Haemophilia B, severe childhood obesity and other extra-haematological features associated with similar 4Mb-deletions on Xq27: Clinical findings, molecular insights and literature update.

INTRODUCTION: Haemophilia B (HB) is associated with pathogenic variants in F9. Hemizygous deletions encompassing the entire F9 and proximate genes may express extra-haematological clinical phenotypes. AIM: To analyse the genotype/phenotype correlations in two unrelated boys with severe early childhood obesity (SCO), global developmental delay (GDD) and similar bleeding phenotype associated with comparable Xq27 deletions spanning the entire F9 and proximate genes, and characterise the pathogenic events estimating the most likely mutational mechanism involved. METHODS: Entire F9-deletions were detected in three hemizygous unrelated probands with HB: two cases, C#1/C#2, presented SCO and GDD and a control patient (Co), who only had severe bleeding symptoms. Dense SNP-array and case-specific STS walking scan allowed characterisation of the deletion breakpoints. Extensive use of bioinformatics, statistics and clinical databases allowed the investigation of genotype-phenotype associations. RESULTS: Patients C#1/C#2 and Co resulted in a complete F9 and additional gene deletions of variable extensions on Xq26.3-Xq27.2 (C#1/C#2/Co: 4.3Mb/3.9Mb/160Kb). C#1/C#2 common deleted gene SOX3 is directly associated with SCO, GDD and pituitary hypothyroidism (PH) whilst C#2 extra-deleted gene MAGEC2 indirectly relates to anal atresia (AA). Breakpoint analysis revealed the involvement of the mechanisms of Alu/Alu recombination for the first time in HB and non-homologous or alternative end-joining. CONCLUSION: Our results represent the first report of unrelated patients with HB, SCO and GDD. This study and the literature update expand the spectrum of clinical findings and molecular insights observed in patients with HB caused by complete F9 and nearby SOX3 and MAGEC2 gene deletions, which may configure a contiguous gene syndrome.

Rabbit antithymocyte globulin dose does not affect response or survival as first-line therapy for acquired aplastic anemia: a multicenter retrospective study.

In a prospective randomized study, treatment for aplastic anemia (AA) with rabbit antithymocyte globulin (r-ATG) and cyclosporine showed inferior hematological response and survival in comparison to horse antithymocyte globulin (h-ATG) and cyclosporine. However, h-ATG was discontinued in most Asian, South American, and European countries, where r-ATG became the only ATG formulation available. We retrospectively evaluated consecutive patients with acquired AA who received either rabbit (n = 170) or horse (n = 85) ATG and cyclosporine for first-line treatment from 1992 to 2014 in seven referral centers in Brazil and Argentina. Overall response at 3 months was 17% (95%CI, 11-23%) for r-ATG and 44% (95%CI, 33-55%) for h-ATG (p < 0.001). At 6 months, it was 31% (95%CI, 34-39%) for r-ATG and 59% (95%CI, 48-69%) for h-ATG (p < 0.001). Overall survival at 5 years was 57% (95%CI, 47-65%) for r-ATG and 80% (95%CI, 69-87%) for h-ATG (log-rank = 0.001). Relapse was significantly higher in patients receiving h-ATG (28%; 95%CI, 17-43%) as compared to r-ATG (9.4%; 95%CI, 4-21%; log-rank, p = 0.01). The type of ATG was the only factor associated with both response and survival. The r-ATG dose varied from 1 to 5 mg/kg/day, but it did not correlate with outcomes. In summary, this is the largest multicenter study comparing the two ATG formulations in AA. Our results indicate that the dose of r-ATG does not influence hematologic response or survival in first-line therapy for acquired AA. Considering the toxicity and costs of r-ATG, our findings challenge its aggregate benefit to cyclosporine therapy and further strengthen that h-ATG should remain standard therapy in AA.

Predictors of early mortality after rabbit antithymocyte globulin as first-line treatment in severe aplastic anemia.

