Glycosylphosphatidylinositol Deficiency Research Articles

Release of Alkaline Phosphatase Caused by PIGV Mutations In Patients with Hyperphosphatasia-Mental Retardation Syndrome (HPMR), a Recently Found Second Inherited GPI Anchor Deficiency.

AbstractAbstract 2031Recent whole exome sequencing of three sibs of non-consanguineous parents demonstrated PIGV mutations in Hyperphosphatasia-Mental Retardation syndrome (HPMR), an autosomal recessive disease characterized by mental retardation and elevated serum alkaline phosphatase (ALP) levels (Eur J Med Genet, vol.53, issue2, p85, 2010). PIGV is the second mannosyltransferase essential for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations found in four families caused amino acid substitutions A341E, A341V, Q256K and H385P. We have shown that these mutant PIGV proteins are unstable and the mutant cDNAs restored only subnormal GPI biosynthetic activity after transfection into PIGV deficient CHO cells. The backbone of GPI-anchor is synthesized in the endoplasmic reticulum (ER) and is transferred to the proteins which have GPI attachment signal at the C-terminal. It is known that GPI-anchored proteins are not expressed on the surface of GPI deficient cells due to degradation within the cells or secretion. ALP, a GPI anchored protein, was efficiently secreted into medium from PIGV deficient CHO cells, in which incomplete GPI bearing one mannose was accumulated. In contrast, ALP was degraded in PIGL, DPM2 or PIGX deficient CHO cells, in which GPIs lacking mannose were accumulated. Secretion of ALP required GPI transamidase that cleaves the C-terminal GPI attachment signal peptide and replaces it with GPI. It seems that GPI transamidase is activated by GPI bearing at least one mannose, cleaving the hydrophobic signal peptide and resulting in secretion of soluble ALP. It is well known that hypophosphatasia is caused by a deficiency of liver-, bone-, kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. This inherited disorder is characterized by defective bone mineralization and some patients develop epilepsy caused by the disturbance of pyridoxal-5-phosphate (PLP) dependent metabolism of neurotransmitters due to the defect in ALPL which converts PLP to pyridoxal (PL) thereby facilitating passive uptake of this freely diffusible form at the cell surface. Some of the HPMR patients have seizures and the patients with previously reported inherited GPI deficiency caused by defective PIGM encoding the first mannosyltransferase (Nature Med. 12, p846 2006) also have seizures. This common symptom may be caused by the loss of membrane bound ALPL. On the other hand, patients with HPMR do not have the portal vein thrombosis seen in the patients with PIGM deficiency. The affected cell types and the degree of deficiencies in various GPI-anchored proteins might be the cause of the variability of the clinical features among GPI deficiencies. Disclosures:No relevant conflicts of interest to declare.

Use of Blood Transfusions In Paroxysmal Nocturnal Hemoglobinuria Patients with and without Aplastic Anemia Enrolled In the Global PNH Registry

Abstract 2241 Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal hematopoietic stem cell disease characterized by complement-mediated hemolysis which can lead to life-threatening complications including thrombosis, kidney disease, pulmonary hypertension, pain, anemia, and severe fatigue. PNH clones occur in a number of patients with concomitant aplastic anemia (AA) and other bone marrow disorders (BMD). Some patients with PNH and/or BMD may require red blood cell (RBC) transfusions to treat anemia due to hemolysis or bone marrow hypoplasia. Aims: To characterize the use of RBC transfusions among PNH patients with and without underlying AA. Methods: Enrollment data from 117 clinical sites participating in the observational PNH Registry in 16 countries on 5 continents was analyzed. Patients are included in the Registry regardless of the proportion of cells with deficiency of glycosylphosphatidyl-inositol anchored proteins (GPI-AP), bone marrow pathology, symptoms, or treatments. Sites collect clinical history at enrollment and additional data every 6 months. Demographic data, use of transfusions and other treatments, lab results, and symptoms are summarized for PNH patients with and without AA. Results: As of June 30, 2010 there were 655 enrolled patients in the Registry (53% female, median age 43). At enrollment, 28% of patients had history of AA and 45% had no history of BMD. Patients with underlying AA had lower median GPI-AP deficient granulocytes than those without BMD (median = 53% vs. 86%, respectively, p Conclusions: A substantial proportion (56%) of patients with PNH, but not all, required at least one transfusion in the year prior to enrolling in the registry, independent of underlying AA. The proportion of patients who had at least one transfusion increased with clone size. Among patients who had at least one transfusion, there was a trend for patients with underlying AA to receive more RBC units than patients without BMD. PNH patients with AA have fewer GPI-AP deficient granulocytes but demonstrate elevated hemolysis similar to patients without history of BMD when patients with similar clone sizes were compared. This Global PNH Registry, which remains open to accrual (pnhregistry@iconplc.com), should help to redefine prospectively the long-term natural history of PNH, its treatments, and the outcomes of treatment. Disclosures: Schrezenmeier: Alexion Pharmaceuticals: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Brodsky: Alexion Pharmaceuticals, Inc.: Membership on an entity9s Board of Directors or advisory committees. Muus: Celgene: Membership on an entity9s Board of Directors or advisory committees; Alexion: Membership on an entity9s Board of Directors or advisory committees; Amgen: Membership on an entity9s Board of Directors or advisory committees; Novartis: Membership on an entity9s Board of Directors or advisory committees. Bessler: Alexion Pharmaceutical Inc: Consultancy; Novartis: Membership on an entity9s Board of Directors or advisory committees; Taligen: Consultancy. Szer: Alexion Pharmaceuticals, Inc.: Membership on an entity9s Board of Directors or advisory committees. Maciejewski: Celgene: Research Funding; Eisai: Research Funding; Alexion: Consultancy. Socie: Alexion: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Urbano-Ispizua: Alexion: Membership on an entity9s Board of Directors or advisory committees. Kanakura: Alexion: Membership on an entity9s Board of Directors or advisory committees. Hoechsmann: Alexion: Speakers Bureau. Rosse: Alexion: Consultancy, Membership on an entity9s Board of Directors or advisory committees. Khursigara: Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Karnell: Alexion Pharma International: Employment, Equity Ownership. Bedrosian: Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Hillmen: Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding.

Association of HLA Haplotypes with Paroxysmal Nocturnal Hemoglobinuria

The pathologies of paroxysmal nocturnal hemoglobinuria (PNH) are primarily caused by somatic mutation in the PIG-A gene in hematopoietic stem cells resulting in glycosyl phosphatidylinositol deficiency and accumulation of phosphatidylinositol (PI) in plasma membranes. The mechanism of pathologic clone domination over normal hematopoietic clones in PNH patients is not yet understood. Forty-four PNH patients, including 9 with aplastic anemia traits (AA/PNH), 31 without full aplasia in bone marrow (de novo PNH, or dn/PNH), and 4 with unclassified PNH, and 200 ethnically matched controls were tested for the HLA A, B, C, DRB1, and DQB1 alleles and haplotype associations. The top block association analysis showed the primary association of PNH with 3 haplotype fragments: the class I fragment A*2501-Cw*1203-B*1801 (risk ratio [RR], 6.64; P = .00012), and 2 class II fragments: DRB1*1501-DQB1*0602 (RR, 7.09; P = .0000015) and DRB1*0401-DQB1*0301 (RR, 6.76, P = .0093). The stratified analysis revealed that the A*2501-Cw*1203-B*1801 haplotype associated preferentially with AA/PNH, and its component HLA molecule showed immunodominant antiapoptotic peptides derived from PI-activated phospholipase D; whereas the DRB1*1501-DQB1*0602 haplotype was associated strongly with dn/PNH and presented immunodominant class II–derived autopeptides. We concluded that certain HLA haplotypes were associated with PNH much more strongly than their allelic components. At least 3 HLA haplotype blocks ( A*2501-Cw*1203-B*1801, DRB1*1501-DQB1*0602, and DRB1*0401-DQB1*0301) were primarily associated with PNH. Our results supported the hypothesis of the roles in AA/PNH of antiapoptotic and in dn/PNH of autoimmune mechanisms.

Inherited GPI deficiency: A disorder of histone hypoacetylation

Co-operative interaction of transcription factors (TF) with epigenetic processes, such as chromatin remodeling and modification (acetylation or methylation), as well as DNA methylation, determine transcriptional activity, activation or repression of a given gene. Mutations disrupting binding of TF to their cognate DNA motifs would be expected to alter the epigenetic landscape of the promoter and selectively affect transcription of the given gene. We review here the transcriptional, epigenetic, biochemical, and clinical consequences of a constitutional mutation in the promoter of PIGM, a housekeeping gene that disrupts binding of the general TF, SP1, thus causing the autosomal recessive disease, inherited glycosylphosphatidylinositol (GPI) deficiency. We suggest that detailed dissection of the function of the mutated PIGM promoter provides important lessons pertinent to the transcriptional and epigenetic control of housekeeping genes as a whole and might have wider therapeutic implications.

Global Differences in RBC Membrane Protein Expression Between Normal and PNH Individuals.

Abstract 1986Poster Board I-1008Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia that results from the deficiency of glycosyl phosphatidylinositol (GPI)-linked proteins on the surface of blood cells carrying a PIGA gene mutation. The absence of 2 GPI-linked complement regulatory proteins, CD59 and CD55, on PNH red blood cells (RBCs) accounts for their increased susceptibility to complement-mediated lysis. The pathophysiology of other PNH complications, including thrombophilia and the association with aplastic anemia, is less well understood. We hypothesized that the differences between PNH and normal RBCs are not restricted to the lack of GPI-linked proteins. To test this hypothesis we have developed a label-free proteomics platform to identify global differences in RBC membrane protein expression between normal and PNH individuals. Six control samples and 17 PNH samples from 13 different patients were analyzed. PNH patients were categorized as high (>80%), medium (between 20% and 80%), and low (<20%), based on their combined percentage of type II and type III PNH RBCs. RBC membranes were prepared, and proteins were extracted and subjected to trypsin digest. Peptides were then analysed by nano-liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). All 23 nanoLC-MS/MS runs (6 control and 17 PNH) were aligned, and peptide ion currents were extracted and normalized for relative quantitation of peptide abundances and inference of relative protein abundance. ANOVA analyses of the different sample classes were performed in order to distinguish differences in the relative abundances at the peptide level (from a dataset containing ∼30,000 peptides). Comparison of control vs. high PNH samples yielded a set of 142 peptides showing patterns of expression that clearly separated the two groups. Targeted nanoLC-MS/MS identified the differential expression of peptides from 5 of the 8 known GPI-linked proteins expressed on human RBCs. As expected, the relative abundances of these peptides were inversely related to the percentage of PNH RBCs in the samples. In addition, we found peptides from complement protein C3 increased on PNH RBCs from patients receiving the complement C5 inhibitor eculizumab. We have developed a statistical model that uses peptide ion intensities to infer relative abundance at the protein level, and allows for significance testing between healthy and disease groups. RBC membranes from the 6 normal control and 7 high PNH samples were processed using MS with ‘data-dependant acquisition', which is biased towards annotation of the more abundant peptides. 1676 peptides from 119 proteins were annotated with high confidence. Nine of these showed a significant difference between control and PNH, including CD59 and semaphorin-7A, the 2 known GPI-linked proteins annotated in this experiment. The remaining 7 non-GPI-linked proteins were peroxiredoxin-2 (PXDN2), valosin-containing protein, gamma-actin, catalase (CATA), heat shock protein 90A, flotilin-1, and RAP1A. Several of these non-GPI-linked proteins are of potential interest to the PNH phenotype or PNH pathophysiology. The anti-oxidative enzymes CATA and PXDN2 both showed significantly lower levels associated with PNH RBC membranes compared to control, and may contribute to an increased susceptibility of the PNH RBC plasma membrane to oxidative damage. The small G-protein RAP1A showed a significantly elevated expression in PNH RBC membrane preparations. RAP1A activation mediates increased sickle cell RBC adhesion to laminin (Blood, 105: 3322), suggesting that elevated RAP1A levels may also contribute to the increased propensity of thrombosis in PNH patients. In summary, differential protein expression analysis indicates that PNH and normal RBCs differ by more than only the lack of GPI-linked proteins. Differences in protein expression are likely to contribute to disease manifestations and are likely to provide insights into additional unexpected underlying disease processes. Future studies will investigate longitudinal changes in the RBC proteome during the course of the disease and in response to treatment, potentially providing new biomarkers that help to distinguish patients at risk for complications and patients likely to benefit from specific forms of treatment. Our comparative label-free proteomics platform should be broadly applicable to the analysis of other RBC disorders. Disclosures:Bessler:Alexion Pharaceuticals Inc: Membership on an entity's Board of Directors or advisory committees.

GPIHBP1 VARIANTS CONTRIBUTE TO LIPID LEVELS IN FAMILIAL COMBINED HYPERLIPIDEMIA

Familial combined hyperlipidemia (FCHL) is a complex genetic disorder, which predisposes to premature atherosclerosis. In mice, deficiency of glycosylphosphatidylinositol (GPI)-anchored high-density lipoprotein-binding protein (GPIHBP1) causes severe chylomicronemia. GPIHBP1 binds lipoprotein lipase and triglyceride-rich lipoproteins on the luminal surface of endothelial cells. Data examining the role of GPIHBP1 variation in human dyslipidemia are sparse. We therefore performed re-sequencing and association analyses to determine the contribution of GPIHBP1 variation to the aetiology of FCHL in white-British families.

PNH revisited: Clinical profile, laboratory diagnosis and follow-up

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by intravascular hemolysis, marrow failure, nocturnal hemoglobinuria and thrombophila. This acquired disease caused by a deficiency of glycosylphosphatidylinositol (GPI) anchored proteins on the hematopoietic cells is uncommon in the Indian population. Data of patients diagnosed with PNH in the past 1 year were collected. Clinical data (age, gender, various presenting symptoms), treatment information and follow-up data were collected from medical records. Results of relevant diagnostic tests were documented i.e., urine analysis, Ham's test, sucrose lysis test and sephacryl gel card test (GCT) for CD55 and CD59. A total of 5 patients were diagnosed with PNH in the past 1 year. Presenting symptoms were hemolytic anemia (n=4) and bone marrow failure (n=1). A GCT detected CD59 deficiency in all erythrocytes in 4 patients and CD55 deficiency in 2 patients. A weak positive PNH test for CD59 was seen in 1 patient and a weak positive PNH test for CD55 was seen in 3 patients. All patients were negative by sucrose lysis test. Ham's test was positive in two cases. Patients were treated with prednisolone and/or androgen and 1 patient with aplastic anemia was also given antithymocyte globulin. A total of 4 patients responded with a partial recovery of hematopoiesis and 1 patient showed no recovery. None of the patients received a bone marrow transplant. The study highlights the diagnostic methods and treatment protocols undertaken to evaluate the PNH clone in a developing country where advanced methods like flowcytometry immunophenotyping (FCMI) and bone marrow transplants are not routinely available.

Neutral evolution in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria is an acquired hematopoietic stem cell (HSC) disorder characterized by the partial or complete deficiency of glycosyl-phosphatidylinositol (GPI)-linked membrane proteins, which leads to intravascular hemolysis. A loss of function mutation in the PIG-A gene, required for GPI biosynthesis, explains how the deficiency of many membrane proteins can result from a single genetic event. However, to date the mechanism of expansion of the GPI(-) clone has not been fully understood. Two hypotheses have been proposed: A selective advantage of GPI(-) cells because of a second mutation or a conditional growth advantage of GPI(-) cells in the presence of an immune attack on normal (GPI(+)) HSCs. Here, we explore a third possibility, whereby the PNH clone does not have a selective advantage. Simulations in a large virtual population accurately reproduce the known incidence of the disease; and the fit is optimized when the number of stem cells is decreased, reflecting a component of bone marrow failure in PNH. The model also accounts for the occurrence of spontaneous cure in PNH, consequent on clonal extinction. Thus, a clonal advantage may not be always necessary to explain clonal expansion in PNH.

GATA-1 Enhances the Transcription of PIGM and Is Responsible for Increased GPI Expression in Erythrocytes in Inherited Glycosylphosphatidylinositol Deficiency: A Model for Tissue-Specific Housekeeping Gene Transcription Regulation

Inherited glycosylphophatidylinositol (GPI) deficiency (IGD), characterized by thrombosis and epilepsy but minimal hemolysis, is caused by a c>g mutation at position −270 that disrupts binding of the transcription factor (TF) Sp1 to the core promoter of the mannosyltransferase-encoding gene PIGM. In IGD, the level of GPI expression varies considerably between hematopoietic cells of different lineages; for example, while in erythrocytes it is near normal, accounting thus for the absence of significant intravascular hemolysis, GPI synthesis in granulocytes is severely deficient suggesting erythroid-specific transcriptional regulation of PIGM. We tested the hypothesis that the erythroid-megakaryocytic-specific TF GATA-1 is critical regulator of PIGM transcription and it can overcome the transcriptional block imposed by the c>g mutation. Consistent with this hypothesis, we found that in luciferase assays a 2kb PIGM promoter construct had at least 3-fold higher activity when transfected in erythroid (K562) rather than myeloid (HL60), B lymphoid or epithelial (HeLA) cell lines. Co-transfection of the 2kb WT or c>g mutated construct with GATA-1 resulted in proportional increase of the transcriptional activity which was completely abolished when shorter, 0.6kb WT or c>g constructs were used, implying the presence of GATA-1 binding elements (BE) between −0.6 and −2kb. The functional significance of 3 such GATA-1 BE initially identified by bioinformatic analysis was investigated next. In vitro binding to each of these BE was shown in electromobility shift assays using appropriate oligos, nuclear extracts from K562 cells and anti-GATA-1 antibody while in vivo binding was demonstrated by chromatin immune-precipitation assays. The relative transcriptional activity of the 3 GATA-1 BE was tested in luciferase assays by co-transfecting into HeLA cells WT or c>g 2 kb constructs with single, double or triple BE disrupted by site-directed mutagenesis. Overall the results suggested that the proximal of the 3 GATA-1 BE is the most active and that the transcription-enhancing effect of GATA-1 is diminished in the presence of the disease causing c>g mutation. Thus, as well as explaining the near normal synthesis of GPI in erythrocytes in IGD, our results also provide an important paradigm of co-operative function of a generic (Sp1) and tissue-specific (GATA-1) TF in the transcriptional regulation of a house-keeping gene such as PIGM.

Development and evaluation of a stabilized whole‐blood preparation as a process control material for screening of paroxysmal nocturnal hemoglobinuria by flow cytometry

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder in which correct diagnosis is essential for effective patient management. Demonstration of deficiency of glycosylphosphatidylinositol (GPI)-linked antigens from red cells and/or granulocytes by flow cytometry represents a highly specific diagnostic test for PNH. Currently, no external quality assessment (EQA) programme or reference material is available for whole-blood PNH testing (red cells and leucocytes) by flow cytometry. In order to address this issue, we report the development of a stabilized whole-blood PNH sample. We present the results of a detailed time course study by flow cytometry that demonstrates the stability of GPI-linked antigen expression on granulocytes and red cells in a stabilized PNH peripheral blood sample, using a previously described method. The PNH cells, as well as the coexisting normal red cell and granulocyte populations, remained stable for up to 120 days, both in terms of immunophenotypic and light scatter characteristics. Subsequent samples were used for a PNH EQA programme and issued to 92 laboratories worldwide. This study has highlighted that PNH testing by flow cytometry has significant problems with regard to false-positive and -negative results. In addition, the variation in GPI-linked antigen detection methods has highlighted the urgent need for standardized protocols.

Glycosylphosphatidylinositol (GPI)-anchored surface antigens in the allogeneic activation of T cells.

GPI-linked surface molecules have recently been described as structures with an activation potential for human T lymphocytes. To study the role of these molecules in T cell activation we analysed GPI-deficient or normal T cells from patients with paroxysmal nocturnal haemoglobinuria (PNH). On activation with allogeneic Epstein-Barr virus (EBV)-transformed B cell lines GPI-deficient freshly separated T cells or continuously growing T cell lines exhibited a significantly lower proliferation or cytokine production compared with their normal counterparts. In contrast, stimulation via the T cell receptor-associated CD3 structure resulted in a comparable response. There was no difference in activation of normal T lymphocytes when GPI-deficient B cells were used as stimulators compared with normal B cells obtained from the same PNH patient. We conclude from these data that GPI deficiency in PNH leads to a functional deficiency of GPI-deficient T cells. In contrast, no difference in activation of T lymphocytes for GPI-deficient cells on the stimulator cell level was observed.

Recent advances in the diagnosis, monitoring, and management of patients with paroxysmal nocturnal hemoglobinuria

Until recently, there has been no specific therapy for PNH with clinical management mainly supportive in terms of cytopenias and control of thrombotic risk. Currently, the only curative procedure for PNH is bone marrow transplantation although for the majority of patients the associated risks are too great to justify transplantation. The pioneering use of the therapeutic monoclonal antibody eculizumab, which binds to and prevents the activation of the complement protein C5, represents a significant advance in treatment for patients with PNH and is set to become the future standard therapy for hemolytic PNH. In both an initial pilot study and two phase III clinical trials, eculizumab has been shown to dramatically reduce intravascular hemolysis, hemoglobinuria, and transfusion requirements thus improving the quality of life in patients with PNH. As a clinical entity, PNH is synonymous with glycosylphosphatidylinositol (GPI) deficiency, and is an acquired clonal disorder associated with somatic mutations of the X-linked PIGA gene in hematopoietic stem cells. A recent study identified a novel autosomal recessively inherited form of GPI-deficiency involving a mutation in a promotor component of the pig-m gene and characterized by a thrombotic tendency and seizures. In both these developments, flow cytometry played a critical role. In the first instance, in monitoring direct response to a new therapeutic agent; second, in demonstrating the phenotypic/genotypic link in a new form of GPI deficiency.

Targeted Therapy for Inherited GPI Deficiency

Disrupted binding of the transcription factor Sp1 to the mutated promoter region of the mannosyl transferase-encoding gene PIGM causes inherited glycosylphosphatidylinositol (GPI) deficiency characterized by splanchnic vein thrombosis and epilepsy. We show that this results in histone hypoacetylation at the promoter of PIGM. The histone deacetylase inhibitor butyrate increases PIGM transcription and surface GPI expression in vitro as well as in vivo through enhanced histone acetylation in an Sp1-dependent manner. More important, the drug caused complete cessation of intractable seizures in a child with inherited GPI deficiency.

Molecular basis of clonal expansion of hematopoiesis in 2 patients with paroxysmal nocturnal hemoglobinuria (PNH)

AbstractSomatic mutation of PIGA in hematopoietic stem cells causes deficiency of glycosyl phosphatidylinositol–anchored proteins in paroxysmal nocturnal hemoglobinuria (PNH) that underlies the intravascular hemolysis but does not account for expansion of the PNH clone. Immune mechanisms may mediate clonal selection but appear insufficient to account for the clonal dominance necessary for PNH to become clinically apparent. Herein, we report 2 patients with PNH whose PIGA-mutant cells had a concurrent, acquired rearrangement of chromosome 12. In both cases, der(12) had a break within the 3′ untranslated region of HMGA2, the architectural transcription factor gene deregulated in many benign mesenchymal tumors, that caused ectopic expression of HMGA2 in the bone marrow. These observations suggest that aberrant HMGA2 expression, in concert with mutant PIGA, accounts for clonal hematopoiesis in these 2 patients and suggest the concept of PNH as a benign tumor of the bone marrow.

Targeted Molecular Therapy for Inherited Glycosylphosphatidylinositol Deficiency.

Inherited glycosylphosphatidylinositol (GPI) deficiency (IGD) is an autosomal recessive disease characterized by splanchnic vein thrombosis and epilepsy. The partial GPI deficiency in the affected children is due to disrupted addition of the first mannose residue onto the GPI intermediate PI-GlcN, a step catalyzed by the α1>4 mannosyltranferase PIGM. IGD is caused by a −270C>G substitution in the core promoter of PIGM which disrupts binding of the transcription factor (TF) Sp1 to its cognate motif resulting in markedly reduced transcription. Sp1 regulates transcription through interactions an with the basal transcriptional complex or with other TF. In some genes, Sp1 is also required for locus histone acetylation. We investigated whether histone acetylation of the PIGM locus was Sp1-dependent and whether histone deacetylase (HDAC) inhibitors such as sodium butyrate (SB) could be used to enhance PIGM transcription even in the presence of the mutated Sp1 binding motif associated with IGD. Using ChIP assays we first showed that histone 4 (H4) at the PIGM locus is constitutively acetylated in GPI+ EBV B cell lines (LBCL), consistent with the housekeeping function of PIGM. By contrast, the same H4 in GPI- LBCL from individuals with IGD was hypoacetylated indicating that efficient PIGM promoter acetylation requires an intact Sp1 core promoter motif. Upon exposure to SB, acetylation was completely restored in the affected cell lines. Consistent with this, in luciferase reporter assays performed in the presence of mithramycin, an agent that specifically inhibits Sp binding, the enhancing effect of SB on transcription driven by both wild type and mutant (−270C>G) promoter constructs was largely (but not exclusively) Sp1-dependent. The increased transcriptional activity of the mutated PIGM promoter in the presence of SB was accompanied by a more than 100-fold increase in the PIGM mRNA and almost complete restoration of surface GPI expression in the affected LBCL.Over a period of 10 years, one of the children with IGD developed progressive, intractable and incapacitating seizures, despite a multitude of different anti-epileptic treatments including sodium valproate. At the age of 14, she was wheelchair-bound with global hypotonia, extreme drowsiness, poorly responsive, unable to feed herself and almost permanently in minor status with approximately 5 tonic-clonic seizures/day. Extensive CNS imaging showed no structural abnormalities or evidence of thrombosis. An EEG was consistent with minor status epilepticus. Guided by the effectiveness of SB in restoring PIGM transcription and GPI expression in vitro, the patient was commenced on sodium phenylbutyrate (SPB) at an oral dose of 20mg/kg tds. SPB treatment induced a progressive increase in the proportions of GPI+ granulocytes and an increase in PIGM mRNA levels in primary mononuclear blood cells from a pre-SPB level of 1.65% to 26%. A marked clinical improvement was noted after only 2 weeks of treatment, in that the patient could walk, interact with others and feed herself and became entirely seizure-free; she remains so 6 months later. No side effects were noted. These data suggest that SPB may be an effective therapeutic option for diseases caused by Sp1-dependent histone hypoacetylation and also for diseases caused by non-inactivating mutations in the coding region of genes where transcription is regulated by Sp1-dependent histone acetylation.

Molecular Complete Remission of Clonal Hematopoiesis in Paroxysmal Nocturnal Hemoglobinuria (PNH) after Syngeneic Transplantation.

PNH is a clonal hematopoietic stem cell disorder and transplantation is potentially curative. However, the pathobiology of PNH is different from malignant clonal myelopathies such as acute leukemia, and the optimal approach to transplant is unresolved. Somatic mutation of PIGA underlies deficiency of glycosyl phosphatidylinositol anchored proteins (GPI-APs) that accounts for the hemolytic anemia of PNH but does not explain the clonal expansion required for clinical manifestation of the disease to become apparent. Recently we identified two patients whose PIGA mutant cells had a concurrent rearrangement of chromosome 12 that resulted in ectopic expression of HMGA2, the architectural transcription factor deregulated in many benign tumors. These observations suggest that aberrant HMGA2 expression, in concert with mutant PIGA, accounted for clonal hematopoiesis in these patients and support the view of PNH as a benign tumor of the bone marrow. One of the patients (a 31 y.o. female) underwent syngeneic SCT because of persistent transfusion-dependent hemolysis and thromboembolic complications that occurred despite prophylactic anticoagulation. At presentation, flow cytometry showed that 95% of PMN were GPI-AP deficient. Marrow analysis revealed erythroid hyperplasia and a karyotype of 46,XX,ins(12)(p13q15q13) in 20/20 (100%) metaphases. The cytogenetic abnormality was shown to be an intrachromosomal insertion that deregulated expression of HMGA2. A 14 bp deletion in exon 2 of PIGA was identified in PMN DNA. Quantitative PCR showed mutant PIGA and der(12) in 91.8% and 93.9%, respectively of bone marrow cells, demonstrating that the two mutations coexisted in the same cells. Neither mutant PIGA nor der(12) was identified in hematopoietic elements of the syngeneic donor, confirming their somatic origin. Donor CD34+ cells were mobilized with filgrastim to yield 5 × 106 cells/kg recipient weight. Because of the complex molecular abnormalities and the anticipated absence of graft vs. tumor effect, a preparative regimen of targeted busulfan (1mg/kg × 16 doses) and cyclophosphamide (50 mg/kg × 4 doses) was given. Engraftment occurred on Day 9 with no subsequent hemolysis. At 6 and 15 months post-transplant, high sensitivity flow cytometry showed no GPI-AP deficient RBCs or PMNs. Moreover, PCR revealed no mutant PIGA or der(12) post-transplant, demonstrating, for the first time, a molecular complete remission of PNH following SCT. These results reveal important concepts in PNH pathobiology and transplant efficacy. 1) As the donor was a syngeneic twin, graft vs. tumor effect was not required for eradication of the PNH clone. 2) Prior to transplant, essentially all hematopoiesis was derived from the double mutant clone, but the marrow ablative regimen completely eradicated the clone without the need for pretransplant cytoreduction. Together, these observations add further support to the concept of PNH as a benign tumor of the marrow and suggest a rational approach to SCT.

A point mutation in an Sp1 binding motif in the promoter of the mannosyltransferase-encoding PIG-M gene causes inherited glycosylphosphatidylinositol deficiency

A point mutation in an Sp1 binding motif in the promoter of the mannosyltransferase-encoding PIG-M gene causes inherited glycosylphosphatidylinositol deficiency

Hypomorphic promoter mutation in PIGM causes inherited glycosylphosphatidylinositol deficiency

Attachment to the plasma membrane by linkage to a glycosylphosphatidylinositol (GPI) anchor is a mode of protein expression highly conserved from protozoa to mammals. As a clinical entity, deficiency of GPI has been recognized as paroxysmal nocturnal hemoglobinuria, an acquired clonal disorder associated with somatic mutations of the X-linked PIGA gene in hematopoietic cells. We have identified a novel disease characterized by a propensity to venous thrombosis and seizures in which deficiency of GPI is inherited in an autosomal recessive manner. In two unrelated kindreds, a point mutation (c --> g) at position -270 from the start codon of PIGM, a mannosyltransferase-encoding gene, disrupts binding of the transcription factor Sp1 to its cognate promoter motif. This mutation substantially reduces transcription of PIGM and blocks mannosylation of GPI, leading to partial but severe deficiency of GPI. These findings indicate that biosynthesis of GPI is essential to maintain homeostasis of blood coagulation and neurological function.

Reticulocyte-Gated Flow Cytometric Analysis of Red Blood Cells in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder characterized by the deficiency of glycosyl phosphatidylinositol (GPI)-anchored proteins in the affected blood cell membranes. Analysis of blood cells by flow cytometry is useful to identify the affected blood cells with PNH-specific phenotypes. Because PNH-affected red blood cells (RBC) have shortened life-spans in the circulation, ratios of PNH-affected populations analyzed by flow cytometry in whole RBC are lower than those in PNH-affected erythropoiesis. We developed a reticulocyte-gated 2-color flow cytometry of RBC and revealed that the percentages of PNH-affected, CD59--populations in reticulocytes were higher than in whole RBC in patients with PNH. A serial analysis of a patient with PNH who underwent peripheral blood stem cell transplantation confirmed the usefulness of this method to evaluate PNH-affected RBC populations with high sensitivity; ie, the presence of CD59- reticulocytes in the circulation could be a sensitive marker for PNH-affected erythropoiesis.

Paroxysmale nächtliche Hämoglobinurie – Folgen eines fehlenden Ankers

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder characterized by the clonal expansion and differentiation of a multi-potent hematopoietic stem cell carrying a somatic mutation in the X-linked PIG-A gene. As a consequence of this mutation, glycosylphosphatidylinositol (GPI)-anchored proteins are lacking on the surface of blood cells derived from the mutated stem cell. This may result clinically in hemolytic anemia and a tendency for venous thrombosis and serious infection. Bone marrow failure is a frequently observed phenomenon associated with PNH. Reliable diagnosis of PNH is currently best achieved by flow cytometric analysis of GPI-anchored proteins on peripheral blood cells. Both the clinically relevant size of the PNH clone and type of GPI deficiency (complete or partial) can be reproducibly determined by this method. Most patients will benefit from supportive measures, albeit that allogeneic hematopoietic stem cell transplantation is currently considered the only potentially curative therapy. The development of a new therapeutic monoclonal antibody that reduces intravascular hemolysis and progress in diagnostic flow cytometry using a new GPI-specific marker may provide further benefit for PNH patients in the future.