PIG-A Research Articles

Rescue of Glycosylphosphatidylinositol-Anchored Protein Biosynthesis Using Synthetic Glycosylphosphatidylinositol Oligosaccharides.

The attachment of proteins to the cell membrane using a glycosylphosphatidylinositol (GPI) anchor is a ubiquitous process in eukaryotic cells. Deficiencies in the biosynthesis of GPIs and the concomitant production of GPI-anchored proteins lead to a series of rare and complicated disorders associated with inherited GPI deficiencies (IGDs) in humans. Currently, there is no treatment for patients suffering from IGDs. Here, we report the design, synthesis, and use of GPI fragments to rescue the biosynthesis of GPI-anchored proteins (GPI-APs) caused by mutation in genes involved in the assembly of GPI-glycolipids in cells. We demonstrated that the synthetic fragments GlcNAc-PI (1), Man-GlcN-PI (5), and GlcN-PI with two (3) and three lipid chains (4) rescue the deletion of the GPI biosynthesis in cells devoid of the PIGA, PIGL, and PIGW genes in vitro. The compounds allowed for concentration-dependent recovery of GPI biosynthesis and were highly active on the cytoplasmic face of the endoplasmic reticulum membrane. These synthetic molecules are leads for the development of treatments for IGDs and tools to study GPI-AP biosynthesis.

Comparison of the in vivo genotoxicity of electronic and conventional cigarettes aerosols after subacute, subchronic and chronic exposures.

Tobacco smoking is classified as a human carcinogen. A wide variety of new products, in particular electronic cigarettes (e-cigs), have recently appeared on the market as an alternative to smoking. Although the in vitro toxicity of e-cigs is relatively well known, there is currently a lack of data on their long-term health effects. In this context, the aim of our study was to compare, on a mouse model and using a nose-only exposure system, the in vivo genotoxic and mutagenic potential of e-cig aerosols tested at two power settings (18 W and 30 W) and conventional cigarette (3R4F) smoke. The standard comet assay, micronucleus test and Pig-a gene mutation assay were performed after subacute (4 days), subchronic (3 months) and chronic (6 months) exposure. The generation of oxidative stress was also assessed by measuring the 8-hydroxy-2'-deoxyguanosine and by using the hOGG1-modified comet assay. Our results show that only the high-power e-cig and the 3R4F cigarette induced oxidative DNA damage in the lung and the liver of exposed mice. In return, no significant increase in chromosomal aberrations or gene mutations were noted whatever the type of product. This study demonstrates that e-cigs, at high-power setting, should be considered, contrary to popular belief, as hazardous products in terms of genotoxicity in mouse model.

Centennial development of paroxysmal nocturnal hemoglobinuria research in Peking Union Medical College Hospital

<p indent=0mm>Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal hematopoietic stem cell (HSC) disease. A somatic phosphatidylinositol glycan-A (PIGA) gene mutation in HSCs translates into abnormal biosynthesis of glycosyl phosphatidylinositol (GPI) anchors on the cell surface. The absence of GPI-anchored complements regulators leads to complement-driven blood cell lysis. Hemolysis and chronic anemia are the most common clinical manifestations, while bone marrow failure is a rare but life-threatening complication. Thrombosis is the leading cause of mortality. Transfusion and supportive care have been introduced as symptomatic treatments since the early decades of the last century. Eculizumab, a monoclonal antibody that blocks terminal complement at C5, is a breakthrough medicine that changed the standard treatment methods of PNH. However, in cases where eculizumab is not accessible, allogeneic HSC transplantation could be an alternative strategy for refractory and aplastic patients. The Peking Union Medical College Hospital (PUMCH) is approaching its centennial anniversary in 2021. The first case of PNH in China was reported by physicians at PUMCH in the 1950s. During the past century, generations of hematologists established the largest cohort of PNH patients in China while continuously developing novel laboratory tests for this disease. They collaborated with investigators of basic science on the pathogenetic mechanisms of bone marrow failure and thrombosis. Although great historical changes have impacted academic progress, the spirit of perseverance, commitment, and innovation in older generations is passed on to the next generations. In this paper, we summarize the clinical evolution of PNH treatment along with basic science at PUMCH, which in turn facilitates great progress in PNH in the era of the 21st century in China.

Identification of Essential Genes Associated With Prodigiosin Production in Serratia marcescens FZSF02.

