Fabry disease: Biochemical, pathological and structural studies of the α-galactosidase A with E66Q amino acid substitution

https://doi.org/10.1016/j.ymgme.2012.01.010Get rights and content

Abstract

Recently, male subjects harboring the c.196G > C nucleotide change which leads to the E66Q enzyme having low α-galactosidase A (GLA) activity have been identified at an unexpectedly high frequency on Japanese and Korean screening for Fabry disease involving dry blood spots and plasma/serum samples. Individuals with the E66Q enzyme have been suspected to have the later-onset Fabry disease phenotype leading to renal and cardiac disease. However, there has been no convincing evidence for this. To determine whether c.196G > C (E66Q) is disease-causing or not, we performed biochemical, pathological and structural studies. It was predicted that the E66Q amino acid substitution causes a small conformational change on the molecular surface of GLA, which leads to instability of the enzyme protein. However, biochemical studies revealed that subjects harboring the E66Q enzyme exhibited relatively high residual enzyme activity in white blood cells, and that there was no accumulation of globotriaosylceramide in cultured fibroblasts or an increased level of plasma globotriaosylsphingosine in these subjects. An electron microscopic examination did not reveal any pathological changes specific to Fabry disease in biopsied skin tissues from a male subject with the E66Q enzyme. These results strongly suggest that the c.196G > C is not a pathogenic mutation but is a functional polymorphism.

Highlights

► We performed to determine whether E66Q is a disease-causing mutation or not. ► There was no accumulation of Gb3 or an increased level of plasma lyso-Gb3. ► An EM examination did not reveal any pathological changes specific to Fabry disease. ► Our results strongly suggest that the E66Q is not a pathogenic mutation.

Introduction

Fabry disease (MIM 301500) is an X-linked genetic disease resulting from the α-galactosidase A (GLA, EC 3.2.1.22) deficient activity [1]. The enzyme defect leads to the progressive accumulation of glycosphingolipids including globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Male patients with little or no GLA activity exhibit the “classic form” of Fabry disease, developing pain in the peripheral extremities, hypohydrosis, angiokeratomas, corneal opacities, and renal, cardiac and cerebrovascular involvement. On the other hand, male patients with the “later-onset” phenotype of the disease have low residual GLA activity and exhibit later clinical manifestations. Heterozygous females exhibit a wide spectrum of clinical forms ranging from asymptomatic to presentation with the classic disease according to the characteristics of an X-linked inherited disease.

Since enzyme replacement therapy (ERT) for Fabry disease with recombinant GLAs has been introduced into clinical medicine [2], [3], early diagnosis is becoming more and more important, and neonatal and high-risk screening for this disease is being widely performed [4], [5], [6], [7], [8].

Recently, subjects harboring the c.196G > C nucleotide change, (amino acid substitution: E66Q) of the GLA gene leading to low GLA activity have been found at an unexpectedly high frequency (0.5–1%) among Korean and Japanese individuals by Fabry screening [9], [10], [11], [12], which has led to interest as to whether c.196G > C is a disease-causing mutation or a functional polymorphism.

In this study, we performed a structural modeling of the GLA with E66Q, and biochemical and pathological examinations of blood and biopsied skin samples from male subjects who had the E66Q enzyme on high-risk screening of patients with renal and cardiovascular disorders for Fabry disease, to assess its characteristics.

Section snippets

Blood and biopsied skin samples

High-risk screening for Fabry disease involving serum samples was performed for male patients with chronic kidney disease and cardiovascular involvement, and the results revealed that there was a group of subjects having low (< 1.5 nmol/h/ml) but residual GLA activity other than Fabry patients who exhibit little or no GLA activity (data not shown). Gene analysis of the GLA revealed that they harbored the c.196G > C base change. White blood cells (WBCs) and plasma samples were obtained for GLA

Activity and stability of GLA

The mean plasma and WBC GLA activities in the normal subjects were 5.3 ± 2.1 nmol/h/ml (n = 101) and 49 ± 20 nmol/h/mg protein (n = 20), respectively. The males harboring the E66Q enzyme exhibited residual enzyme activity, 13–26% of the normal mean for plasma and 24–65% of the normal mean for WBC (Table 1), although Fabry patients exhibited little or GLA activity (< 5% of the normal mean) in either plasma or WBCs.

The stability of the GLA enzyme in plasma from males harboring the E66Q enzyme is shown in

Discussion

The c.196G > C nucleotide change in the GLA gene has been identified at almost the same frequency (~ 0.6%) on both high-risk screening (our experience) and neonatal screening (Nakamura et al. The 53rd Annual Meeting of the Japanese Society for Inherited Metabolic Disorders, November 26, 2011, Chiba) for Fabry disease in Japan. This nucleotide change leading to the E66Q amino acid substitution was first identified in a patient with classic Fabry disease. However, this male also had another GLA

Conflict of interest

None.

Acknowledgments

This work was supported by the Program for Research on Intractable Diseases of Health and Labor Science Research (HS); the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (ID: 09-15, HS); the JAPS Asia/Africa Scientific Platform Program (HS); the Japan Society for the Promotion of Science (JSPS ID: 21390314, HS); and the High-Tech Research Center Project of the Ministry of Education, Culture, Sports, Science and Technology

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