Giant Platelets Research Articles

Bernard-Soulier syndrome (hemorrhagiparous thrombocytic dystrophy).

Bernard-Soulier syndrome (BSS), also known as Hemorrhagiparous thrombocytic dystrophy, is a hereditary bleeding disorder affecting the megakaryocyte/platelet lineage and characterized by bleeding tendency, giant blood platelets and low platelet counts. This syndrome is extremely rare as only ~100 cases have been reported in the literature. Clinical manifestations usually include purpura, epistaxis, menorrhagia, gingival and gastrointestinal bleeding. The syndrome is transmitted as an autosomal recessive trait. The underlying defect is a deficiency or dysfunction of the glycoprotein GPIb-V-IX complex, a platelet-restricted multisubunit receptor required for normal primary hemostasis. The GPIb-V-IX complex binds von Willebrand factor, allowing platelet adhesion and platelet plug formation at sites of vascular injury. Genes coding for the four subunits of the receptor, GPIBA, GPIBB, GP5 and GP9, map to chromosomes 17p12, 22q11.2, 3q29, and 3q21, respectively. Defects have been identified in GPIBA, GPIBB, and GP9 but not in GP5. Diagnosis is based on a prolonged skin bleeding time, the presence of a small number of very large platelets (macrothrombocytopenia), defective ristocetin-induced platelet agglutination and low or absent expression of the GPIb-V-IX complex. Prothrombin consumption is markedly reduced. The prognosis is usually good with adequate supportive care but severe bleeding episodes can occur with menses, trauma and surgical procedures. Treatment of bleeding or prophylaxis during surgical procedures usually requires platelet transfusion.

Assessment of MYH9-Related Syndromes among a Series of Patients with Constitutional Macrothrombocytopenia (MT).

MYH9-related disorders are autosomal dominant giant platelet syndromes with a wide phenotypic variability known under the following names: May Hegglin Anomaly (MHA), Fechtner Syndrome (FTNS), Epstein Syndrome (EPS), Sebastian Syndrome (SBS) and Alport-like Syndrome with MT. The aim of the study was to identify MYH9-related syndromes in a series of 43 propositi with constitutional MT. In this group, 26 have a characteristic MYH9-syndrome phenotype. The 17 other patients have a constitutional MT of uncertain etiology, 8 of them presenting a partial MYH9 syndrome phenotype. The leukocyte repartition of the Non-Muscle Myosin Heavy Chain IIa (NMMHC-IIA) encoded by the MYH9 gene was explored by May-Grunwald-Giemsa staining of blood smears or by immunofluorescence, while platelets were also examined by electron microscopy (EM). The MYH9 gene was studied by genomic DNA amplification and direct sequencing of the 8 exons in which mutations have been published (Dong, 2005). The results are the following: of the 17 patients with constitutional MT of unknown etiology, no MYH9 mutations were found; in contrast, 24/26 (92.3%) patients with a characteristic MYH9-syndrome phenotype were heterozygous for a MYH9 mutation. Among their 29 family members, 5 were heterozygous for the mutated allele. In 4 families, the mutations were found only in the propositus but not in the parents, suggesting that they might be either de novo mutations or the results of somatic mosaicism, as already published ( Kunishima et al, 2005). Six mutations are novel: F1446L, K1937X, A44P, D1424E in 4 MHA patients, W33C in 1 patient with MHA/SBS, and, interestingly, D1447V was associated with 2 different phenotypes, MHA and FTNS. As already published, the majority of the mutations in the C-terminus of the NMMHC-IIA are associated with a pure hematologic disorder. In contrast, the N-terminus mutations were more generally associated with a more severe phenotype with renal manifestations. However the same mutation can be associated with different phenotypes: S96L with FTNS, EPS and SBS/MHA, D1447V with MHA and FTNS. In addition the R702C mutation, which has been identifed in 2 cases with an identical FTNS phenotype, is associated with 2 different leukocyte NMMHC-IIA distributions. No MYH9 mutation could be detected in 2 patients with FTNS. These results, as a whole, are in agreement with the hypothesis that mutations in other genes than MYH9 might be involved in defining the phenotypes of such syndromes. The genotype of the 17 patients with uncertain etiology remains to be identified. The interest to diagnose MYH9 mutations is:to avoid misdiagnosis and inadequate therapy for patients with thrombocytopenia (2 patients initially diagnosed as ITP patients underwent splenectomy);to detect as early as possible the risk and occurrence of renal failure, deafness and cataracts (1 patient initially diagnosed as a SBS patient developed a renal failure characteristic of FTNS).A long-term follow-up of the patients with MYH9 mutations is of a high interest for a better knowledge of the relationship between the mutation and the phenotypic expression.

