Oncogene (1997) 15, 2999 ± 3005  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

SHORT REPORT Analysis of the CDKN2A, CDKN2B and CDK4 in 48 Australian kindreds

Jose F Flores1, Pamela M Pollock2, Graeme J Walker1,2, J Michael Glendening1, Amy H-T Lin1, Jane M Palmer2, Marilyn K Walters2, Nicholas K Hayward2 and Jane W Fountain1

1University of Southern California, Institute for Genetic Medical, Department of Biochemistry and Molecular Biology, 2011 Zonal Ave (HMR601), Los Angeles, CA, 90033, USA; 2Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, Herston, 4029, Australia

Germline mutations within the -dependent kinase 1994, 1996; Walker et al., 1994; Luca et al., 1995; inhibitor 2A (CDKN2A) and one of its targets, the Platz et al., 1996; Flores et al., 1996) and is genetically cyclin dependent kinase 4 (CDK4) gene, have been linked to familial melanoma (Cannon-Albright et al., identi®ed in a proportion of melanoma kindreds. In the 1992; Nancarrow et al., 1993; Gruis et al., 1993, case of CDK4, only one speci®c mutation, resulting in the 1995a; Goldstein et al., 1994; MacGeoch et al., 1994; substitution of a cysteine for an arginine at codon 24 Holland et al., 1995). is a pivotal (R24C), has been found to be associated with melanoma. regulator that binds to the cyclin-dependent kinases 4 We have previously reported the identi®cation of germline (CDK4) and 6 (CDK6) and inhibits their ability to CDKN2A mutations in 7/18 Australian melanoma phosphorylate the retinoblastoma (pRb) kindreds and the absence of the R24C CDK4 mutation (Serrano et al., 1993). Loss (or reduction in the in 21 families lacking evidence of a CDKN2A mutation. amount) of p16 in a cell (a melanocyte, in this case) The current study represents an expansion of these e€orts presumably augments by releasing a and includes a total of 48 melanoma families from `brake' that exists between the G1 to of the Australia. All of these families have now been screened cell cycle. The CDKN2A gene has been shown to for mutations within CDKN2A and CDK4, as well as for ful®ll the requirements of a classical tumor suppressor mutations within the CDKN2A homolog and 9p21 gene (Knudson, 1971), where inactivation of both neighbor, the CDKN2B gene, and the alternative 1 alleles has been detected frequently in tumor cell lines (E1b)ofCDKN2A. Families lacking CDKN2A muta- (Nobori et al., 1994; Kamb et al., 1994a; Hussussian tions, but positive for a polymorphism(s) within this gene, et al., 1994; Liu et al., 1995a; Luca et al., 1995; were further evaluated to determine if their disease was Pollock et al., 1995, 1996; Ohta et al., 1994; Flores et associated with transcriptional silencing of one CDKN2A al., 1996) and, to a lesser extent, in uncultured tumors allele. Overall, CDKN2A mutations were detected in 3/30 (Gruis et al., 1995b; Ohta et al., 1996; Platz et al., (10%) of the new kindreds. Two of these mutations have 1996; Flores et al., 1996) from familial and sporadic been observed previously: a 24 bp duplication at the 5' end melanoma patients. of the gene and a G to C transversion in exon 2 resulting Approximately 50% of all familial melanoma in an M53I substitution. A novel G to A transition in exon kindreds have been found to be genetically linked to 2, resulting in a D108N substitution was also detected. markers located within the 9p21 region (Cannon- Combined with our previous ®ndings, we have now Albright et al., 1992; Nancarrow et al., 1993; Gruis et detected germline CDKN2A mutations in 10/48 (21%) al., 1993, 1995a; Goldstein et al., 1994; MacGeoch et of our melanoma kindreds. In none of the `CDKN2A- al., 1994; Holland et al., 1995). Though germline negative' families was melanoma found to segregate with mutations have been detected in the CDKN2A gene in either an untranscribed CDKN2A allele, an R24C CDK4 many of these kindreds, disparate results between mutation, a CDKN2B mutation, or an E1b mutation. The studies suggest that CDKN2A may not be the only last three observations suggest that these other cell cycle melanoma predisposition within this region (for control genes (or alternative gene products) are either not review, see Dracopoli and Fountain, 1996). The involved at all, or to any great extent, in melanoma detection of CDKN2A mutations in 11/13 unrelated predisposition. 