Relaciones genotipo-fenotipo en Hemofilia: Desarrollo de un esquema de análisis molecular para mejorar la atención médica de las familias con Hemofilia

La Hemofilia es una enfermedad clave para estudiar la genética humana. Los objetivos del trabajo son mejorar las herramientas moleculares diagnósticas y aportar al conocimiento de la relación genotipo-fenotipo en Hemofilia A (HA). Para mejorar la determinación del defecto causal, se desarrolló y ajustó un esquema de análisis del F8 y asignación de patogenicidad, que permitió caracterizar el 96% de las familias con HA (n=911 individuos). Se reportó una familia con HA, con una deleción del promotor del F8 (Del2kb): probando severo, madre portadora, tía no-portadora, y abuelo leve (FVIII:C=35%) mostrando un mosaico genético (somático/germinal). Un abordaje qPCR alelo-específico para Del2KB, sobre sangremesodermo/saliva-ectodermo/orina-endodermo, permitió inferir la producción de FVIII en hepatocitos-endodermo (Normal%≈34%) y gónadas-epiblasto (Del2kb%≈60%) del mosaico, en coincidencia con su fenotipo. Estudios caso/control en pacientes HA-severos (n=404) permitió determinar riesgos absolutos de inhibidor distintos del promedio (18%) para los genotipos del F8: -deleciones multi-exónicas (86,9%, P=0,0006), -inversión intrón 22 (24,7%, P=0,0011) y -missense (1,2%, P<0,0001). Estudios en hermanos afectados indicaron factores predisponentes de inhibidor fuera del F8. Estudios sobre genes inmunoreguladores indicaron alto riesgo de inhibidor para la variante CTLA4:p.Thr17Ala (OR=2,11, P=0,0025). Estos hallazgos aportan a mejorar y extender la atención médica del paciente con HA y su familiaHaemophilia is a key disease to study human genetics. The objectives of the work are to improve the molecular diagnostic tools and contribute to the knowledge of the genotype-phenotype relationship in Haemophilia A (HA). To improve the determination of the causal defect, a scheme including F8-analysis and pathogenicity assignment was developed and adjusted, allowing characterization 96% of the families with HA (n=911 individuals). A family with HA and a deletion of the F8-promoter (Del2kb) was reported: a severe proband, a carrier mother, a non-carrier aunt, and a mild grandfather (FVIII:C=35%) showing a combined somatic/germinal mosaicism. An allele-specific qPCR approach for Del2KB, on bloodmesoderm/saliva-ectoderm/urine-endoderm, allowed inferring the production of FVIII in hepatocytes-endoderm (Normal%≈34%) and gonads-epiblast (Del2kb%≈60%) in the mosaic in close coincidence with his phenotype. Case-control studies in HA-severe patients (n=404) allowed determination of absolute inhibitor risks differing from the average (18%) associated with some F8-genotypes: multi-exonic deletions (86.9%, P=0.0006), -intron 22 inversion (24.7%, P=0.0011) and -missense (1.2%, P<0.0001). Studies in affected siblings showed additional inhibitor-predisposing factors. Studies on immunoregulatory genes indicated high inhibitor risks for the variant CTLA4:p.Thr17Ala (OR=2.11, P=0.0025). These findings contribute to improve and extend the medical care of the patient with HA and his familyFil: Marchione, Vanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Molecular insights into the mechanism of nonrecurrent F8 structural variants: Full breakpoint characterization and bioinformatics of DNA elements implicated in the upmost severe phenotype in hemophilia A

Hemophilia A (HA) provides excellent models to analyze genotype–phenotype relationships and mutational mechanisms. NhF8ld’s breakpoints were characterized using case‐specific DNA‐tags, direct‐ or inverse‐polymerase chain reaction amplification, and Sanger sequencing. DNA‐break’s stimulators (n = 46), interspersed repeats, non‐B‐DNA, and secondary structures were analyzed around breakpoints versus null hypotheses (E‐values) based on computer simulations and base‐frequency probabilities. Nine of 18 (50%) severe‐HA patients with nhF8lds developed inhibitors, 1/8 affecting one exon and 8/10 (80%) affecting multi‐exons. NhF8lds range: 2–165 kb. Five (45%) nhF8lds involve F8‐extragenic regions including three affecting vicinal genes (SMIM9 and BRCC3) but none shows an extra‐phenotype not related to severe‐HA. The contingency analysis of recombinogenic motifs at nhF8ld breakpoints indicated a significant involvement of several DNA‐break stimulator elements. Most nhF8ld’s breakpoint junctions showed microhomologies (1–7 bp). Three (27%) nhF8lds show complexities at the breakpoints: an 8‐bp inverted‐insertion, and the remnant two, inverted‐ and direct‐insertions (46–68 bp) supporting replicative models microhomology‐mediated break‐induced replication/Fork Stalling and Template Switching. The remnant eight (73%) nhF8lds may support nonhomologous end joining/microhomology‐mediated end joining models. Our study suggests the involvement of the retroposition machinery (e.g., Jurka‐targets, Alu‐elements, long interspersed nuclear elements, long terminal repeats), microhomologies, and secondary structures at breakpoints playing significant roles in the origin of the upmost severe phenotype in HA.Fil: 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: 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: Marchione, Vanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Waisman, Karen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Tetzlaff, Guillermo Tomás. Universidad Nacional de General Sarmiento; ArgentinaFil: Neme, Daniela. Fundación de la Hemofilia Alfredo Pavlovsky; ArgentinaFil: 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: 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"; Argentin

F8 intron 22 inversions and SNP rs73563631 in unrelated families with severe haemophilia A: clinical features and gene testing implications

F8 intron 22 inversions (INV22), the commonest cause of severe haemophilia A (HA), are mostly represented by type I (INV22-1) and type II (INV22-2) patterns (4:1) using BclI-fragments´ based techniques such as inverse shifting-PCR (IS-PCR/2008). Using IS-PCR/2008, we identified unusual patterns of the INV22-1 and INV22-2 (characterised by no signals in the diagnostic test and conventional INV22-1/-2 patterns in the complementary test) in patients and carriers from two (0.6%) out of 308 Argentinean families with severe-HA. A theoretical analysis of the 85 SNPs embedded in the relevant BclI-fragments followed by PCR-BclI-RFLP analysis allowed identification of the SNP rs73563631*G allele associated with a new BclI-restriction site in all four patients of Family 1 and 2. Linkage analysis using seven F8-linked-STRs in the two families confirmed two unrelated haplotypes in phase with INV22-1/-2. Screening of 404 X-chromosomes from the Argentinean general population failed to detected SNP rs73563631*G allele (q<0.24%). A new version of the IS-PCR/2008 adding the genotyping of the new patterns (x) of INV22-1/-2 was developed. Clinical/biochemical characteristics (severity and inhibitor risks) of patients with INV22-1x/-2x were virtually equal to canonical INV22s. Our estimations predict the involvement of INV22-1x/-2x in about 2.7% (4/149) of INV22-affected cases worldwide.Fil: 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: 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: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; 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: Marchione, Vanina Daniela. 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; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas ; Argentin

Somatic/Germinal Mosaicism of a F8 Promoter Deletion Confounds Clinical Predictions in a Family with Haemophilia A: Key Role of Genotype Quantitation

Large F8 deletions cause 10-15% of severe-Haemophilia A (HA) cases and associate with the highest clinical/biochemical severity and with significantly augmented risks for developing inhibitors against therapeutic FVIII. Only 45-50% of severe-HA cases present family history of the disease. In the remnant cases (sporadic-HA), the mutation origin defines different clinical scenarios in which the risk of recurrence and thus genetic counselling significantly vary. The origin of the causative mutation may be either pre-zygotic or post-zygotic generating a genetic mosaicism affecting, partially or totally, one or more tissue/organs including the gonads. Furthermore, the technical features of the genotyping approach for detecting and measuring an eventual genetic mosaicism critically affect its diagnosis. The quali-quantitative extent of somatic and germinal mosaicisms is passively assumed to be associated with the phenotypic expression of haemophilia severity and inheritance pattern, respectively. We present a case of a family affected with HA in which the clinical/biochemical severity and inheritance patterns associate with the observed fraction of mosaic cells bearing a F8-promoter deletion.Fil: Abelleyro, Miguel Martin. Academia Nacional de Medicina de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Marchione, Vanina Daniela. 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; ArgentinaFil: Elhelou, L.. Fundacion de la Hemofilia; 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; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: 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; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Nemec, Diego Martín. Fundacion de la Hemofilia; 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; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentin

Inverse PCR to perform long-distance haplotyping: main applications to improve preimplantation genetic diagnosis in hemophilia

Among other applications of long-distance haplotype phasing in clinical genetics, determination of linked DNA markers as surrogate for problematic structural variants (e.g., repeat-mediated rearrangements) is essential to perform diagnosis from low-quality DNA samples. We describe a next-of-kin-independent (physical) phasing approach based on inverse-PCR (iPCR) paired-end amplification (PI). This method enables typing the multialleles of the short tandem repeat (STR) F8Int21[CA]n at the F8-intron 21, as a surrogate DNA marker for the F8-intron 22 inversion (Inv22), the hemophilia A-causative hotspot, within the transmitted haplotype in informative carriers. We provide proof-of-concept by blindly validating the PI approach in 15 carrier mother/affected-son duos. Every F8Int21[CA]n STR allele determined in phase with the Inv22 allele in the female carriers from the informative duos was confirmed in the hemizygous proband (P = 0.00003). A second surrogate STR locus at the F8-IVS22 was obtained by the PI approach improving severe-HA preimplantation genetic diagnosis by augmenting heterozygosity in Inv22 carriers bypassing the requirement for family linkage analysis. The ability of the PI-assay to combine other marker pairs was demonstrated by haplotyping a SNV (F8:c.6118T > C) with a >28kb-distant F8-IVS22 STR. The PI approach has proven flexibility to target different marker pairs and has potential for multiplex characterization of iPCR products by massively parallel sequencing.Fil: 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: Marchione, Vanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Palmitelli, Micaela. 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: Neme, Daniela. Fundación de la Hemofilia Alfredo Pavlovsky; ArgentinaFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Medina Acosta, Enrique. Universidade Estadual Do Norte Fluminense Darcy Ribeiro; BrasilFil: 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: 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; Argentin

