Transfection of the mutant MYH9 cDNA reproduces the most typical cellular phenotype of MYH9-related disease in different cell lines

ABSTRACT: BACKGROUND: Heterozygous mutations of MYH9, encoding the Non-Muscular Myosin Heavy Chain-IIA (NMMHC-IIA), cause a complex disorder named MYH9-related disease, characterized by a combination of different phenotypic features. At birth, patients present platelet macrocytosis, thrombocytopenia and leukocyte inclusions containing NMMHC-IIA. Moreover, later in life some of them develop the additional features of sensorineural hearing loss, cataracts and/or glomerulonephritis that sometimes leads to end stage renal failure. RESULTS: To clarify the mechanism by which the mutant NMMHC-IIA could cause phenotypic anomalies at the cellular level, we examined the effect of transfection of the full-length mutated D1424H MYH9 cDNAs. We have observed, by confocal microscopy, abnormal distribution of the protein and formation of rod-like aggregates reminiscent of the leukocyte inclusions found in patients. Co-transfection of differently labeled wild-type and mutant full-length cDNAs showed the simultaneous presence of both forms of the protein in the intracellular aggregates. CONCLUSION: These findings suggest that the NMMHC-IIA mutated in position 1424 is able to interact with the WT form in living cells, despite part of the mutant protein precipitates in non-functional aggregates. Transfection of the entire WT or mutant MYH9 in cell lines represents a powerful experimental model to investigate consequences of MYH9 mutations

Ophthalmic complications of Lemierre syndrome

PURPOSE: Lemierre syndrome is a life-threatening condition characterized by head/neck bacterial infection, local suppurative thrombophlebitis and septic embolic complications in a range of sites of distant organs. No prior study focused on the course and characteristics of ophthalmic complications of Lemierre syndrome. METHODS: We analysed data of 27 patients with ophthalmic complications from a large cohort of 712 cases with Lemierre syndrome reported globally between 2000 and 2017. We focused on initial manifestations, early (in-hospital) course and long-term ophthalmic deficits at the time of hospital discharge or during postdischarge follow-up. The study protocol was registered in the International Prospective Register of Systematic Reviews PROSPERO (CRD42016052572). RESULTS: Nine (33%) patients were women; the median age was 20 (Q1-Q3: 15-33) years. Fusobacterium spp. was involved in 56% of cases. The most prevalent initial manifestations were decreased vision (35%) and periocular oedema (38%), followed by impaired eye movements/nerve palsy (28%) and proptosis (28%). Venous involvement, notably cerebral vein thrombosis (70%) and ophthalmic vein thrombosis (55%), explained the symptomatology in most cases. Septic embolism (7%), orbital abscesses (2%) and carotid stenosis (14%) were also present. Ophthalmic sequelae were reported in 9 (33%) patients, often consisting of blindness or reduced visual acuity, and nerve paralysis/paresis. CONCLUSION: Ophthalmic complications represent a severe manifestation of Lemierre syndrome, often reflecting an underlying cerebral vein thrombosis. Visual acuity loss and long-term severe complications are frequent. We call for an interdisciplinary approach to the management of patients with Lemierre syndrome and the routine involvement of ophthalmologists

The Large Magellanic Cloud globular cluster NGC 1866: new data, new models, new analysis

We present a new deep (down to V ~ 24) photometry of a wide region (6'x 6') around the LMC globular cluster NGC1866: our sample is complete, down to 3 mag below the brightest MS star. Detailed comparisons with various theoretical scenarios using models computed with the evolutionary code FRANEC have been done reaching the following conclusions: both standard models (i.e. computed by adopting the Schwarzschild criterion to fix the border of the convective core) and models with an enlarged convective core (overshooting) lead to a fair fit of the MS but are not able to reproduce the luminosity and/or the number of He burning giants. Models including a fraction of 30% of binaries leads to a good fit both to the MS luminosity function and to the He clump, if standards models are considered, for a visual distance modulus (m-M)v = 18.8, age t ~ 100 Myr and mass function slope alpha ~ 2.4, thus largely removing the "classical" discrepancy between observed and predicted number of stars in the He burning clump. The fit obtained with models computed with an enlarged convective core gets worse when a binary component is taken into account, because the presence of binary systems increases the existing discrepancy between the observed and predicted clump luminosity. As a consequence of this analysis, we conclude that the next step towards a proper understanding of NGC 1866, and similar clusters, must include the accurate determination of the frequency of binary systems that will be hopefully performed with the incoming Cycle 8 HST observations of NGC~1866.Comment: AASTEX 5.0, 33 pages, 35 figures. Two tables of photometry and full resolution figures available on request from the first author ([email protected]). Accepted on A

