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BMC Cancer
Published in: BMC Cancer 1/2011

Open Access 01-12-2011 | Technical advance

High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene

Authors: Rossella Tricarico, Francesca Crucianelli, Antonio Alvau, Claudio Orlando, Roberta Sestini, Francesco Tonelli, Rosa Valanzano, Maurizio Genuardi

Published in: BMC Cancer | Issue 1/2011

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Abstract

Background

MUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma. High resolution melting analysis (HRMA) is a mutation scanning method that allows detection of heterozygous sequence changes with high sensitivity, whereas homozygosity for a nucleotide change may not lead to significant curve shape or melting temperature changes compared to homozygous wild-type samples. Therefore, HRMA has been mainly applied to the detection of mutations associated with autosomal dominant or X-linked disorders, while applications to autosomal recessive conditions are less common.

Methods

MUTYH coding sequence and UTRs were analyzed by both HRMA and sequencing on 88 leukocyte genomic DNA samples. Twenty-six samples were also examined by SSCP. Experiments were performed both with and without mixing the test samples with wild-type DNA.

Results

The results show that all MUTYH sequence variations, including G > C and A > T homozygous changes, can be reliably identified by HRMA when a condition of artificial heterozygosity is created by mixing test and reference DNA. HRMA had a sensitivity comparable to sequencing and higher than SSCP.

