Are int22h-mediated deletions a common cause of hemophilia?

Hemophilia A (HA) (OMIM 306700) is an X-linked inherited bleeding disorder caused by deleterious mutations in the coagulation factor VIII gene (F8). Even though there is a broad diversity of HA-causative mutations, an uncommon type of rearrangement—a large DNA inversion involving F8 intron 22 (Inv22)—accounts for approximately one half of severely affected patients. Inv22 was formerly described by Lakich et al. [1] and Naylor et al. [2]. A collaborative international effort estimated that Inv22 is the cause of 43% (35%, 7%, and 1% for Inv22 type I, type II, and rare types, respectively) of severe HA cases worldwide with minor geographical or ethnical differences [3], in close agreement with our corresponding Argentinean series (42% of Inv22, and 34% and 7% for type I and type II, respectively) [4]. Naylor et al. [5] indicated that Inv22 originates by homologous recombination between well-defined duplicons (int22h) of 9.5 kb located one copy within F8 intron 22 (int22h-1, h1) and the other, inversely oriented, from a group of two extragenic copies (int22h-3, h3 for Inv22 type I and int22h-2, h2 for type II). It was formerly believed that h2 and h3 were equally oriented (i.e., head to tail). However, Ross et al. [6] determined that h2 and h3 are inversely oriented (i.e., head to head), both embedded in the arms of a large imperfect palindrome (Fig. 1). This finding prompted Bagnall et al. [7] to hypothesize recombination between these arms interchanging the location of the extragenic int22h copies and generating a non-deleterious inversion polymorphism in Xq28, i.e., h123 and h132. In this scenario, Inv22 type I may be generated from intrachromosomal recombination between h1 and h3 on the most frequent variant h123 whereas Inv22 type II may be generated between h1 and h2 on the least frequent h132 (Fig. 1). Moreover, on each of these normal structural variants of the X chromosome, recombination between h1 with either equally oriented copies (h2 or h3) may generate deletions (Del22) or duplications (Dup22) but not inversions [7]. More precisely, Del22 type I would be generated by recombination between h1 and h3 on variant h132 whereas Del22 type II by recombination between h1 and h2 on variant h123 [8] (Fig. 1).Fil: Abelleyro, Miguel Martin. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rossetti, Liliana Carmen. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Radic, Claudia Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Candela, Miguel. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: de Brasi, Carlos Daniel. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms

Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2V617F, is highly desirable. In this study, we present an approach to assess the JAK2V617F burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2V617F spaced from one template for JAK2Wild Type were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2V617F burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.5364.2% and 51.4664.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2V617F expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2V617F from heterozygosity to homozygosity.Fil: Gonzalez, Mariana Selena. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental;Fil: de Brasi, Carlos Daniel. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental;Fil: Bianchini, Michele. DTO. DE GENETICA;Fil: Gargallo, Patricia Martha. INST. DE INVEST. HEMATOLOGICAS;Fil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematologicas "mariano R. Castex";Fil: Zalcberg, Ilana. Molecular Biology, Laboratory, Instituto Nacional do Caˆncer, Rio de Janeiro, Brazil;Fil: Larripa, Irene Beatriz. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental

Dystrophinopathy patients with non-contiguous molecular alterations: diagnosis and characterization of the genetic mechanisms involved

