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CDKN2gene
Molecularand CellularProbes(1995) 9, 465-466
Polymorphism Report
A frequent polymorphism in exon 1 of the gene
p16/CDKN2
J. Fueyo,* C. Gomez-Manzano, W. K. A. Yung, W. Zhang, P. S. Y. Lee, S. Majumder, M. Pershouse, V. A. Levin and A. P. Kyritsis Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 100, Houston, Texas 77030, USA (Received 12 July 1995, Accepted 18July 1995)
Source/Description
(sense) and 1B, 5"-TCAGAGCCTCTCTCTGGI-rCTTTCA-3' (antisense). The reaction was repeated for 35 cycles on a Gene Amp PCR system 9600 (PerkinElmer Cetus) at 96°C for 1 min, 56°C for 1-5 min, 72°C for 1 min. The PCR reaction included 100 ng of each primer, 2-5 mM each dNTP, 2-5 Ill of PCR buffer 10 x (100 mM Tris-HCl; 15 mM MgCI2; 500 mM KCI; pH 8"3) and 1 U of TaqDNA polymerase (Boehringer Mannheim) in a total volume of 25 Ill. The reaction yielded a 490-bp fragment. One microlitre of this PCR product was used as a template in a second PCR reaction with similar conditions. The nested primers of the second PCR were: 2A, 5'-ATGGAGCCTTC GGCTGACTGG-3' (sense) and 2B, 5'-GGTGCAGCA CCACCAGCGTGT-3' (antisense). The nested PCR reaction resulted in a 271-bp fragment. A portion of the PCR-amplified product (1.5 Ill) was subjected to a BsaH I (New England Biolabs) restriction enzyme digestion at 37°C for 4 h. Some samples (1-5 tll) were subjected to Ban I (New England Biolabs) at 27°C for 4h. All samples were analysed by 3% NuSieve agarose gel electrophoresis.
We describe here a novel intragenic polymorphism at the 110th nucleotide within the coding sequence of the pl6/CDKN2 gene.' The polymorphic variants consist of either a C (allele C) or a T (allele T) of codon 37, resulting in proline or leucine, respectively. Using a simple assay involving PCR amplification and restriction enzyme digestion, we examined the incidence of this polymorphism in lymphocytes of 55 unrelated individuals. Among them, 35 subjects (63.6%) were homozygous for the allele C; 17 (30.9%) were heterozygous, and only three individuals (5.4%) were homozygous for the previously reported allele T. Importantly, the allele C reported here was present in 94.5% of the subjects, either heterozygous or homozygous. The characterization of this polymorphism provides a useful tool to monitor loss of heterozygosity of p 16/CDKN2gene in primary tumors and in families with germline mutations, such as families with 9p melanoma (2,3).
Nested PCR and restriction enzyme digestion conditions
PCR-restriction fragment length polymorphism analysis
Lymphocyte cDNA (100 ng) was used as PCR template. The set of primers of the first PCR were: 1A, 5'-ATGGAGCCGGCGGCGGGGGAGCATGGA-3"
Samples subjected to PCR and enzymatic digestion with BsaH I or Banl revealed the expected fragment
* Author to whom correspondenceshouldbe addressed. 0890-85081951060465 + 02 $12.00/0
465
© 1995 AcademicPressLimited
466
J. Fueyo et aL
lengths, resulting in three possible patterns. The homozygous pattern for allele T: only one band of 271 nucleotides; the homozygous pattern for the allele C: two bands of 186 and 85 nucleotides, respectively, and the heterozygous pattern for allele C: three bands of 271, 186 and 85 bp each. Similar restriction fragments were obtained with both enzymes.
Table 1. P16/CDKN2 codon 37 genotype distribution Codon
Allele
n
%
EGD
Pro/Pro Leu/Leu Pro/Leu
(CJC) (T/T) (C/T)
35 3 17
63.6 5.4 30.9
62.5 4.3 33
EGD, expectedgenotype distribution. observed and expected genotype distributions are shown in Table 1.
Direct automated and manual sequencing of PCR-cDNA products Sequence analyses were automatically performed on the Applied Biosystems 373A automatic sequencer. Direct manual sequencing of PCR product was performed using asS and the Sequenase Kit (US Biochemical, Cleveland, OH, USA). Direct automated sequencing of 12 samples homozygous for the allele C confirmed the presence ofa C in the 110th nucleotide position. Three of these samples were also sequenced with the dideoxynucleotide chain terminating method to confirm the results. Similarly, sequencing of two samples homozygous for the allele T demonstrated a T in the same position.
Frequency Allele frequencies were evaluated in 110 chromosomes from 55 unrelated Caucasian individuals. Among them, there were eight healthy normal subjects and 47 patients with malignant gliomas. The
Mendelian inheritance Mendelian inheritance was observed in four families.
ACKNOWLEDGEMENTS We thank Mrs Kimberly Herrick for editorial assistance. This work was supported in part by a R29 NS32269-01A1, a CCSG-CA16672 and a PO1-CA55261 grant from the National Institutes of Health. Drs J Fueyo and C GomezManzano were supported by grants FIS 94/5583 and 94/ 5372, from Madrid, Spain.
REFERENCES I. Serrano, M., Hannon, G. J. & Beach, D. (I 993). A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366, 704-7. 2. Hussussian, C. J., Struewing, J. P., Goldstein, A. M. etal. (I 994). Germline pl 6 mutations in familial melanoma. Nature Genetics 8, 15-21. 3. Kamb, A., Shattuck-Eidens, D., Eeles, R. et al. (1994) Analysis of the pl 6 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus. Nature Genetics 8, 22-6.
