Effects of switching from agalsidase-α to agalsidase-ß on biomarkers, renal and cardiac parameters, and disease severity in fabry disease forming neutralizing antidrug antibodies: a case report.

Fabry disease is an X-linked hereditary disorder caused by deficient α-galactosidase A (GLA) activity. Patients with Fabry disease are often treated with enzyme replacement therapy (ERT). However, ERT often induces the formation of neutralizing antidrug antibodies (ADAs), which may impair the therapeutic efficacy. Here, we report the case of a 32-year-old man with Fabry disease and resultant neutralizing ADAs who was treated by switching from agalsidase-α to agalsidase-ß. We monitored biomarkers, such as plasma globotriaosylsphingosine (lyso-Gb3), urinary globotriaosylceramide (Gb3), urinary mulberry bodies, renal and cardiac parameters, and disease severity during the treatment period. Although plasma lyso-Gb3 and urinary Gb3 levels quickly decreased within two months after the initiation of ERT with agalsidase-α, they gradually increased thereafter. The urinary mulberry bodies continued to appear. Both the ADA titer and serum mediated GLA inhibition rates started to increase after two months. Moreover, 3.5 years after ERT, the vacuolated podocyte area in the renal biopsy decreased slightly from 23.1 to 18.9%. However, plasma lyso-Gb3 levels increased, and urinary Gb3, mulberry body levels, and ADA titers remained high. Therefore, we switched to agalsidase-ß which reduced, but did not normalize, plasma lyso-Gb3 levels and stabilized renal and cardiac parameters. Disease severity was attenuated. However, urinary Gb3 and mulberry body levels did not decrease noticeably in the presence of high ADA titers. The kidneys take up a small amount of the administered recombinant enzyme, and the clearance of Gb3 that has accumulated in the kidney may be limited despite the switching from agalsidase-α to agalsidase-ß.

In Vivo Delivery of Therapeutic Molecules by Transplantation of Genome-Edited Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) have already been used in transplantation therapies. Currently, cells from healthy people are transplanted into patients with diseases. With the rapid evolution of genome editing technology, genetic modification could be applied to enhance the therapeutic effects of iPSCs, such as the introduction of secreted molecules to make the cells a drug delivery system. Here, we addressed this possibility by utilizing a Fabry disease mouse model, as a proof of concept. Fabry disease is caused by the lack of α-galactosidase A (GLA). We previously developed an immunotolerant therapeutic molecule, modified α-N-acetylgalactosaminidase (mNAGA). We confirmed that secreted mNAGA from genome-edited iPSCs compensated for the GLA activity in GLA-deficient cells using an in vitro co-culture system. Moreover, iPSCs transplanted into Fabry model mice secreted mNAGA and supplied GLA activity to the liver. This study demonstrates the great potential of genome-edited iPSCs secreting therapeutic molecules.

Monitoring of anti-drug antibodies and disease-specific biomarkers in three patients from a Japanese Fabry family treated with enzyme replacement therapy.

We monitored anti-drug antibodies and disease-specific biomarkers in three patients with a nonsense mutation from a Japanese Fabry family treated with enzyme replacement therapy (ERT). In two male patients from the family, neutralizing anti-drug antibodies were induced at an early stage of ERT, the antibody titer peak being found at an earlier stage of ERT in the patient treated with 1.0 mg/kg agalsidase beta than in that treated with 0.2 mg/kg agalsidase alfa. Then, the antibody titers decreased with continuation of ERT. The formation of neutralizing anti-drug antibodies adversely affected the plasma globotriaosylsphingosine (Lyso-Gb3) level and urinary globotriaosylceramide (Gb3) excretion in both patients, the impact being greater in the patient treated with 0.2 mg/kg agalsidase alfa than in that treated with 1.0 mg/kg agalsidase beta. The difference might be explained by the different doses of the infused enzymes based on supersaturation of the antibodies. In a heterozygous Fabry female from the family, no sign of antibody formation was found, and both the plasma Lyso-Gb3 level and urinary Gb3 excretion, which were moderately increased at the baseline, decreased gradually. No deterioration of the manifestations or laboratory findings was observed during ERT in either of the patients. Thus, monitoring of anti-drug antibodies and biomarkers in these Fabry patients provided us with important information on their pathological condition during ERT.

