Somatic Mutation In PIG-A Gene Research Articles

Elucidation of Autoinflammatory Mechanism in Pigt-PNH

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder caused by somatic mutations in PIGA gene (PIGA-PNH). PNH red blood cells are susceptible to complement-mediated intravascular hemolysis because of a loss of the glycosylphosphatidyl-inositol (GPI) anchored proteins including complement regulatory proteins. Recently, a PNH patient caused by the PIGT mutations was reported (PIGT-PNH) and had unusual symptoms similar to autoinflammatory syndrome. Whereas PIGA is essential for the first step in the biosynthesis of GPI anchors, PIGT is involved in transfer of GPI to proteins; thus its defect causes accumulation of free GPI, non-protein linked GPI, in affected cells. It is likely that the free GPI accumulated in affected cells together with activated complement contribute to inflammasome activation. Here, we aimed to investigate whether free GPI together with complement activation can induce inflammasome activation in human monocytic cells in PIGT-PNH.We generated THP-1 human monocytic cells in which PIGT or PIGA gene was deleted using the CRISPR/Cas9 system. Each knockout cell was rescued with a corresponding wild-type cDNA. Cells were differentiated into macrophages under RPMI-1640 medium containing PMA (100 ng/ml, 3h) in the presence of 10% fetal bovine serum. For induction of inflammasome activation, both of the priming signal and the stimulating signal are necessary. PMA-differentiated cells were primed for 5hr with Pam3CSK4 (200 ng/ml), which specifically activates the Toll-like receptor 2 (TLR 2), followed by ATP stimulation (5 mM, 1h), or cells were stimulated by acidified serum (21:1 of 0.4M HCl) alone, which activates alternative complement pathway. Heat-inactivated (56°C for 30 min) serum was used as a negative control. ELISA kits were used to measure the interleukin (IL) -1β levels in supernatants according to the manufacturers' instructions. Total RNA was extracted from cultured cells and quantitative Real-Time PCR (qRT-PCR) was performed for mRNA level of IL-1β and NLRP3.Results are expressed as means ± SD of three independent experiments. Priming of PMA-differentiated WT, PIGT deficient (PIGTKO), and PIGA deficient (PIGAKO) THP-1 cells with Pam3CSK4, followed by ATP stimulation induced similar levels of IL-1β secretion (167.9±61.5, 144.3±19.7, 200.3±48.8 pg/1.6x105 cells). In contrast, when cells were stimulated with acidified serum alone, PIGTKO cells significantly enhanced IL-1β secretion compared to WT and PIGAKO cells, in which PIGTKO cells showed about 50- and 2-fold increase, respectively (23.7±2.2, 1221.8±91.6, 568.2±101 pg/1.6x105 cells; P<0.01 PIGTKO compered to PIGAKO). This elevated levels of IL-1β were decreased comparably to WT cells by the treatments with heat-inactivated acidified serum or anti-C5 monoclonal antibody, indicating IL-1β secretion was enhanced due to complement activation in PIGTKO and PIGAKO cells. PIGT and PIGA rescued cells showed similar levels of IL-1β to WT cells. Interestingly, qPCR revealed that PMA treatment alone induced transcription of proIL-1β and NLRP3 and further incubation of PIGTKO cells with acidified serum showed similar elevation in transcription compered to WT and PIGAKO cells.Thus, our data demonstrated that complement activation works as both a priming signal and a stimulating signal for inflammasome activation in PIGAKO and PIGTKO THP-1 cells. The facts that the PIGT-PNH patients, but not PIGA-PNH patients, showed autoinflammatory symptoms and that PIGTKO THP-1 secreted higher level of L-1β than PIGAKO THP-1 cells upon stimulation with acidified serum, indicate that the free GPI accumulated in PIGTKO cells contribute to inflammasome activation together with complement activation. DisclosuresOsato:Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharma GK: Honoraria, Research Funding. Ueda:Alexion Pharma GK: Honoraria, Research Funding. Kinoshita:Alexion Pharmaceuticals: Honoraria. Kanakura:Kyowa Hakko Kirin: Research Funding; Fujimotoseiyaku: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Toyama Chemical: Research Funding; Bristol Myers: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding.

