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Genetic screening of Fabry patients with EcoTILLING and HRM technology

Caterina Bono1, Domenico Nuzzo1*, Giuseppe Albeggiani1, Carmela Zizzo1, Daniele Francofonte1, Francesco Iemolo2, Enzo Sanzaro2 and Giovanni Duro1

Author Affiliations

1 National Research Council-Institute of Biomedicine and Molecular Immunology (CNR-IBIM) - Palermo, Italy

2 Department of Neurology - Guzzardi Hospital - Vittoria (Ragusa), Italy

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BMC Research Notes 2011, 4:323  doi:10.1186/1756-0500-4-323

Published: 6 September 2011



Anderson-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.


We analyzed 740 samples using EcoTILLING, comparing two mismatch-specific

    endonucleases, 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.


    On the basis of its simplicity, flexibility, repeatability, and sensitivity, we believe that

      HRM 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.

      Anderson-Fabry; haplotype; screening; HRM; EcoTILLING