Fabry disease associated with multiple myeloma: a case report.

Fabry disease (FD) is an X-linked genetic lysosomal disorder caused by alpha-galactosidase A (GLA) deficiency. Multiple myeloma (MM) predominately affects older adults, which ranks as the second commonest hematological malignancy. Their overlap has rarely been reported. We present a case of the coexistence of FD and MM in a patient. We report the case of a 68-year-old woman who was referred to our hospital for the evaluation of thoracic spine tumor with bone destruction. On admission, her serum creatinine (Cr) level was elevated to 12.70 mg/dL from the baseline value of 0.91 mg/dL. Bone marrow aspiration revealed MM. Renal biopsy showed myeloma cast nephropathy, which was the primary cause of acute kidney injury. Renal pathology also showed podocyte swelling and tubule myeloid bodies in a mosaic pattern compatible with female FD. Consequently, the patient was diagnosed as FD based on the germ line mutation in GLA. The patient was treated with bortezomib and dexamethasone therapy, which significantly improved the renal function. This is the second case demonstrating a potential pathogenic relationship between FD and MM. Since FD is one of the few genetic diseases for which there are therapeutic agents with fewer side effects, diagnostic value of FD is high. If an MM patient has multiple organ abnormalities or any familial history, the physician should suspect FD.

Genetically Modified Cell Transplantation Through Macroencapsulated Spheroids with Scaffolds to Treat Fabry Disease.

Cell transplantation is expected to be another strategy to treat lysosomal diseases, having several advantages compared to enzyme replacement therapy, such as continuous enzyme secretion and one-time treatment to cure diseases. However, cell transplantation for lysosomal diseases holds issues to be resolved for the clinical field. In this study, we developed a new ex vivo gene therapy platform using a transplant pack, which consists of a porous membrane made of ethylene-vinyl alcohol in the pack-type and spheroids with scaffolds. These membranes have countless pores of less than 0.1 µm2 capable of secreting proteins, including alpha-galactosidase enzyme, and segregating the contents from the host immune system. When the packs were subcutaneously transplanted into the backs of green fluorescent protein (GFP) mice, no GFP-positive cells migrated to the transplanted pack in either autogenic or allogenic mice. The transplanted cells in the pack survived for 28 days after transplantation. When cells overexpressing alpha-galactosidase were used as donor cells for the packs and implanted into Fabry disease model mice, the accumulation of the alpha-galactosidase enzyme was also observed in the livers. In this study, we reported a new ex vivo therapeutic strategy combining macroencapsulation and cellular spheroids with scaffolds. This pack, macroencapsulated spheroids with scaffolds, can also be applied to other types of lysosomal diseases by modifying genes of interest.

Screening of Fabry disease in patients with chronic kidney disease in Japan.

BACKGROUND: Fabry disease (FD), an X-linked lysosomal storage disorder caused by a deficiency in alfa-galactosidase A (α-Gal A) activity due to mutations in the GLA gene, has a prevalence of 0-1.69% in patients undergoing haemodialysis; however, its prevalence in patients with chronic kidney disease (CKD) Stages 1-5 is unknown. METHODS: Serum α-Gal A activity analysis and direct sequencing of GLA were used to screen for FD in 2122 male patients with CKD, including 1703 patients with CKD Stage 5D and 419 with CKD Stages 1-5. The correlation between serum α-Gal A activity and confounding factors in patients with CKD Stages 1-5 was evaluated. RESULTS: FD prevalence rates in patients with CKD Stage 5D and CKD Stages 1-5 were 0.06% (1/1703) and 0.48% (2/419), respectively. A patient with CKD Stage 5D exhibited a novel GLA mutation, p.Met208Arg, whereas two patients with CKD Stages 1-5 had c.370delG and p.Met296Ile. p. Met208Arg caused moderate structural changes in the molecular surface region near the substituted amino acid residue but did not affect the catalytic residues Asp170 and Asp231 in α-Gal A. Serum α-Gal A activity in patients with CKD Stages 1-5 was inversely correlated with age (P < 0.0001) but directly correlated with estimated glomerular filtration rate (P < 0.0001). CONCLUSIONS: FD prevalence was much higher in male patients with CKD Stages 1-5 than in those with CKD Stage 5D. FD screening in patients with CKD Stages 1-5 may improve patient survival, decreasing the number of patients with CKD Stage 5D.

