Chain Of Non-muscle Myosin IIA Research Articles

MYH9 is a novel cancer stem cell marker and prognostic indicator in esophageal cancer that promotes oncogenesis through the PI3K/AKT/mTOR axis.

MYH9 encodes the heavy chain of nonmuscle myosin IIA, a ubiquitously expressed cytoplasmic myosin that regulates the actin cytoskeleton, cell migration, cell polarization, and signal transduction in cancer cells. Here, we investigated the role of MYH9 in cancer stem cells (CSCs) associated with esophageal cancer (EC).The subcellular localization of MYH9 was investigated in SKGT-4 cells through immunofluorescent analysis. MYH9+ and MYH9- spheroid cells were derived from SKGT-4 cells by flow cytometry and compared for self-renewal capacity, tumorigenicity, CD133 positivity, cisplatin resistance, and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR) activity. MYH9 messenger RNA expression was assessed in 30 EC patients by quantitativereverse transcription-polymerase chain reaction. Kaplan-Meier curves were plotted to explore the influence of MYH9 on EC survival.MYH9 localized to the plasma membrane, cytoplasm, and nucleus of SKGT-4 cells. Spheroid cells displayed higher MYH9 expression and positivity compared to parental SKGT-4 cells. MYH9+ cells showed strong CSC characteristics, including in vivo tumorigenicity, migration, invasion, cisplatin resistance, and CD133+ positivity. MYH9 activated the PI3K/AKT/mTOR axis in CSCs and was upregulated in EC patients with poor survival. Collectively, these data show that MYH9 significantly promotes tumorigenesis by regulating PI3K/AKT/mTOR signaling in EC. MYH9 expression remarkably correlates with poor prognosis and represents a novel biomarker and drug target for the diagnosis and treatment of EC.

Defective VWF secretion due to expression of MYH9-RD E1841K mutant in endothelial cells disrupts hemostasis

AbstractMutations in MYH9, the gene encoding the heavy chain of nonmuscle myosin IIa (NMII-A), cause MYH9-related disease (MYH9-RD), which is an autosomal-dominant thrombocytopenia with bleeding tendency. Previously, we showed that NMII-A in endothelial cells (ECs) is critical for hemostasis via regulating von Willebrand factor (VWF) release from Weibel-Palade bodies (WPBs). The aim of this study was to determine the role of the expression of MYH9 mutants in ECs in the pathogenesis of the MYH9-RD bleeding symptom. First, we expressed the 5 most common NMII-A mutants in ECs and found that E1841K mutant-expressing ECs secreted less VWF than the controls in response to a cyclic adenosine monophosphate (cAMP) signaling agonist. Then, we generated 2 knockin mouse lines, 1 with Myh9 E1841K in ECs and the other in megakaryocytes. Endothelium-specific E1841K mice exhibited impaired cAMP-induced VWF release and a prolonged bleeding time with normal platelets, whereas megakaryocyte-specific E1841K mice exhibited macrothrombocytopenia and a prolonged bleeding time with normal VWF release. Finally, we presented mechanistic findings that E1841K mutation not only interferes with S1943 phosphorylation and impairs the peripheral distribution of Rab27a-positive WPBs in Ecs under quiescent condition but also interferes with S1916 phosphorylation by disrupting the interaction with zyxin and CKIIα and reduces actin framework formation around WPBs and subsequent VWF secretion under the stimulation by a cAMP agonist. Altogether, our results suggest that impaired cAMP-induced endothelial VWF secretion by E1841K mutant expression may contribute to the MYH9-RD bleeding phenotype.

MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis.

The accepted notion of dNTP transport following cytoplasmic biosynthesis is ‘facilitated diffusion’; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a ‘reservoir’. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.

Cochlear implantation is safe and effective in patients with MYH9-related disease.

