Genetic screening of Fabry patients with EcoTILLING and HRM technology

被引：17
作者：
Bono C. [1 ]
Nuzzo D. [1 ]
Albeggiani G. [1 ]
Zizzo C. [1 ]
Francofonte D. [1 ]
Iemolo F. [2 ]
Sanzaro E. [2 ]
Duro G. [1 ]
机构：
[1] National Research Council-Institute of Biomedicine and Molecular Immunology (CNR-IBIM), Palermo
[2] Department of Neurology, Guzzardi Hospital, Vittoria (Ragusa)
来源：
BMC Research Notes | / 4卷 / 1期
关键词：
Anderson-Fabry; EcoTILLING; haplotype; HRM; screening;
D O I：
10.1186/1756-0500-4-323
中图分类号：
学科分类号：
摘要：
Background: Anderson-Fabry disease (FD) is caused by a deficit of the -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 -galactosidase A enzyme in subjects with probable FD in order to test analysis strategies which include or rely on initial pre-screening. Findings. 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. Conclusion: 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. © 2011Nuzzo et al; licensee BioMed Central Ltd.
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