[Glial cells and pharmacological targets in Sandhoff disease].

Sandhoff disease (SD) is a genetic disorder caused by a mutation in the ß-hexosaminidase B (HexB) gene in humans. This results in the massive accumulation of GM2 gangliosides in the nervous system, causing progressive neurodegeneration. The symptoms of SD include muscle weakness, seizures, and mental illness;along with loss of muscle coordination, vision, and hearing. In the most severe form, the onset begins during early infancy, and death usually occurs within 3-5 years of age. The established animal model, Hexb-deficient (Hexb-/-) mouse, shows abnormalities that resemble the severe phenotype found in human infants. We have previously reported that activated microglia causes astrogliosis in Hexb-/- mouse at the early stage of development that can be ameliorated via immunosuppression. Moreover, within the cerebral cortices of Hexb-/- mouse, reactive astrocytes were found to express adenosine A2A receptors in later inflammatory phases. Inhibiting this receptor with istradefylline decreases the number of activated microglial cells and inflammatory cytokines/chemokines. Thus, we underline the importance of the astrocytic A2A receptor as a sensor, in regulating microglial activation in the late phase of inflammation.

A cDNA analysis disclosed the discordance of genotype-phenotype correlation in a patient with attenuated MPS II and a 76-base deletion in the gene for iduronate-2-sulfatase.

We previously showed that the genotype-phenotype correlation in MPS II is well-conserved in Japan (Kosuga et al., 2016). Almost all of our patients with attenuated MPS II have missense variants, which is expected to result in residual activity of iduronate-2-sulfatase. In contrast, our patients with severe MPS II have so-called null-type disease-associated variants, such as nonsense variants, frame-shifts, gene insertions, gene deletions and rearrangement with pseudogene (IDS2), none of which are expected to result in residual activity. However, we recently encountered a patient with attenuated MPS II who had a presumable null-type disease-associated variant and 76-base deletion located in exon 1 that extended into intron 1. To investigate this discordance, we extracted RNA from the leukocytes of the patient and performed reverse transcription polymerase chain reaction. One of the bands of the cDNA analysis was found to include a nucleotide sequence whose transcript was expected to generate an almost full-length IDS mature peptide lacking only part of its signal peptide as well as only one amino acid at the end of the N-terminus. This suggests that an alternative splicing donor site is generated in exon 1 upstream of the deleted region. Based on these observations, we concluded that the phenotype-genotype discordance in this patient with MPS II was due to the decreased amount of IDS protein induced by the low level of the alternatively spliced mRNA, lacking part of the region coding for the signal peptide but including the region coding almost the full mature IDS protein. The first 25 amino acids at the N-terminus of IDS protein are a signal peptide. The alternative splice transcript has only 13 (1 M-13 L) of those 25 amino acids; 14G-25G are missing, suggesting that the exclusively hydrophobic 1 M-13 L of the signal peptide of IDS might have a crucial role in the signal peptide.

Cell Transplantation Combined with Recombinant Collagen Peptides for the Treatment of Fabry Disease.

Fabry disease is caused by a decrease in or loss of the activity of alpha-galactosidase, which causes its substrates globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3) to accumulate in cells throughout the body. This accumulation results in progressive kidney injury due to glomerulosclerosis and in heart failure due to hypertrophy. Enzyme replacement therapy (ERT) has been used as the standard therapy for Fabry disease, but it causes a significant financial burden, and regular administration is inconvenient for patients. Because of the short half-life of alpha-galactosidase in vivo, therapeutic methods that can supplement or replace ERT are expected to involve continuous release of alpha-galactosidase, even at low doses. Cell transplantation therapy is one of these methods; however, its use has been hindered by the short-term survival of transplanted cells. CellSaic technology, which utilizes cell spheroids that form after cells are seeded simultaneously with a recombinant collagen peptide scaffold called a µ-piece, has been used to improve cell survival upon implantation. In this study, syngeneic murine embryonic fibroblasts were used to generate CellSaic that were transplanted into Fabry mice. These spheroids survived for 28 days in the renal subcapsular space with forming blood vessels. These results indicate CellSaic technology could be a platform to promote cellular graft survival and may facilitate the development of cell transplantation methods for lysosomal diseases.