BackgroundMYH9-related disease (MYH9-RD) is a rare syndromic disorder deriving from mutations in MYH9, the gene for the heavy chain of non-muscle myosin IIA. Patients present with congenital thrombocytopenia and giant platelets and have a variable risk of developing sensorineural deafness, kidney damage, presenile cataract, and liver abnormalities. Almost all MYH9-RD patients develop the hearing defect, which, in many individuals, progresses to severe to profound deafness with high impact on quality of life. These patients are potential candidates for cochlear implantation (CI), however, no consistent data are available about the risk to benefit ratio of CI in MYH9-RD. The only reported patient who received CI experienced perisurgery complications that have been attributed to concurrent platelet defects and/or MYH9 protein dysfunction.MethodsBy international co-operative study, we report the clinical outcome of 10 patients with MYH9-RD and severe to profound deafness who received a CI at 8 institutions.ResultsNine patients benefited from CI: in particular, eight of them obtained excellent performances with restoration of a practically normal hearing function and verbal communication abilities. One patient had a slightly worse performance that could be explained by the very long duration of severe deafness before CI. Finally, one patient did not significantly benefit from CI. No adverse events attributable to MYH9-RD syndrome were observed, in particular no perisurgery bleeding complications due to the platelet defects were seen. Patients’ perioperative management is described and discussed.ConclusionsCI is safe and effective in most patients with MYH9-RD and severe to profound deafness and should be offered to these subjects, possibly as soon as they develop the criteria for candidacy.

Abstract 19281: Chemokine-Coupled β2 Integrin-Dependent mRNA Stabilization in Macrophages Modulates VEGF-A Protein Levels During Ischemia-Induced Arteriogenesis

VEGF-A directs both developmental and inducible arteriogenesis through the maturation of smaller vessels and the recruitment of collateral networks. Our lab has demonstrated that integrin-induced mRNA stability plays an important role in macrophage production of VEGF-A protein through translocation and activation of the mRNA stabilizing protein, HuR. It is well known that macrophages may serve as a source of VEGF-A; however, it remains unclear whether macrophages are the principle source of VEGF-A during ischemia-induced arteriogenesis and whether this integrin signaling mechanism modulates VEGF-A protein levels in ischemic muscle tissue in vivo. Here we demonstrate that chemokine-coupled β2 integrin adhesion signaling results in the formation of a complex between the low molecular weight G-protein, Rac2, and the heavy chain of Nonmuscle Myosin IIA (MyH9), leading to translocation of the message stabilizing protein, HuR, and the subsequent prolongation of VEGF-A mRNA half-life. Macrophage-specific gene deletion of MyH9 reduces chemokine-coupled β2 integrin adhesion-stimulated HuR translocation and consequent stabilization of VEGF-A message. Moreover, during hindlimb ischemia, there is global loss of VEGF-A protein in the arteriogenic muscle tissue along with decreased perfusion recovery secondary to decreased small vessel arteriogenesis in the macrophage-specific MyH9-/- mice. These defects appear specific to the induced inflammatory response as these mice demonstrate adequate capillary angiogenic, arteriogenic and arteriolar collateral development at baseline. This chemokine-coupled, adhesion-based signaling pathway provides a unique, rapid molecular switch in gene expression whereby inflammatory monocyte-derived macrophages become the key in vivo source of VEGF-A protein during ischemia-induced arteriogenesis. We anticipate this pathway to be a novel target for the therapeutic induction or inhibition of VEGF-A-mediated arteriogenesis where clinically relevant.

MYH9-related disease: Five novel mutations expanding the spectrum of causative mutations and confirming genotype/phenotype correlations

MYH9-related disease (MYH9-RD) is a rare autosomal dominant syndromic disorder caused by mutations in MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA (myosin-9). MYH9-RD is characterized by congenital macrothrombocytopenia and typical inclusion bodies in neutrophils associated with a variable risk of developing sensorineural deafness, presenile cataract, and/or progressive nephropathy. The spectrum of mutations responsible for MYH9-RD is limited. We report five families, each with a novel MYH9 mutation. Two mutations, p.Val34Gly and p.Arg702Ser, affect the motor domain of myosin-9, whereas the other three, p.Met847_Glu853dup, p.Lys1048_Glu1054del, and p.Asp1447Tyr, hit the coiled-coil tail domain of the protein. The motor domain mutations were associated with more severe clinical phenotypes than those in the tail domain.