Paroxyzmální noční hemoglobinurie

Paroxysmal nocturnal hemoglobinuria (PNH) results from a deficiency in inhibitors of activated complement. This lack leads to complement mediated intravascular hemolysis, to activation of coagulation system with increased risk of thrombotic complications and to various degree of bone marrow failure. A molecular basis of PNH is a somatic mutation of PIGA gene causing a lack of glycosyl phosphatidyl inositol which binds many important antigens to cell surface membrane including inhibitors of activated complement CD59 and CD55 antigens. Presence of CD59 and CD55 deficient cells in peripheral blood detected by flow cytometry is essential for diagnosis of PNH. Standard treatment of PNH includes transfusions of leukocyte depleted red blood cells, anticoagulation prophylaxis and administration of folinic acid. PNH patients with predominant signs of bone marrow failure and profound cytopenia may by candidates for stem cell transplantation or combination immunosuppression. Patients with severe hemo-lysis and/or thrombotic complications are indicated for administration of eculizumab - a monoclonal antibody against C5 part of complement. Key words: diagnosis - eculizumab - immunosuppression - paroxysmal nocturnal hemoglobinuria - pathogenesis - transplantation - treatment.

Paroxysmal nocturnal hemoglobinuria: When delay in diagnosis and long therapy occurs.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disorder characterized by hemolytic anemia, bone marrow failure and thrombosis, caused by a somatic mutation in PIG-A gene that results in the absence of CD55 and CD59, two important complement regulatory proteins. In this paper, a case of PNH is retrospectively examined looking for clinical and laboratory features, and the entire course of the disease from the onset of the symptoms is described, together with an adequate follow- up over a 7-years treatment period. In this case, the not specificity and the limited clinical relevance of the symptoms led to a delay in diagnosis. After thrombosis, Eculizumab therapy has been shown to be effective, and during seven years of followup no events have occurred that put the patient’s life at risk. A multidisciplinary approach is crucial in cases like this, in order to allow early diagnosis and minimize the risks for the patients.

Extent and Clinical Implications of Subclonal Diversity in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal hemoglobinuria (PNH) has been traditionally considered a monogenic disease due to somatic mutations in PIGA gene. While selective immune pressure was implicated in PNH evolution via expansion of a PIGA mutant clone in a privileged environment, history of aplastic anemia (AA) is not always present in a manifest hemolytic PNH. Similarly, expansion of PNH clone after AA therapy does not seem to correlate with the strength of immunosuppression and the quality of response. Our initial studies (J.Clin.Invest.2014; 124 :4529-4538) indicated that additional mutational events may be present in PNH and act as intrinsic factors modifying clinical features and leading to a differential expansion. We hypothesize that subclonal hits may be present in some patients with PNH to explain their expansion dynamics and clinical features. To that end we have collected 319 patients with bone marrow failure of whom 202 had PNH clone including 70 hemolytic PNH cases. For a subgroup of these patients (n=197), deep multi-targeted NGS has been performed to identify mutations in 78 myeloid genes of which 38 genes were found to be mutated (≥1) in this PNH spectrum disease. 116 serial samples for 33 patients were also sequenced. As a control group, we have included AA stably negative for PNH clone (n=113). For analytic purposes, on clinical grounds we have sub grouped patients with PNH clone into AA/PNH (31%) defined as patients with presence of bone marrow failure but no clinical or laboratory evidence of hemolysis and WBC PNH clone <20%, and PNH group (69%). The latter (69%) consisted of patients with primary hemolytic PNH (pPNH; 60%) or secondary PNH (sPNH;40%). sPNH is defined as a group showing hemolysis with an antecedent history of AA and clone size >20%.Our analysis focused on driver mutations. In cross-sectional analysis, in addition to PIGA we have identified somatic hits in 10/26 (38%) of AA/PNH pts. (On average 2.1 hits/per positive patient) compared to 27/58(47%) of PNH patients (on average 1.22 hits per positive patients. Subdividing PNH patients, 20/35(57%) had a mutation in primary PNH compared to 7/23(30%) in the sPNH group. In contrast, 42/113(37%) patients had a mutation in the control AA group with a (2.1 mutations per positive patient). The mean VAF for AA is 29% compared to 23% in AA/PNH vs. 34% in PNH vs. 31%in sPNH.In all patients with PNH clone, 44% had a mutation vs. 37% in the AA group. AA/PNH subcohort contained hits in cohesins genes (RAD21-SMC3-STAG2 (8 %), ZRSR2 (8%), BCORs (4%) and TET2 (4%). PNH group had mutations in BCOR/BCORL1 (BCORs) (11%), cohesions, ZRSR2 and TET2 (6% each). In sPNH, a similar trend was observed with the most common hits in EZH2-SUZ12 (9%), ZRSR2 (9%), and BCORs, TET2, LUC7L2 (4% each). In contrast, AA without PNH, the 5 most frequently mutated genes were TET2, ASXL1, EZH2-SUZ12 AND BCORs (5% each) and RUNX 1 (4%). BCORs mutations were more frequent in PNH group compared to AA, (11% vs. 5%). In summary, BCOR/L and ZRSR genes showed more mutations in PNH clone cohort while AA cohort did not show any dominant hits.To determine whether somatic hits observed cross-sectionally led to establishment of stable subclones or were merely transient, we performed a longitudinal analysis. In PNH, 3/4 clones were transient which included EZH2, PHF6 and STAG2. In AA/PNH patients, 9/14 mutational hits appeared in the course of their disease (TET2, CEBPA, CUX1, STAG2, BCOR, SMC3) but 10/14 faded away in subsequent course and thus only 4 were stable clones. In AA cohort, 32 additional mutational hits were noted in 77 serial samples and 34 mutations vanished in subsequent course.In sum, our results show that in PNH, similar to AA, additional somatic hits are common and involve some of the commonly mutated myeloid genes seen in MDS. While likely, many of these subclonal events are transient, seemingly lacking the proper ancestral context, some of the hits resulted in significant expansions (sweeping mutations) and thus may modify the clinical course including speed of the PNH clone evolution. DisclosuresNo relevant conflicts of interest to declare.

