Rapid Diagnosis of Germline p53 Mutation Using the Enzyme Mismatch Cleavage Method
C. Giunta, Ph.D., R. Youil, Ph.D, D. Venter, M.D.,
C.W. Chow, M.D., G. Somers, M.D., A. Lafferty, M.D.,
B. Kemper, Ph.D., and R.G.H. Cotton, D.Sc.
The p53 tumor suppressor gene is the most commonly altered gene in human cancers. Germline mutations in p53 are the genetic alteration underlying predisposition to multiple cancers in Li-Fraumeni syndrome and Li- Fraumeni-like syndrome. We describe a patient who pre- sented with developed adrenocortical carcinoma at age 19 months and a cerebral primitive neuroectodermal tumor at age 5 years. The patient did not have a family history of cancer. We used the enzyme mismatch cleavage (EMC) method to screen for mutations in the p53 gene and found a germline mutation in exon 7 (codon 248). Loss of het- erozygosity analysis in one tumor revealed loss of the wild-type p53 allele. In our report we demonstrate the EMC method to be a rapid and sensitive method for mu- tation detection.
Key Words: Germline p53 mutation-Enzyme mismatch cleavage-Li-Fraumeni syndrome.
Diagn Mol Pathol 5(4): 265-270, 1996.
From the Murdoch Institute (C.G. R.Y. R.G.H.C) and the Royal Children’s Hospital, Departments of Anatomical Pathol- ogy (D.V. C.W.C, G.S) and Endocrinology (A.L), Melbourne. Australia, and the Institute for Genetics, University of Cologne, Germany (B.K).
Address correspondence and reprint requests to Dr. Rima Youil, Avitech Diagnostics. Ltd., 30 Spring Mill Drive. Malvern, PA 19355. U.S.A.
Li-Fraumeni syndrome (LFS) is a rare autosomal dominant syndrome characterized by a high suscep- tibility to cancers (7). LFS is typified by the asso- ciation of sarcoma diagnosed in the proband under the age of 45 with sarcoma, breast cancer, brain tumors, and adrenal cortical carcinoma in a first- degree relative (under the age of 45) and cancer and sarcoma in a close relative. The genetic defect un- derlying the predisposition to cancers in 50% of Li- Fraumeni families was found to be a germline mu- tation in the p53 tumor suppressor gene (8). Germ- line p53 mutations were subsequently found in families with a pattern of cancers similar to LFS but where the proband had a brain tumor or an adrenal cortical carcinoma rather than a sarcoma (1). These germline mutations were observed in the heterozy- gous state in noncancerous cells.
Mutations in the p53 gene are the most common genetic alterations detected in human cancer. They occur mainly within the highly conserved regions (central DNA binding domain of the protein) en- compassing exons 5-8 inclusive, of the gene. Mu- tations outside this region have been found primar- ily in exons 4 and 10 (3). Mutations causing loss of tumor suppressor function may activate p53 to an oncogene in a dominant negative fashion, which in the presence of a cooperating oncogene could lead to cellular transformation (5,15). Loss of the wild- type allele of p53 is found in tumors of Li- Fraumeni-affected individuals, suggesting that in the germline the wild-type allele is functioning even in the presence of the dominant negative mutant allele (5).
Germline mutations have been found in only half of Li-Fraumeni-affected families, suggesting that alterations in other genes rather than p53 may be involved in the disease or the current mutation de- tection strategies have failed in detecting mutations
in the other half of the families (12). We present a fast, easy, reliable, and highly sensitive mutation detection protocol with the use of the enzyme mis- match cleavage (EMC) method (13,14). The EMC method relies on the use of a T4 phage resolvase, T4 endonuclease VII, previously shown in model ex- periments to cut mismatches (11), to detect and cleave at mismatches in hybrids of normal DNA probe with DNA strands containing single base mis- matches.
Unlike existing mutation detection techniques, the EMC method (see Fig. 1) allows for the identi- fication of point mutations (with a detection rate of near 100%) in kilobase lengths of DNA using a sin- gle set of conditions. It has, so far, been described only in the detection of known mutations. This re- port is the first attempt at using this novel approach in the molecular diagnosis of a germline p53 muta- tion in a 5-year-old patient with two primary tumors and no family history of cancer.
PCR
WT genomic DNA
Tumour DNA
purify and End-label
purify
G
C
Mix, denature und anneal
G
T
Digest with enzyme to cleave at mismatch
G
T
Resolve on denaturing gel and autoradiograph
No Enzyme
With Enzyme
Uncleaved probe →
Cleavage products
MATERIALS AND METHODS
Clinical Data
The patient, a Caucasian girl, presented at 19 months of age with virilism. Investigations showed an adrenocortical carcinoma (Fig. 2), which was re- sected. Two years later she developed a single pul- monary metastasis, which was also resected. Two years after this she presented with left-sided weak- ness. Imaging showed a large tumor in the right posterior frontal lobe, which biopsy showed to be a primitive neuroectodermal tumor (Fig. 3). The pa- tient was treated with systemic chemotherapy com- prising VP-16, carboplatin, and methotrexate, but her tumor continued to grow and she died 3 months later. Due to the unusual combination of these tu- mors in a young child, a mutation in the p53 gene was suspected. Blood samples were collected from the patient, parents, and siblings. Frozen samples of the adrenal tumor and autopsy samples of the spleen and kidney were also examined.
DNA Isolation
DNA extraction from peripheral blood, kidney, and spleen specimens and fresh tumor tissue (0.1-1 mg) was homogenized in TNE [0.1 M NaCl, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 1% (wt/vol) sodium dodecyl sulfate SDS] and digested with proteinase K/SDS followed by phenol/chloroform extraction and sodium acetate/ethanol precipitation (10).
PCR Amplification and Template Purification
Amplification of exons 5-6 (408 bp) and 7-9 (780 bp) inclusive of the p53 gene was carried out on 100 ng of genomic DNA using 50 pmol of primers and 0.1 mM dNTPs in a reaction buffer containing 1.5 mM MgCl2. The total reaction volume was 100 ul, with 0.5 U of Taq DNA polymerase (Boehringer). The DNA was amplified in a thermal cycler (Cor- bett Research, Sydney, Australia) at 95℃ for 5 min, followed by 35 cycles of 95℃ for 50 s, 56°℃ (exons 7-9) or 58℃ (exons 5 and 6) for 50 s, and 72℃ for 90 s. The final products were further extended at 72℃ for 5 min. The oligonucleotides used to amplify ex- ons 5 and 6 of p53 and exons 7-9 were as described previously (9). The products were separated by electrophoresis on a 1% agarose gel and then the bands were excised and purified using the Qiaex gel extraction kit (Qiagen), yielding 1-1.8 µg of product in 30 ul of TE. DNA concentrations were estimated by analysis of 2 ul on a 1% agarose gel in compari-
son with standards. The polymerase chain reaction (PCR) amplification and purification took about 8.5 h.
EMC Analysis
Detection of point mutations was performed us- ing the EMC method (13,14). For each reaction, 5 ng of PCR-amplified wild-type DNA labeled with 1 ul of 3,000 mCi [y-32PJATP using polynucleotide kinase as described previously (13) was mixed with 50 ng of unlabeled PCR-amplified patient DNA and
then boiled for 5 min. The mixture was immediately placed in a 65℃ heating block and incubated for 1 h to allow for heteroduplex formation. The heterodu- plex was ethanol precipitated and resuspended to give 1,000 cpm/5 ul in distilled water. Samples (50- 60 ng) of this labeled duplex DNA were digested with 1,000 U of T4 endonuclease VII enzyme per reaction. Incubation was performed at 37℃ for 45- 60 min. A control tube without enzyme was in- cluded for each sample. The reaction was stopped by Na acetate/ethanol precipitation, and the pellet resuspended in denaturing loading dye and kept
Control
Germ Line
Gierm Line
Tumour
M
234 bp →
194 bp
118 bp -
€ :90 bp
72 bp
overnight at -20℃. The EMC procedure took about 3.5 h.
Electrophoresis and Autoradiography
A sample (6 pl) of the digested DNA was sepa- rated on an 8% polyacrylamide-urea sequencing gel
and detected by autoradiography ( - 70℃ for 16 h). The electrophoresis and autoradiography took about 18 h.
Direct Sequencing
Characterization of the mutation was carried out by direct sequencing of two independent purified PCR products. The dideoxy chain termination method and the Sequenase sequencing kit (USB) were used.
Loss of Heterozygosity (LOH) Analysis
LOH at the p53 gene locus was detected by am- plification of a highly polymorphic dinucleotide re- peat polymorphism (the TP53 CA locus) (4).
