title: MALDI-TOF MS and TaqMan assisted SNP genotyping of DNA isolated from formalin-fixed and paraffin-embedded tissues (FFPET).
abstract: Formalin-fixed paraffin-embedded tissues (FFPET) from archived clinical samples provide an invaluable source for large-scale molecular genetic studies. Pharmacogenetic investigations that require long-term clinical follow-up data of patients may particularly benefit from FFPET analysis. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and TaqMan-based (Thermus aquaticus polymerase) methodologies have become standard genotyping procedures. However, no data are available on the applicability of MALDI-TOF MS to the genotyping of low quality DNA, as it is usually obtained from FFPET, and data from TaqMan genotyping are limited. We isolated constitutional DNA from 274 FFPET samples (229 patients with breast cancer and 45 patients with benign breast diseases) and genotyped 15 polymorphic loci in 10 genes. Nine SNPs were genotyped by MALDI-TOF MS, and six were genotyped by the TaqMan methodology. We established rates for successful allele assignment for all FFPET, for FFPET prepared prior to 1990, and for FFPET prepared post-1990. Both methodologies showed high success rates ranging between 70.9 and 99.6% (mean: 91.8%) for MALDI-TOF MS and between 82.3 and 97.7% (mean: 91.0%) for TaqMan genotyping. No significant differences in genotyping performances for FFPET prepared prior to 1990 or post-1990 were observed. With the exception of one, all other genotype frequencies were in Hardy-Weinberg equilibrium. Furthermore, genotype frequencies matched those observed in a German breast cancer population and other Caucasian populations. Our study shows for the first time that MALDI-TOF MS and TaqMan genotyping procedures provide reliable data, and are therefore applicable in studies that require large scale FFPET genotyping.
authors.affiliation: Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Jaremko
authors.forename: Christina
authors.initials: C
authors.lastname: Justenhoven
authors.forename: Benny K
authors.initials: BK
authors.lastname: Abraham
authors.forename: Werner
authors.initials: W
authors.lastname: Schroth
authors.forename: Peter
authors.initials: P
authors.lastname: Fritz
authors.forename: Sandra
authors.initials: S
authors.lastname: Brod
authors.forename: Caren
authors.initials: C
authors.lastname: Vollmert
authors.forename: Thomas
authors.initials: T
authors.lastname: Illig
authors.forename: Hiltrud
authors.initials: H
authors.lastname: Brauch
mesh_terms: D000483:Alleles; D001941:Breast Diseases / Q000235:genetics / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D005557:Formaldehyde; D005838:Genotype; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019914:Taq Polymerase; D016707:Tissue Fixation
chemical_list: D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D005557:Formaldehyde; D004247:DNA; D019914:Taq Polymerase