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Identifying Mutations for MYH‐Associated Polyposis

Thomas W. Prior¹, Scott J. Bridgeman¹

¹Ohio State University, Columbus, Ohio

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 10.13

DOI:

10.1002/0471142905.hg1013s64

Online Posting Date:

January, 2010

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Abstract

Polyposis associated with mutations in the gene MYH is an autosomal recessive syndrome characterized by the development of colorectal adenomas and cancer. Two common mutations, p.Tyr165Cys (exon 7) and p.Glu382Asp (exon 13), have been shown to account for the majority of the mutations occurring in individuals of Caucasian ancestry. Other mutations have been found throughout the gene and many have been shown to have very low frequencies. Ethnic differences in the mutation spectrum have also been observed. Thus, in order to achieve the highest clinical sensitivity, it is necessary to perform whole-gene sequencing of the MYH gene. The sequencing protocol described allows one to identify mutations throughout the MYH gene. Curr. Protoc. Hum. Genet. 64:10.13.1-10.13.14. © 2010 by John Wiley & Sons, Inc.

Keywords: colorectal cancer; multiple colorectal adenomas; genetic mutation; mutation detection; DNA sequencing

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Introduction
Basic Protocol 1: Preparation of DNA from Peripheral Blood for MYH Testing
Basic Protocol 2: Polymerase Chain Reaction (PCR) Amplification of the MYH Gene and PCR Product Purification
Basic Protocol 3: Cycle Sequencing of Purified PCR Products
Basic Protocol 4: Sequence Analysis of MYH Gene
Reagents and Solutions
Commentary
Literature Cited
Tables

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Materials

Basic Protocol 1: Preparation of DNA from Peripheral Blood for MYH Testing

Materials

2× sucrose-Triton (see recipe), ice cold
Blood sample in ACD or EDTA tube
Nuclei lysis buffer (see recipe)
10 mg/ml proteinase K
Sodium chloride, saturated
100% (prechilled) and 70% ethanol
TE buffer, pH 8.0 (appendix 2D)

15- and 50-ml conical polypropylene centrifuge tubes
Refrigerated centrifuge
Cryotubes
0.5- and 1.5-ml microcentrifuge tubes, sterile
Disposable inoculating loop (Nunc, cat. no. 254410/253287)
Plastic disposable transfer pipets, wide bore
Vortex, with multi-head and timer

Additional reagents for quantitation of DNA (appendix 3D)

Basic Protocol 2: Polymerase Chain Reaction (PCR) Amplification of the MYH Gene and PCR Product Purification

Materials

PCR-grade H₂O
100 mM MgCl₂
10× PCR ammonium sulfate buffer, pH 8.8 (see recipe) 25 mM dNTPs (25 mM each of G, C, A, and T)
AmpliTaq Gold DNA polymerase (Applied Biosystems)
2.5 pmol/µl PCR primers for MYH (Table 10.13.1)
100 ng/µl DNA (Basic Protocol 1)
2.5% (w/v) agarose gel in 1× TAE buffer (unit 2.7)
100-bp DNA ladder
10 U/µl exonuclease I enzyme (USB)
1 U/µl Rapid Alkaline Phosphatase, bovine (Roche)

200-µl PCR tubes
Thermal cycler (BioRad Tetrad 2 or equivalent)

Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Table 10.13.1 MYH PCR/Sequence Primers^a

Exon	Primer	Primer Sequence 5¢ – 3¢	Primer length	Fragment size

1.1	MYH EX1.1F	TACCTCTGGGAAGCCGCTC	19	407 bp
	MYH EX1.1R	AGACAGCAGAACACGGAGGC	20
1.2	MYH EX1.2F	CAGCCTGGCGCGCGCTAATTGCCT	24	516 bp
	MYH EX1.2R	TTGTCCCTTCCTCGCCATCC	20
2	MYH EX2F	GGCCTCAAAATTTGGCCTC	19	222 bp
	MYH EX2R	CTTTCATGGCCAATGAGCCT	20
3/4	MYH EX3/4F	CTGTGTCCCAAGACCCTGAT	20	377 bp
	MYH EX3/4R	AAAGTGGCCCTGCTCTCAG	19
5/7	MYH EX5/7F	GCAGGTCAGCAGTGTCCTCAT	19	530 bp
	MYH EX5/7R	CTACCACCTGATTGGAGTGCAA	20
8/9	MYH EX8/9F	CACAGGAGGTGAATCAACTCTGG	23	504 bp
	MYH EX8/9R	AGCAGAGCTCCTTTGCAGACA	21
10/11	MYH EX10/11F	CAGCTCTGGTAGGATGTTGGG	21	476 bp
	MYH EX10/11R	ACCTAGAGAGTGGGCTTTGGC	21
12	MYH EX12F	CTTGGCTTGAGTAGGGTTCG	20	286 bp
	MYH EX12R	ATTCCCTCCATTCTCTCTTG	20
13/14	MYH EX13/14F	AAGAGAGAATGGAGGGAATCG	21	496 bp
	MYH EX13/14R	TTCATGTAGAACATGTAGGAAACAC	25
15	MYH EX15F	GCCTGGCCAAAAAAGCATATT	21	288 bp
	MYH EX15R	GAACTATTCCTCCCTCCAGTGAAG	24
16	MYH EX16F	GATTTGCATTTTTAGGACAATTCGA	25	495 bp
	MYH EX16R	TCACTTGAGGCCAGAATCACCT	22

