Gen Nakamura

Novel mutations of the GLA gene in Japanese patients with Fabry disease and their functional characterization by active site specific chaperone

Fabry disease is an X‐linked recessive inborn metabolic disorder caused by a deficiency of the lysosomal enzyme α‐galactosidase A (EC 3.2.1.22). The causative mutations are diverse, include both large rearrangements and single‐base substitutions, and are dispersed throughout the 7 exons of the α‐galactosidase A gene (GLA). Mutation hotspots for Fabry disease do not exist. We examined 62 Fabry patients in Japan and found 24 GLA mutations, including 11 novel ones. A potential treatment reported for Fabry disease is active site specific chaperone (ASSC) therapy using 1‐deoxygalactonojirimycin (DGJ), an inhibitor of α‐galactosidase A, at subinhibitory concentrations. We transfected COS‐7 cells with the 24 mutant GLAs and analyzed the α‐galactosidase A activities. We then treated the transfected COS‐7 cells with DGJ and analyzed its effect on the mutant enzyme activities. The activity of 11 missense mutants increased significantly with DGJ. Although ASSC therapy is useful only for misfolding mutants and therefore not applicable to all cases, it may be useful for treating many Japanese patients with Fabry disease.

Naked Plasmid DNA-Based α-Galactosidase A Gene Transfer Partially Reduces Systemic Accumulation of Globotriaosylceramide in Fabry Mice

Fabry disease is an X-linked recessive inborn metabolic disorder in which a deficiency in lysosomal enzyme α-galactosidase A (Gal A) causes the systemic accumulation of globotriaosylceramide (Gb3). Although many investigators have attempted to treat α-Gal A knock-out mice (Fabry mice) with gene therapy, no report has demonstrated therapeutic effects by the retrograde renal vein injection of naked DNA. We recently developed a naked plasmid vector-mediated kidney-targeted gene transfer technique. A solution containing naked plasmid DNA encoding human α-Gal A (pKSCX-α-Gal A) was rapidly injected into the left kidney of Fabry mice (pKSCX-α-Gal A mice). pKSCX was used for mock transfections (pKSCX mice). We confirmed that vector-derived human α-Gal A mRNA was present in the left kidney but not in other tissues, by reverse transcriptase polymerase chain reaction. Compared with the pKSCX mice, the pKSCX-α-Gal A mice showed partial therapeutic effects: increased α-Gal A activity in the injected kidney and in the liver, heart, and plasma, and decreased Gb3 in the injected kidney, contralateral kidney, liver, heart, and spleen. Our results demonstrated that, although further studies are needed to improve the outcome, this method has promise as a potential treatment option for Fabry disease.

Rat kidney-targeted naked plasmid DNA transfer by retrograde injection into the renal vein

Kidney-targeted gene transfer is expected to revolutionize the treatment of renal diseases. Recently, we demonstrated that naked plasmid deoxyribonucleic acid (DNA) can be transferred into renal interstitial fibroblasts near the peritubular capillaries (PTCs) in normal rats, by retrograde injection into the renal vein with the renal vein and artery clamped. The PTC network is a main target of kidney transplant rejection and of progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. We retrogradely injected a lacZ expression plasmid in Ringer’s solution into the renal vein of rats using a 24-gage catheter. We detected lacZ expression exclusively in the interstitial fibroblasts near the PTCs of the kidney by immunoelectron microscopy. Nephrotoxicity from the gene transfer was not apparent. We then used a rat erythropoietin (Epo) expression plasmid vector pCAGGS-Epo in a reporter assay. We obtained maximal Epo expression when the DNA solution was injected within 5 s in a volume of 1.0 mL. We detected transgene-derived Epo messenger ribonucleic acid by reverse transcriptase polymerase chain reaction only in the kidneys receiving pCAGGS-Epo. In this article, protocols for naked plasmid DNA transfer into rat kidney using this hydrodynamics-based transfection method and the immunoelectron microscopic technique to determine the lacZ gene transfer site are described in detail.

EFFECT OF CHARCOAL HEMOPERFUSION FOR REMOVAL OF PLASMA METHOTREXATE IN A PATIENT WITH ACUTE RENAL FAILURE

Strategies effective for accelerating methotrexate removal in delayed methotrexate excretion have not been universally accepted. The authors report a case of a 12-year-old girl with osteosarcoma who developed acute renal failure immediately after the first administration of high-dose methotrexate. Plasma methotrexate was effectively removed with repeated charcoal hemoperfusion in addition to plasma exchange and leucovorin rescue. Charcoal hemoperfusion was most effective for reducing plasma methotrexate with approximately 50% of methotrexate being reduced during each of the procedures. No rebound increase in MTX levels was observed. The patient received further therapy with other cancer drugs and has been well for 3.5 years.

Magnetic resonance imaging of bone marrow for TAFRO syndrome

Abstract We report two cases of TAFRO syndrome, which is characterized by thrombocytopenia, anasarca, fever, renal insufficiency, and organomegaly. Magnetic resonance imaging (MRI) of the spine showed a dark medullary pattern in the bone marrow on the T1- and T2-weighted images of both patients. One patient showed complete resolution after treatment. Serial MRIs of the improved patient revealed a transition to a normal marrow pattern on both images, which might represent resolution of the disease.

Giant cell arteritis presenting with ANCA-associated glomerulonephritis

A 71-year-old woman was admitted to our hospital complaining of fever, headache, and stiffness affecting the neck and shoulders for 2 weeks. Laboratory data were as follows: white blood cell count 11,700/μL, C-reactive protein 19.3 mg/dL, erythrocyte sedimentation rate 102 mm, creatinine 2.04 mg/dL, and myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (ANCA) 120 U/mL (normal level, < 3.5 U/mL). Because induration of the temporal artery was palpable, temporal artery biopsy was performed. Inflammatory cell infiltration was identified in the vascular wall (Fig. 1a). Necrotizing arteritis was found in the branches, accompanied by giant cells (Fig. 1b). A diagnosis of giant cell arteritis was made. Renal biopsy demonstrated necrotizing crescentic glomerulonephritis (Fig. 2a, b). In addition, venulitis with perivascular inflammatory infiltrates and giant cells (Fig. 2c), and necrotizing vasculitis in small arteries were observed (Fig. 2d). Therefore, this vasculitis appeared to be associated with MPO-ANCA [1]. Oral prednisolone improved her renal function and symptoms.

Fabry disease associated with chronic meningitis and cerebral infarction

A 48‐year‐old man receiving enzyme replacement therapy for Fabry disease was admitted to Niigata Prefectural Shibata Hospital, Shibata, Japan, for a fever, headache, dysarthria and right hemiparesis. Aseptic meningitis and lacunar infarction were diagnosed, and symptomatic therapy and antiplatelet therapy improved the symptoms. As he had had a sub‐ever and headache once a month for 1 year, and developed a fever and headache again 2 months after discharge from our hospital, chronic meningitis was diagnosed. Prednisolone was introduced, and there was no relapse of meningitis thereafter. It is hypothesized that glycosphingolipids accumulated at the leptomeninges could induce aseptic meningitis and result in a shift to chronic meningitis. Chronic meningitis is implicated as the cause of a chronic headache in Fabry disease, and steroids could be a potential effective therapy.