Peter Carey

IRF8 Mutations and Human Dendritic-Cell Immunodeficiency

BACKGROUND The genetic analysis of human primary immunodeficiencies has defined the contribution of specific cell populations and molecular pathways in the host defense against infection. Disseminated infection caused by bacille Calmette-Guérin (BCG) vaccines is an early manifestation of primary immunodeficiencies, such as severe combined immunodeficiency. In many affected persons, the cause of disseminated BCG disease is unexplained. METHODS We evaluated an infant presenting with features of severe immunodeficiency, including early-onset disseminated BCG disease, who required hematopoietic stem-cell transplantation. We also studied two otherwise healthy subjects with a history of disseminated but curable BCG disease in childhood. We characterized the monocyte and dendritic-cell compartments in these three subjects and sequenced candidate genes in which mutations could plausibly confer susceptibility to BCG disease. RESULTS We detected two distinct disease-causing mutations affecting interferon regulatory factor 8 (IRF8). Both K108E and T80A mutations impair IRF8 transcriptional activity by disrupting the interaction between IRF8 and DNA. The K108E variant was associated with an autosomal recessive severe immunodeficiency with a complete lack of circulating monocytes and dendritic cells. The T80A variant was associated with an autosomal dominant, milder immunodeficiency and a selective depletion of CD11c+CD1c+ circulating dendritic cells. CONCLUSIONS These findings define a class of human primary immunodeficiencies that affect the differentiation of mononuclear phagocytes. They also show that human IRF8 is critical for the development of monocytes and dendritic cells and for antimycobacterial immunity. (Funded by the Medical Research Council and others.).

A comparative study of combination chemotherapy versus marrow transplant in first remission in adult acute lymphoblastic leukaemia

Summary. The results of conventional chemotherapy in adult acute lymphoblastic leukaemia (ALL) have not improved substantially in recent years. The present study is based on a flexible policy of marrow transplantation (allograft and autograft without marrow purging) in first remission compared with a group treated with standard maintenance therapy after a common induction sequence. The actuarial disease free survival (DFS) and actuarial overall survival (OS) at 3 years for autologous marrow grafted patients was 30% and 65% respectively. The allogeneic transplant group had DFS of 30% and OS at 3 years of 38% compared with DFS (12%) and OS (12%) for patients on 6‐mercaptopurine and methotrexate maintenance. The actuarial disease free survival calculations include patients on protocol not entering remission, therefore, giving the worst possible result.

A novel 33-Gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias.

Accurate 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.

Reversal of Type 2 diabetes after bariatric surgery is determined by the degree of achieved weight loss in both short‐ and long‐duration diabetes

To define the impact of duration of diabetes and extent of weight loss on the reversibility of Type 2 diabetes after bariatric surgery.

The use of anthracyclines in the treatment of endemic Burkitt lymphoma

Burkitt lymphoma is the most common malignancy in children in Malawi, the worlds poorest country, where there is a long history of treating this disease using a 28‐day cyclophosphamide‐based protocol. Stage III/IV disease has had poor outcomes. In an attempt to improve the outcome for higher stage disease, anthracyclines were added to the existing protocol. The disease‐free (DFS) and overall survival (OS) of 58 children with cytologically confirmed Burkitt lymphoma admitted during 2012–2014 and treated using this protocol were calculated. Six (10%) children had stage I disease, ten (17%) stage II and 42 stage III or IV (73%). Overall 12‐month DFS (OS) was 68·5% (72·9%); for stage I disease 100% (100%), stage II 56·2% (60%), stage III/IV 66·3% (72·2%). The DFS was significantly improved from the previous protocol (P = 8 × 10−4). The addition of doxorubicin to stage III and IV disease resulted in a markedly improved DFS. Anthracyclines are deliverable in resource‐poor settings and possibly improve the survival of children with Burkitt lymphoma.

Haemophagocytic lymphohistiocytosis following fludarabine/cyclophosphamide chemotherapy for chronic lymphocytic leukaemia

Antonella Anastasia* Carmelo Carlo-Stella* Paolo Corradini Flavia Salvi Chiara Rusconi Alessandro Pulsoni Stefan Hohaus Patrizia Pregno Simonetta Viviani Ercole Brusamolino Stefano Luminari Laura Giordano Armando Santoro Department of Oncology and Haematology, Humanitas Cancer Centre, Humanitas Clinical and Research Centre, Rozzano (MI), Department of Medical Biotechnology and Translational Medicine, University of Milano, Department of Haematology and Bone Marrow Transplantation, Fondazione IRCCS Istituto Nazionale Tumori, Chair of Haematology, University of Milano, Milano, Division of Haematology, AO SS Antonio e Biagio e C. Arrigo, Alessandria, Division of Haematology, Niguarda Ca’ Granda Hospital, Milano, Department of Cellular Biotechnologies and Haematology, “Sapienza” University, Institute of Haematology, Universit a Cattolica S. Cuore, Roma, Haematology, A.O. Citt a della Salute e della Scienza, Torino, Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Division of Haematology, Policlinico S Matteo, Pavia, and Department of Diagnostic Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy. E-mail: carmelo.carlostella@cancercenter.humanitas.it

