Daria V. Babushok

Current topics in genome evolution: Molecular mechanisms of new gene formation

Abstract.Comparative genome analyses reveal that most functional domains of human genes have homologs in widely divergent species. These shared functional domains, however, are differentially shuffled among evolutionary lineages to produce an increasing number of domain architectures. Combined with duplication and adaptive evolution, domain shuffling is responsible for the great phenotypic complexity of higher eukaryotes. Although the domain-shuffling hypothesis is generally accepted, determining the molecular mechanisms that lead to domain shuffling and novel gene creation has been challenging, as sequence features accompanying the formation of known genes have been obscured by accumulated mutations. The growing availability of genome sequences and EST databases allows us to study the characteristics of newly emerged genes. Here we review recent genome-wide DNA and EST analyses, and discuss the three major molecular mechanisms of gene formation: (1) atypical spicing, both within and between genes, followed by adaptation, (2) tandem and interspersed segmental duplications, and (3) retrotransposition events.

Genetic predisposition to myelodysplastic syndrome and acute myeloid leukemia in children and young adults

Abstract Myelodysplastic syndrome (MDS) is a clonal blood disorder characterized by ineffective hematopoiesis, cytopenias, dysplasia and an increased risk of acute myeloid leukemia (AML). With the growing availability of clinical genetic testing, there is an increasing appreciation that a number of genetic predisposition syndromes may underlie apparent de novo presentations of MDS/AML, particularly in children and young adults. Recent findings of clonal hematopoiesis in acquired aplastic anemia add another facet to our understanding of the mechanisms of MDS/AML predisposition. As more predisposition syndromes are recognized, it is becoming increasingly important for hematologists and oncologists to have familiarity with the common as well as emerging syndromes, and to have a systematic approach to diagnosis and screening of at risk patient populations. Here, we provide a practical algorithm for approaching a patient with a suspected MDS/AML predisposition, and provide an in-depth review of the established and emerging familial MDS/AML syndromes caused by mutations in the ANKRD26, CEBPA, DDX41, ETV6, GATA2, RUNX1, SRP72 genes. Finally, we discuss recent data on the role of somatic mutations in malignant transformation in acquired aplastic anemia, and review the practical aspects of MDS/AML management in patients and families with predisposition syndromes.

Recent advances in understanding clonal haematopoiesis in aplastic anaemia

Acquired aplastic anaemia (AA) is an immune‐mediated bone marrow failure disorder inextricably linked to clonal haematopoiesis. The majority of AA patients have somatic mutations and/or structural chromosomal abnormalities detected as early as at diagnosis. In contrast to other conditions linked to clonal haematopoiesis, the clonal signature of AA reflects its immune pathophysiology. The most common alterations are clonal expansions of cells lacking glycophosphotidylinositol‐anchored proteins, loss of human leucocyte antigen alleles, and mutations in BCOR/BCORL1, ASXL1 and DNMT3A. Here, we present the current knowledge of clonal haematopoiesis in AA as it relates to aging, inherited bone marrow failure, and the grey‐zone overlap of AA and myelodysplastic syndrome (MDS). We conclude by discussing the significance of clonal haematopoiesis both for improved diagnosis of AA, as well as for a more precise, personalized approach to prognostication of outcomes and therapy choices.

Clonal evolution and clinical significance of copy number neutral loss of heterozygosity of chromosome arm 6p in acquired aplastic anemia

Acquired aplastic anemia (aAA) results from the T cell-mediated autoimmune destruction of hematopoietic stem cells. Factors predicting response to immune suppression therapy (IST) or development of myelodysplastic syndrome (MDS) are beginning to be elucidated. Our recent data suggest most patients with aAA treated with IST develop clonal somatic genetic alterations in hematopoietic cells. One frequent acquired abnormality is copy-number neutral loss of heterozygosity on chromosome 6p (6p CN-LOH) involving the human leukocyte antigen (HLA) locus. We hypothesized that because 6p CN-LOH clones may arise from selective pressure to escape immune surveillance through deletion of HLA alleles, the development of 6p CN-LOH may affect response to IST. We used single nucleotide polymorphism array genotyping and targeted next-generation sequencing of HLA alleles to assess frequency of 6p CN-LOH, identity of HLA alleles lost through 6p CN-LOH, and impact of 6p CN-LOH on response to IST. 6p CN-LOH clones were present in 11.3% of patients, remained stable over time, and were not associated with development of MDS-defining cytogenetic abnormalities. Notably, no patient with 6p CN-LOH treated with IST achieved a complete response. In summary, clonal 6p CN-LOH in aAA defines a unique subgroup of patients that may provide insights into hematopoietic clonal evolution.

