Abstract
BackgroundAnderson-Fabry disease (FD) is caused by a deficit of the α-galactosidase A enzyme which leads to the accumulation of complex sphingolipids, especially globotriaosylceramide (Gb3), in all the cells of the body, causing the onset of a multi-systemic disease with poor prognosis in adulthood. In this article, we describe two alternative methods for screening the GLA gene which codes for the α-galactosidase A enzyme in subjects with probable FD in order to test analysis strategies which include or rely on initial pre-screening.FindingsWe analyzed 740 samples using EcoTILLING, comparing two mismatch-specificendonucleases, CEL I and ENDO-1, while conducting a parallel screening of the same samples using HRM (High Resolution Melting). Afterwards, all samples were subjected to direct sequencing. Overall, we identified 12 different genetic variations: -10C>T, -12G>A, -30G>A, IVS2-76_80del5, D165H, C172Y, IVS4+16A>G, IVS4 +68 A>G, c.718_719delAA, D313Y, IVS6-22C>T, G395A. This was consistent with the high genetic heterogeneity found in FD patients and carriers. All of the mutations were detected by HRM, whereas 17% of the mutations were not found by EcoTILLING. The results obtained by EcoTILLING comparing the CEL I and ENDO-1 endonucleases were perfectly overlapping.ConclusionOn the basis of its simplicity, flexibility, repeatability, and sensitivity, we believe thatHRM analysis of the GLA gene is a reliable presequencing screening tool. This method can be applied to any genomic feature to identify known and unknown genetic alterations, and it is ideal for conducting screening and population studies.
Highlights
Fabry disease (FD) is a lysosomal storage disease caused by a congenital error in glycosphingolipid metabolism resulting from the deficient or absent activity of the lysosomal enzyme a-galactosidase A [1]
EcoTILLING The need to perform simultaneous analyses of many samples led to the adoption of a high-throughput screening strategy based on pools of 8 samples, as reported by Till et al [15]
High Resolution Melting In order to evaluate the results obtained by EcoTILLING, we conducted another screening with High Resolution Melting
Summary
Fabry disease (FD) is a lysosomal storage disease caused by a congenital error in glycosphingolipid metabolism resulting from the deficient or absent activity of the lysosomal enzyme a-galactosidase A [1]. This enzyme is essential for the sphingolipid recycling process that occurs within cells. The enzyme deficiency causes the interruption of the process of waste product demolition, leading to the intracellular accumulation of complex sphingolipids, especially globotriaosylceramide (Gb3) [2]. The accumulation of these products is the basis of FD symptomology that includes acroparesthesias, angiokeratoma, corneal and. We describe two alternative methods for screening the GLA gene which codes for the a-galactosidase A enzyme in subjects with probable FD in order to test analysis strategies which include or rely on initial pre-screening
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