Despite being recommended as first-line immunosuppressive therapy in severe aplastic anemia (SAA), horse antithymocyte globulin (ATG) is still unavailable in many countries outside the USA. Rabbit ATG is more lymphocytoxic than horse ATG, and this might result in a higher incidence of severe infections and early mortality. This study was designed to identify the risk factors for early mortality and overall survival (OS) after rabbit ATG in patients with SAA. We retrospectively reviewed 185 patients with SAA who underwent rabbit ATG and cyclosporine. The incidence of death in 3 months following rabbit ATG therapy was 15.1% (28/185). Early mortality was mainly related to infectious complications, despite adequate antibiotic and/or antifungal treatment. Age > 35 years (odds ratio [OR] 5.06, P = 0.001) and baseline absolute neutrophil count (ANC) ≤ 0.1 × 109/L (OR 7.64, P < 0.001) were independent risk factors for early mortality after immunosuppressive therapy with this agent. Hematological response at 6 months was observed in only 29.7% of all patients. OS at 1 year after rabbit ATG was 75.3%; and age > 35 years (OR 1.88, P = 0.03), baseline ANC ≤ 0.1 × 109/L (OR 2.65, P < 0.001), and lack of response to rabbit ATG (OR 11.40, P < 0.001) were independently associated with mortality. Alternative strategies are needed for the treatment of SAA patients in countries were horse ATG is unavailable, particularly for those at high risk for early mortality after rabbit ATG due to a higher age and very low pre-treatment neutrophil count.

A new ß(0) frameshift mutation, HBB: c.44delT (p.Leu14ArgfsX5), identified in an Argentinean family associated with secondary genetic modifiers of ß-thalassemia.

ß-Thalassemia intermedia (ß-TI) patients present with a wide spectrum of phenotypes depending on the presence of primary, secondary, and tertiary genetic modifiers which modulate, by different mechanisms, the degree of imbalance between α and ß chains. Here we describe a new ß(0) frameshift mutation, HBB: c.44delT (p.Leu14ArgfsX5), identified in four members of a family, associated with secondary genetic modifiers in three of them. The different genotype present in this family was suspected after hematological analysis and thorough observation of blood smears highlighting their importance in the identification of ß-TI patients among members of the same family.

HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation.

The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1-associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1-/- cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.

Von Willebrand disease in children: diagnosis and management of a pediatric cohort in one single center in Argentina.

Clinical and laboratory data of children with von Willebrand disease (VWD) types have been derived from retrospective studies and small case series. This article reports on the clinical and laboratory data of a large pediatric cohort in one single Argentinian center. The biological and clinical responses to desmopressin and replacement therapies are also described. Over a 15-year period, 194 of 1150 children (16.9%) were diagnosed as having type 1 VWD (80%), type 2 VWD (19%), and type 3 VWD (1%). The distribution of the different type 2 VWD subtypes was type 2A VWD, 43%; type 2B VWD, 32%; type 2M VWD, 19%; and type 2N VWD, 6%. Eighty patients with type 1 VWD and 12 patients with type 2 VWD were prospectively evaluated to desmopressin (DDAVP) response. A complete response was observed in all children with type 1 VWD, whereas 40% of the children with severe type 1 VWD and with type 2 VWD achieved a complete response. All the children who received DDAVP as prophylaxis or treatment for bleeding had good clinical evolution. Considering the restricted availability of specialized hemostasis centers, we believe our clinical and laboratory approach appropriate for the detection of patients with different types of VWD. Further studies are necessary to determine epidemiological aspects of VWD in Argentina to estimate the necessary facilities and trained personnel for the diagnosis and management of patients with VWD.

Clinical experience with Berlin Heart Excor in pediatric patients in Argentina: 1373 days of cardiac support.

The objective of this study was to describe our experience (1373 days of support) with the Berlin Heart Excor (BH) ventricular-assist device (VAD) as bridging to cardiac transplantation in pediatric patients with end-stage cardiomyopathy. This study involved a retrospective observational cohort. Records of patients supported with the BH VAD were reviewed. Data regarding age, sex, weight, diagnosis, preoperative condition, single versus biventricular support, morbidity, and mortality were collected. Criteria for single versus biventricular support and intensive care unit management were registered. The procedure was approved by our Institutional Ethics Committee, and informed consent was obtained. Between March 2006 and March 2010, 12 patients with diagnosis of dilated (n = 10) and restrictive (n = 2) cardiomyopathy were supported. Median age was 56.6 months (range 20.1-165.9); mean weight was 18.3 kg (range 8.5-45); and nine patients were female. Every patient presented with severe heart failure refractory to pharmacological therapy. Biventricular support was necessary in four patients. Nine patients underwent heart transplantation. No child was weaned off the BH VAD because of myocardial recovery. Mean length of support was 73 days (range 3-331), and the total number of days of support was 1373. Three patients had fatal complications: 2 had thrombo-hemorrhagic stroke leading to brain death, and one had refractory vasoplegic shock. The BH VAD is a useful and reasonable safe device for cardiac transplantation bridging in children with end-stage heart failure. Team experience resulted in less morbidity and mortality, and time for implantation, surgical procedure, anticoagulation monitoring, and patient care improved.