MYH9-related Disorders

Myosin heavy chain-9 (MYH9)-related disorders represent a heterogenous group of hereditary diseases caused by mutations in the gene encoding the heavy chain of nonmuscle myosin IIA. May-Hegglin anomaly and Fechtner, Sebastian, and Epstein syndromes are the four phenotypes of the disease, characterized by congenital macrothrombocytopenia and distinguished by different combinations of clinical signs that may include glomerulonephritis, sensorineural hearing loss, and presenile cataract. The spectrum of mutations responsible for the disease is wide and the existence of genotype-phenotype correlation remains a critical issue. We report the first case of an MYH9-RD in a patient of Greek origin presenting with macroscopic hematuria and presenile cataract caused by a p.R1165C mutation. The same mutation was present in the patient's father, who exhibited no extrahematological features of the disease. The p.R1165C mutation is one of the MYH9 alterations whose prognostic significance is still poorly defined. Thus, the patients described add to the limited existing data on the MYH9 mutations and their resultant phenotypes.

Recent advances in the understanding and management of MYH9‐related inherited thrombocytopenias

MYH9-related disease (MYH9-RD) is one of the most frequent forms of inherited thrombocytopenia. It is transmitted in an autosomal dominant fashion and derives from mutations of MYH9, the gene for the heavy chain of non-muscle myosin IIA. Patients present with congenital macrothrombocytopenia with mild bleeding tendency and may develop kidney dysfunction, deafness and cataracts later in life. The term MYH9-RD encompasses four autosomal-dominant thrombocytopenias that were previously described as distinct disorders, namely May-Hegglin Anomaly, Sebastian, Fechtner and Epstein syndromes. Thrombocytopenia is usually mild and derives from complex defects of megakaryocyte maturation and platelet formation. It is easily diagnosed, in that the presence of giant platelets in peripheral blood raises the suspicion of MYH9-RD and a simple immunofluorescence test on blood films confirms the diagnostic hypothesis. However, genotype/phenotype correlations have been recognized and mutation screening is therefore required to define the risk of acquiring extra-haematological defects. Results of a small clinical study suggested that a non-peptide thrombopoietin mimetic might greatly benefit both thrombocytopenia and bleeding tendency of MYH9-RD patients.

Eltrombopag for the Treatment of the Inherited Thrombocytopenia Deriving From MYH9 Mutations

AbstractAbstract 2533MYH9-Related Disease (MYH9-RD) is one of the less rare forms of inherited thrombocytopenia. It derives from mutations of the gene MYH9 for the heavy chain of nonmuscle myosin IIA and is characterized by congenital macrothrombocytopenia variably associated with young-adult onset of hearing loss, cataract, and a severe proteinuric nephropathy.Only platelet transfusions are available for increasing platelet counts in this condition, but they expose to the risks of acute reactions, transmission of infectious diseases, and refractoriness to subsequent platelet transfusions. Moreover, this treatment suffers from scarceness of blood donors.Novel thrombopoiesis-stimulating agents have been developed and 2 of them, eltrombopag and romiplostin, have been approved for increasing platelet count in a few forms of acquired thrombocytopenia. Since it has been recently shown that megakaryocytes of patients with MYH9-RD respond in vitro to TPO stimulation, we reasoned that TPO-mimetics could be effective also in this condition and decided to test the effect of eltrombopag.Therefore, we performed a phase II, multicentre, open-label, dose escalation trial. Twelve adult patients with a platelet count lower than 50×10e9/L and a diagnosis of MYH9-RD confirmed by identification of the causative MYH9 mutations received orally eltrombopag 50 mg daily for 21 days (Revolade®, GSK). Patients with platelet counts lower than 100×10e9/L at day 21 increased eltrombopag to 75 mg daily for 21 additional days. Patients with platelet counts between 100 and 150×10e9/L at day 21 continued eltrombopag 50 mg daily for the following 21 days, while patients with more than 150×10e9 platelets/L stopped therapy.The primary endpoints were the achievement of a platelet count over 100×10e9/L or at least three times the baseline value (major response), or at least twice the baseline value but less than major response (minor response). Secondary end points included safety and tolerability, and the reduction of bleeding tendency.After 3 weeks at the eltrombopag dose of 50 mg daily, 3 patients achieved platelet counts of 150×10e9/L or more and stopped therapy. Two had a platelet counts between 100 and 150×10e9/L and continued treatment at the same dosage, while 7 had less than 100×10e9 platelets/L and received eltrombopag 75 mg daily for 3 weeks.A major response was obtained in 8 patients (67%), in 5 of them after 3 weeks of eltrombopag 50 mg daily, and in 3 cases after 3 additional weeks at the dose of 75 mg. Three patients (25%) achieved a minor response, 1 after 3 weeks at 50 mg daily, and 2 after 3 additional weeks at 75 mg. In one patient the treatment resulted in no response.Mean platelet count at the end of treatment was significantly higher than at baseline (105 versus 31×10e9 platelets/L, p=0.0022). In the 11 patients that achieved major or minor responses, mean platelet count was still higher than baseline 15 days after discontinuation of the drug, while it returned to levels near baseline 15 days later.Platelet size did not change either during treatment or after its cessation, and this indicates that the increases in platelet count were paralleled by corresponding increases in total platelet mass.The extent of platelet aggregation was within the normal range after all tested agonists in 5 of the 7 patients that achieved platelet counts higher than 100×10e9/L, while it was slightly reduced after ADP and collagen in 2 patients.Mean serum TPO level was higher than the normal range and this figure did not change neither during treatment with eltrombopag nor after its discontinuation.Ten of 12 patients had grade 1 or 2 bleeding symptoms, as measured by the WHO bleeding scale, at baseline, while 2 were asymptomatic. Upon treatment, bleeding diathesis quickly ameliorated and disappeared in 8 cases, while it remained unchanged in the only patient with no response to eltrombopag and in another one with a minor response. The benefit in terms of bleeding diathesis lasted well beyond treatment discontinuation.Treatment was well tolerated in all cases, with only two patients reporting mild and transient headache and one patient suffering from transient dry mouth at the beginning of treatment.In conclusion, 50–75 mg of eltrombopag per day increased platelet count and reduced bleeding tendency in most patients with MYH9-RD. Further research is required to ascertain whether TPO mimetics are effective also in other forms of inherited thrombocytopenia. Disclosures:Off Label Use: Eltrombopag for MYH9-related disease.

