Macrothrombocytopenia Research Articles

Structural impact of pathogenic SNPs on β-tubulin using molecular dynamics study

Single nucleotide polymorphisms (SNPs) in the TUBB1 (β-tubulin) gene have been implicated as the primary cause of macro thrombocytopenia. Therefore it is essential to identify the potential SNPs which are harmful to cause diseases such as macro thrombocytopenia. The impact caused by these variants on β-tubulin is twofold, both structural and functional. Multiple in-silico tools were used to scrutinise the most deleterious nsSNPs (non-synonymous SNPs) via sequence and structure-based approaches. Further, the β-tubulin protein model incorporating identified mutants was subjected to MD (molecular dynamic) simulations to analyse the impact on protein structure. A total of 2974 SNPs of TUBB1 were retrieved from various sources, and 32 nsSNPs were identified. By screening through sequence-based technique, 13 variants were detected as deleterious and further structure-based filtration was carried out to find thermally destabilising variants. Finally, three variants have been detected as highly destabilising by the mCSM server and chosen for the MD study. All three variants are present in the N-terminal, Intermediate, and C-terminal regions, breaking the spatial arrangement required for microtubule assembly. The spatial arrangement of these variants is in deviation with respect to WT (wild type) β-tubulin. The protein model was subjected to a simulation period of 100 ns. The FEL analysis revealed multiple clusters with minor populations indicating the unstable conformation adapted by the β-tubulin. The normal mode vector analysis exhibited high-intensity flexible motions at the C-terminal end, responsible for binding with MAPs (microtubule-associated proteins), an essential region in microtubule assembly. All these results reveal that the SNP’s predicted eventually influence the spatial arrangement of β-tubulin, which would disturb the stacking arrangement of αβ tubulin dimer in microtubule assembly. The present study may set a path to cure the diseases like macro thrombocytopenia. Communicated by Ramaswamy H. Sarma

Identification By Whole Exome Sequencing of the Molecular Defect in a Novel Gene Related to Glycosylation in Two Unrelated Families with Syndromic Macrothrombocytopenia

