Mutations In CD59 Research Articles

Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system

BackgroundHomozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59 leading to loss of function have been described and, overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the localization and possible role of membrane-bound complement regulators, including CD59, in the peripheral nervous systems (PNS) of mice and humans.MethodsWe examined the localization of membrane-bound complement regulators in the peripheral nerves of healthy humans and a CD59-deficient patient, as well as in wild-type (WT) and CD59a-deficient mice. Cross sections of teased sciatic nerves and myelinating dorsal root ganglia (DRG) neuron/Schwann cell cultures were examined by confocal and electron microscopy.ResultsWe demonstrate that CD59a-deficient mice display normal peripheral nerve morphology but develop myelin abnormalities in older age. They normally express myelin protein zero (P0), ankyrin G (AnkG), Caspr, dystroglycan, and neurofascin. Immunolabeling of WT nerves using antibodies to CD59 and myelin basic protein (MBP), P0, and AnkG revealed that CD59 was localized along the internode but was absent from the nodes of Ranvier. CD59 was also detected in blood vessels within the nerve. Finally, we show that the nodes of Ranvier lack other complement-membrane regulatory proteins, including CD46, CD55, CD35, and CR1-related gene-y (Crry), rendering this area highly exposed to complement attack.ConclusionThe Nodes of Ranvier lack CD59 and are hence not protected from complement terminal attack. The myelin unit in human PNS is protected by CD59 and CD55, but not by CD46 or CD35. This renders the nodes and myelin in the PNS vulnerable to complement attack and demyelination in autoinflammatory Guillain-Barré syndrome, as seen in CD59 deficiency.

Correction: Absence of pathogenic mutations in CD59 in chronic inflammatory demyelinating polyradiculoneuropathy

[This corrects the article DOI: 10.1371/journal.pone.0212647.].

Absence of pathogenic mutations in CD59 in chronic inflammatory demyelinating polyradiculoneuropathy.

ObjectiveMutations in CD59 cause CIDP-like polyneuropathy in children with inherited chronic hemolysis. We hypothesized that mutations in CD59 might be found in a subset of sporadic CIDP patients.Methods35 patients from two centers, fulfilling the EFNS/PNS diagnostic criteria for CIDP were included. CD59 coding region was amplified by PCR and Sanger sequenced.ResultsOne rare variant was detected in a patient which resulted in a synonymous change and predicted to be neutral. Pathogenic variants were absent in our cohort.InterpretationOur pilot study suggests that mutations in CD59 are absent in adult-onset sporadic CIDP.

Prothrombotic mechanisms in patients with congenital p.Cys89Tyr mutation in CD59

BackgroundThrombosis is the prognostic factor with the greatest effect on survival in patients with paroxysmal nocturnal hemoglobinuria (PNH), who lack dozens of membrane surface proteins. We recently described a primary homozygous Cys89Tyr congenital nonfunctioning CD59 in humans with clinical manifestation in infancy, associated with chronic hemolysis, recurrent strokes, and relapsing peripheral demyelinating neuropathy. Here we investigated hypercoagulability mechanisms characterizing the syndrome. MethodsMembrane attack complex (MAC) deposition (anti-SC5b-9) and free hemoglobin (colorimetric assay) were assessed. Platelet activation was identified (anti-CD61, anti-CD62P), and microparticles (MPs) of 0.5–0.9 μm, were characterized (Annexin V, anti-human GlyA, anti-CD15, anti-CD14, anti-CD61). Platelet-monocyte aggregation was assessed with FlowSight. Findings2/7 patients (29%) with homozygosity for Cys89Tyr and 6/12 (50%) with any of four described CD59 mutations had recurrent strokes. In plasma samples from four patients carrying identical mutations, MAC deposition was increased on RBCs (p < 0.0003), neutrophils (p < 0.009), and platelets (p < 0.0003). Free-plasma hemoglobin levels were abnormally high, up to 100 mg/dl. Patients with CD59 mutation had RBC-derived MP levels 9-fold higher than those in healthy controls (p < 0.01), and 2–2.5 fold higher than PNH patients (p < 0.09). Leukocyte-activated platelet aggregation was increased (p < 0.0062). Loss of CD59 was shown in the endothelium of these patients. InterpretationNonfunctioning CD59 is a major risk factor for stroke and hypercoagulability. Uncontrolled hemolysis causes massive MP release and endothelial heme damage. MAC attack on unprotected endothelium and platelet activation and aggregation with leukocytes mediate additional mechanisms leading to vascular occlusion. It is suggested that CD59 loss represents a major arterial prothrombotic factor in PNH and additional diseases.