Prodigiosin is a promising secondary metabolite produced mainly by Serratia strains. To study the global regulatory mechanism of prodigiosin biosynthesis, a mutagenesis library containing 23,000 mutant clones was constructed with the EZ-Tn5 transposon, and 114 clones in the library showed altered prodigiosin production ability. For 37 of the 114 clones, transposon insertion occurred on the prodigiosin biosynthetic cluster genes; transposon inserted genes of the 77 clones belonged to 33 different outside prodigiosin biosynthetic cluster genes. These 33 genes can be divided into transcription-regulating genes, membrane protein-encoding genes, and metabolism enzyme-encoding genes. Most of the genes were newly reported to be involved in prodigiosin production. Transcriptional levels of the pigA gene were significantly downregulated in 22 mutants with different inserted genes, which was in accordance with the phenotype of decreased prodigiosin production. Functional confirmation of the mutant genes involved in the pyrimidine nucleotide biosynthesis pathway was carried out by adding orotate and uridylate (UMP) into the medium. Gene complementation confirmed the regulatory function of the EnvZ/OmpR two-component regulatory system genes envZ and ompR in prodigiosin production.

Mutational signatures in T-lymphocytes of rats treated with N-propyl-N-nitrosourea and procarbazine.

We have used whole genome sequencing (WGS) to determine mutational signatures induced in the T-cells of rats treated in vivo with N-propyl-N-nitrosourea (PNU) or procarbazine (PCZ). The signatures from the treated rats were different from the signature of background mutations. The main component of the spontaneous T-cell mutational signature was C➔T transition with all other single base substitutions evenly distributed. The PNU-induced mutational signature showed relatively equal contributions from C➔T and T➔C transitions, and T➔A transversions. The PCZ-induced signature was characterized by T➔C transitions, T➔A and, to a smaller extent, T➔G transversions. C➔G transversions were infrequent in either the PNU or PCZ signatures. WGS not only allowed mutational signature detection, but also measured quantitative responses to mutagen treatment: 10-40× increases in the number of mutations per clone were detected in T-cell clones from treated rats. The overall strand specificity of induced mutations for annotated rat genes was comparable to the strand specificity of mutations determined previously for the endogenous X-linked Pig-a gene. Our results provide valuable reference data for future applications of WGS in safety research and risk assessment.

