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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 detection 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.

Objective We investigated the contribution to sporadic focal epilepsies (FE) of ultrarare variants in genes coding for the components of complexes regulating mechanistic Target Of Rapamycin (mTOR)complex 1 (mTORC1). Methods We collected genetic data of 121 Italian isolated FE cases and 512 controls by Whole Exome Sequencing (WES) and single‐molecule Molecular Inversion Probes (smMIPs) targeting 10 genes of the GATOR1, GATOR2, and TSC complexes. We collapsed “qualifying” variants (ultrarare and predicted to be deleterious or loss of function) across the examined genes and sought to identify their enrichment in cases compared to controls. Results We found eight qualifying variants in cases and nine in controls, demonstrating enrichment in FE patients (P = 0.006; exact unconditional test, one‐tailed). Pathogenic variants were identified in DEPDC5 and TSC2, both major genes for Mendelian FE syndromes. Interpretation Our findings support the contribution of ultrarare variants in genes in the mTOR pathway complexes GATOR and TSC to the risk of sporadic FE and a shared genetic basis between rare and common epilepsies. The identification of a monogenic etiology in isolated cases, most typically encountered in clinical practice, may offer to a broader community of patients the perspective of precision therapies directed by the underlying genetic cause.

Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) is a rare autosomal dominant form of thrombocytopenia associated with an increased risk of myelodysplastic syndrome and acute myeloid leukemia (AML) caused by germline mutations in the hematopoietic transcription factor RUNX1. Molecular testing allowed us to identify mutations in 13 individuals from three families with inherited thrombocytopenia (IT). They had thrombocytopenia with platelet normal volume and variable expressivity of other morphological and functional defects of platelets, such as reduction of alpha-granules and expression of GPIa-IIa, decreased aggregation, increased level of serum thrombopoietin. In this cohort, only three patients developed AML, with an incidence relative lower than that reported in literature. Since this discrepancy could be explained by different criteria of enrolment (RUNX1 is regarded as a candidate gene only when thrombocytopenia is associated with AML), a systematic screening of RUNX1 in IT families would allow us to identify carriers and more precisely determine the leukemic risk. Yet, considering that recognition of families with FPD/AML is of fundamental importance in the choice of donors for hematopoietic stem cell transplantation, current recommendation includes molecular genetic testing of genes (RUNX1 but also ANKRD26, and ETV6) whose mutations not only are responsible for thrombocytopenia with normal platelet volume but also increase the risk of hematological cancers. Fil: De Rocco, Daniela. Institute for Maternal and Child Healt; Italia Fil: Melazzini, Federica. Policlinico San Matteo Foundation; Italia. University of Pavia; Italia Fil: Marconi, Caterina. Universidad de Bologna; Italia Fil: Pecci, Alessandro. Policlinico San Matteo Foundation; Italia. University of Pavia; Italia Fil: Bottega, Roberta. Università degli Studi di Trieste; Italia Fil: Gnan, Chiara. Institute for Maternal and Child Healt; Italia Fil: Palombo, Flavia. Universidad de Bologna; Italia Fil: Giordano, Paola Florencia. Universita Degli Studi Di Bari; Italia Fil: Coccioli, Maria Susanna. Hospital “D. Camberlingo; Italia 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; Argentina Fil: 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; Argentina Fil: Seri, Marco. Universidad de Bologna; Italia Fil: Savoia, Anna. Università degli Studi di Trieste; Italia. Institute for Maternal and Child Healt; Italia Fil: Noris, Patrizia. University of Pavia; Italia