A new complement evasion strategy: How S. pyogenes exploits IgGs to recruit more C4BP

Neurological symptoms of patients with p.Cys89Tyr mutation in the CD59 gene include recurrent peripheral neuropathy resembling Guillain-Barré syndrome, characterized by sensory-motor demyelinating neuropathy with secondary axonal damage and moderate enhancement of the nerve roots on spine MRI, together with recurrent strokes and retinal involvement. Three additional mutations in CD59, leading to loss of function, have been described, and overall, 12/12 (100%) of patients with any mutation presented with neurological symptoms; 11/12 (92%) patients presented with recurrent peripheral neuropathy, 6/12 (50%) with recurrent strokes, and 1/12 (8%) with retinal involvement. We review the possible thrombophilic profile associated with the mutations. In these patients, excessive intravascular hemolysis saturates scavenger mechanisms resulting in free hemoglobin in plasma that irreversibly reacts with nitric oxide to form nitrate and methemoglobin, leading to arterial thrombosis. CD59 loss of function is also one of the major thrombophilic mechanisms in patients with paroxysmal nocturnal hemoglobinuria. We then describe the relationship with demyelination. The lack of CD59 allows uncontrolled complement amplification following low-level spontaneous-, viral-, or post viral-induced complement activation, resulting in severe demyelination in the peripheral nervous system. It is interesting, and certainly encouraging, that after 3 years, following 4 patients with Cys89Tyr mutations who are treated with eculizumab, no strokes occurred and non-permanent neurological insults underwent resolution without any new neurological exacerbations.

P.188 - A further case with chronic hemolysis, CIDP and lethal cerebral vasculitis due to a CD59 mutation

P.188 - A further case with chronic hemolysis, CIDP and lethal cerebral vasculitis due to a CD59 mutation

CD59 Deficiency Is Associated with Chronic Hemolysis and Childhood Chronic Immune Mediated Neuropathy

Abstract 3187CD59 deficiency is a common finding in red and white blood cells in patients with chronic hemolysis suffering from paroxysmal nocturnal hemoglobinuria (PNH) where acquired mutation in the PIGA gene leads to membrane loss of GPI-anchored membrane proteins, including CD59. The current study objective was the elucidation of the molecular basis of childhood familial chronic Coombs' negative hemolysis and relapsing polyneuropathy, mimicking chronic inflammatory demyelinating polyradiculoneuropathy in infants of North-African Jewish origin from four unrelated families. A founder mutation was searched using homozygosity mapping followed by exome sequencing. The expression of CD59, CD55 and CD14 was examined in blood cells by flow cytometry followed by western blot of the CD59 protein. A homozygous missense mutation in CD59, was identified in all the patients. The mutation segregated with the disease in the families and had a carrier rate of 1:66 among Jews of North-African origin. The mutated protein was present in the patients' cells in normal amount but was undetectable on the membrane surface.Conclusion: This is the first clinical and molecular characterization of a novel clinical syndrome. CD59 mutation is associated with a failure of proper localization of CD59 protein in the cell surface. This is clinically manifested in infancy by chronic hemolysis and relapsing peripheral demyelinating disease. Disclosures:No relevant conflicts of interest to declare.

Cellular ATP content was decreased by a homogeneous 8.5 T static magnetic field exposure: Role of reactive oxygen species

The literature on the impact of strong static magnetic fields (SMF) on human health is vast and contradictory. The present study focused on the cellular effects of strong homogeneous SMF in human-hamster hybrid (A(L) ) cells, mitochondria-deficient (ρ(0) A(L) ) cells, and double-strand break (DSB) repair-deficient (XRS-5) cells. Adenosine triphosphate (ATP) content was significantly decreased in A(L) cells exposed to 8.5 Tesla (T) but not 1 or 4 T SMF for either 3 or 5 h. In addition, ATP content significantly decreased in the two deficient cell lines exposed to 8.5 T SMF for 3 h. With further incubation of 12 or 24 h without SMF exposure, ATP content could retrieve to the control level in the A(L) cells but not ρ(0) A(L) and XRS-5 cells. Under a fluorescence reader, the levels of reactive oxygen species (ROS) in the three cell lines were significantly increased by exposure to 8.5 T SMF for 3 h. Concurrent treatment with ROS inhibitor, DMSO, dramatically suppressed the ATP content in exposed A(L) cells. However, the CD59 mutation frequency and the cell cycle distribution were not significantly affected by exposure to 8.5 T SMF for 3 h. Our results indicated that the cellular ATP content was reduced by 8.5 T SMF for 3 h exposure, which was partially mediated by mitochondria and the DNA DSB repair process. Moreover, ROS were involved in the process of the cellular perturbations from the SMF.

