Caratterizzazione Molecolare delle Macrotrombocitopenie Ereditarie a Trasmissione Autosomica Dominante

Patients with an autosomal dominant inheritance of macrothrombocytopenia and not fitting any other known classification such as Myosin Related Diseases (MRD) or Bernard-Soulier Syndrome (BSS), were in the past years assigned to the group of Mediterranean forms, that we also named as Chronic Isolated Macrothrombocytopenia (CHMT). We performed a systematic review of patients with Autosomal Dominant Macrothrombocytopenia in order to better characterize the disease and investigate the molecular defects responsible for the unclassified forms of macrothrombocytopenia. This work was carried out with the new methods (flow cytometry, genetic tests) and diagnostic tools (specific monoclonal antibodies - mAbs) now available also in our laboratory. Here we described the results obtained from the investigations performed on 5 families (TP1, TP2, TP3, TP4, TP5). Family TP1. The propositus was a 49 years old man, whose thrombocytopenia was at the beginning misdiagnosed as ITP. Some year later, his daughter also was recognized as affected. May-Grunwald staining of peripheral blood and Ristocetin aggregation of both patients didn't showed any defect, and they were fist assigned to the CHMT group. When the new methods were implemented, the immunofluorescent staining with mAbs anti Non Muscular Myosin Heavy Chain (NMMCH-IIA) revealed in patients' neutrophil granulocytes an abnormal cytoplasmic distribution of the protein, which is a typical sign of MRD, one of the most common hereditary macrothrombocytopenia. Genetic analysis revealed in these patients a heterozygous mutation R1162T in the exon 25 of the myh9 gene (coding for the NMMCH-IIA) never described before. After these results, the diagnosis for TP1 family was changed in MRD. Family TP2. The proposita, her father and her sister showed lifelong story of macrothrombocytopenia without spontaneous bleeding. Only the proposita refers metrorrhagia without other hemorrhagic symptoms. The immunostaining of blood smears with anti NMMCH-IIA mAbs showed normal distribution in the proposita, while in her sister, different experiments revealed an ambiguous distribution of the protein. Sequencing analysis of the myh9 exons seat of known mutation gave negative results. So we performed on both patients flow cytometry tests in order to study platelets surface glycoproteins expression, and exclude BSS deriving from their anomalies, but these tests also didn't revealed any abnormality. Sequence analysis of GPIba chain of GPIb/V/IX complex (von Willebrand - vWF - Receptor), in which are located the most of the mutations responsible for BSS was normal. TP2 at the moment remains assigned to the group of uncharacterized forms of CHMT, and the investigations will continue with the sequencing of the remaining exons of myh9 gene and the other chains of vWF receptor in order to definitively exclude MRD and BSS. Family TP3. The propositus was a 64 year-old man with no hemorrhagic history. His mother and his son was thrombocytopenic too. In the first test performed in the propositus, Ristocetin aggregation (RIPA) at 1,5 mg/ml was normal, as well as MRD-oriented tests. This lead us toward a provisional diagnosis of CHMT. Conversely, RIPA was reduced lowering the agonist at 1,2 mg/ml. Moreover, in propositus'affected son this test resulted completely impaired both at 1,5 and 1,2 mg/ml. This suggested for this family the diagnosis of BSS, which was confirmed by flow cytometry data on GPIba mAbs binding, reduced in the father and completely absent in the son. Genetic analysis showed a four bases deletion (TGAG) in gpIba gene already described in a case of compound heterozygosis, bearing a putative truncated protein of 50 aminoacids. Our patient showing absence of GPIba was the first exemple described of homozygous state of this TGAG deletion. It is interesting to note that after genetical screening, also the unaffected mother of the homozygous subject (she and her husband are consanguineous of 3rd generation) carried the mutation at heterozygous state. This result underlines that the autosomal dominance of the trait shows an incomplete penetrance in one of the heterozygous and consanguineous carriers. Family TP4 and TP5. Following the previous scheme, we found that the affected members of this two unrelated families were actually heterozygous BSS patients. Sequencing analysis in this cases revealed a novel mutation, a heterozygous A>C transversion at nucleotide +169, resulting in an N41H substitution in the GPI? protein sequence. The amino acid substitution, named Padova variant of BSS, is located in the first leucine-rich repeat (LRR) of the protein. Replacement of the asparagine 41 with a histidine (N41H) drastically disturbs the conformational behavior of the first portion of the N-terminal region of GPIb?, which is directly involved in vWF binding. In fact, the mutant N41H lost two of the three stabilizing interactions during the molecular dynamics simulation. The two unrelated families described here represent a form of heterozygous BSS with an autosomal dominant inheritance never described before. We can conclude that the new diagnostic tools allowed to better characterize our patients affected by Autosomal Dominant Macrothrombocytopenia. Four Families out of five were assigned to the more characterized areas of MRD (1 Family) and BSS (3 Families), but the defects found responsible for the disease are new mutations (families TP1, TP4 and TP5) or are present in an homozygous condition (Family TP3) never described before. One Family (TP2) remains to be characterized and will be matter of further investigations. The study also allowed to identified conformational mAbs able to differentiate the newly identified BSS-Padova Variant, from other BSS forms.