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HGNC Approved Gene Symbol: CDKN2A
SNOMEDCT: 717968005;
Cytogenetic location: 9p21.3 Genomic coordinates (GRCh38): 9:21,967,752-21,995,324 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 9p21.3 | {Melanoma and neural system tumor syndrome} | 155755 | Autosomal dominant | 3 |
| {Melanoma-pancreatic cancer syndrome} | 606719 | Autosomal dominant | 3 | |
| {Melanoma, cutaneous malignant, 2} | 155601 | Autosomal dominant | 3 |
The CDKN2A gene encodes proteins that regulate 2 critical cell cycle regulatory pathways, the p53 (TP53; 191170) pathway and the RB1 (614041) pathway. Through the use of shared coding regions and alternative reading frames, the CDKN2A gene produces 2 major proteins: p16(INK4), which is a cyclin-dependent kinase inhibitor, and p14(ARF), which binds the p53-stabilizing protein MDM2 (164785) (Robertson and Jones, 1999).
Using CDK4 (123829) as bait in a yeast 2-hybrid screen, Serrano et al. (1993) cloned human CDKN2A, which they designated p16(INK4). The deduced 148-amino acid protein contained 4 ankyrin repeats and had a calculated molecular mass of 15.8 kD.
Kamb et al. (1994) identified a putative tumor suppressor locus in chromosome band 9p21, within a region of less than 40 kb, by means of analyzing homozygous deletions in melanoma cell lines. The region was found to contain a gene, called MTS1 (for multiple tumor suppressor-1), that encodes a previously identified inhibitor (p16) of CDK4. The sequence of the MTS1 gene as determined by Kamb et al. (1994) was identical to that of the p16 gene as determined by Serrano et al. (1993).
Using a p16 probe, Stone et al. (1995) isolated 2 cDNAs that differed in their first exons, which they called E1-alpha and E1-beta. E1-alpha encodes the first 43 amino acids of the p16 protein. E1-beta is utilized by a second open reading frame that encodes a protein of at least 180 amino acids, in a different reading frame relative to p16. This protein was designated p14(ARF) (alternative reading frame). Stone et al. (1995) also cloned 2 p16 cDNAs from mouse. The mouse and human p16 proteins share 60% identity, but the p14(ARF) proteins share only 28% identity. Northern blot analysis detected different ratios of both transcripts in all human tissues examined. The ratio of the 2 variants changed dramatically through the cell cycle in human peripheral blood lymphocytes. As stimulated T cells entered the cell cycle, the ratio of beta-to-alpha splice variants increased.
Stott et al. (1998) stated that the alpha transcript of CDKN2A encodes p16(INK4a), a recognized tumor suppressor that induces a G1 cell cycle arrest by inhibiting the phosphorylation of the Rb protein by the cyclin-dependent kinases CDK4 and CDK6. The beta transcript of CDKN2A encodes p14(ARF). The predicted 132-amino acid p14(ARF) is shorter than the corresponding mouse protein, p19(ARF), and the 2 proteins share only 50% identity. However, both proteins have the ability to elicit a p53 (191170) response, manifest in the increased expression of both CDKN1A (116899), also called CIP1, and MDM2 (164785), and resulting in a distinctive cell cycle arrest in both the G1 and G2/M phases.
Robertson and Jones (1999) detected a theretofore unrecognized splice variant of INK4a, termed p12, that arises through use of an alternative splice donor site within intron 1. The p12 transcript produced a 12-kD protein composed of INK4a exon 1-alpha and a novel intron-derived C terminus. p12 contains only 1.5 of the 4 ankyrin repeats found in p16(INK4A). Robertson and Jones (1999) showed that p12 did not interact with CDK4, but its overexpression suppressed growth in cultured human cervical carcinoma or pancreatic cell lines. Northern blot analysis detected p12 transcripts in pancreas only.
Lin et al. (2007) stated that p16(INK4A) contains 156 amino acids and has a calculated molecular mass of 16.5 kD. By RT-PCR of a human neuroblastoma cell line, they cloned a splice variant of p16(INK4A), p16-gamma. The transcript includes a 197-bp cryptic exon (exon 2-gamma) from intron 2 that is spliced in-frame with p16(INK4A) exons 2 and 3. The stop codon for p16-gamma lies within exon 2-gamma. The deduced p16-gamma protein contains 167 amino acids and has a calculated molecular mass of 17.9 kD. The first 152 amino acids of p16(INK4A) and p16-gamma are identical. Biophysical analysis indicated that p16-gamma, like p16(INK4A), is an ankyrin repeat protein. Lin et al. (2007) stated that p14(ARF) does not contain ankyrin repeats. RT-PCR detected p16-gamma transcripts in primary T-cell and B-cell acute lymphoblastic leukemia patient samples and in other p16(INK4A)-expressing tumor samples. Lower levels were detected in normal mononuclear cells and non-tumor tissues.
Using RT-PCR, Burdon et al. (2011) demonstrated expression of CDKN2A in human ocular tissues, including in the iris, ciliary body, retina, and optic nerve.
Kamb et al. (1994) stated that the CDKN2 gene consists of 3 coding exons: exon 1 (E1), containing 125 basepairs; E2, containing 307 basepairs; and E3, containing just 12 basepairs.
Stone et al. (1995) determined that the CDKN2A gene spans 30 kb. Exon E1-beta is the most 5-prime of the p16 exons. Lin et al. (2007) identified an alternatively spliced exon, called 2-gamma, between exon E1-alpha and exon 2 of the CDKN2A gene.
Quelle et al. (1995) found that in mouse, as in human, the INK4 locus gives rise to 2 distinct transcripts from different promoters. Each transcript has a specific 5-prime exon, E1-alpha or E1-beta, which is spliced into common exons E2 and E3. The E1-alpha-containing transcript encodes p16(INK4a) and the E1-beta-containing transcript encodes p19(ARF) from a different AUG initiated in the E1-beta exon. Both p19(ARF) and p16(INK4a) induced growth arrest in mammalian fibroblasts.
The p16 gene (CDKN2A) was mapped to 9p21 (Kamb et al., 1994; Nobori et al., 1994). This same region has frequently been involved in deletions and rearrangements in dysplastic nevi (Cowan et al., 1988), a major precursor lesion of melanoma, and in cutaneous malignant melanoma, or CMM (Fountain et al., 1992), and was shown by Petty et al. (1993) to be involved in a constitutional deletion in a patient with multiple primary melanomas. A locus for familial malignant melanoma, symbolized CMM2 (155601), has been mapped to 9p21. Kamb et al. (1994) noted that chromosome region 9p21 is involved in chromosomal inversions, translocations, heterozygous deletions, and homozygous deletions in a variety of malignant cell lines including those from glioma, nonsmall cell lung cancer, leukemia, and melanoma. Deletion of 9p21 markers is found in more than half of all melanoma cell lines. These findings suggested to Kamb et al. (1994) that 9p21 contains a tumor suppressor locus that may be involved in the genesis of several tumor types.
Quelle et al. (1995) mapped the p16(INK4a) and p15(INK4b) genes to position C3-C6 on mouse chromosome 4 in a region syntenic with human chromosome 9p.
Kamb et al. (1994) found that MTS1 was homozygously deleted at high frequency in cell lines derived from tumors of lung, breast, brain, bone, skin, bladder, kidney, ovary, and lymphocyte. Melanoma cell lines carried at least one copy of MTS1 in combination with a deleted allele. Melanoma cell lines that carried at least 1 copy of MTS1 frequently showed nonsense, missense, or frameshift mutations in the gene. Thus, MTS1 may rival p53 (191170) in the universality of its involvement in tumorigenesis. Furthermore, it illustrates, as does p53, the relationship between the tumor suppressor genes and the regulation of the cell cycle.
Quelle et al. (1995) showed that the protein p19(ARF) arises in major part from an alternative reading frame of the mouse INK4a gene, and that its ectopic expression in the nucleus of rodent fibroblasts induces G1 and G2 phase arrest. The authors noted that economical reutilization of coding sequences in this manner is practically without precedent in mammalian genomes, and they speculated that the unitary inheritance of p16(INK4a) and p19(ARF) may underlie their dual requirement in cell cycle control. A somewhat similar situation was reported by Labarriere et al. (1995) who found that transcripts originating from a novel promoter in the human growth hormone gene GH1 (139250) have the potential to specify a 107-amino acid protein, the C-terminal half of which arises from a second reading frame in GH exons 1 and 2. Antibodies to the C terminus of this predicted polypeptide histochemically stained a subpopulation of pituitary cells, arguing for limited focal translation of this mRNA.
The frequent deletion or mutation of CDKN2A in tumor cells suggests that p16 acts as a tumor suppressor. Lukas et al. (1995) showed that wildtype p16 arrests normal diploid cells in late G1, whereas a tumor-associated mutant of p16 does not. Significantly, the ability of p16 to induce cell cycle arrest was lost in cells lacking functional RB1 protein (614041). Thus, loss of p16, overexpression of D-cyclins, and loss of retinoblastoma have similar effects on G1 progression, and may represent a common pathway to tumorigenesis. The mutation used by Lukas et al. (1995) in their studies was a C-to-T transition changing proline-114 to leucine and had been observed in 3 independent melanoma cell lines. Koh et al. (1995) reported similar results. They demonstrated that p16 can act as a potent and specific inhibitor of progression through the G1 phase of the cell cycle and that several tumor-derived alleles of p16 encode functionally compromised proteins. In vivo, the presence of functional retinoblastoma protein appeared to be necessary but may not be sufficient to confer full sensitivity to p16-mediated growth arrest. In addition to the P114L allele, they used an asp74-to-asn (D74N) mutant, a de novo somatic mutation isolated independently from tumors of the esophagus and bladder; an asp84-to-asn (D84N) mutation found in a survey of esophageal squamous cell carcinomas; and several other mutations associated with melanoma.
Using amplification of polyadenylated mRNA by PCR, Quelle et al. (1995) observed no expression of mouse p16 in many normal tissues, whereas p15 was expressed ubiquitously.
By immunohistochemical analysis of archival paraffin specimens and tumor cell lines, Kratzke et al. (1995) found that p16(INK4) was expressed in a nonsmall cell lung cancer cell line but not in 12 of 12 primary thoracic mesotheliomas (156240) and 15 of 15 mesothelioma cell lines. All tumor specimens and the tumor cell lines showed expression of wildtype retinoblastoma protein. In addition, transfection of CDKN2 suppressed the growth of 2 independent mesothelioma cell lines. The authors concluded that inactivation of the CDKN2 gene is an essential step in the etiology of malignant mesotheliomas.
Serrano et al. (1996) proposed that the absence of p16(INK4a) contributes significantly to the tumor susceptibility phenotype. Kamijo et al. (1997) used a conventional targeting vector to ablate the mouse p19(ARF) exon 1b in mouse embryonic stem cells, replacing it with a neomycin-resistant gene. The expression of the p16(INK4a) gene was not abolished. Mice lacking p19(ARF) but expressing functional p16(INK4a) developed tumors early in life. Their embryo fibroblasts did not senesce and were transformed by oncogenic Ha-ras alone. Conversion of p16(ARF)+/+ or p16(ARF)+/- mouse embryo fibroblasts to continuously proliferating cell lines involved loss of either p19(ARF) or p53. Checkpoint control mediated by p53 was unperturbed in p19(ARF)-null fibroblasts, whereas p53-negative cell lines were resistant to p19(ARF)-induced growth arrest. Kamijo et al. (1997) concluded that INK4a encodes growth inhibitory proteins that act upstream of the retinoblastoma protein and p53. They suggested that mutations and deletions targeting this locus in cancer cells are unlikely to be functionally equivalent.