9p21-linked families from the NIH provides the strongest evidence in support of this gene being the Keywords: CDKN2A; CDKN2B; CDK4; melanoma; only familial melanoma locus on 9p (Hussussian et al., familial; mutation 1994; Goldstein et al., 1995). Similarly, we have also detected CDKN2A mutations in 7/18 (39%) of our melanoma kindreds (both 9p21-linked and unlinked) The CDKN2A (or p16) gene is located on chromo- from Australia (Walker et al., 1995). In contrast, some 9p21 within a region that has been found to be other analogous studies have failed to identify a high frequently deleted in sporadic (Fountain et frequency of CDKN2A mutations, even where large al., 1992; Holland et al., 1994; Isshiki et al., 1994; numbers of melanoma kindreds were analysed (Kamb Kamb et al., 1994a; Nobori et al., 1994; Ohta et al., et al., 1994b; MacGeoch et al., 1994; Ohta et al., 1994; Gruis et al., 1995c (views 13 families with a common founder as one); Holland et al., 1995; Liu et Correspondence: JW Fountain Received 27 November 1996; revised 1 August 1997; accepted 1 al., 1995b; Borg et al., 1996; Fitzgerald et al., 1996; August 1997 Platz et al., 1997). CDKN2A mutations in familial melanoma JF Flores et al 3000 Evidence of locus heterogeneity has been demon- 9p21 linkage/haplotype analysis strated previously in genetic linkage studies performed on familial melanoma (Goldstein et al., 1993, 1994; Lymphoblastoid DNAs from available members of the MacGeoch et al., 1994). The recent identi®cation of 30 new Australian melanoma families (some pedigree germline mutations within the CDK4 gene on and background information provided in Nancarrow et 12q15 that cosegregated with melanoma al., 1993; Walker et al., 1994, 1995; others are in two NIH families (Zuo et al., 1996) now provides unpublished) were initially analysed with 9p21 micro- proof that more than one melanoma predisposition satellite markers (e.g., IFNA, D9S942, D9S171, and gene exists within the genome. The ®nding of the same D9S126) to determine the likelihood of linkage to 9p. CDK4 mutation (resulting in a cysteine substitution for Haplotypes were constructed and then used to group an arginine at codon 24; abbreviated R24C) in both of a€ected cases (where indicated) with a common 9p these families, as well as its occurrence as a somatic haplotype. Only 8/30 of the families (41119, 41109, alteration in two sporadic melanomas (WoÈ lfel et al., 40594, 41095, 41102, 41112, 40999, 41096) were found 1995), suggests that this may be the only CDK4 to have a€ected members that all shared the same mutation that is relevant to melanoma predisposition haplotype and were, therefore, assumed to be the or tumorigenesis. This mutation has also been shown families with the highest likelihood of inheriting a to functionally disrupt the binding of p16 (and p15) to mutation within the CDKN2A gene (Table 1). The CDK4, while the binding of other cyclin-dependent remaining 22 families were deemed `indeterminant' for kinase inhibitors to CDK4 remains unaltered (WoÈ lfel et linkage to 9p21 since one to three of the a€ected cases al., 1995). in each of these families did not share the same 9p Additional potential candidate genes for involve- haplotype. If some of these latter families are actually ment in familial melanoma include the CDKN2B linked to 9p, then the non-segregating individuals with (or p15) gene, which is a CDKN2A homolog that melanoma in these families are presumed to be resides 535kb proximal to CDKN2A on 9p21 (Kamb sporadic cases. et al., 1994a; Hannon and Beach, 1994), and an alternative gene product, encoding another cell cycle CDKN2A mutations and polymorphisms inhibitor (ARF), produced at the CDKN2A locus (Stone et al., 1995a; Mao et al., 1995; Quelle et al., SSCP and DNA sequencing