先天性グリコシルホスファチジルイノシトール（ＧＰＩ）欠損症

Thirty genes are involved in the biosynthesis and modification of glycosylphosphatidylinositol (GPI)-anchored proteins. Defects in these genes cause inherited GPI deficiency, whose main clinical features include intellectual disability, developmental delay, and intractable seizures, and sometimes hyperphosphatasia and multiple congenital anomalies. The mechanisms of its systemic, especially neurological, manifestations largely remain unclear, and no fundamental therapy has yet been established. In this study, we report the development of effective adeno-associated virus (AAV)-mediated gene therapy using a mouse model of inherited GPI deficiency caused by phosphatidylinositol glycan anchor biosynthesis class O (PIGO) mutations, with the aim of developing gene therapy applicable to human patients. By optimizing AAV vectors with the most effective and safest promoter, we achieved significant amelioration of neuronal phenotype and growth impairment, with no cases of liver cancer observed. We further determined the optimal administration route and therapeutic dose required for effective systemic delivery. These results provide proof of concept for the therapeutic efficacy of AAV-based gene replacement therapy for inherited GPI deficiency.

OBJECTIVES: Inherited glycosylphosphatidylinositol (GPI) deficiencies (IGDs) impair the expression of GPI-anchored proteins and produce diverse clinical phenotypes that complicate diagnosis. Since we had previously identified CD16b as a useful diagnostic biomarker, we aimed to establish a standardized flow cytometric assay suitable for routine clinical application. METHODS: We analyzed granulocyte expression of CD16b in 29 IGD patients and 101 controls (21 non-IGD and 80 healthy adults). Granulocyte CD16b expression was quantified as geometric mean fluorescence intensity (gMFI) by flow cytometry in our laboratory and a commercial testing company. Receiver operating characteristic (ROC) analysis was used to define diagnostic cut-offs. RESULTS: CD16b on granulocytes showed the largest decrease (approximately 85%) among GPI-anchored proteins in IGD patients. Mean gMFI was 41,660 ± 36,286 in IGD versus 88,370 ± 24,856 in healthy controls (p < 0.001). ROC analysis yielded an optimal cut-off of 53,431 (sensitivity 0.73, specificity 0.96, area under the curve (AUC) 0.84, Youden index 0.69). We defined 40,000 as the definitive diagnostic threshold and 60,000 as the borderline value. These cut-offs were validated with the commercial assay, which provides results within 2-3 days and is reimbursed by Japan's national health insurance. CONCLUSIONS: Measurement of granulocyte CD16b provides a reliable and rapid screening method for IGD that complements genetic testing. Implementation of this assay by a commercial provider, together with validated cutoffs, enables nationwide, insurance-covered diagnostic testing and functional confirmation of GPI pathway gene variants, representing a significant advance in IGD diagnosis.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are attached to the cell surface via a glycolipid anchor, GPI, whose conserved core is synthesized from phosphatidylinositol (PI) in the endoplasmic reticulum through a series of enzymatic reactions. Most PI species in mammalian cells contain diacylglycerol, whereas GPI-APs predominantly possess 1-alkyl-2-acylglycerol. The basis for this characteristic lipid structure has remained unclear. Lipidomic analysis revealed that 1-alkyl-2-acyl PIs, although minor components of cellular PI, are preferentially used by GPI-N-acetylglucosaminyltransferase, which catalyzes the first step of GPI biosynthesis. GPI intermediates containing 1-alkyl-2-acylglycerol were further enriched in subsequent biosynthetic steps, resulting in mature GPIs primarily harboring this lipid species. We demonstrate that a 1-alkyl-containing precursor lipid derived from peroxisomes, likely 1-alkyl-glyceronephosphate, contributes to the formation of 1-alkyl-2-acyl PIs. Disruption of glyceronephosphate O-acyltransferase (GNPAT) or alkylglycerone phosphate synthase (AGPS), the first two enzymes of the peroxisomal ether-lipid pathway, abolished 1-alkyl-2-acyl PI, yielding GPI-APs containing only diacylglycerol. Lipidomic profiling of GPI biosynthetic intermediates in GPI-defective cells revealed accumulation of defective-step-specific intermediates, enabling the use of this approach for diagnosing inherited GPI deficiency (IGD).

