Valeria Bafunno

Mutation of the angiopoietin-1 gene (ANGPT1) associates with a new type of hereditary angioedema

Background: Hereditary angioedema (HAE) is a rare genetic disease usually caused by mutation in the C1 inhibitor or the coagulation Factor XII gene. However, in a series of patients with HAE, no causative variants have been described, and the pathophysiology of the disease remains unknown (hereditary angioedema with yet unknown genetic defect [U‐HAE]). Identification of causative genes in patients with U‐HAE is valuable for understanding the cause of the disease. Objective: We conducted genetic studies in Italian patients with U‐HAE to identify novel causative genes. Methods: Among patients belonging to 10 independent families and unrelated index patients with U‐HAE recruited from the Italian Network for C1‐INH‐HAE (ITACA), we selected a large multiplex family with U‐HAE and performed whole‐exome sequencing. The angiopoietin‐1 gene (ANGPT1) was investigated in all patients with familial or sporadic U‐HAE. The effect of ANGPT1 variants was investigated by using in silico prediction and plasma and transfected cells from both patients and control subjects. Results: We identified a missense mutation (ANGPT1, c.807G>T, p.A119S) in a family with U‐HAE. The ANGPT1 p.A119S variant was detected in all members of the index family with U‐HAE but not in asymptomatic family members or an additional 20 patients with familial U‐HAE, 22 patients with sporadic U‐HAE, and 200 control subjects. Protein analysis of the plasma of patients revealed a reduction of multimeric forms and a reduced ability to bind the natural receptor tunica interna endothelial cell kinase 2 of the ANGPT1 p.A119S variant. The recombinant mutated ANGPT1 p.A119S formed a reduced amount of multimers and showed reduced binding capability to its receptor. Conclusion: ANGPT1 impairment is associated with angioedema, and ANGPT1 variants can be the basis of HAE.

Polymorphisms in genes involved in autoimmune disease and the risk of FVIII inhibitor development in Italian patients with haemophilia A.

Summary.  One of the most severe and important complication in the treatment of patients with haemophilia A is the formation of neutralizing antibodies (FVIII inhibitors) that inhibit the clotting activity of substituted FVIII. Both genetic and environmental factors influence the susceptibility of patients to develop inhibitors. The objective of this study was to evaluate whether polymorphisms in different genes involved in the regulation of the immune system may confer susceptibility to inhibitor development in patients with HA. We analysed the distribution of polymorphisms in the CTLA4, PTPN22, IL10, TNFα, FOXP3 and IRF5 genes that have been reported to be associated with a number of autoimmune disease. In addition, we evaluated the distribution of IL10 haplotypes in haemophilic patients and healthy controls to assess whether specific polymorphisms in IL10 gene were associated to the risk of inhibitor development. We focused on a cohort of Italian unrelated haemophilic patients with and without a history of inhibitors. Genotyping was carried out with standard methods including RFLP, real time PCR and direct DNA sequencing. Our data show that, considering single nucleotide variations, genotype frequencies in patients with inhibitors were not significantly different from those observed in patients without inhibitors, suggesting a lack of association between these polymorphisms and the development of inhibitors. Moreover, no relationship was found between specific combinations of IL10 alleles and the antibody production. Previous contradictory association studies may depend on the different genetic background of the population examined. Further studies may contribute to a clearer understanding of this process.

The JAK2 rs12343867 CC genotype frequently occurs in patients with splanchnic venous thrombosis without the JAK2V617F mutation: a retrospective study

