Evolving patterns and clinical outcome of genetic studies performed at diagnosis in acute myeloid leukemia patients: Real life data from the PETHEMA Registry.

BACKGROUND: There are no studies assessing the evolution and patterns of genetic studies performed at diagnosis in acute myeloid leukemia (AML) patients. Such studies could help to identify potential gaps in our present diagnostic practices, especially in the context of increasingly complex procedures and classifications. METHODS: The REALMOL study (NCT05541224) evaluated the evolution, patterns, and clinical impact of performing main genetic and molecular studies performed at diagnosis in 7285 adult AML patients included in the PETHEMA AML registry (NCT02607059) between 2000 and 2021. RESULTS: Screening rates increased for all tests across different time periods (2000-2007, 2008-2016, and 2017-2021) and was the most influential factor for NPM1, FLT3-ITD, and next-generation sequencing (NGS) determinations: NPM1 testing increased from 28.9% to 72.8% and 95.2% (p < .001), whereas FLT3-ITD testing increased from 38.1% to 74.1% and 95.9% (p < .0001). NGS testing was not performed between 2000-2007 and only reached 3.5% in 2008-2016, but significantly increased to 72% in 2017-2021 (p < .001). Treatment decision was the most influential factor to perform karyotype (odds ratio [OR], 6.057; 95% confidence interval [CI], 4.702-7.802), and fluorescence in situ hybridation (OR, 2.273; 95% CI, 1.901-2.719) studies. Patients ≥70 years old or with an Eastern Cooperative Oncology Group ≥2 were less likely to undergo these diagnostic procedures. Performing genetic studies were associated with a favorable impact on overall survival, especially in patients who received intensive chemotherapy. CONCLUSIONS: This unique study provides relevant information about the evolving landscape of genetic and molecular diagnosis for adult AML patients in real-world setting, highlighting the increased complexity of genetic diagnosis over the past 2 decades.

A multicentre ambispective observational study into the incidence and clinical management of aplastic anaemia in Spain (IMAS study).

Aplastic anemia (AA) is a rare, life-threatening hematological disease, with a poorly defined incidence. As the data available on AA varies substantially worldwide, a multicenter, ambispective, observational study was carried out between 2010 and 2019 to assess the incidence, clinical management and survival of AA at seven Spanish hospitals. The incidence of AA was 2.83 per million inhabitants per year, consistent with that reported previously in Europe, with a median age at diagnosis of 61 years-old (range 12-86), and a similar number of males and females. The initial diagnosis was severe or very severe AA in 55.8% of cases and 93.7% required transfusion. The most frequent first line therapy was anti-thymocyte globulin (ATG) plus cyclosporin A (CsA, 44.2%), followed by other CsA-based regimes (46.3%), with hematopoietic stem cell transplantation an infrequent 1st line therapy. The 6-month response rate was 68.2%, which then increased over a median follow-up of 3.9 years. The 5-year overall survival (5OS) was 73.6%, similar in severe (78.6%) and very severe AA patients (74.6%) but lower in moderate AA (MAA) patients (68.4%). The 5OS was 100% in 0-25 year-old patients but dropping to 58.3% in patients ≥ 60 years-old. At the last contact, 75.8% of the patients were alive. In conclusion, the incidence, characteristics and management of AA in our study are consistent with that reported previously. In terms of survival, although the global long-term OS rate was good, there is room for improvement, particularly in older patients. Finally, what appears to be a worse long-term survival of MAA patients, as reported previously, reinforces the importance of not underestimating this condition when diagnosed as MAA.

Incidence, risk factors, and outcomes of second neoplasms in patients with acute promyelocytic leukemia: the PETHEMA-PALG experience.

