Phosphatidylinositol Glycan Class A Mutations Research Articles

PIG-a Mutations in a Brazilian Cohort of Patients with Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a disorder due to an acquired loss-of-function mutation in the phosphatidylinositol glycan class A (PIG-A) gene. A large spectrum of acquired PIG-A mutations has been described, like insertions or deletions involving a single base or several bases, and single base substitution that are the most common. Usually there are more than one PIGA mutations and one clone is predominant. The clinical manifestations of PNH are intrinsically related to clonal expansion of hematopoietic stem cell deficient in GPI-anchored proteins. Some hypothesis failed to explain alone this clonal expansion. Here we try to identify and to correlate PIG-A gene mutations with clinical manifestations in a series of patients with PNH. Methods: We analyzed 31 patients with classical PNH (n=23) or aplastic anemia and PNH clone (n=8). The sequencing of the PIG-A gene was performed using the Sanger technique. The electropherograms were aligned against the reference sequence of the PIG-A gene deposited in GenBank (Accession number NG_009786), and analyzed using the Geneious R10 software (Biomatters). After analysis, a search was performed in the Clinvar, dbSNP and HGMD databases to verify the pathogenicity of the mutations. For variants without description in the literature, a pathogenicity prediction analysis was performed using Mutation Taster, Polyphen 2 and Human Splicing Finder software. Results: We found 29 different variants of the PIG-A gene in 27 patients: 23 were new mutations, with no previous description in the literature, 3 were previously described mutations, and 3 were single nucleotide polymorphism (SNP). There was great variation in the type and location of somatic mutations. Mutations were predominantly small deletions and simple base changes; 42% of the mutations were described as frameshift mutations and 31% missense mutations. We did not find any specific correlation between the clinical characteristics of hemolytic PNH patients and their mutations, due to the wide variety of mutations. According the pathogenicity prediction programs, the majority (22 of 29) of the variants found were classified as probably pathogenic. Among the 23 patients with hemolytic PNH, 19 patients had at least one mutation classified as pathogenic. In patients with subclinical PNH, only SNPs were found. Fifteen patients with hemolytic PNH had more than one concomitant mutation, most of which were probably pathogenic mutations associated with a polymorphism. Conclusion: We described PIG-A mutations in a series of PNH patients in Brazil and observed no correlation exists between mutation types and clinical features in hemolytic patients. Among subclinical PNH patients, only SNPs were observed, probably because of small clone sizes. Disclosures No relevant conflicts of interest to declare.

Invited editorial comment—the human phenotype of germline PIGA mutations

Two research groups have published reports on PIGA (phosphatidylinositol glycan class A) mutations that validate and extend our understanding of the range of phenotypes of this phenotypic spectrum. One report is primarily confirmatory of the discovery in 2012 that mutations in this gene cause a phenotype of dysmorphic features, neurologic manifestations, and biochemical perturbations. The second report describes an intriguing family with a phenotypically distinct neurological picture, distinguished primarily by CNS iron accumulation. These reports address important lessons in judging causality in the exome age and bear on the question of syndrome nomenclature.

The Phenotype of a Germline Mutation in PIGA: The Gene Somatically Mutated in Paroxysmal Nocturnal Hemoglobinuria

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.

Co-Existance of JAKV617F and PIG-A mutations in Primary Budd-Chiari Syndrome.