(617V>F) mutation. Blood 2007; 110: 485–9. 6 Melillo L, Tieghi A, Candoni A, Radaelli F, Ciancia R, Specchia G, Martino B, Scalzulli PR, Latagliata R, Palmieri F, Usala E, Valente D, Valvano MR, Cedrone M, Comitini G, Martinelli V, Cascavilla N, Gugliotta L. Outcome of 122 pregnancies in essential thrombocythemia patients: a report from the Italian registry. Am J Hematol 2009; 84: 636–40. 7 Gangat N, Wolanskyj AP, Schwager S, Tefferi A. Predictors of pregnancy outcome in essential thrombocythemia: a single institution study of 63 pregnancies. Eur J Haematol 2009; 82: 350–3. 8 Tefferi A, Passamonti F. Essential thrombocythemia and pregnancy: observations from recent studies and management recommendations. Am J Hematol 2009; 84: 629–30. 9 Harrison C. Pregnancy and its management in the Philadelphia negative myeloproliferative diseases. Br J Haematol 2005; 129: 293–306. 10 CLASP (Collaborative Low-dose Aspirin Study in Pregnancy) Collaborative Group. CLASP: a randomised trial of low-dose aspirin for the prevention and treatment of pre-eclampsia among 9364 pregnant women. Lancet 1994; 343: 619–29. 11 Niittyvuopio R, Juvonen E, Kaaja R, Oksanen K, Hallman H, Timonen T, Ruutu T. Pregnancy in essential thrombocythaemia: experience with 40 pregnancies. Eur J Haematol 2004; 73: 431–6. 12 Falanga A, Marchetti M, Vignoli A, Balducci D, Russo L, Guerini V, Barbui T. V617F JAK-2 mutation in patients with essential thrombocythemia: relation to platelet, granulocyte, and plasma hemostatic and inflammatory molecules. Exp Hematol 2007; 35: 702–11. 13 Arellano-Rodrigo E, Alvarez-Larran A, Reverter JC, Villamor N, Colomer D, Cervantes F. Increased platelet and leukocyte activation as contributing mechanisms for thrombosis in essential thrombocythemia and correlation with the JAK2 mutational status.Haematologica 2006; 91: 169–75. 14 PassamontiF,RumiE,PietraD,DellaPortaMG,BoveriE,PascuttoC, VanelliL,ArcainiL,Burcheri S,MalcovatiL,LazzarinoM,CazzolaM. Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders. Blood 2006; 107: 3676–82. 15 Erez O, Gotsch F, Mazaki-Tovi S, Vaisbuch E, Kusanovic JP, Kim CJ, Chaiworapongsa T, Hoppensteadt D, Fareed J, Than NG, NhanChang CL, Yeo L, Pacora P, MazorM, Hassan SS, Mittal P, Romero R. Evidence of maternal platelet activation, excessive thrombin generation, and high amniotic fluid tissue factor immunoreactivity and functional activity in patients with fetal death. JMatern Fetal Neonatal Med. 2009; 22: 672–87. 913457925 [pii] 10.1080/14767050902853117. 16 Lok CA, Jebbink J, Nieuwland R, Faas MM, Boer K, Sturk A, van der Post JA. Leukocyte activation and circulating leukocyte-derived microparticles in preeclampsia. Am J Reprod Immunol 2009; 61: 346– 59. AJI701 [pii] 10.1111/j.1600-0897.2009.00701.x. 17 FalangaA,MarchettiM, Vignoli A, Balducci D, Barbui T. Leukocyteplatelet interaction in patients with essential thrombocythemia and polycythemia vera. Exp Hematol. 2005; 33: 523–30. 18 Vaughan JE, Walsh SW, Ford GD. Thromboxane mediates neutrophil superoxide production in pregnancy. Am J Obstet Gynecol 2006; 195: 1415–20. S0002-9378(06)00297-3 [pii] 10.1016/j.ajog.2006.02.053.