The most important challenges in acute promyelocytic leukemia (APL) is preventing early death and reducing long-term events, such as second neoplasms (s-NPLs). We performed a retrospective analysis of 2670 unselected APL patients, treated with PETHEMA "chemotherapy based" and "chemotherapy free" protocols. Only de novo APL patients who achieved complete remission (CR) and completed the three consolidation cycles were enrolled into the analysis. Out of 2670 APL patients, there were 118 (4.4%) who developed s-NPLs with the median latency period (between first CR and diagnosis of s-NPL) of 48.0 months (range 2.8-231.1): 43.3 (range: 2.8-113.9) for s-MDS/AML and 61.7 (range: 7.1-231.1) for solid tumour. The 5-year CI of all s-NPLs was of 4.43% and 10 years of 7.92%. Among s-NPLs, there were 58 cases of s-MDS/AML, 3 cases of other hematological neoplasms, 57 solid tumours and 1 non-identified neoplasm. The most frequent solid tumour was colorectal, lung and breast cancer. Overall, the 2-year OS from diagnosis of s-NPLs was 40.6%, with a median OS of 11.1 months. Multivariate analysis identified age of 35 years (hazard ratio = 0.2584; p < 0.0001) as an independent prognostic factor for s-NPLs. There were no significant differences in CI of s-NPLs at 5 years between chemotherapy-based vs chemotherapy-free regimens (hazard ratio = 1.09; p = 0.932). Larger series with longer follow-up are required to confirm the potential impact of ATO+ATRA regimens to reduce the incidence of s-NPLs after front-line therapy for APL.

Combined Immune Defect in B-Cell Lymphoproliferative Disorders Is Associated with Severe Infection and Cancer Progression.

B cell chronic lymphoproliferative diseases (B-CLPD) are associated with secondary antibody deficiency and other innate and adaptive immune defects, whose impact on infectious risk has not been systematically addressed. We performed an immunological analysis of a cohort of 83 B-CLPD patients with recurrent and/or severe infections to ascertain the clinical relevance of the immune deficiency expression. B-cell defects were present in all patients. Patients with combined immune defect had a 3.69-fold higher risk for severe infection (p = 0.001) than those with predominantly antibody defect. Interestingly, by Kaplan-Meier analysis, combined immune defect showed an earlier progression of cancer with a hazard ratio of 3.21, than predominantly antibody defect (p = 0.005). When B-CLPD were classified in low-degree, high-degree, and plasma cell dyscrasias, risk of severe disease and cancer progression significantly diverged in combined immune defect, compared with predominantly antibody defect (p = 0.001). Remarkably, an underlying primary immunodeficiency (PID) was suspected in 12 patients (14%), due to prior history of infections, autoimmune and granulomatous conditions, atypical or variegated course and compatible biological data. This first proposed SID classification might have relevant clinical implications, in terms of predicting severe infections and cancer progression, and might be applied to different B-CLPD entities.

Characteristics and Outcomes of Adult Patients in the PETHEMA Registry with Relapsed or Refractory FLT3-ITD Mutation-Positive Acute Myeloid Leukemia.

This retrospective study investigated outcomes of 404 patients with relapsed/refractory (R/R) FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) acute myeloid leukemia (AML) enrolled in the PETHEMA registry, pre-approval of tyrosine kinase inhibitors. Most patients (63%) had received first-line intensive therapy with 3 + 7. Subsequently, patients received salvage with intensive therapy (n = 261), non-intensive therapy (n = 63) or supportive care only (n = 80). Active salvage therapy (i.e., intensive or non-intensive therapy) resulted in a complete remission (CR) or CR without hematological recovery (CRi) rate of 42%. More patients achieved a CR/CRi with intensive (48%) compared with non-intensive (19%) salvage therapy (p < 0.001). In the overall population, median overall survival (OS) was 5.5 months; 1- and 5-year OS rates were 25% and 7%. OS was significantly (p < 0.001) prolonged with intensive or non-intensive salvage therapy compared with supportive therapy, and in those achieving CR/CRi versus no responders. Of 280 evaluable patients, 61 (22%) had an allogeneic stem-cell transplant after they had achieved CR/CRi. In conclusion, in this large cohort study, salvage treatment approaches for patients with FLT3-ITD mutated R/R AML were heterogeneous. Median OS was poor with both non-intensive and intensive salvage therapy, with best long-term outcomes obtained in patients who achieved CR/CRi and subsequently underwent allogeneic stem-cell transplant.

Engineering Erg10 Thiolase from Saccharomyces cerevisiae as a Synthetic Toolkit for the Production of Branched-Chain Alcohols.

Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.

Rabbit antithymocyte globulin versus horse antithymocyte globulin for treatment of acquired aplastic anemia: a retrospective analysis.