Abstract 3193Poster Board III-130Paroxysmal nocturnal hemogloginuria (PNH) is a clonal genetic disorder associated with multiple mutations in the X-linked phosphatidylinositol glycan class A (PIG-A) gene that causes glycosyl phosphatidylinositol (GPI) anchor protein (AP) deficiency in patients with PNH. Conditional PIG-A gene inactivation (PIG-A (-/-)) in hematopoietic cells of mice does not induce overt proliferative advantage to the HPC compartment. In humans, the molecular basis of clonal expansion of GPI-AP deficient cells is unknown but may relate to selective pressure from the bone marrow microenvironment conducive to GPI-AP survival. Alternatively, coexistence of additional genetic anomalies that confer growth potential has been speculated. A single mutation in the Janus Kinase (JAK)-2 gene has been reported to be the underlying molecular mechanism for myeloproliferative diseases including polycythemia vera (PV), essential thrombocythemia, and myelofibrosis. Substitution of a valine for phenylalanine destabilizes the auto-inhibitory activation domain of JAK2 leading to myeloproliferation. Genetic evidence and in vitro functional studies indicate that V617F gives hematopoietic precursors proliferative and survival advantages. A high proportion of patients with myeloproliferative disorders and myelodysplastic syndrome (MDS) carry this dominant gain-of-function mutation of JAKV617F. The clinical syndromes of PNH and JAKV617F mutations often present as overlapping clinical syndromes raising the possibility that acquired JAKV617F mutations confer growth and survival potential to PNH clones in at least a subset of patients. To address this possibility, 21 PNH patients were screened for the presence of JAKV617F mutations. We show here that the features of Budd-Chiari syndrome (BCS) as well as a predisposition to myelodysplastic syndrome can be caused by an acquired co-existing PIGA and JAKV617F activating mutation. Of the 21 cases examined, PNH with JAK2V617F mutations co-existed in three cases (14.3%). Thus far, two patients have been well characterized. Both cases were male, ages 51 (case 1) and 65 year-old (case 2) with normal cytogenetics and de novo classic PNH manifested by Budd-Chiari syndrome. Flow cytometry assay using CD55, CD59, and CD48 (GPI-APs) and FLAER to detect PNH type cells showed that 90% of granulocytes (case 1) and almost 100% of granulocytes (case 2) were GPI-AP deficient cells. Interestingly, T-cells in both cases displayed a normal phenotype suggesting that the PIGA gene mutation was selectively acquired in a common myeloid progenitor (CMP). From sorted populations of PNH+ and normal type granulocytes (case 1 only) or T cells (case 2), the JAK2V617F mutant clone was found to co-exist with the PIGA mutation using allele-specific polymerase chain reaction (PCR). Real-time quantitative PCR using primers and probe to the junction of exon 5-6 coding region demonstrated drastically low to absent mRNA transcript expression of PIGA only in PNH-type granulocytes in both cases. Individual RT-PCR of the coding region of each exon (2-6) confirmed that transcription was absent in case 1 but limited to regions encoded by exons 2,4, and 6 in case 2. These results suggest that case 2 possesses a splicing mutation involving exon 5 to 6. In conclusion, JAK2V617F mutation is known to cause the constitutive activation of the JAK-STAT signaling pathway, and leads to autonomous cell growth in a cytokine-independent expansion of HPCs. While it has been reported that JAK2V617F mutation occurs in roughly 50% of primary Budd-Chiari syndrome and in 15 patients of 163 (9.2%) (Hoekstra et al, Journal of Hepatology, 6:19, 2009) of patients with PNH, these findings implicate co-existing JAK2V617F /PIG-A mutations may contribute clinically to specific manifestations of splenic venous thrombosis or hepatic vein thrombosis in association with intravascular coagulation and myelodysplasia. DisclosuresNo relevant conflicts of interest to declare.

Budd-Chiari syndrome in a paroxysmal nocturnal hemoglobinuria patient with previous cerebral venous thrombosis

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal stem cell disorder characterized by intravascular hemolytic anemia that results from the clonal expansion of hematopoietic stem cells harboring somatic mutations in an X-linked gene, termed PIG-A (phosphatidylinositol glycan class A). PIG-A mutations block glycosylphosphatidylinositol (GPI) anchor biosynthesis, resulting in a deficiency or absence of all GPI-anchored proteins on the cell surface. CD55 and CD59 are GPI-anchored complement regulatory proteins. Their absence on PNH red cells is responsible for the complement-mediated intravascular hemolysis, leading to the release of free hemoglobin, which contributes to many of the clinical manifestations of PNH including fatigue, pain, esophageal spasm and possibly serious thrombotic episodes [1]. Allogeneic hematopoietic stem cell transplantation is the only curative therapy for PNH. The complement inhibitor eculizumab, a humanized monoclonal antibody against C5, that inhibits terminal complement activation, recently approved in the US, has been shown to reduce hemolysis, decrease the erythrocyte transfusion requirements and the risk for thrombosis, and to improve quality of life of PNH patients [2, 3]. Major morbidity and mortality with PNH are often ascribed to the development of venous thromboembolism (VTE) [1–6], and several patients have recurrent thromboses [7]. VTE in PNH patients occur mostly at unusual sites. Data from a recent review report hepatic vein thrombosis, leading to Budd-Chiari syndrome (BCS), as the most frequent (40.7%) thrombotic complication, accounting for the majority of deaths [8], followed by cerebral vein and sinus thromboses, superior sagittal sinus being the most frequently involved site [8]. Inherited thrombophilia may increase the risk of serious thromboembolic events in PNH patients [9]. In patients experiencing VTE, early anti-thrombotic therapy (heparin, thrombolysis) aimed to limit the extension of thrombosis, or to dissolve formed thrombi, should improve the prognosis of this severe complication. PNH patients suffering from VTE should be treated with anticoagulant drugs indefinitely [1]. Thrombocytopenia often complicates PNH, and this issue must be addressed when an anticoagulation management plan is formulated [1]. Recurrent, life-threatening thrombosis merits consideration for bone marrow transplantation (BMT) [1]. Over the past few years significant advances in other therapeutic approaches, such as transjugular intrahepatic portosystemic shunt (TIPS), have contributed to the improvement of survival in this setting [8]. We report the case of a 29-year-old woman, R. D. The family history was negative for VTE and cardiovascular disease. She was a non-smoker. And had been slightly overweight since childhood. When she was 19 years old, an isolated moderate thrombocytopaenia (50,000/mmc) was detected although in the absence of bleeding. As idiopathic thrombocytopaenia was diagnosed, she was treated with long-term steroids, with an improvement in the platelet count (120,000/mmc). At the age of 27, while on oral contraceptives for 3 months, she experienced a sudden occurrence of headache and visual loss, followed by seizures and coma. A computed tomography (CT scan) showed multiple cerebral venous thromboses. Screenings A. Tufano (&) N. Macarone Palmieri E. Cimino A. M. Cerbone Regional Reference Centre for Coagulation Disorders, Department of Clinical and Experimental Medicine, Federico II University of Naples, Via S. Pansini, 5, 80131 Naples, Italy e-mail: atufano@unina.it