IntroductionInherited thrombocytopenias (ITs) are a heterogeneous group of rare platelet disorders. which lead not only to increased bleeding, but also to syndromic forms. ITs are caused by genetic alterations in megakaryopoiesis-related genes. In the last years, whole-exome sequencing (WES) has allowed the identification of novel genes involved in IT.AimTo perform the molecular, clinical and platelet characterization of two unrelated families with syndromic IT, to unveil the underlaying alteration leading to the disease. To explore the functional role of the identified alterations during megakaryocytic (Mk) differentiation.MethodsWES was performed in two unrelated non-consanguineous families with lifelong severe macrothrombocytopenia (MCT), bleeding, and extra-hematological manifestations. Bleeding score (BS) was recorded by ISTH-BAT. Platelet phenotyping included platelet count (P), blood film, aggregometry (LTA) and flow cytometry (FC). UDP-galactose-4-epimerase enzymatic activity was measure by HPLC/MS/MS. In vitro functional studies were performed through overexpression of GALE genetic variants in human K562 cell line to elucidate its role in Mk differentiation, by measuring cell ploidy and expression of CD41, CD61 and CD42b surface markers after 7-days of PMA treatment.ResultsFamily pedigrees are shown in Figure 1. Three patients (A.II.1, A.II.2 and B.II.1) were referred due to lifelong severe MCT and moderate-severe bleeding tendency (Figure 1). Moreover, they presented mental retardation, mitral insufficiency, and increased bilirubin levels. Blood film revealed enlarged, giant, and grey platelets (A.II.1: 36%, 6% and 54%, respectively; A.II.2: 56%, 4%, 34%, respectively; B.II.1: 32%, 46%, 12%).LTA showed moderate/severe impaired aggregation with ADP, TRAP-6, CRP, epinephrine, arachidonic acid, and ristocetin. FC confirmed null secretion of alpha and dense granules in A.II.1, A.II.2 and reduced levels in B.II.1 (7.8%, 8.1%, 28.3% respectively, vs. 51.7% control platelets with ADP 10µM; 10.8%, 7.8%, 36.7% respectively, vs. 95.6% control platelets with TRAP6 25µM).WES revealed that both pedigrees carried compound heterozygous variants in GALE (NM_001127621.2): c.230_231insTGTT; p.Lys78Valfs*32 (exon 3), and c.449C>T; p.Thr150Met (exon 5) in A.II.1 and A.II.2 patients; and, c.668T>C, p.Leu223Pro (exon 7), and c.382G>A, p.Val128Met (exon 5) in B.II.1 (Figure 1). Enzymatic activity of the GALE-encoded protein UDP-galactose-4-epimerase was severely reduced in the affected patients: both A.II.1, A.II.2 patients had 1.3 μmol/h/g hemoglobin (control: 8.8 μmol/h/g hemoglobin), and B.II.1 patient had 0.6 μmol/h/g hemoglobin (control: 8 μmol/h/g hemoglobin).Furthermore, in vitro overexpression assays between wild-type GALE and p.Thr150Met, p.Leu223Pro and p.Val128Met variants, confirmed a delayed maturation of Mks upon PMA treatment (at 3, 5 and 7 days). characterized by a significant reduction in the expression of the megakaryocytic surface markers CD41, CD61 and CD42b.ConclusionWES has allowed us to identify pathogenic variants in GALE, which were associated with syndromic IT characterized by severe macrothrombocytopenia. Patients harboring these pathogenic variants presented moderate to severe bleeding tendency associated with cardiovascular and neurological abnormalities. Regarding the platelet phenotype, the presence of giant and grey platelets and the absence of both platelet granules were the most remarkable features reported. Moreover, these GALE variants led to an alteration in in vitro Mk maturation, supporting the thrombocytopenic phenotype observed in patients.FundingISCIII (PI17/01966, PI 17/01311, PI20/00926), GRS (GRS2061A/19, GRS2135/A/2020), Fundación Séneca (19873/GERM/15), Fundación Mutua Madrileña (AP172142019), Premio López Borrasca (2019), Grupo Trabajo Patología Hemorrágica-SETH (2020). [Display omitted] DisclosuresHernández-Rivas: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

3120 - Dominant-Negative GFI1B Mutations are Causal in Rare Inherited Platelet Disorders and Cause Defects in Stress Thrombopoiesis

Inherited platelet disorders represent a heterogeneous group of rare diseases often characterized by lifelong bleeding diatheses. Specific therapies are lacking because the affected molecular processes are poorly understood. Previously, heterozygous nonsense mutations in Growth Factor Independence 1b (GFI1B) in families with autosomal-dominant bleeding disorders were identified. This disease is characterized by a plethora of megakaryocyte (MK) and platelet abnormalities, including macro thrombocytopenia, dysmorphic megakaryocytes, paucity of platelet /-granules, CD34 expression on MKs and platelets, increased MK numbers and their dislocation in the marrow. GFI1B is a transcription factor that contains an evolutionary conserved N-terminal SNAG domain and six C-terminal zinc fingers of which zinc finger three to five are crucial for DNA binding. Through the SNAG domain GFI1B recruits chromatin-modifying enzymes to promoters of target genes to regulate gene expression. Many mutations in the GFI1B gene found in inherited platelet disorders are located in zinc finger five, resulting in a DNA binding defective protein. To study the consequences of these mutations in vivo, we used Crispr/Cas technology and established three independent mouse lines that carry one Gfi1b allele in which the 5th and 6th zinc fingers are truncated or eliminated. All three lines of GFI1B DN mice had a mild but significant reduction of platelet counts resembling the situation found in patients. Moreover, GFI1B DN mice showed an expansion MKs and Mk precursors as well as hematopoietic stem cells. To test whether the DN mutations affect platelet recovery, we injected GFI1B DN mice and controls with an anti GP-IB antibody, which leads to an almost complete depletion of platelets. Clearly, GFI1B DN mice showed a delayed recovery of platelet numbers compared to controls. In addition, in many cases the platelets of DN mice were larger and appeared less granular after recovery than their counterparts in wt mice. Our data provide evidence for a causal relationship between inherited mutations in GFI1B in patients with bleeding disorders and reveal a new role for GFI1B in stress thrombopoiesis.

