title: A flexible multiplex bead-based assay for detecting germline CDKN2A and CDK4 variants in melanoma-prone kindreds.
abstract: The presence of recurrent high-risk mutations in cyclin-dependent kinase inhibitor 2A/cyclin-dependent kinase 4 (CDKN2A/CDK4) among melanoma-prone families suggests that a high-throughput, multiplex assay could serve as an effective initial screening tool. To this end, we have developed a multiplex bead-based assay for high-throughput CDKN2A/CDK4 genotyping in the context of familial melanoma. Genomic DNA from 1,603 subjects (1,005 in training set and 598 in validation set) were amplified by multiplex PCR using five CDKN2A/CDK4 primer sets followed by multiplex allele-specific primer extension for 39 distinct germline variants. The products were then sorted and analyzed using the Luminex xMAP system. Genotypes were compared with previously determined sequence data. In the Toronto training cohort, all 145 samples with known variants were detected by the bead assay (100% concordance). Analysis of the 598 samples from the GenoMEL validation set led to identification of 150/155 expected variants (96.77%). Overall, the bead assay correctly genotyped 1,540/1,603 (96.07%) of all individuals in the study and 1,540/1,545 (99.68%) of individuals whose variants were represented in the probe set. Out of a total of 62,517 allelic calls, 62,512 (99.99%) were correctly assigned. The multiplex bead-based assay is an accurate method for genotyping CDKN2A/CDK4 variants and is potentially useful in genotyping low-to-moderate melanoma risk single-nucleotide polymorphisms.
authors.affiliation: Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Julie M
authors.initials: JM
authors.lastname: Lang
authors.forename: Michael
authors.initials: M
authors.lastname: Shennan
authors.forename: Jenny C-N
authors.initials: JC
authors.lastname: Njauw
authors.forename: Su
authors.initials: S
authors.lastname: Luo
authors.forename: Julia N
authors.initials: JN
authors.lastname: Bishop
authors.forename: Mark
authors.initials: M
authors.lastname: Harland
authors.forename: Nicholas K
authors.initials: NK
authors.lastname: Hayward
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Tucker
authors.forename: Alisa M
authors.initials: AM
authors.lastname: Goldstein
authors.forename: Maria T
authors.initials: MT
authors.lastname: Landi
authors.forename: Susana
authors.initials: S
authors.lastname: Puig
authors.forename: Nelleke A
authors.initials: NA
authors.lastname: Gruis
authors.forename: Wilma
authors.initials: W
authors.lastname: Bergman
authors.forename: Giovanna
authors.initials: G
authors.lastname: Bianchi-Scarra
authors.forename: Paola
authors.initials: P
authors.lastname: Ghiorzo
authors.forename: David
authors.initials: D
authors.lastname: Hogg
authors.forename: Hensin
authors.initials: H
authors.lastname: Tsao
mesh_terms: D000483:Alleles; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D018095:Germ-Line Mutation / Q000235:genetics*; D006801:Humans; D008545:Melanoma / Q000175:diagnosis / Q000235:genetics*; D008863:Microspheres; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012878:Skin Neoplasms / Q000175:diagnosis / Q000235:genetics*
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D051358:Cyclin-Dependent Kinase 4