Primers (AC strand primer, ACTGCCACTCCT- TGCCCCATTC; GT strand primer, AGGGATAC- TATTCAGCCCGAGGTG) (4) were added to a 25- ul PCR reaction mixture containing 10 pmol of each primer, 200 umol each of dATP, dTTP, and dGTP, 50 mM dCTP, standard PCR reaction buffer con- taining 1.5 mM MgCl2, 0.5 U of Taq polymerase, 10-20 ng of DNA, and 0.05 pCi of [@-32P]dCTP. PCR was performed at an initial temperature of 94℃ for 3 min, followed by 35 cycles consisting of 2 min at 60℃ and 1 min at 94℃, with a final exten- sion of 5 min at 72℃ (4). PCR products were ana- lyzed on a sequencing gel (6% polyacrylamide, 8 M urea) using a sequencing reaction as size markers. Autoradiography was done at room temperature for 1 day without intensifying screens. Products were in the 96- to 116-bp range. LOH was detected as a decrease in the relative intensity of the bands in comparison with the germline control.
Control
AB
AGCTAGCT
Msp I site
€ G/A
G/A→
Adrenal Cortical Tissue
Spleen
Kidney
Control Blood
AGCTAGCTAGCTAGCT
RESULTS
EMC and Direct Sequencing
Exons 5-9 inclusive of the p53 gene was scanned for mutations by the EMC method in each of the patient, parents, and siblings (two brothers). The analysis showed a novel band present in the pa- tient’s germline and tumor DNA but not present in the normal control germline DNA and suggested a mutation about 90 bp from either end of the 780-bp fragment (Fig. 4). EMC analysis of parents’ and sib- lings’ germline DNA did not show this novel band (data not shown). Direct sequencing to characterize the mutation was performed with a 5’-primer lo- cated in exon 7 and a 3’-primer located in exon 9. A mutation was found 85 bp from the 5’ end of the 780-bp fragment (codon 248). This resulted in a CGG → CAG transition (Fig. 5) causing an Arg-to- GIn amino acid change. The mutation was then con- firmed on a second independent purified PCR prod- uct to exclude false positives due to Taq polymer- ase infidelity. The adrenal cortical carcinoma, spleen and kidney sections of the patient, who had since died, revealed the presence of the same mu- tation (Fig. 6). Direct sequencing of exon 7 of the parents’ and siblings’ genomic DNA confirmed the absence of the mutation as previously indicated by the EMC analysis. Direct sequencing of exon 7 from spleen, kidney, and tumor revealed the muta- tion to be heterozygous in the normal tissues and homozygous in the adrenocortical carcinoma (Fig. 6). The possibility of LOH for the p53 gene locus was investigated. The analysis was performed by PCR amplification of the TP53 CA repeat polymor- phism (microsatellite) within the p53 locus in nor- mal and tumor tissues. Only the adrenal tumor was examined and revealed LOH (Fig. 7). Each member
of the family was subsequently investigated for the presence of the same mutation. The EMC gave a negative result revealing the mutation to be a de novo germline mutation.
DISCUSSION
The data presented in this report show the finding of a de novo germline mutation in a patient diag- nosed with two primary malignant tumors who did not have a family history of cancer. The mutation was in codon 248, a known mutational hot spot pre- viously described in LFS. The diagnosis of a germ- line p53 mutation in this patient using the EMC method was rapid and efficient. The method has been shown to be capable of screening kilobase lengths of DNA (14), which facilitates analysis of large genes. The procedure can be performed in just over 20 h following DNA amplification and involves only two manipulations, excluding heteroduplex formation. The EMC detection rate of 98% is based on 2 false negatives of >100 known mutations tested (14,16) making it competitive with methods such as single-strand conformation polymorphism (SSCP) and the chemical cleavage of mismatch (CCM) method (2). Using a single set of conditions, the SSCP method (despite being the most com- monly used mutation detection method) will detect only 35-80% of mutations (2). The CCM method will detect near 100% of mutations, however, the use of four noxious chemicals and the multiple ma- nipulations involved have hindered its widespread use. The introduction of T4 endonuclease VII is an attempt to retain the positive features of the chem- ical cleavage method (scanning kilobase lengths of DNA and localizing mutations) and replace the neg- ative aspects with a safer alternative. We believe
Adrenocortical
Spleen
Tumour
Kidney
Blood
(Mother)
€ 116 bp
< 106 bp
LOH→
€ 96 bp
that the EMC method described here is a useful and worthwhile approach in the study of cancer genet- ics. ☐
Acknowledgment: This work was supported by a grant from the Italian Association for Cancer Research (AIRC;
Associazione Italiana per la Ricerca sul Cancro) held by C.G. and the National Health and Research Foundation (R.Y. and R.G.H.C.).
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