Abstract
Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.
Highlights
Human a-galactosidase A (GLA, EC 3. 2. 1. 22) is a lysosomal hydrolase involved in the catabolism of a-D-galactosyl (GLA) conjugates
The samples consisted of recombinant human GLAs (rhGLAs) serially diluted into GLAnegative serum and plasma samples collected from Fabry patients, and the assays were optimized to yield a linear response over the biological range (Fig. 2A and B)
We compared the sensitivities of these methods with that of enzyme-linked immunosorbent assay (ELISA), by using rhGLA as a standard and found that the MUSTag assays could detect as little as 6.4 pg/mL of GLA, making them apparently more sensitive than ELISA
Summary
Human a-galactosidase A (GLA, EC 3. 2. 1. 22) is a lysosomal hydrolase involved in the catabolism of a-D-galactosyl (GLA) conjugates. Gross alterations, nonsense mutations, and most of the splicing mutations of the GLA gene lead to a deficiency of the GLA protein, but missense mutations comprising the majority of mutations cause heterogeneous pathogenesis, i.e., some of them affect the active site, while others decrease the stability of the enzyme molecule [2,3]; the molecular basis of Fabry disease is complex Functional polymorphisms, such as p.E66Q [4,5] and p.D313Y [6], have been frequently found in Japanese/Korean and Caucasian populations, respectively; this further increases the difficulty of understanding the basis of the disease
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