Saverio Scianguetta

Mutations in ANKRD26 are responsible for a frequent form of inherited thrombocytopenia: analysis of 78 patients from 21 families

Until recently, thrombocytopenia 2 (THC2) was considered an exceedingly rare form of autosomal dominant thrombocytopenia and only 2 families were known. However, we recently identified mutations in the 5-untranslated region of the ANKRD26 gene in 9 THC2 families. Here we report on 12 additional pedigrees with ANKRD26 mutations, 6 of which are new. Because THC2 affected 21 of the 210 families in our database, it has to be considered one of the less rare forms of inherited thrombocytopenia. Analysis of all 21 families with ANKRD26 mutations identified to date revealed that thrombocytopenia and bleeding tendency were usually mild. Nearly all patients had no platelet macrocytosis, and this characteristic distinguishes THC2 from most other forms of inherited thrombocytopenia. In the majority of cases, platelets were deficient in glycoprotein Ia and α-granules, whereas in vitro platelet aggregation was normal. Bone marrow examination and serum thrombopoietin levels suggested that thrombocytopenia was derived from dysmegakaryopoiesis. Unexplained high values of hemoglobin and leukocytes were observed in a few cases. An unexpected finding that warrants further investigation was a high incidence of acute leukemia. Given the scarcity of distinctive characteristics, the ANKRD26-related thrombocytopenia has to be taken into consideration in the differential diagnosis of isolated thrombocytopenias.

Mutations in the 5′ UTR of ANKRD26, the Ankirin Repeat Domain 26 Gene, Cause an Autosomal-Dominant Form of Inherited Thrombocytopenia, THC2

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 19xa0bp 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.xa0Watsons 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.

Clinical and laboratory features of 103 patients from 42 Italian families with inherited thrombocytopenia derived from the monoallelic Ala156Val mutation of GPIbα (Bolzano mutation)

Background Bernard-Soulier syndrome is a very rare form of inherited thrombocytopenia that derives from mutations in GPIbα, GPIbβ, or GPIX and is typically inherited as a recessive disease. However, some years ago it was shown that the monoallelic c.515C>T transition in the GPIBA gene (Bolzano mutation) was responsible for macrothrombocytopenia in a few Italian patients. Design and Methods Over the past 10 years, we have searched for the Bolzano mutation in all subjects referred to our institutions because of an autosomal, dominant form of thrombocytopenia of unknown origin. Results We identified 42 new Italian families (103 cases) with a thrombocytopenia induced by monoallelic Bolzano mutation. Analyses of the geographic origin of affected pedigrees and haplotypes indicated that this mutation originated in southern Italy. Although the clinical expression was variable, patients with this mutation typically had a mild form of Bernard-Soulier syndrome with mild thrombocytopenia and bleeding tendency. The most indicative laboratory findings were enlarged platelets and reduced GPIb/IX/V platelet expression; in vitro platelet aggregation was normal in nearly all of the cases. Conclusions Our study indicates that monoallelic Bolzano mutation is the most frequent cause of inherited thrombocytopenia in Italy, affecting 20% of patients recruited at our institutions during the last 10 years. Because many people from southern Italy have emigrated during the last century, this mutation may have spread to other countries.

A mutation in the acyl-coenzyme A binding domain-containing protein 5 gene (ACBD5 ) identified in autosomal dominant thrombocytopenia

*Department of Clinical Genetics, Erasmus Medical Centre, Rotterdam, the Netherlands; Department of Paediatrics, Second University ofNaples, Naples; Ematologia-Oncologia Pediatrica, Ospedale di Nocera Inferiore, Salerno, Italy; and §CBG-Department of Clinical Genetics,Erasmus Medical Centre, Rotterdam, the NetherlandsTo cite this article: Punzo F, Mientjes EJ, Rohe CF, Scianguetta S, Amendola G, Oostra BA, Bertoli-Avella AM, Perrotta S. A mutation in the acyl-coenzyme A binding domain-containing protein 5 gene (ACBD5) identified in autosomal dominant thrombocytopenia. J Thromb Haemost 2010; 8:2085–7.