Elucidation of Autoinflammatory Mechanism in Pigt-PNH

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

Paroxysmal Nocturnal Hemoglobinuria Results from Initial Clonal PIG-a Mosaicism

The pathogenesis of PNH involves alteration of at least one HSC clone, characterized by a somatic mutation in the PIG-A gene. The resulting phenotype appears to offer a growth advantage over other unaffected HSCs, yet the failure of PIG-A mutant clones to expand in animal models suggests that additional factors are necessary for the development of PNH. The association of PNH with aplastic anemia (AA) is evidence of selection pressure by immune attack, but secondary hits in additional genes may also be clonally acquired to render PNH clones competitive irrespective of immune pressure. With the advent of NGS, clonal architecture analysis has been revolutionized. Yet, despite increased resolution, NGS is hampered by the presence of normal/non-clonal cells at varying frequencies in the sequenced sample, often decreasing the detection threshold and precluding recognition of subclonal mosaicism.To study these issues in PNH, 133 patients with PNH (N=33), AA (N=33), and AA/PNH (N=67) were sequenced using deep targeted NGS covering all exons of the PIG-A gene. All patients had a detectable PNH clone by flow cytometry, with the mean WBC clone size of 0.22% in AA, 27.4% in AA/PNH, and 77.1% in PNH. 223 PIG-A mutations were identified in this cohort; 35% were frameshift mutations, 34% missense, 14% splice site, 13% nonsense, and 3% non-frameshift. In unpurified blood, PIG-A mutations were found in 27/33 (82%) of PNH patients, 36/67 (54%) of AA/PNH, and 2/33 (6%) of AA, with a clear association between PNH clone size and the ability to detect mutations by NGS (P<0.0001). In DNA extracted from cells flow sorted by FLAER negativity, detection rates were 26/29 (90%), with an average of nearly 3 mutations per patient, indicating that mechanisms such as microdeletions may affect as many as 10% of PNH or AA/PNH patients and that clonal mosaicism is common. Our results also show that indeed type III and II PNH cells are derived from 2 distinct clones, each with a distinct mutation.To examine disease pathogenesis, we studied 47 patients longitudinally using both PNH flow and deep sequencing. Serial testing illustrates possible modes of evolution: mutations can fluctuate in the contribution to clonal burden over time with the variant allelic frequency of some mutations increasing while others decrease. Linear regression of the number of mutations vs . time from diagnosis suggests that in AA/PNH (N=29), the number of mutant clones detected increases over time (P=.012) while in classic PNH (N=18) the trend is reversed, as the number of mutations detected decreases (P<.0001) and a dominant clone emerges to supply a majority of blood cells.Traditionally, PNH has been considered a monoclonal disease in which a PIG-A mutant HSC clone can outcompete normal hematopoiesis within the context of permissive conditions. Using deep NGS, our results clearly indicate that while a single clone ultimately may dominate, PNH begins as oligoclonal process with the eventual emergence of the most competitive HSC clone(s) populating hematopoietic space. Initially, an environment that “favors” a state of GPI-anchor deficiency thereby allows multiple clones to evolve. Over time, the most “fit” PNH HSC clones dominate, resulting in monoclonality. In addition, our results confirm the presence of distinct mutations with corresponding phenotype through sequencing of sorted type II and type III PNH cells. Clonal drift may also be observed, likely through a newly acquired advantage of one clone within the available pool of GPI-deficient PIGA mutant clones. Our previous work indicated that acquisition of additional enabling mutations results in a more complex clonal architecture comparable to that seen in MDS, an intrinsic mechanism of the selection process that involves mutations in genes altered in MDS. Additional intrinsic hits also explain patient-specific kinetics of PNH evolution and apparent independence of established PNH from the effect of immunosuppression. Thus, while immune selection may provide initial conditions favorable for oligoclonal PNH evolution, once pressure is relieved, intrinsic factors can lead to PNH monoclonality. Alternative mechanisms could include loss of dominance by exhaustion, senescence, or telomere shortening.In sum, our study clearly demonstrates initial polyclonality early in the pathogenesis of PNH with selection of dominant clones during disease duration. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

Fibrinogen and Clot Structure Is Affected By Eculizumab in Patients with Paroxysmal Nocturnal Haemoglobinuria

Background Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic stem cell disorder in which an acquired somatic mutation of the PIGA gene in a haematopoietic stem cell results in the deficiency of key complement regulatory molecules, namely CD59 and CD55, from the surface of blood cells leading to an increased susceptibility of blood cells to complement-mediated activation +/- intravascular haemolysis and thrombosis. Thrombosis has been the leading cause of death in PNH. Multiple mechanisms of thrombosis in PNH have been proposed, including cross talk between the coagulation and complement system. This may lead to denser clots which are more resistant to fibrinolysis. Treatment with eculizumab, which inhibits complement factor C5 and thereby prevents terminal complement activation, reduces thromboembolic events in PNH, indicating that complement activation plays a significant role in the pathophysiology of thrombosis in this disease. Patients with other thrombotic disorders are known to form denser fibrin clots that incur greater resistance to fibrinolysis. It has not previously been explored whether changes to clot architecture contribute to thrombosis in PNH. Furthermore, the role of eculizumab in clot architecture is undocumented.Aims Characterise fibrin clot structure in patients with PNH and determine the effect of eculizumab on clot structure.Methods Informed consent was obtained from all participants. 53 patients from the PNH National Service (Leeds) were recruited. They were grouped according to the proportion of PNH granulocytes, <40% (n=18), 40> - < 90 %(n=18) and >90% (n=17) and reanalysed according to treatment with (n=19) or without (n=32) eculizumab. Plasma samples were obtained and ex vivo fibrin clot structure and function was analysed by permeation analysis of clot pore structure, turbidity analysis of clot formation and fibrinolysis, and fibrin ultrastructure by laser scanning confocal microscopy and scanning electron microscopy, according to methodology and protocols well established in our laboratory. The proportion of PNH granulocytes was measured by flow cytometry and fibrinogen levels by Clauss. P values of <0.05 were taken to indicate significance.Results The proportion of PNH granulocytes correlated with fibrinogen (r= -0.35, p=0.048), maximum absorbency (r= -0.34, p=0.046) and clot density (r= -0.36, p=0.04). Seventy per cent of patients with large proportions of PNH granulocytes were on eculizumab. Analysis of patients not on eculizumab showed a trend towards proportion of PNH granulocytes correlating positively with fibrinogen levels (r=0.42, p=ns). Patients on eculizumab had lower fibrinogen levels (3.1±0.2 v 3.6±0.1g/L, P=0.02), maximum absorbance (0.31±0.1 v 0.40±0.1, p=0.028) and fibre density/100µm (18.5±4.4 v 23.9±4.2, p=0.006), indicative of a less dense clot.Summary/Conclusions This is the first study to demonstrate that patients on eculizumab showed reduced fibrinogen, fibre thickness and fibre density. Our data indicate that the beneficial effects of eculizumab in PNH is partly due to reduced complement activation and attenuated inflammatory response, leading to lower fibrinogen levels and a less thrombotic fibrin clot structure. These novel data help to explain the impact of complement inhibition in this disease. [Display omitted] DisclosuresArnold:Alexion Pharmaceuticals, Inc.: Honoraria. Griffin:Alexion Pharmaceuticals, Inc.: Honoraria. Munir:Janssen: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria; Roche: Honoraria; AbbVie: Honoraria; Gilled: Honoraria. Hillmen:Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Celgene: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding. Richards:Alexion Pharmaceuticals, Inc.: Honoraria. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