Pyridoxine-sensitive X-linked ‘sideroblastic’ anaemia in the absence of ring sideroblasts – molecular diagnosis

A 2-year-old boy presented with a persistent productive cough and a 6-month history of worsening pallor and lethargy. He had been born by emergency Caesarean section at 41 weeks gestation for fetal distress but had subsequently been healthy. Examination showed no hepatosplenomegaly. He was small for age with height and weight on the 2nd and 0 4th centiles respectively. His full blood count showed a haemoglobin concentration (Hb) of 27 g/l, MCV 48 fl and MCH 13 5 pg, with reticulocytes 0 40 9 10/l. White cell count was 6 62 9 10/l, neutrophil count 2 23 9 10/l, platelet count 311 9 10/l and ferritin 262 lg/l. A peripheral blood film demonstrated dimorphic red cells with one population of normocytic, normochromic cells and a second population with marked poikilocytosis, target cells, irregular microcytes and fragmented red cells (left). Despite the dimorphic red cell morphology the clinical presentation was felt to be suggestive of hereditary pyropoikilocytosis. An eosin-50maleimide (EMA) binding test, however, was normal and a haemolysis screen was negative (bilirubin 6 lmol/l, lactate dehydrogenase 214 iu/l). A bone marrow aspirate showed normocellular erythropoiesis. A Perls’ stain (right) showed storage iron to be present; there were very occasional ring sideroblasts, constituting less than 1% of all sideroblasts. Both parents had normal Hb, red cell indices and EMA binding test. His mother’s blood film showed occasional elliptocytes but no other abnormalities. Further enquiry into the family history revealed a maternal male cousin with congenital sideroblastic anaemia. Subsequently targeted genetic analysis identified an unusual combination of abnormalities. The patient was hemizygous for a C to A nucleotide substitution in exon 7 of ALAS2 (c.791 C>A), which, although not previously reported, was predicted to result in the replacement of the amino acid serine with tyrosine at residue 264 (p.Ser264Tyr) and was therefore consistent with Xlinked sideroblastic anaemia, inherited from his mother. In addition, he was heterozygous for SPTA1 (c.2608G>A; p.Val870Met) and SPTB (c.2519G>A; p.Arg840His) variants. Although not previously described, this digenic effect was felt to be potentially consistent with hereditary pyropoikilocytosis. The mother was a carrier of the SPTA1 genetic variant and had some elliptocytes, suggesting a spectrinopathy. At this stage, the predominant abnormality responsible for his phenotype was unclear. Due to the family history of congenital sideroblastic anaemia and the identification of a potential pathogenic ALAS2 variant a trial of pyridoxine therapy was commenced. His Hb rose dramatically to 112 g/l within 3 months without further red cell transfusion. Despite the lack of overt sideroblastic change, we postulate a primary diagnosis of pyridoxine-sensitive X-linked sideroblastic anaemia. Additional mutations associated with red cell membrane abnormalities may have contributed to the morphological abnormalities although this remains speculative. However, the absence of overt haemolysis and the dimorphic blood film were inconsistent with a significant pathological red cell membrane abnormality, such as hereditary pyropoikilocytosis. This case highlights the importance of correlation between genetic and phenotypic features in the diagnosis of congenital anaemia. It suggests that the absence of significant ring sideroblasts does not preclude a diagnosis of congenital sideroblastic anaemia. A thorough family history and appropriate genetic analysis were particularly important in this patient’s management.

Assessing Patients with Asymptomatic Retinal Emboli Detected at Retinal Screening

IntroductionAsymptomatic retinal emboli have been associated with diabetes, the presence of significant carotid artery stenosis (≥70%) and an increased risk of stroke. However, there is no clear guidance on how best to investigate and manage patients found to have asymptomatic retinal emboli. Therefore, this study aimed to assess the incidence of significant carotid artery stenosis in patients found to have asymptomatic retinal emboli at diabetic retinopathy screening, and to examine disease management approaches among these patients.MethodsPatients with new retinal emboli visible at diabetic retinopathy screening were referred to a medical retinopathy clinic and underwent examinations according to a standardized protocol, including carotid Doppler ultrasound and echocardiography. Case notes of patients referred between January 2013 and April 2014 were reviewed. Results of investigations, medication changes and the number of patients who underwent relevant surgical interventions were noted.ResultsRetinal emboli were present in 44 of 13,643 people screened (0.32%). Full data were available for 39 patients. Twenty-two patients (56%) had relevant medication changes. Nine (23%) patients had significant carotid artery stenosis. One underwent carotid endarterectomy, and eight received maximal medical therapy.ConclusionSignificant carotid artery stenosis was not uncommon in patients with incident retinal emboli at retinal screening. The referral and investigation protocols identified individuals at risk of cerebrovascular events and led to optimized management. Pathways utilizing Doppler ultrasound and physician referral should be more widely implemented.