Velocardiofacial Syndrome (VCF): Presentation with and Management of Immune Cytopenias.

Background: VCF is a 22q deletion syndrome which occurs in 1:4000 live births. It consists of dysmorphic facies, cleft palate, neurodevelopmental disorders and conotruncal cardiac defects. In addition to 22q11.2 deletion, other loci, e.g.10p4, have been implicated. Autoimmune cytopenias are prominent in this condition and present specific issues in diagnosis and therapy. Method: With IRB approval, we retrospectively reviewed patients referred to our center for evaluation of thrombocytopenia between 1999 and the present. Five children were suspected to have the VCF phenotype.Results: Two of the 5 patients were confirmed to have 22q11.2 deletion by genetic testing (Table1). The other 3 also tested negative for the 10p4 defect. The universal features were immune cytopenias and neuropsychiatric disorders manifesting as developmental delay and/or abnormal behavior. Three patients had cardiac defects, of which 2 were right sided aortic arches; 3 had plastic repair of cleft palates. All had 2 or 3 immune cytopenias, including Evans syndrome, presented between ages 2 to 8 years, and were difficult to treat. Prolonged steroid therapy was avoided and Rituximab was generally effective. Patient 1 was evaluated on the basis of cytopenias with coexisting psychiatric disease but had no cardiac or palatal findings, leaving the diagnosis uncertain. His subsequent genetic testing was negative. Patient 2 presented with Evans syndrome, and, on review of chest xrays, a previously unreported right aortic arch was noted. This patient developed acute deterioration in preexisting behavioral features during an initial steroid course for ITP.Discussion: In view of the implications for treatment of the immune cytopenias, it is important to suspect VCF, but how many phenotypic features of VCF are required to make the diagnosis is uncertain. The adverse impact of prolonged steroid therapy on neuropsychiatric disease warrants earlier use of Rituximab, especially in autoimmune hemolysis. Rituximab therapy avoids T cell immunosuppression in patients who may have concurrent immunologic defects and it is now accepted that splenectomy should be avoided in children with Evans syndrome. In patients 2 and 5, Rituximab therapy was implemented early in the course; patient 4 presented prior to its availability. All our patients manifested thrombocytopenia without giant platelet morphology. An individual with 22q11.2 deletion is haploinsufficient for Gp1bβ, also carried at this locus, and is therefore a Bernard Soulier syndrome heterozygote. A patient not responding to standard therapy for ITP may have thrombocytopenia related to this rare condition. In summary, VCF patients without significant cardiac or palatal disease may be undiagnosed at the time a cytopenia manifests. Pitfalls in diagnosis may occur with inadequate history, good cosmetic repair of a palatal defect, and missed right aortic arches. Prolonged steroid therapy often exacerbates coexisting neuropsychiatric disorders and immunosuppression and is not recommended in these difficult to treat patients. Rituximab is a useful treatment, especially for autoimmune hemolysis.Table1:Clinical featuresPatientGenetic confirmationThrombocytopeniaNeutropeniaAutoimmune hemolytic anemiaCardiac defectsImmunologic defectsPalatal defects1NegativeYesYesNoNoNoNo2NegativeYesNoYesYesNoYes3PositiveYesYesNoNoYesYes4PositiveYesYesYesYesYesYes5NegativeYesYesYesYesYesNoAll patients had neuropsychiatric disorders.

Platelet Pathology in GATA-1 Dyserythropoietic Macrothrombocytopenia.