In Vitro PIG-A Gene Mutation Assay in Human B-Lymphoblastoid TK6 Cells

AbstractThe X-linked PIG-A gene is involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD59 and CD55). In recent years, in vitro PIG-A assay has been established based on the high conservation of PIG-A/Pig-a loci among different species and the large data from the in vivo system. The purpose of this study was to extend the approach for PIG-A mutation assessment to in vitro human B-lymphoblastoid TK6 cells by detecting the loss of GPI-linked CD55 and CD59 proteins. TK6 cells were treated with three mutagens 7,12-dimethylbenz[a]anthracene (DMBA), N-ethyl-N-nitrosourea (ENU), etoposide (ETO), and two nonmutagens: cadmium chloride (CdCl2) and sodium chloride (NaCl). The mutation rate of PIG-A gene within TK6 cells was determined on the 11th day with flow cytometry analysis for the negative frequencies of CD55 and CD59. The antibodies used in this production were APC mouse-anti-human CD19 antibody, PE mouse anti-human CD55 antibody, PE mouse anti-human CD59 antibody, and nucleic acid dye 7-AAD. An immunolabeling method was used to reduce the high spontaneous level of preexisting PIG-A mutant cells. Our data suggested that DMBA-, ENU-, and ETO-induced mutation frequency of PIG-A gene was increased by twofold compared with the negative control, and the effects were dose-dependent. However, CdCl2 and NaCl did not significantly increase the mutation frequency of PIG-A gene, with a high cytotoxicity at a dose of 10 mmol/L. Our study suggested that the novel in vitro PIG-A gene mutation assay within TK6 cells may represent a complement of the present in vivo Pig-a assay, and may provide guidance for their potential use in genotoxicity even in cells with a significant deficiency of GPI anchor.

Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination.

Despite the great potential of Serratia marcescens in industrial applications, lack of powerful genetic modification tools limits understanding of the regulatory networks of the useful metabolites and therefore restricts their mass production. To meet the urgent demand, we established a genome-editing strategy for S. marcescens based on Red recombineering in this study. Without host modification in advance, nucA and pigA were substituted by PCR-amplified resistance genes. No long homologous arms were required at the two sides of resistance genes. Using this procedure, the fragment at the S. marcescens as large as 20 kb was easily deleted. Then we constructed a counter-selection gene kil constructed under the control of inducible PBAD operon, which demonstrates obvious lethality to S. marcescens. Subsequently, GmR-kil double selection cassette was inserted into the CDS of pigA gene. Using single-stranded DNA-mediated recombination, this insertion mutation was efficiently repaired through kil counter-selection. A powerful genetic modification platform based on Red recombineering system was successfully established for S. marcescens. Multiple types of modification and multiple recombination strategies can all be performed easily in this species. We hope this study will be useful for the theoretical research and the research of metabolic engineering in S. marcescens.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

CD52/GPI− T-Cells Are Enriched for Alloreactive Specificity and Predict Acute Graft-Versus-Host-Disease After Stem Cell Transplantation