Studies comparing rabbit antithymocyte globulin (rATG) and horse ATG (hATG) in patients with aplastic anemia (AA) have shown conflicting results. These studies included fewer than 60 subjects in the rATG arm with relatively short follow-up. A total of 169 patients treated with rATG and 62 treated with hATG were included in this retrospective analysis, across 33 centers. Patients were treated with rATG or hATG plus cyclosporine A. Over half were classified, as having severe AA (SAA) or very severe AA (VSAA), and the mean follow-up was 45 months. There was no significant difference detected in cumulative response to treatment or survival between the rATG and hATG groups. The response to treatment was 63 % in the rATG group versus 66 % in the hATG group at 3 months. By 12 months, this pattern had reversed, and 84 % of rATG patients had responded to treatment versus 76 % in the hATG group (n.s.). Early mortality due to infection tended to be higher with rATG compared to hATG (n.s). rATG and hATG would seem to be therapeutically equivalent in SAA and VSAA. However, patients treated with rATG may take longer to respond than those treated with hATG and may also require more active prevention of early infections.

Serum galactomannan versus a combination of galactomannan and polymerase chain reaction-based Aspergillus DNA detection for early therapy of invasive aspergillosis in high-risk hematological patients: a randomized controlled trial.

BACKGROUND: The benefit of the combination of serum galactomannan (GM) assay and polymerase chain reaction (PCR)-based detection of serum Aspergillus DNA for the early diagnosis and therapy of invasive aspergillosis (IA) in high-risk hematological patients remains unclear. METHODS: We performed an open-label, controlled, parallel-group randomized trial in 13 Spanish centers. Adult patients with acute myeloid leukemia and myelodysplastic syndrome on induction therapy or allogeneic hematopoietic stem cell transplant recipients were randomized (1:1 ratio) to 1 of 2 arms: "GM-PCR group" (the results of serial serum GM and PCR assays were provided to treating physicians) and "GM group" (only the results of serum GM were informed). Positivity in either assay prompted thoracic computed tomography scan and initiation of antifungal therapy. No antimold prophylaxis was permitted. RESULTS: Overall, 219 patients underwent randomization (105 in the GM-PCR group and 114 in the GM group). The cumulative incidence of "proven" or "probable" IA (primary study outcome) was lower in the GM-PCR group (4.2% vs 13.1%; odds ratio, 0.29 [95% confidence interval, .09-.91]). The median interval from the start of monitoring to the diagnosis of IA was lower in the GM-PCR group (13 vs 20 days; P = .022), as well as the use of empirical antifungal therapy (16.7% vs 29.0%; P = .038). Patients in the GM-PCR group had higher proven or probable IA-free survival (P = .027). CONCLUSIONS: A combined monitoring strategy based on serum GM and Aspergillus DNA was associated with an earlier diagnosis and a lower incidence of IA in high-risk hematological patients. Clinical Trials Registration. NCT01742026.

Impact of prior rituximab on outcomes of autologous stem-cell transplantation in patients with relapsed or refractory aggressive B-cell lymphoma: a multicentre retrospective Spanish group of lymphoma/autologous bone marrow transplant study.

The use of highly effective rituximab-containing therapy for treating diffuse large B-cell lymphoma (DLBCL) makes it more difficult to salvage relapsed or refractory patients. Autologous stem-cell transplantation (ASCT) is the reference treatment for these patients, but the impact of previous exposure to rituximab on the subsequent results of ASCT remains unknown. We analysed 248 patients with relapsed or refractory DLBCL or grade 3B follicular lymphoma pre-treated with rituximab as part of first-line therapy (R+ group) who received ASCT, in comparison with a control group of 127 patients without previous exposure to rituximab (R- group). The complete remission (CR) rates were similar in both groups. Multivariate analysis identified age-adjusted International Prognostic Index at diagnosis, extranodal involvement and disease status at transplant, and the number of previous chemotherapy lines as independent factors with a negative influence on CR rate. Compared with R- patients, those in the R+ group had a significantly better progression-free survival (63% vs. 48% at 5 years) and overall survival (72% vs. 61% at 5 years). This observation was independent of other prognostic factors that affected these outcomes. In conclusion, ASCT is no less effective in patients with relapsed or refractory aggressive B-cell lymphoma pre-treated with first-line rituximab-containing therapy than in rituximab-naive patients.

Maintenance therapy with bortezomib plus thalidomide or bortezomib plus prednisone in elderly multiple myeloma patients included in the GEM2005MAS65 trial.

Maintenance therapy has become a hot field in myeloma, and it may be particularly relevant in elderly patients because the major benefit results from the initial therapy. We report the results of a randomized comparison of maintenance with bortezomib plus thalidomide (VT) or prednisone (VP) in 178 elderly untreated myeloma patients who had received 6 induction cycles with bortezomib plus either melphalan and prednisone or thalidomide and prednisone. The complete response (CR) rate increased from 24% after induction up to 42%, higher for VT versus VP (46% vs 39%). Median progression-free survival (PFS) was superior for VT (39 months) compared with VP (32 months) and overall survival (OS) was also longer in VT patients compared with VP (5-year OS of 69% and 50%, respectively) but the differences did not reach statistical significance. CR achievement was associated with a significantly longer PFS (P < .001) and 5-year OS (P < .001). The incidence of G3-4 peripheral neuropathy was 9% for VT and 3% for VP. Unfortunately, this approach was not able to overcome the adverse prognosis of cytogenetic abnormalities. In summary, these maintenance regimens result in a significant increase in CR rate, remarkably long PFS, and acceptable toxicity profile. The trial is registered at www.clinicaltrials.gov as NCT00443235.

Caracterización molecular de dos nuevas mutaciones de α ° talasemia en 2 familias españolas (mutación - - ED y - - GP) / Molecular characterization of two new mutations of α ° thalassemia in two Spanish families (mutation -- ED and -- GP)

Fundamento y objetivo: La hemoglobina es un tetrámero constituido por dos cadenas α y dos cadenas β. Los dos genes estructurales que codifican la cadena α se encuentran localizados en el brazo corto del cromosoma 16. Los individuos normales tienen 4 genes α (αα/αα). Las α talasemias se producen generalmente por la deleción de uno, dos, tres o cuatro de los genes α. La deleción de ambos genes α dentro del mismo cromosoma (α° talasemia) se observa en individuos del área mediterránea y en el sudeste asiático. Material y método: Se estudian dos familias naturales de Madrid con anemia microcítica e hipocroma. El ADN extraído de los leucocitos de sangre periférica se digiere con diferentes enzimas de restricción e hibridación con sondas del cluster de genes α. Los extremos de la deleción se han caracterizado combinando las técnicas de Southern blot, reacción en cadena de la polimerasa (PCR) e hibridación in situ fluorescente (FISH). Resultados: En este trabajo presentamos dos nuevas mutaciones de α° talasemia en dos familias españolas, no descritas previamente en la bibliografía. La mutación (--ED) presenta una deleción de alrededor de 80kb con el punto de rotura 5’ en la coordenada +100 (± 3kb), mientras que el extremo 3’ HVR se sitúa en la coordenada 178±750bp. La segunda mutación (--GP) es más extensa, con pérdida de 145kb, situándose la deleción en la región 5’, entre las coordenadas 34 y 37, respetando el telómero. En la región centromérica la rotura se sitúa también en la coordenada 178±1,4kb. En ambas mutaciones se pierden los dos genes α, el gen θ y la región reguladora HS40. Conclusiones: La exacta identificación de estas mutaciones es esencial para determinar la función de los genes α en caso de consejo genético (AU)

Background and objetives: The two structural genes encoding the human α-globin chains are located on the short arm of chromosome 16. Normal individuals have four genes α (αα/αα). α-thalassemias are usually produced by the deletion of one, two, three, or four α genes. Deletion of both α genes within the same chromosome (α° thalassemia) is commonly observed in individuals from the Mediterranean basin and Southeast Asia. Material and methods: We study two natural families of Madrid with microcytic hypochromic anemia. The DNA extracted from peripheral blood leukocytes was digested with different restriction enzymes and hybridization with probes of gene cluster α. The ends of the deletion were characterized by combining the techniques of Southern blot, PCR and FISH. Results: We present two new mutations of α° thalassemia in two Spanish families, not previously described in the literature. The deletion (--ED) is ∼80kb with the point of bread 5’ in the coordinate +100 (± 3kb), whereas the end 3’HVR places in the coordinate 178±750bp. The second deletion (--GP) is more extensive, with loss of 145kb, placing the deletion in the end 5’ between the coordinates 34 and 37, respecting therefore the telomere. In the centromeric region the point of break places as the previous one in the coordinate 178±1.4bp. Conclusions: In both mutations both alpha genes were deleted, the gene θ and the region HS40. The exact identification of these deletions is essential to determine the function of the genes α with a view to a possible genetic diagnosis (AU)