analyses were performed on 1995). The CDKN2B gene is an obvious candidate 1a, 2, and 3 of the CDKN2A gene as previously gene for melanoma predisposition both due to its described (Walker et al., 1995). Three mutations were location and its high homology to the CDKN2A gene. identi®ed after examining one (in some families with a Inactivation of this gene has also been observed either shared 9p21 haplotype) or more a€ected members from (at a high frequency) along with the CDKN2A gene each of the 30 new families. Only one (family 41119) out (Kamb et al., 1994a; Flores et al., 1996), or (at a low of eight of the families initially presumed by segregation frequency) independent of the CDKN2A gene (Stone analysis to be a likely carrier for a CDKN2A mutation et al., 1995a; Glendening et al., 1995; Flores et al., was positive for a mutation (Table 1). In this case, all 1996) in sporadic melanoma tumors and cell lines. ®ve of the a€ected individuals genotyped from this Similarly, the ARF (alternative reading frame) family were found to carry a 24 bp repeat mutation protein, which is produced from a transcript present at the 5' end of the original exon 1 (E1a) of the composed of a di€erent exon 1 (E1b) spliced to CDKN2A gene (Table 1 and Figure 1a). The other two exons 2 and 3 of the CDKN2A gene, is also mutations were both in exon 2 and were detected in two consistently disrupted in sporadic melanomas and, of the `9p-indeterminate' families (41031 and 41105). while bearing little or no resemblance to the p16 or The mutation in family 41031, a G to C transversion at p15 , is another potent inhibitor of cell cycle nucleotide position 159, resulting in a M53I substitution progression that functions independent of the CDKs in p16, was identi®ed in 2/3 of the a€ected individuals (Quelle et al., 1995). analysed from this family (Table 1 and Figure 1b). The The current study was aimed at determining the last mutation, a novel G to A transition at nucleotide overall frequency of CDKN2A (including the ARF position 322, resulting in a D108N substitution in p16, product), CDKN2B, and CDK4 mutations in 48 was detected in 2/4 a€ected members from family 41105 melanoma kindreds from Australia. The 9p21 linkage (Table 1, Figure 1c and Figure 2). and mutational status for CDKN2A in 18 families and Both the 24 bp duplication and the M53I substitution for CDK4 (speci®cally the R24C mutation) in 21 have been reported previously by ourselves (Walker et families has already been reported (Nancarrow et al., al., 1995), as well as by others (reviewed in Dracopoli 1993; Walker et al., 1994, 1995; Zuo et al., 1996). Thus, and Fountain, 1996; Foulkes et al., 1997; Fitzgerald et our mutational screens (using single-strand conforma- al., 1996). The detection of these same mutations in tion polymorphism (SSCP), followed by direct DNA independent studies suggests that either (i) there are sequencing) of CDKN2A and CDK4 were con®ned to mutational `hotspots' within the CDKN2A gene, or (ii) 30 and 27 of the families, respectively, while all the families collected in these studies are related through `CDKN2A-negative' families (a total of 38) were ancestry. A comparison of haplotypes between the analysed for mutations in ARF and CDKN2B. `24 bp duplication' or `M53I' families suggests the latter Additional analyses were also performed to determine is the case and that these families may descend from a if any of our new melanoma kindreds (n=30) showed limited number of founders (P Pollock et al., manuscript evidence of linkage to 9p21 and, when possible, if submitted). The recurrent identi®cation of these two melanoma segregated with a silent (untranscribed) mutations in melanoma kindreds, and not in una€ected CDKN2A allele in families determined to be negative individuals within the general population, implies they for a mutation within CDKN2A. are indeed responsible for the increased incidence of CDKN2A mutations in familial melanoma JF Flores et al 3001 Table 1 9p linkage and CDKN2A mutation status on 30 Australian melanoma