P.E1549K: A NEW MUTATION RESPONSIBLE OF VON WILLEBRAND DISEASE TYPE 2M

WOODS AI; SÁNCHEZ LUCEROS A; KEMPFER AC; BERMEJO EI; CALDERAZZO JC; BLANCO AN; MESCHENGIESER SS; LAZZARI MA

LIVERPOOL

Congreso; 58th Annual Meeting of the Scientific and Standardization Committee of the ISTH; 2012

INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS

Objectives: von Willebrand disease 2M variant (VWD2M) is characterized by a loss of VWF binding to GP1b, VWF:RCo/VWF:Ag<0,6, absent 1.2 mg/mL ristocetin induced platelet aggregation (RIPA) and normal multimers.von Willebrand disease 2M variant (VWD2M) is characterized by a loss of VWF binding to GP1b, VWF:RCo/VWF:Ag<0,6, absent 1.2 mg/mL ristocetin induced platelet aggregation (RIPA) and normal multimers. Methods: We genotypically studied 9 available patients (5 females) out of 23 members of a family diagnosed as VWD2M with mucocutaneous bleeding history. Bleeding score (BSS) (ISHT) was calculated. Following phenotypic studies were made: bleeding time (BT Ivy), platelet count, FVIII:C, VWF:Ag, VWF:RCo, multimeric profile, 1.2, 1.5 and 2 mg/mL RIPA, VWFpp/VWF:Ag. The exon 28 of the patients and 100 healthy donnors was amplified by PCR in fragments, and sequenced (ABI Prism 310). We designed primers avoiding pseudogen amplification.We genotypically studied 9 available patients (5 females) out of 23 members of a family diagnosed as VWD2M with mucocutaneous bleeding history. Bleeding score (BSS) (ISHT) was calculated. Following phenotypic studies were made: bleeding time (BT Ivy), platelet count, FVIII:C, VWF:Ag, VWF:RCo, multimeric profile, 1.2, 1.5 and 2 mg/mL RIPA, VWFpp/VWF:Ag. The exon 28 of the patients and 100 healthy donnors was amplified by PCR in fragments, and sequenced (ABI Prism 310). We designed primers avoiding pseudogen amplification. Results: Phenotypic studies: all the patients had prolonged BT, normal platelet count, normal or slightly low FVIII:C and VWF:Ag. VWF:RCo was <10 IU/dL in 6 patients and within 10-20 IU/dL in three. Normal multimers. 1.2 and 1.5mg/mL RIPA absent; 2 mg/mL RIPA normal. BSS: males= 3.7±2.1; females= 4.8±1.5. VWFpp/VWF:Ag was within normal range. The heterozygous g.4645G>A substitution resulting in p.E1549K was found in the 9 patients but not in normals. This new missense mutation was associated with VWD2M. It was reported by us, to the ISTH SSC VWF database. In silico analysis was performed by using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) to search for possible impact of the amino acid substitution on the structure and function of the VWF. According to this, the p.E1549K was predicted to be probably damaging with score of 0.999 (sensitivity: 0.14; specificity: 0.99).Phenotypic studies: all the patients had prolonged BT, normal platelet count, normal or slightly low FVIII:C and VWF:Ag. VWF:RCo was <10 IU/dL in 6 patients and within 10-20 IU/dL in three. Normal multimers. 1.2 and 1.5mg/mL RIPA absent; 2 mg/mL RIPA normal. BSS: males= 3.7±2.1; females= 4.8±1.5. VWFpp/VWF:Ag was within normal range. The heterozygous g.4645G>A substitution resulting in p.E1549K was found in the 9 patients but not in normals. This new missense mutation was associated with VWD2M. It was reported by us, to the ISTH SSC VWF database. In silico analysis was performed by using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) to search for possible impact of the amino acid substitution on the structure and function of the VWF. According to this, the p.E1549K was predicted to be probably damaging with score of 0.999 (sensitivity: 0.14; specificity: 0.99). Conclusion: We described here a new heterozygous missense substitution responsible for VWD2M phenotype. Given that the substitution was not found in the 200 normal alleles and considered as probably damaging by Polyphen-2, we consider it is not a polymorphism.We described here a new heterozygous missense substitution responsible for VWD2M phenotype. Given that the substitution was not found in the 200 normal alleles and considered as probably damaging by Polyphen-2, we consider it is not a polymorphism.