Splicing of alternative first exons (1-alpha vs 1-beta) to a common second exon within the INK4A gene generates mRNAs in which exon 2 sequences are translated in 2 different reading frames. One of the products, the cyclin D-dependent kinase inhibitor p16, is functionally inactivated by mutations or deletions in a wide variety of cancers; however, because many such mutations occur in exon 2, they also affect the alternative reading frame (ARF) protein. To determine whether such mutations disrupt p19(ARF) function, Quelle et al. (1997) introduced naturally occurring missense mutations into mouse Ink4a exon 2 sequences and tested mutant p16 and p19 proteins for their ability to inhibit cell cycle progression. Six p19(ARF) point mutants remained fully active in mediating cell cycle arrest in NIH 3T3 fibroblasts, whereas 2 of the corresponding mutations within p16 resulted in complete loss of activity. Analysis of p19(ARF) deletion mutants indicated that the unique N-terminal domain encoded by exon 1-beta was both necessary and sufficient for inducing cell cycle G1 arrest. Therefore, they concluded that cancer-associated mutations within exon 2 of the INK4A gene specifically target p16 and not p19 for inactivation.
Zhang et al. (1998) stated that the 2 unrelated proteins encoded by the INK4A-ARF locus function in tumor suppression. Zhang et al. (1998) showed that ARF binds to MDM2 (164785) and promotes the rapid degradation of MDM2. This interaction is mediated by the E1-beta-encoded N-terminal domain of ARF and a C-terminal region of MDM2. ARF-promoted MDM2 degradation is associated with MDM2 modification and concurrent p53 (191170) stabilization and accumulation. The functional consequence of ARF-regulated p53 levels via MDM2 proteolysis is evidenced by the ability of ectopically expressed ARF to restore a p53-imposed G1 cell cycle arrest that is otherwise abrogated by MDM2. Thus, Zhang et al. (1998) concluded that deletion of the ARF-INK4A locus simultaneously impairs the INK4A--cyclin D/CDK4--RB and the ARF--MDM2--p53 pathways.
Pomerantz et al. (1998) showed that p19(ARF) potently suppresses oncogenic transformation in primary cells and that this function is abrogated when p53 is neutralized by viral oncoproteins and dominant-negative mutants but not by the p53 antagonist MDM2. This finding, coupled with the observations that p19(ARF) and MDM2 physically interact and that p19(ARF) blocks MDM2-induced p53 degradation and transactivational silencing, suggests that p19(ARF) functions to prevent the neutralization of p53 by MDM2. Pomerantz et al. (1998) suggested that INK4A has a potent tumor suppressor activity to the cooperative actions of its 2 protein products and their relation to the 2 central growth control pathways, Rb and p53.
Zhang and Xiong (1999) reported that the human ARF protein predominantly localizes to the nucleolus via a sequence within the exon 2-encoded C-terminal domain and is induced to leave the nucleolus by MDM2. ARF forms nuclear bodies with MDM2 and p53 and blocks p53 and MDM2 nuclear export. Tumor-associated mutations in exon 2 of the ARF-INK4a locus disrupted the nucleolar localization of ARF and reduced its ability to block p53 nuclear export and to stabilize p53. These results suggested an ARF-regulated MDM2-dependent p53 stabilization and linked the human tumor-associated mutations in the ARF-INK4a locus with a functional alteration.
Vivo et al. (2001) performed coimmunoprecipitation and transfection experiments demonstrating that the C-terminal region of spinophilin (PPP1R9B; 603325) interacts with ARF in vitro and in mammalian cells. Studies with deletion mutants showed that the first 65 amino acids in the N terminus of ARF are necessary for this interaction. Ectopic expression in different human and mouse cell lines showed that spinophilin reduced the number of G418-resistant colonies with an efficiency similar to or higher than that of ARF; this effect was independent of the status of p53, Rb, and ARF. Coexpression of ARF/spinophilin in Saos-2 cells suggested synergistic activity.
The p16(INK4A) cyclin-dependent kinase inhibitor is implicated in replicative senescence, the state of permanent growth arrest provoked by cumulative cell divisions or as a response to constitutive Ras-Raf-MEK signaling in somatic cells. Ohtani et al. (2001) demonstrated a role for the ETS1 (164720) and ETS2 (164740) transcription factors in regulating the expression of p16(INK4A) in these different contexts based on their ability to activate the p16(INK4A) promoter through an ETS binding site and their patterns of expression during the life span of human diploid fibroblasts. The induction of p16(INK4A) by ETS2, which is abundant in young human diploid fibroblasts, is potentiated by signaling through the Ras-Raf-MEK kinase cascade and inhibited by a direct interaction with the helix-loop-helix protein ID1 (600349). In senescent cells, where the ETS2 levels and MEK signaling decline, the marked increase in p16(INK4A) expression is consistent with the reciprocal reduction of ID1 and accumulation of ETS1.
Linggi et al. (2002) demonstrated that p14(ARF) is a direct transcriptional target of the AML1-ETO (133435) fusion gene that results from the t(8;21) translocation associated with acute leukemia. Repression of p14(ARF) may explain why p53 is not mutated in t(8;21)-containing leukemias and suggests that p14(ARF) is an important tumor suppressor in a large number of human leukemias.
In mouse embryo fibroblasts, Qi et al. (2004) showed that p19(Arf) can inhibit c-Myc (190080) by a unique and direct mechanism that is independent of p53. When c-Myc increased, p19(Arf) bound with c-Myc and dramatically blocked c-Myc's ability to activate transcription and induce hyperproliferation and transformation. In contrast, c-Myc's ability to repress transcription was unaffected by p19(Arf), and c-Myc-mediated apoptosis was enhanced. These differential effects of p19(Arf) on c-Myc function suggested that separate molecular mechanisms mediate c-Myc-induced hyperproliferation and apoptosis. This direct feedback mechanism represents a p53-independent checkpoint to prevent c-Myc-mediated tumorigenesis.
Reef et al. (2006) identified mouse and human smARF, a short mitochondrial form of p19(ARF) that results from initiation of translation at met45 in mice and met48 in humans. smARF lacks the N-terminal nucleolar localization signal and several functional domains of p19(ARF) and represents a small fraction of total p19(ARF) in vivo. Mouse smARF localized to mitochondria and caused dissipation of mitochondrial membrane potential independent of p53 (191170) and Bcl2 (151430) family members without releasing cytochrome c. Overexpression of smARF led to induction of massive autophagy and to caspase-independent cell death.
Janzen et al. (2006) reported that the cyclin-dependent kinase inhibitor p16(INK4a), the level of which increases in other cell types with age, accumulates and modulates specific age-associated hematopoietic stem cell functions. Notably, in the absence of p16(INK4a), hematopoietic stem cell repopulating defects and apoptosis were mitigated, improving the stress tolerance of cells and the survival of animals in successive transplants, a stem cell-autonomous tissue regeneration model. Janzen et al. (2006) suggested that inhibition of p16(INK4a) may ameliorate the physiologic impact of aging on stem cells and thereby improve injury repair in aged tissue.
Molofsky et al. (2006) showed that progenitor proliferation in the subventricular zone and neurogenesis in the olfactory bulb, as well as multipotent progenitor frequency and self-renewal potential, all decline with age in the mouse forebrain. These declines in progenitor frequency and function correlate with increased expression of p16(INK4a), which encodes a cyclin-dependent kinase inhibitor linked to senescence. Aging p16(INK4a)-deficient mice showed a significantly smaller decline in subventricular zone proliferation, olfactory bulb neurogenesis, and the frequency and self-renewal potential of multipotent progenitors. p16(INK4a) deficiency did not detectably affect progenitor function in the dentate gyrus or enteric nervous system, indicating regional differences in the response of neural progenitors to increased p16(INK4a) expression during aging. Molofsky et al. (2006) concluded that declining subventricular zone progenitor function and olfactory bulb neurogenesis during aging are caused partly by increasing p16(INK4a) expression.
Krishnamurthy et al. (2006) showed that p16(INK4a) constrains islet proliferation and regeneration in an age-dependent manner. Expression of the p16(INK4a) transcript is enriched in purified islets compared with the exocrine pancreas, and islet-specific expression of p16(INK4a), but not other cyclin-dependent kinase inhibitors, increases markedly with aging. To determine the physiologic significance of p16(INK4a) accumulation on islet function, Krishnamurthy et al. (2006) assessed the impact of p16(INK4a) deficiency and overexpression with increasing age and in the regenerative response after exposure to a specific islet beta-cell toxin. Transgenic mice that overexpress p16(INK4a) to a degree seen with aging demonstrated decreased islet proliferation. Similarly, islet proliferation was unaffected by p16(INK4a) deficiency in young mice, but was relatively increased in p16(INK4a)-deficient old mice. Survival after toxin-mediated ablation of beta-cells, which requires islet proliferation, declined with advancing age; however, mice lacking p16(INK4a) demonstrated enhanced islet proliferation and survival after beta-cell ablation. Krishnamurthy et al. (2006) concluded that these genetic data supported the view that an age-induced increase of p16(INK4a) expression limits the regenerative capacity of beta-cells with aging.
Lin et al. (2007) showed that human p16-gamma interacts with CDK4 and inhibited its kinase activity. Using a reporter gene assay, transfection of p16-gamma repressed the E2F response (see E2F1, 189971), the downstream target of RB1, with an efficacy equivalent to that of p16(INK4A). Moreover, p16-gamma, like p16(INK4A), induced cell cycle arrest at G0/G1, and inhibited cell growth in a human osteosarcoma cell line in a colony formation assay.
Li et al. (2009) showed that the Ink4/Arf locus, comprising Cdkn2a-Cdnk2b (600431), is completely silenced in induced pluripotent stem (iPS) cells as well as in embryonic stem cells, acquiring the epigenetic marks of a bivalent chromatin domain, and retaining the ability to be reactivated after differentiation. Cell culture conditions during reprogramming enhance the expression of the Ink4/Arf locus, further highlighting the importance of silencing the locus to allow proliferation and reprogramming. Indeed, Oct4 (164177), Klf4 (602253), and Sox2 (184429) together repress the Ink4/Arf locus soon after their expression and concomitant with the appearance of the first molecular markers of 'stemness.' This downregulation also occurs in cells carrying the oncoprotein simian virus-40 'large-T' antigen, which functionally inactivates the pathways regulated by the Ink4/Arf locus, thus indicating that the silencing of the locus is intrinsic to reprogramming and not the result of a selective process. Genetic inhibition of the Ink4/Arf locus has a profound positive effect on the efficiency of iPS cell generation, increasing both the kinetics of reprogramming and the number of emerging iPS cell colonies. In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 and p21 (CDKN1A; 116899) whereas in human fibroblasts, INK4a is more important than ARF. Furthermore, organismal aging upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms, but this defect can be rescued by inhibiting the locus with a short hairpin RNA. Li et al. (2009) concluded that the silencing of Ink4/Arf locus is rate-limiting for reprogramming, and its transient inhibition may significantly improve the generation of iPS cells.
Utikal et al. (2009) noted that the reprogramming potential of primary murine fibroblasts into iPS cells decreases after serial passaging and the concomitant onset of senescence. They demonstrated that cells with low endogenous p19(Arf) protein levels and immortal fibroblasts deficient in components of the Arf-Trp53 pathway yield iPS cell colonies with up to 3-fold faster kinetics and at a significantly higher efficiency than wildtype cells, endowing almost every somatic cell with the potential to form iPS cells. Notably, the acute genetic ablation of p53 in cellular subpopulations that normally fail to reprogram rescues their ability to produce iPS cells. The results of Utikal et al. (2009) showed that the acquisition of immortality is a crucial and rate-limiting step towards the establishment of a pluripotent state in somatic cells and underscored the similarities between induced pluripotency and tumorigenesis.