Congenital Dyserythropoietic Anemia Type II: molecular analysis and expression of the SEC23B Gene

BackgroundCongenital dyserythropoietic anemia type II (CDAII), the most common form of CDA, is an autosomal recessive condition. CDAII diagnosis is based on invasive, expensive, and time consuming tests that are available only in specialized laboratories. The recent identification of SEC23B mutations as the cause of CDAII opens new possibilities for the molecular diagnosis of the disease. The aim of this study was to characterize molecular genomic SEC23B defects in 16 unrelated patients affected by CDAII and correlate the identified genetic alterations with SEC23B transcript and protein levels in erythroid precursors.MethodsSEC23B was sequenced in 16 patients, their relatives and 100 control participants. SEC23B transcript level were studied by quantitative PCR (qPCR) in peripheral erythroid precursors and lymphocytes from the patients and healthy control participants. Sec23B protein content was analyzed by immunoblotting in samples of erythroblast cells from CDAII patients and healthy controls.ResultsAll of the investigated cases carried SEC23B mutations on both alleles, with the exception of two patients in which a single heterozygous mutation was found. We identified 15 different SEC23B mutations, of which four represent novel mutations: p.Gln214Stop, p.Thr485Ala, p.Val637Gly, and p.Ser727Phe. The CDAII patients exhibited a 40-60% decrease of SEC23B mRNA levels in erythroid precursors when compared with the corresponding cell type from healthy participants. The largest decrease was observed in compound heterozygote patients with missense/nonsense mutations. In three patients, Sec23B protein levels were evaluated in erythroid precursors and found to be strictly correlated with the reduction observed at the transcript level. We also demonstrate that Sec23B mRNA expression levels in lymphocytes and erythroblasts are similar.ConclusionsIn this study, we identified four novel SEC23B mutations associated with CDAII disease. We also demonstrate that the genetic alteration results in a significant decrease of SEC23B transcript in erythroid precursors. Similar down-regulation was observed in peripheral lymphocytes, suggesting that the use of these cells might be sufficient in the identification of Sec23B gene alterations. Finally, we demonstrate that decreased Sec23B protein levels in erythroid precursors correlate with down-regulation of the SEC23B mRNA transcript.

LEOPARD syndrome: clinical dilemmas in differential diagnosis of RASopathies

BackgroundDiagnosis within RASopathies still represents a challenge. Nevertheless, many efforts have been made by clinicians to identify specific clinical features which might help in differentiating one disorder from another. Here, we describe a child initially diagnosed with Neurofibromatosis-Noonan syndrome. The follow-up of the proband, the clinical evaluation of his father together with a gene-by-gene testing approach led us to the proper diagnosis.Case presentationWe report a 8-year-old male with multiple café-au-lait macules, several lentigines and dysmorphic features that suggest Noonan syndrome initially diagnosed with Neurofibromatosis-Noonan syndrome. However, after a few years of clinical and ophthalmological follow-up, the absence of typical features of Neurofibromatosis type 1 and the lack of NF1 mutation led us to reconsider the original diagnosis. A new examination of the patient and his similarly affected father, who was initially referred as healthy, led us to suspect LEOPARD syndrome, The diagnosis was then confirmed by the occurrence in both patients of a heterozygous mutation c.1403 Cu2009>u2009T, p.(Thr468Met), of PTPN11. Subsequently, the proband was also found to have type-1 Arnold-Chiari malformation in association with syringomyelia.ConclusionOur experience suggests that differential clinical diagnosis among RASopathies remains ambiguous and raises doubts on the current diagnostic clinical criteria. In some cases, genetic tests represent the only conclusive proof for a correct diagnosis and, consequently, for establishing individual prognosis and providing adequate follow-up. Thus, molecular testing represents an essential tool in differential diagnosis of RASophaties. This view is further strengthened by the increasing accessibility of new sequencing techniques.Finally, to our knowledge, the described case represents the third report of the occurrence of Arnold Chiari malformation and the second description of syringomyelia with LEOPARD syndrome.

Early-onset central diabetes insipidus is associated with de novo arginine vasopressin–neurophysin II or Wolfram syndrome 1 gene mutations