Paroxysmal Nocturnal Hemoglobinuria Caused By Pigt Mutations; Atypical PNH

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired GPI-anchor deficiency caused by the somatic mutation of PIGA gene in a hematopoietic stem cell (PIGA-PNH). Loss of function of PIGA causes loss of GPI anchored proteins including complement regulatory proteins. Main symptoms are hemolytic anemia and venous thrombosis caused by the uncontrolled complement activation, and bone marrow failure. For X-linked PIGA, one hit of somatic mutation causes GPI-deficiency. All other genes involved in GPI-anchor biosynthetic pathway are autosomal and hits to two alleles are required to cause GPI-deficiency. In 2013, a PNH patient caused by the PIGT mutations was reported. This patient had a splice site mutation in one germ line allele and a somatic 8Mb deletion including entire PIGT. Recently, we found a Japanese patient who has a nonsense mutation in a germ line allele and a somatic 18Mb deletion in chromosome 20q including PIGT. Here we compare two patients and report that these cases of PNH are atypical in two points. One is that PIGT-PNH cases had unusual symptoms similar to auto inflammatory syndrome. Both had long lasting chronic urticaria and joint pain. Additionally, the Japanese case had recurrent aseptic meningitis and the German patient had ulcerative colitis. It should be noted that these symptoms had lasted over ten years before hemolytic attacks occurred and diagnosis as PNH was made. Urticaria, joint pain and recurrent meningitis were controllable by eculizumab, anti-C5 antibody, showing involvement of complement activation. These inflammatory symptoms are specific to PIGT-PNH and are not seen in PIGA-PNH. PIGT is involved in transfer of GPI to proteins and its defect causes accumulation of free GPI, non-protein linked GPI. In fact, FACS analysis using the antibody which recognizes free GPI revealed that GPI negative granulocytes, monocytes, and B cells from the PIGT-PNH patient, but not from PIGA-PNH patients, highly expressed free GPI. We speculate that this free GPI together with complement activation cause inflammasome activation and are establishing assay system. The other atypical nature is a mechanism of clonal expansion. In typical PNH, autoimmunity against hematopoietic stem cells causing bone marrow failure is thought to positively select GPI-deficient cells. In contrast, the somatically deleted 18Mb included a region commonly deleted in some patients with myeloproliferative noeplasms. This region contains several maternally imprinted genes, defective expression of which is likely causally related to clonal expansion in atypical PNH cases. Thus, the symptoms and mechanism of clonal expansion of PIGT-PNH are quite different from those of PIGA-PNH, so that PIGT-PNH should be categorized as an atypical PNH. Screening of PIGT-PNH among the PNH patients by detecting free GPI using FACS analysis should be feasible. DisclosuresUeda:Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharma: Honoraria, Research Funding. Kanakura:Bristol Myers: Research Funding; Toyama Chemical: Research Funding; Kyowa Hakko Kirin: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Fujimotoseiyaku: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Alexionpharma: Research Funding; Astellas: Research Funding. Kinoshita:Alexion Pharma: Honoraria.