Hb S-San Martin: a new sickling hemoglobin with two amino acid substitutions [ß6(A3)Glu→Val;ß105(G7)Leu→Pro] in an Argentinean family.

A new sickling hemoglobin (Hb) detected in an Argentinean family from San Martín, Buenos Aires, Argentina, is hereby described. Two mutations were identified on the same ß-globin gene resulting in a new variant named Hb San Martin. One mutation was found on exon 1, corresponding to Hb S [ß6Glu→Val, GAG>GTG] and the second one on exon 3 at ß105(G7)Leu→Pro, CTC>CCC. The replacement of leucine by proline will likely impair the structure breaking helix G and causing instability of the molecule and the clinical manifestations typical of unstable Hbs. The mutation at ß105 seemed to be a de novo one in our patients, arising on a previously mutated gene, due to the fact that Hb S is the most frequent structural variant.

The complex genetic landscape of familial MDS and AML reveals pathogenic germline variants.

The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.

Phenotypic and genotypic characterization of glucose-6-phosphate dehydrogenase deficiency in Argentina. Retrospective and descriptive study. / Caracterización fenotípica y genotipica de la deficiencia de glucosa-6-fosfato deshidrogenasa en Argentina. Estudio retrospectivo y descriptivo.

Glucose-6-phosphate dehydrogenase deficiency is an erythrocyte enzyme disorder caused by mutations in the G6PD gene, which has an X-linked inheritance. Here we analyze the clinical and laboratory characteristics of 24 subjects with G6PD deficiency over 25 years. Their median age at diagnosis was 10.2 years (range: 0.6-56.4). No symptoms were observed in 54.2 % of patients, whereas 25 % had chronic non-spherocytic hemolytic anemia; 12.5 %, neonatal jaundice and postinfectious hemolytic anemia; and 8.3 %, acute hemolytic anemia after ingestion of fava beans. The 24 studied patients had variants that had been previously described in the bibliography. The clinical characteristics observed here were consistent with the variants found. A total of 21 women from the maternal line of affected subjects were identified as deficiency carriers using molecular biology techniques, so they received the corresponding genetic counseling.

La deficiencia de glucosa-6-fosfato deshidrogenasa es la enzimopatía eritrocitaria causada por mutaciones en el gen G6PD, cuya herencia está ligada al cromosoma X. Se analizan las características clínicas y de laboratorio de 24 individuos con deficiencia de G6PD durante 25 años. La edad mediana al momento del diagnóstico fue 10,2 años (rango: 0,6-56,4). El 54,2 % de los pacientes fueron asintomáticos, mientras que el 25 % presentó anemia hemolítica crónica no esferocítica; el 12,5 %, ictericia neonatal y anemia hemolítica posinfecciones, y el 8,3 %, anemia hemolítica aguda pos ingesta de habas. Los 24 pacientes estudiados presentaron variantes descritas previamente en la literatura. Las características clínicas observadas estuvieron acordes con las variantes encontradas. Veintiuna mujeres, pertenecientes a la rama materna de los individuos afectados, pudieron ser identificadas por biología molecular como portadoras de la deficiencia, por lo que recibieron el consejo genético correspondiente.

Recurrent activating STAT5B N642H mutation in myeloid neoplasms with eosinophilia.

Determining the underlying cause of persistent eosinophilia is important for effective clinical management but remains a diagnostic challenge in many cases. We identified STAT5B N642H, an established oncogenic mutation, in 27/1715 (1.6%) cases referred for investigation of eosinophilia. Of the 27 mutated cases, a working diagnosis of hypereosinophilic syndrome (HES; n = 7) or a myeloid neoplasm with eosinophilia (n = 20) had been made prior to the detection of STAT5B N642H. Myeloid panel analysis identified a median of 2 additional mutated genes (range 0-4) with 4 cases having STAT5B N642H as a sole abnormality. STAT5B N642H was absent in cultured T cells of 4/4 positive cases. Individuals with SF3B1 mutations (9/27; 33%) or STAT5B N642H as a sole abnormality had a markedly better overall survival compared to cases with other additional mutations (median 65 months vs. 14 months; hazard ratio = 8.1; P < 0.001). The overall survival of STAT5B-mutated HES cases was only 30 months, suggesting that these cases should be reclassified as chronic eosinophilic leukemia, not otherwise specified (CEL-NOS). The finding of STAT5B N642H as a recurrent mutation in myeloid neoplasia with eosinophilia provides a new diagnostic and prognostic marker as well as a potential target for therapy.