[Molecular characterization of two new mutations of α° thalassemia in two Spanish families (mutation --(ED) and --(GP))]. / Caracterización molecular de dos nuevas mutaciones de α° talasemia en 2 familias españolas (mutación --(ED) y --(GP)).

BACKGROUND AND OBJECTIVES: The two structural genes encoding the human α-globin chains are located on the short arm of chromosome 16. Normal individuals have four genes α (αα/αα). α-thalassemias are usually produced by the deletion of one, two, three, or four α genes. Deletion of both α genes within the same chromosome (α° thalassemia) is commonly observed in individuals from the Mediterranean basin and Southeast Asia. MATERIAL AND METHODS: We study two natural families of Madrid with microcytic hypochromic anemia. The DNA extracted from peripheral blood leukocytes was digested with different restriction enzymes and hybridization with probes of gene cluster α. The ends of the deletion were characterized by combining the techniques of Southern blot, PCR and FISH. RESULTS: We present two new mutations of α° thalassemia in two Spanish families, not previously described in the literature. The deletion (--(ED)) is ∼80 kb with the break point 5' in the coordinate +100 (± 3 kb), whereas the end 3'HVR places in the coordinate 178±750 bp. The second deletion (--(GP)) is more extensive, with loss of 145 kb, placing the deletion in the end 5' between the coordinates 34 and 37, respecting therefore the telomere. In the centromeric region the breakpoint places as the previous one in the coordinate 178±1.4 bp. CONCLUSIONS: In both mutations both alpha genes were deleted, the gene θ and the region HS40. The exact identification of these deletions is essential to determine the function of the genes α with a view to a possible genetic diagnosis.

[Hemoglobin Stanleyville II [alpha78(EF7)Asn --> Lys]. First case described in Spain]. / Hemoglobina Stanleyville II [ alpha78(EF7)Asn --> Lys]. Primer caso descrito en España.

BACKGROUND AND OBJECTIVE: Structural hemoglobinopathies are the result of mutations in the genes of globin, which determine a qualitative alteration in the expression of these genes. Most alterations do not originate any significant change, and correspond to silent or asymptomatic forms. This study proves a new case of hemoglobin (Hb) Stanleyville II. PATIENTS AND METHOD: The propositus was a 72 years old Caucasian woman, from the Canary Islands. Her hematological data were: Hb 14.3 g/dl; hematocrit 44.4%; mean corpuscular volume 85.8 fl; mean corpuscular hemoglobin 27.7 pg; red cell distribution width 15.1%; reticulocytes 1.2%; HbA2 3.1% and HbF 1.6%. Electrophoretic studies in cellulose acetate electrophoresis at alkaline pH = 8.6 and isoelectrofocusing showed an anomalous Hb similar to HbS. The anomalous Hb did not appear in agar citrate electrophoresis (pH 6.0). The analysis by reverse phase high performance liquid chromatography for globin chains showed an X anomalous after A. RESULTS: Molecular analysis by sequentiation of the polymerase chain reaction products genes 1 and 2 showed the mutation AAC --> AAA at CD78 of second gene 2 in heterozygote state, which leads the change of asparagine to lysine. CONCLUSIONS: The substitution of an amino acid with neutral charge like asparagine for another one with positive charge like lysine in the segment EF, which corresponds to the external surface of the tertiary structure of the chain of globin, determines the change of charge in the chain. This allows an easy differentiation by electrophoretic and chromatographic methods. Nevertheless, owing to its position in the chain, which is not critique for the stability, solubility and affinity for the oxygen allows for silent or asymptomatic forms. The Hb Stanleyville II had been described before in black families of the Congo, Uganda, USA, Alsace and Brazil. This case represents the first case described in Spain.

