Genetic screening of Fabry patients with EcoTILLING and HRM technology

BMC Research Notes

Giovanni Duro²  Enzo Sanzaro¹  Francesco Iemolo¹  Daniele Francofonte²  Carmela Zizzo²  Giuseppe Albeggiani²  Domenico Nuzzo²  Caterina Bono² 

[1] Department of Neurology - Guzzardi Hospital - Vittoria (Ragusa), Italy;National Research Council-Institute of Biomedicine and Molecular Immunology (CNR-IBIM) - Palermo, Italy

关键词: EcoTILLING;    HRM;    screening;    haplotype;    Anderson-Fabry;   

Others  :  1167228
DOI  :  10.1186/1756-0500-4-323

 received in 2011-03-01, accepted in 2011-09-06,  发布年份 2011

【 摘 要 】

Background

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.

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.

【 授权许可】

   
2011 Nuzzo et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Hoffman Bjorn: Fabry disease: recent avances in pathology, diagnosis, treatment and monitoring. Orphanet J Rare Dis 2009, 4:21. BioMed Central Full Text
• [2]Aerts JohannesM, et al.: Elevated globotriaosylsphingosine is a hallmark of Fabry disease. Proc Natl Acad Sci USA 2008, 105(8):2812-2817.
• [3]Shen Jin-Song, et al.: Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells. Mol Genet Metab 2008, 95(3):163-168.
• [4]Linhart Aleš, Elliott PerryM: The heart in AndersonıFabry disease and other lysosomal storage disorders. Heart 2007, 93(4):528-535.
• [5]Schiffmann Raphael, et al.: Fabry disease: progression of nephropathy, and prevalence of cardiac and cerebrovascular events before enzyme replacement therapy. Nephrol Dial Transplant 2009, 24(7):2102-2111.
• [6]Shows TB, et al.: Assignment of a-Galactosidase (aGal) to the q22-qter region of the X chromosome in man. Cytogenet Cell Genet 1978, 22:541-4.
• [7]Eng CM, et al.: Nature and frequency of mutations in the alpha-galactosidase A gene that cause Fabry disease. Am J Hum Genet 1993, 53(6):1186-1197.
• [8]Filoni C, et al.: Functional studies of new GLA gene mutations leading to conformational fabry disease. Biochimica et Biophysica Acta 2010, 1802:247-252.
• [9]Lin HY, et al.: Novel human pathological mutations. Gene symbol: GLA. Disease: Fabry disease. Hum Genet 2010, 127(1):124..
• [10]Eng CM, et al.: Nature and frequency of mutations in the α-galactosidase A gene that cause Fabry disease. Am J Hum Genet 1993, 53:1186-97.
• [11]Davies JP, et al.: Sequence variations in the first exon of α-galactosidase A. J Med Genet 1993, 30:658-63.
• [12]Shabbeer J, et al.: Detection of α-galactosidase a mutations causing Fabry disease by denaturing high performance liquid chromatography. Hum Mutat 2005, 25:299-305.
• [13]Barkley NA, Wang M: Application of TILLING and EcoTILLING as Reverse Genetic Approaches to Elucidate the Function of Genes in Plants and Animals. Curr Genomics 2008, 9(4):212-26.
• [14]Montgomery JL, et al.: High-resolution DNA melting analysis in clinical research and diagnostics. Expert Rev Mol Diagn 2010, 10(2):219-40.
• [15]Till BJ, et al.: High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling. Nucleic Acids Res 2006, 34(13):e99..
• [16]Coassin S, et al.: An optimized procedure for the design and evaluation of Ecotilling assays. BMC Genomics 2008, 9:510. BioMed Central Full Text
• [17]Till BJ, et al.: High-throughput TILLING for functional genomics. Methods Mol Biol 2003, 236:205-20.
• [18]McCallum CM, et al.: Targeted screening for induced mutations. Nat Biotechnol 2000, 18(4):455-7.
• [19]Greene EA, et al.: Spectrum of chemically induced mutations from a large-scale reversegenetic screen in Arabidopsis. Genetics 2003, 164:731-740.
• [20]Till BJ, et al.: Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 2007, 7:19. BioMed Central Full Text
• [21]Raghavan C, et al.: Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping. Mol Breed 2007, 19:87-101.
• [22]Comai L, et al.: Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J 2004, 37:778-786.
• [23]Gilchrist EJ, et al.: Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa. Mol Ecol 2006, 15:1367-1378.
• [24]Wittwer CT, et al.: High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 2003, 49:853-860.
• [25]Erali M, et al.: SNP genotyping by unlabeled probe melting analysis. Methods Mol Biol 2008, 429:199-206.
• [26]Reed GH, et al.: Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 2004, 50:1748-1754.
• [27]Liew M, et al.: Genotyping of single-nucleotide polymorphisms by high-resolution melting of mall amplicons. Clin Chem 2004, 50:1156-1164.
• [28]Erali Maria, et al.: High Resolution Melting Applications for Clinical Laboratory Medicine. Exp Mol Pathol 2008, 85(1):50-58.
• [29]Krypuy M, et al.: High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer 2006, 6:295. BioMed Central Full Text
• [30]Takano EA, et al.: Rapid detection of carriers with BRCA1 and BRCA2 mutations using high resolution melting analysis. BMC Cancer 2008, 8:59. BioMed Central Full Text
• [31]Krenková P, et al.: Evaluation of high-resolution melting (HRM) for mutation scanning of selected exons of the CFTR gene. Folia Biol (Praha) 2009, 55(6):238-42.
• [32]Laurie AD, et al.: Detection of factor VIII gene mutations by high-resolution melting analysis. Clin Chem 2007, 53(12):2211-4.
• [33]Janavicius R, et al.: Microsatellite instability detection by high-resolution melting analysis. Clin Chem 2010, 56(11):1750-7.
• [34]Kummalue T, et al.: Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis. Diagn Pathol 2010, 5:31. BioMed Central Full Text
• [35]Pornprasert S, Sukunthamala K: SYTO9 and SYBR GREEN1 with a high-resolution melting analysis for prenatal diagnosis of β-thalassemia/hemoglobin-E. Eur J Haematol 2010, 85(5):424-9.
• [36]Wong EM, Dobrovic A: Assessing gene-specific methylation using HRM-based analysis. Methods Mol Biol 2011, 687:207-17.
• [37]Castellanos E, et al.: Rapid identification and differentiation of Mycobacterium avium subspecies paratuberculosis types by use of real-time PCR and high-resolution melt analysis of the MAP1506 locus. J Clin Microbiol 2010, 48(4):1474-7.
• [38]Ong DC, et al.: Rapid detection of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis by high-resolution melting analysis. J Clin Microbiol 2010, 48(4):1047-54.
• [39]Tanislav C, et al.: Frequency of Fabry disease in patients with small-fibre neuropathy of unknown aetiology: a pilot study. Eur J Neurol 2010.