Dominant inheritance of a novel integrin beta3 mutation associated with a hereditary macrothrombocytopenia and platelet dysfunction in two Italian families.

BACKGROUND: Defects of integrin alpha(IIb)beta(3) are typical of Glanzmann's thrombasthenia, an inherited autosomal recessive bleeding disorder characterized by the failure of platelets to aggregate in response to all physiological agonists, but with no abnormalities in the number or size of platelets. Although large heterogeneity has been described for Glanzmann's thrombasthenia, no family has so far been described as having an autosomal dominant form of this disease. DESIGN AND METHODS: We describe two Italian families with moderate thrombocytopenia with large platelets, defective platelet function and moderate/severe mucocutaneous bleeding, transmitted as an autosomal dominant trait and associated with a novel integrin beta(3)-gene (ITGB3) mutation. RESULTS: The characteristics of our families are moderate macrothrombocytopenia and defective platelet function associated with a mild reduction of surface alpha(Ib) beta(3), impaired platelet aggregation to physiological agonists but not to ristocetin, normal clot retraction, reduced fibrinogen binding and expression of activated alpha(IIb)beta(3) upon stimulation, normal platelet adhesion to immobilized fibrinogen but reduced platelet spreading and tyrosine phosphorylation, indicating defective alpha(IIb)beta(3)-mediated outside-in signaling. Molecular analysis revealed a novel mutation of ITGB3 that determines an in-frame deletion producing the loss of amino acids 647-686 of the betaTD ectodomain of integrin beta(3). Haplotype analysis indicated that the two families inherited the mutation from a common ancestral chromosome. CONCLUSIONS: This novel autosomal dominant macrothrombocytopenia associated with platelet dysfunction raises interesting questions about the role of integrin beta(3), and its betaTD domain, in platelet formation and function.

Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease.

MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. No consistent correlations have been identified between the 27 different MYH9 mutations identified so far and the variable clinical evolution of the disease. We have evaluated 108 consecutive MYH9-RD patients belonging to 50 unrelated pedigrees. The risk of noncongenital manifestations associated with different genotypes was estimated over time by event-free survival analysis. We demonstrated that all subjects with mutations in the motor domain of NMMHC-IIA present with severe thrombocytopenia and develop nephritis and deafness before the age of 40 years, while those with mutations in the tail domain have a much lower risk of noncongenital complications and significantly higher platelet counts. We also evaluated the clinical course of patients with mutations in the four most frequently affected residues of NMMHC-IIA (responsible for 70% of MYH9-RD cases). We concluded that mutations at residue 1933 do not induce kidney damage or cataracts and cause deafness only in the elderly, those in position 702 result in severe thrombocytopenia and produce nephritis and deafness at a juvenile age, while alterations at residue 1424 or 1841 result in intermediate clinical pictures. These findings are relevant not only to patients' clinical management but also to the elucidation of the pathogenesis of the disease.

Congenital amegakaryocytic thrombocytopenia: clinical and biological consequences of five novel mutations.

BACKGROUND AND OBJECTIVES: Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare, autosomal recessive disorder induced by mutations of the gene coding for thrombopoietin (TPO) receptor (c-MPL). Patients initially present with isolated thrombocytopenia that subsequently progresses into pancytopenia. Although the mechanisms leading to aplasia are unknown, the age of onset has been reported to depend on the severity of the c-MPL functional defect. To improve our knowledge in this field, we studied clinical and biological features of five new patients. DESIGN AND METHODS: We diagnosed five CAMT patients, identified c-MPL mutations, including five novel alterations and investigated relationships between mutations and their clinical-biological consequences. RESULTS: In all cases, platelet c-MPL and bone marrow colonies were reduced, while serum TPO levels were elevated. We also documented that the percentage of bone marrow cells expressing tumor necrosis factor-a and interferon-g was increased during pancytopenia as compared to in controls, suggesting that, as in other bone marrow failure diseases, these inhibitory cytokines contributed to the pancytopenia. Contrary to previously published data, we found no evidence of correlations between different types of mutations and the clinical course. INTERPRETATION AND CONCLUSIONS: These results suggest that therapies, such as hematopoietic stem cell transplantation, which are potentially curative although associated with a risk of treatment-related mortality, should not be postponed even in those CAMT patients whose c-MPL mutations might predict residual activity of the TPO receptor.

Cleft lip with or without cleft palate: implication of the heavy chain of non-muscle myosin IIA.

Non-syndromic cleft lip with or without palate (CL/P) is one of the most common malformations among live births, but most of the genetic components and environmental factors involved remain to be identified. Among the different causes, MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA, was considered a potential candidate, because it was found to be abundantly and specifically expressed in epithelial cells of palatal shelves before fusion. After fusion, its expression level was shown to decrease and to become limited to epithelial triangles before disappearing, as fusion is completed. To determine whether MYH9 plays a role in CL/P aetiology, a family-based association analysis was performed in 218 case/parent triads using single-nucleotide polymorphism (SNP) markers. Pairwise and multilocus haplotype analyses identified linkage disequilibrium between polymorphism alleles at the MYH9 locus and the disease. The strongest deviation from a null hypothesis of random sharing was obtained with two adjacent SNPs, rs3752462 and rs2009930 (global p value = 0.001), indicating that MYH9 might be a predisposing factor for CL/P, although its pathogenetic role needs to be investigated more accurately.

Dissecting clinical findings: platelet defects segregate independently of deafness and cataract in a family affected by an apparent syndromic form of macrothrombocytopenia.