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

Background: MYH9-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. Methods: By 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. Results: Nine 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. Conclusions: CI 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

MYH9 related disease : a novel missense Ala95Asp mutation of the MYH9 gene

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 nephritis leading to end-stage renal failure. In a family with eight individuals suffering from macrothrombocytopenia and hearing impairment we identified a novel c.Ala95Asp mutation. Affecting the motor domain of the protein, the mutation is likely to be associated with a severe phenotype. Therefore, this family should be carefully monitored to follow-up the renal status even though the affected members do not seem to be at risk of early kidney disease.Fil: de Rocco, Daniela. Università degli Studi di Trieste; ItaliaFil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Girotto, Giorgia. Università degli Studi di Trieste; ItaliaFil: Pastore, Annalisa. National Institute for Medical Research; Reino UnidoFil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Marta, Rosana Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Bozzi, Valeria. Universita Degli Studi Di Pavia; ItaliaFil: Pecci, Alessandro. Universita Degli Studi Di Pavia; ItaliaFil: Molinas, Felisa Concepción. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Savoia, Anna. Università degli Studi di Trieste; Itali

5′UTR point substitutions and N-terminal truncating mutations of ANKRD26 in acute myeloid leukemia

Thrombocytopenia 2 (THC2) is an inherited disorder caused by monoallelic single nucleotide substitutions in the 5'UTR of the ANKRD26 gene. Patients have thrombocytopenia and increased risk of myeloid malignancies, in particular, acute myeloid leukemia (AML). Given the association of variants in the ANKRD26 5'UTR with myeloid neoplasms, we investigated whether, and to what extent, mutations in this region contribute to apparently sporadic AML. To this end, we studied 250 consecutive, non-familial, adult AML patients and screened the first exon of ANKRD26 including the 5'UTR. We found variants in four patients. One patient had the c.-125T>G substitution in the 5'UTR, while three patients carried two different variants in the 5' end of the ANKRD26 coding region (c.3G>A or c.105C>G). Review of medical history showed that the patient carrying the c.-125T>G was actually affected by typical but unrecognized THC2, highlighting that some apparently sporadic AML cases represent the evolution of a well-characterized familial predisposition disorder. As regards the c.3G>A and the c.105C>G, we found that both variants result in the synthesis of N-terminal truncated ANKRD26 isoforms, which are stable and functional in cells, in particular, have a strong ability to activate the MAPK/ERK signaling pathway. Moreover, investigation of one patient with the c.3G>A showed that mutation was associated with strong ANKRD26 overexpression in vivo, which is the proposed mechanism for predisposition to AML in THC2 patients. These data provide evidence that N-terminal ANKRD26 truncating mutations play a potential pathogenetic role in AML. Recognition of AML patients with germline ANKRD26 pathogenetic variants is mandatory for selection of donors for bone marrow transplantation

Common laboratory tests and their correlation with the clinical presentation and prognosis of lemierre syndrome

INTRODUCTION Lemierre syndrome is a thromboembolic complication following an acute bacterial infection of the head/neck area, often due to anaerobes. Data on the prognostic role of laboratory parameters is lacking. METHODS We analyzed individual-patient level data from a multinational cohort of patients with Lemierre-syndrome. Patients had an infection in the head/neck area, and contiguous vein thrombosis or septic embolism, irrespective of the causal pathogen. We studied the patterns of white blood cell count, platelet count, and C-reactive protein concentration investigating their association with baseline characteristics and in-hospital clinical outcomes (septic embolism, major bleeding, all-cause death). RESULTS A total of 447 (63%) patients had complete data for analysis. White blood cells were elevated across all subgroups (median 17 × 10$^{3}$/μL; Q1-Q3:12-21). Median platelet count was 61 × 10$^{3}$/μL (Q1-Q3:30-108) with decreasing levels with increasing age. Males, patients with renal failure or cardiopulmonary impairment, and those with typical Lemierre syndrome (tonsillitis, septic thromboembolism, positivity for Fusobacterium spp.) had the lowest platelet count. Median C-reactive protein was 122 (Q1-Q3:27-248) mg/L with higher values in patients who also had more severe thrombocytopenia. The overall risk of complications was similar across subgroups of patients stratified according to white blood cell and C-reactive protein levels. Patients in the lowest third of platelet count (<42 × 10$^{3}$/μL) had the highest rate of complications (26%), as opposed to those in the highest third (11%), notably septic embolic events. CONCLUSIONS Common laboratory tests correlate with the clinical presentation of Lemierre syndrome. However, extreme values did not appear to be prognostically relevant for in-hospital complications and potentially able to improve clinical management