Introduction: Dystrophinopathies are neuromuscular X-linked recessive diseases caused by DMD mutations. Molecular alterations in this gene are large deletions/duplications in 80% of cases and small mutations in the remaining. Several authors reported the occurrence of non-contiguous rearrangements within the same DMD allele, with frequencies up to 4%. The present work aims to characterize the incidence of complex rearrangements in an Argentinian dystrophinopathy cohort and unravel the causing molecular mechanisms.Materials and Methods: We analyzed 437 boys with clinical diagnosis of Dystrophinopathy. The following techniques were implemented: MLPA, WES, WGS, PCR-Sanger Sequencing, CGH Array and HUMARA assay. In 2 cases, breakpoints were precisely determined, so we performed a bioinformatic screening of microhomologies, interspersed repeats, secondary structures and recombinogenic motifs 50pb surrounding each breakpoint. Results: We detected 6 patients carrying complex rearrangements in DMD: 2 deletions-duplications, 3 non-contiguous duplications and 1 large deletion plus a 20pb insertion. These accounted for 1.4% of our cohort. In a deletion-duplication case, familial segregation and bioinformatics analysis suggested that the duplication was the first mutagenic event caused by Fork Stalling and Template Switching (FoSTeS), while the deletion occurred secondly by Non-homologous end joining. Furthermore, bioinformatic screening of the deletion plus insertion propose that the deletion was due to Microhomology-mediated end joining, while the insertion arose by FoSTeS. Conclusions: Our findings widen the understanding of the molecular events that may take place in DMD and characterize the occurrence of complex rearrangements in our dystrophinopathy cohort.This study was supported by PTC Therapeutics and University of Buenos Aires.Fil: Luce, Leonela Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; ArgentinaFil: Carcione, María Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; ArgentinaFil: Mazzanti, Chiara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; ArgentinaFil: Szijan, Irena. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; ArgentinaFil: Menazzi, Sebastian. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Francipane, Liliana. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Nevado, Julian. No especifíca;Fil: Lapunzina, Pablo. No especifíca;Fil: Rossetti, Liliana Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Radic, Claudia Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Abelleyro, Miguel Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Giliberto, Florencia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina53rd European Society of Human Genetics ConferenceViennaAlemaniaWiener Medizinische AkademieEuropean Society of Human Genetic

Sphingosine Kinase 1 Participates In The Activation, Proliferation And Survival Of Chronic Lymphocytic Leukemia Cells

Sphingosine kinases (SKs) have received the most attention as important enzymes in cancer biology. They participate in the regulation of bioactive sphingolipid metabolism by producing sphingosine-1 phosphate (S1P) which mediates several biological functions, including cell growth, differentiation, cell survival, migration, and angiogenesis among other tasks.1 S1P generation depends on the conversion of sphingosine to S1P, in a reaction catalyzed by two isoforms of SKs, SK1 and SK2, and its levels are tightly controlled via a rapid degradation by intracellular S1P lyases (S1PL) or dephosphorylated by S1P phosphatases.1 Once produced, S1P may function as an intracellular second messenger and/or can be exported outside the cells, where it binds to specific S1P receptors (S1PRs) and initiates downstream signaling pathways, in a paracrine or autocrine manner, in a process known as “inside-out” signaling.Fil: Almejún, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Borge, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Colado, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Elías, Esteban Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Podaza, Enrique Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Risnik, Denise Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Cabrejo, María. Ciudad Autónoma de Buenos Aires. Sanatorio Municipal "Dr. Julio Méndez"; ArgentinaFil: Fernández Grecco, Horacio. Ciudad Autónoma de Buenos Aires. Sanatorio Municipal "Dr. Julio Méndez"; ArgentinaFil: Bezares, Raimundo Fernando. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; ArgentinaFil: Cranco, Santiago. Instituto Alexander Fleming; ArgentinaFil: Burgos, Rubén Ángel. Instituto Alexander Fleming; ArgentinaFil: Sánchez Ávalos, Julio César Américo. Instituto Alexander Fleming; ArgentinaFil: Oppezzo, Pablo. Instituto Pasteur de Montevideo; UruguayFil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Genetic markers linked to the coagulation factor VIII gene. : Their utility for molecular diagnosis in haemophilia a and for the study of the molecular mechanism of homologous recombination in human meiosis.