Polymorphism Report
A frequent polymorphism in exon 1 of the gene
p16/CDKN2
J. Fueyo,* C. Gomez-Manzano, W. K. A. Yung, W. Zhang, P. S. Y. Lee, S. Majumder, M. Pershouse, V. A. Levin and A. P. Kyritsis Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 100, Houston, Texas 77030, USA (Received 12 July 1995, Accepted 18July 1995)
Source/Description
(sense) and 1B, 5"-TCAGAGCCTCTCTCTGGI-rCTTTCA-3' (antisense). The reaction was repeated for 35 cycles on a Gene Amp PCR system 9600 (PerkinElmer Cetus) at 96°C for 1 min, 56°C for 1-5 min, 72°C for 1 min. The PCR reaction included 100 ng of each primer, 2-5 mM each dNTP, 2-5 Ill of PCR buffer 10 x (100 mM Tris-HCl; 15 mM MgCI2; 500 mM KCI; pH 8"3) and 1 U of TaqDNA polymerase (Boehringer Mannheim) in a total volume of 25 Ill. The reaction yielded a 490-bp fragment. One microlitre of this PCR product was used as a template in a second PCR reaction with similar conditions. The nested primers of the second PCR were: 2A, 5'-ATGGAGCCTTC GGCTGACTGG-3' (sense) and 2B, 5'-GGTGCAGCA CCACCAGCGTGT-3' (antisense). The nested PCR reaction resulted in a 271-bp fragment. A portion of the PCR-amplified product (1.5 Ill) was subjected to a BsaH I (New England Biolabs) restriction enzyme digestion at 37°C for 4 h. Some samples (1-5 tll) were subjected to Ban I (New England Biolabs) at 27°C for 4h. All samples were analysed by 3% NuSieve agarose gel electrophoresis.
We describe here a novel intragenic polymorphism at the 110th nucleotide within the coding sequence of the pl6/CDKN2 gene.' The polymorphic variants consist of either a C (allele C) or a T (allele T) of codon 37, resulting in proline or leucine, respectively. Using a simple assay involving PCR amplification and restriction enzyme digestion, we examined the incidence of this polymorphism in lymphocytes of 55 unrelated individuals. Among them, 35 subjects (63.6%) were homozygous for the allele C; 17 (30.9%) were heterozygous, and only three individuals (5.4%) were homozygous for the previously reported allele T. Importantly, the allele C reported here was present in 94.5% of the subjects, either heterozygous or homozygous. The characterization of this polymorphism provides a useful tool to monitor loss of heterozygosity of p 16/CDKN2gene in primary tumors and in families with germline mutations, such as families with 9p melanoma (2,3).
Nested PCR and restriction enzyme digestion conditions
PCR-restriction fragment length polymorphism analysis
Lymphocyte cDNA (100 ng) was used as PCR template. The set of primers of the first PCR were: 1A, 5'-ATGGAGCCGGCGGCGGGGGAGCATGGA-3"
Samples subjected to PCR and enzymatic digestion with BsaH I or Banl revealed the expected fragment
* Author to whom correspondenceshouldbe addressed. 0890-85081951060465 + 02 $12.00/0
465
© 1995 AcademicPressLimited
466
J. Fueyo et aL
lengths, resulting in three possible patterns. The homozygous pattern for allele T: only one band of 271 nucleotides; the homozygous pattern for the allele C: two bands of 186 and 85 nucleotides, respectively, and the heterozygous pattern for allele C: three bands of 271, 186 and 85 bp each. Similar restriction fragments were obtained with both enzymes.
Table 1. P16/CDKN2 codon 37 genotype distribution Codon
Allele
n
%
EGD
Pro/Pro Leu/Leu Pro/Leu
(CJC) (T/T) (C/T)
35 3 17
63.6 5.4 30.9
62.5 4.3 33
EGD, expectedgenotype distribution. observed and expected genotype distributions are shown in Table 1.
Direct automated and manual sequencing of PCR-cDNA products Sequence analyses were automatically performed on the Applied Biosystems 373A automatic sequencer. Direct manual sequencing of PCR product was performed using asS and the Sequenase Kit (US Biochemical, Cleveland, OH, USA). Direct automated sequencing of 12 samples homozygous for the allele C confirmed the presence ofa C in the 110th nucleotide position. Three of these samples were also sequenced with the dideoxynucleotide chain terminating method to confirm the results. Similarly, sequencing of two samples homozygous for the allele T demonstrated a T in the same position.
Frequency Allele frequencies were evaluated in 110 chromosomes from 55 unrelated Caucasian individuals. Among them, there were eight healthy normal subjects and 47 patients with malignant gliomas. The
Mendelian inheritance Mendelian inheritance was observed in four families.
ACKNOWLEDGEMENTS We thank Mrs Kimberly Herrick for editorial assistance. This work was supported in part by a R29 NS32269-01A1, a CCSG-CA16672 and a PO1-CA55261 grant from the National Institutes of Health. Drs J Fueyo and C GomezManzano were supported by grants FIS 94/5583 and 94/ 5372, from Madrid, Spain.
REFERENCES I. Serrano, M., Hannon, G. J. & Beach, D. (I 993). A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366, 704-7. 2. Hussussian, C. J., Struewing, J. P., Goldstein, A. M. etal. (I 994). Germline pl 6 mutations in familial melanoma. Nature Genetics 8, 15-21. 3. Kamb, A., Shattuck-Eidens, D., Eeles, R. et al. (1994) Analysis of the pl 6 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus. Nature Genetics 8, 22-6.