Role of the M2 haplotype within the annexin A5 gene in the occurrence of pregnancy-related venous thromboembolism

OBJECTIVE Knowledge about risk factors for venous thromboembolism (VTE) is still limited. A recently found haplotype within the natural anticoagulant protein annexin A5 (ANXA5) exerts an important modulating effect on gene expression. STUDY DESIGN Eighty-three nonanticoagulated patients with a documented pregnancy-related VTE and 195 controls were investigated. The presence of the ANXA5 haplotypes was determined. RESULTS Twenty-seven patients (32.5%) carried the M2 haplotype. Among them, 17 (63.0%) had a history of VTE in puerperium and 10 (37.0%) during pregnancy. The prevalence of the M2 haplotype was different as compared with that recorded among controls (odds ratio, 2.7; 95% confidence interval, 1.5-4.9, P < .001). A logistic regression analysis, correcting for potential confounders (age at which the thrombotic event occurred, factor V Leiden, and factor IIA20210 variants) showed a significant increase (odds ratio, 3.4; 95% confidence interval, 1.7-6.7) of the occurrence of VTE in carriers of the M2 haplotype as compared with noncarriers. CONCLUSION The M2 haplotype within the ANXA5 gene may represent a new thrombophilic risk factor for pregnancy-related VTE.

Mutational Spectrum of the C1 Inhibitor Gene in a Cohort of Italian Patients with Hereditary Angioedema: Description of Nine Novel Mutations

Hereditary angioedema (HAE) is an autosomal dominant disease due to mutations in the C1 inhibitor gene (C1NH) that affects protein synthesis (HAE type I) or function (HAE type II). In 45 subjects affected by HAE diagnosed through clinical features and C1 inhibitor deficiency from the south of Italy (38 with type I and 7 with type II HAE), the whole C1NH coding region was screened for mutations by direct DNA sequencing. A severity score based on clinical manifestation, age at disease onset, and need for long‐term prophylaxis was used to investigate possible genotype‐phenotype correlations. A series of 22 different mutations was identified: nine missense (40.9%), five nonsense (22.7%), six frameshift (27.3), one small deletion (4.5%), and one splicing defect (4.5%). Nine C1NH mutations have not been previously described. No correlation was found between C1 inhibitor function level and severity score or age at first attack. Moreover, there was no correlation between different types of mutations and clinical phenotype. The number of different mutations identified highlights the heterogeneity of C1 inhibitor deficiency and supports the hypothesis that HAE clinical phenotype is not strictly related to the type of mutation but rather depends on unknown factors.

The risk of occurrence of venous thrombosis: focus on protein Z

Protein Z (PZ) is a vitamin K-dependent factor identified in human plasma in 1984 characterized by an homology with other vitamin K-dependent factors. PZ acts as the cofactor of the PZ dependent inhibitor (ZPI), in the inhibition of activated factor X bound on phospholipid surface. In humans, PZ is characterized by an unusual wide distribution in plasma partly explained by a genetic control. Several PZ gene polymorphisms influencing plasma concentration have been described. In mice, the disruption of PZ gene is asymptomatic, but in association with homozygous FV Leiden produced a severe prothrombotic phenotype. This review analyzes the results obtained from different studies so far published in order to understand whether PZ deficiency could be considered as a risk factor for venous thrombosis. The roles of PZ plasma level and PZ gene polymorphisms remain debated with conflicting results. Many of these studies reported low PZ levels in association with an increased risk of venous thrombosis. On the other side, some studies did not observe an association between low levels of PZ and thrombotic events. A relationship between PZ deficiency and pregnancy complications was also described but not confirmed by all studies. These discrepancies can be explained by the heterogeneity of populations chosen as control, by the PZ interindividual variability and by the small size of the cohorts in mainly retrospective studies. Large prospective studies remain to be done to investigate its possible role in thrombosis.

Characterization of patients with angioedema without wheals: The importance of F12 gene screening

Sporadic and familiar forms of non-histaminergic angioedema and normal C1 inhibitor encompass a group of disorders possibly caused by bradikinin. We aimed to study the subgroups of hereditary angioedema with FXII mutation (FXII-HAE), unknown genetic defect (U-HAE) and idiopathic non-histaminergic acquired angioedema (InH-AAE). We screened the F12 locus in our cohort and delineated the clinical, laboratory and genetic features. Four families carried the p.Thr309Lys mutation in F12 gene. Haplotyping confirmed the hypothesis of a common founder. Six families were affected by U-HAE and 13 patients by sporadic InH-AAE. C4 levels were significantly lower in FXII-HAE than in InH-AAE. In the FXII-HAE group, none had attacks exclusively in high estrogenic states; acute attacks were treated with icatibant. Prophylaxis with tranexamic acid reduced the attack frequency in most patients. Our study provides new data on the diagnosis, clinical features and treatment of non-histaminergic angioedema, underlying the role of the screening for F12 mutations.

Sex modulation of the occurrence of jak2 v617f mutation in patients with splanchnic venous thrombosis.

BACKGROUND The JAK2 V617F mutation is an independent risk factor for MPN and SVT. Gender-related differences in MPN distribution have been reported and, recently, variability in the JAK2 V617F allele burden between sexes has been suggested. We wondered whether gender would modulate the role of the JAK2 V617F mutation as susceptibility risk factor for SVT. MATERIALS AND METHODS In 180 patients presenting with SVT, medical history was collected. The presence of the JAK2 V617F mutation and 46/1 haplotype was determined by polymerase chain reaction followed by TaqMan SNP genotyping assays. RESULTS Among patients with SVT, 43 (23.9%; 95%-CI: 18.2-30.7) carried the JAK2 V617F mutation. The JAK2 V617F mutation was found more frequently in women (29/95: 30.5%; 95%-CI: 22.1-40.4) than in men (14/85: 16.5%; 95%-CI: 10.0-25.9; OR: 2.2; 95%-CI: 1.1-4.5). The distribution of 46/1 haplotype frequencies did not differ significantly between men and women. In women carrying the rs12343867 CC genotype, the frequency observed for the occurrence of the V617F mutation was significantly higher than that observed in those not carrying (60.0% [95% CI: 31.2-83.3] vs. 26.8% [95% CI: 18.4-37.4]; OR: 4.1; 95%-CI: 1.1-14.9). In men, a similar prevalence was found among carriers of the rs12343867 CC genotype (16.7% [95% CI: 3.5-46.0]) and in non carriers (16.4% [95% CI: 9.3-27.2]). The V617F allele burden was unrelated to clinical characteristics and significantly higher in carriers of the rs12343867 CC genotype. CONCLUSIONS Present findings suggest that, in patients presenting with SVT, the JAK2 V617F mutation is frequently found in women and, possibly by interacting with the 46/1 haplotype, may represent a gender-related susceptibility allele for SVT.

New TET2 gene mutations in patients with myeloproliferative neoplasms and splanchnic vein thrombosis