Cefiderocol, a novel β-lactam antibiotic, exhibits potent activity against carbapenemase-producing Enterobacterales (CPE). While its clinical efficacy has been reported for infections caused by KPC-type CPE and Stenotrophomonas maltophilia, evidence regarding its effectiveness against IMP-type CPE remains primarily derived from in vitro studies, with limited clinical data available. This is the first pediatric case successfully treated with cefiderocol for IMP-type CPE bacteremia. He was a 6-year-old boy with inherited glycosylphosphatidylinositol deficiency and acute lymphoblastic leukemia undergoing chemotherapy. He developed bacteremia that blood culture multiplex PCR identified Klebsiella pneumoniae with IMP gene. The combination therapy of cefiderocol 60mg/kg/dose every 8 hours and gentamicin 5mg/kg/dose once daily sterilized blood culture, and subsequent monotherapy with cefiderocol was continued for a total of 14 days. Additional molecular test in the strain detected IMP-1 carbapenemase, SHV extended spectrum β-lactamase and EBC-type AmpC β-lactamase. Cefiderocol was susceptible at minimum inhibitory concentration 0.5μg/mL. Further study is needed for cefiderocol treatment for IMP-type CPE infection in children.

BACKGROUND: Inherited glycosylphosphatidylinositol (GPI) deficiencies are a heterogeneous group of inherited disorders of glycosylation, caused by mutations in genes involved in GPI-anchored proteins (GPI-AP) biosynthesis. PIGW is a gene known to be involved in the early steps of the GPI-anchor biosynthesis, as well as functional studies for most patients. Biallelic mutations in PIGW have been previously linked to hyperphosphatasia with mental retardation syndrome 5, also known as glycosylphosphatidylinositol biosynthesis defect 11 (GPIBD11). METHODS: We report seven individuals, including two fetuses from six unrelated families. Whole exome sequencing and chromosome analysis were performed, with variant interpretation based on ACMG and AMP guidelines. Magnetic resonance imaging (MRI) was also conducted on some of the patients. Blood samples were collected from patients to analyze GPI-AP expression using flow cytometry on markers like CD16, CD24, and FLAER. Functional analyses were performed using PIGW KO HEK 293 cells generated with CRISPR/Cas9 technology. The cells were transfected with rat Pigw cDNA that contained patient variants. The restoration of GPI-AP expression was measured by flow cytometry. Western blotting was used to assess protein expression. RESULTS: Affected patients exhibited a wide range of clinical features. Some patients presented classic GPIBD11 symptoms like developmental delay, hyperphosphatasia, and intellectual disability. Other patients showed atypical or milder phenotypes. The magnetic resonance imaging scans revealed variable neurological abnormalities in the affected individuals. Whole exome sequencing results identified PIGW mutations in all patients, which confirms the genetic basis of the disorder. Flow cytometry analysis of blood samples from patients P1, P4, and P5 using various markers showed a significant reduction in GPI-AP expression. The CD16 marker decreased to 1.8% in P1 and 21% in P5 compared to controls. CD24 was reduced to 22% in P1 granulocytes. Also, a minor decrease in CD14 on monocytes was observed in P4, as well as a slight reduction in the expression of FLAER in lymphocytes. Functional studies on PIGW-deficient CHO cells and HEK293 cells, using flow cytometry, showed that GPI-AP expression is affected by the PIGW variants. Western blotting showed reduced PIGW protein expression, except for P153L and R36G, which were similar to wild-type levels. CONCLUSIONS: To date, six patients and two fetuses with biallelic variants in PIGW have been reported. Here, we describe five new patients and two fetuses harboring homozygous or compound heterozygous variants in the PIGW gene. Our results illustrate the clinical variability of GPIBD11, highlighting the importance of broad genomic sequencing assays for patients who do not show typical symptoms. Therefore, our study expands the clinical and molecular spectrum of PIGW-associated disorder.