Molecular pathogenesis of human CD59 deficiency

Objective To characterize all 4 mutations described for CD59 congenital deficiency. Methods The 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality. Results Immunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation. Conclusions All 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non–MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis

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

Background: Homozygous 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. Methods: We 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. Results: We 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. Conclusion: The 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

Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells

In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine the role of HBD in the clearance of apoptotic cells, and whether the phagocytic and tolerizing state of DCs is mediated by the HBD itself, or whether the entire TSP-1 is needed. Therefore, we have cloned the human HBD, and compared its interactions with DC to those with TSP-1. Here we show that rHBD by itself is not directly responsible for immune paralysis and tolerizing phenotype of DCs, at least in the monomeric form, but has a significant role in rendering DCs phagocytic. Binding of TSP-1-C-terminal domain on the other hand induces a tolerizing phenotype in dendritic cells

Bi-allelic loss-of-function CACNA1B mutations in progressive epilepsy-dyskinesia

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

The Natural History of Homozygous Cys89Tyr CD59 DefiCiency in Infancy: Differences and Similarities to PNH

Abstract Background. CD59 encodes a 77-amino acid glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein synthesized as a 128-amino acid protein that includes a signal sequence and the sequence for a GPI anchor replacement. The CD59 protein inhibits the final and most important step of membrane attack complex (MAC) formation. A novel primary homozygous Cys89Tyr CD59 deficiency was recently reported by us in 5 children. The mutation resulted in amino acid substitution of p.Cys89Tyr and function failure to of CD59 protein on the cell surface. In this study, we characterized an additional 10 children, and summarized outcomes from a clinical study of eculizumab in these patients (NCT01579838). Methods. Participants were recruited from our new patient registry for homozygosity of the p.Cys89Tyr mutation on CD59. 15 homozygous and 42 heterozygous patients were identified and characterized using clinical history, lab results, medical treatment, hospitalizations, biopsies, and in one case post mortem analysis. 4 patients were recruited for a clinical study where participants received repeated treatment with intravenous eculizumab. In this 24-month open-label phase IIa study we aimed to determine whether eculizumab reduces chronic hemolysis and cumulative doses of steroids and IV IgG, and ameliorates neurological deficits compared to those observed before treatment. Treatment response was evaluated every 2-4 weeks over 34 weeks, including examination with gross motor scoring, ASIA score, INCAT disability score, laboratory examination, and SF-12 fulfillment. Neurological relapses and the cumulative dose of IVIG and/or corticosteroids before and after treatment initiation were documented. RBCs and neutrophils were stained to evaluate C5b-9 deposition. Results. All 15 homozygous patients suffered from chronic Coombs' negative hemolysis with hemoglobin levels of 6.5-12 gr%. All patients had post infectious peripheral neuropathy resembling recurrent Gillian Barre syndrome (GBS) and 2 patients suffered from recurrent strokes. A dramatic and significant neurological amelioration in the upper limbs and trunk, with a more modest amelioration in the lower limbs was observed in all patients. Corticosteroid and immunoglobulin treatment, which had been used extensively prior to eculizumab initiation, was completely stopped. No patient suffered a relapse during the treatment period despite infections, and there were no hospital admissions. The SF-12 health questionnaires indicated significant improvement (p&lt;0.003). Decreased deposition of C3bi and C5b-9 on RBCs and neutrophils was documented (p&lt;0.0001). Conclusions. The natural history of patients with homozygous Cys89Tyr CD59 deficiency includes chronic hemolysis and recurrent peripheral neuropathy resembling GBS. Recurrent strokes were observed in 2/15 patients. Eculizumab was safely given to 4 patients. The clinical improvement suggests this is a lifesaving treatment for patients with primary CD59 deficiency. Cys89Tyr CD59 homozygous deficiency is similar to PNH with regard to chronic hemolysis and the risk for stroke, but differs from recurrent GBS episodes that are eliminated once eculizumab was used. Heterozygous patients seem to have normal life expectancy and no hemolysis or any clinical syndromes. Disclosures Mevorach: Enlivex: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties, Research Funding. </jats:sec