Chen et al. (2010) reported that ARF is very unstable in normal human cells but that its degradation is inhibited in cancer cells. Through biochemical purification, Chen et al. (2010) identified a specific ubiquitin ligase for ARF and named it ULF (604506). ULF interacts with ARF both in vitro and in vivo and promotes the lysine-independent ubiquitylation and degradation of ARF. ULF knockdown stabilizes ARF in normal human cells, triggering ARF-dependent p53-mediated growth arrest. Moreover, nucleophosmin (NPM; 164040) and c-Myc (190080), both of which are commonly overexpressed in cancer cells, are capable of abrogating ULF-mediated ARF ubiquitylation through distinct mechanisms, and thereby promote ARF stabilization in cancer cells. Chen et al. (2010) concluded that their findings revealed the dynamic feature of the ARF-p53 pathway and suggested that the transcription-independent mechanisms are critically involved in ARF regulation during responses to oncogenic stress.
Baker et al. (2011) used a biomarker for senescence, p16(Ink4a), to design a novel transgene, INK-ATTAC, for inducible elimination of p16(Ink4a)-positive senescent cells upon administration of a drug. Baker et al. (2011) showed that in the BubR1 (602860) progeroid mouse background, INK-ATTAC removes p16(Ink4a)-positive senescent cells upon drug treatment. In tissues such as adipose tissue, skeletal muscle, and eye, in which p16(Ink4a) contributes to the acquisition of age-related pathologies, lifelong removal of p16(Ink4a)-expressing cells delayed onset of these phenotypes. Furthermore, late-life clearance attenuated progression of already established age-related disorders. Baker et al. (2011) concluded that cellular senescence is causally implicated in generating age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend 'healthspan.'
Watari et al. (2012) found that ectopic expression of PANO (PANO1; 620541) induced apoptosis in HeLa cells in a low-serum medium. PANO colocalized with p14ARF in nucleolus, and PANO expression increased the expression level of p14ARF by stabilizing the p14ARF protein. Knockdown analysis in Hela cells confirmed that PANO was necessary for stable expression of p14ARF protein and showed that PANO-induced apoptosis was mediated by p14ARF. PANO also suppressed tumorigenicity in vivo, as PANO-expressing Hela cell clones did not form tumors in nude mice.
Braumuller et al. (2013) showed that the combined action of the T helper-1-cell cytokines IFN-gamma (IFNG; 147570) and tumor necrosis factor (TNF; 191160) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumor immunity from oncogene-induced senescence, Braumuller et al. (2013) used a mouse model in which the Simian virus-40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumors by attenuating p53 (191170)- and Rb (614041)-mediated cell cycle control. When combined, Ifng and Tnf drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16Ink4a, and downstream Rb hypophosphorylation at ser795. This cytokine-induced senescence strictly requires Stat1 (600555) and Tnfr1 (TNFRSF1A; 191190) signaling in addition to p16Ink4a. In vivo, Tag-specific T-helper-1 cells permanently arrest Tag-expressing cancers by inducing Ifng- and Tnfr1-dependent senescence. Conversely, Tnfr1-null Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in Tnfr1-expressing hosts. Braumuller et al. (2013) concluded that as IFNG and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.
Sousa-Victor et al. (2014) reported that geriatric satellite cells are incapable of maintaining their normal quiescent state in muscle homeostatic conditions, and that this irreversibly affects their intrinsic regenerative and self-renewal capacities. In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible presenescence state, caused by derepression of p16(INK4a). On injury, these cells fail to activate and expand, undergoing accelerated entry into a full senescence state (geroconversion), even in a youthful environment. Silencing of p16(INK4a) in geriatric satellite cells restores quiescence and muscle regenerative functions. Sousa-Victor et al. (2014) concluded that maintenance of quiescence in adult life depends on the active repression of senescence pathways.
To explore the physiologic relevance and consequences of naturally occurring senescent cells, Baker et al. (2016) used the INK-ATTAC transgene (Baker et al., 2011) to induce apoptosis in p16(Ink4a)-expressing cells of wildtype mice by injection of AP20187 twice a week starting at 1 year of age. Baker et al. (2016) showed that compared to vehicle alone, AP20187 treatment extended median life span in both male and female mice of 2 distinct genetic backgrounds. The clearance of p16(Ink4a)-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart, and fat, where clearance preserved the functionality of glomeruli, cardioprotective K(ATP) channels, and adipocytes, respectively. The authors concluded that p16(Ink4a)-positive cells that accumulate during adulthood negatively influence life span and promote age-dependent changes in several organs.
Reyes et al. (2022) identified a population of Cdkn2-expressing fibroblasts in the basement membrane adjacent to epithelial stem cells in lungs of young mice. When injured, these cells increased secretion and stimulated airway stem cells to regenerate epithelium. The authors concluded that, rather than being Cdkn2-expressing senescent cells, these Cdkn2-expressing cells represent sentinel stem cells that respond to inflammation to promote tissue regeneration.
The p16 protein binds to CDK4 (123829) and inhibits the ability of CDK4 to interact with cyclin D and stimulate passage through the G1 phase of the cell cycle (Serrano et al., 1993). Deletions or mutations in the p16 gene may affect the relative balance of functional p16 and cyclin D, resulting in abnormal cell growth. Kamb et al. (1994) and Nobori et al. (1994) observed a high frequency of p16 deletions and mutations in many tumor cell lines, which supported the above model and suggested that p16 has a pivotal role in inhibiting the development of human cancers. On the other hand, studies by Cairns et al. (1994), demonstrating a much lower frequency of mutations in primary tumors, suggested that the high frequency of p16 mutations observed in cell lines are in vitro artifacts and not evidence of a major role of the gene in the development of a wide variety of malignancies.
Inactivation of tumor suppressor genes by large deletions, intragenic mutations, altered splicing and promoter mutations may not be the only mechanism leading to tumorigenesis. Merlo et al. (1995) showed that although LOH on 9p21 is one of the most frequent genetic alterations identified in human cancer, point mutations of p16 on the other chromosome are relatively rare. They showed that monosomic cell lines with structurally unaltered p16 contained methylation of the 5-prime CpG island of the p16 gene. This distinct methylation pattern was associated with a complete transcriptional block that was reversible upon treatment with 5-deoxyazacytidine. Moreover, de novo methylation of the 5-prime CpG island of p16 was found in approximately 20% of different primary neoplasms, but not in normal cells, potentially representing a common pathway of tumor suppressor gene inactivation in human cancers. Little and Wainwright (1995) raised the possibility that the aberrant methylation of p16 may reflect the activity of a p16-specific 'imprintor.' The isolation of a p16 imprintor gene would be an exciting development in cancer biology. As tumorigenesis often apes embryology, such an imprintor may be necessary to 'switch off' p16-driven inhibition of cell proliferation during certain stages of development. The findings of Merlo et al. (1995) suggested that gene-specific methylation is another way to 'suppress the suppressors.' In their brief review, Little and Wainwright (1995) speculated: 'If methylation of (the) p16 (gene) is the driving force behind the tumour, could p16 be specifically demethylated?'
Hinshelwood et al. (2009) investigated the temporal progression of DNA methylation and histone remodeling in the p16(INK4A) CpG island in primary human mammary epithelial cell (HMEC) strains during selection, as a model for early breast cancer. Gene silencing occurred prior to de novo methylation and histone remodeling. An increase in DNA methylation was associated with a rapid loss of both histone H3K27 trimethylation and H3K9 acetylation and a gradual gain of H3K9 dimethylation. Regional-specific 'seeding' methylation occurred early after post-selection, and the de novo methylation pattern observed in HMECs correlated with the apparent footprint of nucleosomes across the p16(INK4A) CpG island. Hinshelwood et al. (2009) concluded that CDKN2A gene silencing is a precursor to epigenetic suppression; subsequent de novo methylation initially occurs in nucleosome-free regions across the p16(INK4A) CpG island, and this is associated with a dynamic change in histone modifications.
McKenzie et al. (2010) assessed the utility of different assays in predicting functional effects of CDKN2A mutations. They evaluated 28 different mutations, including missense, truncating, and intragenic deletions for binding affinity to CDK4 and CDK6, cell-cycle inhibitory activity, and subcellular distribution. There were varying results, with some mutant proteins showing features similar to wildtype, and others causing disruption of normal function. Almost all mutant proteins showed a decrease in binding to CDK4, and most showed altered subcellular localization in a punctate or speckled pattern. McKenzie et al. (2010) concluded that a combination of assays to determine CDK4 binding affinity and assays to determine subcellular distribution in mammalian cells can provide a rapid and accurate assessment of CDKN2A mutation function. CDK6-binding activity, in silico analysis, and Ki67 (176741) expression were not useful adjuncts.
Role in Pancreatic Cancer
Caldas et al. (1994) concluded that the CDKN2A gene is frequently the site of mutations causing pancreatic adenocarcinoma. They had noted a high frequency of allelic loss at 9p, including 9p21, in cases of pancreatic adenocarcinoma. This tumor characteristically generates an intense host desmoplastic reaction, and primary tumor tissue contains a high admixture of contaminating nonneoplastic inflammatory and stromal cells. For this reason, and because of the difficulty in establishing pancreatic adenocarcinoma cells in culture, Caldas et al. (1994) took advantage of xenograft explants. The analysis of MTS1 in 27 xenografted pancreatic carcinomas and 10 pancreatic carcinoma cell lines showed homozygous deletions in 15 (41%) and sequence changes in 14 (38%). Sequencing of MTS1 from primary tumors confirmed the mutations. Coexistent inactivation of both MTS1 and p53 (191170) was common. Bartsch et al. (1995) found somatic CDKN2 mutations in 11 of 32 pancreatic adenocarcinomas. One tumor appeared to have a homozygous deletion of CDKN2.
Liu et al. (1995) found that exons 1 and 2 of the MTS1 gene were deleted in 50% of pancreatic cancer cell lines. The region 9p22-p21, where the MTS1 gene is located, had been observed to show frequent loss of heterozygosity (LOH) in esophageal squamous cell carcinomas and pancreatic ductal adenocarcinomas.
Mutation in CDKN2A can cause a syndrome of pancreatic cancer and melanoma (see 606719).
In a population-based study, Ghiorzo et al. (2012) identified CDKN2A mutations in 13 (5.7%) of 225 Italian patients with pancreatic cancer. Six patients carried the common G101W mutation (600160.0005), which was the most common mutation. Among the 16 probands with a family history of cancer, including pancreatic and melanoma, 5 (31%) were found to carry CDKN2A mutations. The mutation frequency ranged from 20% in families with 2 affected members to 50% in families with 3 affected members. The findings suggested that CDKN2A is the main susceptibility gene in Italian families with pancreatic cancer.
Harinck et al. (2012) identified CDKN2A mutations in 6 (21%) of 28 families ascertained for familial clustering of pancreatic cancer. Five of the 6 families were of Caucasian origin and carried the same Dutch founder Leiden mutation (19-bp del; 600160.0003). Four of the 5 families with the Leiden mutation had members who developed melanoma. In the fifth family, only pancreatic cancer segregated with mutation status. A sixth family, of Indonesian origin, with only pancreatic cancer and no melanoma carried a different heterozygous mutation in the CDKN2A gene (600160.0022). Harinck et al. (2012) concluded that the CDKN2A gene should be analyzed in families with pancreatic cancer, even if melanoma is not present.
Zhen et al. (2015) tested germline DNA from 727 unrelated probands with pancreatic cancer and a positive family history for mutations in BRCA1 (113705) and BRCA2 (600185) (including deletions and rearrangements), PALB2 (610355), and CDKN2A. Among these probands, 521 met criteria for familial pancreatic cancer (FPC; at least 2 affected first-degree relatives). The prevalence of deleterious mutations, excluding variants of unknown significance, among FPC probands was BRCA1, 1.2%; BRCA2, 3.7%; PALB2, 0.6%; and CDKN2A, 2.5%. Four novel deleterious mutations were detected. FPC probands carried more mutations in the 4 genes (8.0%) than nonfamilial pancreatic cancer probands (3.5%; OR = 2.40, 95% CI 1.06-5.44, p = 0.03). The probability of testing positive for deleterious mutations in any of the 4 genes ranged up to 10.4%, depending on family history of cancers.
Role in Esophageal and Gastric Cancers
Igaki et al. (1994) found homozygous deletion of p16 in 12 of 13 esophageal cancer cell lines and in 2 of 9 gastric cancer cell lines. They also found that p16 gene loss, cyclin D1 (168461), and p53 gene mutations occurred independently in these cell lines. They interpreted these results as indicating that changes in the p16 gene are involved in most esophageal cancers and play a critical role in the development of this type of malignancy.
Liu et al. (1995) found that exons 1 and 2 of the MTS1 gene were deleted in 67% of esophageal squamous cancer cell lines examined.
Serrano et al. (2000) analyzed gastrinomas from 44 patients for CDKN2A gene mutations and correlated the results to the tumor's biologic behavior, growth pattern, and aggressiveness. No gastrinomas had mutations of exon 1 or exon 2 of CDKN2A, although polymorphisms were found in 54%. No homozygous deletions were found. In 52% of the gastrinomas, hypermethylation of a 5-prime CpG island of the CDKN2A promoter was found. The presence or absence of methylation of the CDKN2A gene did not correlate with clinical characteristics of the gastrinoma, biologic behavior (gastrin release and basal or maximal acid output), the presence or absence of known prognostic factors (tumor size, gastrinoma location, lymph node metastases, liver metastases, and curability), or growth pattern of the gastrinoma postresection. The authors concluded that methylation of the CDKN2A gene is probably a central process in the molecular pathogenesis of these tumors.
Role in Leukemia
By Southern blot analysis, Ogawa et al. (1994) found that both alleles of the CDK4 inhibitor gene were completely or partially deleted in human leukemia cells derived from leukemia patients and from established cell lines. Homozygous deletion was found in 14 of 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia, and 13 with chronic myelocytic leukemia in blastic crisis. All 4 leukemia patients with homozygous deletion of CDKN2A had acute lymphocytic leukemia; 2 of them had no cytogenetic abnormality of chromosome 9. Hebert et al. (1994) likewise found homozygous MTS1 deletions in 20 of 24 cases of T-cell acute lymphoblastic leukemia; homozygous MTS1 deletions were found in only 2 of 31 B-lineage cases (P less than 0.001). The deletions involved MTS1 and MTS2 (CDKN2B; 600431) in most cases. In only 5 cases (4 T and 1 B), deletions involving MTS1 spared the MTS2 gene.
Hemizygous deletions and rearrangements of 9p21 are among the most frequent cytogenetic abnormalities detected in pediatric acute lymphoblastic leukemia (ALL), occurring in approximately 10% of cases. To determine if the p16(INK4a) locus and the tandemly linked p15(INK4b) locus might be the target of these chromosomal lesions, Okuda et al. (1995) analyzed both genes in primary clinical samples from 43 pediatric ALL patients using interphase fluorescence in situ hybridization, Southern blot analysis, and PCR. Deletion of the 2 genes was identified in 18 of 20 cases with cytogenetically observed abnormalities of 9p and in 5 of 23 cases with apparently normal chromosomes 9p, with the majority containing biallelic deletions (16 homozygous and 7 hemizygous). Although most homozygous deletions involved both genes, Southern blot analysis showed an interstitial deletion in a single case that was confined to p16(INK4a), suggesting that p15(INK4b) was not the critical target gene in this case. Sequence analysis of both genes in all 7 cases with hemizygous deletions failed to show mutations within the coding regions of the retained alleles.
Using data from a genomewide association study of 907 individuals with childhood acute lymphoblastic leukemia and 2,398 controls and with validation in samples totaling 2,386 cases and 2,419 controls, Sherborne et al. (2010) demonstrated that common variation at 9p21.3 (rs3731217, intron 1 of CDKN2A) influences acute lymphoblastic leukemia risk with an odds ratio = 0.71, p = 3.01 x 10(-11), irrespective of cell lineage.
Role in Bladder Cancer
In a screen for deletions and sequence variants of p16 in 140 bladder tumors and 16 bladder tumor cell lines, Williamson et al. (1995) found homozygous deletion of p16 in 8 cell lines and small sequence variations in 2. All 13 tumors with small defined deletions of 9p21, 18/31 (58%) of tumors with monosomy 9, and 9/91 (10%) of tumors with no chromosome 9 LOH had homozygous deletion of p16. No tumor-specific sequence variants were identified. Deletion mapping revealed a nested set of deletions focused on p16. The p16 gene, but not the related adjacent p15 gene, was involved in 6 deletions, and 1 tumor had an intragenic deletion of p16. All other deletions involved both p16 and p15 (CDKN2B). Williamson et al. (1995) concluded that p16 represents the major target for deletion at 9p21 in bladder cancer. Cairns et al. (1995) likewise found that homozygous deletions represent the predominant mechanism of inactivation of 9p21 in bladder tumors and are present in other tumor types, including breast and prostate cancer. Moreover, fine mapping of these deletions implicated a 170-kb minimal region that includes p16 and excludes p15. Of 285 bladder cancers studied, 177 (62%) had loss of 9p material; of these, they found LOH in 51 and homozygous deletion in 126.
Tsutsumi et al. (1998) studied p16/p19 deletion and p16 promoter methylation, as well as loss of 9p21 heterozygosity, in pure squamous cell carcinomas (SCC) and in transitional cell carcinomas (TCC) with SCC of the bladder. Homozygous deletion of p16/p19 was detected in 11 of 21 (52%) cases of pure SCC and in 3 of 10 (30%) cases of TCC with SCC. Three cases of TCC with SCC had p16/p19 deletion, hypermethylation of the p16 promoter, or loss of heterozygosity on 9p21 only in the SCC components, suggesting that these molecular alterations occurred preferentially in SCC. Interestingly, homozygous deletion of p16/p19 was observed in squamous metaplasia from 5 of 11 (45%) bladder cancer patients, showing that this change occurred in preneoplastic cells. On the other hand, these deletions were not found in squamous metaplasias from noncancerous patients. Tsutsumi et al. (1998) concluded that p16/p19 deletion is associated with early carcinogenesis of SCC of the bladder, and squamous metaplasia of the bladder cancer patient has already sustained genetic changes found in cancer, and that genetic mosaicism occurs in cases of TCC with SCC, with the SCC component showing more frequent 9p21 alterations than the TCC component.
Role in Cutaneous Melanoma
Kamb et al. (1994) found mutations or homozygous deletions in approximately 75% of melanoma cell lines. Studying 18 familial melanoma kindreds, Hussussian et al. (1994) identified 6 probable disease-related mutations in the CDKN2A gene. Among families with sufficient linkage data, the disease-related CDKN2A germline mutations were detected in families linked to 9p21 and not in families linked to 1p36 (155600), thus providing support for genetic heterogeneity for this disease. Hussussian et al. (1994) admitted that the data did not provide definitive proof that CDKN2A mutation or deletion is required for the development of melanoma. Functional studies of the mutated p16 protein and gene transfer experiments to determine the ability of wildtype and mutant CDKN2A to suppress tumorigenicity in melanoma cells with homozygous deletions of 9p21 were considered necessary to provide that proof.
Ranade et al. (1995) described biochemical analyses of the missense germline mutations and a single somatic mutation detected in the melanoma families by Hussussian et al. (1994). The melanoma-related mutants were impaired in their ability to inhibit the catalytic activity of the cyclin D1/CDK4 (123829) and cyclin D1/CDK6 (603368) complexes in vitro. The data of Ranade et al. (1995) were thought to provide a biochemical rationale for the hypothesis that carriers of certain CDKN2 mutations are at increased risk of developing melanoma.
Kamb et al. (1994) concluded that either the majority of mutations in the CDKN2A gene causing malignant melanoma fall outside the CDKN2A coding sequence or that CDKN2A is not the chromosome 9p melanoma susceptibility locus that they symbolized MLM. They screened the gene (referred to as CDKN2) for mutations in 8 American and 5 Dutch families in which linkage studies appeared to indicate mapping of a susceptibility locus to 9p21. Sequence analysis of the 3 coding exons and the adjacent splice junctions revealed only 3 heterozygous nucleotide substitutions among the 8 American probands and none in the Dutch probands. A population frequency analysis was then conducted in unrelated individuals who had married into high risk cancer kindreds in Utah. Two of the variants were not detected in a set of 100 normal samples; the third was present in 6 out of 163 samples, suggesting that it is a polymorphism present in roughly 4% of the Utah population. By allele-specific oligonucleotide (ASO) experiments, Kamb et al. (1994) sought the other 2 mutations in 30 affected individuals with a positive family history for melanoma but with unknown linkage status, and in 66 affected individuals with unknown family history. No other occurrence of the mutations was detected.
Wainwright (1994) referred to the uncertainty about the relationship of familial melanoma and p16 as 'a hung jury.' He suggested that the familial melanoma story 'contains a sobering, perhaps slightly depressing lesson for those attempting to isolate genes which do not show a fully penetrant, single locus inheritance' such as the hereditary breast and ovarian cancer locus at chromosome 17q21 (113705).
Puig et al. (1995) analyzed 12 microsatellite markers on 9p in 54 CMM tumors and paired normal tissues from the same subjects. In 46% of the tumors, including 2 in situ CMMs, LOH was found at 9p. Only one tumor was homozygously deleted for 9p markers. The smallest deleted region was defined by 5 tumors and included markers D9S126 to D9S259. Loss of 8 or more markers correlated significantly with a worse prognosis (P less than 0.002). Among the primary tumors, 87.5% of those with large deletions had a high risk of metastasis, as compared with only 18% of those without deletions or with loss of fewer than 8 markers (P = less than 0.001). It was not possible for Puig et al. (1995) to demonstrate homozygous deletions of p16 in any of the CMM tumors. In 4 tumors, the LOH for 9p markers did not involve p16. Thus, the data suggested the existence of several tumor suppressor genes on 9p that are involved in the predisposition to and/or progression of CMM and exclude p16 from involvement in the early development of some melanoma tumors.
Liu et al. (1995) described a family with inherited melanoma in which a novel mutation in exon 2 of the p16(INK4A) gene (600160.0004) segregated with disease. The mutant allele encoded a protein with an in-frame deletion of 2 amino acids (asp96 and leu97). They showed that the mutant protein is functionally abnormal: it was unable to bind CDK4 in vitro and did not inhibit colony formation in tertiary passage rat embryo fibroblasts. Moreover, in a metastatic lesion from 1 patient, the wildtype allele was deleted and the mutant allele retained. Liu et al. (1995) concluded that family members carrying the germline mutation in this gene are predisposed to melanoma.
Walker et al. (1995) found that in 7 of 18 Australian melanoma kindreds, including the 6 largest, CDKN2 mutations segregated with the putative melanoma chromosome previously assigned by 9p haplotype analysis. The mutations included duplication of a 24-bp repeat, a deleted C residue resulting in the introduction of a premature stop codon, and 4 single basepair changes causing amino acid substitutions. Mutations segregated to 46 of 51 affected persons in these 7 kindreds, with 3 apparent sporadic cases in 1 family and 1 in each of another 2 families. Penetrance was variable (55-100%) among the different mutations. These data were presented as additional strong support that the CDKN2 gene is the chromosome 9p21 familial melanoma locus.
FitzGerald et al. (1996) screened for germline mutations in p16 and in 2 other candidate melanoma genes, p19ARF and CDK4 (123829), in 33 consecutive patients treated for melanoma; these patients had at least 1 affected first- or second-degree relative (28 independent families). Five independent, definitive p16 mutations were detected, including 1 nonsense, 1 disease-associated missense, and 3 small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame from p16, were detected in patients who also had mutations inactivating p16.
