Plasma mutant α-galactosidase A protein and globotriaosylsphingosine level in Fabry disease

Tsukimura, Takahiro; Nakano, Sachie; Togawa, Tadayasu; Tanaka, Toshie; Saito, Seiji; Ohno, Kazuki; Shibasaki, Futoshi; Sakuraba, Hitoshi

doi:10.1016/j.ymgmr.2014.07.005

Cited by 15 publications

(25 citation statements)

References 44 publications

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“…The aim of the study was to analyze GLA mutations in‐depth on the basis of a holistic approach involving in vitro enzyme activity measurement of the mutant α‐gal A enzyme in cell culture (over‐expression) as well as determination of the biomarker globotriaosylsphingosine (lyso‐Gb3) from either plasma or dried blood spots (DBS). Lyso‐Gb3 is a deacylated metabolite of Gb3 with a higher sensitivity than Gb3 and a good correlation to the FD phenotype [Tsukimura et al., , Smid et al., (II)]. Furthermore, we introduce outline data for 61 male and 116 female patients with atypical FD (including age of diagnosis and symptomatic spectrum) in concurrence with one of 26 GVUS described in this study, in an attempt to elucidate the pathogenesis of these specific cases.…”

Section: Introductionmentioning

confidence: 80%

Functional and Clinical Consequences of Novel α-Galactosidase A Mutations in Fabry Disease

et al. 2015

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Fabry disease (FD) is a rare metabolic disorder of glycosphingolipid storage caused by mutations in the GLA gene encoding lysosomal hydrolase α-galactosidase A (α-gal A). Recently, the diagnostic procedure for FD has advanced in several ways, through the development of a specific biomarker (lyso-Gb3) and the implementation of newborn screenings, which acted as a catalyst to augment general awareness of the disease. Heterologous over-expression of α-gal A variants and subsequent in vitro measurement of enzyme activity provided molecular data to elucidate the relationship between mutation, enzyme damage, lyso-Gb3 biomarker levels, and clinical phenotype. This knowledge is the foundation for improved counseling with regard to prognosis and therapeutic decisions. Herein, we resume the approach of in vitro characterization, with a further 73 mainly novel GLA gene mutations. Patient lyso-Gb3 data were available for most of the mutations. All mutations were tested for responsiveness to pharmacological chaperone treatment and phenotypic data for 61 hemizygous male and 116 heterozygous female patients carrying a mutation associated with ≥ 20% residual activity, formerly classified as "mild" variant, were collected in order to evaluate the pathogenicity. We conclude that a mild GLA variant is typically characterized by high residual enzyme activity and normal biomarker levels. We found evidence that these variants can still be classified as a distinctive, but milder, sub-type of FD.

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Section: Introductionmentioning

confidence: 80%

Functional and Clinical Consequences of Novel α-Galactosidase A Mutations in Fabry Disease

et al. 2015

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“…Patients with this mutation have residual GLA activity, and usually manifest no childhood symptoms, such as acroparesthesia, angiokeratomas, hypohidrosis, or corneal opacities, but develop renal and/or cardiac disease [ 12 , 29 ]. Previous examinations revealed that the plasma lyso-Gb3 concentrations in male patients with R112H and M296I were below or near the limit of determination [ 7 , 8 , 12 , 30 ]. However, our new sensitive method allowed successful determination of the lyso-Gb3 concentrations in such patients.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2015

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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.

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“…For measurement of GLA activity in leukocytes, 10 μl of a leukocyte homogenate (about 10 μg protein) was mixed with 40 μl of the substrate solution in a 1.5 mL micro-tube, followed by incubation at 37 °C for 30 min [ 20 , 23 ]. Then, the reaction was stopped by adding 950 μL of the stopping solution, and the released MU was measured using a spectrofluorometer (F2700; Hitachi Ltd., Tokyo, Japan) at excitation and emission wavelengths of 365 nm and 450 nm, respectively.…”

Section: Methodsmentioning

confidence: 99%

Fabry disease in a Japanese population-molecular and biochemical characteristics

Sakuraba

Tsukimura

Togawa

et al. 2018

Molecular Genetics and Metabolism Reports

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We had experienced 117 Japanese Fabry patients (72 males and 45 females) from 1977 to 2006, and then we generated an improved Fabry analysis system in 2007 and have found 196 ones (95 males and 101 females) since then. In this study, we summarized the data of the patients and tried to elucidate the molecular and biochemical characteristics of Japanese Fabry patients. Gene analysis revealed various GLA mutations, including missense mutations (56.5%, 48 types); nonsense mutations (15.9%, 13 types); deletions (12.6%, 13 types); splicing defects (10.1%, 6 types); insertions (1.0%, 2 types), and insertions/deletions (0.5%, 1 type), in the patients that were tested. Amino acid substitutions resulting from the missense mutations found in the classic form patients tended to be localized in the core of the GLA protein, and those in the later-onset ones in the peripheral region. The most commonly identified pathogenic mutations are c.888G > A (p.M296I), c.936 + 919G > A, c.679C > T (p.R227X), c.335G > A (p.R112H), c.334C > T (p.R112C), and c.902G > A (p.R301Q). Among them, c.888G > A (p.M296I) is unique to Japanese Fabry patients. On the other hand, c.936 + 919G > A is a variant that has been frequently detected in Taiwan Chinese Fabry patients, and c.335G > A (p.R112H) in various countries. These are found in later-onset patients, and c.679C > T (p.R227X) and c.334C > T (p.R112C) classic ones. c.902G > A (p.R301Q) is found in both classic and later-onset form patients. A possible functional polymorphism, c.196G > C (p.E66Q), was identified in 0.4% of the subjects who underwent high-risk screening. The biochemical findings including leukocyte α-galactosidase A activity, plasma globotriaosylsphingosine level and urinary globotriaosylceramide in the individual phenotypic groups well reflected the phenotypic differences in this disease. The results will be useful for understanding the basis of Fabry disease in Japan.

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Plasma mutant α-galactosidase A protein and globotriaosylsphingosine level in Fabry disease

Cited by 15 publications

References 44 publications

Functional and Clinical Consequences of Novel α-Galactosidase A Mutations in Fabry Disease

Functional and Clinical Consequences of Novel α-Galactosidase A Mutations in Fabry Disease

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

Fabry disease in a Japanese population-molecular and biochemical characteristics

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