Mutations of the integrin αIIb/β3 intracytoplasmic salt bridge cause macrothrombocytopenia and enlarged platelet α-granules.

Rare gain-of-function mutations within the ITGA2B or ITGB3 genes have been recognized to cause macrothrombocytopenia (MTP). Here we report three new families with autosomal dominant (AD) MTP, two harboring the same mutation of ITGA2B, αIIbR995W, and a third family with an ITGB3 mutation, β3D723H. In silico analysis shows how the two mutated amino acids directly modify the salt bridge linking the intra-cytoplasmic part of αIIb to β3 of the integrin αIIbβ3. For all affected patients, the bleeding syndrome and MTP was mild to moderate. Platelet aggregation tended to be reduced but not absent. Electron microscopy associated with a morphometric analysis revealed large round platelets; a feature being the presence of abnormal large α-granules with some giant forms showing signs of fusion. Analysis of the maturation and development of megakaryocytes reveal no defect in their early maturation but abnormal proplatelet formation was observed with increased size of the tips. Interestingly, this study revealed that in addition to the classical phenotype of patients with αIIbβ3 intracytoplasmic mutations there is an abnormal maturation of α-granules. It is now necessary to determine if this feature is a characteristic of all mutations disturbing the αIIb R995/β3 D723 salt bridge.

Pathogenic Variants in Thrombocytopenia Genes in 119 Cases with Undiagnosed Heritable Thrombocytopenia

The incidence of inherited rare bleeding, thrombotic and platelet disorders (BPD) is estimated to be 200-250 per million individuals. A considerable portion have heritable thrombocytopenia (HT), a genetically heterogeneous group of disorders with which 27 of the 76 ISTH-approved Tier 1 BPD genes have been causally associated. It is important to distinguish cases because variants in some HT genes confer risk of malignancy and pathologies outside hemopoiesis. This can be seldom achieved by considering clinical and laboratory characteristics alone. In order to improve the precision of diagnosis for HT subtypes and to identify novel genes underlying HT, we have developed two complementary high throughput sequencing (HTS) approaches. The first is for BPD cases with an assumed molecular diagnosis; 500 DNA samples have been sequenced with the ThromboGenomics HTS gene panel test for the 76 Tier 1 BPD genes. The second is for patients with rare disorders of unclear molecular aetiology enrolled in the NIHR BioResource. To identify the genetic basis of these rare disorders 10,000 DNA samples have been whole genome sequenced, including samples from 1,378 BPD probands, 123 affected and 41 unaffected relatives. Upon consenting, clinical parameters, laboratory results and pedigree history were deposited in a shared single study database and human phenotype ontology (HPO) terms appended to each patient. Novel statistical methods have been deployed to identify causal variants. Here we report on the pathogenic and likely pathogenic variants (PV and LPV) in 27 Tier 1 genes identified in cases with HT who had completed evaluation by either approach by July 2016.We defined PV as either high impact variants or variants present in 4 or more independent HT cases reported in the literature or our study database, and LPV as other credible variants with fewer than 4 independent cases previously observed. All cases were reviewed at a multi-disciplinary team meeting before being reported as "explained", "unexplained" or "partially explained" if components of the broader phenotype could not be explained by the variants observed. Between January 2011 and July 2016, 119 cases from 104 pedigrees with HT completed analysis with the identification of a PV or LPV in a Tier 1 HT gene believed to explain or partially explain the described phenotype. Variants were identified in 18 of the 27 HT genes (Table 1), including genes encoding transcription regulators (11/104 pedigrees) and platelet membrane proteins (8/104 pedigrees). However, variants in 5 genes encoding cytoskeletal components or regulators comprised the largest single functional group (57/104 pedigrees).We identified 18 different PV or LPV (8 novel) in ACTN1 in 29 pedigrees with non-syndromic macrothrombocytopenia (MTP) and 8 different PV or LPV (6 novel) in TUBB1 in 8 further pedigrees with non-syndromic MTP. PV or LPV in Tier 1 cytoskeletal HT genes associated with thrombocytopenia and variable non-haematological syndromic features were identified in 20 pedigrees, including 3 pedigrees with 2 different gain-of-function variants in DIAPH1 causing MTP, intermittent neutropenia and sensorineural hearing impairment. One variant has recently been reported by our Consortium, the other is novel. The remainder comprised 2 PV in WAS in 2 pedigrees and 14 different PV or LPV (4 novel) in MYH9 in 15 pedigrees, some of whom had isolated MTP without neutrophil inclusion bodies. Of note, common variants in four of these five genes have been found to be associated with the count and volume of platelets in genome wide association studies, confirming the notion that platelet-GWAS loci are enriched for genes underlying Mendelian platelet disorders.In conclusion, HT are genetically diverse disorders for which there is an unmet need for precision diagnosis. For HT cases with clinical and laboratory characteristics similar to previously reported ones for HT, the ThromboGenomics test is a valuable and affordable approach to obtain an accredited genetic diagnosis. Whole genome sequencing is a powerful approach to identify novel genes implicated in HT for the rapid enhancement of the ThromboGenomics HTS panel test. The high prevalence of variants causal of HT in Tier 1 BPD genes that encode cytoskeletal elements highlights the pivotal role of the cytoskeleton in platelet formation by mediating pro-platelet protrusion and budding from megakaryocytes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vitamin B12 and/or Folate Deficiency is a Cause of Macro Thrombocytopenia