A G to C transversion at the last nucleotide of exon 25 of the MYH9 gene results in a missense mutation rather than in a splicing defect

MYH9-related disease ( MYH9-RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. Patients present with congenital macrothrombocytopenia and inclusion bodies in neutrophils and might develop sensorineural deafness, presenile cataract, and/or progressive nephropathy leading to end-stage renal failure. In two families with macrothrombocytopenia we identified a novel c.3485G > C mutation in the last nucleotide of exon 25. Bioinformatic tools for splice site prediction and minigene functional test predicted splicing anomalies of exon 25. However, analysis of RNA purified from patient’s peripheral blood did not allowed us to detect any anomalies, suggesting that RNA processing is correct at least in this tissue. Therefore, we concluded that c.3485G > C leads to a novel missense mutation (p.Arg1162Thr) of myosin-9, which resulted to be slightly degraded in patient platelets. A precise definition of the effect of mutations is fundamental to improve our knowledge into the pathogenetic mechanisms responsible for the disease.

MYH9 related disease: four novel mutations of the tail domain of myosin‐9 correlating with a mild clinical phenotype

MYH9-related disease (MYH9-RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. All patients present congenital macrothrombocytopenia and inclusion bodies in neutrophils. Some of them can also develop sensorineural deafness, presenile cataract, and/or progressive nephropathy leading to end-stage renal failure. We report four families, each with a novel mutation: two missense mutations, in exons 31 and 32, and two out of frame deletions in exon 40. They were associated with no bleeding diathesis, normal, or only slightly reduced platelet count and no extra-hematological manifestations, confirming that alterations of the tail domain cause a mild form of MYH9-RD with no clinically relevant defects.

Dense mapping of MYH9 localizes the strongest kidney disease associations to the region of introns 13 to 15

Admixture mapping recently identified MYH9 as a susceptibility gene for idiopathic focal segmental glomerulosclerosis (FSGS), HIV-associated nephropathy (HIVAN) and end-stage kidney disease attributed to hypertension (H-ESKD) in African Americans (AA). MYH9 encodes the heavy chain of non-muscle myosin IIA, a cellular motor involved in motility. A haplotype and its tagging SNPs spanning introns 12–23 were most strongly associated with kidney disease (OR 2–7; P < 10−8, recessive). To narrow the region of association and identify potential causal variation, we performed a dense-mapping study using 79 MYH9 SNPs in AA populations with FSGS, HIVAN and H-ESKD (typed for a subset of 46 SNPs), for a total of 2496 cases and controls. The strongest associations were for correlated SNPs rs5750250, rs2413396 and rs5750248 in introns 13, 14 and 15, a region of 5.6 kb. Rs5750250 showed OR 5.0, 8.0 and 2.8; P = 2 × 10−17, 2 × 10−10 and 3 × 10−22, respectively, for FSGS, HIVAN and H-ESKD; OR 5.7; P = 9 × 10−27 for combined FSGS and HIVAN, recessive. An independent association was observed for rs11912763 in intron 33. Neither the highly associated SNPs nor the results of resequencing MYH9 in 40 HIVAN or FSGS cases and controls revealed non-synonymous changes that could account for the disease associations. Rs2413396 and one of the highly associated SNPs in intron 23, rs4821480, are predicted splicing motif modifiers. Rs5750250 combined with rs11912763 had receiver operator characteristic (ROC) C statistics of 0.80, 0.73 and 0.65 for HIVAN, FSGS and H-ESKD, respectively, allowing prediction of genetic risk by typing two SNPs.

