Autosomal Dominant Macrothrombocytopenia Research Articles

αIIbβ3 variants in ten families with autosomal dominant macrothrombocytopenia: Expanding the mutational and clinical spectrum.

BackgroundRare pathogenic variants in either the ITGA2B or ITGB3 genes have been linked to autosomal dominant macrothrombocytopenia associated with abnormal platelet production and function, deserving the designation of Glanzmann Thrombasthenia-Like Syndrome (GTLS) or ITGA2B/ITGB3-related thrombocytopenia.ObjectivesTo describe a series of patients with familial macrothrombocytopenia and decreased expression of αIIbβ3 integrin due to defects in the ITGA2B or ITGB3 genes.MethodsWe reviewed the clinical and laboratory records of 10 Portuguese families with GTLS (33 patients and 11 unaffected relatives), including the functional and genetic defects.ResultsPatients had absent to moderate bleeding, macrothrombocytopenia, low αIIbβ3 expression, impaired platelet aggregation/ATP release to physiological agonists and low expression of activation-induced binding sites on αIIbβ3 (PAC-1) and receptor-induced binding sites on its ligand (bound fibrinogen), upon stimulation with TRAP-6 and ADP. Evidence for constitutive αIIbβ3 activation, occurred in 2 out of 9 patients from 8 families studied, but also in 2 out of 12 healthy controls. We identified 7 missense variants: 3 in ITGA2B (5 families), and 4 in ITGB3 (5 families). Three variants (αIIb: p.Arg1026Trp and p.Arg1026Gln and β3: p.Asp749His) were previously reported. The remaining (αIIb: p.Gly1007Val and β3: p.Thr746Pro, p.His748Pro and p.Arg760Cys) are new, expanding the αIIbβ3 defects associated with GTLS. The integration of the clinical and laboratory data allowed the identification of two GTLS subgroups, with distinct disease severity.ConclusionsPreviously reported ITGA2B and ITGB3 variants related to thrombocytopenia were clustered in a confined region of the membrane-proximal cytoplasmic domains, the inner membrane clasp. For the first time, variants are reported at the outer membrane clasp, at the transmembrane domain of αIIb, and at the membrane distal cytoplasmic domains of β3. This is the largest single-center series of inherited macrothrombocytopenia associated with αIIbβ3 variants published to date.

First case report of perioperative management in a novel entity of macrothrombocytopenia: the autosomal dominant macrothrombocytopenia with platelet dysfunction associated with reduction of surface αIIbβ3

Autosomal dominant macrothrombocytopenia with platelet dysfunction associated with reduction of surface αIIbβ3 (ADM) is an autosomal dominant form of Glanzmann’s thrombasthenia reported in few families in the literature. This case presents the first report of perioperative approach of a patient with ADM successfully submitted to a surgical procedure. The multidisciplinary approach including immunohaemotherapy consultation was considered the best clinical approach to avoid bleeding. The use of desmopressin and tranexamic acid in the preoperative period followed by desmopressin plus aminocaproic acid in the postoperative period showed to be safe and effective in the perioperative approach of the patient with ADM.

ACTN1 mutations lead to a benign form of platelet macrocytosis not always associated with thrombocytopenia

The inherited thrombocytopenias (IT) are a heterogeneous group of diseases resulting from mutations in more than 30 different genes. Among them, ACTN1-related thrombocytopenia (ACTN1-RT; Online Mendelian Inheritance in Man: 615193) is one of the most recently identified forms. It has been described as a mild autosomal dominant macrothrombocytopenia caused by mutations in ACTN1, a gene encoding for one of the two non-muscle isoforms of α-actinin. We recently identified seven new unrelated families with ACTN1-RT caused by different mutations. Two of them are novel missense variants (p.Trp128Cys and p.Pro233Leu), whose pathogenic role has been confirmed by invitro studies. Together with the 10 families we have previously described, our cohort of ACTN1-RT now consists of 49 individuals carrying ACTN1 mutations. This is the largest case series ever collected and enabled a critical evaluation of the clinical aspects of the disease. We concluded that ACTN1-RT is the fourth most frequent form of IT worldwide and it is characterized by platelet macrocytosis in all affected subjects and mild thrombocytopenia in less than 80% of cases. The risk of bleeding, either spontaneous or upon haemostatic challenge, is negligible and there are no other associated defects, either congenital or acquired. Therefore, ACTN1-RT is a benign form of IT, whose diagnosis provides affected individuals and their families with a good prognosis.

Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia

AbstractThe von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibβ, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB. By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.

An Autosomal-Recessive GFI1B Mutation Defines the Splice Isoform p37 As Essential for Biogenesis of Functional Human Platelets, but Dispensable for Erythropoiesis

Growth factor independent 1 (GFI1) and Growth Factor Independent 1B (GFI1B) are zinc finger transcriptional repressors that share about 90% homology on amino acid sequence and are expressed during hematopoiesis. While GFI1 is most important for granulocyte-monocyte lineage commitment, GFI1B is an essential master regulator of erythroid and megakaryocytic lineages. Mice lacking Gfi1b are embryonic lethal due to anemia and thrombocytopenia. In humans, alternative splicing leads to a shorter p32 isoform that lacks the first 2 of 6 zinc fingers. GFI1B germline mutations have been reported to cause autosomal-dominant macrothrombocytopenia with a grey-platelet syndrome phenotype, implying that the mutant protein acts in a dominant-negative manner.We report on a Chechen family from eastern Georgia whose affected family members all present with severe, life-threatening bleeding diathesis. The female index patient had recurrent hematomata and multiple petechiae since childhood. Both of her children (age 9 and 7) present with very low platelet counts (below 45/nL) and a similar cutaneous bleeding pattern like her mother. The brother also had thrombocytopenia and died at age 33 in reponse to a spontaneous cerebral hemorrhage. In contrast, the index patient's husband, her parents and the children of the deceased brother were clinically unaffected.Blood smears of affected patients showed macrothrombocytopenia with reduced May-Grünwald-Giema staining and decreased staining for alpha-granule markers von Willebrand factor (vWF) and P-selectin (CD62P). Platelet function testing revealed reduced responses to ADP, collagen, TRAP-6 and arachidonic acid. White and red blood cell parameters were overall normal in the index patient and the two affected children. We analyzed DNA from the index patient by targeted next generation sequencing for 59 genes relevant for platelet formation or function. We found a novel homozygous single nucleotide insertion in GFI1B (NM_004188.5; c.551insG), which was confirmed by Sanger sequencing and is expected to cause a premature stop-codon. The homozygous mutation co-segregated with the phenotype. The unaffected mother, the husband and two unaffected nephews were heterozygous, suggesting a local founder variant and an unexpected autosomal-recessive trait. Bone marrow analysis showed unaffected myeloid and erythroid cells, but dysplastic micromegakaryocytes with increased CD34 staining. Peripheral blood platelets were also positive for CD34. We performed quantitative real-time PCR of platelet RNA and found residual homozygous c.551_G insertion in the p37 transcript and an unexpected expression of the p32 variant. The p37 transcript was markedly reduced in context with an increased p32/p37 ratio compared to controls. Our findings indicate that the mutated transcript was not completely degraded by nonsense-mediated decay, but mostly subjected to alternative splicing skipping the mutated exon 9.Our findings imply that the first two zinc fingers of GFI1B are dispensable for human erythropoiesis, but essential for normal megakaryopoiesis and the production of functional platelets. While previous mutations affect both isoforms, the insertion variant presented here, results in a premature stop-codon and affects only the p37 isoform due to alternative splicing. This splice variant defines an important node at the megakaryocytic-erythroid progenitor stage and we conclude that the transcriptional regulation of erythropoiesis is uncoupled from that of megakaryopoiesis through alternative splicing of GFI1B. DisclosuresNo relevant conflicts of interest to declare.