Alemtuzumab is a CD52-specific lympho-depleting antibody. CD52- T cells emerge under alemtuzumab selection pressure. We sought to investigate the phenotype and function of the CD52- T cell fraction and related their presence to clinical outcome. We obtained longitudinal peripheral blood samples from 67 consecutive patients undergoing allo-HSCT between 2013-2016. Forty-seven patients (70%) had a myeloid disease (acute myelogenous leukemia or myelodysplastic syndrome) whereas 20 patients had lymphoid disease. All patients received in vivo alemtuzumab (10 mg/d from day -5 for 5 days) as part of their conditioning protocol. Sixty-three (94%) received reduced-intensity conditioning chemotherapy, whereas 4 (6%) received a myeloablative regimen. All patients received post-transplantation cyclosporine A for graft-versus-host disease (GVHD) prophylaxis. Six (9%) also received methotrexate, whereas 2 (3%) patients also received mycophenolate mofetil. Overall survival at 2 years was 68%, and relapse-free survival was 48%. Twenty-none percent of patients experienced acute GVHD (grade 2 or above), and 15% developed chronic GVHD. CD52- T cells were detectable in 66 of 67 consecutive patients. CD52- T cells demonstrated low binding of fluorescent aerolysin, indicating downregulation of the glycophosphatidylinositol anchor, although we did not detect any mutations in the PIG-A gene as is typically seen in patients with paroxysmal nocturnal hemoglobinuria. CD52- T cells were almost exclusively CD4+ and exhibited a dominant memory phenotype with only small numbers of CD25+ CD127low Foxp3+ regulatory T cells. CD52- T cells exhibited alloreactive specificity in vitro and have a distinct TCR repertoire to CD52+ T cells. Early after allo-hematopoietic stem cell transplantation, the presence of a significant population of CD52- T cells (comprising >51% of the T cell fraction) was found to be an independent risk factor for acute GvHD. This was confirmed in a validation cohort of 28 patients obtained between 2017-2018. These data suggest that the CD52- T cell fraction may represent a residual "footprint" of an early CD4+ T cell alloreactive response and may have been rescued from alemtuzumab-mediated lysis by antigen engagement in vivo. These data help to delineate the nature of T cell escape from alemtuzumab surveillance and contribute to increasing interest in the importance of CD4+ T cells in alloreactive immune responses, which could help inform immunotherapy protocols.

Limb-clasping, cognitive deficit and increased vulnerability to kainic acid-induced seizures in neuronal glycosylphosphatidylinositol deficiency mouse models.

Posttranslational modification of a protein with glycosylphosphatidylinositol (GPI) is a conserved mechanism exists in all eukaryotes. Thus far, >150 human GPI-anchored proteins have been discovered and ~30 enzymes have been reported to be involved in the biosynthesis and maturation of mammalian GPI. Phosphatidylinositol glycan biosynthesis class A protein (PIGA) catalyzes the very first step of GPI anchor biosynthesis. Patients carrying a mutation of the PIGA gene usually suffer from inherited glycosylphosphatidylinositol deficiency (IGD) with intractable epilepsy and intellectual developmental disorder. We generated three mouse models with PIGA deficits specifically in telencephalon excitatory neurons (Ex-M-cko), inhibitory neurons (In-M-cko) or thalamic neurons (Th-H-cko), respectively. Both Ex-M-cko and In-M-cko mice showed impaired long-term fear memory and were more susceptible to kainic acid-induced seizures. In addition, In-M-cko demonstrated a severe limb-clasping phenotype. Hippocampal synapse changes were observed in Ex-M-cko mice. Our Piga conditional knockout mouse models provide powerful tools to understand the cell-type specific mechanisms underlying inherited GPI deficiency and to test different therapeutic modalities.

Application of counter-selectable marker PIGA in engineering designer deletion cell lines and characterization of CRISPR deletion efficiency.

The CRISPR/Cas9 system is a technology for genome engineering, which has been applied to indel mutations in genes as well as targeted gene deletion and replacement. Here, we describe paired gRNA deletions along the PIGA locus on the human X chromosome ranging from 17 kb to 2 Mb. We found no compelling linear correlation between deletion size and the deletion efficiency, and there is no substantial impact of topologically associating domains on deletion frequency. Using this precise deletion technique, we have engineered a series of designer deletion cell lines, including one with deletions of two X-chromosomal counterselectable (negative selection) markers, PIGA and HPRT1, and additional cell lines bearing each individual deletion. PIGA encodes a component of the glycosylphosphatidylinositol (GPI) anchor biosynthetic apparatus. The PIGA gene counterselectable marker has unique features, including existing single cell level assays for both function and loss of function of PIGA and the existence of a potent counterselectable agent, proaerolysin, which we use routinely for selection against cells expressing PIGA. These designer cell lines may serve as a general platform with multiple selection markers and may be particularly useful for large scale genome engineering projects such as Genome Project-Write (GP-write).