[Erythrocyte indexes in hereditary spherocytosis]. / Indices eritrocitarios en la esferocitosis hereditaria.

Hereditary spherocytosis is a group of heterogenous disorders characterized by variability in its clinical manifestations, membrane protein defects and inheritance. We analysed the sensitivity and specificity of mean corpuscular hemoglobin concentration (MCHC) and red cell distribution width (RDW) in the diagnostic screening of hereditary spherocytosis. Ninety-four patients were compared to equal number of healthy, age-matched children. All indexes were derived from measurements obtained by aperture impedance (Coulter Counter Model JT). In patients with hereditary spherocytosis, MCHC (35.67+/-1.33 g/dl) and RDW (20.60+/-4.5%) were significantly higher than in normal control subjects (MCHC 33.48+/-0.68 g/dl, p: 0.000; RDW 13.22+/-0.9%, p: 0.000). By using a cutoff for the MCHC of 34.5 g/dl and for the RDW of 14.5%, both indexes showed a sensitivity of 81% and a specificity of 98.9%. The combination of the two test is an excellent predictor for the diagnosis of hereditary spherocytosis.

[Beta thalassemia intermedia: clinical characteristics and molecular analysis. Case series]. / Beta talasemia intermedia: características clínicas y estudio molecular. Serie de casos clínicos.

Beta thalassemia intermedia is a quantitative haemoglobinopathy covering a broad clinical spectrum, that results from the presence of one or two HBB gene mutations associated with secondary and/or tertiary genetic modifiers. We analyze the clinical and laboratory features of 29 patients with beta thalassemia intermedia, assessed over a period of 23 years. Median age was 10.8 years (range: 0.34-60.4). Hypochromic microcytic anemia was seen in 100% of the patients, while only 17.2% had splenomegaly and occasional transfusion requirement. The molecular analysis of patients detected: 3 with two HBB affected genes; 2 with one HBB affected gene and alpha quadruplicate/triplicate genes; 23 with one HBB affected gene and alpha triplicate genes and 1 with two HBB affected genes and polymorphisms of gamma genes. The adequate identification of these patients enables us to give appropriate genetic counseling and implementation of regular clinical follow up.

Factor V Leiden and prothrombin gene G20210A mutation in children with venous thromboembolism.

To determine whether factor V Leiden (FVL) and/or prothrombin gene G20210A mutation (PT20210A) are risk factors for venous thromboembolism (VTE) in Argentinean children. One hundred and thirty consecutive children with VTE were prospectively assisted at a single centre. Blood samples were available from 110 of them for detailed haematological analysis. The prevalence of both mutations was compared with a control group. The odds ratio for VTE was significantly increased in patients with FVL (OR 3.64; 95% CI: 1.14-11.6, p < 0.029) whereas odds ratio for VTE was not significantly increased in patients with PT20210A (OR 1.06; 95% CI: 0.24-4.73, p = 0.938). Combined disorders were found in 5 of the 10 children with the aforementioned mutations. In 21 children (19%) without these mutations other inherited and acquired disorders were detected. Our data show that FVL is a risk factor for VTE whereas PT20210A does not seem to be a risk factor in our paediatric population.

[Effect of hydroxyurea on hemoglobin S]. / Efecto de la hidroxiurea en hemoglobina S.

Previous studies have determined the laboratory alterations, clinical efficacy and toxicity profile associated with hydroxyurea (HU) therapy in patients with severe sickle cell anemia. We report the efficacy of HU treatment in the prevention of vaso-occlusive crises in an 11-years-old boy with severe sickle cell disease. The number of vaso-occlusive crises, hospital days and blood transfusions in the year before HU treatment were compared with the same parameters at 6, 12, 24, 36 and 72 months of treatment. A decrease in the frequency of vaso-occlusive crises, blood transfusions and days spent in hospital were demonstrated during the HU treatment period compared to the same period before hand. The clinical and laboratory response to HU was dramatic in this severely affected patient, allowing him a normal schooling and social life. The adverse effects observed were not serious and reversed after transient discontinuation of HU. We conclude that long-term chronic treatment with HU for seriously ill sickle cell patients appears feasible, and devoid of any major toxicity.

[Beta thalassemia major in Argentina]. / Beta talasemia mayor en la Argentina.