Conclusions

The availability of a rapid and inexpensive method for the identification of MUTYH sequence variants is relevant for the diagnosis of colorectal cancer susceptibility, since the MAP phenotype is highly variable.
Appendix
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Literature
1.
Parker AR, Eshleman JR: Human MutY: gene structure, protein functions and interactions, and role in carcinogenesis. Cell Mol Life Sci. 2003, 60 (10): 2064-2083. 10.1007/s00018-003-3053-4.CrossRefPubMed
2.
Sampson JR, Jones S, Dolwani S, Cheadle JP: MutYH (MYH) and colorectal cancer. Biochem Soc Trans. 2005, 33 (Pt 4): 679-683.CrossRefPubMed
3.
Miyaki M, Iijima T, Yamaguchi T, Hishima T, Tamura K, Utsunomiya J, Mori T: Germline mutations of the MYH gene in Japanese patients with multiple colorectal adenomas. Mutat Res. 2005, 578 (1-2): 430-433.CrossRefPubMed
4.
Cheadle JP, Sampson JR: MUTYH-associated polyposis--from defect in base excision repair to clinical genetic testing. DNA Repair (Amst). 2007, 6 (3): 274-279. 10.1016/j.dnarep.2006.11.001.CrossRef
5.
Nielsen M, de Miranda NF, van Puijenbroek M, Jordanova ES, Middeldorp A, van Wezel T, van Eijk R, Tops CM, Vasen HF, Hes FJ, Morreau H: Colorectal carcinomas in MUTYH-associated polyposis display histopathological similarities to microsatellite unstable carcinomas. BMC Cancer. 2009, 9: 184-10.1186/1471-2407-9-184.CrossRefPubMedPubMedCentral
6.
Terdiman JP: MYH-associated disease: attenuated adenomatous polyposis of the colon is only part of the story. Gastroenterology. 2009, 137 (6): 1883-1886. 10.1053/j.gastro.2009.10.017.CrossRefPubMed
7.
Vogt S, Jones N, Christian D, Engel C, Nielsen M, Kaufmann A, Steinke V, Vasen HF, Propping P, Sampson JR, Hes FJ, Aretz S: Expanded extracolonic tumor spectrum in MUTYH-associated polyposis. Gastroenterology. 2009, 137 (6): 1976-1985. 10.1053/j.gastro.2009.08.052.CrossRefPubMed
8.
Isidro G, Laranjeira F, Pires A, Leite J, Regateiro F, Castro e Sousa F, Soares J, Castro C, Giria J, Brito MJ, Medeira A, Teixeira R, Morna H, Gaspar I, Marinho C, Jorge R, Brehm A, Ramos JS, Boavida MG: Germline MUTYH (MYH) mutations in Portuguese individuals with multiple colorectal adenomas. Hum Mutat. 2004, 24 (4): 353-354.CrossRefPubMed
9.
Piccioli P, Serra M, Gismondi V, Pedemonte S, Loiacono F, Lastraioli S, Bertario L, De Angioletti M, Varesco L, Notaro R: Multiplex tetra-primer amplification refractory mutation system PCR to detect 6 common germline mutations of the MUTYH gene associated with polyposis and colorectal cancer. Clin Chem. 2006, 52 (4): 739-743. 10.1373/clinchem.2005.060137.CrossRefPubMed
10.
Russell AM, Zhang J, Luz J, Hutter P, Chappuis PO, Berthod CR, Maillet P, Mueller H, Heinimann K: Prevalence of MYH germline mutations in Swiss APC mutation-negative polyposis patients. Int J Cancer. 2006, 118 (8): 1937-1940. 10.1002/ijc.21470.CrossRefPubMed
11.
Montgomery J, Wittwer CT, Palais R, Zhou L: Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. Nat Protoc. 2007, 2 (1): 59-66.CrossRefPubMed
12.
Erali M, Voelkerding KV, Wittwer CT: High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol. 2008, 85 (1): 50-58. 10.1016/j.yexmp.2008.03.012.CrossRefPubMedPubMedCentral
13.
Kennerson ML, Warburton T, Nelis E, Brewer M, Polly P, De Jonghe P, Timmerman V, Nicholson GA: Mutation scanning the GJB1 gene with high-resolution melting analysis: implications for mutation scanning of genes for Charcot-Marie-Tooth disease. Clin Chem. 2007, 53 (2): 349-352.CrossRefPubMed
14.
Laurie AD, Smith MP, George PM: Detection of factor VIII gene mutations by high-resolution melting analysis. Clin Chem. 2007, 53 (12): 2211-2214. 10.1373/clinchem.2007.093781.CrossRefPubMed
15.
de Juan I, Esteban E, Palanca S, Barragán E, Bolufer P: High-resolution melting analysis for rapid screening of BRCA1 and BRCA2 Spanish mutations. Breast Cancer Res Treat. 2009, 115 (2): 405-414. 10.1007/s10549-008-0073-7.CrossRefPubMed
16.
Dobrowolski SF, McKinney JT, Amat di San Filippo C, Giak Sim K, Wilcken B, Longo N: Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene. Hum Mutat. 2005, 25 (3): 306-313. 10.1002/humu.20137.CrossRefPubMed
17.
Montgomery J, Wittwer CT, Kent JO, Zhou L: Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis. Clin Chem. 2007, 53 (11): 1891-1898. 10.1373/clinchem.2007.092361.CrossRefPubMed
18.
Audrezet MP, Dabricot A, Le Marechal C, Ferec C: Validation of high-resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. J Mol Diagn. 2008, 10 (5): 424-434. 10.2353/jmoldx.2008.080056.CrossRefPubMedPubMedCentral
19.
Ebberink MS, Kofster J, Wanders RJ, Waterham HR: Spectrum of PEX6 mutations in Zellweger syndrome spectrum patients. Hum Mutat. 2010, 31 (1): E1058-1070. 10.1002/humu.21153.CrossRefPubMed
20.
López-Villar I, Ayala R, Wesselink J, Morillas JD, López E, Marín JC, Díaz-Tasende J, González S, Robles L, Martínez-López J: Simplifying the detection of MUTYH mutations by high resolution melting analysis. BMC Cancer. 2010, 10: 408-10.1186/1471-2407-10-408.CrossRefPubMedPubMedCentral
21.
Santonocito C, Paradisi A, Capizzi R, Torti E, Lanza-Silveri S, Penitente R, Zuppi C, Capoluongo E: Common genetic variants of MUTYH are not associated with cutaneous malignant melanoma: application of molecular screening by means of high-resolution melting technique in a pilot case-control study. Int J Biol Markers. 2011, 26 (1): 37-42. 10.5301/JBM.2011.6285.CrossRefPubMed
22.
Al-Tassan N, Chmiel NH, Maynard J, Fleming N, Livingston AL, Williams GT, Hodges AK, Davies DR, David SS, Sampson JR, Cheadle JP: Inherited variants of MYH associated with somatic G:C-->T:A mutations in colorectal tumors. Nat Genet. 2002, 30 (2): 227-232. 10.1038/ng828.CrossRefPubMed