Novel therapeutics for paroxysmal nocturnal hemoglobinuria

Following the reports of paroxysmal nocturnal hemoglobinuria (PNH) due to PIGT and PIGB gene mutations, the definition of PNH was changed to a hematopoietic stem cell disease with complement-mediated intravascular hemolysis as a result of clonal expansion of hematopoietic stem cells with mutations in genes involved in glycosylphosphatidylinositol (GPI) anchor synthesis, including PIGA. Treatment with eculizumab (Soliris®), a humanized anti-C5 monoclonal antibody, significantly improved the quality of life in patients with PNH, remarkably reduced hemolysis, improved symptoms associated with hemolysis, and prevented thrombosis. Although the administration interval has been extended and convenience has been greatly improved using the technique of recycling antibodies (ravulizumab and crovalimab), extravascular hemolysis has become another issue. Although attempts have been made to overcome this issue with proximal complement (C3, factor D, and factor B) inhibitors, the optimal therapeutic agent is expected to be selected after evaluating for not only efficacy and safety but also convenience.

Advances in research for pathogenesis of paroxysmal nocturnal hemoglobinuria

Paroxysmal Nocturnal hemoglobinuria, PNH is usually caused by the somatic mutation of X-linked PIGA gene followed by the clonal expansion of the GPI (glycosylphosphatidylinositol) anchor defective hematopoietic stem cell clone. There are two hypotheses for the mechanism of clonal expansion, one is selection theory, in which GPI deficient cells escape from attacks of cytotoxic cells, and another is benign tumor theory in which GPI deficient cells get the additional mutations and acquire proliferative nature. Recently, we identified two types of PNH patients caused by the biallelic mutation of PIGT on chromosome 20 and PIGB on chromosome 15. Both PNH clones had the germ-line mutation in one allele and another allele was somatically mutated in a hematopoietic stem cell. Both somatic mutations were loss of heterozygosity (LOH), deletion in PIGT-PNH and copy neutral LOH (CN-LOH) in PIGB-PNH. These PNH patients had typical PNH symptoms, but they have in addition auto-inflammatory features. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. Similarly, in PIGB-PNH cells, GPI intermediates are accumulated and expressed on the cell surface. Those GPIs together with complement activation cause the inflammasome activation.

Benchmark dose analysis of multiple genotoxicity endpoints in gpt delta mice exposed to aristolochic acid I.

As the carcinogenic risk of herbs containing aristolochic acids (AAs) is a global health issue, quantitative evaluation of toxicity is needed for the regulatory decision-making and risk assessment of AAs. In this study, we selected AA I (AAI), the most abundant and representative compound in AAs, to treat transgenic gpt delta mice at six gradient doses ranging from 0.125 to 4 mg/kg/day for 28 days. AAI-DNA adduct frequencies and gpt gene mutation frequencies (MFs) in the kidney, as well as Pig-a gene MFs and micronucleated reticulocytes (MN-RETs) frequencies in peripheral blood, were monitored. The dose-response (DR) relationship data for these in vivo genotoxicity endpoints were quantitatively evaluated using an advanced benchmark dose (BMD) approach with different critical effect sizes (CESs; i.e., BMD5, BMD10, BMD50 and BMD100). The results showed that the AAI-DNA adduct frequencies, gpt MFs and the MN-RETs presented good DR relationship to the administrated doses, and the corresponding BMDL100 (the lower 90% confidence interval of the BMD100) values were 0.017, 0.509 and 3.9 mg/kg/day, respectively. No positive responses were observed in the Pig-a MFs due to bone marrow suppression caused by AAI. Overall, we quantitatively evaluated the genotoxicity of AAI at low doses for multiple endpoints for the first time. Comparisons of BMD100 values across different endpoints provide a basis for the risk assessment and regulatory decision-making of AAs and are also valuable for understanding the genotoxicity mechanism of AAs.

Ultradeep Sequencing Analysis of Paroxysmal Nocturnal Hemoglobinuria Clones Detected by Flow Cytometry: PIG Mutation in Small PNH Clones.