Germline mutations in the X-linked hematopoietic transcription factor, GATA-1, have been associated with dyserythropoietic anemia (DEA), macrothrombocytopenia (MTP), and congenital erythropoietic porphyria. Recently, morphological features suggestive of the Gray Platelet Syndrome (GPS) were described in a pedigree with a germline R216Q GATA-1 mutation (Blood 106:6a, 2005). The present study has evaluated platelets (Pl) in the electron microscope from members of a previously described pedigree (Blood 98:2681–2688, 2001) with GATA-1 G208S, MTP, and dyserythropoiesis without anemia, for whom detailed platelet morphology studies have not been reported. The presence of a hemizygous GATA-1 mutation was confirmed by conventional fluorescent dye chemistry sequencing of DNA from patient peripheral blood mononuclear cells. Their Pl revealed wide variations in size, shape, internal and external structure. Some normal sized and giant Pl contained usual numbers of alpha granules and dense bodies, while most of the large cells were hypogranular. Some contained no membrane systems or organelles. Others were filled with masses of dense tubular system (DTS) channels, and others contained the tubular inclusions found in the Medich Giant Platelets Disorder (Platelets 15:345–353, 2004). Many of the hypogranular cells contained small vacuoles that may have been enclosing membranes of alpha granules. Except for the tubular inclusions, all of these structural variations are observed in GPS Pl (Am. J. Pathol. 95:445–462, 1979). However, additional striking abnormalities were seen in GATA-1 Pl that are not characteristic of GPS Pl. Many GATA-1 Pl contained unusual, closely associated dense double membranes differing from normal elements of rough endoplasmic reticulum, smooth endoplasmic reticulum, DTS or surface connected open canalicular system (OCS), but resembling channels of the OCS after exposure to EDTA. They were observed previously only in megakaryocytes (Mk) from one family with GATA-1 DEA-MTP (Nat. Genet , 24:266–270, 2000). The dense double membranes were often found in parallel association and, in some examples, isolating areas of cytoplasm. However, the isolated areas were not undergoing autophagic degradation as such areas enclosed by elements of the DTS do in White Platelet Syndrome Pl (Platelets 15:173,2004). Instead, they proved to be another unique feature of GATA-1 platelets, the presence of Pl within Pl. The sequestered cells were usually mature in appearance and often discoid in form with circumferential coils of microtubules. In some GATA-1 Pl there were two Pl within the same cell, and on rare occasions, a Pl within a Pl within a Pl. Pl within Pl have never been observed previously in any human platelet disorder. Another unique feature, related to Pl within Pl, was the frequent Pl to Pl surface attachment of non-activated cells forming large macrothrombocytes with no similarity to aggregates of stimulated normal platelets.Conclusion: The substructural abnormalities of Pl from patients with germline GATA-1 mutations share some overlap with those seen in GPS Pl, but are distinct and unique. The MTP, Pl within Pl and surface Pl to Pl attachments suggest a major defect in the formation and separation of GATA-1 Pl from pro-Pl of the parent Mk, resulting in the MTP characteristic of the disorder.

Two Mutations (1717 T〉C, GP IX;124del145, GPIb beta) Occur Frequently among Patients with Bernard Soulier Syndrome in India.

Bernard Soulier syndrome (BSS) is a rare (1:1000000) hereditary bleeding disorder of platelet adhesion, caused by a qualitative or quantitative defect of glycoprotein (GP)Ib/IX/V complex. We report here the molecular basis of BSS in eight unrelated patients from India. The diagnosis was based on low platelet count, presence of giant platelets and aggregometry studies (absence of response to ristocetin) in eight unrelated patients who presented with mucocutaneous bleeding at 17.9 years of age (range:9 months–60yrs). Flow cytometry to assess surface GPIb/IX/V complex using anti CD42b antibodies showed reduced (7.7%–21.1%, n=6) expression. Genomic DNA was screened for mutations in the GPIb alpha, GPIb beta, GP IX genes by a novel PCR and conformation sensitive gel electrophoresis method. Mutations were identified in all the 8 patients. Six different mutations, including missense (n=4) mutations, a deletion and a duplication were identified of which 5 are novel. Three novel missense mutations were identified in a patient (CD42b:21.1%) of which, Tyr492His in GPIb alpha gene was in heterozygosity and the Glu129His, Leu132Pro mutations in GPIb beta was detected in a double homozygous condition. All these substitutions occur at a highly conserved codon in the transmembrane region of GPIb alpha and beta chains, a region critical for anchoring the glycoprotein subunits into the platelet membrane. A novel homozygous deletion of 22 bp in exon 2 of GPIb beta (124del145, p.Arg17CysfsX14) gene was identified in 3 different patients (CD42b: range, 10.6–15.9%). This deletion occurs at a sequence flanking the leucine rich repeat at the NH2-side of GPIb beta protein results in a premature termination of translation (PTC) and a truncated mutant with 30 amino acids. A novel homozygous duplication of 38 nucleotides (2119_2120 dup38) in the transmembrane region resulted in a frameshift (142AlafsX76) in one of the patient. This frameshift predicts termination after the addition of 75 amino acids. A previously reported homozygous transition (1717 T >C) in the GP IX gene resulting in a Cys8Arg substitution was identified in the remaining 3 patients (CD42b:10.02%, 7.7%). The frequent presence of GP IX 1717 T>C and GPIb beta 124del145 in 6 out of 8 (75%) patients studied, suggests that this could be due to a founder effect. This data adds significantly to the mutation database of this condition and will be useful for genetic diagnosis of this condition in India.

