p53 Testing for Li‐Fraumeni and Li‐Fraumeni‐Like Syndromes

Kelly Gonzalez1, Cindy Fong1, Carolyn Buzin1, Steve S. Sommer1, Juan‐Sebastian Saldivar1

1 City of Hope National Medical Center, Duarte, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 10.10
DOI:  10.1002/0471142905.hg1010s57
Online Posting Date:  April, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Li‐Fraumeni Syndrome (LFS; OMIM #151623) is an autosomal dominant cancer predisposition syndrome characterized by early onset tumors including sarcomas, breast cancer, leukemia, brain tumors, and adrenocortical carcinoma. Li‐Fraumeni syndrome is primarily attributed to germline mutations in the p53 tumor suppressor gene, which encodes tumor protein 53. In addition to germline p53 mutations, the p53 gene is the most commonly mutated gene in human cancers, with as much as 50% of tumors containing somatic p53 mutations. This unit provides a protocol to perform germline mutation analysis of the p53 gene. The protocol includes steps for amplification and sequencing of the entire coding region of the p53 gene (exons 2 to 11). The protocol was designed for detecting germline alterations from DNA extracted from blood; however, with some additional optimization, it could also be used to detect somatic mutations in DNA extracted from tumors. Curr. Protoc. Hum. Genet. 57:10.10.1‐10.10.11. © 2008 by John Wiley & Sons, Inc.

Keywords: p53; TP53; Li‐Fraumeni Syndrome

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: PCR Amplification of Exons 2 to 11 of the p53 Gene and PCR Product Purification
  • Basic Protocol 2: Cycle Sequencing of Purified PCR Products
  • Basic Protocol 3: Sequence Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: PCR Amplification of Exons 2 to 11 of the p53 Gene and PCR Product Purification

  Materials
  • 10× PCR buffer with 15 mM MgCl 2 (comes with AmpliTaq enzyme from Roche)
  • 1.25 mM dNTP mix (1.25 mM each dNTP)
  • 2.5 µM PCR primer mixes (see Table 10.10.1): mixes of upstream and downstream primers for each amplicon, with each primer at 2.5 µM
  • 5 U/µl AmpliTaq Gold polymerase (Roche)
  • PCR‐grade H 2O
  • 50% DMSO
  • 25 mM MgCl 2
  • 50 ng/ml genomic DNA ( appendix 3B)
  • Sterile water
  • 1× TAE buffer ( appendix 2D)
  • 10 U/µl exonuclease I enzyme (Exo; from USB)
  • 2 U/µl shrimp alkaline phosphatase enzyme (SAP; from USB)
  • 200 µl PCR tubes
  • Thermal cycler (Applied Biosystems 9700 or equivalent)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
    Table 0.0.1   Materialsp53 PCR Primers

    Exon Primer Primer sequence 5′→3′ Primer length Fragment size
    2‐4 P53E2‐4PD GGG TTG TGG TGA AAC ATT GGA AGA 24 1030 bp
    P53E2‐4PU GCT GAG GGT GTG ATG GGA TGG 21
    5‐6 P53E5‐6PD GCT CCT GAG GTG TAG ACG CCA A 22 586 bp
    P53E5‐6PU GCC ACT GAC AAC CAC CCT TAA C 22
    7 P53E7PD CAA GGC GCA CTG GCC TCA 18 194 bp
    P53E7PU GGC ACA GCA GGC CAG TGT 18
    8‐9 P53E8‐9PD TGG GAC AGG TAG GAC CTG AT 20 505 bp
    P53E8‐9PU CCA GGA GCC ATT GTC TTT GAG GCA TCA CT 29
    10‐11 P53E10‐11PD GCT GTA TAG GTA CTT GAA GTG CAG TTT CT 29 1416 bp
    P53E10‐11PU AGG CCA ACT TGT TCA GTG GAG C 22

Basic Protocol 2: Cycle Sequencing of Purified PCR Products

  Materials
  • ABI BigDye Terminator Kit v1.1 and 5× sequencing buffer (Applied Biosystems)
  • Milli‐Q H 2O
  • Purified PCR products (see protocol 1)
  • 1.5 mM sequencing primers (see Table 10.10.2)
  • CleanSeq kit (Agencourt) including:
    • Magnetic bead solution
    • Magnetic purification beads
  • 85% ethanol (freshly made)
  • 0.3 mM EDTA (elution buffer)
  • Thermal cycler (Applied Biosystems 9700 or equivalent)
  • Magnetic purification tray (Agencourt)
    • 200‐µl pipet
    • 10‐µl tip
      Table 0.0.2   Materialsp53 Sequencing Primers