We aimed to determine whether small paroxysmal nocturnal hemoglobinuria (PNH) clones detected by flow cytometry (FCM) harbor PIG gene mutations with quantitative correlation. We analyzed 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM. We performed ultradeep sequencing for the PIGA, PIGM, PIGT, and PIGX genes in these specimens. A strong positive correlation between PNH clone size by FCM and variant allele frequency (VAF) of PIG gene mutation was identified (RBCs: r = 0.77, P < .001; granulocytes: r = 0.68, P < .001). Granulocyte clone size of 2.5% or greater and RBCs 0.4% or greater by FCM always harbored PIG gene mutations. Meanwhile, in patients with clone sizes of less than 2.5% in granulocytes or less than 0.4% in RBCs, PIG gene mutations were present in only 15.9% and 12.2% of cases, respectively. In addition, there was not a statistically significant positive correlation between FCM clone size and VAF or the presence or absence of a PIG mutation. Our results showed that in small PNH clones PIG gene mutations were present in only a small portion without significant correlation to VAF or the presence or absence of a PIG mutation.

In vitro genotoxicity study of silver nanoparticles and titanium dioxide nanoparticles.

Nanoparticles are widely used in cosmetic, pharmaceutical, and food industries, but their safety and genetic toxicity are still unclear. In this study, the genotoxicity of silver nanoparticles (AgNPs) and titanium dioxide nanoparticles (titanium dioxide nanoparticles) were evaluated by in vitro comet assay and PIG-A assay in TK6 cells. We exposed TK6 cells to two types of nanoparticles at the highest concentration of 200 μmol/L for 4 h and conducted the in vitro comet assay. We examined the mutation results of PIG-A gene in vitro after 4 h, 24 ho and 10 days of exposure, respectively. We also examined the endocytosis of nanoparticles in TK6 cells exposed to nanoparticles for 24 h. In the endocytosis assay, with the increase of nano-material concentration, the side scatter (SSC) of TK6 cells in flow cytometry showed a concentration-dependent and time-dependent increase, indicating that TK6 cells could uptake both types of nanoparticles. In the comet assay, AgNPs could induce a concentration-dependent increase in DNA tail intensity. However, titanium dioxide NPs could not induce the concentration-dependent increase of DNA fluorescence intensity of comet tail. In the PIG-A assay, both AgNPs and TiO2NPs did not induce PIG-A gene mutation frequency in TK6 cells. The results showed that AgNPs could induce DNA damage in TK6 cells, but could not induce increase of PIG-A gene mutation frequency. TiO2NPs neither induce DNA damage in TK6 cells nor increase PIG-A mutation frequency. Further tests are needed to determine whether TiO2NPs are genotoxic.

Paroxysmal nocturnal hemoglobinuria caused by CN-LOH of constitutional PIGB mutation and 70-kbp microdeletion on 15q

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell (HSC) disorder characterized by defective synthesis of the glycosylphosphatidylinositol (GPI) anchors as a result of somatic mutations in the X-linked PIGA gene. The disease is acquired. No constitutional PNH has been described. Here, we report familial PNH associated with unusual inflammatory symptoms. Genetic analysis revealed a germline heterozygous PIGB mutation on chromosome 15 without mutations in PIGA or any of the other genes involved in GPI biosynthesis. In vitro data confirmed that transfection of the mutant PIGB could not restore the surface expression of GPI-anchored proteins (APs) in PIGB-deficient Chinese hamster ovary cells. Homozygosity was caused by copy number–neutral loss of heterozygosity (CN-LOH) of the germline PIGB mutation, leading to deficient expression of GPI-APs in the affected blood cells of the index patient and her mother. The somatic event leading to homozygosity of the germline mutant PIGB gene involved a 70-kbp microdeletion of chromosome 15q containing the TM2D3 and TARSL2 genes, which was implicated in chromosome 15q mosaicism. Interestingly, we detected the deletion in both the patient and her mother. A sister of the mother, who carried the same germline PIGB mutation but without this microdeletion involving TM2D3 and TARSL2, did not have a PNH clone or CN-LOH. In conclusion, we describe PNH caused by CN-LOH of a germline heterozygous PIGB mutation in a patient and her mother and hypothesize that the 70-kbp microdeletion may have contributed to the PNH clone in both.