Molecular genetic analysis of a variant Bernard–Soulier syndrome due to compound heterozygosity for two novel glycoprotein Ibβ mutations

Bernard-Soulier syndrome (BSS) is a rare bleeding disorder characterized by giant platelets, thrombocytopenia, and prolonged bleeding time. It is caused by abnormalities in the glycoprotein (GP) Ib/IX/V complex, the receptor for von Willebrand factor (vWF). Most cases of BSS described so far involve quantitative rather than qualitative defects in the complex. In this study, we investigated the effects of two naturally occurring mutations in the GPIbbeta gene, C122S and 443delG, on the expression of the GPIb/IX complex identified in a variant type of BSS in which the platelets had severely reduced GPIbalpha ( approximately 10%) and less markedly reduced GPIbbeta and GPIX ( approximately 20%) expression. Immunoblot analysis showed the absence of non-reduced GPIb (GPIbalpha/GPIbbeta) in the patient's platelets. Transient transfection experiments in 293T cells revealed the expression of GPIbbeta Ser122 polypeptide and absence of GPIbbeta 443delG polypeptide. Although no disulfide-linked association was observed between GPIbbeta Ser122 and GPIbalpha, GPIbbeta Ser122 was non-covalently associated with both GPIbalpha and GPIX subunits on the cell surface when cotransfected with wild-type GPIbalpha and GPIX. Chinese hamster ovary cells stably expressing GPIbalpha/Ibbeta Ser122/IX had the ability to bind soluble vWF and to aggregate in the presence of ristocetin. These results suggest that despite disruption of the disulfide linkage between GPIbalpha and GPIbbeta, GPIb/IX is formed, but its stability may be impaired, resulting in low levels of the complex on the platelet membranes.

Identification of inherited macrothrombocytopenias based on mean platelet volume among patients diagnosed with idiopathic thrombocytopenia

Inherited macrothrombocytopenia is a rare illness that is often misdiagnosed as idiopathic thrombocytopenia (ITP), a more widespread acquired disease. Automated blood cell counters in routine clinical use usually miss giant platelets and underestimate mean platelet volume (MPV). Incorrect diagnoses might expose patients to a risk of unnecessary treatment. The ADVIA 120 hematology counter efficiently detects large platelets based on two-dimensional laser light scatter. The present study measures and re-evaluates MPV using the ADVIA 120 in 112 patients who had initially been diagnosed with ITP. We identified 11 unrelated patients as having probable macrothrombocytopenia (average MPV of 19.2 ± 3.8 fL; normal range 7.8–10.2). Functional, phenotypical and DNA analyses confirmed that three of these patients had Bernard–Soulier syndrome and one had MYH9-related disease, both of which are the most common forms of inherited macrothrombocytopenia. We stress that a conventional automated hematology analyzer had overlooked giant platelets in these patients, and that all of them had received high-dose steroid therapy and/or splenectomy before this study according to a diagnosis of ITP. Thus, checking MPV using the ADVIA 120 in thrombocytopenic patients is a useful method of correctly diagnosing inherited macrothrombocytopenia, and thus avoiding patient exposure to unnecessary and sometimes toxic treatment.

Le syndrome MYH9 : à propos d'une nouvelle observation et de la mise en évidence d'une nouvelle mutation du gène MYH9

Introduction Familial macrothrombocytopenias are a group of rare autosomal dominant platelet disorders including many syndromes in particular the May-Hegglin anomaly. They are characterized by thrombocytopenia with giant platelets and in some cases neutrophilic inclusions in peripheral blood granulocytes. Recently these different clinical entities have been demonstrated to be linked to mutations in the same gene, MYH9. Case report We report in a young African woman presenting as a May-Hegglin anomaly a new mutation of the MYH9 gene. In regard of this case we present a brief review of the MYH9 syndrome. Conclusion The MYH9 syndrome includes now several clinical entities who share some common clinical and biological characteristics such as a thrombocytopenia with giant platelets, presence or absence of other manifestations including Dohle like bodies, nephritis, sensineural hearing loss, cataract. We report a new case in which a new mutation of the MYH9 gene was evidenced.

