The CDKN2A gene, which encodes the proteins p16INK4a and p14ARF, is located on chromosome 9p21. Germline mutations at this locus increase susceptibility to cutaneous malignant melanoma (CMM). In general, missense and nonsense mutations are primarily responsible for defective p16INK4a and possibly p14ARF protein function and account for ∼20% of inherited CMM cases. We report a G>T transversion mutation in the last nucleotide of exon 2, affecting the aspartic acid residue at position 153 of CDKN2A-p16INK4a in a proband with melanoma. If splicing were unaffected, this mutation would change Asp to Tyr. RT–PCR analysis, however, revealed that this mutation, which we have termed D153spl(c.457G>T), and a previously described mutation at the next nucleotide, IVS2+1G>T, result in identical aberrant splicing affecting both p16INK4a and p14ARF. The two main alternate splice products for each of the two normal transcripts includes a 74 bp deletion in exon 2, revealing a cryptic splice site, and the complete skipping of exon 2. The dual inactivation of p16INK4a and p14ARF may contribute to the CMM in these families.
Cutaneous malignant melanoma (CMM) is the most fatal form of skin cancer and its etiology is varied and complex. Approximately 5–12% of malignant melanomas occur in individuals who have at least one first-degree relative with CMM (Goldstein and Tucker, 2001). CDKN2A is located on chromosome 9p21 (Kamb et al., 1994; Nobori et al., 1994) and mutations in this gene are present in 10–25% of melanoma-prone families (Goldstein et al., 2000). The likelihood of detecting a CDKN2A mutation in a melanoma patient increases as the number of cases within the family increases. Mutations in the CDKN2A gene have been found in 50% of the families with six or more affected members, but drops to 20–40% in families with three or more affecteds, and to <5% in families with two or more CMM members (Kefford et al., 1999).
CDKN2A is a complex gene that encodes two distinct proteins, p16INK4a and p14ARF. Despite arising from the same gene, there is no protein sequence similarity between these products (Quelle et al., 1995). p16INK4a is encoded by exons 1α, 2, and 3 and functions as a cyclin-dependent kinase inhibitor of the cell cycle by inhibiting the activity of the cyclin-dependent kinase complex, CDK4/CDK6/cyclin D, thereby blocking the passage from G1 into S by inhibiting the pRB phosphorylation (Serrano et al., 1993; Rocco and Sidransky, 2001). The alternate reading frame product, p14ARF, is encoded by a different first exon (exon 1β) that is 15 kb upstream of exon 1α, using the same second exon as p16INK4a but in a different reading frame (Rocco and Sidransky, 2001). The amino-acid coding sequence of p14ARF ends in exon 2, with the remainder of exon 2 and exon 3 comprising the 3′-untranslated region (reviewed in Haber, 1997). p14ARF functions by preventing p53 degradation, thereby allowing p53-mediated apoptosis or cell cycle arrest (Pomerantz et al., 1998).
As part of an ongoing IRB-approved genetic epidemiology study of familial melanoma (Genetic Epidemiology Branch, National Cancer Institute), probands from families with at least two living first-degree relatives with invasive melanoma are screened for mutations in the CDKN2A gene. In one new family (AY), we identified a mutation in the donor splice site for exon 2. The family included three patients with confirmed CMM in three generations (one child and one grandchild of the index case). Only one patient from this family was available for testing. This mutation and a previously identified (but uncharacterized) one in the next nucleotide, IVS2+1G>T, from family Q (Hussussian et al., 1994) were analysed further to determine their effect on splicing.
Genomic DNA sequencing revealed a G>T transversion mutation in Family AY in the last nucleotide of exon 2, which is the first base of codon 153 (Figure 1). Once this mutation was identified, we also included a member from Family Q in the analysis for comparison. Family Q harbors the IVS2+1G>T mutation and includes two siblings with invasive melanoma, one of their parents with both melanoma and pancreatic cancer, and a grandparent with pancreatic cancer (Hussussian et al., 1994; Goldstein et al., 2000). All patients from this family had the IVS2+1G>T mutation or were obligate carriers.
On initial inspection of the DNA sequence only, the G>T mutation from family AY resembled a missense mutation, changing the aspartic acid residue at codon 153 to a tyrosine residue. Upon RT–PCR and sequencing analysis, however, a D153Y mutation was not evident. Instead, the mutation created aberrantly spliced products, thus we have termed this mutation D153spl(c.457G>T) to indicate a splice mutation in codon 153 of p16INK4a, which is nucleotide 457 of p16INK4a when counting from the ATG initiation codon (nucleotide 728 of the p16INK4a reference mRNA sequence NM_000077.2 and nucleotide 785 of the p14ARF sequence NM_058195.1). This mutation, initially thought to be novel, appears to be the same as a previously published but uncharacterized mutation called D145C (Moskaluk et al., 1998).