Effectiveness of immunosuppressive therapy for nephrotic syndrome in a patient with late-onset Fabry disease: a case report and literature review.

BACKGROUND: Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations of the GLA gene, followed by deficiency in α-galactosidase A (α-gal) activity. Nephrotic syndrome, as the renal phenotype of FD, is unusual. Here, we report the rare case of a patient with FD with nephrotic syndrome whose proteinuria disappeared by immunotherapy. CASE PRESENTATION: A 67-year-old Japanese man was admitted to our hospital because of emesis, abdominal pain, and facial edema due to nephrotic syndrome. The patient was diagnosed with focal segmental glomerulosclerosis (FSGS) by renal biopsy before being diagnosed with FD, and immunotherapy was initiated. After treatment, the kidney biopsy results showed typical glycosphingolipid accumulation in the podocytes of this patient. The white blood cell α-gal activity was very low, and genetic analysis revealed a GLA gene variant (M296I), which is known as a late-onset genetic mutation of FD. Immunotherapy (steroids and cyclosporine A) dramatically improved the massive proteinuria. Currently, he has been undergoing enzyme replacement therapy, and his proteinuria has further decreased. There is the possibility that other nephrotic syndromes, such as minimal change nephrotic syndrome or FSGS, may co-exist in this patient. CONCLUSIONS: We experienced the rare case of a FD patient whose nephrotic syndrome disappeared by immunotherapy. These findings suggest that immunosuppressive treatment may be considered if nephrotic syndrome develops, even in patients with FD.

Inhibition of astrocytic adenosine receptor A2A attenuates microglial activation in a mouse model of Sandhoff disease.

Astrocyte-microglia communication influences the onset and progression of central nervous system (CNS) disorders. In this study, we determined how chronic inflammation by activated astrocytes affected and regulated CNS functions in Sandhoff disease (SD), a CNS lysosomal storage disorder. SD triggers intense CNS inflammation such as microglial activation and astrogliosis. It is caused by mutation of the HEXB gene, which reduces ß-hexosaminidase (Hex) enzymatic activity in lysosomes, leading to accumulation of the substrate GM2 ganglioside in neuronal cells. Hexb-/- mice display a phenotype similar to human patients that suffer from chronic inflammation characterized by activation of astrocytes and microglia. In Hexb-/- mice, tremors and loss of muscle coordination begins at ~12 weeks. Interestingly, we found that reactive astrocytes expressed adenosine A2A receptor in the cerebral cortices of Hexb-/- mice at the later inflammatory phase. In cultured astrocytes, expression of A2A receptor could be induced by astrocyte defined medium, and then the activation of the A2A receptor induced ccl2 expression. In Hexb-/- mice, inhibition of the A2A receptor antagonized by istradefylline decreased the number of activated microglial cells and inflammatory cytokines/chemokines at 13 weeks. Thus, the astrocytic A2A receptor is an important sensor that regulates microglial activation in the late phase of inflammation.

Plasma lyso-Gb3: a biomarker for monitoring fabry patients during enzyme replacement therapy.

BACKGROUND: Recently, globotriaosylsphingosine (lyso-Gb3) has attracted interest as a biomarker of Fabry disease. However, little is known regarding its utility for the evaluation of the therapeutic efficacy. METHOD: We measured plasma lyso-Gb3 concentration in Japanese healthy subjects and Fabry patients by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS). We determined the reference interval in Japanese (UMIN000016854), and examined the effect of enzyme replacement therapy (ERT) with recombinant α-galactosidase A (GLA) and the influence of antibodies against the enzyme on the plasma lyso-Gb3 level in Fabry patients (UMIN000017152). RESULTS: The reference interval was determined to be 0.35-0.71 nmol/L, this being almost the same as the normal range in a non-Japanese population previously reported. The analysis revealed that the plasma lyso-Gb3 level was strikingly increased in classic Fabry males, and to a lesser extent in later-onset Fabry males and Fabry females. The elevation of the plasma lyso-Gb3 level was related to renal involvement in the Fabry females. ERT gave a rapid reduction in the elevated plasma lyso-Gb3 level in the classic Fabry males, and a gradual one or stabilization in most of the later-onset Fabry males and Fabry females. However, formation of antibodies against the recombinant GLA had a negative effect on the reduction of plasma lyso-Gb3. CONCLUSIONS: Regular observation of plasma lyso-Gb3 and antibodies is useful for monitoring of Fabry patients during ERT.