Analysis of clinical manifestations, mutant gene and encoded protein in two Chinese MYH9-related disease families

Background MYH9-related disease is a rare autosomal dominant disorder characterized by the triad of giant platelet, thrombcytopenia and inclusion bodies in neutrophil. In recent years, much progress has been made in the investigation of its clinical feature and pathogenesis. Methods Clinical manifestations were analyzed in two Chinese MYH9-related disease families. Polymerase chain reaction (PCR), DNA sequencing and CpoI restrictive endonuclease map analysis were used to identify spot mutation in nonmuscle myosin heavy chain 9 (MYH9) gene. Indirect immunofluence combined propidium iodine (PI) nuclei count-staining technology was applied to probe nonmuscle myosin heavy chain IIA (NMMHC-A) in MYH9-related disease neutrophils and platelets. Western blot was undergone to examine the expression of NMMHC-A in MYH9-related disease patients. Results All of the patients manifested with the typical triad, mild to moderate bleeding tendency were their common clinical feature, some patients were accompanied by renal lesion. G5521A mutation in MYH9 gene was identified in both families. Spindle-like inclusions with yellow fluorescence in MYH9-related disease neutrophils were clearly revealed by indirect immunofluence combined PI nuclei count-staining technology, which matched very well with the inclusions, detected by Wright–Giemsa's stain. An upregulation of NMMHC-A in MYH9-related disease neutrophils was observed by Western blotting analysis. Conclusion Mutation of MYH9 gene exists in cases of Chinese MYH9-related disease. In the two families, the point mutation was located in exon 38(G5521A), and the transference rule of the MYH9 gene mutation is corresponding with clinical phenotype distribution. Indirect immunofluorescence combining with PI nuclei staining technology is sensitive and more specific than Wright–Giemsa's staining in detecting MYH9-related disease inclusions, with which we might easily distinguish MYH9-related disease inclusions from infection-associated inclusions. The expression of the NMMHC-A in MYH9-related disease neutrophils was upregulated than normal control.

Novel heterozygous missense mutation in the second leucine rich repeat of GPIbα affects GPIb/IX/V expression and results in macrothrombocytopenia in a patient initially misdiagnosed with idiopathic thrombocytopenic purpura

Recent studies have shown that heterozygous carriers of the bleeding disorder Bernard-Soulier syndrome are occasionally identified as isolated case of giant platelet disorder/macrothrombocytopenia or misdiagnosed with idiopathic thrombocytopenic purpura (ITP). We describe here a patient with congenital macrothrombocytopenia who had been diagnosed with ITP. On peripheral blood smears, platelet diameter was approximately 30% larger than normal controls. In the patient's platelets, the expression level of the GPIbIX complex was slightly decreased (70-80% of normal control). Densitometric analysis of immunoblots showed GPIbalpha to be approximately 52% of normal. DNA sequencing analysis revealed a novel heterozygous missense mutation in the GPIbalpha gene that converts Tyr to Asp at residue 54 (Y54D) in the second leucine-rich repeat. Mutant GPIbalpha protein was not detected in the patient's platelets. Transient transfection studies demonstrated that mutant GPIbalpha affects complex expression. These findings suggest that null expression of the mutant GPIbalpha causes decreased density of the complex and results in macrothrombocytopenia.

Clinical Characteristics of Autosomal Dominant Giant Platelet Syndromes and Mutation Analysis of MYH9

Background: The autosomal dominant giant platelet syndromes (GPS), characterized by triads of giant platelets, thrombocytopenia, and Döhle-like leukocyte inclusions are caused by MYH9 mutation, a gene encoding the nonmuscle myosin heavy chain-IIA. This study was aimed to identify the Korean GPS patients and to define clinical findings and molecular characteristics on them. Methods: After taking a family history, platelets were counted using hematologic autoanalyzer and peripheral blood smear (PBS) was examined for platelet size and number, and the presence of leukocyte inclusions. Mutation of MYH9 was studied from mononuclear cells from PB by direct sequencing of previously known 8 exons after PCR amplification of genomic DNA. Results: Twenty patients from 5 unrelated families were diagnosed as GPS. Giant platelets, greater than red cells on PBS, were found to be 3.1% of platelet (range, 1～11%). The median platelet count was 61,000/μL. Inclusion bodies were found in 3 families. Two families had previously reported mutations. Family I had Arg1944Ter in exon 40, located in the tail portion of myosin, while Family IV had Lys373Asn in exon 10, located in the proximal portion of myosin head. The mutations were found only in affected patients, but not in normal siblings or unrelated families. Conclusion: In this study, we identified several families with autosomal dominant GPS. Two families had known MYH9 mutations, Arg1944Ter and Lys373Asn. The search for unknown mutations in the remaining families as well as study of protein structural and functional alteration seems to be necessary for further delineation of these rare genetic disorders.

