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

GWAS meta-analysis of over 29,000 people with epilepsy identifies 26 risk loci and subtype-specific genetic architecture

Epilepsy is a highly heritable disorder affecting over 50 million people worldwide, of which about one-third are resistant to current treatments. Here we report a multi-ancestry genome-wide association study including 29,944 cases, stratified into three broad categories and seven subtypes of epilepsy, and 52,538 controls. We identify 26 genome-wide significant loci, 19 of which are specific to genetic generalized epilepsy (GGE). We implicate 29 likely causal genes underlying these 26 loci. SNP-based heritability analyses show that common variants explain between 39.6% and 90% of genetic risk for GGE and its subtypes. Subtype analysis revealed markedly different genetic architectures between focal and generalized epilepsies. Gene-set analyses of GGE signals implicate synaptic processes in both excitatory and inhibitory neurons in the brain. Prioritized candidate genes overlap with monogenic epilepsy genes and with targets of current antiseizure medications. Finally, we leverage our results to identify alternate drugs with predicted efficacy if repurposed for epilepsy treatment

High throughput sequencing analysis for the molecular diagnosis of Inherited Thrombocytopenias

Inherited thrombocytopenias are a heterogenous group of rare genetic disorders characterized by reduced platelet count sometimes combined with bleeding tendency and/or other clinical defects. The molecular diagnosis of ITs is essential to make clinical decision and infer personalized prognosis and risks. More than 30 genes have been identified that harbor mutations responsible for ITs (Balduini et al., 2017). In addition, ITs often show phenotypic overlaps that hamper the correct diagnosis with the traditional diagnostic algorithm based on step-wise specialized investigations. However, the advent of next generation sequencing has changed the diagnostic approach of diseases characterized by high genetic heterogeneity like ITs. In order to improve the diagnosis of IT, we designed a targeted next generation sequencing panel (IT-NGS) to screen the 28 genes more commonly mutated in ITs. Ninety-seven consecutive probands with a suspicious of ITs had been sequenced. The analysis led us to reach a definite diagnosis for 37 probands. In these probands we identified known or novel likely pathogenic mutations causing specific diseases, including monoallelic Bernard Soulier syndrome (N=14), biallelic Bernard Soulier syndrome (N=4), ACTN1-related thrombocytopenia (N=4), MYH9-related disease (N=7), ANKRD26-related thrombocytopenia (N=4), congenital amegakaryocytic thrombocytopenia (N=1), grey platelet syndrome (N=1), Wiskott-Aldrich syndrome (N=1) and Acute Myelogenous Leukemia (N=1). In another 34 cases we identified variants of uncertain significance (VUS) whose pathogenic role has to be supported by segregation analysis and in-depth functional studies. Since 17 probands had no potential candidate variant impacting IT-NGS genes, they are eligible for whole exome sequencing (WES) to clone novel genes involved in ITs. In conclusion, since some IT forms predispose to additional acquired disease during life, an accurate diagnosis is essential to infer personalized prognosis and define proper treatments and follow-up. Because of clinical and genetic heterogeneity, the molecular diagnosis of ITs represents a lengthy and expensive challenge using conventional technologies. The use of IT-NGS in clinical practice aided by specific investigations clarifying the role of variant of uncertain significance, overcomes these issues facilitating a definite diagnosis in patients with a suspicious of known ITs forms

A new form of inherited thrombocytopenia due to monoallelic loss of function mutation in the thrombopoietin gene

Pubblicazione senza abstrac

A new form of inherited thrombocytopenia due to monoallelic loss of function mutation in the thrombopoietin gene

