Elena Cela

Chromosome fragility in patients with Fanconi anaemia: diagnostic implications and clinical impact

Background Fanconi anaemia (FA) is a rare syndrome characterized by bone marrow failure, malformations and cancer predisposition. Chromosome fragility induced by DNA interstrand crosslink (ICL)-inducing agents such as diepoxybutane (DEB) or mitomycin C (MMC) is the ‘gold standard’ test for the diagnosis of FA. Objective To study the variability, the diagnostic implications and the clinical impact of chromosome fragility in FA. Methods Data are presented from 198 DEB-induced chromosome fragility tests in patients with and without FA where information on genetic subtype, cell sensitivity to MMC and clinical data were available. Results This large series allowed quantification of the variability and the level of overlap in ICL sensitivity among patients with FA and the normal population. A new chromosome fragility index is proposed that provides a cut-off diagnostic level to unambiguously distinguish patients with FA, including mosaics, from non-FA individuals. Spontaneous chromosome fragility and its correlation with DEB-induced fragility was also analysed, indicating that although both variables are correlated, 54% of patients with FA do not have spontaneous fragility. The data reveal a correlation between malformations and sensitivity to ICL-inducing agents. This correlation was also statistically significant when the analysis was restricted to patients from the FA-A complementation group. Finally, chromosome fragility does not correlate with the age of onset of haematological disease. Conclusions This study proposes a new chromosome fragility index and suggests that genome instability during embryo development may be related to malformations in FA, while DEB-induced chromosome breaks in T cells have no prognostic value for the haematological disease.

Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function

Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function.

Association in Cis of the Mutations +20 (C>T) in the 5′ Untranslated Region and IVS-II-745 (C>G) on the β-Globin Gene

In the 5′ untranslated region (5′UTR), which is transcribed but not translated and is involved in posttranscriptional regulation of the gene promoting and stabilizing the mRNA translation, several mutations associated with mild β-thalassemia (β-thal) have been described. One of these, the +20 (C>T) mutation, is described in the HbVar database as a mutation responsible for β+-thal. In heterozygote cases, it gives rise to a phenotype of β-thal minor and β-thal intermediate (β-TI) when the mutation is associated with β+ IVS-II-745 (C>G). To clarify if this mutation is responsible for β+-thal, we studied nine cases where we found an association of the +20 and IVS-II-745 mutations. All patients were carriers of four α genes. Three patients carried β-thal major (β-TM), two were compound heterozygotes for IVS-II-745 and codon 8 (–AA) or codon 39 (C>T), and the third was homozygous for IVS-II-745; all had the +20 mutation in the 5′UTR. The remaining patients showed the mutation IVS-II-745 associated with a replacement of C>T at nucleotide (nt) +20 of the 5′UTR. Contrary to reports in the HbVar database, the +20 mutation should be considered as an innocuous single nt polymorphism associated with the IVS-II-745 mutation in cis.

National registry of hemoglobinopathies in Spain (REPHem)

Although highly prevalent throughout the world, the accurate prevalence of hemoglobinopathies in Spain is unknown.

Diagnóstico prenatal de hemoglobinopatías y talasemias

BACKGROUND AND OBJECTIVE Thalassemias are the most common single-gene disorders in the world population and the most important health problem in several countries. The best program of prevention of new births is prenatal diagnosis. Here we gather the experience from 1996 of the Spanish Group of Erythropathology related to the prenatal diagnosis of hemoglobinopathies in Spain. SUBJECT AND METHOD 36 couples carriers of beta-thalassemia or Hb S were studied. Fetal material was obtained by amniocentesis and BCV. The genotype was determined by molecular biology technologies. RESULTS We observed 3 spontaneous abortions (8.3%), 6 interruptions of pregnancy (16.7%) and a case of maternal contamination (2.8%). Prenatal diagnosis could be completed in 97.2% of the cases (35). CONCLUSIONS In our experience, with regards to hemoglobinopathies, it is essential to provide a good genetic advice in order to identify the molecular alteration in the progenitors before the first pregnancy. This would allow a prenatal diagnosis during the first quarter and, in case of a positive result, to perform an early interruption of the pregnancy without risks.