kindreds No. of No. of a€ecteds Total a€ecteds sharing no. of genotyped common 9p Nucleotide Consequence Consequence Family a€ectedsa (or inferred) haplotype change Exon within p16b within ARFc 41119d 8 5 5 24 bp ins.e 1a 8 add'1. a.a. none 41109d 7 5 5 40594d 3 3 3 41095d 3 3 3 41102d 3 3 3 41112d 3 3 3 40999d 3 3 3 41096d 3 2 2 41116 5 5 4 41114 5 4 3 40993 4 4 3 41107 4 4 3 41113 4 4 3 40978 6 3 2 40374 5 3 2 40972 4 3 2 40605 3 3 2 41063 3 3 2 41106 3 3 2 41110 3 3 2 41111 3 3 2 41031 5 2 1 159 G4C 2 M53I DtoH 41057 3 2 1 40449 6 5 3 40872 5 5 3 40928 6 4 2 41036 4 4 2 41105 4 4 2 322 G4A 2 D108N RtoQ 40254 3 3 1 41015 6 5 2 aOnly individuals with pathologically con®rmed melanoma, including melanoma in situ, were considered a€ected. bNucleotide and codon numbering for CDKN2A have been modi®ed from Serrano et al. (1993); the corrected numbering system alters the `original' nucleotide positions by six and amino acid codons by eight. For example, the G to C transversion at nucleotide position 153, previously designated as a M45I substitution in p16 (Walker et al., 1995), is now referred to as a mutation at nucleotide position 159 and is a M53I substitution. cPrimer sequences used to analyze E1b were 5'-CACCTCTGGTGCCAAAG-3' (forward primer) and 5'-CAAAACAAGTGCCGAATGCG- 3' (reverse primer). dFamilies in which all a€ected cases shared a common 9p21 haplotype. eTwenty-four insertion, representing the additional copy of a 24 bp repeat naturally present as two copies at the 5' end of the CDKN2A gene; predictably, this mutation leads to the insertion of an additional eight amino acids (a.a.) at the N terminus of p16 (see Walker et al., 1995 for a more detailed description)

melanoma in these families. While the third mutation, a opportunity to distinguish between CDKN2A alleles D108N substitution, has not been reported previously in 11 families that had been deemed non-mutant, but in any other melanoma kindreds, it has, similarly, only were polymorphic, for CDKN2A (Table 2). Speci®cally, been identi®ed in individuals a€ected with melanoma we were interested in determining if transcriptional and is, therefore, presumed to have a deleterious e€ect silencing of a CDKN2A allele cosegregated with on the p16 protein. melanoma in these families. This was assessed by All other SSCP variants identi®ed in these 30 families reverse transcribing RNA isolated from lymphoblas- were determined to result from three established toid cell lines (LCLs) derived from appropriate (i.e. polymorphisms within the CDKN2A gene. Two of a€ected and heterozygous) family members, followed these polymorphisms reside in exon 2 (both G to A by PCR ampli®cation and sequencing of products that transitions at nucleotide positions 318 and 442, contained both the nucleotide 442 (in exon 2) and 500 respectively), while the third, a C to G transversion at (in exon 3) polymorphic sites. Theoretically, this assay nucleotide 500, resides in the 3' untranslated region of served as a gross screen for promoter mutations that exon 3 (reviewed in Pollock et al., 1996). Table 2 shows might a€ect the transcription of a CDKN2A allele or, a compilation of the CDKN2A polymorphisms detected alternatively, could indicate transcriptional silencing of in all 48 of the families. Overall, one family (41015) was a CDKN2A allele via DNA methylation or imprinting. positive for the polymorphism at nucleotide position In no instance was loss of expression of either 318 (see also Figure 2), four carried the G to A CDKN2A allele observed in any of these 11 families transition at position 442, and 16 were positive for the C (data not shown). to G transversion at nucleotide 500. ARF mutation screen Transcription of CDKN2A alleles All families determined to be negative for mutations The identi®cation of polymorphisms within exons 2 within the `original' CDKN2A coding/promoter and 3 of the CDKN2A gene a€orded us the sequences were screened by SSCP for mutations CDKN2A mutations in familial melanoma JF Flores et al 3002 a b

c