Prevalence and clinical features of Fabry disease in Japanese male patients with diagnosis of hypertrophic cardiomyopathy.

BACKGROUND: The prevalence of Fabry disease (FD) in Japanese patients presenting with unexplained left ventricular hypertrophy (LVH) has remained unclear. METHODS: We measured plasma α-galactosidase A activity in 177 men with a diagnosis of hypertrophic cardiomyopathy (HCM) (maximum LV wall thickness ≥15mm). RESULTS: Two patients (1.1%) showed very low α-galactosidase A activity [0.0 and 0.3nmol/hr/ml (normal range: 3.6-17.6nmol/hr/ml)], and a clinical diagnosis of cardiac variant of FD was finally made. One patient was a 55-year-old man who came to our hospital because of abnormal results of electrocardiography and showed concentric LVH in echocardiography. A missense mutation, R112L, was identified. The other was a 74-year-old man who had been diagnosed with HCM at the age of 60 years in another hospital and was referred for evaluation of repeated hospitalization for heart failure. Although echocardiography revealed asymmetric septal hypertrophy (ASH) with interventricular septal wall thickness of 16mm and posterior wall thickness of 11mm and reduced LV ejection fraction with hypokinetic posterior wall motion, his echocardiographic findings at the initial diagnosis of HCM were not ASH but concentric LVH with normal LV systolic function. A splicing mutation, IVS4+919G>A, was identified. CONCLUSIONS: The prevalence of FD in Japanese male patients with a clinical diagnosis of HCM was found to be 1.1%. These patients showed late onset and concentric LVH at initial presentation.

Differences in cleavage of globotriaosylceramide and its derivatives accumulated in organs of young Fabry mice following enzyme replacement therapy.

In Fabry disease, large amounts of globotriaosylceramide (Gb3) and related glycosphingolipids accumulate in organs due to a deficiency of α-galactosidase A (GLA) activity. Enzyme replacement therapy (ERT) with recombinant GLA is now available, and it has been reported that ERT is beneficial for patients with Fabry disease, especially those who start treatment at an early stage of the disease. However, it seems that the efficacy of ERT differs with each organ, and Gb3 accumulated in the kidneys shows resistance to ERT when it is started at a late stage. In this study, we examined the differences in cleavage of Gb3 isoforms, and lyso-Gb3 and its analogues in the kidneys, liver, and heart in young Fabry mice subjected to ERT. The results revealed that recurrent administration of recombinant GLA had prominent effects in terms of degradation of Gb3 and its derivatives accumulated in the organs. However, particular Gb3 isoforms, i.e., Gb3 (C20:0) and Gb3 (C24OH), accumulated in the kidneys largely escaped from degradation. Such Gb3 isoforms may gradually accumulate in the kidneys from a young age, which results in a reduction in the efficacy of ERT for Fabry disease.

Distributions of Globotriaosylceramide Isoforms, and Globotriaosylsphingosine and Its Analogues in an α-Galactosidase A Knockout Mouse, a Model of Fabry Disease.

Fabry disease is caused by deficient activity of α-galactosidase A (GLA) and characterized by systemic accumulation of glycosphingolipids, substrates of the enzyme. To gain insight into the pathogenesis of Fabry disease based on accumulated substrates, we examined the tissue and plasma distributions of globotriaosylceramide (Gb3) isoforms, and globotriaosylsphingosine (lyso-Gb3) and its analogues in a GLA knockout mouse, a model of Fabry disease, by means of liquid chromatography-mass spectrometry and nano-liquid chromatography-tandem mass spectrometry, respectively. The results revealed that the contents of these substrates in the liver, kidneys, heart, and plasma of GLA knockout mice were apparently higher than in those of wild-type ones, and organ specificity in the accumulation of Gb3 isoforms was found. Especially in the kidneys, accumulation of a large amount of Gb3 isoforms including hydroxylated residues was found. In the GLA knockout mice, the proportion of hydrophobic Gb3 isoforms was apparently higher than that in the wild-type mice. On the other hand, hydrophilic residues were abundant in plasma. Unlike that of Gb3, the concentration of lyso-Gb3 was high in the liver, and the lyso-Gb3/Gb3 ratio in plasma was significantly higher than those in the organs. The concentration of lyso-Gb3 was apparently higher than those of its analogues in the organs and plasma from both the GLA knockout and wild-type mice. This information will be useful for elucidating the basis of Fabry disease.