Clonal Hematopoiesis in Patients with Dyskeratosis Congenita

Dyskeratosis congenita (DC) is a rare inherited telomeropathy most frequently caused by mutations in a number of genes all thought to be involved in telomere maintenance. The main causes of mortality in DC are bone marrow failure as well as malignancies including leukemias and solid tumors. The clinical picture including the degree of bone marrow failure is highly variable and factors that contribute to this variability are poorly understood. Based on the recent finding of frequent clonal hematopoiesis in related bone marrow failure syndromes, we hypothesized that somatic mutations may also occur in DC and may contribute at least in part to the variability in blood production. To evaluate for the presence of clonal hematopoiesis in DC, we used a combination of X‐inactivation, comparative whole exome sequencing (WES) and single nucleotide polymorphism array (SNP‐A) analyses. We found that clonal hematopoiesis in DC is common, as suggested by skewed X‐inactivation in 8 out of 9 female patients compared to 3 out of 10 controls, and by the finding of acquired copy neutral loss‐of‐heterozygosity on SNP‐A analysis. In addition, 3 out of 6 independent DC patients were found to have acquired somatic changes in their bone marrow by WES, including a somatic reversion in DKC1, as well as missense mutations in other protein coding genes. Our results indicate that clonal hematopoiesis is a common feature of DC, and suggest that such somatic changes, though commonly expected to indicate malignancy, may lead to improved blood cell production or stem cell survival. Am. J. Hematol. 91:1227–1233, 2016.

Allogeneic transplantation for myelofibrosis: for whom, when, and what are the true benefits?

Purpose of reviewAllogeneic hematopoietic stem cell transplantation (HSCT) is the only potentially curative therapy for myelofibrosis. Despite improved outcomes, morbidity and mortality of HSCT remain high. Here we examine recent data on patient selection, timing, and outcomes of HSCT in myelofibrosis. Recent findingsWhile there is a general effort to restrict HSCT to transplant-eligible intermediate-2 and high-risk patients, this group has comparatively worse HSCT outcomes, largely driven by their high transplant-related mortality (TRM). When adjusted for age, reduced intensity conditioning (RIC) has shown superior outcomes compared with myeloablative conditioning (MAC), making RIC-HSCT a viable option for older patients. Emerging concepts include the use of ruxolitinib pretransplant, optimizing MAC to decrease toxicity, and use of posttransplant JAK2-mutant allele burden to guide prophylactic immunotherapy to prevent relapse. The recognition of prognostic significance of somatic mutations in the ASXL1, EZH2, SRSF2, and IDH1/2 genes, and the improved assessment of risk of leukemic transformation have added a new dimension to risk stratification. SummaryImproving our understanding of molecular genetics and leukemic transformation holds promise for more precise patient selection for HSCT. Although RIC-HSCT may reduce TRM, further studies are needed to optimize conditioning regimens and to define the optimal timing of HSCT.

Clonal Replacement Underlies Spontaneous Remission in Paroxysmal Nocturnal Haemoglobinuria

Casella, J.F., King, A.A., Barton, B., White, D.A., Noetzel, M.J., Ichord, R.N., Terrill, C., Hirtz, D., McKinstry, R.C., Strouse, J.J., Howard, T.H., Coates, T.D., Minniti, C.P., Campbell, A.D., Vendt, B.A., Lehmann, H. & Debaun, M.R. (2010) Design of the silent cerebral infarct transfusion (SIT) trial. Pediatric Hematology Oncology, 27, 69–89. Colombatti, R., De Bon, E., Bertomoro, A., Casonato, A., Pontara, E., Omenetto, E., Saggiorato, G., Steffan, A., Damian, T., Cella, G., Teso, S., Manara, R., Rampazzo, P., Meneghetti, G., Basso, G., Sartori, M.T. & Sainati, L. (2013) Coagulation activation in children with sickle cell disease is associated with cerebral small vessel vasculopathy. PLoS ONE, 8, e78801. DeBaun, M.R., Gordon, M., McKinstry, R.C., Noetzel, M.J., White, D.A., Sarnaik, S.A., Meier, E.R., Howard, T.H., Majumdar, S., Inusa, B.P., Telfer, P.T., Kirby-Allen, M., McCavit, T.L., Kamdem, A., Airewele, G., Woods, G.M., Berman, B., Panepinto, J.A., Fuh, B.R., Kwiatkowski, J.L., King, A.A., Fixler, J.M., Rhodes, M.M., Thompson, A.A., Heiny, M.E., ReddingLallinger, R.C., Kirkham, F.J., Dixon, N., Gonzalez, C.E., Kalinyak, K.A., Quinn, C.T., Strouse, J.J., Miller, J.P., Lehmann, H., Kraut, M.A., Ball, W.S. Jr, Hirtz, D. & Casella, J.F. (2014) Controlled trial of transfusions for silent cerebral infarcts in sickle cell anemia. New England Journal of Medicine, 371, 699–710. van der Land, V., Hijmans, C.T., de Ruiter, M., Mutsaerts, H.J., Cnossen, M.H., Engelen, M., Majoie, C.B., Nederveen, A.J., Grootenhuis, M.A. & Fijnvandraat, K. (2015) Volume of white matter hyperintensities is an independent predictor of intelligence quotient and processing speed in children with sickle cell disease. British Journal of Haematology, 168, 553–556. van der Land, V., Mutsaerts, H.J., Engelen, M., Heijboer, H., Roest, M., Hollestelle, M.J., Kuijpers, T.W., Nederkoorn, P.J., Cnossen, M.H., Majoie, C.B., Nederveen, A.J. & Fijnvandraat, K. (2016) Risk factor analysis of cerebral white matter hyperintensities in children with sickle cell disease. British Journal of Haematology., 172, 274–284. Montanaro, M., Colombatti, R., Pugliese, M., Migliozzi, C., Zani, F., Guerzoni, M.E., Manoli, S., Manara, R., Meneghetti, G., Rampazzo, P., Cavalleri, F., Giordan, M., Paolucci, P., Basso, G., Palazzi, G. & Sainati, L. (2013) Intellectual function evaluation of first generation immigrant children with sickle cell disease: the role of language and sociodemographic factors. Italian Journal of Pediatrics, 39, 36.

