Complex gene regulation is one of the key requirements for the evolution of higher eukaryotes. 1 In these organisms, many genes are regulated by enhancers that are 10 4 -10 6 base pairs (bp) distant from the promoter. Enhancer sequences usually contain multiple small transcription factor binding sites (typically ~10bp), and physical contact between the promoter and enhancer is thought to be required to modulate gene expression. 2 Current methods have extensively defined chromatin architecture at scales above 1 kb but until now it has not been possible to define physical contacts at the scale of the key proteins determining gene expression. Here we define the interactions between different classes of regulatory elements (enhancers, promoters and boundary elements) in unprecedented detail, using a novel chromosome conformation capture method (Micro Capture-C (MCC)), which allows physical contacts to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show, using base pair resolution plots of ligation junctions, that transcription factors generate a key component of the contacts between enhancers and promoters. Our data show that contacts from CTCF sites highly correlate with cooccupancy of cohesin and that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. We also find that promoters make the strongest contacts with both enhancers and CTCF sites and that while CTCF sites contact promoters strongly they only make weak contacts with enhancers. The highly punctate nature of the contacts is an unexpected finding because the current view is that physical contacts are constrained by much larger domains such as topological associated domains (TADs). 3 Our results support a model in which chromatin loop extrusion 4-6 is dependent on cohesin loading at active promoters and enhancers, explaining the formation of tissue-specific chromatin domains without changes in CTCF binding. The data suggest that a separate mechanism to loop extrusion underlies enhancer-/promoter contacts, which likely involves DNA binding proteins at enhancers and promoters. The unprecedented

β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with β-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for β-thalassemia.

Diffuse large B-cell lymphoma with secondary involvement of the central nervous system (SCNS-DLBCL) is a rare condition carrying a poor prognosis. No optimal therapeutic regimen has been identified. We retrospectively analysed 23 patients with SCNS-DLBCL treated with R-IDARAM (rituximab 375 mg/m(2) IV day 1; methotrexate 12·5 mg by intrathecal injection day 1; idarubicin 10 mg/m(2) /day IV days 1 and 2; dexamethasone 100 mg/day IV infusion over 12 h days 1-3; cytosine arabinoside 1000 mg/m(2) /day IV over 1 h days 1 and 2; and methotrexate 2000 mg/m(2) IV over 2 h day 3. Ten out of 23 (44%) patients had CNS involvement at initial presentation ('new disease'), 10/23 (44%) had relapsed disease and 3/23 (13%) had primary refractory disease. 14/23 (61%) of patients responded - 6 (26%) complete response, 8 (35%) partial response. Grade 3-4 haematological toxicity was seen in all cycles, with no grade 3-4 or long-term neurological toxicity. Median follow-up for surviving patients was 49 months. At 2 years, estimated progression-free survival (PFS) was 39% and overall survival (OS) was 52%. Encouraging outcomes were reported in patients with new disease, with 5-year estimated PFS of 50% and OS 75%. R-IDARAM is a well-tolerated regimen with encouraging efficacy in patients with SCNS-DLBCL, although patients with relapsed or refractory disease continue to fare poorly.

(2013) High-dose cyclophosphamide compared with antithymocyte globulin for treatment of acquired severe aplastic anemia. Experimental Hematology, 41, 328-334.Combination-therapy with concurrent deferoxamine and deferiprone is effective in treating resistant cardiac iron-loading in aceruloplasminaemia Aceruloplasminaemia is a rare disorder of iron metabolism with a prevalence of around 1 in 2 million people (Miyajima, 2015). Ceruloplasmin, a plasma ferroxidase, is involved in the oxidation of ferrous (Fe 2+ ) iron so that it can be released from intracellular stores to be transported by transferrin. Absence or dysfunction of ceruloplasmin leads to iron accumulation, mainly in the liver, pancreas and central nervous system (CNS). We report the combined use of two iron-chelating drugs, deferiprone (DFP) and deferoxamine for the management of iron overload with resistant cardiac ironloading in a patient with aceruloplasminaemia. Although potentially beneficial, treatment with DFP has not hitherto been described in managing this condition.A 28-year-old man with a provisional diagnosis of Ehlers Danlos syndrome Type III with Raynaud syndrome was referred after persistently high alanine aminotransferase levels (109 u/l) were noted. Neurological examination was normal. Blood tests showed a high serum ferritin (1766 lg/l), markedly low serum copper [0Á09 lmol/l, normal range (NR) 11-20] and ceruloplasmin levels <0Á07 g/l (NR 0Á2-0Á6), leading to a diagnosis of aceruloplasminaemia. A T2 magnetic resonance imaging (MRI) brain scan demonstrated hypointensities within the dentate nuclei, red nuclei, substantia nigra and globus pallidi, reflecting increased iron deposition. T2* MRI showed moderate iron loading in the liver (1Á86 ms) and heart (11Á9 ms). A liver biopsy revealed severe iron loading (19 mg/g dry weight). There was no clinical evidence of endocrine dysfunction. Thyroid function tests (thyroid-stimulating hormone 0Á35 mu/l, free thyroxine 14Á4 pmol/l) and a fasting blood glucose (5Á4 mmol/l) test were normal.The oral iron-chelating agent DFP was commenced at 1Á5 g/d and was increased over 1Á5 months to a target dose of 5Á5 g/d given in 3 divided doses (75 mg/kg/d). As there was CNS involvement DFP was used due to its ability to cross the blood-brain barrier and intracellular membranes (Pandolfo & Hausmann, 2013). After 6 months on this dose the serum ferritin had decreased to 1309 lg/l. The patient had weekly blood checks for the first month, then monthly thereafter, to monitor for agranulocytosis. DFP was well tolerated during this phase, with no gastrointestinal disturbance, or arthropathy. Blood tests showed stable haematological parameters and improving transaminase levels. A T2* MRI performed 18 months after the commencement of treatment showed improving mild liver iron-loading (3Á35 ms), but unimproved moderate cardiac ironloading (13Á0 ms) with normal cardiac function. This was surprising given the efficacy of DFP in alleviating cardiac iron-loading in patients with thalassemia (Pennell et al, 2006). In...

Haemoglobin E (HbE) β-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE β-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe β-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (β-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.

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