Eculizumab treatment modifies the immune profile of PNH patients

Paroxysmal Nocturnal Haemoglobinuria (PNH) is due to pathological expansion of a stem progenitor bearing a somatic mutation of PIG-A gene involved in the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor. Numerous data suggest a role for immune-mediated mechanisms in the selection/expansion of GPI-defective clone. Haemolytic anaemia in PNH is dependent on the effect of complement against GPI-defective red cells. Eculizumab, an anti-C5 monoclonal antibody, is dramatically effective in controlling haemolysis and thrombosis, in reducing fatigue and in improving quality of life of patients. However, this therapy presents new challenges that need to be properly faced.Here, we report the decrease in B, Natural Killer (NK) and regulatory T cells (Treg), an altered cytokine profile of invariant-NKT cells (NKTi) and the increasing of C-X-C chemokine receptor type 4 (CXCR4) receptor in PNH patients before the Eculizumab therapy. Treatment significantly affects some of these alterations: after Eculizumab, the number of B lymphocytes, the cytokine secretion of NKTi and CXCR4 expression on CD8 T cells became similar to healthy donors. No effects were observed on NK and Treg. The amplitude of the GPI-defective compartment remained unchanged.

The Somatic Mutation of PIG-A Gene and the Expressions of EGR-1 and WT1 Genes in Bone Marrow Cells of the Patients with Paroxysmal Nocturnal Hemoglobinuria

Abstract 4379 ObjectiveTo investigate the somatic mutations of PIG-A gene and the expressions of EGR-1 and WT1 genes in the bone marrow cells of the patients with Paroxysmal nocturnal hemoglobinuria, then explore the characteristics of PIG-A gene mutation in Chinese patients with PNH and the possible mechanism of PNH clonal expansion related to EGR-1 and WT1 genes. MethodsThe quantities of CD55− cells and CD59− cells in both peripheral blood and bone marrow were examined with flow cytometric assay(FCM), and then sorting CD59− cells from bone marrow mononuclears (BMMNCs). The mutant segments of PIG-A gene were identified by DNA sequencing; The expressions of EGR-1 and WT1 mRNA in CD59− and CD59+ BMMNC were measured with semiquantitive RT-PCR. ResultsNine in sixteen patients with PNH and one in four patients with AA-PNH were measured for their PIG-A gene mutation. Five single base substitution were found, including A→G, G→T, A→T, T→G and G→C, resulting in one consense and seven missense mutations. Each of 3 PNH patients had more than two point mutations of PIG-A gene. Insert or deletion mutation of PIG-A gene were not found. The relative mRNA expressions of EGR-1and WT1 genes in CD59− BMMNC, CD59+ BMMNC of PNH or AA-PNH patient and BMMNC of normal controls were (1.00±0.11), (0.86±0.14), (0.85±0.06) and (1.06±0.16), (0.90±0.14), (0.88±0.06), the expression in CD59− BMMNC was significantly higher than that in CD59+ BMMNC or normal controls (p<0.05). There was no significant difference between the expression in CD59+ BMMNC and normal controls(p>0.05). The relative mRNA expressions of EGR-1 and WT1 in BMMNC of PNH and AA-PNH cases were positively correlated with the proportion of CD59− cells(coefficient correlation was 0.509 and 0.481 respectively, p<0.05), but not related to the proportion of CD59+ cells. ConclusionsThe PIG-A gene mutation in some Chinese patients mainly manifested as single base substitution, occurred at random sites and without hot spot, the overexpression of EGR-1 and WT1 genes in PNH clone could promote the abnormal clone expansion. Disclosures:No relevant conflicts of interest to declare.