Figure 1 Pedigree structures of families incorporating results of 9p21 haplotype and CDKN2A mutation analyses. Individual identi®cation numbers are shown beneath each pedigree symbol and age of onset of melanoma or age at last contact is given above each symbol. Pedigrees have been condensed to conserve space and preserve a degree of anonymity. (a) Family 41119: all a€ecteds analysed share a common (boxed) haplotype which cosegregates with a 24 bp duplication (+ ins) in exon 1a of the CDKN2A gene and melanoma. (b) Family 41031: individuals 001 and 005 are carriers of a missense mutation associated with a M531 substitution in p16. The three other individuals analysed in this pedigree are wild-type (wt) at the CDKN2A locus. An asterisk represents an inferred haplotype from individual 001 inherited from his decreased father; haplotype analysis was not performed on 005. A more extensive number of markers was typed in this family to determine that the a€ected individual 002 did not share the same CDKN2A (disease) allele as 001. The location of CDKN2A is indicated by an arrow and the boxed regions in 002 and 004 mark haplotypes that are partially shared with 001. Individual 002 is presumed to be a sporadic case of melanoma. (c) Family 41105: individuals 001 and 002 carry a mutation which is associated with a D108N substitution in p16, whereas the remaining family members analysed (regardless of disease status) were determined to be wild type (wt) for CDKN2A. The `disease' haplotype is indicated by either solid or dashed boxes; unable to conclusively assign in this pedigree. The presumed sporadic occurrence of melanoma in a 19 year old individual (006) in this family also illustrates the necessity to dermatologically screen all individuals in a kindred regardless of mutational status at the CDKN2A locus. This discovery suggests that other genetic and/or environmental risk factors may in¯uence the development of melanoma in a kindred

within the alternative exon 1 (E1b) using similar 1996; Platz et al., 1997). It should be noted, however, methods as for exon 1a (Walker et al., 1995). No that a proportion of the mutations identi®ed in exon 2 variant SSCP bands were observed on any of the of the CDKN2A gene also a€ect the protein translated individuals analysed from these families, indicating that from the ARF transcript. For example, the G to A melanoma predisposition mutations, as well as benign transition at nucleotide position 322 (associated with polymorphisms, are rare (or non-existent) within this the D108N substitution in family 41119) produces a exon of CDKN2A. Three other groups have also signi®cant change (arginine for glutamine) in ARF reported a similar lack of exon 1b mutations in their (Table 1). Likewise, the G to C transversion at melanoma kindreds, implying that this alternative nucleotide position 159, associated with the M53I CDKN2A product is not involved in melanoma substitition in p16, changes a negatively-charged amino predisposition (Stone et al., 1995a; Fitzgerald et al., acid (aspartic acid) to a positively charged one CDKN2A mutations in familial melanoma JF Flores et al 3003 (histidine) in the ARF protein. The signi®cance of protein (Walker et al., 1995; this study). The strongest these latter ®ndings, if any, remains uncertain evidence against the involvement of ARF in familial especially since the carriers of these mutations do not melanoma comes from our original analyses where we display any phenotypic features that distinguish them reported the identi®cation of ®ve independent muta- from other familial melanoma patients who have tions in exon 1a of the CDKN2A gene in our inherited mutations that do not a€ect the ARF melanoma kindreds (Walker et al., 1995). These mutations would, predictably, have no e€ect on ARF since they reside within an exon that is not shared in common between the CDKN2A and ARF transcripts. A A > > CDKN2B mutation screen 322 G 318 G Mutations in exons 1 and 2 of the CDKN2B gene were screened for by SSCP in families where melanoma had not been found to segregate with