The May-Hegglin anomaly in a kidney transplant recipient.

We report a 37-year-old renal transplanted patient with thrombocytopaenia. He had May–Hegglin anomaly (MHA) inherited from his father, who died due to myocardial infarction subsequent to two renal transplantations. At the age of 15 years, proteinuria was found, but he was not biopsied. In 1982, he underwent splenectomy because of trauma. In 1989, when he was 17, renal function was found to be decreased. Five years later, he started haemodialysis and at 24 years he underwent live related kidney transplantation. Serum creatinine was always found stable around 1.9 mg/dl on cyclosporine, azathioprine and steroids. Automated blood count reported a number of platelets around 8000/μl with a mean volume of 20.3 fl (normal range 7.2– 11.1 fl). However, blood cell counters have a defined volume window for platelet counting and large macrothrombocytes are classified as red blood cells, meaning that platelet number is underestimated (Figure 1). The true value of platelets after count with the microscope was around 30 000/μl. He was completely asymptomatic, without any bleeding tendency. MHA is a rare autosomal dominant platelet disorder, characterized by macrothrombocytopaenia and granulocyte inclusion bodies. The mutation involves the MYH9 gene, localized in chromosome 22q, which encodes a heavy chain of non-muscle myosin IIA. MYH9-related hereditary macrothrombocytopaenia disorders include: MHA, Ebstein, Fechtner and Sebastian syndromes, which are characterized by nephritis, hearing loss and cataract [1]. In 1992, Nel et al. [2] reported two cases of MHA incidentally discovered in a patient and his brother during investigation for end-stage renal failure and workup for renal transplantation. Alhindawi and Al-Jbour [3] described an association between Epstein syndrome and chronic nephropathy. Recently, Singh et al. [4] suggested that MYH9 gene alterations are associated with a greater risk of focal segmental glomerulosclerosis and hypertensive nephrosclerosis in African Americans.

Identification of the first duplication in MYH9-related disease: A hot spot for unequal crossing-over within exon 24 of the MYH9 gene

MYH9-related disease (MYH9RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. All patients present with congenital macrothrombocytopenia and inclusion bodies in neutrophils. Some of them can also develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. The spectrum of mutations so far identified is peculiar, consisting of mostly missense mutations. Others are nonsense and frameshift mutations, all localized in the COOH terminus of the protein, or in-frame deletions. We report a family with three affected members carrying a novel mutation, the first duplication (p.E1066_A1072dup), of MYH9. The mutation was localized within exon 24, where the presence of a 16 nucleotide repeat was likely to be responsible for unequal crossing-over. Of note, a deletion of the same amino acids 1066_1072 was also identified in another MHY9RD family. Since two of the four patients with the duplication or the deletion in exon 24 were affected with bilateral neonatal cataracts, we speculate that these mutations might correlate with the ocular defect, which is reported only in 16% of MYH9RD patients.

Clinical manifestation and molecular genetic characterization of MYH9 disorders

Currently, the May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndrome are considered to be distinct clinical manifestations of a single disease caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). Manifestations of these disorders include giant platelets, thrombocytopenia and combinations of the presence of granulocyte inclusions, deafness, cataracts and renal failure. We examined 15 patients from 10 unrelated families on whom we performed immunostaining of NMMHC-IIA in blood samples. Polymerase chain reaction (PCR) analysis of selected exons of the MYH9 gene revealed mutations in nine samples with one novel mutation. Results of fluorescence and mutational analysis were compared with clinical manifestations of the MYH9 disorder. We also determined the number of glycoprotein sites on the surface of platelets. Most patients had an increased number of glycoproteins, which could be due to platelet size.