OBJECTIVEnIdiopathic early-onset central diabetes insipidus (CDI) might be due to mutations of arginine vasopressin-neurophysin II (AVP-NPII (AVP)) or wolframin (WFS1) genes.nnnDESIGN AND METHODSnSequencing of AVP and WFS1 genes was performed in nine children with CDI, aged between 9 and 68 months, and negative family history for polyuria and polydipsia.nnnRESULTSnTwo patients carried a mutation in the AVP gene: a heterozygous G-to-T transition at nucleotide position 322 of exon 2 (c.322G>T) resulting in a stop codon at position 108 (p.Glu108X), and a novel deletion from nucleotide 52 to 54 (c.52_54delTCC) producing a deletion of a serine at position 18 (p.Ser18del) of the AVP pre-prohormone signal peptide. A third patient carried two heterozygous mutations in the WFS1 gene localized on different alleles. The first change was A-to-G transition at nucleotide 997 in exon 8 (c.997A>G), resulting in a valine residue at position 333 in place of isoleucine (p.Ile333Val). The second novel mutation was a 3u200abp insertion in exon 8, c.2392_2393insACG causing the addition of an aspartate residue at position 797 and the maintenance of the correct open reading frame (p. Asp797_Val798insAsp). While similar WFS1 protein levels were detected in fibroblasts from healthy subjects and from the patient and his parents, a major sensitivity to staurosporine-induced apoptosis was observed in the patient fibroblasts as well as in patients with Wolfram syndrome.nnnCONCLUSIONSnEarly-onset CDI is associated with de novo mutations of the AVP gene and with hereditary WFS1 gene changes. These findings have valuable implications for management and genetic counseling.

Congenital erythrocytosis associated with gain-of-function HIF2A gene mutations and erythropoietin levels in the normal range

Hypoxia-inducible factor 2α (HIF-2α) plays a pivotal role in the balancing of oxygen requirements throughout the body. The protein is a transcription factor that modulates the expression of a wide array of genes and, in turn, controls several key processes including energy metabolism, erythropoiesis and angiogenesis. We describe here the identification of two cases of familial erythrocytosis associated with heterozygous HIF2A missense mutations, namely Ile533Val and Gly537Arg. Ile533Val is a novel mutation and represents the genetic HIF2A change nearest to Pro-531, the primary hydroxyl acceptor residue, so far identified. The Gly537Arg missense mutation has already been described in familial erythrocytosis. However, our patient is the only described case of a de novo HIF2A mutation associated with the development of congenital polycythemia. Functional in vivo studies, based on exogenous expression of hybrid HIF-2α transcription factors, indicated that these genetic alterations lead to the stabilization of HIF-2α protein. All the identified polycythemic subjects with HIF2A mutations show serum erythropoietin in the normal range, independently of the hematocrit values and phlebotomy frequency. The erythroid precursors obtained from the peripheral blood of patients showed an altered phenotype, including an increased rate of growth and a modified expression of some HIF-2α target genes. These results suggest the novel proposal that polycythemia observed in subjects with HIF2A mutations might also be due to primary changes in hematopoietic cells and not only secondary to increased erythropoietin levels.

β-spectrinBari: a truncated β-chain responsible for dominant hereditary spherocytosis

This report describes a β-spectrin variant, named β-spectrinBari, characterized by a truncated chain and associated with hereditary spherocytosis. We describe a β-spectrin variant, named β-spectrin Bari, characterized by a truncated chain and associated with hereditary spherocytosis. The clinical phenotype consists of a moderately severe hemolytic anemia, splenomegaly, and spherocytes and acanthocytes in the blood smear. The occurrence of the truncated protein, that represents about 8% of the total β-spectrin occurring on the membrane, results in a marked spectrin deficiency. The altered protein is due to a single point mutation at position −2 (A->G) of the acceptor splice site of intron 16 leading to an aberrant β-spectrin message skipping exons 16 and 17 indistinguishable from that reported for β-spectrin Winston-Salem. We provide evidence that the mutated gene is transcribed but its mRNA is less abundant than either its normal counterpart or β-spectrin Winston-Salem mRNA. Our findings are an example of how mutations in different splice sites, although causing the same truncating effect, result in clearly different clinical pictures.

HNF-1β mutation affects PKD2 and SOCS3 expression causing renal cysts and diabetes in MODY5 kindred.

BACKGROUNDnMaturity onset diabetes of young (MODY) type 5 is a form of non-insulin-dependent diabetes mellitus associated with renal cysts. It is an autosomal dominant disorder caused by mutations in the gene encoding hepatocyte nuclear factor-1β (HNF-1β).nnnMETHODSnWe performed molecular screening of HNF-1β in a 13-year-old patient and his affected father, and analyzed polycystic kidney disease 2 (PKD2) gene and suppressor of cytokine signaling 3 (SOCS3) expression in lymphoblastoid cell lines and lymphocytes from both patients.nnnRESULTSnWe found a novel HNF-1β frameshift mutation (c.C1304del) that results in a truncated protein (p.I434IfsX1). The genetic change is localized in the transactivated protein domain.nnnCONCLUSIONSnWe demonstrated that this novel HNF-1β mutation strongly influences the expression of both PKD2, responsible for the formation of the renal cysts, and SOCS3, which is associated with early diabetes onset.