Expression of HMGA2 In Blood Cells From Patients with Paroxysmal Nocturnal Hemoglobinuria

AbstractAbstract 4242Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation of PIG-A gene in one or few hematopoietic stem cells and subsequent clonal expansion of mutant stem cells. It is known that PIG-A mutation is insufficient to account for the clonal expansion required for clinical manifestation of PNH. We proposed a 3-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Based on the close association of PNH with aplastic anemia, it has been suggested that the selection pressure is immune mediated. However, in spite that over 60% of patients with aplastic anemia have subclinical population of GPI-deficient hematopoietic cells at diagnosis, only 10% develop clinical PNH, suggesting that steps-1 and 2 are insufficient to cause PNH. Under immune mediated selection pressure, GPI-deficient cells not only survive, but also must proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate may increase a chance that additional mutations are acquired, in turn leads to step 3. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative phenotype. According to this hypothesis, we searched for the candidate gene for step 3. We reported 2 patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome12, generating the break within the 3′ untranslated region in HMGA2. This gene encodes an architectural transcription factor which is deregulated in many benign mesenchymal tumors (Blood. 2006 vol.108 no.13, p4232). Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation of PIG-A mutant cells. We reported that the expression of HMGA2 in peripheral blood from PNH patients was significantly higher than that from normal volunteers (relative mRNA expression, 4.8±2.4 vs 1.3±0.3, p<0.05) but this was not the case in the bone marrow. We investigated whether over expression of HMGA2 really causes the clonal expansion using the mouse model. We transduced the mouse bone marrow cells with retrovirus vectors, pMYs-HMGA2-IRES-EGFP or pMY-IRES-EGFP as a control, and transplanted them into lethally irradiated mice. The percentage of HMGA2 expressing cells in peripheral blood cells of each lineage from transplanted mice gradually increased during 4-months' observation, suggesting that over expression of HMGA2 caused clonal expansion of multipotent hematopoietic cells. This result is consistent with a recent report on the gene therapy of beta-thalathemia that clonal expansion of rescued hematopoietic cells occurred due to lentivaral insertion into and ectopic expression of HMGA2 (Science. 2009, 326, p1468). Investigation of the somatic mutation which causes upregulation of HMGA2 is being conducted. The dysregulation of Wnt pathway is one of the candidate mechanisms (Blood. 2009, vol.114, no.22, p786). These results are consistent with our 3-step model of PNH pathogenesis, that is, clonal expansion is caused not only by survival advantage from immunological attack but also by benign-tumor-like proliferation. Disclosures:No relevant conflicts of interest to declare.

Wnt Pathway Is Upregulated in Blood Cells From Patients with Paroxysmal Nocturnal Hemoglobinuria.

Abstract 1987Poster Board I-1009Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation of PIG-A gene in one or few hematopoietic stem cells and subsequent clonal expansion of mutant stem cells that leads to development of symptoms. It is known that PIG-A mutation is insufficient to account for the clonal expansion required for clinical manifestation of PNH. We are proposed a 3-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Based on the close association of PNH with aplastic anemia, it has been suggested that the selection pressure is immune mediated. However, in spite that over 60% of patients with aplastic anemia have subclinical population of GPI-deficient hematopoietic cells at diagnosis, only 10% develop clinical PNH, suggesting that step-1 and 2 are insufficient to cause PNH. Under immune mediated selection pressure, GPI-deficient cells not only survive, but also must proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate may increase a chance that additional genetic mutations are acquired, in turn leads to step 3. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative phenotype. According to this hypothesis, we searched for the candidate gene for step 3. We reported 2 patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome12, making the break within the 3' untranslated region in HMGA2. This gene encodes an architectural transcription factor which is deregulated in many benign mesenchymal tumors (Blood. 2006 vol.108 no.13, p4232). Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation of PIG-A mutant cells. We have analyzed the expression of HMGA2 by quantitative RT-PCR in peripheral blood and bone marrow from healthy volunteers and PNH patients. The samples from patients had significantly higher expression of HMGA2 than those from normal volunteers in the peripheral blood (relative mRNA expression, 4.8±2.4 vs 1.3±0.3, p<0.05) but this was not the case in the bone marrow samples. To investigate the upstream and downstream of HMGA2 upregulation, we used oligonucleotide microarrays in analysis of peripheral blood from PNH patients and normal volunteers. Bioinformatic analyses showed Wnt pathway is significantly upregulated in PNH patients. To confirm this result, we analyzed the expression of cyclin D1 and β-catenin by quantitative RT-PCR and Western blotting, respectively. Expression of cyclin D1 and β-catenin in patients' bone marrow were higher than normal volunteers'. It was reported that Wnt signaling is required for normal growth of HSCs but dysregulation of this pathway leads to various cancers such as chronic myelocytic leukemia. It is possible that HMGA2 is upregulated by enhanced expression of c-Myc, which is the main target of Wnt pathway. How Wnt pathway is dysregulated is the big question, which is common to other hematopoietic malignancies. These results are consistent with our 3-step model of PNH pathogenesis, that is, clonal expansion is caused not only by survival advantage from immunological attack but also by tumorigenic proliferation. Disclosures:Shichishima:Alexion Pharmaceuticals: Research Funding. Kinoshita:Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Good Pregnancy Outcome in a Patient Affected by Paroxysmal Nocturnal Hemoglobinuria Treated with Eculizumab.