^aFrom http://frodo.wi.mit.edu/primer3/, http://www.ensembl.org/Homo_sapiens/Gene/Summary g=ENSG00000132781, and http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi id=604933.

Basic Protocol 3: Cycle Sequencing of Purified PCR Products

Materials

BigDye Terminator v1.1 cycle sequencing kit and 5× sequencing buffer (Applied Biosystems)
Nuclease-free H₂O
Purified PCR products (Basic Protocol 2)
0.8 pmol/µl primers (Table 10.13.1)
Hi-Di formamide (Applied Biosystems; optional)

200-µl PCR tubes
Thermal cycler (BioRad Tetrad 2)
Edge BioSystems V3 96-well Short Plate, and rubber cushions (http://www.edgebio.com/)
Centrifuge with 96-well deep-well plate carrier
96-well receiver plate: ABI MicroAmp Optical 96-Well Reaction Plate (Applied Biosystems, cat. no. N801-0560)
ABI 3130 capillary sequencer (Applied Biosystems; see Basic Protocol 4)

Additional reagents and equipment for cycle sequencing (unit 7.4) and sequence analysis of MYH gene (Basic Protocol 4)

Basic Protocol 4: Sequence Analysis of MYH Gene

Materials

Purified cycle sequence products (Basic Protocol 3)
Hi-Di Formamide (Applied Biosystems)

Automated Sequencer (ABI 3130 from Applied Biosystems or equivalent)
Mutation Surveyor Software (SoftGenetics; or any other analysis software)