Vitamin B12 and/or Folate Deficiency is a Cause of Macro Thrombocytopenia

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.

Clinical and molecular genetic analysis of a family with macrothrombocytopenia and early onset sensorineural hearing loss

A kindred with inherited macrothrombocytopenia (MTCP) and sensorineural hearing loss (SNHL) from Ghent, Belgium was identified. Currently, joint expression of MTCP and hearing loss are linked to mutations within MYH9 only. Thus, we tested the hypothesis that a mutation within MYH9 is responsible for the autosomal dominant inheritance of MTCP and hearing loss in the Ghent family. A mutation screen of MYH9 coding region including its intron–exon junctions, as well as common hearing loss genes GJB2, GJB3, and GJB6, was performed. However, no pathogenic sequence alteration was identified. Patients' leukocytes were determined to be normal for NMMHC-A distribution via immunofluorescence analysis and free of Döhle body-like inclusions, identified as aggregates of mutant NMHC-IIA in MYH9 disorders. Also, western blot analysis with anti-NMHC-IIA antibody identified a single 220 kDa immunoreactive band with normal expression level of NMHC-IIA within the platelets and leukocytes of the affected family members. The immunoblot analysis eliminates the possibility of a large deletion within MYH9 that can escape detection by direct sequencing. Collectively, these results suggest that molecular genetic etiology of the Ghent family disorder may be due to as yet unidentified gene whose mutation(s) yields a phenocopy of the MYH9-related disease.

Unexpected Consequences of Sitosterolemia on Platelet Formation.