Abstract 4222Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired disorder, consequence of a clonal expansion of haematopoietic stem cells that have acquired a somatic mutation of PIGA gene and is characterized by intravascular haemolysis, susceptibility to infections and arterial and venous thromobophilia. PNH frequently arises in association with disorders of bone marrow failure, particularly aplastic anaemia. Venous thrombo-embolism (VTE) is a major cause of morbidity and mortality in accounting for about 1/3 of all death. Pathogenesis of VTE is multifactorial but may be related to the ongoing haemolysis and the increased platelets activation by release of ADP and consumption of nitric oxide. Thromboembolic risk is known to be related to the clone size being higher in patients with PNH granulocytes > 50%, and probably to the ongoing haemolysis. PNH often affects the middle aged adults, of reproductive age, but women with PNH have to be generally discouraged from becoming pregnant because of the increased risk of complication both for the mother and the foetus. Twenty-six published clinical reports describe pregnancy outcome in 43 women with PNH: in the majority of cases no prophylactic anticoagulant therapy was administrated. Major maternal complication (mainly thromboembolic events) are reported in 16,3% of cases during pregnancy and in 30.2% of cases during puerperium. Five patients died as a consequence of thromboembolic events (n=3) and infections (n=2). The pregnancy terms with a pre-term delivery in 38% of cases, usually as a consequence of maternal complication: no foetal abnormalities were reported. Anticoagulation with low molecular weight heparin (LMWH) is generally recommended during pregnancy and puerperium, however, it provide only partial protection from thrombotic complications. Eculizumab is a humanized monoclonal antibody directed against the C5 complement protein, with inhibition of complement-mediated cell lysis. This drug is effective in controlling intravascular haemolysis, stabilizes haemoglobin levels, reduces transfusion requirements and thrombotic events and improves quality of life of PNH patients. It is composed by a hybrid IgG2-IgG4 constant fraction portion without antibody effector mechanism, moreover IgG2 isotypes do not cross placenta but few and partial experiences have been reported about its effects during pregnancy. We report the case of a 35-year old woman with a diagnosis of PNH establish in 2003 evolved from aplastic anaemia (1995). She needed about one unit of packed red blood cells monthly. She started eculizumab treatment at standard dosage (900 mg every other week) in April 2005 obtaining a rapid normalization of haemoglobin and LDH levels. She hadn't further haemolytic events and she never presented vascular complications. The patient became pregnant in October 2008: flow-cytometry analysis demonstrated that 35% of neutrophils and monocytes had loss of CD55 and CD59. Although the patient had a relatively small PNH clone and she hadn't previous thrombotic event, we decided to continue eculizumab during pregnancy in order to avoid haemolysis and the consequent VTE risk. Moreover, we started a prophylactic anticoagulant therapy with LMWH and anti-platelet therapy with acetylsalicylic acid at 16 weeks of gestation. During the entire pregnancy the patient never required transfusional support and the minimal haemoglobin dosage was 9 g/dl. Anti-platelet therapy was stopped at 36 weeks of gestation. At 38th weeks of gestation the patient delivered vaginally a child male, 3430 g, with Apgar score of 9 and 10. Both the mother and the newborn didn't experience any complication in particular the mother didn't need transfusional support and haemoglobin level maintained stable in spite of a mild LDH increase. Eculizumab treatment was continued also in the puerperium. In absence of data about the possible presence of eculizumab in breast milk and its possible effect on the newborn, we decided to avoid breast-feeding. At this time, after 2 months from the childbirth, both the mother and the newborn haven't medical complication. This is the first report of a pregnancy carried out during eculizumab treatment without significant toxicity for both the mother and the baby. The role of eculizumab in pregnancy and puerperium has to be further investigate in order to reduce the disease related maternal and foetal risk. Disclosures:No relevant conflicts of interest to declare.

Expression of HMGA2 in Blood Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation of PIG-A gene in one or few hematopoietic stem cells and subsequent clonal expansion of mutant stem cells that leads to development of symptoms. It is known that PIG-A mutation is insufficient to account for the clonal expansion required for clinical manifestation of PNH. We are proposing a 3-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Based on the close association of PNH with aplastic anemia, it has been suggested that the selection pressure is immune mediated. However, in spite that over 60% of patients with aplastic anemia have subclinical population of GPI-deficient hematopoietic cells at diagnosis, only 10% develop clinical PNH, suggesting that step-1 and 2 are insufficient to cause PNH. Under immune mediated selection pressure, GPI-deficient cells not only survive, but also proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate increases the chance that additional genetic mutations are acquired, in turn leads to Step 3. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative phenotype. According to this hypothesis, we searched for the candidate gene for Step 3. We reported 2 patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome12, making the break within the 3' untranslated region in HMGA2. This gene encodes an architectural transcription factor which is deregulated in many benign mesenchymal tumors (Blood. 2006 vol.108 no.13, p4232). Recently, many reports show that truncation of 3' untranslated region of HMGA2 disrupts binding of miRNA, let-7, which regulates both transcription and translation of HMGA2. In fact, these two PNH patients with chromosomal abnormalities had ectopic expression of HMGA2 in the bone marrow. Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation. We have established the method for stable isolation of mRNA and miRNA from blood and bone marrow cells from PNH patients and analyzed the expression of HMGA2 and let-7 by quantitative RT-PCR. We have analyzed the peripheral blood from 8 healthy volunteers and 12 PNH patients. The samples from patients had significantly higher expression of HMGA2 than those from normal volunteers (relative mRNA expression, 4.8±2.4 vs 1.3±0.3, p<0.05). We analyzed the genomic sequence of three patients including one who has highest HMGA2 expression and found no mutation in 3' untranslated region. We also analyzed the expression of miRNA and found significantly lower expression of let7b and c in patients. Surprisingly, truncated form without 3' untranslated region is predominantly expressed in patients. There maybe deregulation of alternative splicing of HMGA2 gene in patients, which needs further investigations. We are now analyzing more PNH samples including bone marrow, where proliferation of stem cells takes place, to investigate whether high expression of HMGA2 contributes to the pathogenesis of PNH. In addition we are going to analyze whether high expression of HMGA2 causes the clonal expansion of PNH cells using PNH mouse model.

Isolated Granulocytopenia in Patients with Unclassifiable MDS May Be Related to a Mutation of PIG-A Gene Leading to GPI-APs Deficiency Expressed Predominantly on Granulocytes.

An acquired somatic mutation of PIG-A gene leads to a deficiency in glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) in affected cells. A typical clinical manifestation of the GPI-APs deficiency is paroxysmal nocturnal hemoglobinuria (PNH) with intravascular hemolysis, various degree of cytopenia reflecting marrow failure and trombophilia. Besides so-called „classic“ PNH, clinical and laboratory features of PNH may also be present in some patients with aplastic anemia (AA) or myelodysplasia (MDS). Moreover, a small subset of AA or MDS patients may exhibit in peripheral blood small populations of GPI-APs deficient red blood cells (RBC) and granulocytes, not accompanied by clinical and laboratory signs of hemolysis. This subset of patients with a small PNH clone may respond to immunosuppressive therapy. Herein, we report on three patients who were referred to our hospital with the diagnosis of unclassified MDS (RA unclassifiable according to the WHO classification). In all patients, bone marrow aspiration and biopsy revealed normocellular marrow with only minimal to mild dysplasia with no excess of blasts and no karyotype abnormalities. The main laboratory feature was moderate to severe granulocytopenia (absolute neutrophil count: 0.23x109/l, 1.39x109/l and 0.81x109/l, respectively). All patients had normal Hb level, RBC and platelet counts and none of them exhibited any clinical and laboratory signs of hemolysis (reticulocyte count, serum bilirubin, LDH and haptoglobin were within normal limits). Flow cytometric analysis of peripheral blood revealed a significant number of GPI-AP-deficient granulocytes in all patients, but no CD59 or CD55 deficient RBC except for a small PNH RBC clone in patient Nr.3. In patient Nr.1 (50 years old male) who exhibited 30–35% of GPI-AP-deficient granulocytes, sequencing analysis of PIG-A gene discovered transition C/T in exon 3, causing a missense substitution at codon 248 (P248L). Patient Nr.2 was a 28 years female with 20% of GPI-AP-deficient granulocytes, with sequentially identified transition G/A in exon 6 of PIG-A gene resulting in replacement of glutamic acid by lysine at codon 482 (E482K). In patient Nr.3 (34 years old male), flow cytometric analysis revealed 40–45% of PNH granulocytes and 8–12% of CD59 and CD55 deficient RBC. In this patient, sequencing analysis of PIG-A gene revealed transversion A/T in exon 5, leading to a non-sense mutation at codon 367, forming a preterminal STOP codon. This mutation resulted in the loss of 117 aminoacids from the C-terminus of the protein. Our results suggest that unexplained profound granulocytopenia may be in some patients related to mutation of PIG-A gene leading to GPI-APs deficiency expressed predominantly on granulocytes. MDS patients with isolated granulocytopenia who are mostly diagnosed as unclassifiable RA according to the WHO criteria should be screened by sensitive flow cytometry methods for presence of GPI-APs deficient granulocytes. Evidence of a PNH clone may have a prognostic value and may be helpful for decision of an optimal treatment strategy, including immunosuppression.