Giant platelet syndrome

Giant platelet syndrome is a group of unique disorders characterized by the presence of abnormally large platelets, and usually accompanied by thrombocytopenia. Most cases of giant platelets are encountered in idiopathic thrombocytopenic purpura(ITP). In contrast, inherited giant platelet disorders, a group of heterogeneous diseases, are rare. Bernard-Soulier syndrome and its variants, and MYH9 related diseases have been defined at the molecular level. Abnormalities in transcription factors are implicated in a couple of macrothrombocytopenia syndromes. However, the molecular defects are unknown in gray platelet syndrome. It is important to make a proper diagnosis of congenital macrothrombocytopenia to avoid unnecessary medications and potentially dangerous treatment for presumed ITP

An inherited macrothrombocytopenic disorder with abnormal large granules

Inherited giant platelet syndromes are a heterogeneous group of rare bleeding disorders. In the current study, a patient was reported with prolonged bleeding time, thrombocytopenia and giant platelets. Both the patient's red and white cell counts were normal without morphological abnormalities. The electron microscopy of platelets showed abnormal large electron-dense granules in the cytoplasm with exocytosis being easily observed. Her fathers platelets had the same abnormalities. The expression of glycoprotein (GP)Ib, GPIIb and GPIIIa on the surface of the patient's platelets was normal, and her platelet aggregation in response to ADP and ristocetin was also normal. Immunogold probes combined to monoclonal antibodies against GPIIIa, P-selectin or CD63 could not identify any relationship between the aberrant structures and alpha granules or lysosomes of platelet. Serotonin level was normal in the platelets of the patient, indicating the electron opaque granules are not dense bodies. The morphological abnormalities of the platelets from the patient are clearly distinguishable from other hereditary giant platelet disorders. We propose that the abnormal large granules from the patient's platelets probably represent a novel inherited thrombocytopenic disorder.

Von Willebrand disease type 2B must be always considered in the differential diagnosis of genetic thrombocytopenias with giant platelets

Type 2B von Willebrand's disease (VWD) is an inherited bleeding disorder characterized by spontaneous binding of large von Willebrand factor (VWF) multimers to platelets in vivo. This phenomenon induces the clearance of both large multimers and platelets, usually resulting in thrombocytopenia with slightly increased platelet size. We describe a newborn with a VWD type 2B due to the heterozygous missense mutation V1316M who presented the atypical feature of giant platelets in peripheral blood. Based on this observation and literature review, we suggest that the diagnosis of VWD 2B should be always considered in patients with chronic thrombocytopenia and giant platelets.

Genetic Diagnosis of Congenital Giant Platelet Disorders

Genetic Diagnosis of Congenital Giant Platelet Disorders

Unusual cause of thrombocytopenia in neonatal period: MYH‐9 related giant platelet disorders

Unusual cause of thrombocytopenia in neonatal period: MYH‐9 related giant platelet disorders

Clinical Characteristics of Autosomal Dominat Giant Platelet Syndromes (GPS) and Mutation Analysis of MYH9 Encoding Non-Muscle Myosin Heavy Chain from 6 Unrelated Korean Patients.

Autosomal dominant GPS is characterized by thrombocytopenia, giant platelets and Doehle-like inclusion bodies. Studies have indicated that platelet structure and function are normal, suggesting a defect in megakaryocyte fragmentation. This study was aimed to identify Korean patients with GPS and define clinical findings and molecular characteristics on them. Twenty-two patients from 6 unrelated families were identified. Giant platelets, measuring greater than red cells on blood smear were found to be 3.1% (1–11%). The median platelet count was 61,000/μL. Doehle-like inclusion bodies were found in 4 families, while renal impairment in 2. MYH9 in each family was analyzed by direct sequencing of exons or cDNAs. Among 6 families, 5 families were found to have the mutations: Arg1933Ter, Trp33Cys, Lys373Asn, Ile1816Val, Arg1165Cys located in 41st, 2nd, 11th, 38th, 27th exons, respectively. All of the mutations are found only in the affected patients, but not in the normal siblings. Arg1933Ter and Lys373Asn were reportedly associated with GPS and Arg1933Ter is known to make the protein unstable. The novel mutations, Trp33Cys and Ile1816Val, were placed in highly conserved amino acids in mammals and Drosophila, and even in other types of myosin heavy chain. Computer-aided modeling analysis shows that Trp33Cys is located in the proximal portion of the myosin head, serving as a dock for actin binding, while Ile1816Val is in the distal portion of myosin tail, which may act to form polymer with other myosin heavy chains. Isolated mononuclear cells or the stable cell lines originated from Trp33Cys, Lys373Asn and Arg1165Cys patients were subjected to immunoblot. Compared to their normal siblings, the 220 kDa bands in the soluble fraction were decreased in those patients, while the bands were increased in the insoluble pellet. However, Northern blot analysis revealed that transcript levels were not altered in the samples from patients. Trp33Cys, Lys373Asn, and Arg1165Cys also produced aberrant 140 kDa bands in addition to normal 220 kDa MYH9 band in the insoluble fractions. After immunoprecipitation with MYH9 antibody, the 140 kDa bands were analyzed and turned to be actinin. These results suggest that novel mutations found in GPS are directly associated with the disease and that the phenotype might be caused by the solubility or instability of the proteins.