      Exon Primer Primer sequence 5′→3′ Primer length
      2‐3 P53E2‐3SD TGT CTC AGA CAC TGG CAT GG 20 nt
      P53E2‐3SU TGA AAA GAG CAG TCA GAG GAC 21 nt
      4 P53E4SD AAG GGT TGG GCT GGG GAC CT 20 nt
      P53E4SU AGT TCC AAA CAA AAG AAA TGC AG 23 nt
      5‐6 P53E5‐6SD GCT CCT GAG GTG TAG ACG CCA A 22 nt
      P53E5‐6SU ACT TTG CAC ATC TCA TGG GG 20 nt
      7 P53E7SD CAA GGC GCA CTG GCC TCA 18 nt
      P53E7SU GGC ACA GCA GGC CAG TGT 18 nt
      8‐9 P53E8‐9SD TGG GAC AGG TAG GAC CTG AT 20 nt
      P53E8‐9SU CCA GGA GCC ATT GTC TTT GAG GCA TCA CT 29 nt
      10 P53E10SD CAA TTG TAA CTT GAA CCA TCT TT 23 nt
      P53E10SU GGC AGG ATG AGA ATG GAA TC 20 nt
      11 P53E11SD GCA CAG ACC CTC TCA CTC A 19 nt
      P53E11SU CAA AAT GGC AGG GGA GGG A 19 nt