Poor outcome with hematopoietic stem cell transplantation for bone marrow failure and MDS with severe MIRAGE syndrome phenotype

Key Points Success of hematopoietic stem cell transplantation for MIRAGE syndrome may be limited by syndrome-specific comorbidities. SAMD9 mutations associated with MIRAGE syndrome are a newly described cause of congenital amegakaryocytic thrombocytopenia.

Multicenter analysis of the use of transjugular intrahepatic portosystemic shunt for management of MPN‐associated portal hypertension

BCR‐ABL1‐negative myeloproliferative neoplasms (MPNs) are clonal stem cell disorders defined by proliferation of one or more myeloid lineages, and carry an increased risk of vascular events and progression to myelofibrosis and leukemia. Portal hypertension (pHTN) occurs in 7‐18% of MPN patients via both thrombotic and nonthrombotic mechanisms and portends a poor prognosis. Transjugular intrahepatic portosystemic shunt (TIPS) has been used in the management of MPN‐associated pHTN; however, data on long‐term outcomes of TIPS in this setting is limited and the optimal management of medically refractory MPN‐associated pHTN is not known. In order to assess the efficacy and long‐term outcomes of TIPS in MPN‐associated pHTN, we performed a retrospective analysis of 29 MPN patients who underwent TIPS at three academic medical centers between 1997 and 2016. The majority of patients experienced complete clinical resolution of pHTN and its clinical sequelae following TIPS. One, two, three, and four‐year overall survival post‐TIPS was 96.4%, 92.3%, 84.6%, and 71.4%, respectively. However, despite therapeutic anticoagulation, in‐stent thrombosis occurred in 31.0% of patients after TIPS, necessitating additional interventions. In conclusion, TIPS can be an effective intervention for MPN‐associated pHTN regardless of etiology. However, TIPS thrombosis is a frequent complication in the MPN population and indefinite anticoagulation post‐TIPS should be considered.

Diagnosis and Treatment of Aplastic Anemia

Opinion statementAcquired aplastic anemia (AA) is a rare, life-threatening bone marrow failure (BMF) disorder that affects patients of all ages and is caused by lymphocyte destruction of early hematopoietic cells. Diagnosis of AA requires a comprehensive approach with prompt evaluation for inherited and secondary causes of bone marrow aplasia, while providing aggressive supportive care. The choice of frontline therapy is determined by a number of factors including AA severity, age of the patient, donor availability, and access to optimal therapies. For newly diagnosed severe aplastic anemia, bone marrow transplant should be pursued in all pediatric patients and in younger adult patients when a matched sibling donor is available. Frontline therapy in older adult patients and in all patients lacking a matched sibling donor involves immunosuppressive therapy (IST) with horse antithymocyte globulin and cyclosporine A. Recent improvements in upfront therapy include encouraging results with closely matched unrelated donor transplants in younger patients and the emerging benefits of eltrombopag combined with initial IST, with randomized studies underway. In the refractory setting, several therapeutic options exist, with improving outcomes of matched unrelated donor and haploidentical bone marrow transplantation as well as the addition of eltrombopag to the non-transplant AA armamentarium. With the recent appreciation of frequent clonal hematopoiesis in AA patients and with the growing use of next-generation sequencing in the clinic, utmost caution should be exercised in interpreting the significance of somatic mutations in AA. Future longitudinal studies of large numbers of patients are needed to determine the prognostic significance of somatic mutations and to guide optimal surveillance and treatment approaches to prevent long-term clonal complications.