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Literature Cited

Literature Cited
	Al-Tassan, N., Chimel, N.H., Maynard, J., Fleming, N., Livinngston, A.L., Williams, G.T., Hodges, A.K., Davies, D.R., David, S.S., Sampson, J.R., and Cheadle, J.P. 2002. Inherited variants of MYH associated with somatic G:C>T:A mutations in colorectal tumors. Nat. Genet. 30:227-232.
	Chow, E., Thirlwell, C., Macrae, F., and Lipton, L. 2004. Colorectal cancer and inherited mutations in base-excision repair. Lancet Oncol. 5:600-606.
	Jenkins, M.A., Croitoru, M.E., Monga, N., Clearly, S.P., Cotterchi, M., Hopper, J.L., and Gallinger, S. 2006. Risk of colorectal cancer in monoallelic and biallelic carriers of MYH mutations: A population based Case-Family Study. Cancer Epidemiol. Biomarkers Prev. 15:312-314.
	Jo, W.S., Bandipalliam, P., Shannon, K.M., Niendorf, K.B., Chan-Smutko, G., Hur, C., Syngal, S., and Chung, D.C. 2005. Correlation of polyp number and family history of colon cancer with germline MYH mutations. Clin. Gastroenterol. Hepatol. 3:1022-1028.
	Jones, S., Emmerson, P., Maynard, J., Best, J.M., Jordan, S., Williams, G.T., Sampson, J.R., and Cheadle, J.P. 2002. Biallelic germline mutations in MYH predispose to multiple colorectal adenoma and somatic G:C–>T:A mutations. Hum. Mol. Genet. 11:2961-2967.
	Kairupan, C.F., Meldrum, C.J., Crooks, R., Milward, E.A., Spigelman, A.D., Burgess, B., Groombridge, C., Kirk, J., Tucker, K., Ward, R., Williams, R.M., and Scott, R.J. 2005. Mutation analysis of the MYH gene in an Australian series of colorectal polyposis patients with or without germline APC mutations. Int. J. Cancer 10:73-77.
	Lynch, H.T. and de la Chapelle, A. 2003. Hereditary colorectal cancer. New Engl. J. Med. 348:919-932.
	Miller, S.A., Dykes, D.D., and Polesky, H.F. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16:1215.
	Miyaki, M., Iijima, T., Yamaguchi, T., Hishima, T., Tamura, K., Utsumomiya, J., and Mori, T. 2005. Germline mutations of the MYH gene in Japanese patients with multiple colorectal adenomas Mutat. Res. 578:430-433.
	Riegiert-Johnson, D.L., Johnson, R.A., Rabe, K.G., Wang, L., Thomas, B., Baudhuin, L.M., Thibodeau, S.N., and Boardman, L.A. 2007. The value of MUTYH testing in patients with early onset microsatellite stable colorectal cancer referred for hereditary nonpolyposis colon cancer syndrome testing. Genet. Test. 11:361-365.
	Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491.
	Sampson, J.R., Dolwani, S., Jones, S., Eccles, D., Ellis, A., Evans, D.G., Frayling, I., Jordan, S., Maher, E.R., Mak, T., Maynard, J., Pigatto, F., Shaw, J., and Cheadle, J.P. 2003. Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH. Lancet 362:39-41.
	Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467.
	Sieber, O.M., Lipton, L., Crabtree, M., Heinimann, K., Fidalgo, P., Phillips, R.K., Bisgaard, M.L., Orntoft, T.F., Aaltonen, L.A., Hodgson, S.V., Thomas, H.J., and Tomlinson, I.P. 2003. Multiple colorectal adenomas, classic adenomatous polyposis and germ-line mutations in MYH. New Engl. J. Med. 348:791-799.
Internet Resources
	http://agvgd.iarc.fr/
Align GVGD (International Agency for Research on Cancer). Align GVGD is a freely available, Web-based program that combines the biophysical characteristics of amino acids and protein multiple sequence alignments to predict where missense substitutions in genes of interest fall in a spectrum ranging from enriched deleterious to enriched neutral.
	http://frodo.wi.mit.edu/primer3/
Primer3 (v.0.4.0). Primer3 is a widely used program for designing PCR primers on the basis of the DNA sequence to be amplified by PCR, which is an essential and ubiquitous tool in genetics and molecular biology. Primer3 can also design hybridization probes and sequencing primers.
	http://genes.mit.edu/burgelab/rescue-ese/
RESCUE-ESE, an online tool for identifying candidate ESEs in vertebrate exons. Specific short oligonucleotide sequences that enhance pre-mRNA splicing when present in exons, termed exonic splicing enhancers (ESEs), play important roles in constitutive and alternative splicing. A hybrid computational/experimental method, RESCUE-ESE. was developed for identifying sequences with ESE activity.
	http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi process=home
ESE Finder (v. 3.0), an online tool for identifying candidate ESEs, which are specific short oligonucleotide sequences that enhance pre-mRNA splicing when present in exons. These play important roles in constitutive and alternative splicing. A hybrid computational/experimental method, ESE Finder was developed for identifying sequences with ESE activity.
	http://www.edgebio.com/techinfo/protocols/63887_89939v2.pdf
Edge Biotech protocol for using Performa DTR V3 96-well short plates. These are gel-filtration plates that consist of 440-µl columns in a standardized array. This plate provides optimal performance for removal of BigDye v. 3.0 and 3.1 as well as other dye terminators, dNTPs, salts, and other low-molecular-weight materials from sequencing reactions.
	http://www.ensembl.org/Homo_sapiens/Gene/Summary g=ENSG00000132781
(MUTYH-001; ENST00000372115; ENSP00000361187); Ensembl Gene Based Display; A/G-specific adenine DNA glycosylase (MutY homolog) (hMYH); chromosome 1: 45,794,914-45,806,142 reverse strand. The Ensembl project produces genome databases for vertebrates and other eukaryotic species, and makes this information freely available online. Ensembl is a joint project of EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system that produces and maintains automatic annotation on selective eukaryotic genomes. ENSGGene; ENSTTranscript; ENSPProtein.
	http://www.hgvs.org/mutnomen/
Human Genome Variation Society (HGVS)—nomenclature for the description of sequence variations. The Society aims to foster discovery and characterization of genomic variations including population distribution and phenotypic associations. HGVS promotes collection, documentation, and free distribution of genomic variation information and associated clinical variations and endeavors to foster the development of the necessary methodology and informatics.
	http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi id=604933
NCBI–OMIM–604933; MutY, E. coli, homolog of; MUTYH. Online Mendelian Inheritance in Man (OMIM) is a comprehensive, authoritative, and timely compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM focuses on the relationship between phenotype and genotype. It is updated daily, and the entries contain copious links to other genetics resources.
	http://www.softgenetics.com/mutationSurveyor.html
Mutation Surveyor—a unique tool for DNA variant analysis from sequence traces. Mutation Surveyor uses patented anti-correlation technology to provide highly accurate discovery of DNA variants from Sanger sequencing traces. Compatible with outputs from Applied Biosystems, Beckman-Coulter, and MegaBACE systems, Mutation Surveyor rapidly locates all variants and SNP/indels between reference traces and sample/patient traces with excellent accuracy and sensitivity.
	http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_061441.pdf
The AmpliTaq 360 DNA Polymerase protocol provides all the information needed to perform PCR over a wide range of DNA templates, including some of the most challenging GC-rich sequences.