Molecular diagnosis of familial adenomatous polyposis (FAP): genotyping of adenomatous polyposis coli (APC) alleles by MALDI-TOF mass spectrometry.
OBJECTIVE
Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited colorectal cancer predisposition syndrome caused by germ line mutations in the adenomatous polyposis coli gene (APC). For prophylactic colectomy, timely identification of patients at risk is urgent. Here, matrix assisted laser desorption ionization - time of flight - mass spectrometry (MALDI-TOF-MS) genotyping is offered for an efficient molecular diagnosis of APC germline mutations.


METHODS
The four most frequent APC germ line mutations (three deletions, one point mutation) were genotyped by allele specific elongation and termination of extension primers. The extension products generated were analyzed by MALDI-TOF-MS.


RESULTS
Following PCR amplification and allele specific primer extension reactions MALDI-TOF-MS allowed the unambiguous identification of informative nucleic acid fragments corresponding to distinct genotypes or mutants even in duplex assays. Results were confirmed by DNA-sequencing.


CONCLUSIONS
Due to its high molecular resolution and accuracy, this method is highly suitable as an alternative for clinical APC genotyping.
Bonk
Thomas
T
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Fahrstrasse 17 D-91054, Erlangen, Germany.
Humeny
Andreas
A
Sutter
Christian
C
Gebert
Johannes
J
von Knebel Doeberitz
Magnus
M
Becker
Cord-Michael
CM
D011125:Adenomatous Polyposis Coli / Q000175:diagnosis / Q000235:genetics*; D000483:Alleles; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis / Q000097:blood; D017491:Genes, APC; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012307:Risk Factors; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D017931:DNA Primers; D004273:DNA, Neoplasm