The D153spl(c.457G>T) mutation generated similar splicing products as the previously identified intronic mutation IVS2+1G>T, affecting not only p16INK4a, but also p14ARF. Figure 2 is a representative gel of an RT–PCR experiment with amplified products from both CDKN2A transcripts. The arrows indicate the similar wild-type bands and four main aberrant splice products in an individual with the IVS2+1G>T and the D153spl(c.457G>T) mutations. These bands were excised from the gel and sequenced. Some smaller products can be seen in the p14ARF; however, these were not reproducible and sequence information either did not work or showed no similarity to CDKN2A. The samples with the D153spl(c.457G>T) and the IVS2+1G>T mutations (lanes 1, 2 and 7, 8) produced the same products, which were of lower molecular weight than the wild-type allele (lanes 3 and 9).
In normal splicing conditions, the last base of exon 2 is spliced to the first two bases of exon 3 to create the aspartic acid (GAC) at position 153 in the p16INK4a protein coding sequence (indicated by the solid double-headed arrow and the solid bar over the codon sequence, Figure 3). Sequence analysis of the excised bands of splice product 1α (Figure 2) from both the D153spl(c.457G>T) and the IVS2+1G>T mutations showed a deletion of 74 bp because of a cryptic splice site within exon 2. Figure 3 shows the cryptic donor splice site, indicated by the underlined nucleotides within the sequence of exon 2. The result is a frameshift from the aberrantly spliced site at amino acid R128 to the new encoded sequence (underlined): DVAR-HPRLKEPERL*.
Based on our sequence analyses of the aberrant splice products, Figure 4 shows a schematic of the four main spliced products (two for each normal transcript) resulting from the mutations at either D153spl (c.457G>T) or IVS2+1G>T. At the nucleotide level, the same aberrant splice products result for both p16INK4a and p14ARF, but the effects on the protein coding sequences differ. For p16INK4a, in the bottom half of Figure 4, the mRNA of splice product 1α stops 74 bases short of the full-length exon 2 (indicated by the white line, Figure 4). The cryptic splice site 74 bases from the end of exon 2 splices to exon 3 and causes a frameshift. Splice product 2α skips exon 2 entirely, creating a product that splices from exon 1α to exon 3. The amino-acid sequence of exon 3 normally splices to the last base of exon 2 to create the aspartic acid residue, but exon 1α remains intact with its sequence of 50 amino acids, thus causing a frameshift in exon 3. This frameshift creates an additional 39 residues before the stop codon (new sequence underlined): IndexTermRPIQ-TSPIERTREALRNLGKLRSSVTEGPTGPQLPPPQP TPLS*. (Figure 4, bottom).
The top half of Figure 4 shows the effects of the splice mutations on p14ARF. Splice product 1β does not have an effect on the amino-acid coding portion of p14ARF. The stop site in exon 2 is located just upstream of the cryptic splice site (indicated by the black line in exon 2). The effect of the mutation does however delete 74 bases of the 3′-UTR. The consequence of this deletion is not known. We used the UTRScan program (Pesole et al., 1999) to search the UTR functional elements for the patterns defined in the UTR site collection. We examined the 74 bases as a whole, and then examined approximately 20 bp segments separately. No obvious 3′-UTR patterns were found (not shown), but the UTRScan database is limited and the effect(s) of this deletion will need to be determined experimentally. Splice product 2β, like splice product 2α, skips exon 2 entirely. It forms a true p14ARF/p16INK4a chimera by splicing to and expressing exon 3 of p16INK4a. The last four amino-acid residues for exon 1β and the chimeric sequence of the normal reading frame of exon 3 of p16INK4a is as follows: PRRP-DIPD*.
There is a possibility that there are other aberrantly spliced products in individuals with the IVS2+1G>T or the D153spl(c.457G>T) mutation that would be tissue specific. Material from our patients with these mutations was limited to lymphocytes. As expected, the p16INK4a protein products from the lymphocytes were not detectable by Western blot analysis (not shown; Rizos et al., 1997).
In summary, we have identified a mutation in the last nucleotide of exon 2, termed D153spl(c.457G>T), that results in the same splicing patterns as the previously identified IVS2+1G>T mutation (Hussussian et al., 1994). Based on nonexperimental analysis of the genomic sequence, the initial report indicated that the IVS2+1G>T mutation would result in a splicing defect that caused translation through the splice site until a stop codon was reached (from DIPD* to VGD*) (Hussussian et al., 1994). Our experimental data does not provide evidence for this; instead, both of these mutations reveal a cryptic splice site within exon 2 and also cause the splicing machinery to skip exon 2 entirely (Figure 4).
