A Flexible Multiplex Bead-Based Assay for Detecting Germline CDKN2A and CDK4 Variants in Melanoma-Prone Kindreds

Abstract

Background The presence of recurrent high-risk mutations in CDKN2A and CDK4 among melanoma-prone kindreds suggests that a high-throughput, multiplex assay could serve as an effective initial screening tool. Moreover, with the emergence of new melanoma risk single nucleotide polymorphisms (SNPs) through genome-wide association studies, a flexible platform that can easily accommodate these new risk alleles is needed for more accurate genetic risk profiling. To this end, we have developed a novel melanoma-associated mutation detection method using a multiplex bead-based assay. This assay is suitable for high-throughput CDKN2A and CDK4 genotyping and can be eventually adapted to multiple loci across various constituent populations. Methods Genomic DNA from a 1603 subjects (1005 in training set, 598 in validation set) were amplified by multiplex PCR using five primer sets followed by multiplex allele-specific primer extension for 39 different known germline variants. The products were then sorted on an xMAP™ (formerly Tag-It™) array and detected by use of the Luminex xMAP™ system. Genotypes were compared to previously-determined sequence data. Results In the Toronto training cohort, variants were detected in 145 samples, giving complete concordance between the bead assay and direct sequencing results. Analysis of the 598 samples from the GenoMEL validation set led to identification of 150/155 expected variants (96.77% concordance). Overall, the bead assay correctly genotyped 1540/1603 (96.07%) of all individuals in the study and 1540/1545 (99.68%) of individuals whose mutations were represented in the probe set. Out of a total of 62,512 SNP calls, 62,517 (99.99%) were correctly assigned. Conclusions In this initial evaluation, the multiplex bead-based assay for familial melanoma appears to be a highly accurate method for genotyping CDKN2A and CDK4 variants.

Comprehensive mutational analysis of CDKN2A and CDK4 in Greek patients with cutaneous melanoma

“…7, 8, 9 DNA was extracted from peripheral blood leucocytes and subjected to the same mutational analysis as listed above. Statistical analyses were performed with SigmaStat.…”

Screening and Identification of Distant Metastasis‐Related Differentially Expressed Genes in Human Squamous Cell Lung Carcinoma

“…Some scholars found, at DNA level, these two genes simultaneously or at least one of their downstream target objects changed in malignant melanoma cell lines (Walker et al, 1998). CDKN2A gene transcripts can inhibit CDK4 (Lang et al, 2011) and stabilize the p53 gene (Huschtscha et al, 2009); CDKN2B can inhibit the activity of CDK4 and CDK6 and stimulate cells' passing-through of G1/S check point and replication (Soto et al, 2005). The down-regulation of CDKN2A and CDKN2B in metastasis group of this study was probably through decreasing the inhibition of CDKs, which pushed cell cycle to pass G1/S monitoring point, resulting in rapid growth and uncontrolled proliferation of tumor cells.…”

T-Cell Receptor Gene Therapy: Critical Parameters for Clinical Success

“…It is important to realize though that certain driver mutations in cancer development are hereditary. As an example, the CDK4 mutation that results in a novel HLA-A2-restricted T-cell epitope (Wolfel et al, 1995) may seem a perfect target for TCR gene therapy but is sometimes observed in familial melanoma (Lang et al, 2011). For these patients, the targeting of this antigen would certainly not result in selective tumor cell recognition.…”