Japanese Journal of Clinical Oncology 30:204-206 (2000)
© 2000 Foundation for Promotion of Cancer Research
A Novel Non-pathogenetic Polymorphism of the APC Gene in a Patient with Familial Adenomatous Polyposis Coli
1Department of Laboratory Medicine, 2Division of Clinical and Molecular Genetics, 3Second Department of Surgery and 4Department of Hygiene and Medical Genetics, Shinshu University School of Medicine, Matsumoto and 5Division of Clinical Pathology, Nagano Children’s Hospital, Nagano, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT CASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
|---|
Disorder: Familial adenomatous polyposis coli
Ethnicity of patient: Japanese
Gene: APC
GenBank accession number: M 74088
Chromosomal assignment: 5q21
Type of DNA variant: A germline missense mutation
A germline nonsense mutation
Mutation: CGG (Arg, wild type) to TGG (Trp) substitution at codon 88 in exon 3 of the APC gene
CGA (Arg, wild type) to TGA (term.) at codon 213 in exon 5 of the APC gene
Allelic frequency: <0.014 (missense mutation, TGG at codon 88)
Method of mutation detection: PCR–SSCP/direct sequencing
|
CASE REPORT AND GENETIC ANALYSIS |
|---|
|
TOPABSTRACTCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
|---|
Germline mutations of the APC gene predispose individuals to development of multiple colorectal adenomas which frequently lead to carcinomas (1–4). Most of those genetic lesions generate the termination codon or the frameshifts which yield the truncated form of APC gene products. In some pedigrees, missense mutations in the APC gene had been identified as the pathogenetic mutations of familial adenomatous polyposis (FAP) (5,6), suggesting that such minimum peptide alterations are also feasible in causing multiple colorectal adenomas. In the case of missense mutations, however, it may be difficult to exclude completely the possibility that they are normal DNA polymorphisms, even if the unaffected individuals possess none of those nucleotide sequences. Here, we report a case in which a FAP patient had both a rare non-pathogenic polymorphism of the APC gene and a nonsense mutation which the first-step screening, PCR–SSCP assay, failed to detect.
The client was a 55-year-old man who was affected by adenomatous polyposis coli (Fig. 1, P). We screened the coding regions of the APC gene with PCR–SSCP and observed that only one PCR fragment of exon 3 showed a significant mobility shift (Fig. 2). Subsequent direct nucleotide sequencing of this fragment identified a missense mutation which caused a substitution of tryptophan for arginine (CGG to TGG) at codon 88 (Fig. 3a). To determine whether this genetic diversity is a pathogenetic missense mutation or a normal polymorphism, we examined the nucleotide sequences surrounding this site on more than 70 alleles of non-polyposis individuals and all alleles were the CGG type at codon 88. Since this result showed that the TGG type sequence is uncommon and raised the possibility that the TGG type was a pathogenetic missense mutation, we attempted to test a blood sample of another affected relative in the same pedigree who lived in a remote area (Fig. 1, asterisk). However, we found that the genome of this affected relative was a homozygote of the CGG-type alleles (CGG/CGG), indicating that the single nucleotide substitution (CGG/TGG) was not a pathogenetic missense mutation. Next, we determined the nucleotide sequence of the whole coding regions of the APC gene and finally identified a nonsense mutation at codon 213 in exon 5 (CGA/TGA) (Fig. 3b). Because both patients in this pedigree shared the same nucleotide sequence at codon 213, we concluded that the nonsense mutation is a causative genetic factor of FAP.
|
|
|
TGA:termination). Another affected relative also possessed the same nucleotide sequence.After detection of the nonsense mutation, we checked again all the stored gels of PCR–SSCP and confirmed that no PCR product except for a fragment containing codon 88 (CGG/TGG) exhibited an obvious additional band. In the PCR–SSCP assay, we could hardly recognize a significant mobility shift corresponding to the nonsense mutation at codon 213 (Fig. 2). Our previous study demonstrated that the PhastSystem could effectively separate single-stranded PCR products, but it missed some single nucleotide substitutions that generate minimal conformational changes (8). Therefore, if possible, it is necessary to prepare another set of primers in order to avoid missing small genetic alterations.