Thrombopoietin (THPO) is an essential regulator of haemopoiesis that is required for the maintenance of haemopoietic progenitors and their differentiation into megakaryocytes (Mks). Moreover, it modulates the events that drive Mk maturation and allows the release of platelets into bone marrow sinusoids. THPO plays these roles by binding the MPL receptor, which is expressed in bone marrow stem cells, Mks, platelets and many other human cells. Until recently, no inherited THPO defect was known to cause thrombocytopenia or bone marrow aplasia. However, it was recently shown that microdeletions encompassing the THPO gene in chromosome 3 result in a complex clinical picture, including mild congenital thrombocytopenia. Moreover, a Micronesian family carrying the homozygous c.112C>T (p.Arg38Cys or p.Arg17Cys in the mature protein) missense mutation in THPO presents inherited bone marrow aplasia. No THPO mutation associated with isolated thrombocytopenia has been reported to date, but the application of whole exome sequencing (WES) in a cohort of patients with inherited thrombocytopenias (ITs) of unknown origin revealed that monoallelic changes in this gene identify a new form of IT. In fact, WES identified two unrelated individuals carrying the heterozygous variant c.91C>T (p. Arg31*), which is expected to result in mutant protein degradation and THPO haploinsufficiency

MYH9 -Related Thrombocytopenia: Four Novel Variants Affecting the Tail Domain of the Non-Muscle Myosin Heavy Chain IIA Associated with a Mild Clinical Evolution of the Disorder

MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA). Patients present congenital macrothrombocytopenia and inclusions of NMMHC-IIA in leukocytes, and have a variable risk of developing kidney damage, sensorineural deafness, presenile cataracts and/or liver enzymes abnormalities. The spectrum of mutations found in MYH9-RD patients is limited and the incidence and severity of the non-congenital features are predicted by the causative MYH9 variant. In particular, different alterations of the C-terminal tail domain of NMMHC-IIA associate with remarkably different disease evolution. We report four novel MYH9 mutations affecting the tail domain of NMMHC-IIA and responsible for MYH9-RD in four families. Two variants cause amino acid substitutions in the coiled-coil region of NMMHC-IIA, while the other two are a splicing variant and a single nucleotide deletion both resulting in frameshift alterations of the short non-helical tailpiece. Characterization of phenotypes of affected individuals shows that all of these novel variants are associated with a mild clinical evolution of the disease

MYH9 -Related Thrombocytopenia: Four Novel Variants Affecting the Tail Domain of the Non-Muscle Myosin Heavy Chain IIA Associated with a Mild Clinical Evolution of the Disorder

MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA). Patients present congenital macrothrombocytopenia and inclusions of NMMHC-IIA in leukocytes, and have a variable risk of developing kidney damage, sensorineural deafness, presenile cataracts and/or liver enzymes abnormalities. The spectrum of mutations found in MYH9-RD patients is limited and the incidence and severity of the non-congenital features are predicted by the causative MYH9 variant. In particular, different alterations of the C-terminal tail domain of NMMHC-IIA associate with remarkably different disease evolution. We report four novel MYH9 mutations affecting the tail domain of NMMHC-IIA and responsible for MYH9-RD in four families. Two variants cause amino acid substitutions in the coiled-coil region of NMMHC-IIA, while the other two are a splicing variant and a single nucleotide deletion both resulting in frameshift alterations of the short non-helical tailpiece. Characterization of phenotypes of affected individuals shows that all of these novel variants are associated with a mild clinical evolution of the disease

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

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

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

Thrombopoietin mutation in congenital amegakaryocytic thrombocytopenia treatable with romiplostim

Congenital amegakaryocytic thrombocytopenia (CAMT) is an inherited disorder characterized at birth by thrombocytopenia with reduced megakaryocytes, which evolves into generalized bone marrow aplasia during childhood. Although CAMT is genetically heterogeneous, mutations of MPL, the gene encoding for the receptor of thrombopoietin (THPO), are the only known disease-causing alterations. We identified a family with three children affected with CAMT caused by a homozygous mutation (p.R119C) of the THPO gene. Functional studies showed that p.R119C affects not only ability of the cytokine to stimulate MPL but also its release, which is consistent with the relatively low serum THPO levels measured in patients. In all the three affected children, treatment with the THPO-mimetic romiplostim induced trilineage hematological responses, remission of bleeding and infections, and transfusion independence, which were maintained after up to 6.5 years of observation. Recognizing patients with THPO mutations among those with juvenile bone marrow failure is essential to provide them with appropriate substitutive therapy and prevent the use of invasive and unnecessary treatments, such as hematopoietic stem cell transplantation or immunosuppression