23.
Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C: Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem. 2004, 50 (7): 1156-1164. 10.1373/clinchem.2004.032136.CrossRefPubMed
24.
Palais RA, Liew MA, Wittwer CT: Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping. Anal Biochem. 2005, 346 (1): 167-175. 10.1016/j.ab.2005.08.010.CrossRefPubMed
25.
Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ: High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 2003, 49 (6 Pt 1): 853-860.CrossRefPubMed
26.
Budowle B, Chakraborty R, Giusti AM, Eisenberg AJ, Allen RC: Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE. Am J Hum Genet. 1991, 48 (1): 137-144.PubMedPubMedCentral
27.
Reed GH, Wittwer CT: Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem. 2004, 50 (10): 1748-1754. 10.1373/clinchem.2003.029751.CrossRefPubMed
28.
Dobrowolski SF, Ellingson CE, Caldovic L, Tuchman M: Streamlined assessment of gene variants by high resolution melt profiling utilizing the ornithine transcarbamylase gene as a model system. Hum Mutat. 2007, 28 (11): 1133-1140. 10.1002/humu.20558.CrossRefPubMed
29.
Garritano S, Gemignani F, Voegele C, Nguyen-Dumont T, Le Calvez-Kelm F, De Silva D, Lesueur F, Landi S, Tavtigian SV: Determining the effectiveness of High Resolution Melting analysis for SNP genotyping and mutation scanning at the TP53 locus. BMC Genet. 2009, 10: 5-CrossRefPubMedPubMedCentral
30.
Wittwer CT: High-resolution DNA melting analysis: advancements and limitations. Hum Mutat. 2009, 30 (6): 857-859. 10.1002/humu.20951.CrossRefPubMed
31.
Aguirre-Lamban J, Riveiro-Alvarez R, Garcia-Hoyos M, Cantalapiedra D, Avila-Fernandez A, Villaverde-Montero C, Trujillo-Tiebas MJ, Ramos C, Ayuso C: Comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning in the ABCA4 gene. Invest Ophthalmol Vis Sci. 2010, 51 (5): 2615-2619. 10.1167/iovs.09-4518.CrossRefPubMed
32.
Lin CW, Er TK, Tsai FJ, Liu TC, Shin PY, Chang JG: Development of a high-resolution melting method for the screening of Wilson disease-related ATP7B gene mutations. Clin Chim Acta. 2010, 411 (17-18): 1223-1231. 10.1016/j.cca.2010.04.030.CrossRefPubMed
33.
Sergouniotis PI, Li Z, Mackay DS, Wright GA, Borman AD, Devery SR, Moore AT, Webster AR: A Survey of DNA Variation of C2ORF71 in Probands with Progressive Autosomal Recessive Retinal Degeneration and Controls. Invest Ophthalmol Vis Sci. 2011, 52 (3): 1880-1886. 10.1167/iovs.10-6043.CrossRefPubMed
34.
Reed GH, Kent JO, Wittwer CT: High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics. 2007, 8 (6): 597-608. 10.2217/14622416.8.6.597.CrossRefPubMed
35.
Out AA, Tops CM, Nielsen M, Weiss MM, van Minderhout IJ, Fokkema IF, Buisine MP, Claes K, Colas C, Fodde R, Fostira F, Franken PF, Gaustadnes M, Heinimann K, Hodgson SV, Hogervorst FB, Holinski-Feder E, Lagerstedt-Robinson K, Olschwang S, van den Ouweland AM, Redeker EJ, Scott RJ, Vankeirsbilck B, Grønlund RV, Wijnen JT, Wikman FP, Aretz S, Sampson JR, Devilee P, den Dunnen JT, Hes FJ: Leiden Open Variation Database of the MUTYH gene. Hum Mutat. 2010, 31 (11): 1205-1215. 10.1002/humu.21343.CrossRefPubMed
36.
37.
Tindall EA, Petersen DC, Woodbridge P, Schipany K, Hayes VM: Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments. Hum Mutat. 2009, 30 (6): 876-883. 10.1002/humu.20919.CrossRefPubMed
38.
Chow E, Thirlwell C, Macrae F, Lipton L: Colorectal cancer and inherited mutations in base-excision repair. Lancet Oncol. 2004, 5 (10): 600-606. 10.1016/S1470-2045(04)01595-5.CrossRefPubMed
39.
Lipton L, Tomlinson I: The multiple colorectal adenoma phenotype and MYH, a base excision repair gene. Clin Gastroenterol Hepatol. 2004, 2 (8): 633-638. 10.1016/S1542-3565(04)00286-1.CrossRefPubMed
40.
Croitoru ME, Cleary SP, Di Nicola N, Manno M, Selander T, Aronson M, Redston M, Cotterchio M, Knight J, Gryfe R, Gallinger S: Association between biallelic and monoallelic germline MYH gene mutations and colorectal cancer risk. J Natl Cancer Inst. 2004, 96 (21): 1631-1634. 10.1093/jnci/djh288.CrossRefPubMed
41.
Fleischmann C, Peto J, Cheadle J, Shah B, Sampson J, Houlston RS: Comprehensive analysis of the contribution of germline MYH variation to early-onset colorectal cancer. Int J Cancer. 2004, 109 (4): 554-558. 10.1002/ijc.20020.CrossRefPubMed
42.
Jenkins MA, Croitoru ME, Monga N, Cleary SP, Cotterchio M, Hopper JL, Gallinger S: Risk of colorectal cancer in monoallelic and biallelic carriers of MYH mutations: a population-based case-family study. Cancer Epidemiol Biomarkers Prev. 2006, 15 (2): 312-314. 10.1158/1055-9965.EPI-05-0793.CrossRefPubMed
43.
Theodoratou E, Campbell H, Tenesa A, Houlston R, Webb E, Lubbe S, Broderick P, Gallinger S, Croitoru EM, Jenkins MA, Win AK, Cleary SP, Koessler T, Pharoah PD, Küry S, Bézieau S, Buecher B, Ellis NA, Peterlongo P, Offit K, Aaltonen LA, Enholm S, Lindblom A, Zhou XL, Tomlinson IP, Moreno V, Blanco I, Capellà G, Barnetson R, Porteous ME, et al: A large-scale meta-analysis to refine colorectal cancer risk estimates associated with MUTYH variants. Br J Cancer. 2010, 103 (12): 1875-1884. 10.1038/sj.bjc.6605966.CrossRefPubMedPubMedCentral
44.
Metadata
Title
High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene
Authors
Rossella Tricarico
Francesca Crucianelli
Antonio Alvau
Claudio Orlando
Roberta Sestini
Francesco Tonelli
Rosa Valanzano
Maurizio Genuardi
Publication date
01-12-2011
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2011
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/1471-2407-11-305

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