Identification of Inherited Macrothrombocytopenias Based on Mean Platelet Volume among Patients Diagnosed with Idiopathic Thrombocytopenia.

Inherited macrothrombocytopenia is a rare illness that is often misdiagnosed as idiopathic thrombocytopenia (ITP), a more widespread acquired disease. The hallmark of a diagnosis of these diseases is the presence of giant platelets. Automated blood cell counters in routine clinical use usually miss giant platelets and underestimate mean platelet volume (MPV). Incorrect diagnoses might expose patients to a risk of unnecessary treatment. The ADVIA 120 hematology counter efficiently detects large platelets based on two-dimensional laser light scatter. This two-dimensional approach provides a more accurate estimation of platelet volume than conventional one-dimensional analysis. The present study measures and re-evaluates MPV using the ADVIA 120 in 112 patients who had initially been diagnosed with ITP.We identified 11 unrelated patients as having probable macrothrombocytopenia (average MPV of 19.2 ± 3.8 fL; normal range 7.8–10.2). A diagnosis of inherited macrothrombocytopenia was immediately ruled out in 5 of the 11 patients, because their medical records indicated that their platelet counts had once been normal or became normalized (>100x199/L) in response to steroid therapy. Of the remaining 6 patients, platelets aggregated abnormally in response to ristocetin in 3 patients and MYH9-RD was suspected in one patient since Döhle-like bodies were apparent in granulocytes on blood films. DNA analyses confirmed that 3 of these patients had Bernard-Soulier syndrome and 1 had MYH9-related disease, both of which are the most common forms of inherited macrothrombocytopenia. We detected 3 homozygous mutations in the 3 patients with Bernard-Soulier syndrome: A3171T and 4444insT in the GPIbα gene that created new premature termination codons, and the missense mutation, G1913A in the GPIX gene, that caused the loss of GPIX surface expression. In one patient with MYH9-related disease, we found a heterozygous missense mutation of C5797T in exon 40 of the MYH9 gene. We stress that all four patients had received high-dose steroid therapy and/or splenectomy before this study according to a diagnosis of ITP.Checking MPV using the ADVIA 120 in thrombocytopenic patients is a useful method of correctly diagnosing inherited macrothrombocytopenia, and thus avoiding patient exposure to unnecessary and sometimes toxic treatment.

Two Candidate Genes for Low Platelet Count Identified by Genome-Wide Linkage Analysis: Glycoprotein IX and Thrombopoietin.

Although the 'normal' range of platelet count in a healthy person is quite broad (150 to 400 ×109/L), an individual's platelet count is maintained within a much narrower range, constantly balanced between thrombopoiesis and senescence. Platelet counts within the normal range have been shown to be largely determined by genetic factors with heritability estimates from twin studies ranging from 0.57 to 0.80. A genome-wide linkage analysis was carried out in a large Asian Indian kindred with β thalassemia to search for genes influencing variation in normal platelet count. Significant linkage is shown at two locations on chromosome 3q with age, sex and β thalassemia adjusted platelet counts. Linkage analysis showed one marker (D3S1309) on chromosome 3q with a lod score of 3.26 and another marker (D3S1282) approximately 30 cM centromeric, with a lod score of 2.52. Multipoint linkage analysis across 90 cM of chromosome 3q identified two peaks across the region with maximum multipoint lod scores of 3.52 and 4.11 under markers D3S1309 and D3S1282, respectively. Two strong candidate genes for platelet variation were identified in the linked region; thrombopoietin (THPO) and glycoprotein IX (GPIX). Resequencing of four individuals revealed five single nucleotide polymorphisms (SNPs) in THPO and one mutation in the transmembrane region of GPIX. Analysis of variance showed that the GPIX mutation and one THPO SNP accounted for 6% and 4% of the variation in platelet count in the kindred, respectively. The THPO SNP lies in the 3′ untranslated region (3′ UTR) of the gene and has not been previously reported.Although no function could be attributed to the THPO SNP it could potentially influence post-transcriptional control of gene expression. The G to A transition in nucleotide 653 (Ref seq NM_000174) resulted in an Ala 156 (GCC) to Thr (ACC) replacement in the GPIX protein. The GPIX mutation was recently identified in a Chinese patient with Bernard-Soulier syndrome (BSS), a rare bleeding disorder characterized by severe thrombocytopenia and giant platelets. The Chinese patient was homozygous for the mutation; however, heterozygous relatives did not show the characteristic BSS symptoms, leading the authors to conclude that the mutation acted under a recessive model of inheritance. Transient transfection studies by the same group confirmed that the mutation prevents GP Ib/IX complex insertion in the cytoplasmic membrane of platelets, and suppresses GPIbα and GPIX expression, but does not cause its intracellular degradation. One copy of the GPIX mutation was found in 300 European individuals with platelet counts within the normal clinical range.Conclusion: Although the genetic determinants of many thrombocythemias and congenital thrombocytopenias have been identified, very little is known about the genetic determinants of normal platelet count variation. The results suggest that two QTLs on chromosome 3q influence platelet count variation in the Asian Indian kindred, with the GPIX transmembrane mutation and the 3′ UTR SNP in THPO, being strong candidates.