Renin-angiotensin system blockade is effective in reducing proteinuria of patients with progressive nephropathy caused by MYH9 mutations (Fechtner-Epstein syndrome)

Fetchner syndrome (FTNS, OMIM 153640) is an autosomal dominant disorder characterized by the association of macrothrombocytopaenia, leukocyte inclusions, sensorineural deafness, cataracts and nephropathy often leading to end-stage renal disease (ESRD) [1]. Epstein syndrome (EPTS, OMIM 153650) presents the same features of FTNS, with the exception of leukocyte inclusions and cataracts [2]. Recently, it was shown that both FTNS and EPTS derive from mutations of MYH9, the gene for the heavy chain of non-muscle myosin IIA [3,4]. The same gene was found to be also responsible for two non-syndromic forms of inherited macrothrombocytopaenia with leukocyte inclusions, May-Hegglin anomaly (MHA, OMIM 155100) and Sebastian syndrome (SBS, OMIM 605249) [3]. Further studies demonstrated that patients initially diagnosed as having MHA/SBS can subsequently develop nephropathy, deafness and/or cataracts, while affected relatives of FTNS or EPTS patients present for all their lifetime a clinical picture indistinguishable from that of MHA/SBS subjects [5]. Moreover, immunofluorescence studies showed that leukocyte inclusions containing the MYH9 protein are present not only in MHA/SBS and FTNS, but also in all patients with EPTS [5]. Therefore, it was clear that FTNS, EPTS, MHA and SBS are not distinct entities, but rather they represent different clinical presentations of a single disease, characterized by the constant finding of congenital thrombocytopaenia and leukocyte inclusions, and possible development of nephropathy, deafness and/or cataracts in

Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history ofMYH9-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.

High-resolution melting analysis for detection of MYH9 mutations

May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.

Metastasis-Associated Protein S100A4: Spotlight on its Role in Cell Migration

S100A4 (also known as Mts1, metastasin, p9Ka, pEL98, CAPL, calvasculin, Fsp-1, placental calcium-binding protein) belongs to the family of EF-hand calcium-binding proteins, whose expression is elevated in a number of pathological conditions. Although it is well documented that S100A4 is expressed in cancer cells and contributes to tumor cell motility and metastatic progression, the exact underlying mechanisms remain elusive. An important characteristic feature of S100 proteins is their dual function, inside and outside the cell. In this review, we focus on the intracellular function of S100A4. The review contains structural analysis of S1004 in comparison with other members of S100 proteins. Possible modes of the interaction of S100 proteins with targets are described. Several examples of best-studied molecular interactions involving S100A4 with heavy chain of nonmuscle myosin IIA, LAR-interacting protein liprin beta1 and tumor suppressor protein p53 are provided. We suggest that the binding of S100A4 to these molecules is critical for the S100A4 function. Further studies of the implications of these interactions in different molecular pathways may shed additional light on the role of S100A4 protein in the control of tumor cell motility and migration. We discuss the approaches for down-regulation of S100A4 expression and their potential for application in the clinics.

Altered cytoskeleton organization in platelets from patients with MYH9‐related disease

MYH9-related disease (MYH9-RD) is an autosomal dominant disorder deriving from mutations in the MYH9 gene encoding for the heavy chain of non-muscle myosin IIA, and characterized by thrombocytopenia and giant platelets. Isoform IIA of myosin is the only one expressed in platelets, but the possibility that MYH9 mutations affect the organization of contractile structures in these blood elements has never been investigated. In this work we have analyzed the composition and the agonist-induced reorganization of the platelet cytoskeleton from seven MYH9-RD patients belonging to four different families. We found that an increased amount of myosin was constitutively associated with actin in the cytoskeleton of resting MYH9-RD platelets. Upon platelet stimulation, an impaired increase in the total cytoskeletal proteins was observed. Moreover, selected membrane glycoproteins, tyrosine kinases, and small GTPases failed to interact with the cytoskeleton in agonist-stimulated MYH9-RD platelets. These results demonstrate for the first time that mutations of MYH9 result in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton. We suggest that these abnormalities may represent the biochemical basis for the previously reported functional alterations of MYH9-RD platelets, and for the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets.