Haemophilia 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 family. RESUMEN RESUMEN La Hemofilia A (HA) es una coagulopatía hereditaria ligada al cromosoma-X, que afecta 1:5000 varones sin distinciones étnico-geográficas. La HA puede clasificarse clínica-bioquímicamente en severa (FVIII:C< 1%), moderada (FVIII:C 1-5%) y leve (FVIII:C 5-40%) según los niveles residuales del FVIII plasmático. La severidad fenotípica está asociada al defecto genotípico en el F8 (gen complejo con 26 exones sobre 187 kb de ADN genómico en Xq28). Alrededor del 45% de los casos severos tienen historia familiar de Hemofilia y el resto son esporádicos surgidos por una mutación de novo sea sobre una célula germinal (80-90%) o en una mitosis postcigótica generando un mosaico genético. La HA puede ser tratada eficientemente con concentrados de FVIII, sin embargo, entre un 20-30% de los pacientes severos desarrollan anticuerpos neutralizantes contra el FVIII exógeno (inhibidor), haciendo inefectiva la terapia de reemplazo, reduciendo la calidad de vida del paciente y aumentando, aún más, los costos terapéuticos involucrados. En este escenario, este trabajo se propone avanzar tanto en el área molecular diagnóstica como en el conocimiento de la relación causal genotipo-fenotipo en HA. Para caracterizar la mutación causal de la HA, se desarrolló un algoritmo molecular para análisis del F8 que involucra la aplicación ordenada de abordajes convencionales de mediana/baja complejidad. En pacientes con HA-severa, primero se investigan las inversiones recurrentes del F8, la inversión del intrón 22 (Inv22) (tipos, variantes y deleciones/duplicaciones aso...

Introduction: Up to 20% of patients (pts) with essential thrombocythemia (ET) and up to 10-15% of pts with polycythemia vera (PV) are diagnosed before the age of 40 years and the percentage is even lower in pts with primary myelofibrosis (PMF). Pregnancy (Pcy) in MPN is associated with an increased incidence of thrombotic and bleeding events and obstetric complications with live birth rates of 50% to 70%. There are no evidence-based guidelines for the management of these pts and most of the data come from case series of few pts. There are no validated variables that can predict outcomes in pregnant women with MPN. Objetives: Primary: To learn the incidence of thromboembolic, hemorrhagic and OC in pregnant women with MPN in Argentina. Secondary: To evaluate parameters such as mutational status, platelet count, history of complications in previous Pcy, use of aspirin (ASA), low molecular weight heparin (LMWH) or interferon (IFN) and their relationship with obstetric outcomes. Materials and methods: Retrospective evaluation of medical records of pts with diagnosis of MPN and Pcy. Quantitative variables were expressed as median and interquartile range (IQR) and qualitative variables as total number and percentage (%). Fisher's exact test was used to analyze variables and their association with events and Wilcoxon test for platelet counts. Results: A total of 30 Pcy in 23 women with a diagnosis of MPN were recorded, 20 Pcy in pts with ET, 4 Pcy in pts with PV and 6 MF pregnancies. Pcy was planned in 56.6% of cases. There were OC in 15/19 previous Pcy. The median age was 32.9 years (IC25-75% 30-36 years). Mutational status was assessed in 22 pts: 9/22 JAK2 +, 7/22 CALR +, 1/22 triple-negative, 1/22 JAK2-CALR- and MPL not performed, 6/22 JAK2- CALR and MPL without data. The overall number of live births and first trimester spontaneous abortions (SA) were 26(86%) and 4(13.3%) respectively, without cases of second (2T) and third trimester (3T) fetal loss or stillbirths. There was a case of neonatal death in a patient with MF. We detected 13 obstetric events (ET: 9/20, PV: 2/4, and MF 2/6): 4 SA, 5 cases of fetal growth retardation, 1 partial placental abruption and 3 placental hematomas. There was no significant association between JAK2 mutational state, history of complications in previous Pcy, platelet count, use of ASA, LMWH or IFN and obstetric outcomes. Two of the pts who had SA presented smoking as a cardiovascular risk factor. There was heterogeneity among hematologist regarding therapeutic management: ASA was indicated in 24 Pcy and LMWH in prophylactic doses during Pcy in 15. In 21/25 cases LMWH was indicated as postpartum prophylaxis, only in 3/21 it was extended for 6 weeks(wks). Before Pcy 17 cases received cytoreductive treatment: 2 with HU and 1 with anagrelide that were discontinued at the time Pcy was confirmed, 5 with peg IFN, and 9 conventional IFN. During Pcy 8 continued with cytoreductive treatment (6/9 conventional IFN, 2/9, pegIFN, including 1 patient previously receiving HU) and 5 initiated IFN (1/5 conventional, 4/5 pegIFN). Platelet counts in pts who did not require cytoreduction dropped from 600 x 109/L (290-1596 x 109/L) at the beginning of Pcy to 470 (240-1548) x 109/L, p=0.0068, and 423 (240-843) x 109/L, p=0.0005, in the 2T and 3T, respectively, with non-significant increase after delivery to 494 x 109/L (122-1600 x 109/L). No cases of maternal thrombosis and 2 episodes of major postpartum bleeding who required surgical intervention one of them with hysterectomy were found.The median time of delivery was at 38 wks of gestation (IC25-75% 36.5-38.5). Vaginal delivery was performed in 6 Pcy, caesarean section (CS) was required due to emergency in 2, because of lack of fetal progression in 2, and 16 were scheduled CS. The median birth weight was 2900g (IC25-75% 2500-3300g). No congenital malformations in live births were detected. Conclusions: Pcy is a rare but high-risk event in MPN pts with 11/30 OC in this cohort. The live birth rate was high compared to the literature. The risk of MC was low, without thrombotic complications and 2/30 episodes of major postpartum bleeding. There was no correlation between mutational status, platelet count or treatments received and obstetric outcomes. There was a significant reduction in the number of platelets in pts with thrombocytosis without requiring cytoreduction. The high percentage of pts who finished their pregnancy with scheduled CS is highlighted. Disclosures No relevant conflicts of interest to declare.