Hemoglobina Fannin-Lubbock II [b111(G13)Val ->Leu y b119(GH2)Gly -> Asp]: descripción de 4 nuevos casos

Fundamento y objetivo El objetivo de este trabajo es la caracterizacion molecular de 4 nuevos casos de hemoglobina Fannin-Lubbock II, que presenta la sustitucion de 2 aminoacidos en la misma cadena beta de globina. Pacientes y metodo Se han estudiado 4 casos pertenecientes a 3 familias de raza blanca y origen espanol. El analisis molecular se realizo con la secuenciacion de los productos de amplificacion por reaccion en cadena de la polimerasa del gen beta. Resultados El estudio molecular demostro la mutacion GTC → CTC en el codon 111, que determina un cambio de valina por leucina, y la mutacion GGC → GAC en el codon 119, que determina un cambio de glicina por aspartico, ambas en estado heterocigoto. Conclusiones Hasta la actualidad se han descrito 17 hemoglobinopatias con 2 sustituciones de aminoacidos en la cadena beta de globina. La hemoglobina Fannin-Lubbock II habia sido descrita en 5 familias espanolas, por lo que la comunicacion de estos nuevos casos indica que podria tratarse de una hemoglobinopatia relativamente frecuente y circunscrita a nuestra poblacion.

Erythrocytosis in a Child due to Hb Andrew-Minneapolis [β144(HC1)Lys→Asn (AAG>AAT or AAC)] Associated with a Spanish (δβ)0-Thalassemia

We report a rare association of δβ-thalassemia (δβ-thal) and a hemoglobin (Hb) variant with high oxygen affinity in a Spanish newborn. The proband had no Hb A and showed microcytosis and hypochromia; the peripheral blood smear was compatible with a thalassemia trait. Molecular studies revealed that the proband had a Spanish (δβ)0-thal (inherited from his father) and also carried a de novo variant (Hb Andrew-Minneapolis) because from the point of hematology, his mother was quite normal. The hemoglobinopathies with high affinity for oxygen constitute an infrequent cause of secondary congenital erythrocytosis. The degree of erythrocytosis and the resulting clinical manifestations are highly variable, depending on the degree of altered oxygen affinity and the presence of thalassemic genes. Thus, when these variants are associated with β0- or δβ-thal, as in our case, the proportion of abnormal Hb is ∼100.0%, which may cause polycythemia, hyperviscosity, and iron deficiency. This type of association is very rare and few have been described, especially in children, as they would normally be detected in adults as the increased packed cell volume (PCV) also increases blood viscosity and causes the typical symptoms (cephalalgia, drowsiness, dizziness). The association of a high oxygen affinity Hb and a δβ-thal presents a greater degree of erythrocytosis than when this same variant is associated with a β0-thal, mainly because the Hb F percentage is usually greater in the δβ-thal, and Hb F normally shows a greater affinity for oxygen and a reduced P50, although one must always take into account the degree of oxygen affinity of the Hb variant. Familial erythrocytosis and an abnormal electrophoresis finding are indicative of a high affinity Hb. However, the absence of these findings does not reject the possibility of hemoglobinopathies, and in these cases, functional and molecular studies would be justified and should be mandatory for the differential diagnosis of erythrocytosis.