1 Roberts HR, Escobar M, White GC. Hemophilia A and Hemophilia B. In: Lichtman MA, Beutler E, Kaushansky K, Kipps TJ, Seligsohn U, Prchal J, eds. Williams Hematology. New York: McGraw-Hill, 2006: 1867–86. 2 NicholsWC, AmanoK, Cacheris PM, FigueiredoMS,Michaelides K, Schwaab R, Hoyer L, Kaufman RJ, Ginsburg D. Moderation of hemophilia A phenotype by the factor VR506Qmutation.Blood 1996; 88: 1183–7. 3 Arbini AA,Mannucci PM, Bauer KA. Low prevalence of the factor V Leiden mutation among ‘‘severe’’ hemophiliacs with a ‘‘milder’’ bleeding diathesis. Thromb Haemost 1995; 74: 1255–8. 4 Alberio L, Safa O, Clemetson KJ, Esmon CT, Dale GL. Surface expression and functional characterization of alpha-granule factor V in human platelets: effects of ionophore A23187, thrombin, collagen and convulxin. Blood 2000; 95: 1694–702. 5 Dale GL, Friese P, Batar P, Hamilton SF, Reed GL, Jackson KW, Clemetson KJ, Alberio L. Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface. Nature 2002; 415: 175–9. 6 Dale GL. Coated-platelets: an emerging component of the procoagulant response. J Thromb Haemostasis 2005; 3: 2185–92. 7 Remenyi G, Szasz R, Friese P, Dale GL. Role of mitochondrial permeability transition pore in coated-platelet formation. Arterioscler Thromb Vasc Biol 2005; 25: 467–71. 8 Jobe SM, Leo L, Eastvold JS, Dickneite G, Ratliff TL, Lentz SR, Di PJ. Role of FcR-gamma and factor XIIIA in coated platelet formation. Blood 2005; 106: 4146–51. 9 Brooks MB, Catalfamo JL, Friese P, Dale GL. Scott syndrome dogs have impaired coated-platelet formation and calcein release but normal mitochondrial depolarization. J Thromb Haemostasis 2007; 5: 1972–4. 10 Jobe SM, Wilson KM, Leo L, Raimondi A, Molkentin JD, Lentz SR, Di Paola J. Critical role for the mitochondrial permeability transition pore and cyclophilin D in platelet activation and thrombosis. Blood 2008; 111: 1257–65. 11 Prodan CI, Joseph PM, Vincent AS, Dale GL. Coated-platelets in ischemic stroke: differences between lacunar and cortical stroke. J Thromb Haemostasis 2008; 6: 609–14. 12 Prodan CI, Joseph PM, Vincent AS, Dale GL. Coated-platelet levels are influenced by smoking, aspirin and selective serotonin reuptake inhibitors. J Thromb Haemostasis 2007; 5: 2149–51. 13 Valaydon ZS, Lee P, Dale GL, Januszewski AS, Rowley KG, Nandurkar H, Karschimkus C, Best JD, Lyons TJ, Jenkins AJ. Increased coated-platelet levels in chronic haemodialysis patients. Nephrology 2009; 14: 148–54. 14 Prodan CI, Vincent AS, Padmanabhan R, Dale GL. Coated-platelet levels are low in patients with spontaneous intracerebral hemorrhage. Stroke 2009; 40: 2578–80. 15 Prodan CI, Ross ED, Vincent AS, Dale GL. Coated-platelets are higher in amnestic versus non-amnestic patients with mild cognitive impairment. Alzheimer Dis Assoc Disord 2007; 21: 259–61.

Fatal pulmonary thromboembolism. A retrospective autopsy study: Searching for genetic thrombophilias (Factor V Leiden (G1691A) and FII (G20210A) gene variants) and dating the thrombus

The accuracy of antemortem diagnosis of pulmonary embolism is within the range of just 10-30%, so representing one of the most frequent missed diagnosis in sudden, unexpected death. We describe 43 fatal cases of pulmonary embolism as confirmed by post-mortem examination. The aim of our study was to verify the systematic search for the most common genetic thrombophilias (Factor V Leiden (G1691A) and FII (G20210A) gene variants) and dating the thrombus. As a whole, 41 patients (95.3%) had at least one risk factor. Pre-existing symptoms are described just before fatal embolism in 18 (41.9%) out 43 patients. In 18 out of 43 (41.9%) it was not possible to find the thrombotic site. In 24 out of the remaining 25 cases the involvement of the deep veins of one leg was shown; in 1 case the thrombus was localised in the inferior caval vein. 10 (41.7%) were iliac vein thromboses, 7 (29.1%) femoral, 2 (8.3%) popliteal, 3 (12.6%) posterior-tibial, 1 (4.1%) anterior-tibial and 1 (4.1%) peroneal vein thromboses. In our cohort of patients, 4 (10%) out of 40 cases carried the 20210A prothrombin gene variant in heterozygosis. One (2.5%) out of 40 carried the Factor V Leiden (G1691A) gene variant in heterozygosis. Patients carrying these gene variants in homozygosis or carrying both were not present in our case-series. We strongly underline the relevance of a complete methodological approach, integrating clinical data by means of autopsy findings and histological study. On the contrary, investigating common inherited thrombophilia is not warranted.