a CDKN2A mutation (n=38). Methods were similar to those employed for the analysis of CDKN2A (Walker et al., 1995) with the following primers: exon 1, forward primer=5'- CGTTAAGTTTACGGCCAACGG-3' and reverse primer=5'-ATCTAGGTTCCAGCCCCGAT-3'; exon 2, initial ampli®cation with primers 89F and 50R (Kamb et al., 1994a) followed by secondary amplifica- tions with either (i) forward primer=5'- CACTCTGCTCTCTCTGGCAG-3' and reverse pri- Figure 2 Examples of SSCP shifts for exon 2 (mid region) of the mer=5'-CACCAGCGTGTCCAGGAA-3' (5' region); CDKN2A gene. The two variant bands are indicative of the silent (ii) forward primer=5'-CCCGACCGGTGCATGAT-3' G to A substitution at nucleotide position 318 (codon V106) that and reverse primer=5'-AGGTACCCTGCAACGTCG- was detected in 2/5 a€ected individuals in family 41015 (Table 2), and the G to A mutation at nucleotide 322 (D108N) harbored by 3' (mid region); or (iii) forward primer=5'- 2/4 a€ected individuals in family 41105 (Table 1 and Figure 1c) TGGACTTGGCCGAGGAG-3' and reverse pri-

Table 2 CDKN2A polymorphisms present in 48 Australian melanoma kindreds Family Polymorphism Family Polymorphism 40138b 40978a 500 C4G 40193b 40993 40254 500 C4G 40999 40354b 41001b 500 C4G 40374a 500 C4G 41015a 318 G4A, 442 G4A, 500 C4G 40449 41031 40566b 41036 40575b 41057a 500 C4G 40582b 41063a 500 C4G 40594 41095 40598b 41096 40599b 41102 40605 41105 40750b 41106a 500 C4G 40787b 41107a 442 G4A, 500 C4G 40792b 41109 40800b 41110 500 C4G 40804a,b 500 C4G 41111 40823b 442 G4A, 500 C4G 41112 40870b 41113 40872a 442 G4A, 500 C4G 41114 40928 41116 40929a,b 500 C4G 41119 500 C4G 40972a 500 C4G 60001b aEleven families that were assessed for transcriptional silencing of one CDKN2A allele by RT-PCR and direct sequencing. In brief, this involved extraction of RNAs from LCLs (RNeasy kits; Qiagen) which were then reverse transcribed with Superscript II reverse transcriptase (Gibco BRL) and ampli®ed (2 ml) with primers 305F and 530R (Hussussian et al., 1994). A `touchdown' thermal cycling routine of 2 cycles at each degree between 658C and 568C, followed by 15 cycles at 558C, was used. Each cycle consisted of 45 seconds at 948C, 90 seconds at the annealing temperature and 90 seconds at 728C. Reactions (in duplicate) were electrophoresed on 1.5% agarose gels and bands were excised and puri®ed using a Qiagen gel extraction kit. Automated sequencing, using an Applied Biosystems Incorporated (ABI) Dye-Terminator cycle sequencing kit, was performed on both strands as previously described (Pollock et al., 1995). Sequencing of LCL DNA from these patients, as well as `control' cDNAs which were derived from individuals known to be homozygous or heterozygous for these polymorphisms, was also performed to enable comparison of automated DNA sequencer traces. Those familes that harbored polymorphisms but were not analyzed further either carried a coding region mutation within CDKN2A or were homozygous for the polymorphism. bEighteen families originally described in Walker et al., 1995. Re-evaluation of family 40929 indicated that inividuals within this kindred also carried the C to G polymorphism at nucleotide 500 (this study). The ten families indicated in bold-face type are ones in which melanoma cosegregates with a CDKN2A mutation CDKN2A mutations in familial melanoma JF Flores et al 3004 mer=5'-CCTTCATCGAATTAGGAGGG-3' (3' re- has arisen in these families primarily due to shared gion). No SSCP variants were observed, indicating a environmental factors. The identi®cation of up to three lack of mutations or polymorphisms within CDKN2B sporadic cases of melanoma in 6 of our 10 families in the individuals analysed. These ®ndings concur with with CDKN2A mutations supports the latter conclu- those of others (Stone et al., 1995b; Platz et al., 1997), sion and suggests that a number of our smaller families where no mutations have been identi®ed in the coding (with 4four a€ecteds and no evidence of a CDKN2A region of this gene in other unrelated melanoma mutation) may just be clusters of sporadic