EMS mutant spectra generated by multi-parameter flow cytometry

The CHO A L cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59 − regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59 − clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.

Mutant spectra of irradiated CHO A L cells determined with multiple markers analyzed by flow cytometry

We have previously developed a sensitive and rapid mammalian cell mutation assay which is based on a Chinese hamster ovary cell line that stably incorporates human chromosome 11 (CHO A L) and uses flow cytometry to measure mutations in CD59. We now show that multiparameter flow cytometry may be used to simultaneously analyze irradiated CHO A L cells for mutations in five CD genes along chromosome 11 ( CD59, CD44, CD90, CD98, CD151) and also a GPI-anchor gene. Using this approach, 19 different mutant clones derived from individual sorted mutant cells were analyzed to determine the mutant spectrum induced by ionizing radiation. All clones analyzed were negative for CD59 expression and PCR confirmed that at least CD59 exon 4 was also absent. As expected, ionizing radiation frequently caused large deletions along chromosome 11. This technology can readily be used to rapidly analyze the mutant yield as well as the spectrum of mutations caused by a variety of genotoxic agents and provide greater insight into the mechanisms of mutagenesis.

High frequency of several PIG-A mutations in patients with aplastic anemia and myelodysplastic syndrome

To clarify some characteristics of phosphatidylinositol glycan-class A gene (PIG-A) mutations in aplastic anemia (AA) and myelodysplastic syndrome (MDS) patients compared with those in paroxysmal nocturnal hemoglobinuria (PNH) patients, we investigated PIG-A mutations in CD59- granulocytes and CD48- monocytes from seven AA, eight MDS, and 11 PNH Japanese patients. The most frequent base or type abnormalities of the PIG-A gene in AA and MDS patients were base substitutions or missense mutations, respectively, and deletions or frameshift mutations, respectively, in PNH patients. Several PIG-A mutations, most of which were statistically minor, were found in glycosylphosphatidylinositol-negative cells from all AA and MDS patients but not from all PNH patients. However, the common PIG-A mutations during the clinical course between CD59- granulocytes and/or CD48- monocytes from each AA or MDS patient, except for Case 5, were not found. PIG-A mutations were different between the granulocytes and monocytes from five AA and five MDS patients. Our results indicate that there were some characteristics of PIG-A mutations in AA and MDS patients compared with PNH patients and that several minor PNH clones in these patients occurred at random during the clinical course. This partly explains the transformation of AA or MDS to PNH at intervals.

Mutagenic effect of a keV range N + beam on mammalian cells

The radiobiological effects of a keV (5–20 keV) range nitrogen ion (N +) beam on mammalian cells were studied, particularly with regard to the induction of mutation in the cell genome. The experiment demonstrated that the 20 keV N + beam, which resulted in cell death to a certain extent, induced a 2–3 fold increase in the mutation rates at the CD59 gene locus of the mammalian A L cells as compared to the control. Within certain fluence ranges (0–6 × 10 14 N +/cm 2), the cell survival displayed a down–up–down pattern which is similar to the phenomenon known as ‘hyper-radiosensitivity’ manifested under low-dose irradiation; the CD59 mutation rate firstly showed a gradual rise up to a 3-fold increment above the background level as the ion fluence went up to 4 × 10 14 N +/cm 2, after this peak point however, a downtrend appeared though the ion fluence increased further. It was also observed that the fraction of CD59 mutation bears no proportional relation to ion energy in further experiments of mutation induction by N + beams with the incident energies of 5, 10, 15 and 20 keV at the same fluence of 3 × 10 14 N +/cm 2. Analyses of the deletion patterns of chromosome 11 in CD59 − mutants induced by 5–20 keV N + beams showed that these ions did not result in large-size chromosome deletions in this mammalian cell system. A preliminary discussion, suggesting that the mutagenic effect of such low-energy ion influx on mammalian cells could result from multiple processes involving direct collision of particles with cellular DNA, and cascade atomic and molecular reactions due to plentiful primary and secondary particles, was also presented. The study provided the first glimpse into the roles low-energy ions may play in inducing mutagenesis in mammalian cells, and results will be of much value in helping people to understand the contribution of low-energy ions to radiological effects of various ionising radiations.