Hemoglobina Stanleyville II [alfa78(EF7)Asn → Lys]. Primer caso descrito en España / Hemoglobin Stanleyville II [alpha78(EF7)Asn → Lys]. First case described in Spain

FUNDAMENTO Y OBJETIVO: Las hemoglobinopatías estructurales son el resultado de mutaciones enlos genes de globina que determinan una alteración cualitativa en la expresión de dichos genes.En la mayoría de ellas la alteración estructural no condiciona ningún cambio significativo,por lo que cursan de forma silente o asintomática. En este trabajo presentamos un nuevo casode hemoglobina (Hb) Stanleyville II.PACIENTES Y MÉTODO: El probando es una mujer de 72 años, raza blanca y origen canario. En laanalítica presentaba Hb de 14,3 g/dl, hematocrito del 44,4%, volumen corpuscular medio de85,8 fl, Hb corpuscular media de 27,7 pg y concentración de Hb corpuscular media de 32,2g/l; el índice de anisocitosis era del 15,1%, reticulocitos del 1,2%, HbA2 del 3,1% y HbF del1,6%. En la electroforesis en acetato de celulosa a pH alcalino y en el isoelectroenfoque se separóuna Hb anormal a la altura de la HbS. En agar citrato a pH ácido la Hb anormal no se separabade la HbA. Por cromatografía líquida de alta resolución de fase reversa se eluyó una cadenaalfa anormal más precoz que la alfa normal.RESULTADOS: En el análisis molecular, que se completó con la secuenciación de los productos deamplificación por reacción en cadena de la polimerasa de los genes alfa1 y alfa2, se demostró lamutación AAC → AAA en el codón 78 del segundo exón del gen alfa2 en estado heterocigoto, quedetermina un cambio de asparagina por lisina.CONCLUSIONES: La sustitución de un aminoácido con carga neutra, como la asparagina, por otrocon carga muy positiva, como la lisina, en el segmento EF, que corresponde a la superficie externade la estructura terciaria de la cadena de globina, determina un cambio neto en la cargade la cadena. Esto permite su fácil diferenciación por métodos electroforéticos y cromatográficos.Sin embargo, como la localización no es fundamental para la estabilidad, solubilidad y afinidadpor el oxígeno del tetrámero, cursa de forma silente o asintomática. La Hb Stanleyville IIse había descrito hasta ahora en familias de raza negra del Congo, Uganda, Zaire, EE.UU., Alsaciay Brasil. Este caso representa el primero descrito en España

BACKGROUND AND OBJECTIVE: Structural hemoglobinopathies are the result of mutations in the genesof globin, which determine a qualitative alteration in the expression of these genes. Mostalterations do not originate any significant change, and correspond to silent or asymptomaticforms. This study proves a new case of hemoglobin (Hb) Stanleyville II.PATIENTS AND METHOD: The propositus was a 72 years old Caucasian woman, from the Canary Islands.Her hematological data were: Hb 14.3 g/dl; hematocrit 44.4%; mean corpuscular volume85.8 fl; mean corpuscular hemoglobin 27.7 pg; red cell distribution width 15.1%; reticulocytes1.2%; HbA2 3.1% and HbF 1.6%. Electrophoretic studies in cellulose acetate electrophoresisat alkaline pH = 8.6 and isoelectrofocusing showed an anomalous Hb similar to HbS. The anomalousHb did not appear in agar citrate electrophoresis (pH 6.0). The analysis by reverse phasehigh perfomance liquid chromatography for globin chains showed an alphaX anomalous after alphaA.RESULTS: Molecular analysis by sequentiation of the polymerase chain reaction products genesalpha1 and alpha2 showed the mutation AAC → AAA at CD78 of second gene alpha2 in heterozygote state,which leads the change of asparagine to lysine.CONCLUSIONS: The substitution of an amino acid with neutral charge like asparagine for anotherone with positive charge like lysine in the segment EF, which corresponds to the external surfaceof the tertiary structure of the chain of globin, determines the change of charge in the chain.This allows an easy differentiation by electrophoretic and chromatographic methods. Nevertheless,owing to its position in the chain, which is not critique for the stability, solubility and affinityfor the oxygen allows for silent or asymptomatic forms. The Hb Stanleyville II had beendescribed before in black families of the Congo, Uganda, USA, Alsace and Brazil. This case representsthe first case described in Spain

Hb La Coruña [beta38(C4)Thr-->Ile]: a new hemoglobin variant leading to familial polycythemia.