In the case of a rare amino acid substitution of the APC gene, such as arginine/tryptophan (CGG/TGG) at codon 88, it is sometimes difficult to distinguish a causative point mutation from a mere non-pathogenetic polymorphism. This case suggests that the molecular analysis also involves a risk of misjudgement of genetic diseases and it requires more knowledge concerning single nucleotide polymorphisms (SNPs) to reduce such risks.
|
METHODS FOR MUTATION DETECTION |
|---|
|
TOPABSTRACTCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
|---|
PCR–SSCP and PCR/direct sequencing were performed with the following conditions and parameters:
Exon 3:
PCR primer, forward: 5'ATG GAA TTC CAT TAA GAA TAT TTT AGA CTG CT3'
PCR primer, reverse: 5'TTA AAG CTT AAC AAT AAA CTG GAG TAC ACA3'
Size of PCR product: 340 bp
Thermal cycle profile:
Initial denaturation: 96°C, 3 min
35 cycles of 96°C, 30 s/58°C, 1 min/72°C, 30 s
Final extension: 72°C, 3 min
PCR–SSCP: PhastSystem (Pharmacia Biotech, Uppsala, Sweden) (8)
Electrophoresis: PhastGel 12.5%, 15°C
Sequencing primer: same as the PCR primer
Exon 5:
PCR primer, forward: 5'CAG GAA TTC TTT ATT GGT TCT TAT ATG CT3'
PCR primer, reverse: 5'CTG AAG CTT CCT AAT AGC TCT TCG CTG3'
Size of PCR product: 262 bp
Thermal cycle profile: same as exon 3
Electrophoresis: same as exon 3
Sequencing primer: same as the PCR primer
|
FOOTNOTES |
|---|
+ For reprints and all correspondence: Yoshifumi Ogiso, Division of Clinical Pathology, Nagano Children’s Hospital, 3100 Toyoshina, Nagano 399-8288, Japan. E-mail: yogiso@coral.ocn.ne.jp
|
REFERENCES |
|---|
|
TOPABSTRACTCASE REPORT AND GENETIC...METHODS FOR MUTATION DETECTIONREFERENCES |
|---|
1 Groden J, Thliveris A, Samowitz W, Carlson M, Gelbert L, Albertsen H, et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 1991;66:589–600.[Web of Science][Medline]
2 Joslyn G, Carlson M, Thliveris A, Albertsen H, Gelbert L, Samowimowitz W, et al. Identification of deletion mutations and three new genes at the familial polyposis locus. Cell 1991;66:601–13.[Web of Science][Medline]
3 Kinzler KW, Nilbert MC, Su LK, Vogelstein B, Bryan TM, Levy DB, et al. Identification of FAP locus genes from chromosome 5q21. Science 1991;253:661–5.
4 Nishisho I, Nakamura Y, Miyoshi Y, Miki Y, Ando H, Horii A, et al. Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients. Science 1991;253:665–9.
5 Miyoshi Y, Ando H, Nagase H, Nishisho I, Horii A, Miki Y, et al. Germ-line mutations of the APC gene in 53 familial adenomatous polyposis patients. Proc Natl Acad Sci USA 1992;89:4452–6.
6 Nagase H, Miyoshi Y, Horii A, Aoki T, Ogawa M, Utsunomiya J, et al. Correlation between the location of germ-line mutations in the APC gene and the number of colorectal polyps in familial adenomatous polyposis patients. Cancer Res 1992;52:4055–7.
7 Bennett RL, Steinhaus KA, Uhrich SB, O’Sullivan CK, Resta RG, Lochner-Doyle D, et al. Recommendations for standardized human pedigree nomenclature. Pedigree standardization task force of the National Society of Genetic Counselors. Am J Hum Genet 1995;56:745–52.[Web of Science][Medline]
8 Furuwatari C, Yagi A, Yamagami O, Ishikawa M, Hidaka E, Ueno I, et al. Comprehensive system to explore p53 mutations. Am J Clin Pathol 1998;110:368–73.[Web of Science][Medline]
Received December 8, 1999; accepted January 26, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||