Sitosterolemia is a rare autosomal recessive disorder characterized by the accumulation of plant and animal sterols in blood and tissues. The disease is caused by a mutation in either of the ATP-binding cassette half transporters Abcg5 or Abcg8, which function as obligate heterodimers that are highly expressed in the liver and intestine where they transport plant sterols into bile or into the intestinal lumen, respectively. Patients with sitosterolemia develop xanthomas, arthritis, hemolysis, thrombocytopenia, short stature and premature cardiovascular disease. Recently, patients with Mediterranean stomatocytosis or Mediterranean macro thrombocytopenia were characterized as sitosterolemia patients. In this study we addresed the underlying mechanisms for thrombocytopenia in sitosterolemia using the Abcg5 deficient mouse as a model. Abcg5−/− mice demonstrated macro thrombocytopenia with reduced platelets counts (262±58×109/L) compared to wt controls (805±65×109/L, p<0.001) and increased platelet size as determined visually on blood smears and by forward scatter analysis on a flow cytometer (48±11 A.U. in Abcg5−/− mice versus 6.2±0.4 A.U. in wt controls, p<0.01). Red blood cell counts were only slightly decreased in Abcg5−/− mice (8.7×109/L) versus 9.4×109/L in controls, whereas MCV was not affected. The decreased platelet levels correlated inversely with increased plasma TPO levels in Abcg5−/− mice (528±136 pg/ml versus 338±57 pg/ml in wt, p=0.02) and increased megakaryocyte (MK) progenitors in the bone marrow (36±1 CFU-MK/1×105 cells versus 17±2 CFU-MK in wt controls, p<0.01). Furthermore, Abcg5−/− mice had slightly decreased body weight, but a significantly larger spleen which contained increased numbers of MKs (16.3% CD41+ cells versus 4.5% in wt controls). The MKs from Abcg5−/− mice showed a normal morphology, with no difference in DNA ploidy distribution and were able to form proplatelet structures in vitro. Quantitative PCR analysis revealed decreased expression levels of the cholesterol transporters Abca1 and Abcg1 and increased expression of the HDL receptor SR-B1 in MKs from Abcg5−/− mice, indicative for altered cellular cholesterol homeostasis. Interestingly, Abcg5 nor Abcg8 were expressed in MKs. To establish whether macro thrombocytopenia was due to the Abcg5 deficiency per se or caused by too high plant sterol levels, bone marrow (BM) transplantation studies were performed in lethally irradiated mice. Wt mice receiving Abcg5−/− BM cells displayed normal platelet size and only slightly decreased platelet levels (639±87×109/L) at 13 weeks after transplantation, whereas Abcg5−/− mice receiving wt BM cells showed macro thrombocytopenia with decreased platelets counts (353±65×109/L) and increased platelet size. These studies unequivocally demonstrate that macro thrombocytopenia associated with sitosterolemia is not related to a genetic defect in MKs, but a consequence of high plant sterol levels that have profound effects on platelet formation.

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.

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.

Expression of the nonmuscle myosin heavy chain IIA in the human kidney and screening for MYH9 mutations in Epstein and Fechtner syndromes.

Mutations in the MYH9 gene, which encodes the nonmuscle myosin heavy chain IIA, have been recently reported in three syndromes that share the association of macrothrombocytopenia (MTCP) and leukocyte inclusions: the May-Hegglin anomaly and Sebastian and Fechtner syndromes. Epstein syndrome, which associates inherited sensorineural deafness, glomerular nephritis, and MTCP without leukocyte inclusions, was shown to be genetically linked to the same locus at 22q12.3 to 13. The expression of MYH9 in the fetal and mature human kidney was studied, and the 40 coding exons of the gene were screened by single-strand conformation polymorphism in 12 families presenting with the association of MTCP and nephropathy. MYH9 is expressed in both fetal and mature kidney. During renal development, it is expressed in the late S-shaped body, mostly in its lower part, in the endothelial and the epithelial cell layers. Later, as well as in mature renal tissue, MYH9 is widely expressed in the kidney, mainly in the glomerulus and peritubular vessels. Within the glomerulus, MYH9 mRNA and protein are mostly expressed in the epithelial visceral cells. Four missense heterozygous mutations that are thought to be pathogenic were found in five families, including two families with Epstein syndrome. Three mutations were located in the coiled-coil rod domain of the protein, and one was in the motor domain. Two mutations (E1841K and D1424N) have been reported elsewhere in families with May-Hegglin anomaly. The two others (R1165L and S96L) are new mutations, although one of them affects a codon (R1165), found elsewhere to be mutated in Sebastian syndrome.