A novel CDKN2A variant (p16L117P ) in a patient with familial and multiple primary melanomas

Abstract

Germline mutations in CDKN2A (p16) are commonly found in patients with family history of melanoma or personal history of multiple primary melanomas. The p16 tumor suppressor gene regulates cell cycle progression and senescence through binding of cyclin-dependent kinases (CDK) and also regulates cellular oxidative stress independently of cell cycle control. We identified a germline missense (c.350T>C, p.Leu117Pro) CDKN2A mutation in a patient who had history of four primary melanomas, numerous nevi, and self-reported family history of melanoma. This particular CDKN2A mutation has not been previously reported in prior large studies of melanoma kindreds or patients with multiple primary melanomas. Compared with wild-type p16, the p16^L117P mutant largely retained binding capacity for CDK4 and CDK6 but exhibited impaired capacity for repressing cell cycle progression and inducing senescence, while retaining its ability to reduce mitochondrial reactive oxygen species. Structural modeling predicted that the Leu117Pro mutation disrupts a putative adenosine monophosphate (AMP) binding pocket involving residue 117 in the fourth ankyrin domain. Identification of this new likely pathogenic variant extends our understanding of CDKN2A in melanoma susceptibility and implicates AMP as a potential regulator of p16.

Keywords: AMP binding; CDKN2A; germline; melanoma; p16.

FIGURE 1

Identification and characterization of the p16^L117P mutant. Saliva was collected and genetic testing was performed under protocol 96762, approved by the University of Utah Institutional Review Board. Genomic DNA was isolated using an OGR-500 kit (DNA Genotek), and exons 1 and 2 of CDKN2A (Hashemi et al., 2000) were sequenced as described. (a) Chromatogram showing T → C substitution at residue 350 in CDKN2A. (b) A FLAG-tagged p16^L117P construct was prepared by primer-directed site-directed mutagenesis (QuikChange II XL Kit, Agilent Technologies) using a wild-type human CDKN2A cDNA cloned into a Gateway (Thermo Fisher Scientific) vector with an N-terminal FLAG tag as a template. Both wild-type and p16^L117P FLAG constructs were confirmed by DNA sequencing. Western blotting of anti-FLAG immunoprecipitates (1 mg eluates, Pierce Co-Immunoprecipitation Kit) from 293T cells (American Type Culture Collection) transfected with GFP (control vector), wild-type p16, or the p16^L117P mutant using antibodies against CDK4 (Santa Cruz Biotechnology), CDK6 (Santa Cruz), or FLAG (Sigma). Numbers indicate densitometry values, normalized to FLAG signal for each construct. Representative of three experiments performed. (c) Western blotting of lysates of p16-deficient fibroblasts derived from p16^−/−Arf^+/+ mice (Jenkins et al., 2011) and obtained 48 h following lentiviral infection (Jenkins et al., 2011) with empty construct (GFP control) or constructs expressing wild-type p16 or the p16^L117P mutant (site-directed mutagenesis as above, confirmed by sequencing). Representative of three experiments performed. (d) Cell cycle analysis by flow cytometry of propidium iodide-stained cells in (c). Error bars correspond to SEM of triplicate determinations. ***p < 0.001, 2-sided t tests. Representative of three experiments performed. (e) p16-deficient fibroblasts were infected with lentiviruses as in (c) for 2 days and then were plated into 6-well dishes (10⁵ cells/well). Cell counts performed after 4 days. Error bars correspond to SEM of triplicate determinations. **p < 0.01, 2-sided t test. Representative of two experiments performed. (f) β-galactosidase staining of cells in (c). Error bars correspond to SEM of triplicate determinations. ***p < 0.001, 2-sided t tests. Representative photographs are shown. Representative of three experiments performed. (g) Detection of mitochondrial ROS by flow cytometry of cells in (c) stained with Mitosox Red (Life Technologies). Error bars correspond to SEM of triplicate determinations. ***p < 0.001, **p < 0.01, 2-sided t tests. Representative of three experiments performed

FIGURE 2

Structural modeling of the AMP binding site in the proximity of the L117P mutation in p16. Predicted structural elements of wild-type p16 (left panel) and p16^L117P mutant (right panel). Structures of the wild-type and mutant were predicted using I-TASSER (Roy et al., 2010). Side chain of residue 117 is shown in red and AMP in blue. The other residues are depicted using a conventional color scheme (carbon in brown, nitrogen in blue, oxygen in red). The binding pocket for AMP was identified using RaptorX (Kallberg et al., 2012). The AMP binding site predictions were both found to be statistically significant (p < 0.05), and both the uSeqID/SeqID (unnormalized/normalized sequence identity) and uGDT/GDT (unnormalized/normalized global distance test) scores indicated high model and prediction quality, respectively (http://raptorx.uchicago.edu/documentation/#goto2)