Basic Protocol 3: Sequence Analysis

  Materials
  • Purified cycle sequence products (see protocol 2)
  • Automated sequencer (ABI 3730 from Applied Biosystems or equivalent)
  • Sequence analysis software (Sequencher by Gene Codes or equivalent)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Bartek, J., Falck, J., and Lukas, J. 2001. CHK2 kinase‐a busy messenger. Nat. Rev. Mol. Cell Biol. 2: 877‐886.
   Bell, D.W., Varley, J.M., Szydlo, T.E., Kang, D.H., Wahrer, D.C., Shannon, K.E., Lubratovich, M., Verselis, S.J., Isselbacher, K.J., Fraumeni, J.F., Birch, J.M., Li, F.P., Garber, J.E., and Haber, D.A. 1999. Heterozygous germ line hCHK2 mutations in Li‐Fraumeni syndrome. Science 286: 2528‐2531.
   Birch, J.M., Hartley, A.L., Tricker, K.J., Prosser, J., Condie, A., Kelsey, A.M., Harris, M., Morris Jones, P.H., Binchy, A., Crowther, D., Craft, A.W., Eden, O.B., Evans, D.G.R., Thompson, E., Mann, J.R., Martin, J., Mitchell, E.L.D., and Santibáñez‐Koref, M.F. 1994. Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li‐Fraumeni families. Cancer Res. 54: 1298‐1304.
   Birch, J.M., Alston, R.D., McNally, R.J., Evans, D.G., Kelsey, A.M., Harris, M., Eden, O.B., and Varley, J.M. 2001. Relative frequency and morphology of cancers in carriers of germline TP53 mutations. Oncogene 20: 4621‐4628.
   Carmichael, P.L., Mills, J.J., Campbell, M., Basu, M., and Caldwell, J. 2001. Mechanisms of hormonal carcinogenesis in the p53± hemizygous knockout mouse: studies with diethylstilbestrol. Toxicol. Pathol. 29: 155‐160.
   Chompret, A., Brugieres, L., Ronsin, M., Gardes, M., Dessarps‐Freichey, F., Abel, A., Hua, D., Ligot, L., Dondon, M.G., Bressac‐de Paillerets, B., Frebourg, T., Lemerle, J., Bonaiti‐Pellie, C., and Feunteun, J. 2000. P53 germline mutations in childhood cancers and cancer risk for carrier individuals. Br. J. Cancer 82: 1932‐1937.
   Chompret, A., Abel, A., Stoppa‐Lyonnet, D., Brugieres, L., Pages, S., Feunteun, J., and Bonaiti‐Pellie, C. 2001. Sensitivity and predictive value of criteria for p53 germline mutation screening. J. Med. Genet. 38: 43‐47.
   Eeles, R.A. 1995. Germline mutations in the TP53 gene. Cancer Surv. 25: 101‐124.
   Eng, C., Hampel, H., and de la Chapelle, A. 2001. Genetic testing for cancer predisposition. Annu. Rev. Med. 52: 371‐400.
   Field, M., Shanley, S., and Kirk, J. 2007. Inherited cancer susceptibility syndromes in paediatric practice. J. Paediatr. Child Health 43: 219‐229.
   Ford, M.J. and Hanawalt, P.C. 1995. Li‐Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription‐coupled repair and enhanced UV resistance. Proc. Natl. Acad. Sci. U.S.A. 92: 8876‐8880.
   Frebourg, T., Barbier, N., Yan, Y.X., Garber, J.E., Dreyfus, M., Fraumeni, J. Jr., Li, F.P., and Friend, S.H. 1995. Germ‐line p53 mutations in 15 families with Li‐Fraumeni syndrome. Am. J. Hum. Genet. 56: 608‐615.
   Garber, J.E., Burke, E.M., Lavally, B.L., Billett, A.L., Sallan, S.E., Scott, R.M., Kupsky, W., and Li, F.P. 1990. Choroid plexus tumors in the breast cancer‐sarcoma syndrome. Cancer 66: 2658‐2660.
   Garber, J.E., Goldstein, A.M., Kantor, A.F., Dreyfus, M.G., Fraumeni, J.F. Jr., and Li, F.P. 1991. Follow‐up study of twenty‐four families with Li‐Fraumeni syndrome. Cancer Res. 51: 6094‐6097.
   Hainaut, P. and Hollstein, M. 2000. p53 and human cancer: The first ten thousand mutations. Adv. Cancer Res. 77: 81‐137.
   Han, Z., Wei, W., Dunaway, S., Darnowski, J.W., Calabresi, P., Sedivy, J., Hendrickson, E.A., Balan, K.V., Pantazis, P., and Wyche, J.H. 2002. Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin. J. Biol. Chem. 277: 17154‐17160.
   Hill, K.A., Buettner, V.L., Heidt, A., Chen, L.L., Li, W., Gonzalez, K.D., Wang, J.C., Scaringe, W.A., and Sommer, S.S. 2006. Most spontaneous tumors in a mouse model of Li‐Fraumeni syndrome do not have a mutator phenotype. Carcinogenesis 27: 1860‐1866.
   Hisada, M., Garber, J.E., Fung, C.Y., Fraumeni, J.F. Jr., and Li, F.P. 1998. Multiple primary cancers in families with Li‐Fraumeni syndrome. J. Natl. Cancer Inst. 90: 606‐611.
   Hussain, S.P. and Harris, C.C. 2006. p53 biological network: At the crossroads of the cellular‐stress response pathway and molecular carcinogenesis. J. Nippon Med. Sch. 73: 54‐64.
   Hwang, S.J., Lozano, G., Amos, C.I., and Strong, L.C. 2003. Germline p53 mutations in a cohort with childhood sarcoma: Sex differences in cancer risk. Am. J. Hum. Genet. 72: 975‐983.