CD34+ cells from paroxysmal nocturnal hemoglobinuria (PNH) patients are deficient in surface expression of cellular prion protein (PrPc).

Cellular prion protein (PrP(c)) is a glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP) constitutively expressed by neurons but also in hematopoietic cells. In trasmissible spongiform encephalopathies, the protease-resistant form of prion (PrP (s c)) converts the host PrP(c) into the pathologic form. We have investigated PrP(c) expression in hematopoietic cells from paroxysmal nocturnal hemoglobinuria (PNH). In this disease, due to somatic mutations in PIG-A gene, biosynthesis of the (GPI)-anchor is impaired and affected cells lack membrane expression of all GPI-AP. Normal and PNH hematopoietic progenitors and paired wild-type (WT) and PIG-A mutant cell lines were used for analysis of intracellular and surface PrP(c) expression using flow cytometry and Western blot.By flow cytometry, PrP(c) was constitutively present on normal CD34(+) cells, including more immature CD38(dim) cells, as well as hematopoietic cell lines. Similar results were obtained in purified CD34(+). Phospholipase C treatment confirmed that PrP(c) was expressed on the membrane via the GPI-anchor. In PNH patients, GPI-AP-deficient CD34(+) cells lacked PrP(c) membrane expression. PIG-A-mutated cell lines (Jurkat, K562, C(EBV), A(EBV)), in contrast to their normal counterparts, did not express surface PrP(c). However, we detected intracellular PrP(c) at approximately equivalent levels in both normal and PIG-A-mutated cells using intracellular flow cytometry and Western blotting. Cells and cell lines with PNH phenotype together with their normal counterparts may be a suitable system to explore the function of membrane PrP(c) in the hematopoietic system. Conversely, PrP(c) is a good model to elucidate the fate of GPI-AP in PIG-A-deficient cells.

PIG-A gene mutations in four Taiwanese patients with paroxysmal nocturnal haemoglobinuria following aplastic anaemia.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic disorder caused by deficient biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor in haemopoietic stem cells. PIG-A, an X-linked gene that participates in the first step of GPI-anchor synthesis, is responsible for PNH. Various abnormalities of the PIG-A gene have been demonstrated in all patients with PNH so far examined. In this study we characterized the somatic mutations in PIG-A gene in four Taiwanese patients with PNH. We identified five novel mutations in the PIG-A gene, three single nucleotide substitution mutations (-342, C-->G, codon 335, GGT-->AGT and codon 405, GCT-->GTT) and two frameshift mutations (codon 22, GGA-->G-A and codon 356, TGT-->TGTT) in the PIG-A gene. The -342 mutation was judged to be a polymorphism. Furthermore, three patients had previous clinicopathologic evidence which suggested aplastic anaemia (AA), before the development of PNH. One of these was found to have thrombocytopenia during follow-up. We suggest that the somatic PIG-A gene mutations highlight a subgroup of AA having a pathogenetic link with PNH.