Urinary globotriaosylceramide excretion correlates with the genotype in children and adults with Fabry disease

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Abstract

Fabry disease is a complex, multisystemic and clinically heterogeneous disease, in which the urinary excretion of globotriaosylceramide (Gb3), the principal substrate of the deficient enzyme, α-galactosidase A, is more prominent than the increased concentrations of the lipid in the plasma of affected hemizygotes and heterozygotes. We have developed and validated a simultaneous analysis of Gb3 and creatinine in a 2.6-min run using filter paper discs saturated with urine and analyzed by LC–MS/MS. Using this method, we studied the relationship between urinary levels of total Gb3/creatinine excretion and four types of mutations in the GLA gene (missense, nonsense, frameshift, and splice-site defects) in 32 children and 78 adult patients with Fabry disease. Forty-one patients were treated by enzyme replacement therapy and 69 were untreated. Our results show that the mean recoveries of Gb3 and creatinine from the urine filter paper standards were 91% and 97%, respectively, with precision, reproducibility, and linearity within acceptable ranges. Statistical analysis using the independent variables of sex, age, types of mutations and treatment showed that the mutation factor has a statistically significant impact on urinary Gb3 excretion (p = 0.0007). This means that the levels of urinary excretion of Gb3/creatinine in children and adults with Fabry disease are directly related to the types of mutations. The same correlation was found for the sex (p < 0.0001) and treatment (p = 0.0011). In conclusion, we studied 35 mutations in 110 children and adults with Fabry disease and found a significant correlation between the types of mutations and total Gb3 excretion in Fabry patients.

Section snippets

Materials and methods

This project was approved by the Research Ethics Board of the Faculty of Medicine and Health Sciences and the Sherbrooke Medical Centre.

LC–MS/MS analysis

Fig. 1 shows the total ion chromatogram (TIC) in MRM mode of a 5-year-old hemizygote with Fabry disease with the R301Q mutation excreting increased levels of total Gb3 and a normal control child. Complete analysis time is 2.6 min.

Comparison of two quantitative creatinine methodologies

Regarding the comparison between creatinine samples analyzed by the spectrophotometric technique and the multiplex LC–MS/MS method, the Friedmann test shows no difference between the two methods (χ2 = 0.19, p-value = 0.6629) for 47 urine filter paper samples.

Gb3

With total Gb3

Discussion

Previous efforts to analyze total Gb3 isoforms over the years were both time and labor intensive, necessitating sample dilution, extraction, ultrasonication, centrifugation and evaporation steps [29], [31], [43]. Creatinine is most frequently analyzed using the Jaffe method, but this is prone to interference by proteins, bilirubin and ketones [44]. On the other hand, specific constituents of blood have been analyzed simultaneously with creatinine [45], [46]. We have previously developed a

Acknowledgments

The authors thank the nurse-coordinators, geneticists and genetic counsellors at each collaborating centre: Carolina Azcona, (Toronto), Kaye Lemoine (Halifax), Carole Fortier (Montreal), Nicole Labbé (Quebec), and Dr. Karelle Benistan (Paris) and Dr. John James Mitchell (Montreal) for their collaboration in providing urine samples from Fabry patients. We acknowledge the assistance of Claude Alie and Dr. René Gagnon for their keen expertise. We are particularly grateful to the patients with

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