La hemofiliaA (HA)es una enfermedad hemorrágica hereditaria, ligada al sexo, causada por deficiencia del factor VIIIde coagulación (FVIII). La ruta preferida para la provisión de datos para diagnóstico molecular es la detección directa de mutaciones. Entre las mutaciones que afectan al gen del FVIII, la inversión del intrón 22 (inv22) constituye la causa de casi la mitad de las HA severas (SHA). Mediante análisis de Southern blot fueron estudiadas 47 familias Argentinas con SHA. En cercano acuerdo a lo reportado series internacionales, 19 de ellas (40%) resultaron informativas para la Inv22 y pudo proveerse diagnóstico molecular por el método directo. Dentro de éstas, 14 (79%) resultaron Inv22 tipo distal y las restantes 5 (21%), proximal. No fueron encontradas significativas asociaciones entre la presencia o no de la Inv22 en SHA, y el origen de la enfermedad (esporádica o familiar), ni tampoco con la propensión o no a desarrollar anticuerpo inhibidor neutralizante contra el FVIII terapéutico. El camino alternativo para el diagnóstico es por vía indirecta usando polimorfismos ligados a la enfermedad. Entre los polimorfismos de longitud de fragmentos de restricción (RFLP) ubicados dentro del gen del FVIII, el marcador Xbal A del intrón 22 a pesar de proveer una alta informatividad en los distintos ganos étnicos analizados, fue casi excluido de la rutina diagnóstica por la lentitud y laboriosidad de su detección. Aquí se describen dos métodos rápidos, no-radioactivos, basados en PCR de larga distancia (LDPCR), para genotipificación del RFLP Xbal A. Los amplimeros de 6,6 y 6,9 kb incluyen el sitio polimórfico y un sitio constante de Xbal que provee control de digestión. La especificidad del método fue desafiada en experimentos a ciegas de muestras previamente genotipificadas por Southern blot, obteniéndose una perfecta correlación entre ambos métodos. Mediante este método, 53 cromosomas X de la población Argentina general fueron genotipificados para Xbal A, lo que permitió proyectar una informatividad de aproximadamente el 47%. Los estudios de asociaciones alélicas entre Xbal A (X) y el polimorfismo Bc/l (B) del intrón 18 del gen del FVIII, determinaron un alto, pero no-completo desequilibrio de ligamiento entre los marcadores, proyectando una informatividad combinada (X+B) del 55%. A 737 bp rio abajo del RFLP Xbal, fue localizado y caracterizado un nuevo polimorfismo bialélico, de cambio de nucleótido que afecta el sitio de reconocimiento de la enzima Mspl (CCg/aG). La genotipificación especifica del RFLP Mspl A se realiza utilizando la LD PCR específica para Xbal A como primera vuelta. seguida por una PCR anidada de 176 bp que incluye al sitio polimórfico y uno constante de Mspl para proveer control de digestión. Su análisis en las poblaciones nomiales Argentina y Británica permitió estimar una heterocigocidad de 49 y 46%, respectivamente. A pesar de su proximidad física y su desequilibrio de ligamiento, X y M proyectan una informatividad conjunta del 62% para población Argentina y 59% para la Británica. Ambos RFLPs (X y M) están incluídos en una región de 9,5 kb dentro del intrón 22 del gen del FVIII(int22h-1), que se encuentra repetida por lo menos 2 veces fuera del gen del FVIII, también sobre Xq28 (int22h-2 y -3). Para investigar los sitios correspondientes de X y M sobre los homólogos extragénicos fueron diseñados abordajes específicos similares a aquellos usados para los sitios intragénicos. Los sitios Mspl extragénicos (B y C) resultaron polimórficos en población Argentina y Británica aunque con frecuencias del alelo [+] mucho menores a las intragénicas. La Inv22 es originada por un evento de entrecruzamiento recíproco intracromosómico por recombinación homóloga entre las secuencias int22h descriptas resultando en la interrupción inactivante del gen del FVIII en su intrón 22. Debido a (1) que los marcadores X y M ubicados dentro de int22h permiten mediante sus frecuencias alélicas diferenciar las copias intra de las extragénicas y (2) que en el cromosoma X de varones de la población normal podemos estimar el estado de estos marcadores antes del evento y en los hemofílicos con la Inv22 podemos analizar el estado de estos marcadores después del evento; se diseño una estrategia para recabar datos del mecanismo molecular que origina la Inv22 como modelo de entrecruzamiento recíproco en meiosis humanas. La aplicación del método planteando nueve hipótesis de posibles intermediarios moleculares de la Inv22 asociadas a la predicción de resultados numéricos utilizando las estimaciones sobre población de varones normales y su comparación con los datos obtenidos en el grupo de afectados con la Inv22, permitió determinar que el evento molecular de recombinación meiótica está acoplado a la conversión génica del marcador Xbal, con la secuencia extragénica como dadora de la información. El mismo análisis sobre Mspl si bien proyecta resultados similares, no permite determinado con significación estadística. El aumento de la población hemofílica para la obtención de resultados más precisos además de los estudios de mapeo de puntos calientes para rupturas meióticas de doble cadena dentro de las secuencias int22h permitirá la elucidación específica del mecanismo de la Inv22 como modelo de recombinación homóloga en meiosis humanas.Haemophilia A (HA) is a sex linked inherited haemorragic disorder characterised by deficiency in the coagulation factor VIII (FVIII). Direct mutation detection in the gene is the best choice to obtain data for molecular diagnosis. Intron 22 FVIII gene inversion (Inv22) is involved in almost half of severe HA (SHA). It has been studied by Southern blot 47 Argentinean families with SHA and found inversions in 19 (40%), 15 (32%) with distal and 4 (8%) with proximal pattern. In this group of Inv22 informative families definitive information for molecular diagnosis could be provided. No significant correlation between the inheritance of the disease (either familiar or sporadic haemophilia) and the presence of inversions was found. The development of therapeutic-FVIII inhibitor in inv22-positive SHA-affected families was found increased but not significantly. The alternative route to obtain molecular information for genetic counselling is gene tracking using DNA polymorphisms. Among the restriction fragment length polymorphisms (RFLPs) in the FVIII gene, the Xbal A (intron 22) (X) despite its high informativity in almost all ethnic groups analysed, has not been frequently used in the routine because its reliable detection is tedious and labour-intensive. Two rapid, non-radioactive, long distance PCR-based methods (LD PCR) to genotype the X RFLP are described. The amplimers of 6.6 and 6.9 kb long include both the polymorphic and a constant Xbal site, which provides a digestion control. The specificity of the method was challenged by a blind experiment with genomic DNA samples previously genotyped using Southern blot analysis: a perfect correlation was obtained using both approaches. Fifty-three alleles from the Argentinean normal population were Xbal A genotyped. The results predict an informativity of 47%. Despite the high degree of allelic associations found between X and Bc/l (intron 18) (B), they predicts a combined heterozygocity (X+B) of 55%. A new polymorphism in the human FVIII gene has been localised and characterised. It is a bi-allelic single nucleotide polymorphism which affects an Mspl site (CCg/aG)located 737 bp downstream frorn the Xbal marker. The specific genotyping of the so termed Mspl A RFLP (M) requires a nested PCR approach using the X-specific LD PCR as first round and a 176 bp-long second round which includes both the polymorphic and a constant control digestion Mspl site. M genotyping from the Argentinean and the British normal population estimate an informativityof 49 and 46%, respectively. Despite their close proximity, the X and M polymorphisms are not in complete linkage disequilibnum: haplotype analysis from Argentinean and British population predict a combined informativity (X+M)of 62 and 59%, respectively. The markers X and Mare located in intron 22 of the FVIII gene within a 9.5 kb int22h-1segment. Int22h is duplicated at least twice extragenically at Xq28. Both extragenic copies (int22h-2 and —3) are also polymorphic with respect to X. There have been designed LDPCR-based methods to specifically genotype these extragenic X and M sites. The extragenic sites of both X and M resulted also polymorphic in Argentinean and British normal populations but with much lower frequencies of the [+] allele than those obtained frorn int22h-1. Inv22 originates by an intrachromosomic crossing over due to homologous recombination between int22h-1 and int22h-2 or —3. The molecular inversion interrupts the normal sequence of the intron 22 of the FVIII gene causing SHA. Due to (1) X and M enable the discrimination of the intra from the extragenic copies of int22h by means of differences in their allelic frequencies, and (2) that there can be estimated these frequencies before the event in males from the normal population and the frequencies after the event in the Inv22-SHA population; it was possible to design an genetic testing strategy to address the molecular mechanism of the Inv22 as a model for homologous recombination in human meiosis. The inspection of the literature prompts the design of 9 possible hypothesis of the intermediaries of the mechanism involved. Each hypothesis was associated with numerical values calculated from the parameters estimated in the normal populations, to predict the results to be obtained from the Inv22-affected population. This analysis allowed us to conclude that the molecular inversion is coupled to the gene conversion from a DNA segment including the Xbal extragenic site to its intragenic homologue. The results obtained using Mspl, although similar than those using Xbal, are not statistically significant. The increase in the number of the haemophilic population in order to gain precision in the test and the investigation of meiotic double-strand-breaks hot spots within int22h would allow further insights in the molecular mechanism of the Inv22 as a model for homologous recombination in human meiosis.Fil:De Brasi, Carlos Daniel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Homeologous recombination between AluSx-sequences as a cause of hemophilia.