melanoma kindreds. cases. This speculation seems feasible given that the lifetime incidence of melanoma in Queensland, Australia (where all but one of our families come CDK4 mutation screen from) is approximately 7% (MacLennan et al., 1992). Exon 2 of the CDK4 gene was screened by SSCP in 27 In fact, we have observed during the course of our out of the 48 families that had not been previously studies that the number of a€ected members in a screened for mutations in CDK4. This exon is the site family is as good a predictive indicator that a family of the only mutation (a C to T transition associated will harbor a CDKN2A mutation as the sharing of a with the R24C substitution) that has been reported in common 9p21 haplotype. Speci®cally, approximately familial, as well as sporadic, melanoma patients half (8/18) of our largest families (with ®ve or more (WoÈ lfel et al., 1995; Zuo et al., 1996). Since this a€ected members) carry a CDKN2A mutation and, of mutation has been determined to create a novel StuI those with mutations, a similar proportion (4/8; 50%) restriction enzyme site within the CDK4 gene (Zuo et share a common 9p21 haplotype among all melanoma al., 1996), DNAs from a€ected members were also cases (Walker et al., 1995; and this study). In contrast, analysed non-isotopically via PCR ampli®cation of CDKN2A mutations were detected in only 2/30 of our exon 2 followed by digestion of products with StuI. smaller families (with 3 or 4 a€ecteds). Overall, these DNA from a melanoma cell line (SK-MEL-28) known ®ndings suggest that the minimal number of a€ected to harbor this particular mutation (unpublished results) cases used to designate a bona ®de Australian served as a positive control in these assays. All 27 melanoma kindred should be raised to ®ve. Under families were determined to be negative for the R24C this new restriction, the number of CDKN2A muta- CDK4 mutation. The identi®cation of this mutation is tions observed in our families (8/18; 46%) approaches only 2/10 NIH families (Zuo et al., 1996) and in 0/48 the mutational frequency of 58% (11/19) reported by of our Australian families suggests it contributes little Dracopoli and co-workers for CDKN2A mutations in to melanoma predisposition. To date, only 2% (2/86; NIH (or U.S.-based) melanoma kindreds (Hussussian includes 28 additional families from Fitzgerald et al., et al., 1994; Dracopoli and Fountain, 1996). The 1996) of all analysed melanoma kindreds have been occurrence of melanoma in the remaining half or so found to carry this mutation. of kindreds from both of these populations presumably arises due to the inheritance of a mutation in another melanoma predisposition locus, one of which (in the Melanoma predisposition in Australia case of the NIH families) is CDK4. Overall, only 10 out of 48 (21%) of our Australian melanoma families have been found to harbor CDKN2A mutations which cosegregate with melano- ma. The possibility that susceptibility to melanoma in Acknowledgements the remaining 38 families arises due to mutations in We are indebted to the family members who participated in either the ARF product produced by the CDKN2A this study and are sincerely grateful to the many clinicians and members of the Epidemiology Unit at the Queensland locus, CDK4,orCDKN2B, or, alternatively, is Institute of Medical Research for referring some of the associated with an untranscribed CDKN2A allele, has families. This work was supported by the Donald E and also been ruled out in the current study. This leaves Delia B Baxter Foundation (JWF), the National Institutes few explanations as to why the mutation frequency of Health grant CA66021 (JWF), and the National Health observed at CDKN2A is not higher; either the majority and Medical Research Council of Australia (NH&MRC). of these families inherit mutations within other, as yet GJW and NKH are recipients of CJ Martin and Research unidenti®ed, melanoma susceptibility loci or melanoma Fellowships of the NH&MRC, respectively.

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