Functional Characterization of a Novel GFI1B Mutation Causing Congenital Macrothrombocytopenia

▪Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of disorders characterized by abnormally large platelets and low platelet counts. So far, genes responsible for CMTP have been identified in approximately half of the cases. The genes currently known are MYH9, GP1BA, GP1BB, GP9, ITGA2B, ITGB3, ACTN1 and other less frequent genes. However, in the remaining half, CMTP-causing genes are unknown. To identify genes causing CMTP, we first performed whole exome sequencing and target sequencing in 46 Japanese CMTP families and identified 1 family with a heterozygous mutation in GFI1B gene. Several groups have recently reported GFI1B as a causative gene for CMTP with alpha-granule deficiency and red cell anisopoikilocytosis. Here, we report on the identification of a novel mutation in GFI1B gene and its functional characterization.We performed whole exome sequencing and additional targeted sequencing in genetically undefined 46 CMTP families and identified 1 family with GFI1B variant (Figure), which co-segregated with CMTP. The proband (arrow in the Figure) of this family was a 15 years old boy with a history of easy bruising and platelet count of 70-115x109/L. His mother also had low platelet count, although details of her bleeding complications are unknown. Morphologic analysis of peripheral blood smear from the affected family members showed gray appeared enlarged platelets and red cell anisocytosis. Subsequent sequencing analysis confirmed a heterozygous point mutation (c.G814+1C, p.G272fsX274) at the splice donor site flanking exon 6 of GFI1B. GFI1B contains six zinc fingers (Znfs), of which Znf 3 to 5 are essential for DNA binding. The mutated transcript predicted a truncation of 58 C-terminal amino acids, which resulted in complete deletion of Znf 5. Since previous reports on GFI1B mutations have also described mutations of Znf 5 (p.H294fsX307 and p.Q287X mutations by Stevenson et al and Monteferrario et al, respectively), we performed biological analyses of these 3 patient-derived mutations affecting essential DNA-binding domain.GFI1B is an essential transcriptional factor for megakaryocyte and erythroid development. Therefore, we tested the effects of the GFI1b mutations on the transcriptional repression of firefly luciferase reporter constructs containing GFI1B promoter as a validated GFI1B target. Whereas wild-type GFI1B repressed the expression of reporter gene, the G272fsX274, H294fsX307 and Q287X mutants were unable to repress the expression. To test whether these patient-derived mutations acted in dominant-negative manner, we measured the effect of increasing the amount of mutant GFI1B cDNA, cotransfected into cells with a set amount of wild-type GFI1B cDNA. All 3 mutants inhibited gene repression mediated by wild-type GFI1B in a dose-dependent manner.To investigate biological effects of mutant GFI1B on proplatelet formation from megakaryocytes, we expressed the mutant GFI1B in mouse fetal liver cells (FLCs) by retrovirus-mediated gene transfer. FLCs harvested from embryonic day 13.5 embryos were transfected with pGCDNsamIRES/EGFP vector containing wild-type or the 3 patient-derived mutant GFI1B and were cultured with thrombopoietin. And we observed proplatelet formation of EGFP positive megakaryocytes. The number of proplatelet-producing megakaryocytes was reduced in mutant GFI1B transduced FLCs on day 3 and 4 after transfection, suggesting that mutant GFI1B inhibited the rate of proplatelet production from megakaryocytes. Next, we investigated the size and number of proplatelet tips from megakaryocytes, and found that proplatelet tips produced from G272fsX274, H294fsX307 and Q287X-transduced megakaryocytes were significantly reduced in numbers, but were increased in the size of the tips. This was compatible with decreased platelet count and enlarged platelet seen in GFI1B mutated patients. These findings indicated that patient-derived mutant GFI1B disrupted proplatelet formation of megakaryocyte.In summary, we have identified a novel GFI1B mutation that causes autosomal dominant macrothrombocytopenia with gray platelets and red cell anisocytosis. The mutant GFI1B acted in a dominant-negative manner over wild-type GFI1B. In addition, we first showed that the mutant-transduced FLCs produced abnormally enlarged proplatelet tips, which was reduced in numbers. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel β3 Intracytoplasmic Domain Mutation Associated with Macrothrombocytopenia: Structural Analysis in Comparison with Previously Reported Cases