   Kaufmann, S.H. and Earnshaw, W.C. 2000. Induction of apoptosis by cancer chemotherapy. Exp. Cell Res. 256: 42‐49.
   Krutilkova, V., Trkova, M., Fleitz, J., Gregor, V., Novotna, K., Krepelova, A., Sumerauer, D., Kodet, R., Siruckova, S., Plevova, P., Bendova, S., Hedvicakova, P., Foreman, N.K., and Sedlacek, Z. 2005. Identification of five new families strengthens the link between childhood choroid plexus carcinoma and germline TP53 mutations. Eur. J. Cancer 41: 1597‐1603.
   Lane, D.P. 1992. p53, guardian of the genome. Nature 358: 15‐16.
   Li, F.P., Fraumeni, J.F. Jr., Mulvihill, J.J., Blattner, W.A., Dreyful, M.G., Tucker, M.A., and Miller, R.W. 1988. A cancer family syndrome in twenty‐four kindreds. Cancer Res. 48: 5358‐5362.
   Libe, R. and Bertherat, J. 2005. Molecular genetics of adrenocortical tumours, from familial to sporadic diseases. Eur. J. Endocrinol. 153: 477‐487.
   Limacher, J.M., Frebourg, T., Natarajan‐Ame, S., and Bergerat, J.P. 2001. Two metachronous tumors in the radiotherapy fields of a patient with Li‐Fraumeni syndrome. Int. J. Cancer 96: 238‐242.
   Liu, Q., Li, X., Chen, J.S., and Sommer, S.S. 2003. Robust dosage‐PCR for detection of heterozygous chromosomal deletions. Biotechnique 34: 558‐570.
   Malkin, D., Li, F.P., Strong, L.C., Fraumeni, J.F. Jr., Nelson, C.E., Kim, D.H., Kassel, J., Gryka, M.A., Bischoff, F.Z., Tainsky, M.A., Friend, S.H. 1990. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250: 1233‐1238.
   Morris, S.M. 2002. A role for p53 in the frequency and mechanism of mutation. Mutat. Res. 511: 45‐62.
   Moule, R.N., Jhavar, S.G., and Eeles, R.A. 2006. Genotype phenotype correlation in Li‐Fraumeni syndrome kindreds and its implications for management. Fam. Cancer 5: 129‐133.
   Nagy, R., Sweet, K., and Eng, C. 2004. Highly penetrant hereditary cancer syndromes. Oncogene 23: 6445‐6470.
   Nguyen, V., Liu, Q., and Sommer, S.S. 2007. A large‐scale validation of dosage analysis by robust dosage‐polymerase chain reaction. Anal. Biochem. 371: 37‐42.
   Petitjean, A., Mathe, E., Kato, S., Ishioka, C., Tavtigian, S.V., Hainaut, P., and Olivier, M. 2007. Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: Lessons from recent developments in the IARC TP53 database. Hum. Mutat. 28: 622‐629.
   Salmon, A., Amikam, D., Sodha, N., Davidson, S., Basel‐Vanagaite, L., Eeles, R.A., Abeliovich, D., and Peretz, T. 2007. Rapid development of post‐radiotherapy sarcoma and breast cancer in a patient with a novel germline ‘de‐novo’ TP53 mutation. Clin. Oncol. 19: 490‐493.
   Sedlacek, Z., Kodet, R., Seemanova, E., Vodvarka, P., Wilgenbus, P., Mares, J., Poustka, A., and Goetz, P. 1998. Two Li‐Fraumeni syndrome families with novel germline p53 mutations: Loss of the wild‐type p53 allele in only 50% of tumours. Br. J. Cancer 77: 1034‐1039.
   Shiloh, Y. 2001. ATM and ATR: Networking cellular responses to DNA damage. Curr. Opin. Genet. Dev. 11: 71‐77.
   Strong, L.C. 2003. General keynote: Hereditary cancer: Lessons from Li‐Fraumeni syndrome. Gynecol. Oncol. 88: S4‐S7.
   Varley, J.M. 2003. Germline TP53 mutations and Li‐Fraumeni syndrome. Hum. Mutat. 21: 313‐320.
   Varley, J.M., Evans, D.G., and Birch, J.M. 1997. Li‐Fraumeni syndrome‐a molecular and clinical review. Br. J. Cancer 76: 1‐14.
   Varley, J.M., McGown, G., Thorncroft, M., James, L.A., Margison, G.P., Forster, G., Evans, D.G., Harris, M., Kelsey, A.M., and Birch, J.M. 1999. Are there low‐penetrance TP53 Alleles? Evidence from childhood adrenocortical tumors. Am. J. Hum. Genet. 65: 995‐1006.
   Wang, J., Gonzalez, K.D., Scaringe, W.A., Tsai, K., Liu, N., Gu, D., Li, W., Hill, K.A., and Sommer, S.S. 2007. Evidence for mutation showers. Proc. Natl. Acad.Sci. U.S.A. 104: 8403‐8408.
   Wong, P., Verselis, S.J., Garber, J.E., Schneider, K., DiGianni, L., Stockwell, D.H., Li, F.P., and Syngal, S. 2006. Prevalence of early onset colorectal cancer in 397 patients with classic Li‐Fraumeni syndrome. Gastroenterology 130: 73‐79.
   Yonish‐Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A., and Oren, M. 1991. Wild‐type p53 induces apopotosis of myeloid leukemic cells that is inhibited by interleukin‐6. Nature 353: 345‐347.
Internet Resources
   http://www‐p53.iarc.fr/
  Information on p53 at the IARC mutation database.
   http://www.genecodes.com
  Seqeuncher software, demo product available for download.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library