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.Fil: 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: Ziegler, Betiana Michelle. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Marchione, Vanina Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Nevado, Julián. Instituto de Investigacion del Hospital de la Paz.; España. Centro de Investigación Biomédica en Red de Enfermedades Raras; EspañaFil: Lapunzina, Pablo. Centro de Investigación Biomédica en Red de Enfermedades Raras; España. Instituto de Investigacion del Hospital de la Paz.; EspañaFil: Sciuccati, Gabriela. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Neme, Daniela. Fundación de la Hemofilia Alfredo Pavlovsky; ArgentinaFil: 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: Bonduel, Mariana. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; 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 "Mariano R. Castex"; Argentin

CTLA4 p.Thr17Ala (c.49A>G) polymorphism associates with inhibitor development in argentine patients with severe hemophilia A

El desarrollo de inhibidores de FVIII en hemofilia A (HA) es considerado un rasgo complejo, ya que involucra factores genéticos y ambientales, siendo el genotipo del gen del factor VIII (F8) el factor condicionante más importante, seguido por factores genéticos secundarios, más débiles. Se estimaron los riesgos de inhibidor asociados a cada genotipo-F8 mediante un estudio de casos/ controles en pacientes HA-severos (n=390), que caracteriza especialmente el estrato con la Inv22 (inversión del intrón 22). El cálculo de la prevalencia de inhibidor (IP) y odds-ratio (OR) con intervalos de confianza del 95% (IC95%) permitió clasificar tres grupos de riesgo (alto, medio y bajo) asociado a cada genotipo-F8. La asociación de factores genéticos secundarios se investigó mediante el análisis de concordancia/discordancia en el estatus de inhibidor en 17 pares de hermanos con la Inv22 vs pares al azar mostrando un OR(IC95%) de 1,83(0,69-4,85) (p=0,33), sugiriendo una tendencia a coincidir con el estatus de inhibidor del hermano. Se realizaron estudios tipo caso/control del riesgo de inhibidor en polimorfismos de IL10, TNFA y CTLA4 en los estratos Inv22-positivo (n=140-148). IL10 c.- 1117A>G, TNFA c.-488G>A y CTLA4 c.-319C>T mostraron tendencias de riesgo o protección similares a las reportadas, con ORs no significativos. CTLA4 c.49A>G p.Thr17Ala mostró un incremento significativo del riesgo de inhibidor con OR de 2,61(1,27-5,36) (p=0,0096). Nuestros resultados concuerdan con especulaciones teóricas previas acerca de diferencias regionales en los factores secundarios no modificables. CTLA4 p.Thr17Ala contribuye a aumentar el riesgo de inhibidor en nuestra población de pacientes con HA-severa.FVIII inhibitor development in hemophilia A (HA) is considered a complex trait as it involves genetic and environmental factors; the causative factor VIII gene (F8) genotype has been established as the most important determining factor, followed by weaker secondary genetic risks factors. Risks of inhibitor development associated with each F8- genotype were estimated by a case/control study in HA-severe patients (n=390), which especially characterizes strata with Inv22 (intron 22 inversion). Inhibitor prevalence (IP) and odds ratio (OR) with confidence intervals of 95% (95%CI) calculations allowed classifying three risk groups (high, medium and low) associated with each F8-genotype. The association of genetic factors was investigated by analyzing concordance/discordance inhibitor-status in 17 pairs of brothers with Inv22 vs random pairs showing 1.83(0.69 to 4.85) (p=0.33), suggesting a trend to agree the inhibitor status between siblings. Case/control studies associated risk inhibitor development with IL10, TNFA and CTLA4 polymorphisms in Inv22-positive strata (n=140- 148) were performed and in IL10 c.-1117A>G, TNFA c.-488G>A & CTLA4 c.-319C>T trends of risks or protection, similar to reported ones with not significant ORs. CTLA4 c.49A>G p.Thr17Ala showed significantly increased inhibitor risks with OR of 2.61(1.27-5.36) (p=0.0096). Our findings agree with previous theoretical speculations about regional differences in non-modifiable secondary factors. CTLA4 p.Thr17Ala, contributes to an increased risk of inhibitor in our population of patients with HA-severFil: Marchione, Vanina Daniela. 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: Primiani, L.. Fundación de la Hemofilia Dr. Alfredo Pavlovsky; ArgentinaFil: Neme, D.. Fundación de la Hemofilia Dr. Alfredo Pavlovsky; ArgentinaFil: Candela, M.. Fundación de la Hemofilia Dr. Alfredo Pavlovsky; ArgentinaFil: de Tezanos Pinto, M.. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex". Departamento de Genética; Argentina. Fundación de la Hemofilia Dr. Alfredo Pavlovsky; 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 "Mariano R. Castex". Departamento de Genética; ArgentinaFil: 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; Argentin