Although large deletions from the coagulation factor VIII gene, F8, are responsible for 5% of severe hemophilia A (seHA), few of them have been fully characterised. A detailed description of a large partial deletion of the F8 caused by unequal recombination between homeologous AluSx-derived sequences is presented. The proband, a case of isolated hemophilia A with a high inhibitor titre (5700 BU), showed a consistent absence of PCR-amplification of exons 4 to 10, EX4_EX10del. Two approaches were used to narrow down the deletion breakpoints: a direct physical analysis based on PCR (that additionally permits carrier detection in the family); and, under the hypothesis that the mutation resulted from homologous recombination, sequence alignments of F8 intron 3 and 10. Both approaches indicate an unequal crossing over (CO) between two Alu-related sequences. Both elements involved were derived from the AluSx-subfamily consensus and demonstrate 86% sequence identity (with only single-base mismatches), with three gaps (of 2, 3 and 14-bases) and two main tracts of perfectly homologous sequence (28 and 24-bp). The short stretch of intron 10 embedded into intron 3 sequence, linked to the CO, represents a typical hallmark of homologous recombination (double-strand break repair model). A detailed description of EX4_EX10del mutation is c.[338+3485delins1687+2223_1687+2225; 338+3551_1687+2291 del]. The common involvement of unequal homologous recombination mediated by repetitive elements allowed us to suggest that our experimental design (based on intron sequence alignments) may be successfully applied to rearrangements involved in other X-linked inherited diseases. Like other Alu-rich genes throughout the human genome, Alu-mediated homologous recombination in F8 may be an important cause of hemophilia by promoting large DNA deletions. Copyright 2004 Wiley-Liss, Inc.Fil: Rossetti, Liliana Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Goodeve, Anne. University of Sheffield; Reino UnidoFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentin

Incidence of BCL-2 gene rearrangements in Argentinean non-Hodgkin lymphoma patients: Increased frequency of breakpoints outside of MBR and MCR

The t(14;18)(q32;q21) is the most frequent cytogenetic abnormality observed in follicular lymphoma (FL), but also occurs in diffuse large B-cell lymphoma (DLBCL). We have evaluated the frequency of this translocation in 106 Argentinean non-Hodgkin lymphoma (NHL) patients: 83 with diagnosis of FL and 23 with DLBCL. Nested (N) and long-distance PCR (LD-PCR) approaches were used. By N-PCR, a total of 42 (51%) FL cases showed BCL-2 rearrangement: 28 (34%) for major breakpoint region (MBR) and 14 (17%) for minor cluster region (mcr). By LD-PCR, additional 23 (28%) new positive cases were found: 10 (12%) for MBR and 13 (16%) for mcr. These data make a total of 65 (78%) positive cases for BCL-2 gene rearrangements. In DLBCL cases, N-PCR detected two (9%) cases with the MBR breakpoint and one (4%) with mcr. Seven (30%) new positive cases by LD-PCR were found: four (17%) for MBR and three (13%) for mcr, showing a total of 10 (43%) positive cases. Thus, both FL and DLBCL had high frequencies of breakpoints located between MBR and mcr clusters. Our N-PCR results in FL (51%; 95% CI, 40-62%) showed statistical differences with respect to the pooled data from USA (P < 0.0001) and overlapped with the frequencies from Asia and Europe. In DLBCL, no significant differences with respect to the literature were found. This data support the idea that FL may be a heterogeneous malignancy with distinct molecular pathogenesis and suggest that the geographic differences may be related with the distribution of breakpoints that are widely spread throughout the sequence stretch between MBR and mcr. © 2003 Elsevier Inc. All rights reserved.Fil: Noriega, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; ArgentinaFil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; ArgentinaFil: Narbaitz, Marina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; ArgentinaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentin

Biclonal follicular lymphoma: Histological, clinical and molecular characteristics

First evidence of the coexistence of MBR and mcr BCL2 rearrangements confirmed by DNA sequencing. This FL patient was negative for the standard Bcl-2 antibody but positive for an alternative Bcl-2 epitope and presented a favourable clinical course, although more reports on the clinical characteristics of similar FL cases with confirmed biclonal BCL2 rearrangements are necessary to determine its prognostic significance.Fil: Noriega, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: de Brasi, Carlos Daniel. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Narbaitz, Marina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Rodrguez, Andrea. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Symptomatic female carriers of Duchenne Muscular Dystrophy (DMD): genetic and clinical characterization

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the dystrophin gene and is characterized by muscle degeneration and death. DMD affects males; females being asymptomatic carriers of mutations. However, some of them manifest symptoms due to a translocation between X chromosome and an autosome or to a heterozygous mutation leading to inactivation of most of their normal X chromosome. Six symptomatic female carriers and two asymptomatic were analyzed by: I) Segregation of STRs-(CA)n and MLPA assays to detect a hemizygous alteration, and II) X chromosome inactivation pattern to uncover the reason for symptoms in these females. The symptomatic females shared mild but progressive muscular weakness and increased serum creatin kinase (CK) levels. Levels of dystrophin protein were below normal or absent in many fibers. Segregation of STRs-(CA)n revealed hemizygous patterns in three patients, which were confirmed by MLPA. In addition, this analysis showed a duplication in another patient. X chromosome inactivation assay revealed a skewed X inactivation pattern in the symptomatic females and a random inactivation pattern in the asymptomatic ones. Our results support the hypothesis that the DMD phenotype in female carriers of a dystrophin mutation has a direct correlation with a skewed X-chromosome inactivation pattern.Fil: Giliberto, Florencia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; Argentina;Fil: Radic, Claudia Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina;Fil: Luce, Leonela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; Argentina;Fil: Ferreiro, Verónica. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín. Instituto de Neurociencias Aplicadas; Argentina; Laboratorio de Genética Molecular Diagnóstica; Argentina;Fil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental; Argentina;Fil: Szijan, Irena. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; Argentina

Genetic testing in bleeding disorders

The aim of molecular genetic analysis in families with haemophilia is to identify the causative mutation in an affected male as this provides valuable information for the patient and his relatives. For the patient, mutation identification may highlight inhibitor development risk or discrepancy between different factor VIII assays. For female relatives, knowledge of the familial mutation can facilitate carrier status determination and prenatal diagnosis. Recent advances in understanding mutations responsible for haemophilia and methods for their detection are presented. For reporting of such mutations, participation in external quality assessment ensures that essential patient and mutation details are routinely included and that pertinent information is incorporated in the interpretation.Fil: de Brasi, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; ArgentinaFil: El Maarri, O.. Universitat Bonn; AlemaniaFil: Perry, D. J.. Universitat Bonn; AlemaniaFil: Oldenburg, J.. Universitat Bonn; AlemaniaFil: Pezeshkpoor, B.. Universitat Bonn; AlemaniaFil: Goodeve, A.. Sheffield Children’s NHS Foundation Trust; Reino Unido. Sheffield University Medical School; Reino Unid