Dysregulation of oncogenic factors by GFI1B p32: investigation of a novel GFI1B germline mutation

GFI1B is a transcription factor essential for the regulation of erythropoiesis and megakaryopoiesis, and pathogenic variants have been associated with thrombocytopenia and bleeding. Analysing thrombocytopenic families by whole exome sequencing, we identified a novel GFI1B variant (c.648+5G>A), which causes exon 9 skipping and overexpression of a shorter p32 isoform. We report the clinical data of our patients and critically review the phenotype observed in individuals with different GFI1B variants leading to the same effect on the p32 expression. Since p32 is increased in acute and chronic leukemia cells, we tested the expression level of genes playing a role in various type of cancers, including hematological tumors and found that they are significantly dysregulated, suggesting a potential role for GFI1B in carcinogenesis regulation. Increasing the number of individuals with GFI1B variants will allow us to better characterize this rare disease and determine whether it is associated with an increased risk of developing malignancies

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.Fil: de Rocco, Daniela. Istituto di Ricovero e Cura a Carattere Scientifico "Burlo Garofolo"; ItaliaFil: Zieger, Barbara. University of Freiburg; AlemaniaFil: Platokouki, Helen. “Aghia Sophia” Children; GreciaFil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Medicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Pastore, Annalisa. National Institute for Medical Research; Reino UnidoFil: Bottega, Roberta. Istituto di Ricovero e Cura a Carattere Scientifico "Burlo Garofolo"; ItaliaFil: Noris, Patrizia. Istituto di Ricovero e Cura a Carattere Scientifico "Burlo Garofolo"; Italia. University of Pavia; ItaliaFil: Barozzi, Serena. Istituto di Ricovero e Cura a Carattere Scientifico "Burlo Garofolo"; Italia. University of Pavia; ItaliaFil: Glembotsky, Ana Claudia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Medicas; ArgentinaFil: Pergantou, Helen. “Aghia Sophia” Children; GreciaFil: Balduini, Carlo L.. Istituto di Ricovero e Cura a Carattere Scientifico "Burlo Garofolo"; Italia. University of Pavia; ItaliaFil: Savoia, Anna. Istituto di Ricovero e Cura a Carattere Scientifico "Burlo Garofolo"; Italia. Universita Degli Studi Di Trieste; ItaliaFil: Pecci, Alessandro. Istituto di Ricovero e Cura a Carattere Scientifico "Burlo Garofolo"; Italia. University of Pavia; Itali

International collaboration as a tool for diagnosis of patients with inherited thrombocytopenia in the setting of a developing country

Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resourcelimited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina. Methods: Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin- 9 in granulocytes and platelet thrombospondin-1 and molecular screening of candidate genes. Results: Thirty-one patients from 14 pedigrees were included; their median age was 32 (4?72) years and platelet count 72 (4?147) · 109 L)1. Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9-related disease in four, while classic and monoallelic Bernard?Soulier syndrome, gray platelet syndrome, X-linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each. Conclusions: Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases.Fil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Marta, Rosana Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Pecci, Alessandro. Universita Degli Studi Di Pavia; ItaliaFil: de Rocco, Daniela. Institute for Maternal and Child Health – IRCCS "Burlo Garofolo"; ItaliaFil: Gnan, Chiara. Institute for Maternal and Child Health – IRCCS "Burlo Garofolo"; ItaliaFil: Espasandin, Yesica Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Goette, Nora Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Negro, F.. Instituto Médico Sagrado Corazón; ArgentinaFil: Noris, Patrizia. Universita Degli Studi Di Pavia; ItaliaFil: Savoia, Anna. Institute for Maternal and Child Health – IRCCS "Burlo Garofolo"; Italia. Università degli Studi di Trieste; ItaliaFil: Balduini, C. L.. Universita Degli Studi Di Pavia; ItaliaFil: Molinas, Felisa Concepción. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin