Phosphatidylinositol Glycan Class A Research Articles

266 POSTER Preclinical evaluation of IL-21 combination therapy with sorafenib and sunitinib in renal cell carcinoma

Pluripotent human embryonic stem (hES) cells can differentiate into various cell types derived from the three embryonic germ layers and extraembryonic tissues such as trophoblasts. The mechanisms governing lineage choices of hES cells are largely unknown. Here, we report that we established two independent hES cell clones lacking a group of cell surface molecules, glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs). The GPI-AP deficiency in these two hES clones is due to the deficiency in the gene expression of PIG-A (phosphatidyl-inositol-glycan class A), which is required for the first step of GPI synthesis. GPI-AP-deficient hES cells were capable of forming embryoid bodies and initiating cell differentiation into the three embryonic germ layers. However, GPI-AP-deficient hES cells failed to form trophoblasts after differentiation induction by embryoid body formation or by adding exogenous BMP4. The defect in trophoblast formation was due to the lack of GPI-anchored BMP coreceptors, resulting in the impairment of full BMP4 signaling activation in the GPI-AP-deficient hES cells. These data reveal that GPI-AP-enhanced full activation of BMP signaling is required for human trophoblast formation.

Paroxysmal Nocturnal Hemoglobinemia: A Molecular Definition of the Clinical Biology of the Disorder

Paroxysmal nocturnal hemoglobinemia (PNH), a hematopoietic stem cell disorder, arises from a somatic mutation of the phosphatidylinositol glycan-class A (PIG-A) gene. The gene product is required in the biosynthesis of a glycosylphosphatidylinositol (GPI) structure and serves as an anchor for a group of membrane proteins. The PNH cells are characterized by a total or partial lack of the GPI-anchored membrane proteins. Without this structure, intravascular hemolysis occurs due to the inability to regulate the lytic and cellstimulatory activities of complement on the membrane surface of hematopoietic cells. Two proteins, CD55-decay accelerating factor and CD59-membrane inhibitor of reactive lysis, are known to be tethered to the cell membrane by the GPI-anchor. Additionally, platelets lacking CD59 may cause venous thrombosis with possible Budd-Chiari syndrome. The development of PNH requires a hypoplastic bone marrow, somatic mutation restricted to the PIG-A gene in the stem cell, and clonal expansion of the hematopoietic stem cell pool. Received 11.9.05 | Revisions Received 12.7.05 | Accepted 12.7.05

A cohort study of the nature of paroxysmal nocturnal hemoglobinuria clones and PIG‐A mutations in patients with aplastic anemia

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the clonal expansion of blood cells, which are deficient in glycosylphosphatidylinositol anchored proteins (GPI-APs). As PNH frequently occurs during the clinical course of acquired aplastic anemia (AA), it is likely that a process inducing bone marrow failure in AA is responsible for the selection of GPI-AP deficient blood cells or PNH clone. To explore the nature and mutation of a PNH clone in AA. We performed regular repeated flow cytometric analyses of CD59 expression on peripheral blood cells from a cohort of 32 patients with AA. Mutation of phosphatidylinositol glycan class A (PIG-A) was also studied. Fifty-one episodes of occurrences of CD59 negative granulocytes out of a total cohort 167 flow cytometric analyses (31%) were observed in 22 patients (69%). CD59 negative erythrocytes were less apparent than the granulocytes. Repeated occurrences of PNH clones were observed in 16 patients. Most of the emerging PNH clones were transient in nature. They were more frequently detected during episodes of lower white blood cell and platelet counts. Persistence and expansion of the GPI-AP deficient blood cell populations to the level of clinical PNH were seen in only four patients (12.5%). Analysis of PIG-A gene demonstrated eight mutations among the four patients, with two and four independent mutations in two patients. Our study indicates that PIG-A mutations of hematopoietic stem cells with resultant PNH clones, are relatively common among AA patients. It also supports the hypothesis of selection of the PNH clone by a process or condition associated with or responsible for bone marrow failure in AA. However, there must be an additional factor favoring expansion or growth of the clone to the level of clinical or florid PNH.

Glycosylphosphatidylinositol (GPI) Protein-Negative Population of Leukemic Cells in Patients with De Novo Acute Leukemia.

Paroxysmal nocturnal hemoglobinuria (PNH) is considered to be an acquired stem cell disorder affecting all hematopoietic lineages, which lack GPI-anchored membrane proteins, such as CD59, because of abnormalities in the phosphatidylinositol glycan-class A (PIG-A) gene. Also, PNH is one disorder of the bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome, which are considered as pre-leukemic states. In this study, to know some condition of pre-leukemic states in patients with de novo acute leukemia, we investigated the expression of CD59 in leukemic cells from 25 patients (female: male=8: 17; mean age, 57.8 ± 19.5 years) with de novo acute leukemia by single-color flow cytometric analysis. In addition, the PIG-A gene from CD59− leukemic cells, sorted by FACS Vantage, in 10 patients with acute leukemia was examined by sequence analysis. All the patients had no past history of PNH. Based on the French-American-British criteria, the diagnosis and subtypes of acute leukemia were determined. The number of patients with subtypes M1, M2, M3, M4, M5, and M7 was 1, 14, 2, 4, 2, and 2, respectively. Two of the patients were classified into acute myeloid leukemia with trilineage myelodysplasia from morphological findings in bone marrow. Chromosomal analyses presented abnormal karyotypes in 14 of 25 patients. Flow cytometric analyses showed that leukemic cells from 16 of 25 patients (64%) had negative populations of CD59 expression and the mean proportion of the populations was 63.3 ± 25.7%, suggesting the possibility that CD59− leukemic cells from patients with de novo acute leukemia might be derived from PNH clones. In fact, the PIG-A gene analyses showed that single (n=4) or multiple (n=6) PIG-A mutations in coding region were found in leukemic cells from 10 patients with CD59− leukemic cells and all of the clones with the PIG-A mutations were statistically minor. Then, various clinacal parameters, including peripheral blood, bone marrow blood, and laboratory findings and the results of chromosomal analyses were statistically compared between 2 groups of patients with (n=16) and without CD59− leukemic cells (n=9). The reticulocyte counts (mean ± standard deviation; 10.5 ± 13.0 x 104/μl) and proportions of bone marrow erythroblast (17.5 ± 13.9%) in patients with only CD59+ leukemic cells were significantly higher than those in patients with CD59− leukemic cells (2.5 ± 1.7 x 10 4/μl; p<0.05 and 5.6 ± 6.2%; p<0.01, respectively). The proportions of bone marrow blasts (69.3 ± 21.1%) in patients with CD59− leukemic cells were significantly higher than that those in patients with only CD59+ leukemic cells (45.5 ± 19.3%; p<0.02). In conclusion, our findings indicate that leukemic cells derived from PNH clones may be fairly common in de novo acute leukemia patients, suggesting that bone marrow failure as pre-leukemic states may have already occurred in localized bone marrow even in de novo acute leukemia.

DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms' tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of >22,000 probe sets (corresponding to >14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P <0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.

PIG-A mutations in normal hematopoiesis

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is caused by phosphatidylinositol glycan–class A (PIG-A) mutations in hematopoietic stem cells (HSCs). PIG-A mutations have been found in granulocytes from most healthy individuals, suggesting that these spontaneous PIG-A mutations are important in the pathogenesis of PNH. It remains unclear if these PIG-A mutations have relevance to those found in PNH. We isolated CD34+ progenitors from 4 patients with PNH and 27 controls. The frequency of PIG-A mutant progenitors was determined by assaying for colony-forming cells (CFCs) in methylcellulose containing toxic doses of aerolysin (1 × 10-9 M). Glycosylphosphatidylinositol (GPI)–anchored proteins serve as receptors for aerolysin; thus, PNH cells are resistant to aerolysin. The frequency of aerolysin resistant CFC was 14.7 ± 4.0 × 10-6 in the bone marrow of healthy donors and was 57.0 ± 6.7 × 10-6 from mobilized peripheral blood. DNA was extracted from individual day-14 aerolysin-resistant CFCs and the PIG-A gene was sequenced to determine clonality. Aerolysin-resistant CFCs from patients with PNH exhibited clonal PIG-A mutations. In contrast, PIG-A mutations in the CFCs from controls were polyclonal, and did not involve T cells. Our data confirm the finding that PIG-A mutations are relatively common in normal hematopoiesis; however, the finding suggests that these mutations occur in differentiated progenitors rather than HSCs.

Characteristics of De Novo Acute Leukemia Patients with Glycosylphosphatidylinositol (GPI) Protein-Negative Population of Leukemic Cells.

Paroxysmal nocturnal hemoglobinuria (PNH) is considered to be an acquired stem cell disorder affecting all hematopoietic lineages, which lack GPI-anchored membrane proteins, such as CD59, because of abnormalities in the phosphatidylinositol glycan-class A (PIG-A) gene. Also, PNH is one disorder of bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome, which are considered as pre-leukemic states. In this study, to know some characteristics of patients with de novo acute leukemia, we investigated expression of CD59 in leukemic cells from 25 patients (female: male=8: 17; mean age ± standard deviation, 57.8 ± 19.5 years) with de novo acute leukemia by single-color flow cytometric analysis. In addition, the PIG-A gene from CD59− leukemic cells sorted by FACS Vantage in 3 patients with acute leukemia was examined by sequence analysis. All the patients had no past history of PNH. Based on the French-American-British criteria, the diagnosis and subtypes of acute leukemia were determined. The number of patients with subtypes M1, M2, M3, M4, M5, and M7 was 1, 14, 2, 4, 2, and 2, respectively. Two of the patients were classified into acute myeloid leukemia with trilineage myelodysplasia from morphological findings in bone marrow. Chromosomal analyses presented abnormal karyotypes in 14 of 25 patients. Flow cytometric analyses showed that leukemic cells from 16 of 25 patients (64%) had negative populations of CD59 expression and the proportion of the populations was 63.3 ± 25.7%, suggesting the possibility that CD59− leukemic cells from patients with de novo acute leukemia might be derived from PNH clones. In fact, the PIG-A gene analyses showed that monoclonal or oligoclonal PIG-A mutations in coding region were found in leukemic cells from 3 patients with CD59− leukemic cells and all of the clones with the PIG-A mutations were minor. Then, various clinical parameters, including rate of complete remission for remission-induction chemotherapy, peripheral blood, bone marrow blood, and laboratory findings, and results of chromosomal analyses were statistically compared between 2 groups of patients with (n=16) and without (n=9) CD59− leukemic cells. The reticulocyte counts (10.5 ± 13.0 x 104/μl) and proportions of bone marrow erythroblasts (17.5 ± 13.9%) in patients with only CD59+ leukemic cells were significantly higher than those (2.5 ± 1.7 x 104/μl, p<0.05; and 5.6 ± 6.2%, p<0.01, respectively) in patients with CD59− leukemic cells. The proportions of bone marrow blasts (69.3 ± 21.1%) in patients with CD59− leukemic cells were significantly higher than those (45.5 ± 19.3%, p<0.02) in patients with only CD59+ leukemic cells. In conclusion, our findings indicate that leukemic cells derived from PNH clones may be common in de novo acute leukemia patients, suggesting that bone marrow failure may have already occurred in localized bone marrow even in de novo acute leukemia.

Natural history of paroxysmal nocturnal haemoglobinuria using modern diagnostic assays.

Paroxysmal nocturnal haemoglobinuria (PNH) is an uncommon, acquired disorder of blood cells caused by mutation of the phosphatidylinositol glycan class A (PIG-A) gene. The disease often manifests with haemoglobinuria, peripheral blood cytopenias, and venous thrombosis. The natural history of PNH has been documented in retrospective series; but there has only been one study that correlated the more sensitive and specific flow cytometric assays that have become available in the last decade with severe symptoms associated with PNH. In a retrospective analysis of 49 consecutive patients with PNH evaluated at Johns Hopkins, large PNH clones were associated with an increased risk for thrombosis as well as haemoglobinuria, abdominal pain, oesophageal spasm, and impotence. Of the 14 (29%) patients that developed thrombosis, nine died; six of these from complications related to thromboses. According to logistic regression modelling, for a 10% change in PNH clone size, the odds ratio for risk of thrombosis was estimated to be 1.64. No patient with <61% PNH granulocytes developed a thrombosis, whereas 12 of 22 patients (54.5%) with > or =61% PNH granulocytes manifested with thrombosis. These data not only confirm that the size of the PNH clone correlates with the risk for thrombosis, but they also suggest a correlation of PNH clone size to more symptomatic PNH.

Production of interferon-gamma by lymphocytes from paroxysmal nocturnal haemoglobinuria patients: relationship with clinical status.

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by the expansion of phosphatidylinositol glycan class A (PIG-A) defective haematopoietic cells, probably due to the immune-mediated alterations of the bone marrow environment selecting PIG-A- stem cells. The present study investigated the presence of alterations of the immune system in a population of 11 PNH patients. The production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), evaluated by intracellular cytokine analysis, and the frequencies of class I and II human leucocyte antigen (HLA) alleles were studied in comparison with healthy human subjects. Similar percentages of lymphocytes produced cytokines in PNH patients and controls after costimulation-independent activation; however, a negative correlation was found between the percentage of IFN-gamma producing cells and white cell or platelets counts. PNH patients showed an higher percentage, compared with controls, of IFN-gamma producing cells after costimulation-dependent activation. The frequency of HLA-A31 was higher in patients than in controls (27.2% vs. 4%), similarly to that of HLA-B7 (27.2% vs. 6%). With regard to class II alleles, 18% of PNH patients expressed DQB1*04 compared with none of 50 control cases. This study supports the hypothesis that immune alteration are present in PNH and that the immunogenetic background could influence the development of the disease.

Phenotypes and phosphatidylinositol glycan-class A gene abnormalities during cell differentiation and maturation from precursor cells to mature granulocytes in patients with paroxysmal nocturnal hemoglobinuria

To define the phosphatidylinositol glycan-class A (PIG-A) gene abnormality in precursor cells and the changes of expression of glycosylphosphatidylinositol-anchored protein and contribution of paroxysmal nocturnal hemoglobinuria (PNH) clones with PIG-A gene abnormalities among various cell lineages during differentiation and maturation, we investigated CD59 expression on bone marrow CD34(+) cells and peripheral granulocytes from 3 patients with PNH and the PIG-A gene abnormalities in the CD59(-), CD59(+/-), and CD59(+) populations by nucleotide sequence analyses. We also performed clonogeneic assays of CD34(+)CD59(+) and CD34(+)CD59(-) cells from 2 of the patients and examined the PIG-A gene abnormalities in the cultured cells. In case 1, the CD34(+) cells and granulocytes consisted of CD59(-) and CD59(+) populations and CD59(-), CD59(+/-), and CD59(+) populations, respectively. Sequence analyses indicated that mutation 1-2 was in the CD59(+/-) granulocyte population (20 of 20) and the CD34(+)CD59(-) population (2 of 38). In cases 2 and 3, the CD34(+) cells and granulocytes consisted of CD59(+) and CD59(-) cells. Sequence analyses in case 3 showed that mutation 3-2 was not in CD34(+)CD59(-) cells and was present in the CD59(-) granulocyte population. However, PIG-A gene analysis of cultured CD34(+)CD59(-) cells showed that they had the mutation. This analysis also revealed that there were some other mutations, which were not found in CD34(+)CD59(-) cells and CD59(-) or CD59(+/-) granulocytes in vivo, and that sometimes they were distributed specifically among different cell lineages. In conclusion, our findings suggest that PNH clones might contribute qualitatively and quantitatively differentially to specific blood cell lineages during differentiation and maturation of hematopoietic stem cells.

Mutation analysis of the PIG-A gene in Korean patients with paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is caused by deficient biosynthesis of the glycosylphosphatidylinositol (GPI) anchor in haemopoietic stem cells. Mutation of the phosphatidylinositol glycan class A (PIG-A) gene, an X linked gene that participates in the first step of GPI anchor biosynthesis, is responsible for PNH. The characteristics of somatic mutation of the PIG-A gene in Korean patients with PNH were studied. Twenty four patients with PNH were selected. Ham tests and sucrose haemolysis tests were carried out on all patients. The expression of CD59 in erythrocytes and granulocytes was investigated in 14 and five patients, respectively, to confirm the diagnosis. Dideoxy fingerprinting (ddF) was used to screen mutations, and direct sequencing of DNA was performed to characterise the mutations. Gene mutation was detected in 12 of the 24 patients. The other 12 patients were negative in ddF screening. Ten new mutations and two known mutations were detected. The mutations consisted of five deletions, six substitutions, and one insertion. These mutations resulted in six premature terminations, three abnormal splicings, one missense mutation in exon 2, and two nonsense mutations. Two patients with venous thrombosis showed mutations in exon 3 only. Substitution mutations were seen in six patients and frameshift mutations in the other six. There were 10 new mutations among the 12 mutations in the Korean patients with PNH and the characteristics of the mutations varied, with no significant hot spots in sites or types.

A New Aspect of the Molecular Pathogenesis of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder which is manifest by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Complement-mediated hemolysis in PNH is explained by the deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59 on erythrocyte surfaces. All the PNH patients had phosphatidylinositol glycan-class A (PIG-A) gene abnormalities in various cell types, indicating that PIG-A gene mutations cause the defects in GPI-anchored proteins that are essential for the pathogenesis of PNH. In addition, a PIG-A gene abnormality results in a PNH clone. Bone marrow failure causes cytopenias associated with a proliferative decrease of its hematopoietic stem cells and appears to be related to a pre-leukemic state. Although it is unclear how a PNH clone expands in bone marrow, it is considered that the most important hypothesis implicates negative selection of a PNH clone, but it does not explain the changes in the clinical features at the terminal stage of PNH. Recently, it has been suggested that an immune mechanism, in an HLA-restricted manner, plays an important role in the occurrence or selection of a PNH clone and GPI may be a target for cytotoxic-T lymphocytes. Also, it has been indicated that the Wilms' tumor gene (WT1) product is related to a PNH clone, but the significance of WT1 expression is not clear because of the functional diversity of the gene. To elucidate this problem, it is important to know the pathophysiology of bone marrow failure in detail and how bone marrow failure affects hematopoietic stem cells and immune mechanisms in bone marrow failure syndromes.

FES-Cre targets phosphatidylinositol glycan class A (PIGA) inactivation to hematopoietic stem cells in the bone marrow.

A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the mutation occurs in a hematopoietic stem cell. In transgenic mice, germline transmission of an inactive Piga gene is embryonic lethal. To inactivate the murine Piga gene in early hematopoiesis we therefore chose conditional gene inactivation using the Cre/loxP system. We expressed Cre recombinase under the transcription regulatory sequences of the human c-fes gene. FES-Cre inactivated PIGA in hematopoietic cells of mice carrying a floxed Piga allele (LF mice). PIGA(-) cells were found in all hematopoietic lineages of definitive but not primitive hematopoiesis. Their proportions were low in newborn mice but subsequently increased continuously to produce for the first time mice that have almost exclusively PIGA(-) blood cells. The loss of GPI-linked proteins occurred mainly in c-kit(+)CD34(+)Lin(-) progenitor cells before the CFU-GEMM stage. Using bone marrow reconstitution experiments with purified PIGA(-) cells we demonstrate that LF mice have long-term bone marrow repopulating cells that lack GPI-linked proteins, indicating that recombination of the floxed Piga allele occurs in the hematopoietic stem cell.

Proliferative capacity of single isolated CD34+ hematopoietic stem/progenitor cells in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations of the X-linked PIG-A (phosphatidylinositol glycan-class A) gene, which occurs on a hematopoietic stem cell level, leading to a proportion of blood cells being deficient in all glycosylphosphatidylinositol (GPI)-anchored surface proteins. Although these GPI-deficient cells can explain many of the clinical symptoms of PNH, the pathogenesis of PNH is still somewhat obscure and many questions remain. To assess the hematopoietic defect involved in PNH, CD34+ CD59+ (normal phenotype hematopoietic stem/progenitor) and CD34+ CD59- (PNH phenotype) cells from PNH patients (n = 16) and CD34+ CD59+ cells from healthy volunteers (n = 10) were sorted as single cells into 96-well flat-bottom culture plates containing culture medium supplemented with stem cell factor, interleukin (IL)-3, erythropoietin, granulocyte-macrophage-colony-stimulating factor (GM-CSF), G-CSF, IL-6, thrombopoietin, and Flt-3 ligand. We found that the single PNH CD34+ CD59- cells had a growth advantage over the single CD34+ CD59+ cells to some extent, but they both had impaired growth abilities compared with CD34+ cells from healthy volunteers.

Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis due to deficiencies of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in subpopulations of blood cells. Acquired mutations in the X-linked phosphatidylinositol glycan–class A (PIG-A) gene appear to be the characteristic and pathogenetic cause of PNH. To develop a gene therapy approach for PNH, a retroviral vector construct, termed MPIN, was made containing the PIG-A complementary DNA along with an internal ribosome entry site and the nerve growth factor receptor (NGFR) as a selectable marker. MPIN transduction led to efficient and stable PIG-A and NGFR gene expression in a PIG-A–deficient B-cell line (JY5), a PIG-A–deficient K562 cell line, an Epstein-Barr virus–transformed B-cell line (TK-14−) established from a patient with PNH, as well as peripheral blood (PB) mononuclear cells from a patient with PNH. PIG-A expression in these cell lines stably restored GPI-AP expression. MPIN was transduced into bone marrow mononuclear cells from a patient with PNH, and myeloid/erythroid colonies and erythroid cells were derived. These transduced erythroid cells restored surface expression of GPI-APs and resistance to hemolysis. These results indicate that MPIN is capable of efficient and stable functional restoration of GPI-APs in a variety of PIG-A–deficient hematopoietic cell types. Furthermore, MPIN also transduced into PB CD34+ cells from a normal donor, indicating that MPIN can transduce primitive human progenitors. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells, thereby providing a potential new therapeutic option in PNH.

Genetic and environmental effects in paroxysmal nocturnal hemoglobinuria: this little PIG-A goes “Why? Why? Why?”

Genetic and environmental effects in paroxysmal nocturnal hemoglobinuria: this little PIG-A goes “Why? Why? Why?”

Chimaeric mice with disruption of the gene coding for phosphatidylinositol glycan class A (Pig-a) were defective in embryogenesis and spermatogenesis.

Mutations in the gene encoding PIG-A (phosphatidylinositol glycan class A) are found in patients with paroxysmal nocturnal haemoglobinuria (PNH), an acquired haematopoietic stem cell disorder. Individuals with hereditary PIG-A mutations have never been identified, which is also manifested by the difficulties in generating Pig-a knockout (KO) mice. This study investigated the effect of Pig-a mutations on the development of visceral and genital organs in addition to the haematopoietic system by the generation of Pig-a KO chimaeric mice. Of a total of 54 live births out of 1684 blastocysts injected, chimaerism for Pig-a knockout was detected in 29 mice, suggesting the importance of Pig-a in embryogenesis and in live birth. Quantification of the degree of chimaerism in different organs of the surviving chimaeric mice revealed extremely low levels of Pig-a KO cells in the liver and spleen. In contrast, high levels of KO signals were usually detected in the brain, heart, lung and kidney. Haematopoiesis proceeded normally in these chimaeric mice (as measured by 'complete blood cell counting') and the Pig-a KO cells were present at low levels in red blood cells and B lymphocytes but at high levels in T lymphocytes, although these KO cells did not gain any growth advantage. The effect of Pig-a knockout was also prominent in the reproductive system, another organ with high mitotic activity. Breeding the male chimaeras revealed a high rate of infertility and abnormality in the male genital organs, including abnormally shaped testes, epididymis and seminal vesicles. Even in the absence of gross abnormalities of the genital organs, low counts of motile sperm were also discernible. Pig-a KO sperm was detected in these organs; however, no transmission of the KO allele was observed. The results suggest a possible mechanism underlying the non-transmission of the Pig-a KO gene in germlines.

Antibody selection against CD52 produces a paroxysmal nocturnal haemoglobinuria phenotype in human lymphocytes by a novel mechanism.

The CD52 antigen is a lymphocyte glycoprotein with an extremely short polypeptide backbone and a single N-linked glycan, and it is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Treatment of rheumatoid arthritis patients with CAMPATH-1H, a humanized monoclonal antibody against CD52, resulted, in a small number of cases, in the appearance and persistence of CD52-negative T cells. Similarly, CD52-negative B cells emerged following in vitro treatment of a CD52-positive human B cell line with CAMPATH-1H. Both the B and T CD52-negative cells were also found to be defective in surface expression of other GPI-anchored proteins. Biochemical analysis revealed a severe defect in the synthesis of a mature GPI precursor in both the B and T cell lines. Therefore the phenotype of these CD52-negative B and T cells closely resembles that of lymphocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH), in which the first step of the GPI-biosynthetic pathway, i.e. synthesis of GlcNAc-phosphatidylinositol, is blocked. In all cases studied to date, this defect maps to a mutation of the phosphatidylinositolglycan class A (PIG-A) structural gene. We therefore amplified the PIG-A gene from both the GPI-negative B and T cells by PCR and determined the nucleotide sequence. No differences from the wild-type sequence were detected; therefore a classical PNH mutation cannot be responsible for the GPI-biosynthesis defect in these cell lines. Significantly, the GPI-negative phenotype of the B cells was reversible upon separation of the positive and negative cells, resulting in a redistribution to a mixed population with either CD52-positive or -negative cells, whereas populations of 100% CD52-negative T cells were stably maintained during culture. Therefore, whereas the GPI-biosynthesis deficiency in the T cell lines may be due to a mutation in another gene required by the GPI-biosynthetic pathway, the reversible nature of this block in the B cell lines suggests a less direct cause, possibly an alteration in a regulatory factor. Overall, these data demonstrate that the PNH phenotype can be generated without a mutation in the PIG-A structural gene, and thereby identify a novel mechanism for the development of GPI deficiency.

Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder in which there is a deficiency in the synthesis by hematopoietic cells of the glycosyl-phosphatidylinositol molecules that anchor proteins to the cell membrane. Recently, we demonstrated that a gene termed PIG-A (for phosphatidylinositol glycan class A), a component of glycosyl-phosphatidylinositol biosynthesis, was responsible for PNH in two patients. The present study was undertaken to elucidate whether PIG-A is the gene responsible for all cases of PNH and to characterize further the somatically acquired abnormalities of this gene. We studied granulocytes from 15 patients with PNH. The cell content of CD55 and CD59 was assessed by fluorescence-activated flow cytometry. PIG-A transcripts were reverse-transcribed, amplified by the polymerase chain reaction, and cloned into plasmids. The structure of the cloned complementary DNA was analyzed by nucleotide sequencing, and its function was assessed on the basis of its ability to restore to normal the abnormal phenotype of a PIG-A-deficient cell line after transfection. Three patients had size abnormalities of PIG-A transcripts with different patterns, and in one patient a very low level of the PIG-A transcript was found. Eleven patients had transcripts of normal size, but the transfection assay revealed that in each patient some of them were nonfunctional. The percentage of nonfunctional PIG-A transcripts was correlated with the percentage of affected granulocytes (P < 0.001). Sequence analysis demonstrated somatic mutations in two of the patients. PIG-A is the gene responsible for PNH in all patients studied to date.

Surfactant protein D (SP-D) binding to alveolar macrophages

Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many proteins, including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA mutations. Although somatic PIGA mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contractures, central nervous system (CNS) structural malformations, and other anomalies. An X chromosome exome next-generation sequencing screen identified a single nonsense PIGA mutation, c.1234C>T, which predicts p.Arg412∗. This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. PIGA-null mutations are thought to be embryonic lethal, suggesting that p.Arg412∗ PIGA has residual function. Transfection of a mutant p.Arg412∗ PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234C>T (p.Arg412∗) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234C>T in PIGA results in the lethal X-linked phenotype recognized in the reported family.