An analysis of beta thalassemia major patients seen at Hospital Juan P. Garrahan was carried out in order to determine the characteristics and outcome of the population. From August 1987 to July 2000, 45 patients were admitted (27 males-18 females). The most common beta globin gene defects were C-39 (30.7%); IVS-I nt 110 (20%); IVS-I nt 6 (13.3%); IVS-I nt 1(4%). alpha globin genes were normal in 42 patients, 1 patient had triplicate and cuadriplicate alpha globin genes and 2 patients were not analyzed. Six patients of 5 families were heterozygous for -158G gamma mutation. Allogeneic stem cell transplantation was performed in 7 patients, with an identical sibling. Transfusion-related infections and alloantibodies were detected in 6.7% patients. Growth assessment showed no significant difference in the stature of girls compared to the reference population, but 5 boys had short stature. There is a tendency to short trunk. Growth velocity was normal at prepubertal age. No X-ray lesions related to desferrioxamine were observed. Delayed puberty and hypogonadotropic hypogonadism were found in 35.7% and abnormalities in GH/IGF-I axis in 12.5% of the patients. Impaired glucose tolerance was found in 2 patients. No patient developed diabetes mellitus, thyroid or adrenal insufficiency. One patient had cardiac complications. Forty-two patients are alive and 3 died (cardiac failure 1, central nervous system bleeding 1, sepsis 1). We conclude that beta thalassemia major, originated mainly from Italian immigrants, has a cumbersome treatment and is severely hindered by the lack of adequate economic resources in our patients.

Caracterización fenotípica y genotipica de la deficiencia de glucosa-6-fosfato deshidrogenasa en Argentina: Estudio retrospectivo y descriptivo / Phenotypic and genotypic characterization of glucose-6-phosphate dehydrogenase deficiency in Argentina: Retrospective and descriptive study

La deficiencia de glucosa-6-fosfato deshidrogenasa es la enzimopatía eritrocitaria causada por mutaciones en el gen G6PD, cuya herencia está ligada al cromosoma X. Se analizan las características clínicas y de laboratorio de 24 individuos con deficiencia de G6PD durante 25 años. La edad mediana al momento del diagnóstico fue 10,2 años (rango: 0,6-56,4). El 54,2 % de los pacientes fueron asintomáticos, mientras que el 25 % presentó anemia hemolítica crónica no esferocítica; el 12,5 %, ictericia neonatal y anemia hemolítica posinfecciones, y el 8,3 %, anemia hemolítica aguda pos ingesta de habas. Los 24 pacientes estudiados presentaron variantes descritas previamente en la literatura. Las características clínicas observadas estuvieron acordes con las variantes encontradas. Veintiuna mujeres, pertenecientes a la rama materna de los individuos afectados, pudieron ser identificadas por biología molecular como portadoras de la deficiencia, por lo que recibieron el consejo genético correspondiente.

Glucose-6-phosphate dehydrogenase deficiency is an erythrocyte enzyme disorder caused by mutations in the G6PD gene, which has an X-linked inheritance. Here we analyze the clinical and laboratory characteristics of 24 subjects with G6PD deficiency over 25 years. Their median age at diagnosis was 10.2 years (range: 0.6-56.4). No symptoms were observed in 54.2 % of patients, whereas 25 % had chronic non-spherocytic hemolytic anemia; 12.5 %, neonatal jaundice and postinfectious hemolytic anemia; and 8.3 %, acute hemolytic anemia after ingestion of fava beans. The 24 studied patients had variants that had been previously described in the bibliography. The clinical characteristics observed here were consistent with the variants found. A total of 21 women from the maternal line of affected subjects were identified as deficiency carriers using molecular biology techniques, so they received the corresponding genetic counseling.

[Severe hemolytic anemia due to hemoglobin Southampton: case report]. / Anemia hemolítica grave causada por hemoglobina Southampton: presentación de caso clínico.

Variant hemoglobins are the result of different types of mutations that occur in the globin genes. In many cases, these hemoglobinopathies are harmless, while in others they determine alterations in the physical and chemical properties raising clinical manifestations of variable severity. In the unstable hemoglobinopathies, the changes reduce solubility, inducing the formation of precipitates of denaturated hemoglobin (Heinz bodies), which damage the membrane and finally destroy the red blood cells prematurely. Up to now, more than 142 different unstable hemoglobins have been described, most of them cause chronic hemolysis, increased by infections or drugs. We report the clinical presentation of an unstable hemoglobin (hemoglobin Southampton) in a girl with severe hemolytic anemia, splenomegaly and red blood cell requirement.