Main clinical and laboratory features of the AML patients with ANKRD26 mutations identified by the present investigation. Table S2. Prediction of translation start codon presence in the ANKRD26 coding sequence and relative protein size. Figure S1. The stability of ANKRD26 mutant proteins is similar to that of the WT counterpart. HeLa cells were transfected by ANKRD26-FLAG WT or mutant constructs and cultured in a 12-well plate. Protein synthesis was blocked 24 h after transfection by addition to the cell culture of cycloheximide 100 mM diluted in DMSO. Control conditions were carried out by adding the same amount of DMSO alone. Cells were then lysed just before the addition of cycloheximide or DMSO (time 0) and 8, 24, and 48 h after the addition of cycloheximide or DMSO and analyzed by immunoblotting with anti-FLAG and anti-tubulin antibodies. The histograms show the amount of proteins expressed as FLAG/tubulin ratio and referred to time 0 of each condition. After the addition of cycloheximide, WT ANKRD26 expression decreased to about 60% at 8 h, to 45% at 24 h, and to 20% at 48 h. The expression was significantly lower after cycloheximide treatment compared with DMSO alone at each time point, indicating that protein synthesis was efficiently blocked by cycloheximide (***P

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.

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients’ megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

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.

Objectives Mutations in ACTA2 have been reported as a cause of familiar thoracic aortic aneurysm (TAA) with associated bicuspid aortic valve (BAV) in some individuals. Our aim is to investigate the role of ACTA2 mutations in BAV associated with TAA in 20 patients. Methods We recruited 20 patients who underwent surgery for BAV and TAA; clinical genetic evaluation and ACTA2 mutation analysis were performed on each patient, along with next-generation sequencing analysis of BAV-related genes. Available first-degree relatives were enrolled and evaluated with echocardiography and clinical genetic examination. Results No mutations were found in ACTA2 or in BAV-related genes in our probands nor any common clinical signs possibly related to their heart disease. One-third of probands did not have any cardiovascular risk factor. Surgery was required at a young age (mean age 47.2 years) and at relatively small ascending aortic diameters (mean size 49.7 mm). In 77 first-degree relatives, 1 new diagnosis of TAA requiring surgery was made and 8 previous BAV/TAA diagnoses (9/77 = 11.7%) were confirmed. The phenotype BAV ± TAA segregated in 25% of our families. Conclusions Although based on a small cohort, our results seemed to justify the conclusion that ACTA2 did not play a significant role in the pathogenesis of BAV aortopathy. The underlying genetic factors of this condition remain elusive and both large association studies and exome or genome sequencing could represent promising tools to unravel its pathogenesis. Aortic resection of TAA at elective surgery in these patients should be recommended as well as echocardiography in their first-degree relatives.

Inherited thrombocytopenias (ITs) are a group of disorders characterized by different degrees of complexity and severity, which, in turn, result from a wide genetic heterogeneity. To date, at least 25 genes responsible for ITs have been identified. Moreover, known genetic defects account for only 50% of patients, indicating that not all the existing forms have yet been recognized. The development of next generation sequencing is favoring the discovery of novel genes causative for IT, and characterization of these “new” forms is important for improving patients’ management as well as for gaining novel information on mechanisms of platelet production. Recently, Fletcher and colleagues identified by exome analysis monoallelic mutations in the SLFN14 gene as a novel cause of IT. SLFN14-related thrombocytopenia (SLFN14-RT) was described in three pedigrees as a non-syndromic thrombocytopenia associated with excessive bleeding phenotype and defective platelet ATP secretion. The reported families carried three different mutations predicted to result in substitutions hitting consecutive residues (Lys218, Lys219, and Val220) within the ATPase-AAA-4 domain of the SLFN14 protein. The function of SLFN family of proteins is poorly known: they have been associated with regulation of cell proliferation and differentiation, and recently SLFN14 has been shown to have endoribonuclease activity in rabbit reticulocytes. However, the role of SLFN14 in platelet biogenesis or survival is completely unknown. Here we report a fourth family with IT caused by a novel SLFN14 mutation.