Clinical and biochemical investigation of male patients exhibiting membranous cytoplasmic bodies in biopsied kidney tissues; a pitfall in diagnosis of Fabry disease.

BACKGROUND: The existence of membranous cytoplasmic bodies in biopsied kidney tissues is one of the important findings when considering Fabry disease as the first choice diagnosis. However, there are possible acquired lysosomal diseases associated with pharmacological toxicity, although less attention has been paid to them. CASE PRESENTATION: We experienced 3 male patients presenting with proteinuria and specific pathological changes strongly suggesting Fabry disease. We sought detailed clinical and biochemical information to avoid a wrong diagnosis. The patients were examined clinically and pathologically, and plasma α-galactosidase A (GLA) activity and the globotriaosylsphingosine (lyso-Gb3) concentrations were measured. Electron microscopic examination revealed numerous membranous inclusion bodies in podocytes, and biochemical analysis revealed normal GLA activity and a normal lyso-Gb3 level in plasma, showing that they did not have Fabry disease. They suffered from hyperlipidemia, myeloma, or lupus nephritis. They had received pitavastatin calcium, clarithromycin, loxoprofen and/or prednisolone, and there was no medication history of cationic amphiphilic drugs. CONCLUSIONS: In this case series, the etiology of the inclusions was not clarified. However, these cases indicate that careful attention should be paid on diagnosis of patients exhibiting inclusion bodies in kidney cells, and it is important to confirm their past and present illnesses, and medication history as well as to measure the GLA activity and lyso-Gb3 level.

Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy.

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.

Chaperone therapy for Krabbe disease: potential for late-onset GALC mutations.

Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant GM1 ß-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations.

Nano-LC-MS/MS for Quantification of Lyso-Gb3 and Its Analogues Reveals a Useful Biomarker for Fabry Disease.

Biomarkers useful for diagnosis and evaluation of treatment for patients with Fabry disease are urgently needed. Recently, plasma globotriaosylsphingosine (lyso-Gb3) and lyso-Gb3-related analogues have attracted attention as promising biomarkers of Fabry disease. However, the plasma concentrations of lyso-Gb3 and its analogues are extremely low or below the detection limits in some Fabry patients as well as in healthy subjects. In this paper, we introduce the novel application of a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) system to the measurement of lyso-Gb3 and its analogues in plasma. Nano-LC-MS/MS requires smaller amounts of samples and is more sensitive than conventional techniques. Using this method, we measured the plasma concentrations of lyso-Gb3 and its analogues in 40 healthy subjects, 5 functional variants (males with E66Q), and various Fabry patients (9 classic Fabry males/9 mutations; 7 later-onset Fabry males/5 mutations; and 10 Fabry females/9 mutations). The results revealed that the mean lyso-Gb3 and lyso-Gb3(-2) concentrations in all the Fabry patient subgroups were statistically higher, especially in the classic Fabry males, than those in the functional variants and healthy subjects. The plasma concentrations of lyso-Gb3 and its analogues in healthy subjects, functional variants, and some Fabry patients with specific mutations (R112H and M296I) that cannot be established by conventional techniques were successfully determined by means of nano-LC-MS/MS. The lyso-Gb3 and lyso-Gb3(-2) concentrations in male patients with these mutations were lower than those in most Fabry patients having other mutations, but higher than those in the functional variants and healthy subjects. This new method is expected to be useful for sensitive determination of the plasma concentrations of lyso-Gb3 and its analogues. This study also revealed that not only lyso-Gb3 but also lyso-Gb3(-2) in plasma is a useful biomarker for the diagnosis of Fabry disease.