Hb La Coruña [beta38(C4)Thr --> Ile] is a new hemoglobin (Hb) variant that has an increased oxygen affinity. Clinically, this Hb leads to erythrocytosis. Hb La Coruña is an electrophoretically silent variant that can be detected by reversed phase high performance liquid chromatography (HPLC) and characterized by DNA sequencing. The patient was a 22-year-old Spanish male whose family lived in La Coruña, in the northwest of Spain. His mother was also a carrier.

Frequency of the C282Y and H63D mutations of the hemochromatosis gene (HFE) in a cohort of 1,000 neonates in Madrid (Spain).

For centuries in Europe, population movements have contributed to ethnic groups, cultures, and consequently, inheritance mixing. There are certain genetic diseases such as hereditary hemochromatosis whose distribution is directly related to the population movements. The objective of the present investigation was to determine the C282Y and H63D mutation frequency of the HFE gene in a cohort study of 1,000 neonates in the Community of Madrid (Spain), thus contributing to the HFE gene mutations distribution research in Europe and establishing the origin of the mutations in Spain. The allelic frequency of C282Y mutation was 1.7% (CI 95% 1.1-2.3) and the H63D allele was present in 16.4% of chromosomes (CI 95% 14.8-18). In Spain, the presence of C282Y mutation and its distribution could be due more to Celtic than to Viking legacy, whereas it is assumed that the one in relation to the H63D variant occurred in the Basque Country during the Paleolithic Period.

First Spanish case of thalassemia major due to a compound heterozygosity for the IVS-II-848 (C --> A) and codon 39 (C --> T) mutations of the beta-globin gene.

This report describes the first case in Spain of a severe form of beta-thalassemia (thal) due to a compound heterozygosity for the IVS-II-848 (C --> A) and the nonsense codon 39 (C --> T) mutations. Five members of a family from Cadiz (southern Spain) were studied. The proband was an 8-year-old girl diagnosed as anemic at the age of 13 months. Her father had the codon 39 (C --> T) mutation and her mother the C --> A change at nucleotide (nt) 848 of IVS-II. Haplotype analysis showed that the proband was a compound heterozygote for haplotypes I [+ --> + +] and VII [+ --> +]. This is the first description in Spain of the IVS-II-848 (C --> A) mutation. It appears, from restriction fragment length polymorphism (RFLP) analysis, that this mutation has a different origin in the various populations, where it was found. This observation shows that in this case the association of a beta(0)- and a beta(+)-thal mutation does not lead to a thalassemia intermedia but to a severe thalassemia with very low hemoglobin (Hb) levels. From a therapeutic point of view, early introduction of a transfusion regimen may improve the clinical picture of these children, allowing for better development and growth.

The first case of Hb E-Saskatoon associated with Hb Lepore-Baltimore found in Spain.

Hb E-Saskatoon [beta22(B4)Glu-->Lys] does not cause any clinical symptoms in the heterozygous state. The homozygous state shows moderate phenotype expression. It has also been detected in association with beta-thalassemia. We present the first case of Hb E-Saskatoon associated with Hb Lepore-Baltimore. This unusual combination of mutations does not aggravate the clinical picture, as only microcytosis and hypochromia have been observed. Hb E-Saskatoon can only be correctly characterized by ion exchange high performance liquid chromatography (HPLC) or by DNA sequencing.

A novel mutation of the alpha2-globin causing alpha(+)-thalassemia: Hb Plasencia [alpha125(H8)Leu--Arg (alpha2).

We describe, in a Spanish family with moderate microcytosis and hypochromia, a novel nondeletional alpha-thalassemia (thal) mutation localized on the alpha2-globin gene. DNA sequencing revealed a point mutation at codon 125 (CTG --> CGG) in the heterozygous state, that was confirmed by restriction analysis. The resulting variant, which causes a nondeletional alpha-thal, was named Hb Plasencia [alpha125(H8)Leu --> Arg (alpha2)] after the place of residence of the affected family.