Lactate Dehydrogenase: A Marker of the Severity of Vaso-Occlusive Crisis in Children with Sickle Cell Disease Presenting at the Emergency Department

Abstract The aim of this study was to describe the characteristics of vaso-occlusive crises (VOC) in children with sickle cell disease and to identify factors associated with greater severity. We performed a prospective observational study from August 2012 to January 2014. The study population comprised patients with sickle cell disease who consulted at the emergency department (ED) for VOC. We recorded demographic variables, history of complications related to the disease, and data on usual treatment. We also assessed pain, analgesia at home, need for admission, length of stay, and analgesia during admission. Analytical parameters were collected. A total of 29 patients with VOC were included. The patient’s usual treatment was hydroxyurea (HU) in 69.0%, and 7.0% required chronic transfusions. In the ED, 90.0% had moderate or severe pain, even though 86.0% had received analgesia at home (41.4% minor opioids). Overall, 27 of the 29 patients were admitted, and 56.0% needed major opioids. Higher lactate dehydrogenase (LDH) levels were related to the use of major opioids during admission (p = 0.038). A significant difference was recorded between the median number of days of admission for patients receiving non-steroidal anti-inflammatory drugs and for those requiring intravenous opioids (p = 0.005). Most patients with VOC were admitted to hospital. Lactate dehydrogenase level in the ED was a predictor of severity and was associated with the need for major opioids during admission and more days of admission.

Cost-effectiveness analysis of newborn screening for sickle-cell disease in Spain

ABSTRACT Objectives: This study assesses the cost-effectiveness of adding sickle-cell disease (SCD) to the Spanish newborn screening (NBS) program, and explores the sensitivity of the results to key model parameters. Methods: A discrete event simulation model was developed that compared NBS for SCD versus clinical detection. The model followed a simulated cohort of newborns for 10 years, and estimated the impact in costs and life expectancy of prophylactic treatments established after early detection. Probabilistic, one-way and two-way sensitivity analysis were performed. Results: NBS was found to be more costly and more effective than clinical detection of SCD. The estimated incremental cost per life year (LY) gained was 34,169.46 €/LY. This result was very sensitive to the cost of the screening test, the birth prevalence and the proportion of severe cases among the affected children. Conclusions: There is uncertainty regarding the cost-effectiveness of NBS for SCD in the Spanish context. Our base case estimate of the cost per LY gained lies near the 30,000€/LY commonly cited in Spain.

Trasplante de médula ósea en pacientes con anemia falciforme. Experiencia en un centro

INTRODUCTION Sickle cell disease (SCD), despite the improvement in the medical management, is still associated with severe morbidity and decreased survival. Allogenic hematopoietic stem cell transplantation (Allo-HSCT) currently provides the only curative therapy. A report is presented on our experience in children with SCD, who underwent Allo-HSCT in a single centre. MATERIAL AND METHOD A single centre descriptive study was conducted on patients with SCD who underwent a bone marrow transplant from an HLA-identical sibling donor between January 2010 and December 2014. Epidemiological, clinical and analytical parameters were collected with a follow-up to December 2015. Data are presented as frequencies, percentages, and medians (range). RESULTS Allo-HCST was performed in 11 patients (8 males) with a median age of 7 years (2-13), all of them with comorbidity prior to the HCST. A stable graft was achieved in 10 out of 11 patients, 9 of them with complete donor chimerism, and one patient with stable mixed chimerism after 1 year of allo-HSCT. One patient has secondary graft failure with re-appearance of symptoms associated with SCD on day 180. Complications of Allo-HSCT are: arterial hypertension 7/11, acute renal failure 3/11, CMV reactivation 9/11, neurological complications 4/11 (subarachnoid haemorrhage, seizure), and acute graft versus host disease (aGVHD) of the skin 6/11, one of whom developed grade iv intestinal aGVHD, causing his death (day 51). None of the patients developed chronic GVHD. The overall survival and event-free survival was 90.9% and 81.9%, respectively, with a median follow-up of 3.1 (1-5.7) years. CONCLUSIONS Allo-HSCT, the only curative therapy, remains associated with morbidity. There was a transplant related mortality in our study, consistent with multicentre studies (1/11), and with aGVHD being the main cause. Other problems still include graft failure (1/11), and neurological complications (4/11), although the permanent sequelae are mild.