Dracopoli and Fountain (1996) reviewed the role of CDKN2 mutations in cutaneous melanoma. They reported that there had been 7 independent studies in which the CDKN2 gene was screened for germline mutations. Germline mutations were found in 34 of 76 families. They emphasized that CDKN2 mutations are only found in a subset of the 9p21-linked families and postulated that failure to detect CDKN2 mutations in these 9p21-linked families may be due to problems of mutation detection by SSCP analysis in a GC-rich region, failure to detect promoter mutations, or genomic imprinting of CDKN2. Dracopoli and Fountain (1996) reviewed the evidence for a second melanoma predisposing gene on 9p21 and for the possible role in melanoma pathogenesis of the CDKN2B gene (600431), which maps in close proximity to the CDKN2 gene. They noted that 14 different germline mutations of CDKN2 have been reported; these include 7 missense mutations, 1 nonsense mutation, 1 insertion, 4 deletions, and a splice donor mutation. Dracopoli and Fountain (1996) stated that the functional significance of the 7 missense mutations was hard to predict. Using in vitro assays, they identified 3 mutants with greatly reduced binding capacity for CDK4/cyclin D1 complexes and reduced inhibition of RB phosphorylation. Based on comparative analysis of sporadic melanomas and melanoma cell lines, Dracopoli and Fountain (1996) determined that small homozygous deletions limited to a region surrounding the CDKN2 gene are approximately 5 to 6 times more common in melanoma cell lines than in uncultured metastatic melanomas. They concluded that the growth potential in culture of a melanoma cell without functional CDKN2 protein is significantly higher than that of a melanoma cell with wildtype CDKN2, and that mutation analysis based solely on analysis of tumor cell lines should be viewed with caution.
In 27 UK families showing evidence of predisposition to melanoma, Harland et al. (1997) sequenced all exons of CDKN2 and analyzed the CDK4 gene, which encodes the protein to which p16 binds, for mutations. Five different germline mutations in CDKN2 were found in 6 families (e.g., 600160.0007); 3 of them had previously been reported.
Monzon et al. (1998) used PCR, SSCP analysis, and direct DNA sequencing to identify germline mutations in the CDKN2A gene in patients with multiple primary melanomas who did not have family histories of the disease. A quantitative yeast 2-hybrid assay was used to evaluate the functional importance of the variants found. Of 33 patients with multiple primary melanomas, 5 (15%) had germline mutations. These included a 24-bp insertion at the 5-prime end of the coding sequence, 3 missense mutations, and a 2-bp deletion at nucleotide position 307-308, resulting in a truncated CDKN2A protein. In 3 families, CDKN2A mutations identical to those in the probands were found in other family members. In 2 families with mutations, previously unknown family histories of melanoma were uncovered.
Fargnoli et al. (1998) screened 10 familial melanoma kindreds for germline mutations in the p16(INK4a) and p19(ARF) genes, alternatively spliced forms of the CDKN2A gene. They identified 4 independent germline mutations in exon 1-alpha and exon 2 of the CDKN2A gene. No disease-associated mutations in exon 1-beta of the p19(ARF) gene were found. From this small study, it appeared that the p19(ARF) gene does not play a role in melanoma susceptibility.
As noted earlier, Dracopoli and Fountain (1996) reviewed the evidence for a second melanoma predisposing gene on 9p21. Puig et al. (1995) found large 9p deletions in 25 of 54 primary and metastatic melanomas. Surprisingly, 4 of those 25 deletions did not include p16 itself, but were located more proximally. Furthermore, Wiest et al. (1997) observed 100% loss of heterozygosity in the 9p region in squamous cell carcinomas of the lung. About half of these tumors were shown to be homozygous for a microdeletion within the area of loss of heterozygosity. Those microdeletions clustered approximately equally in 2 areas, one of which included p16, whereas the other more proximal cluster could have revealed the location of another tumor suppressor gene. Studying Dutch families with familial atypical multiple mole-melanoma syndrome (FAMMM; 155600) with the founder mutation, a 19-bp deletion in exon 2 of the CDKN2A gene, so-called p16-Leiden (600160.0003), van der Velden et al. (1999) found a characteristic haplotype in those carriers of p16-Leiden with melanoma, as opposed to those p16-Leiden persons without melanoma. They interpreted this as indicating the location of a locus linked to p16 that modifies melanoma risk in these families.
Solar ultraviolet radiation is the major environmental risk factor for nevi. However, Zhu et al. (1999) performed a twin study to investigate large differences among individuals living in a small geographic area with uniformly high skin exposure. Nevi may be subclassified as raised or flat. In the sample of Zhu et al. (1999), raised nevi were 27% of the total, and the 2 kinds had a correlation of 0.33. Correlations for total nevus count in 153 monozygotic and 199 dizygotic twin pairs were 0.94 and 0.60, respectively, a finding compatible with a very high degree of genetic determination. The authors hypothesized that some of the genetic variance might be due to variation in the CDKN2A gene. Analysis of linkage to a highly polymorphic marker (D9S942), located close to CDKN2A, detected quantitative trait loci (QTL) effects accounting for 27% of variance in total nevus count, rising to 33% if flat but not raised moles were considered. Total heritability was higher for raised (0.69) than for flat (0.42) moles, but QTL linkage was zero for raised moles, whereas it accounted for 80% of the heritability of flat moles; additionally, family environment accounted for only 15% of variance in raised moles versus 46% in flat moles. These findings suggested that raised and flat nevi have different etiologies. Longer alleles at D9S942 were associated with higher flat mole counts. Since germline mutations in CDKN2A are rare, it was considered likely that variants in the noncoding regions of this gene, or in another gene nearby, are responsible for this major determinant of moles and, hence, of melanoma risk.
In a genomewide study of nevus count using an expanded sample of twins and their families, including 221 pairs of monozygotic twins, Zhu et al. (2007) confirmed linkage to chromosome 9p21 with a maximum lod score of 3.42 after inclusion of fine mapping markers.
Bahuau et al. (1998) identified a germline deletion involving the CDKN2A locus in familial proneness to melanoma and nervous system tumors. Petronzelli et al. (2001) detected a novel splice site mutation in a family with melanomas, neurofibromas, and multiple dysplastic nevi. Both alternative mRNAs produced by the mutant allele lacked shared sequences from exon 2, which encodes a substantial portion (more than 50%) of both p16(INK4) and p14(ARF) proteins. They suggested that the development of neurofibromas may be explained by cooperative effects of combined inactivation of these 2 proteins or, alternatively, of p14(ARF) alone.
The melanoma-astrocytoma syndrome (155755) is characterized by a dual predisposition to melanoma and neural system tumors, commonly astrocytoma. Germline deletions of the region on 9p21 containing the CDKN2A and CDKN2B genes and CDKN2A exon 1-beta have been reported in kindreds, implicating contiguous tumor suppressor gene deletion as a cause of this syndrome. Randerson-Moor et al. (2001) described a family characterized by multiple melanoma and neural cell tumors segregating with a germline deletion of the p14(ARF)-specific exon 1-beta of CDKN2A. The deletion did not affect the coding or minimal promoter sequences of either CDKN2A or CDKN2B. The authors hypothesized that the phenotype is due to either loss of p14(ARF) function, rather than contiguous loss of both CDKN2A and CDKN2B; or disruption of expression of p16.
Mutations in the CDKN2A gene are melanoma-predisposition alleles with high penetrance, although they have low population frequencies. In contrast, variants of the melanocortin-1 receptor gene (MC1R; 155555) confer much lower melanoma risk but are common in European populations. To test for possible modifier effects on melanoma risk, Box et al. (2001) assessed 15 Australian CDKN2A mutation-carrying melanoma pedigrees for MC1R genotype. A CDKN2A mutation in the presence of a homozygous consensus MC1R genotype had a raw penetrance of 50%, with a mean age at onset of 58.1 years. When an MC1R variant allele was also present, the raw penetrance of the CDKN2A mutation increased to 84%, with a mean age at onset of 37.8 years (P = 0.01). The presence of a CDKN2A mutation gave a hazard ratio of 13.35, and a hazard ratio of 3.72 for MC1R variant alleles was also significant. The impact of MC1R variants on risk of melanoma was mediated largely through the action of 3 common alleles, arg151 to cys (R151C; 155555.0004), arg160 to trp (155555.0005), and asp294 to his (155555.0001), known to be associated with red hair, fair skin, and skin sensitivity to ultraviolet light.
Van der Velden et al. (2001) found that the MC1R variant R151C modified melanoma risk in Dutch families with melanoma. They concluded that the R151C variant is overrepresented in patients with melanoma from families with the p16-Leiden mutation (600160.0003). They suggested that the R151C variant may be involved in melanoma tumorigenesis in a dual manner, both as a determinant of fair skin and as a component in an independent additional pathway, because the variant contributed to increased melanoma risk even after statistical correction for its effect on skin type.
In an analysis of 15 Italian melanoma families for germline mutations, Della Torre et al. (2001) gained results supporting the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families.
In North America, Europe, and Australasia, approximately 20% of familial melanoma kindreds carry germline mutations in CDKN2A. There is also an increased risk of pancreatic cancer in a subset of families with mutation in this gene. Using published data, Goldstein (2004) found that 67 different CDKN2A mutations had been identified in 189 melanoma-prone families. In 42 families with 18 different mutations, pancreatic cancer had also been reported. Seventy percent of the mutations were observed only once. Comparison of 147 melanoma-prone families without pancreatic cancer to the 42 families that had pancreatic cancer reported showed no significant differences in the types or locations of mutations.
Kannengiesser et al. (2009) identified 20 novel germline mutations in the CDKN2A gene in patients with familial melanoma or multiple melanomas. Segregation studies, in silico analysis, in vitro functional studies showing loss of interaction with CDK4, and cell proliferation assays indicated that 18 of the 20 variants had clear loss of function, allowing them to be classified as pathogenic. All of the mutations affected the p16(INK4) structure.
Association with Diabetes
In genomewide association studies of type 2 diabetes (125853) involving genotype data from a variety of international consortia, the Diabetes Genetics Initiative of Broad Institute of Harvard and MIT, Lund University, and Novartis Institutes for BioMedical Research (2007), Zeggini et al. (2007), and Scott et al. (2007) detected association of a single-nucleotide polymorphism (SNP) on chromosome 9, rs10811661, and diabetes susceptibility. This SNP is 125 kb upstream from CDKN2A/CDKN2B, the nearest annotated genes. All-data metaanalyses obtained genomewide significance (OR = 1.20, P = 7.8 x 10(-15)).
Helgadottir et al. (2008) replicated the association of the rs10811661 T allele to type 2 diabetes in Icelandic, Danish, and United States case-control groups (OR = 1.29, P = 2.5 x 10(-10)).
Role in Other Cancers
Although pituitary tumors develop at a high frequency in retinoblastoma (Rb)-knockout mice, defects in the RB gene are not common in human pituitary tumors. The inverse correlation of RB and p16 defects in certain human tumors led Woloschak et al. (1996) to investigate the expression of p16 in human pituitary tumors as an indirect mechanism of RB inactivation. By Western blot analysis, the p16 gene product was undetectable in 25 human pituitary tumors, whereas high levels of p16 could be demonstrated in 10 normal human pituitary specimens under the same conditions of protein extraction and immunoblotting. Similar results were obtained at the mRNA level. Quantitative PCR analysis revealed diminished amplification of p16 relative to a control gene in 3 of 25 tumors, suggesting homozygous p16 gene loss. This altered expression is not associated with frequent p16 mutation or gene loss, suggesting to Woloschak et al. (1996) that alternative mechanisms of gene inactivation and/or altered regulation occur in most of these tumors.