We studied a family with a suspected diagnosis of MYH9-related disease, which is one of the most common forms of autosomal dominant macrothrombocytopenias associated with hearing impairment, cataracts and nephritis. No mutation of the MYH9 gene was identified. Moreover, the A156V variant of the GPIbalpha gene, responsible for 30% of macrothrombocytopenias in Italy, was not detected in the family. Therefore, we hypothesized that the clinical symptoms were caused by mutations in different genes. The screening of the candidate genes for deafness and/or cataract allowed us to identify two variants, M34T and S19T, of the GJB2 gene in family members with hearing impairment. Because of the relatively common occurrence of inherited hearing loss and, at least in the Mediterranean area, of platelet macrocytosis, the two traits occurred by chance in the same family and mimicked the MYH9-related disease.

Application of a diagnostic algorithm for inherited thrombocytopenias to 46 consecutive patients.

BACKGROUND AND OBJECTIVES: The Italian Gruppo di Studio delle Piastrine recently developed a diagnostic algorithm to assist clinicians in the diagnosis of inherited thrombocytopenias. This algorithm is based on the simplest possible diagnostic investigations and can also be used in centers that are not highly specialized. The aim of the present study was to validate this diagnostic algorithm by applying it to a case series of genetic thrombocytopenias. DESIGN AND METHODS: The diagnostic algorithm was applied retrospectively to 46 consecutive patients observed during the last five years at a single institution. Twenty-eight were affected by defined illnesses or their variants, while 18 had a disorder that did not fit the criteria for any known genetic thrombocytopenia. The study was based on the evaluation of clinical records and laboratory tests. RESULTS: The diagnostic algorithm recognized: 4 homozygous and 4 heterozygous Bernard-Soulier syndromes, 11 MYH9-related diseases, one von WillebrandOs disease type 2B, one gray platelet syndrome and one X-linked thrombocytopenia with thalassemia. Moreover, it identified 4 patients with the clinical and laboratory features of heterozygous Bernard-Soulier syndrome not caused by mutations in the coding region of the GPIbalpha, GPIbbeta, GPIX or GPV genes, and two patients with the clinical phenotype of MYH9-related disease but without MYH9 mutations. Since the diagnostic flow chart did not allow prompt recognition of two subjects with MYH9-related disease, we introduced a small change to the previously proposed flow chart to obviate this defect. INTERPRETATION AND CONCLUSIONS: The diagnostic algorithm correctly diagnosed 26 of 28 patients with known disorders or phenotypic variants of known disorders. By a simple modification of the investigation sequence, its sensitivity reached 100%. The algorithm also identified 18 patients with new, as yet uncharacterized forms of genetic thrombocytopenia.

MYH9-related disease: May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but represent a variable expression of a single illness.

May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are autosomal dominant macrothrombocytopenias distinguished by different combinations of clinical and laboratory signs, such as sensorineural hearing loss, cataract, nephritis, and polymorphonuclear Döhle-like bodies. Mutations in the MYH9 gene encoding for the nonmuscle myosin heavy chain IIA (NMMHC-IIA) have been identified in all these syndromes. To understand the role of the MYH9 mutations, we report the molecular defects in 12 new cases, which together with our previous works represent a cohort of 19 families. Since no genotype-phenotype correlation was established, we performed an accurate clinical and biochemical re-evaluation of patients. In addition to macrothrombocytopenia, an abnormal distribution of NMMHC-IIA within leukocytes was observed in all individuals, including those without Döhle-like bodies. Selective, high-tone hearing deficiency and cataract was diagnosed in 83% and 23%, respectively, of patients initially referred as having May-Hegglin anomaly or Sebastian syndrome. Kidney abnormalities, such as hematuria and proteinuria, affected not only patients referred as Fechtner syndrome and Epstein syndrome but also those referred as May-Hegglin anomaly and Sebastian syndrome. These findings allowed us to conclude that May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but rather a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to a severe form complicated by hearing loss, cataracts, and renal failure. For this new nosologic entity, we propose the term "MHY9-related disease," which better interprets the recent knowledge in this field and identifies all patients at risk of developing renal, hearing, or visual defects.

Epstein syndrome: another renal disorder with mutations in the nonmuscle myosin heavy chain 9 gene.

Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing loss, and thrombocytopenia with giant platelets. Renal and hearing abnormalities are indistinguishable from those observed in Fechtner syndrome (FTNS), an Alport-like variant. EPTS macrothrombocytopenia is similar to that described in FTNS, May-Hegglin anomaly (MHA), and Sebastian syndrome (SBS), three disorders caused by mutations in the nonmuscle heavy chain myosin IIA ( MYH9). Unlike FTNS, MHA, and SBS, EPTS does not show inclusion bodies in the leukocytes. The clinical features of EPTS and the chromosomal localization of the respective gene in the same region as MYH9 suggest that this disorder is allelic with the other giant platelet disorders. We identified a MYH9 missense mutation in two EPTS familial cases. In both families, an R702H substitution was found, probably inducing conformational changes to the myosin head. A different amino acid substitution at the same codon (R702C) has been previously identified in FTNS. On the basis of predictions from molecular modeling of the X-ray crystallographic structure of chick smooth muscle myosin, the mutated thiol reactive group of R702C may lead to intermolecular disulfide bridges, with the consequent formation of the inclusions typical of FTNS. On the contrary, the R702H mutation does not allow the protein to aggregate and thus to generate "Döhle-like" bodies, which are indeed absent in EPTS. In conclusion, our results extend the allelic heterogeneity of MYH9 mutations to another clinical syndrome and contribute to the clarification of the pathogenesis of the various inherited giant platelet disorders.