Activation of Clotting Factors in Thrombocytopenia Due to Platelets Destruction: A Possible Compensated Mechanism for Hemostasis.

INTRODUCTION. Thrombocytopenia (TP) due to bone marrow failure, as in aplastic anemia (AA), is often associated with frequent and fatal bleeding, whereas fatal bleeding in TP due to platelet destruction, as in ITP, is rare. It has been suggested that giant platelets or increased cell-derived microparticles (MP) act to limit bleeding in ITP. We investigated clotting factors and MP from 3 cell types, platelets (PMP), leukocytes (LMP), and endothelial (EMP), in patients with different causes of their TP.METHODS. Group 1 (platelet destruction) consisted of 2 subgroups, Grp 1a having n=35 pts with active ITP (ITP-A) and Grp 1b having n=15 pts with platelet destruction (PD) by other disorders (TTP, PNH, others). Group 2 (impaired production (IP)) consisted of n=19 pts with AA, MDS/MPD, and others. Group 3 comprised n=28 pts with ITP in remission (ITP-R). Laboratory measures included CBC, platelets, FVIII, FIX, FXI, and 3 kinds of MP: PMP, LMP, EMP. Signs and symptoms of bleeding were graded and recorded.RESULTS. Key data is summarized in Table 1. The percentage of patients with elevated activity of FVIII was significantly more prevalent in Grp 1a vs. Grp 3 (53% vs. 15%, p=0.002) or Grp 1b vs. Grp 2 (73% vs. 11%, p=0.0002). The mean value of FVIII titer was also significantly higher in Grp 1a vs. Grp 3 (1.92 vs. 1.33 U/mL, p=0.0036) or Grp 1b vs. Grp 2 (2.44 vs. 1.18 U/mL, p=0.00007). The mean percentage of patients with elevated FIX or FXI, or the mean value of FIX or FXI titer were also higher in Grp 1a vs. Grp 3 or Grp 1b vs. Grp 2, but the difference did not reach statistical significance. PMP counts were found to correlate with platelet counts in all groups, and were significantly higher in Grp 3 than Grp 1a (p=0.0004). Grp 3 also had higher EMP than Grp 1a (p=0.04). LMP showed no significant differences.REPRESENTATIVE CASE. A patient with chronic ITP who relapsed with severe acute ITP was monitored sequentially. In acute phase of TP, activities of FVIII, FIX, and FXI surged, in parallel with shortening of aPTT, and patient had minimal signs of bleeding. Serial assays revealed gradual fall of factor activities, reaching normal levels after 5 months. A similar pattern was observed in another patient, with acute TTP: clotting factors returned to normal after 2 months.CONCLUSION. TP due to platelet destruction appears to trigger activation of intrinsic clotting pathway which may protect from excessive bleeding. Surge in FVIII was most notable. This compensation effect appears to be absent in TP due to depressed platelet production, and likewise for ITP in remission. The mechanism underlying this phenomenon remains to be elucidated. Further study is in progress to delineate the mechanism.Table 1Grp1a (ITP-A)Grp1b (PD)Grp2 (IP)Grp3 (ITP-R)p valuep valueNumber351519281avs31bvs2FVIII (%)537311150.0020.0002FIX (%)1927613NSNSFXI (%)161408NSNSPMP8315155319768170850.0004NSEMP3283253935780.04NSLMP1388123012551486NSNS