagy inhibition, using either drugs (chloroquine/3-methyladenine) or short-hairpin (sh)RNA targeting the essential autophagy gene ATG7, attenuates myeloid differentiation. Importantly, we show that enhancing autophagy promotes ATRA-induced granulocytic differentiation of an ATRA-resistant derivative of the non-APL AML HL60 cell line (HL60-Diff-R). These data support the development of strategies to stimulate autophagy as a novel approach to promote differentiation in AML.

The loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene tribbles (Trib)2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by CCAAT enhancer-binding protein α (C/EBPα)-p42, occurs in acute myeloid leukemia (AML), resulting in the perturbation of cell cycle and apoptosis, emphasizing its importance in the molecular pathogenesis of AML. Here we show that E2F family members directly regulate Trib2 in leukemic cells and identify a feedback regulatory loop for E2F1, C/EBPα, and Trib2 in AML cell proliferation and survival. Further analyses revealed that E2F1-mediated Trib2 expression was repressed by C/EBPα-p42, and in normal granulocyte/macrophage progenitor cells, we detect C/EBPα bound to the Trib2 promoter. Pharmacological inhibition of the cell cycle or Trib2 knockdown resulted in a block in AML cell proliferation. Our work proposes a novel paradigm whereby E2F1 plays a key role in the regulation of Trib2 expression important for AML cell proliferation control. Importantly, we identify the contribution of dysregulated C/EBPα and E2F1 to elevated Trib2 expression and leukemic cell survival, which likely contributes to the initiation and maintenance of AML and may have significant implications for normal and malignant hematopoiesis.

BackgroundLaboratory testing is an integral part of day-to-day primary care practice, with approximately 30 % of patient encounters resulting in a request. However, research suggests that a large proportion of requests does not benefit patient care and is avoidable. The aim of this systematic review was to comprehensively search the literature for studies evaluating the effectiveness of interventions to improve primary care physician use of laboratory tests.MethodsA search of PubMed, Cochrane Library, Embase and Scopus (from inception to 09/02/14) was conducted. The following study designs were considered: systematic reviews, randomised controlled trials (RCTs), controlled clinical trials (CCTs), controlled before and after studies (CBAs) and interrupted time series analysis (ITSs). Studies were quality appraised using a modified version of the Effective Practice and Organisation of Care (EPOC) checklist. The population of interest was primary care physicians. Interventions were considered if they aimed to improve laboratory testing in primary care. The outcome of interest was a volume of laboratory tests.ResultsIn total, 6,166 titles and abstracts were reviewed, followed by 87 full texts. Of these, 11 papers were eligible for inclusion in the systematic review. This included four RCTs, six CBAs and one ITS study. The types of interventions examined included education, feedback, guidelines, education with feedback, feedback with guidelines and changing order forms. The quality of included studies varied with seven studies deemed to have a low risk of bias, three with unclear risk of bias and one with high risk of bias. All but one study found significant reductions in the volume of tests following the intervention, with effect sizes ranging from 1.2 to 60 %. Due to heterogeneity, meta-analysis was not performed.ConclusionsInterventions such as educational strategies, feedback and changing test order forms may improve the efficient use of laboratory tests in primary care; however, the level of evidence is quite low and the quality is poor. The reproducibility of findings from different laboratories is also difficult to ascertain from the literature. Some standardisation of both interventions and outcome measures is required to enable formal meta-analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13012-015-0356-4) contains supplementary material, which is available to authorized users.