It has been estimated that approximately 15% of all disease-causing point mutations result in mRNA splicing defects, with the majority (60%) of the mutations involving the GT dinucleotide at positions +1 and +2 of the splice donor site (Krawczak et al., 1992). In addition to the D153spl(c.457G>T) and IVS2+1G>T mutations characterized here, a number of other splice site mutations have been described in CDKN2A (Hussussian et al., 1994; Harland et al., 2001; Petronzelli et al., 2001; Lynch et al., 2002). Of those molecularly characterized, the IVS1-1G>C can also cause skipping of exon 2 (Petronzelli et al., 2001), while the IVS2-105A>G mutation retains the coding sequence of exon 2 (Harland et al., 2001). Further studies will be needed to examine the phenotypic consequences of these splice site mutations that affect both CDKN2A transcripts.
It was initially thought that mutations in exon 2 of CDKN2A mainly affected the p16INK4a transcript and not p14ARF (Quelle et al., 1997; Zhang et al., 1998). It has been shown, however, that exon 2 for p14ARF is required for its nucleolar localization (Zhang and Xiong, 1999). Exon skipping caused by these mutations noted here would therefore disrupt this localization. Whether dual inactivation of p14ARF and p16INK4a leads to a lower age at onset and/or susceptibility to other cancers requires further evaluation.
Goldstein AM, Struewing JP, Chidambaram A, Fraser MC and Tucker MA . (2000). J. Natl. Cancer Inst., 92, 1006–1010.
Goldstein AM and Tucker MA . (2001). Arch. Dermatol., 137, 1493–1496.
Haber DA . (1997). Cell, 91, 555–558.
Harland M, Mistry S, Bishop DT and Bishop JA . (2001). Hum. Mol. Genet., 10, 2679–2686.
Hussussian CJ, Struewing JP, Goldstein AM, Higgins PA, Ally DS, Sheahan MD, Clark WJ, Tucker MA and Dracopoli NC . (1994). Nat. Genet., 8, 15–21.
Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day III RS, Johnson BE and Skolnick MH . (1994). Science, 264, 436–440.
Kefford RF, Newton Bishop JA, Bergman W and Tucker MA . (1999). J. Clin. Oncol., 17, 3245–3251.
Krawczak M, Reiss J and Cooper DN . (1992). Hum Genet, 90, 41–54.
Lynch HT, Brand RE, Hogg D, Deters CA, Fusaro RM, Lynch JF, Liu L, Knezetic J, Lassam NJ, Goggins M and Kern S . (2002). Cancer, 94, 84–96.
Moskaluk CA, Hruban H, Lietman A, Smyrk T, Fusaro L, Fusaro R, Lynch J, Yeo CJ, Jackson CE, Lynch HT and Kern SE . (1998). Hum. Mutat., 12, 70.
Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K and Carson DA . (1994). Nature, 368, 753–756.
Pesole G, Liuni S, Grillo G, Ippedico M, Larizza A, Makalowski W and Saccone C . (1999). Nucleic Acids Res., 27, 188–191.
Petronzelli F, Sollima D, Coppola G, Martini-Neri ME, Neri G and Genuardi M . (2001). Genes Chromosomes Cancer, 31, 398–401.
Pomerantz J, Schreiber-Agus N, Liegeois NJ, Silverman A, Alland L, Chin L, Potes J, Chen K, Orlow I, Lee HW, Cordon-Cardo C and DePinho RA . (1998). Cell, 92, 713–723.
Quelle DE, Cheng M, Ashmun RA and Sherr CJ . (1997). Proc. Natl. Acad. Sci. USA, 94, 669–673.
Quelle DE, Zindy F, Ashmun RA and Sherr CJ . (1995). Cell, 83, 993–1000.
Rizos H, Becker TM, Holland EA, Kefford RF and Mann GJ . (1997). Oncogene, 15, 515–523.
Rocco JW and Sidransky D . (2001). Exp. Cell Res., 264, 42–55.
Serrano M, Hannon GJ and Beach D . (1993). Nature, 366, 704–707.
Zhang S, Ramsay ES and Mock BA . (1998). Proc. Natl. Acad. Sci. USA, 95, 2429–2434.
Zhang Y and Xiong Y . (1999). Mol. Cell, 3, 579–591.
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Rutter, J., Goldstein, A., Dávila, M. et al. CDKN2A point mutations D153spl(c.457G>T) and IVS2+1G>T result in aberrant splice products affecting both p16INK4a and p14ARF. Oncogene 22, 4444–4448 (2003). https://doi.org/10.1038/sj.onc.1206564
- alternate reading frame
- cryptic splice site
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