Ohhara et al. (1996) examined the sequence and expression level of the CDKN2A gene in primary colorectal carcinomas. Using RT-PCR, they quantitatively detected the CDKN2A transcript in 14 of 17 tumors, but in only one case of adjacent normal mucosa. Direct sequencing of the amplified CDKN2A gene showed no somatic mutations in the 17 tumors examined. The authors concluded that enhanced expression, rather than inactivation of the CDKN2A gene, may be involved in the early stages of the pathogenesis of primary colorectal carcinomas.
Pilon et al. (1999) investigated inactivation of the p16 tumor suppressor gene in a series of 14 adrenocortical tumors. Using 11 polymorphic microsatellite markers spanning the short arm of chromosome 9, they demonstrated that 3 of 7 adrenocortical carcinomas and 1 of 7 adrenocortical adenomas had LOH within chromosome 9p21, the region containing p16. Immunohistochemistry showed the absence of p16 nuclear staining in all adrenocortical tumors with LOH within 9p21, and positive staining in all remaining tumors without LOH. The authors concluded that LOH within 9p21 associated with lack of p16 expression occurs in a considerable proportion of adrenocortical malignant tumors but is rare in adenomas. Furthermore, they suggested that inactivation of p16 may contribute to the deregulation of cell proliferation in this neoplastic disease.
Honoki et al. (2007) performed a metaanalysis of 6 studies representing 188 patients with Ewing sarcoma (612219). Presence of a p16(INK4a) mutation was associated with a poor prognosis, as assessed by likelihood of 2-year survival. The estimated pooled relative risk for p16(INK4a) alteration for 2-year survival was statistically significant (2.17; 95% confidence interval 1.55-3.03). There was no statistically significant difference in the pooled estimated risk ratios of p16(INK4a) alteration for disease outcome between patients with or without metastasis at diagnosis.
In a study of patients with stage I nonsmall cell lung cancer (NSCLC; see 211980) who underwent curative resection but had a recurrence compared to matched patients who did not have a recurrence, Brock et al. (2008) found that promoter methylation of the CDKN2A, CDH13 (601364), RASSF1A (605082), and APC (611731) genes in tumors and in histologically tumor-negative lymph nodes was independently associated with tumor recurrence. Methylation of the promoter regions of CDKN2A and CDH13 in both tumor and mediastinal lymph nodes was associated with an odds ratio of recurrent cancer of 15.50 in the original cohort and an OR of 25.25 when the original cohort was combined with an independent validation cohort of 20 patients with stage I NSCLC.
Metastatic Cancer
Robinson et al. (2017) performed whole-exome and transcriptome sequencing of 500 adult patients with metastatic solid tumors of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53 (191170), CDKN2A, PTEN (601728), PIK3CA (171834), and RB1 (614041). Putative pathogenic germline variants were present in 12.2% of cases, of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing to identify gene fusions, pathway activation, and immune profiling.
Mutation Databases
Smith-Sorensen and Hovig (1996) reported a database with 146 point mutations in the CDKN2A gene. They also summarized studies of the biochemical and biologic functions of both wildtype and mutant proteins.
Murphy et al. (2004) described an online database describing both germline and somatic variants of the CDKN2A tumor suppressor gene.
Associations Pending Confirmation
For a discussion of a possible association between variation in the CDKN2A gene and glioma, see GLM5 (613030).
Cyclin-dependent kinase inhibitor-2A (CDKN2A) goes by the colloquial designation p16, and is sometimes (e.g., Wainwright, 1994; Ranade et al., 1995) referred to as p16(INK4). The gene was originally symbolized MTS1 (for multiple tumor suppressor-1) by Kamb et al. (1994), who later used the symbol CDKN2 because MTS1 had been preempted by the malignant transformation suppression-1 gene (154280) located on 1p. See also CDKN2B (600431).
Krimpenfort et al. (2001) mutated mice specifically in the Cdkn2a(p16-Ink4a) isoform, generating an allele called Ink4a. This allele is silent in the p16(Arf) reading frame but introduces a stop codon in the p16(Ink4a) transcript at conserved amino acid position 101, resulting in deletion of the fourth ankyrin repeat motif. The analogous human allele is a naturally occurring mutation found in a wide variety of human tumor types and results in an unstable protein that is severely defective in its ability to inhibit phosphorylation of RB1 and to induce cell cycle arrest in transfected cells. Ink4a-homozygous mice do not show a significant predisposition to spontaneous tumor formation within 17 months. Embryo fibroblasts derived from them proliferate normally, are mortal, and are not transformed by oncogenic HRAS (190020). The very mild phenotype of the Ink4a-homozygous mice implies that the very strong phenotypes of the original Ink4a/Arf(delta-2,3) mice was primarily or solely due to loss of Arf. However, mice that are deficient for Ink4a and heterozygous for Arf spontaneously developed a wide spectrum of tumors, including melanoma. Treatment of these mice with the carcinogen 7,12-dimethylbenzanthracene (DMBA) results in an increased incidence of melanoma, with frequent metastases. Krimpenfort et al. (2001) concluded that in the mouse, Ink4a is a tumor-suppressor gene that, when lost, can recapitulate the tumor predisposition seen in humans. Sharpless et al. (2001) generated p16(Ink4a)-specific knockout mice that retained normal p19(Arf) function. Mice lacking p16(Ink4a) were born with expected mendelian distribution and exhibited normal development except for thymic hyperplasia. T cells deficient in p16(Ink4a) exhibited enhanced mitogenic responsiveness, consistent with the established role of p16(Ink4a) in constraining cellular proliferation. In contrast to mouse embryo fibroblasts deficient in p19(Arf), p16(Ink4a)-null mouse embryo fibroblasts possessed normal growth characteristics and remained susceptible to RAS-induced senescence. Compared with wildtype mouse embryo fibroblasts, p16(Ink4a)-null mouse embryo fibroblasts exhibited an increased rate of immortalization, although this rate was less than that observed previously for cells null for Ink4a/Arf, p19(Arf), or p53 (191170). Furthermore, p16(Ink4a) deficiency alone was associated with an increased incidence in spontaneous and carcinogen-induced cancers. Sharpless et al. (2001) concluded that p16(Ink4a) along with p19(Arf), functions as a tumor suppressor in mice.
INK4A/ARF and p53 mutations promote tumorigenesis and drug resistance in part by disabling apoptosis. Schmitt et al. (2002) showed that primary murine lymphomas responded to chemotherapy by engaging a senescence program controlled by p53 and p16(Ink4a). Hence, tumors with p53 or Ink4a/Arf mutations, but not those lacking Arf alone, responded poorly to cyclophosphamide therapy in vivo. Moreover, tumors harboring a Bcl2 (151430)-mediated apoptotic block underwent a drug-induced cytostasis involving the accumulation of p53, p16(Ink4a), and senescence markers, and typically acquired p53 or Ink4a mutations upon progression to a terminal stage. Mice bearing tumors capable of drug-induced senescence had a much better prognosis following chemotherapy than those harboring tumors with senescence defects. Therefore, cellular senescence contributes to treatment outcome in vivo.
Sugimoto et al. (2003) showed that p19(ARF) inhibits production of ribosomal RNA, retarding processing of 47/45S and 32S precursors. These effects correlated with but did not strictly depend upon inhibition of rRNA biosynthesis or cell cycle arrest, were not mimicked by p53, and required neither p53 nor MDM2. ARF mutants lacking conserved amino acid residues 2 to 14 did not block rRNA synthesis and processing or inhibit cell proliferation. The authors proposed that evolution may have linked a primordial nucleolar ARF function to MDM2 and p53, creating a more efficient checkpoint-signaling pathway for coordinating ribosomal biogenesis and cell cycle progression.
The TRP53 and RB pathways are 2 of the principal pathways controlling cell proliferation that have been identified in human and mouse cells. The CDKN2A locus is involved in both pathways by virtue of encoding p16(INK4a), a regulator of RB1 phosphorylation mediated by CDK4 and CDK6 (603368), and p19(ARF), a modulator of p53 degradation mediated by MDM2. Mice deficient for both p16(INK4a) and p19(ARF) are viable but highly prone to tumors, succumbing to lymphomas and fibrosarcomas early in life (Serrano et al., 1996). Lund et al. (2002) used large-scale insertional mutagenesis to screen for loci that can participate in tumorigenesis in collaboration with loss of the Cdkn2a gene in Cdkn2a -/- mice. They infected such mice with Moloney murine leukemia virus (MoMuLV). Insertional mutagenesis by the latent retrovirus was synergistic with loss of Cdkn2a expression, as indicated by a marked acceleration in the development of both myeloid and lymphoid tumors. Lund et al. (2002) isolated 747 unique sequences flanking retroviral integration sites and mapped them against the mouse genome sequence databases from Celera and Ensembl. In addition to 17 insertions targeting gene loci known to be cancer related, the authors identified 37 new common insertion sites, 8 of which encode components of signaling pathways that are involved in cancer.
Tsai et al. (2002) demonstrated that loss of the Arf tumor suppressor gene strongly accelerates intermediate lobe pituitary tumorigenesis in Rb heterozygous mice. The effects in the pituitary were greater than those conferred by loss of p53. Tsai et al. (2002) concluded that inactivation of ARF acts more broadly than that of p53 in connecting abrogation of the RB pathway to tumorigenesis.
Aslanian et al. (2004) found that in wildtype mouse embryonic fibroblasts (MEFs), the Arf promoter was occupied by E2f3 but not by any other E2f family members. In quiescent cells, this role was largely fulfilled by the E2f3b isoform. E2f3 loss was sufficient to derepress Arf, triggering activation of p53 (191170) and expression of p21(Cip1). Thus, E2f3 is a key repressor of the p19(Arf)-p53 pathway in normal cells. Consistent with this, Arf mutation suppressed the activation of p53 and p21(Cip1) in E2f3-deficient MEFs. Arf loss also rescued the cell cycle reentry defect of E2f3-null cells, which correlated with restoration of appropriate activation of classic E2f-responsive genes.
In rodent models of aging, Krishnamurthy et al. (2004) found that expression of p16(Ink4a) and Arf markedly increased in almost all tissues with advancing age, whereas there was little or no change in the expression of other related cell cycle inhibitors. The age-associated increase in expression of p16(Ink4a) and Arf was attenuated in the kidney, ovary, and heart by caloric restriction, and this decrease correlated with diminished expression of an in vivo marker of senescence as well as decreased pathology of those organs. Krishnamurthy et al. (2004) suggested that expression of the INK4A/ARF tumor suppressor locus is a robust biomarker, and possible effector, of mammalian aging.
To investigate the role of oncogenic signaling in p53-mediated protection against cancer, Efeyan et al. (2006) used mice with 2 genetically engineered traits: one had no Arf allele, and the other had a 'super' p53 allele, i.e., they carried a single additional transgenic copy of the intact p53 gene. Efeyan et al. (2006) found that Arf-null mice responded normally to DNA damage and that p53(super) mice showed the same enhancement of apoptosis irrespective of whether ARF was present or absent. However, Arf-null cells were unable to respond effectively to oncogenic signaling and underwent neoplastic transformation by oncogenes in vitro, irrespective of the presence or absence of the p53(super) allele. Efeyan et al. (2006) found that p53(super)/Arf-null mice succumbed to spontaneous tumors at the same rate as wildtype p53/Arf-null mice, producing the same profile of sarcomas, lymphomas, and histiocytic sarcomas. When they treated both classes of mice with a DNA-damaging agent, the p53(super) mice with the Arf-null allele did not benefit from the extra p53 allele. Efeyan et al. (2006) concluded that oncogenic signaling is the critical event that elicits p53-dependent protection, and that DNA damage stimulus is less important.
Using the model developed by Christophorou et al. (2005) in which p53 status can be reversibly switched in vivo between functional and inactive states, Christophorou et al. (2006) found that the p53-mediated pathologic response to whole body irradiation, a prototypic genotoxic carcinogen, is irrelevant for suppression of radiation-induced lymphoma. In contrast, delaying the restoration of p53 function until the acute radiation response subsided abrogated all of the radiation-induced pathology yet preserved much of the protection from lymphoma. Christophorou et al. (2006) found that such protection is absolutely dependent on p19(ARF), a tumor suppressor induced not by DNA damage, but by oncogenic disruption of the cell cycle.
Matheu et al. (2007) showed that genetically manipulated mice with increased but otherwise normally regulated levels of Arf and p53 presented strong cancer resistance and had decreased levels of aging-associated damage. Matheu et al. (2007) concluded that their observations extended the protective role of Arf/p53 to aging, revealing a previously unknown anti-aging mechanism and providing a rationale for the coevolution of cancer resistance and longevity.
Because mice lacking the Arf tumor-suppressor gene develop eye disease reminiscent of persistent hyperplastic primary vitreous (PHPV; see 611308), Thornton et al. (2007) explored mechanisms by which Arf promoted eye development and its absence caused a PHPV-like disease. Chimeric mice were made by fusing wildtype and Arf -/- morulae. Newborn chimeras had primary vitreous hyperplasia, evident as a retrolental mass. The mass was usually present when the proportion of Arf -/- was relatively high and absent when the Arf -/- proportion was low. Thornton et al. (2007) concluded that in the mouse model, loss of Arf in only a subset of cells caused a PHPV-like disease. The data indicated that both cell autonomous and non-cell autonomous effects of Arf might contribute to its role in vitreous development.
Krimpenfort et al. (2007) reported that mice deficient for all 3 open reading frames encoded at the Cdkn2 locus (Cdkn2ab-null) are more tumor-prone and develop a wider spectrum of tumors than Cdkn2a mutant mice, with a preponderance of skin tumors and soft tissue sarcomas (i.e., mesothelioma) frequently composed of mixed cell types and often showing biphasic differentiation. Cdkn2ab-null mouse embryonic fibroblasts were substantially more sensitive to oncogenic transformation than Cdkn2a mutant mouse embryonic fibroblasts (MEFs). Under conditions of stress, p15(Ink4b) (600431) protein levels were significantly elevated in MEFs deficient for p16(Ink4a). Krimpenfort et al. (2007) concluded that p15(Ink4b) can fulfill a critical backup function for p16(Ink4a) and suggested a model that provided an explanation for the frequent loss of the complete CDKN2B-CDKN2A locus in human tumors.
Bmi1 (164831) is necessary for the maintenance of adult hematopoietic stem cells (HSCs) and neural stem cells. Akala et al. (2008) demonstrated that bone marrow cells from mice with triple deletion of p16(Ink4a), p19(Arf), and Trp53, all genetically downstream of Bmi1, have an approximately 10-fold increase in cells able to reconstitute the blood long term. This increase was associated with the acquisition of long-term reconstitution capacity by cells of the phenotype c-kit+Sca1+Flt3+CD150-CD48-Lin-, which defines multipotent progenitors in wildtype mice. The pattern of triple mutant multipotent progenitor response to growth factors resembled that of wildtype multipotent progenitors but not wildtype HSCs. Akala et al. (2008) concluded that p16(Ink4a)/p19(Arf) and Trp53 have a central role in limiting the expansion potential of multipotent progenitors. The authors commented that these pathways are commonly repressed in cancer, suggesting a mechanism by which early progenitor cells could gain the ability to self-renew and become malignant with further oncogenic mutations.
Visel et al. (2010) showed that deletion of the 70-kb noncoding interval on mouse chromosome 4 orthologous to the chromosome 9p21 interval associated with human coronary artery disease (CAD) (see CHD8, 611139) affects cardiac expression of neighboring genes, as well as proliferation properties of vascular cells. Mice with homozygous deletion of the 70-kb interval (delta-70-kb) were viable but showed increased mortality both during development and as adults. Cardiac expression of 2 genes near the noncoding interval, Cdkn2a and Cdkn2b (600431), was severely reduced in delta-70-kb homozygous mice, indicating that distant-acting gene regulatory functions are located in the noncoding CAD risk interval. Allele-specific expression of Cdkn2b transcripts in heterozygous mice showed that the deletion affects expression through a cis-acting mechanism. Primary cultures of aortic smooth muscle cells from homozygous delta-70-kb mice exhibited excessive proliferation and diminished senescence, a cellular phenotype consistent with accelerated CAD pathogenesis. Visel et al. (2010) concluded that, taken together, their results provided direct evidence that the CAD risk interval has a pivotal role in the regulation of cardiac CDKN2A/B expression, and suggested that this region affects coronary artery disease progression by altering the dynamics of vascular cell proliferation.
Huang et al. (2011) demonstrated the direct induction of functional hepatocyte-like (induced hepatocyte, iHep) cells from mouse tail-tip fibroblasts by transduction of Gata4 (600576), Hnf1-alpha (142410), and Foxa3 (602295) and inactivation of p19(Arf). iHep cells showed typical epithelial morphology, expressed hepatic genes, and acquired hepatocyte functions. Notably, transplanted iHep cells repopulated the livers of fumarylacetoacetate hydrolase-deficient (Fah-null; see 613871) mice and rescued almost half of recipients from death by restoring liver functions.
The report of Gonzalez et al. (2006), which concluded that aberrant expression of CDC6 (602627) is oncogenic by directly repressing the INK4/ARF locus through the RD(INK4/ARF) element, was retracted.
Among the melanoma (155601) cell lines that carried at least 1 copy of CDKN2A (the other copy frequently being deleted), Kamb et al. (1994) identified a variety of nonsense, missense, or frameshift mutations. One of these was a G-to-A transition that converted gly259 to ser.
Among 14 melanoma (155601) cell lines in which at least 1 copy of CDKN2A was present (the other copy frequently being deleted) and in which nonsense, missense, or frameshift mutations were identified, Kamb et al. (1994) found the same mutation in 2: a C-to-T transition converting codon 232 from arg to stop.
Gruis et al. (1995) analyzed CDKN2A coding sequences in 15 Dutch FAMMM syndrome pedigrees and identified a 19-bp germline deletion in 13 of them. All 13 families originated from an endogamous population. The deletion caused a reading-frame shift, predicted to result in a severely truncated p16 protein. Homozygosity for the deletion was found in 2 family members, one of whom showed no obvious signs of melanoma. The finding demonstrated that homozygotes for this CDKN2A mutation are viable, and suggested the presence of a genetic mechanism that can compensate for the functional loss of p16. The results strengthened the notion that p16 is the molecular nature of the 9p21-linked form of familial melanoma (CMM2; 155601). Of the 2 homozygous individuals, one was fully examined at the age of 54 and showed as the only possible sign of FAMMM 3 very mildly atypical nevi. Until her death from adenocarcinoma (site not stated) at the age of 55, this subject remained free of melanomas. The second homozygote, a nephew of the first, had a very large number of atypical moles at the age of 11; at the age of 15, an invasive melanoma was found.
(In addition to the 19-bp deletion of p16, which is designated p16-Leiden, there is at least one familial hypercholesterolemia Leiden (143890.0041), factor V Leiden (612309.0001), apoE3 Leiden (107741.0006), and a hemoglobin Leiden (141900.0156).)
Van der Velden et al. (1999) hypothesized that a tentative second tumor-related gene in 9p21 may also act as a modifier of melanoma risk conveyed by known CDKN2A mutations. To identify genetic modifiers for a known, 'primary' susceptibility gene, one would ideally need to study a large number of carriers of a single mutation in that primary gene. Dutch FAMMM families provided them with a unique opportunity for such studies, since the 19-bp founder deletion in exon 2 of the CDKN2A gene, p16-Leiden, segregated in most Dutch FAMMM families. The 36% cumulative incidence for melanoma in p16-Leiden carriers illustrated a high melanoma risk associated with this mutation but also suggested that environmental and/or genetic factors act as risk modifiers. Van der Velden et al. (1999) performed haplotype analysis for 9p21 using microsatellite markers in 6 p16-Leiden families originating from a founder population. In 2 families, p16-Leiden carriers shared an unexpectedly large founder haplotype (approximately 20 cM) around CDKN2A, mostly in the proximal direction. Melanoma-positive p16-Leiden carriers from these families showed this extensive proximal haplotype, compared with melanoma-negative p16-Leiden carriers from the same families. Additional p16-Leiden families less heavily affected with melanoma showed shorter haplotype sharing, excluding the region proximal of CDKN2A. The presence of a gene involved in melanoma susceptibility proximal to CDKN2A was corroborated by somatic deletions of 9p in tumors, which frequently did not include CDKN2A but a more proximal chromosomal region instead. The results provided a candidate region for further gene mapping in p16-negative 9p21-linked melanoma families and guided the search for risk modifiers in melanoma development.
Vasen et al. (2000) performed mutation analysis on 27 families with FAMMM syndrome and identified the CDKN2A-Leiden mutation in 19 families. They identified 86 patients with melanoma, and the second most frequent cancer was pancreatic carcinoma, which was observed in 15 patients from 7 families. The mean age at diagnosis of pancreatic carcinoma was 58 years, with a range from 38 to 77 years. Putative mutation carriers had an estimated cumulative risk of 17% for developing pancreatic carcinoma by age 75 years. In the 8 CDKN2A-Leiden-negative families, no cases of pancreatic carcinoma had occurred. The authors concluded that individuals with the CDKN2A-Leiden mutation show an enormous risk of developing pancreatic cancer (see 606719).
Schneider-Stock et al. (2003) found the p16-Leiden mutation in heterozygous state in the blood and all 3 tumors of a man who was diagnosed at age 54 years with simultaneous development of 3 carcinomas of the pharynx and oral cavity. The patient neither smoked more than 5 cigarettes daily nor abused alcohol. Both his parents and his only sister died of cancer very early (the mother of gynecologic cancer, the father of liver carcinoma, and the sister of leukemia).
Harinck et al. (2012) identified the Leiden mutation (225_243del) in 5 Dutch families ascertained for familial clustering of pancreatic cancer. Four of the families with the Leiden mutation had members who developed melanoma. In the fifth family, only pancreatic cancer segregated with mutation status; no cases of melanoma were noted. Harinck et al. (2012) concluded that the CDKN2A gene should be analyzed in families with pancreatic cancer, even if melanoma is not present.
In a family with melanoma (155601), Liu et al. (1995) found an in-frame deletion of 2 amino acids, asp96 and leu97, in 3 affected and 2 unaffected members. The mutation was a 6-bp deletion of nucleotides 363-368 of their CDKN2A sequence.
In 3 families with melanoma (155601), Hussussian et al. (1994) identified a gly93-to-trp mutation in the CDKN2A gene. (The GLY93TRP mutation is now designated GLY101TRP.)
Whelan et al. (1995) described a kindred with an increased risk of pancreatic cancers, melanomas, and possibly additional types of tumors (see 606719) cosegregating with the gly93-to-trp CDKN2 mutation. Of interest was the occurrence of squamous-cell carcinomas in this family, a rare form, and squamous cell carcinoma of the tongue in the proband. More than half of primary esophageal squamous cell carcinomas have CDKN2 mutations (Mori et al., 1994). The mutation was identified by SSCP analysis and was located in exon 2 where direct sequencing demonstrated a G-to-T nucleotide change at position 295.
Ciotti et al. (1996) indicated that in a small geographic area of Italy (possibly because of founder effect), they had detected the gly93-to-trp mutation in 7 apparently unrelated families and in none of 50 control persons. Nineteen cases of melanoma and 3 of dysplastic nevi were diagnosed at ages ranging from 21 to 70 years in the kindreds with the G93W mutation. In addition, 15 cancers at other sites were found in these kindreds, including 3 pancreatic cancers but no gastric cancers. The pancreatic tumors developed in members of 3 different families at the ages of 48, 51, and 60 years.
Ciotti et al. (2000) stated that gly101-to-trp is the most common CDKN2A missense mutation, having been reported in numerous families from around the world, with a particularly high occurrence in France and Italy. They examined the date of origin of the mutation and its migratory spread in 10 families from Italy, 4 families from the U.S., and 6 families from France. In all families studied, the mutation appeared to have been derived from a single ancestral haplotype. Using maximum likelihood methods, they estimated that the mutation arose 97 generations ago, providing some explanation for the wide geographic spread of this common mutation, particularly in southwestern Europe. All of the Italian families, with one exception, came from a small area on the eastern coast of Liguria.
Auroy et al. (2001) found the G101W mutation in 7 patients with multiple primary melanomas with no known melanoma cases within their families. They stated that the mutation had already been described in more than 20 melanoma-prone families. They genotyped 8 microsatellite markers flanking the CDKN2A gene and found, after allowing for recombination over time, that haplotype sharing provided evidence for an original G101W mutation common to 6 of the 7 sporadic multiple primary melanoma cases.
In Italy, Mantelli et al. (2002) screened for CDKN2A mutations in families with 2 melanoma patients, 1 of whom was younger than 50 years at onset and the other complying with 1 of the following: being a first-degree relative; having an additional relative with pancreatic cancer; or having multiple primary melanomas. Mutations were found in 21 of the 62 families (34%) with a high prevalence of the G101W mutation (18 of 21).
In in vitro functional studies in mammalian cells, McKenzie et al. (2010) found that the G101W mutant protein had decreased binding to CDK4 (123829) (about 20% of wildtype). Cell cycle inhibitory activity was similar to wildtype at 37 degrees Celsius, but was decreased at 40 degrees.
In 10 melanoma (155601) kindreds from southern Sweden, Borg et al. (1996) identified a novel germline mutation in 2 families, constituting an in-frame 3-bp duplication at nucleotide 332 in exon 2. The mutation resulted in an insertion of arg at codon 105, which interrupts the last of the 4 ankyrin repeats of the p16 protein, motifs which have been demonstrated as important in binding and inhibiting the activity of cyclin D-dependent kinases 4 and 6 in cell cycle G1 phase regulation. Other malignancies observed in gene carriers or obligate carriers included cervical, breast, and pancreatic carcinomas, and a non-Hodgkin lymphoma. Analysis of microsatellite markers adjacent to the p16 gene at chromosomal region 9p21 in the 2 families with the mutation showed that they shared a common haplotype, in keeping with a common ancestor.
By haplotype analysis, Hashemi et al. (2001) concluded that the mutation arose 98 generations, or approximately 2,000 years, ago. Thus, the mutation, which they designated 113insR, could be expected to have a more widespread geographic distribution in regions of Europe and North America with ancestral connections to Sweden. Alternatively, CDKN2A may lie in a recombination hotspot region, as suggested by the finding of many meiotic recombinations in a narrow region of approximately 1 cM on 9p21.
Harland et al. (1997) identified a met53-to-ile (M53I) mutation in the CDKN2A gene in affected members of a family with melanoma (155601). They showed that the protein expressed from this previously described mutation did not bind to CDK4/CDK6 (see 123829), confirming its role as a causal mutation in melanoma. Monzon et al. (1998) found the same mutation in a patient with multiple melanomas who was thought to have no family history of melanoma when first investigated.
Pollock et al. (1998) pointed out that the M53I mutation had been described in 5 melanoma families from Australia and North America. Haplotype analysis suggested that there may have been only 1 original M53I mutation.
MacKie et al. (1998) identified this mutation in 4 U.K. melanoma families and also in 1 patient with multiple primary melanomas and a negative family history.
In a patient with multiple primary melanomas (155601), Monzon et al. (1998) identified an arg24-to-pro mutation in the CDKN2A gene. They pointed out that this mutation had previously been reported in melanoma-prone families and was found to cosegregate with cases of melanoma. MacKie et al. (1998) identified this mutation in a U.K. melanoma family.
Pollock et al. (1998) identified 2 new melanoma (155601) kindreds that carried a duplication of a 24-bp repeat present in the 5-prime region of the CDKN2A gene. This brought to a total of 5 the number of melanoma families described with this mutation; the 5 families were from 3 continents: Europe, North America, and Australasia. Previous families were reported by Goldstein et al. (1995), Walker et al. (1995), and Flores et al. (1997). This suggested to Pollock et al. (1998) that there had been at least 3 independent 24-bp duplication events. The duplication was hypothesized to have arisen due to an unequal crossing-over between the two 24-bp repeats naturally present in the wildtype sequence, possibly through polymerase slippage during replication. Further evidence that this repeat region is unstable and therefore prone to both meiotic and mitotic slippage was provided by the identification of a somatic 24-bp deletion of 1 of these normally occurring repeats in a prostate tumor (Komiya et al., 1995).
In in vitro functional expression studies in mammalian cells, McKenzie et al. (2010) found that the 24-bp duplication mutant had slightly decreased affinity for CDK4 (123829) (80% compared to wildtype), but remained fully active in mediating cell cycle arrest. In addition, the 24-bp duplication showed normal subcellular localization.
Though germline CDKN2A coding mutations cosegregated with melanoma (155601) in 25 to 60% of families predisposed to the disease, there remain a number of mutation-negative families that demonstrate linkage of inherited melanoma to 9p21 markers (Hayward, 1996). Liu et al. (1999) showed that a subset of these kindreds possesses a G-to-T transversion at nucleotide -34 of CDKN2A, designated -34G-T. The mutation gives rise to a novel AUG translation initiation codon that decreases translation from the wildtype AUG. The -34G-T mutation was not seen in controls, segregated with melanoma in families, and, on the basis of haplotyping studies, appeared to have arisen from a common founder in the United Kingdom. Liu et al. (1999) suggested that screening for mutations in the promoter region of the CDKN2A gene should be useful in English (MacGeoch et al., 1994), Australian (Holland et al., 1995), and other northern European populations (Borg et al., 1996) in which a low incidence of germline coding mutations of CDKN2A has been found in familial melanoma cases.
Randerson-Moor et al. (2001) described a family characterized by multiple melanoma and neural cell tumors (155755) segregating with a germline deletion of the p14(ARF)-specific exon 1-beta of CDKN2A. The deletion was approximately 14 kb and did not affect the coding or minimal promoter sequences of either CDKN2A or CDKN2B.
One of the most common melanoma (155601)-related CDKN2A mutations reported in North America is val126 to asp (V126D). Goldstein et al. (2001) examined 9 markers surrounding the CDKN2A gene in 3 American and 4 Canadian families carrying this mutation. All 7 families had a haplotype consistent with a common ancestor/founder. The mutation appeared to have originated 34 to 52 generations ago; 1 lod unit supported an interval of 13 to 98 generations.
Harland et al. (2001) reported that affected individuals in 6 of 90 English melanoma (155601) pedigrees screened carried a transition (IVS2-105 A-G) deep in intron 2 of the CDKN2A gene. The mutation creates a false GT splice donor site 105 bases 5-prime of exon 3 and results in aberrant splicing of the mRNA. The authors proposed that this mutation and others similar to it may account for a significant proportion of 9p21-linked melanoma pedigrees with no detectable mutations in the coding region of CDKN2A.
Hewitt et al. (2002) reported a family harboring a splice mutation in exon 1-beta of the CDKN2A gene that resulted in ARF haploinsufficiency. The mutation was observed in a mother and daughter with melanoma and a sib of the mother with breast cancer. The mutation was a 334G-C transversion in exon 1-beta, which predicts a gly122-to-arg substitution. Its position at the 3-prime end of exon 1-beta raised the possibility of interference with splicing. Analysis of the melanoma from 1 individual revealed a 62-bp deletion in exon 3 of the wildtype allele and loss of the mutant allele; these somatic changes would affect both CDKN2A and ARF. The authors suggested that concomitant inactivation of both ARF and CDKN2A may be necessary for melanoma development and that mutations in ARF and CDKN2A possibly confer different levels of susceptibility to melanoma, with the former associated with lesser predisposition.
A val59-to-gly mutation in the CDKN2A gene was found in 4 families segregating cutaneous malignant melanoma (155601): an Israeli family of Moroccan Jewish ancestry (Yakobson et al., 2001), 2 French families (1 of Tunisian Jewish ancestry and another without known Jewish roots) (Soufir et al., 1998), and a Spanish family (Ruiz et al., 1999). Yakobson et al. (2003) found that all but 1 of those affected in these families were heterozygous for the mutation; 1 affected member of the Israeli family was homozygous. Haplotype analysis indicated a single ancestral founder. The mutation, which occurs in a hydrophobic region with the second ankyrin repeat, impairs p16-INK4a function, as shown by studies of protein-protein interactions and cell proliferation assays.
In 4 affected members from 3 families and 1 isolated patient with cutaneous malignant melanoma (155601), Kannengiesser et al. (2007) identified a heterozygous tandem germline 339G-C transversion and 340C-T transition in the CDKN2A gene, resulting in a leu113-to-leu (L113L) and a pro114-to-ser (P114S) substitution, respectively. All families were from southeastern France, and haplotype analysis indicated a founder effect. The sporadic patient had a high sun exposure history and Parkinson disease (168600) and received treatment with levodopa. He subsequently developed 22 primary melanomas, suggesting that levodopa may have contributed to the lesions. Further testing of this individual showed 2 pathogenic variants in the MC1R gene (see, e.g., R151C; 155555.0004), which likely contributed to the severe phenotype.
In affected members of 3 families with cutaneous malignant melanoma (155601), Kannengiesser et al. (2007) identified a 167G-T transversion in the CDKN2A gene, resulting in a ser56-to-ile (S56I) substitution. Two patients were homozygous for the mutation, suggesting remote consanguinity. All families were from southeastern France, and haplotype analysis indicated a founder effect.
Goldstein et al. (2008) identified a gly89-to-asp (G89D) variant in the CDKN2A gene that was associated with significantly increased risk for cutaneous malignant melanoma (155601) in an Icelandic population. The mutation results in a synonymous G143G change in the p14(ARF) protein. The frequency of the G89D variant was 0.7 in melanoma patients compared to 0.08 in controls. The association was strengthened when restricted to invasive melanoma, present in 2% of patients (p = 0.0015). Relatives of affected G89D carriers were at significantly increased risk of melanoma, head and neck cancers, and pancreatic carcinoma compared to relatives of other melanoma patients. Haplotype analysis indicated a founder effect. The common ancestor was determined to be a female who lived in Hunavatnssysla county in northern Iceland from about 1605-1665.
In affected members of 2 unrelated Italian families with malignant melanoma (155601), Binni et al. (2010) identified a heterozygous A-to-G transition in exon 1B of the CDKN2A gene, affecting splicing of the p14(ARF) isoform. The families were ascertained from a large cohort of 155 Italian probands.
In affected members of an Italian family with malignant melanoma (155601), Binni et al. (2010) identified a heterozygous 161G-A transition in exon 1B of the CDKN2A gene, resulting in an arg54-to-his (R54H) substitution in a highly conserved residue of the p14(ARF) isoform. The family was ascertained from a large cohort of 155 Italian probands.
In 3 members of a family of Indonesian descent with pancreatic cancer (606719), Harinck et al. (2012) identified a heterozygous 5-bp duplication (19_23dup) in the CDKN2A gene, resulting in a frameshift and premature termination. Although there were no cases of melanoma in this family, Harinck et al. (2012) noted that the dark skin of the individuals may have offered protection from developing melanoma.
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