Background: Glanzmann thrombasthenia (GT) is a moderate to severe inherited bleeding syndrome associating a lack of platelet aggregation with quantitative or qualitative deficiencies of the αIIbβ3 integrin. Rare patients with mutations in ITGA2B or ITGB3 genes show a variable GT-like phenotype associated with macrothrombocytopenia (MTP). Gain-of-function mutations that localize to the integrin cytoplasmic domains cause autosomal dominant MTP. We now report a novel heterozygous ΔLeu in ITGB3 causing a structural modification of the cytoplasmic domain of β3 in a family with moderate MTP. In silico modeling was performed to obtain structural insights into this and other known mutations affecting cytoplasmic domains of αIIb and β3 and impacting megakaryopoiesis.Aim: To help define structural changes in αIIbβ3 linked to MTP.Results: A novel in-frame heterozygous 3bp deletion (c.2230_2232delCTC) was identified by exome sequencing performed as part of the BRIDGE-BPD consortium project in a French pedigree with lifelong mucocutaneous bleeding. The consequence is the loss of β3 Leu718 (ΔL718) the second L of a LLITIHD motif of the mature protein (nomenclature used throughout). Platelets of the proband have (i) moderate expression of αIIbβ3, (ii) a major reduction in fibrinogen (Fg) and PAC-1 binding after ADP stimulation yet (iii) spontaneous Fg or PAC-1 binding to platelets was not seen. Western blotting showed a slightly slower migration of β3 and normal αIIb. The proband had a moderate MTP with platelet size heterogeneity and frequent enlarged α-granules regrouping several of normal size with Fg bound to the membrane of the α-granules as revealed by immunogold staining and electron microscopy. Platelet aggregation was much reduced with all physiologic agonists including TRAP with a reduced surface expression of P-selectin and diminished secretion. The same 3bp del in ITGB3 co-segregates with the platelet phenotype and bleeding across 3 generations in the family. We then performed molecular modeling (PyMol Molecular Graphics System version 1.3) to compare the structural effects of this mutation on αIIbβ3 with other cytoplasmic tail mutations described in the literature giving GT-like phenotypes and/or MTP. Firstly αIIb-R995Q/W or β3-D723H were confirmed to disrupt a salt bridge between the integrin subunits and induce the separation of their tails. PyMol modeling showed that the DL718 results in D723 being no longer in phase with R995 thereby disrupting the salt bridge. Significantly, introduction of a β-turn by an activating β3 L718P substitution again results in the spontaneous separation of the tails, once more through a conformational change of the mutated β3. In contrast, a previously reported ITGB3 C2268T variant resulting in Arg724*, a truncation coming after the deletion in our patient, or a β3 S752P substitution that completely blocks platelet aggregation and PAC-1 binding (and specific loss of kindlin-3 binding) despite a capacity to secrete a storage pool of Fg bound α-granule αIIbβ3 failed to give MTP despite resulting in a GT-like syndrome.Conclusions: The novel ITGB3 ΔL718 of β3 showed an intermediate phenotype with MTP, a much decreased platelet aggregation, a reduced activity of αIIbβ3 at the platelet surface but a progressive fusion of α-granules. This is despite the theoretical retention of the talin and kindlin-3 binding sites suggesting that transmembrane signaling is the principle defect. Our studies highlight how considerable phenotypic heterogeneity exists in patients with genetic variants affecting αIIb and β3 cytoplasmic domains and suggest different signaling mechanisms for surface and intracellular αIIbβ3 pools.*PN and ET; WHO and ATN made equal contributions,On behalf of the BRIDGE-Bleeding and Platelet Disorder Consortium DisclosuresNo relevant conflicts of interest to declare.

A Missense Mutation in the Alpha-Actinin 1 Gene (ACTN1) Is the Cause of Autosomal Dominant Macrothrombocytopenia in a Large French Family

Inherited thrombocytopenia is a heterogeneous group of disorders characterized by a reduced number of blood platelets. Despite the identification of nearly 20 causative genes in the past decade, approximately half of all subjects with inherited thrombocytopenia still remain unexplained in terms of the underlying pathogenic mechanisms. Here we report a six-generation French pedigree with an autosomal dominant mode of inheritance and the identification of its genetic basis. Of the 55 subjects available for analysis, 26 were diagnosed with isolated macrothrombocytopenia. Genome-wide linkage analysis mapped a 10.9 Mb locus to chromosome 14 (14q22) with a LOD score of 7.6. Candidate gene analysis complemented by targeted next-generation sequencing identified a missense mutation (c.137GA; p.Arg46Gln) in the alpha-actinin 1 gene (ACTN1) that segregated with macrothrombocytopenia in this large pedigree. The missense mutation occurred within actin-binding domain of alpha-actinin 1, a functionally critical domain that crosslinks actin filaments into bundles. The evaluation of cultured mutation-harboring megakaryocytes by electron microscopy and the immunofluorescence examination of transfected COS-7 cells suggested that the mutation causes disorganization of the cellular cytoplasm. Our study concurred with a recently published whole-exome sequence analysis of six small Japanese families with congenital macrothrombocytopenia, adding ACTN1 to the growing list of thrombocytopenia genes.

Mutational analysis of APOL1 in patients with Fechtner and Epstein syndromes: no evidence of a digenic etiology in MYH9-related disorders with renal disease

Mutations in MYH9 result in a group of clinically overlapping autosomal dominant macrothrombocytopenia syndromes, collectively termed the MYH9-related disorders (MYH9RD). Intriguingly, three of these disorders, Fechtner, Alport-like, and Epstein syndromes, are associated with the additional clinical phenotype of glomerulosclerosis. Recently, an abun- dance of studies have demonstrated that the APOL1 gene, contiguous to MYH9, is associated with a form of kidney disease in individuals of African ancestry. Given these findings related to kidney disease arising in mutations in two contiguous genes, this study aimed to determine whether APOL1 mutations could also be present in patients with Fechtner (FTNS) and Epstein (EPS) syndromes. This study used sequence analysis to investigate a discrete discovery set of FTNS patients, but did not identify second hit mutations in APOL1.

Abnormal Proplatelet Formation and Myosin Activation Status in Patients with MYH9-Related Syndrome

Abstract 4826MYH9-related disorders (MYH9-RD) are characterized by an autosomal-dominant macrothrombocytopenia associated or not with glomerular impairment, hearing loss and/or cataracts. It is caused by mutations of the MYH9 gene (encoding for the nonmuscle myosin heavy chain IIA), present at the heterozygous state in patients.In knockout animal models, proplatelet formation (PPF) was found to be increased in certain conditions, but decreased in others (Chen et al, Blood 2007, Eckly et al, Blood 2007 and Thrombosis and Haemostasis 2010) and samples with heterozygosity did not show differences with the wild type. More recently, in a mouse model with a mutated MYH9 gene, mice with the mutation showed a phenotype similar to the one found in MYH9-RD, affecting multiple organs and, interestingly, mice heterozygous for the mutation showed an abnormal phenotype as well, although attenuated (Anderson et al, ASH Meeting 2010, Abstract 2527). However, opposite to the initial reports in mouse models with a knockout, proplatelet formation was found to be impaired in 4 patients (Pecci et al, Thrombosis and Haemostasis 2009). Therefore the question for the exact mechanism for thrombocytopenia still stands, so does the question whether the mutated protein causes disease through a dominant negative effect versus a functional haploinsufficiency. To address these questions, we performed studies on patient samples on one hand and seeked to knock down the gene expression in human megakaryocytes (MKs) on the other hand.To study PPF in these patients, we analyzed MK differentiation in CD34+ cells from 9 patients with mutations in exons encoding for the motor domain (2 patients) or for the coiled coil domain of myosin IIA (7 patients). Compared with cultured MKs derived from healthy donors, a 2 to 5 –fold decrease in PPF was observed in the patients (4.9 ± 1.2% for patients vs. 15.1 ± 1.8% for control) and proplatelet area was decreased in patients as well (threefold decrease, 6156 μm2 for patients versus 18064 μm2 for control). In confocal microscopy with immunofluorescence staining, we observed disorganization of the granules, abnormal spreading and stress fibers, meaning that actin-myosin network is unstable.We next used a shRNA strategy to knock down MYH9 expression during normal MK differentiation and compared shRNA-treated MKs (with 50% residual MYH9 expression) with those derived from patient CD34+ cells. Treatment with shRNA decreased in vitro PPF, as previously observed for cells from patients. Moreover, shRNA-treated MKs exhibited the same ultrastructural abnormalities as MKs from patients. Normal platelet production is dependent on the formation of branched long proplatelets. Surprisingly the defect in PPF formation seen in patients was rescued by blebbistatin, an inhibitor of class II myosin. In its presence the proplatelets increased by 2 to 5 fold, suggesting that the remaining myosin IIA might be hyperactivated and inhibiting PPF. Ultrastructural studies by electron microscopy performed on cells with blebbistatin treatment corrected abnormalities seen in the patients. Parallel to blebbistatin, ROCK (Y27632) or MLCK inhibitors (P18 or ML7) led to a threefold increase in proplatelet formation. We therefore compared by western blot the status of pMLC2 in patients and control MKs. In 3 out of 6 patients we found an increased pMLC2. In addition, we also found an increased pMLC2 in MKs treated with shRNA with 50% residual myosin expression. Altogether our results show that reducing the protein expression by half was sufficient to recreate features characteristic of the disease in megakaryocytes, while the increased PPF, relative to control, in response to rho/ROCK inhibitors and to myosin inhibitors, suggest that the defective PPF in patients may be related to an activated rho/ROCK pathway and/or increase of the contractile force. Disclosures:No relevant conflicts of interest to declare.

Advances in the understanding of MYH9 disorders

MYH9 disorders are autosomal dominant macrothrombocytopenias with leukocyte inclusion bodies caused by mutations in MYH9, the gene for the nonmuscle myosin heavy chain IIA. May-Hegglin anomaly and Sebastian, Fechtner, and Epstein syndromes belong to MYH9 disorders. The present review summarizes the recent advances in genetic diagnosis and our understanding of the pathogenetic mechanisms of MYH9 mutations and the development of nonhematological complications. A genotype-phenotype cohort study showed that patients with an MYH9 mutation in the motor head domain of myosin IIA have severe macrothrombocytopenia and are at a high risk for the development of glomerulonephritis and deafness. Among these, Arg702 mutations are associated with the most severe phenotype. In-vitro studies on cultured megakaryocytes elucidated that myosin IIA inhibits proplatelet formation. The loss of myosin IIA function owing to MYH9 mutations promotes proplatelet formation and may trigger precocious and premature platelet release, resulting in macrothrombocytopenia. Giant platelets only residually express mutant myosin IIA that has a loss of function and cannot participate in the reorganization of cytoskeletal contractile structures. Renal histopathological and immunochemical studies have suggested that glomerulonephritis in MYH9 disorders is caused by podocyte malfunction owing to defects in the myosin IIA structure and MYH9 expression. MYH9 disorders are not merely benign hematological abnormalities, but serious syndromic disorders affecting the kidney, inner ear, and lens. A genetic diagnosis is mandatory for an accurate prognosis of nonhematological complications and management or possibly prophylactic treatment.

Dominant inheritance of a novel integrin 3 mutation associated with a hereditary macrothrombocytopenia and platelet dysfunction in two Italian families

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. 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. 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. 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.

Identification and characterization of the first large deletion of the MYH9 gene associated with MYH9 disorders

MYH9 disorders are autosomal dominant macrothrombocytopenias with leukocyte inclusion bodies. Single point mutations in the protein-coding sequence of the MYH9 gene are the most common cause. So far no large gene deletion/insertion and splicing defects have been reported. Conventional DNA sequencing of each MYH9-coding exon showed no abnormalities in a patient. Reverse transcription- polymerase chain reaction (PCR) amplification and sequencing of neutrophil mRNA identified an inframe deletion of exon 25. Further long-range PCR amplification of genomic DNA revealed a deletion of 1220 nucleotides including entire exon 25. Immunoblot analysis showed a small, abnormal protein in neutrophils but not in platelets. This is the first report of a large deletion of the MYH9 gene leading to the development of MYH9 disorders.

Acute myeloid leukemia in a child with hereditary thrombocytopenia

A child with a known diagnosis of an autosomal dominant macrothrombocytopenia, Fechtner Syndrome, developed acute myeloid leukemia (AML). Recently the disease gene for the inherited macrothrombocytopenias has been identified as MYH9, encoding for non-muscle myosin heavy chain-A. MYH9 has never been associated with the development of acute leukemia, but MYH11 is disrupted in the M4 eosinophilia sub-type of AML (inv16). The patients leukemic blasts did carry the common t(8;21) which yields an AML1-ETO fusion protein that inhibits AML-1. Despite his thrombocytopenia, the patient successfully completed intensive bone marrow cytoreduction without significant bleeding complications and is now in remission for over 3 years.

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.

Detection of unique neutrophil non‐muscle myosin heavy chain‐A localization by immunofluorescence analysis in MYH9 disorder presented with macrothrombocytopenia without leukocyte inclusions and deafness

MYH9 disorders are autosomal-dominant macrothrombocytopenias with leukocyte inclusions caused by mutations in the MYH9 gene, which encodes the non-muscle myosin heavy chain-A (NMMHCA). We report a patient with an MYH9 disorder who presented with macrothrombocytopenia without leukocyte inclusions and severe bilateral sensory deafness. Conventional May-Grunwald-Giemsa staining failed to detect granulocyte cytoplasmic inclusions, whereas immunofluorescence analysis clearly demonstrated abnormal neutrophil NMMHCA localization. Genetic analyses revealed a novel heterozygous 18 base deletion in MYH9, leading to a six-amino acid in-frame deletion (N76_S81del) in NMMHCA. These results further support the usefulness of immunofluorescence analysis in differential diagnosis of MYH9 disorders.

Congenital Macrothrombocytopenia and Defective Localization of the Nonmuscle Myosin Heavy Chain IIA in Leukocytes and Megakaryocytes with a Normal MYH9 Gene.

Autosomal dominant macrothrombocytopenias such as the May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome and Epstein syndrome are all characterized by mutations in the MYH9 gene. It has been proposed that these MYH9-related diseases are not distinct entities but represent a variable expression of a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to more severe conditions encompassing hearing loss, cataracts and renal failure. Mutations in the MYH9 gene cause abnormal expression and/or function of the 224 kDa nonmuscle myosin heavy chain IIA (NMMHC-IIA) protein. Class II myosins are hexameric complexes composed of 2 heavy chains and 2 pairs of light chains forming a structure containing 2 N-terminal globular domains and an elongated α-helical C-terminal tail. Since identical mutations in the MYH9 gene cause variable clinical presentations in different patients, other factors likely modulate the mutant phenotype. We describe a patient with congenital macrothrombocytopenia, mild bleeding problems, Döhle-like leukocyte inclusion bodies, normal hearing, normal renal function and absent cataracts. The patient has macrothrombocytes with an average manual platelet count of 100 x 109/L. Immunofluorescence confocal microscopy using an antibody specific for NMMHC-IIA revealed unique localization of NMMHC-IIA in the patient's leukocytes. Platelets and leukocytes from both parents and one sister are normal. Megakaryocytes cultured from the patient's and one parent's peripheral blood CD34+ cells also demonstrated abnormal distribution of NMMHC-IIA in only the patient's megakaryocytes. Ultrastructural analysis using electron microscopy revealed distinct inclusion bodies in the patient's leukocytes and megakaryocytes that were not observed in either parent. The patient's macrothrombocytopenia together with the abnormal distribution of NMMHC-IIA within leukocytes is highly suggestive of a MYH9-related disorder. The absence of these findings in either parent suggested a de novo MYH9 mutation. We therefore sought to identify the mutation via amplification and DNA sequencing of all MYH9 exons and intron/exon boundaries. Exons 1, 10, 16, 24, 25, 26, 30, 38 and 40 representing all of the previously described mutations in MYH9-related disorders were found to be normal. Surprisingly, the remaining MYH9 gene exons and intron/exon boundaries were also normal. This unexpected finding suggests that another factor is involved in the normal assembly and/or localization of NMMHC-IIA in human leukocytes and megakaryocytes. It also suggests that a deficiency of this factor may lead to congenital macrothrombocytopenia with features indistinguishable from those of MYH9-related disorders. Identification of this factor could enhance our understanding of NMMHC-IIA-related congenital macrothrombocytopenias and would allow us to gain insights into the normal assembly and/or function of NMMHC-IIA in human megakaryocytes and platelets.

Sebastian Platelet Syndrome: A Hereditary Macrothrombocytopenia

Sebastian platelet syndrome is a rare autosomal dominant disorder characterized by macrothrombocytopenia with granulocyte inclusions similar to those in patients with Fechtner platelet syndrome but without evidence of hereditary nephritis and sensorineural hearing loss that characterizes the latter. Although by light microscopy the granulocyte inclusions in these disorders appear morphologically similar to those found in May-Hegglin anomaly, another autosomal dominant macrothrombocytopenia, by electron microscopy the inclusions are distinct. Studies of platelet function usually suggest normal or near-normal platelet function, although mild bleeding symptoms can be associated with each of these disorders. We describe a 38-year-old woman and her 11-year-old daughter who presented with lifelong histories of mild thrombocytopenia and easy bruising. Detailed hemostatic studies showed prolonged bleeding times in the child and the mother, with the child having absent secondary wave platelet aggregation responses to epinephrine, also reflected by testing with the platelet function analyzer (PFA-100 device). The mother's hemostatic studies were normal including platelet aggregometry, PFA-100 testing, and platelet flow cytometry. By light microscopy the blood smears of both individuals showed neutrophil inclusions, and their platelets were mildly enlarged but were not giant. Electron microscopy showed the neutrophil inclusions seen in classic Sebastian platelet syndrome or Fechtner platelet syndrome. These 2 cases expand the description of Sebastian platelet syndrome to include individuals with large but not giant platelets and mild or minimal thrombocytopenia. The differential diagnosis of hereditary thrombocytopenias is reviewed briefly.

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.

Immunofluorescence Analysis of Neutrophil Nonmuscle Myosin Heavy Chain-A in MYH9 Disorders: Association of Subcellular Localization with MYH9 Mutations

The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly, Sebastian syndrome, and Fechtner syndrome, are rare human disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like cytoplasmic inclusions in granulocytes. Epstein syndrome is another autosomal dominant macrothrombocytopenia associated with Alport syndrome but without leukocyte inclusions. These disorders are caused by mutations in the same gene, the MYH9, which encodes the nonmuscle myosin heavy chain-A (NMMHCA). The term, MYH9 disorders, has been proposed, but the clinicopathologic basis of MYH9 mutations has been poorly investigated. In this study, a total of 24 cases with MYH9 disorders and suspected cases were subjected to immunofluorescence analysis by a polyclonal antibody against human platelet NMMHCA. Abnormal subcellular localization of NMMHCA was observed in every neutrophil from individuals with MYH9 mutations. Comparison with May-Grünwald-Giemsa staining revealed that the NMMHCA always coexisted with the neutrophil inclusion bodies, suggesting that NMMHCA is associated with such bodies. In three cases, neutrophil inclusions were not detected on conventional May-Grünwald-Giemsa-stained blood smears but immunofluorescence analysis revealed the abnormal NMMHCA localization. In contrast, cases with Epstein syndrome and the isolated macrothrombocytopenia with normal NMMHCA localization had no MYH9 mutations. An antibody that recognizes the C-terminal 12 mer peptides showed similar immunoreactivity from the patients heterozygous for truncated mutations that abolished the C-terminal epitope, suggesting that normal NMMHCA dimerizes with abnormal NMMHCA to form inclusion bodies. We further propose that the localization pattern can be classified into three groups according to the number, size, and shape of the fluorescence-labeled NMMHCA granule. Immunofluorescence analysis of neutrophil NMMHCA is useful as a screening test for the clear hematopathologic classification of MYH9 disorders.