Plasma globotriaosylsphingosine as a biomarker of Fabry disease

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Abstract

Fabry disease is an X-linked genetic disorder caused by a deficiency of α-galactosidase A (GLA) activity. As enzyme replacement therapy (ERT) involving recombinant GLAs has been introduced for this disease, a useful biomarker for diagnosis and monitoring of therapy has been strongly required. We measured globotriaosylsphingosine (lyso-Gb3) and globotriaosylceramide (Gb3) in plasma samples from ten hemizygous males (six classic and four variant cases) and eight heterozygous females with Fabry disease, and investigated the responses of plasma lyso-Gb3 and Gb3 in a male Fabry patient who had undergone ERT for 4 years to determine whether plasma lyso-Gb3 and Gb3 could be biomarkers of Fabry disease. The results revealed that plasma lyso-Gb3 was apparently increased in male patients and was higher in cases of the classic form than those of the variant one. In Fabry females, plasma lyso-Gb3 was moderately increased in both symptomatic and asymptomatic cases, and there was a correlation between the increase in lyso-Gb3 and the decrease in GLA activity. As to plasma Gb3, the levels in the variant Fabry hemizygotes and Fabry heterozygotes could not be distinguished from those in the controls, although those in the classic Fabry hemizygotes were increased. The plasma lyso-Gb3 level in the Fabry patient who had received ERT was elevated at the baseline and fell more dramatically on ERT than that of Gb3. Plasma lyso-Gb3 could thus be a potential biomarker of Fabry disease.

Introduction

Fabry disease (MIM 301500) is an X-linked genetic disorder due to a deficiency of α-galactosidase A (GLA, EC3.2.1.22) activity [1]. This deficiency causes the lysosomal accumulation of glycolipids, predominantly globotriaosylceramide (Gb3). Male patients with the classic form of this disease having no GLA activity experience pain in the peripheral extremities, cutaneous angiokeratomas, hypohidrosis, corneal opacities, cerebrovascular disorders, and renal and cardiac involvement. On the other hand, male patients with the variant form having residual GLA activity develop milder clinical manifestations, sometimes limited to heart disorders. Heterozygous Fabry females exhibit a quite wide range of clinical phenotypes. They either suffer from a milder form of the disease or are asymptomatic, but sometimes they may exhibit severe clinical features as well as the classic form seen in male patients [1]. A recent clinical study revealed that the vast majority of Fabry heterozygotes are symptomatic and develop cardiac involvement with increasing age [2]. The diagnosis of Fabry hemizygotes is essentially based on the demonstration of decreased GLA activity. However, the GLA assay is inconclusive in heterozygous Fabry females, the activity ranging from relatively low to normal levels.

Recombinant GLAs have been produced in Chinese hamster ovary cells [3], [4] and genetically modified human fibroblasts [5], and they are clinically available for enzyme replacement therapy (ERT) for Fabry disease. Although many patients have been successfully treated with these recombinant GLAs, little or no improvement in cardiac and renal functions can be obtained in some patients, especially in ones in whom the stage of the disease is advanced [6], [7]. This suggests that early diagnosis and early treatment are important, and thus the determination of a useful biomarker for the diagnosis of and monitoring of the response to ERT in Fabry disease has been strongly required.

The levels of Gb3 in plasma and/or urine have been measured for the above purposes [8], [9], [10], [11], but recent systemic analysis revealed that Gb3 was not an ideal marker for a diagnosis or the response to treatment in patients with this disease [12], [13]. Recently, Aerts et al. reported that globotriaosylsphingosine (lyso-Gb3) was increased in the plasma of classically affected male Fabry patients, and in the plasma and tissues of Fabry mice, and they suggested that circulating lyso-Gb3 could be a candidate biomarker for monitoring Fabry disease [13].

In this study, we measured the plasma lyso-Gb3 and Gb3 concentrations in male and female patients with both the classic and variant forms of Fabry disease, and compared them, and examined the response to ERT to determine whether lyso-Gb3 is a useful biomarker of Fabry disease.

Section snippets

Materials

Lyso-Gb3, Gb3 and N-acetyl-d-galactosamine were obtained from Sigma Chemical Co. (St. Louis, MO). o-Phthalaldehyde (OPA) was purchased from Nacalai Tesque (Kyoto, Japan). 4-Methylumbelliferyl-α-d-galactopyranoside was obtained from Calbiochem (LaJolla, CA). All other chemicals used were of analytical grade.

Plasma samples and patients

Plasma samples for measurement of the lyso-Gb3 and Gb3 concentrations, and GLA activity were obtained from ten hemizygous Fabry males (six classic and four variant cases), eight heterozygous

Determination of lyso-Gb3 concentrations in plasma

Fig. 1 shows representative HPLC-chromatograms of lyso-Gb3 in plasma samples from a hemizygous Fabry male, a heterozygous Fabry female, and a control subject. In this study, the extraction procedure and chromatographic system for the measurement of lyso-Gb3 were basically carried out according to Aerts, and standard solutions were made by adding authentic lyso-Gb3 to normal plasma, the lyso-Gb3 being extracted by the same procedure. Peak identification was performed by comparison of the

Discussion

Newborn screening studies performed in Italy and Taiwan revealed that Fabry disease is one of the most prevalent sphingolipidoses, the estimated incidence of the disease being 1 in 1250–4000 male live births [15], [16]. The results suggest that the true incidence is unexpectedly higher than originally thought [1]. Actually, a lot of Fabry patients have been found among patients with stroke [17], left ventricular hypertrophy [18], and renal failure [19]. So, the clinical importance of this

Conflict of interest

None.

Acknowledgments

This work was partly supported by Grants from the Ministry of Health, Labor and Welfare of Japan, the JSPS AA Science Platform Program (FY2010), the High-Tech Research Center Project of the Ministry of Education, Science, Sports and Culture of Japan (S0801043), the Japan Society for the Promotion of Science (18390303), and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (09-15).

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