Fabry disease: Biochemical, pathological and structural studies of the α-galactosidase A with E66Q amino acid substitution
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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2020, Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease: Volume 1Effectiveness of plasma lyso-Gb3 as a biomarker for selecting high-risk patients with Fabry disease from multispecialty clinics for genetic analysis
2019, Genetics in MedicineCitation Excerpt :Of the 11 males, 7 declined gene analysis. Of the four males who underwent ordinary gene analysis, three presented the p.E66Q class 2 mutation—a nonpathogenic, functional polymorphism that was found at an unexpectedly high frequency in the GLA analysis of Japanese patients selected through the abnormal α-Gal A activity in the FD screening.14,27 One male with normal plasma lyso-Gb3 levels exhibited a gene promoter variant (c.−10C>T) in the 5'-untranslated region of exon 1 that was associated with decreased α-Gal A expression;28 this is a nonpathogenic class 2 mutation (Supplementary Table S6 online).28
Fabry disease in a Japanese population-molecular and biochemical characteristics
2018, Molecular Genetics and Metabolism ReportsCitation Excerpt :Their GLA activity level was 59% of the control mean, and there was no statistical difference in the plasma Lyso-Gb3 concentration between the cases with c.196G > C (p.E66Q) and the healthy subjects, strongly suggesting that this is a functional polymorphism. The evidence that no pathological findings specific to Fabry disease have been observed in biopsied tissues supports it [34]. The biochemical data including leukocyte GLA activity, plasma Lyso-Gb3 concentration, and urinary Gb3 excretion in the individual phenotypic groups well reflected the phenotypic differences in this disease.
Screening for Fabry Disease in Japanese Patients with Young-Onset Stroke by Measuring α-Galactosidase A and Globotriaosylsphingosine
2018, Journal of Stroke and Cerebrovascular DiseasesCitation Excerpt :After the p.E66Q was first identified in a male patient with classic Fabry disease,23 it was considered pathogenic. However, based on the results of a biochemical study, the p.E66Q does not affect enzymatic activity; rather, it leads to instability in the enzyme structure, and no pathological changes specific to Fabry disease have been observed in biopsied tissue specimens.19-24 In addition, the allele frequency of the p.E66Q has been reported to range from .1% to 1% in various cohort studies including general populations.25-33