Changes of Clonality of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones during Clinical Courses in Patients with PNH.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder, in which almost all hematopoietic cells lack glycosylphosphatidylinositol (GPI)-anchored proteins due to phosphatidylinositol glycan-class A (PIG-A) gene abnormalities at the level of hematopoietic stem cells. It has been reported that several minor PNH clones in patients with aplastic anemia and myelodysplastic syndromes occur at random during the clinical course (Okamoto M et al, Leukemia, 2006), while one or more major PNH clones in patients with typical PNH continue to exist during their clinical courses (Nishimura J et al, Blood, 2002). To clarify how PNH clones in patients with PNH expand during their clinical courses, we examined sequence analyses of the PIG-A gene using peripheral blood granulocytes from PNH patients with high (>10%; n=5) and low (<10%; n=3) proportions of CD16− or CD59− granulocytes, which were determined by flow cytometry using CD16b or CD59 monoclonal antibody, respectively. A patient with over 1% of CD59− erythrocytes was judged to have PNH erythrocytes (Shichishima T et al, Br J Haematol, 1993). PNH clones were defined as GPI-negative hematopoietic cells with PIG-A mutations. Twenty to 40 bacterial clones of PCR or RT-PCR products of the PIG-A gene with GPI-negative or unsorted populations of peripheral blood granulocytes were analyzed by nucleotide sequencing. We examined sequence analyses of the PIG-A gene in peripheral blood granulocytes at least twice during the clinical course (the period of 1995–2007). Major PNH clones were decided by the Chi2 test or the Fisher's exact probability test. In all of 5 PNH patients with high proportions of PNH clones, major PNH clones were found at least once and were common during their clinical courses. However, the PNH clones in 3 of 5 PNH patients were transiently major during their clinical courses. In 2 of 3 PNH patients with low proportions of PNH clones, there were common PIG-A mutations during their clinical courses, but were not major clones. The PNH clone from one of the 2 PNH patients acquired a new PIG-A mutation, which was also minor. Another patient of the 3 patients with low proportions of PNH clones had a major PNH clone at the time of the first examination, but disappeared at the time of the next analysis. In conclusion, our findings suggest that the clonality of PNH clones in PNH patients with high and low proportions of PNH clones could change during their clinical courses and that especially, the clonality of PNH clones in PNH patients with low proportions of PNH clones is not necessarily definitive.PIG-A mutations in PNH patients with high and low proportions of PNH clonesCase No.Common mutationUncommon mutationPNH patients with high proportions of PNH clonesCase 1214 C to T, 5′ splicing site del G (Major)NDCase 2698 G to A (Major), 1152 del CNDCase 3548 G to A (Major)982 G to TCase 4ND491 C to TCase 5ND407 A to GPNH patients with low proportions of PNH clonesCase 5ND148 del G, 142 G to A (Major), 460 C to TCase 6ND11 G to C, 91 ins C, 916 del CCase 6ND67 del G, 72 del 13bp, 72 del 13bp/188 G to A, 358 G to A, 365 A to C, 952 del AAbbreviations: del , deletion; ND, not dectected; and ins, insertion

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.

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.

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.

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.