We acknowledge Dr. years. Because many people from southern Italy have emigrated during the last century, this mutation may have spread to other countries. gene;7 this genetic mutation results in a p.Ala156Val substitution (Bolzano mutation), which reduces the ability of GPIb to interact with vWF.8 Since 2001, we have included the search for monoallelic Bolzano mutation among the diagnostic tests for subjects with non-syndromic, inherited thrombocytopenias. We report here 103 new patients with the monoallelic Bolzano mutation and describe their clinical symptoms and laboratory findings. Moreover, we present data suggesting that this mutation originated in an ancestral individual in southern Italy. Design and Methods Patients We enrolled 216 patients in this study who were referred to the IRCCS Policlinico San Matteo Foundation of Pavia or to the Department of Pediatrics of the Second University of Naples between 2001 and 2010 because of a dominant, non-syndromic form of inherited thrombocytopenia of unknown origin. The institutional review boards of both institutions approved the study, and all patients gave written informed consent in accordance with the Declaration of Helsinki. Genetic analyses DNA isolation Genomic DNA was isolated from blood samples that were anticoagulated with ethylene-diamine-tetra-acetic acid (EDTA) EMR2 according to standard blood collection procedures. Mutation screening To detect the c.515C T transition, polymerase chain reaction (PCR) products were amplified from genomic DNA with the primers 1F (5-CCCTGCGTGGTCTTGGCA-3) and 1R MZ1 (5-ATA-GAGGATCTCACAGTTGC-3); the amplicons were subsequently digested with the restriction enzyme (Fermentas, Vilnius, Lithuania) restriction digestion and electrophoresis on a 10% acrylamide gel. Bleeding tendency The bleeding tendency was measured according to the World Health Organization (WHO) bleeding scale (grade 0, no bleeding; grade 1, petechiae; grade 2, mild blood loss; grade 3, gross blood loss; and grade 4, debilitating blood loss). Platelet count EDTA-anticoagulated blood samples were used to determine the platelet count, which was performed within 2 h of drawing blood. The Sysmex XE-2100? (Sysmex Corporation, Kobe, Japan) analyzer was used to measure the platelet count based on the impedance method. In some patients the platelet count was also measured with an ADVIA 120? counter (Bayer, Tarrytown, NY, USA), which uses two-dimensional laser lightCscatter, as well as by manual platelet counting with an optical microscopy in a Neubauer chamber as recommended by the International Committee for Standardization in Haematology.10 Platelet size The mean platelet volume was measured in parallel with the platelet count with the two analyzers described above. The platelet diameter was measured by optical microscopy on May-Grnwald-Giemsa-stained peripheral blood films, and the pictures were analyzed with Axio-vision 4.5? software (Carl Zeiss, G?ttingen, Germany).11 The blood smears were prepared from non-anticoagulated blood, and the largest diameter of each platelet was measured. The mean platelet diameter was calculated from 200 different cell measurements. Platelet aggregation To analyze platelet MZ1 aggregation, blood was collected in 3.8% (w/v) sodium citrate (blood:anticoagulant ratio 9:1). To minimize the loss of denser platelets, platelet-rich plasma (PRP) was obtained by MZ1 sedimentation of the blood at 1 g for 20 to 30 min. Platelet aggregation was evaluated by the densitometric method according to Born,12 after stimulating the PRP with.