ETV6-related thrombocytopenia is an autosomal dominant thrombocytopenia that has been recently identified in a few families and has been suspected to predispose to hematologic malignancies. To gain further information on this disorder, we searched for ETV6 mutations in the 130 families with inherited thrombocytopenia of unknown origin from our cohort of 274 consecutive pedigrees with familial thrombocytopenia. We identified 20 patients with ETV6-related thrombocytopenia from seven pedigrees. They have five different ETV6 variants, including three novel mutations affecting the highly conserved E26 transformation-specific domain. The relative frequency of ETV6-related thrombocytopenia was 2.6% in the whole case series and 4.6% among the families with known forms of inherited thrombocytopenia. The degree of thrombocytopenia and bleeding tendency of the patients with ETV6-related thrombocytopenia were mild, but four subjects developed B-cell acute lymphoblastic leukemia during childhood, resulting in a significantly higher incidence of this condition compared to that in the general population. Clinical and laboratory findings did not identify any particular defects that could lead to the suspicion of this disorder from the routine diagnostic workup. However, at variance with most inherited thrombocytopenias, platelets were not enlarged. In vitro studies revealed that the maturation of the patients' megakaryocytes was defective and that the patients have impaired proplatelet formation. Moreover, platelets from patients with ETV6-related thrombocytopenia have reduced ability to spread on fibrinogen. Since the dominant thrombocytopenias due to mutations in RUNX1 and ANKRD26 are also characterized by normal platelet size and predispose to hematologic malignancies, we suggest that screening for ETV6, RUNX1 and ANKRD26 mutations should be performed in all subjects with autosomal dominant thrombocytopenia and normal platelet size.

Inherited thrombocytopenias (ITs) are a heterogeneous group of syndromic and nonsyndromic diseases caused by mutations affecting different genes. Alterations of ACTN1, the gene encoding for α-actinin 1, have recently been identified in a few families as being responsible for a mild form of IT (ACTN1-related thrombocytopenia; ACTN1-RT). To better characterize this disease, we screened ACTN1 in 128 probands and found 10 (8 novel) missense heterozygous variants in 11 families. Combining bioinformatics, segregation, and functional studies, we demonstrated that all but 1 amino acid substitution had deleterious effects. The clinical and laboratory findings of 31 affected individuals confirmed that ACTN1-RT is a mild macrothrombocytopenia with low risk for bleeding. Low reticulated platelet counts and only slightly increased serum thrombopoietin levels indicated that the latest phases of megakaryopoiesis were affected. Given its relatively high frequency in our cohort (4.2%), ACTN1-RT has to be taken into consideration in the differential diagnosis of ITs.

Gnathodiaphyseal dysplasia (GDD) is an autosomal dominant syndrome characterized by frequent bone fractures at a young age, bowing of tubular bones and cemento-osseus lesions of the jawbones. Anoctamin 5 (ANO5) belongs to the anoctamin protein family that includes calcium-activated chloride channels. However, recent data together with our own experiments reported here add weight to the hypothesis that ANO5 may not function as calcium-activated chloride channel. By sequencing the entire ANO5 gene coding region and untranslated regions in a large Italian GDD family, we found a novel missense mutation causing the p.Thr513Ile substitution. The mutation segregates with the disease in the family and has never been described in any database as a polymorphism. To date, only two mutations on the same cysteine residue at position 356 of ANO5 amino-acid sequence have been described in GDD families. As ANO5 has also been found to be mutated in two different forms of muscular dystrophy, the finding of this third mutation in GDD adds clues to the role of ANO5 in these disorders.

THC2, an autosomal-dominant thrombocytopenia described so far in only two families, has been ascribed to mutations in MASTL or ACBD5. Here, we show that ANKRD26, another gene within the THC2 locus, and neither MASTL nor ACBD5, is mutated in eight unrelated families. ANKRD26 was also found to be mutated in the family previously reported to have an ACBD5 mutation. We identified six different ANKRD26 mutations, which were clustered in a highly conserved 19 bp sequence located in the 5' untranslated region. Mutations were not detected in 500 controls and are absent from the 1000 Genomes database. Available data from an animal model and Dr. Watson's genome give evidence against haploinsufficiency as the pathogenetic mechanism for ANKRD26-mediated thrombocytopenia. The luciferase reporter assay suggests that these 5' UTR mutations might enhance ANKRD26 expression. ANKRD26 is the ancestor of a family of primate-specific genes termed POTE, which have been recently identified as a family of proapoptotic proteins. Dysregulation of apoptosis might therefore be the pathogenetic mechanism, as demonstrated for another thrombocytopenia, THC4. Further investigation is needed to provide evidence supporting this hypothesis.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0382-y) contains supplementary material, which is available to authorized users.