familial melanoma in the Polish population �

Mutations in CDKN2A have been found in sporadic cutaneous malignant (CMM), in familial CMM and in other syndromes associated with melanoma. In this study DNA was obtained from 207 individuals and five cell lines. There were 157 CMM patients and 50 healthy members of melanoma patients families. The CMM group included patients with one or two melanoma cases in the family, families with dysplastic nevus syndrom (DNS) and patients with a spectrum of other types of cancers in the family. PCR-SSCP analysis and sequencing identified: six substitutions in codon 58 CGA/TGA (Arg/Stop), 16 substitutions GAC/GAT in codon 84 (Asp/Asp), six substitutions CGA/TGA in codon 148 (Arg/Thr), 14 substitutions G/C in 3'UTR and 4 double changes (two in codon 84 and 3'UTR; two in codon 148 and 3'UTR). The mutation identified in codon 58 was found in tissue only. Other substitutions were polymorphisms found in DNA from tissue and blood samples. Most of them were identified in sporadic CMM (six in codon 148 Ala/Thr, 12 in codon 84 Asp/Asp and six in 3'UTR). The frequency of the polymorphisms was also high in DNS and CMM/DNS families (four in codon 84 Asp/Asp and six in 3'UTR). No mutations or polymorphisms were found in CMM patients with one or two melanoma cases and CMM patients, with other cancers in family history. The analysis of the CDKN2A gene mutations in the Polish population demonstrated: (i) no germline mutations; (ii) a relatively high number of genetic changes in sporadic melanoma; (iii) a high number of polymorphisms in DNS and CMM/DNS families.

The progression of mammalian cells from G 1 to the S phase is regulated by a cascade of protein interactions and protein phosphorylation.One of the regulatory proteins is p16, an inhibitor of cyclin-dependent kinases (CDK) 4 and 6 (Sherr, 1996).p16 by binding to CDKs complexes inhibits their ability to interact with cyclin D (Lukas et al., 1995).Cyclin D/CDK4-6 complex phosphorylates the retinoblastoma protein (pRb), which leads to the activation of a group of transcription factors essential for cell cycle progression (Serrano et al., 1993) and DNA replication (Stone et al., 1995).The p16 gene (CDKN2A) was mapped to chromosome region 9p21 (Kamb et al., 1994b).Later studies demonstrated that from this locus two distinct gene transcripts are derived from different promoters.The second transcript, referred to as p14/ARF, arises from the alternative exon 1 (exon 1 beta) and is spliced into common (with p16) exons 2 and 3 but is translated in a different reading frame (Quelle et al., 1995).A common feature of cancer cell is unrestrained division, that might be related to changes in genes encoding components of the cell cycle regulatory machinery.Mutations in the p16 locus, whose protein products are involved in the Rb pathway, have been implicated in the pathogenesis of many tumor types.The high frequency of mutations observed in melanoma cell lines suggested an important role of p16 in carcinogenesis.Further studies revealed a relatively lower frequency of CDKN2A mutations in sporadic melanoma (0-15%) (Ruiz et al., 1998;Fujimoto et al., 1999) compared to cell lines (16-40%) (Castellano et al., 1997).Moreover, a number of other mechanisms of CDKN2A inactivation, such as point mutations, small deletions, large hetero-and homozygous deletions, and silencing by methylation of CpG islands in the promoter region have been characterized.They have been analyzed in melanoma patients, but their frequency and role in pathogenesis and progression of melanoma are still unclear.
Genetic predisposition plays an important role in the development of nearly 10% of cutaneous malignant melanomas (CMM) (Holuska & Hodi, 1998).Inherited and acquired deletions or point mutations in CDKN2A increase the likelihood that a mutagenic DNA damage would escape repair before cell division.The best known genetic syndrome predisposing to melanoma is referred to as familial malignant melanoma (FMM) (Koph et al., 1986).Beyond melanoma other types of cancer, such as breast, pancreas and colon were observed in FMM (Ciotti et al., 1996).However, only 20% of FMM patients were shown to carry germline mutations in CDKN2A.Other genetic syndromes associated with melanoma are familial atypical mole-malignant melanoma syndrome-pancreatic cancer (FAMMM-PC) (Lynch et al., 2002), dysplastic nevus syndrome (DNS) (Lee et al., 1997), and melanoma-astrocytoma syndrome (Hayward, 2000).In addition, melanoma may appear in the cancer spectrum of other syndromes such as Lynch syndrome (Kamb et al., 1994a) and Li-Fraumeni syndrome (Burt et al., 1999).In these syndromes mutations in the CDKN2A gene were also reported.
Here we summarize the results of the CDKN2A analysis carried out in sporadic and familial melanoma patients in the Polish population.

Materials
Genomic DNA obtained from 207 individuals was analyzed.There were 157 patients with CMM and 50 healthy members of the melanoma patients families.Moreover DNA from five melanoma cell lines (WM9, WM35, WM239, WM902b and A375) was also studied.DNA was extracted from melanoma tissue only (31 patients), from tissue and peripheral blood samples (24 patients), or from peripheral blood only (78 patients).The CMM group included 16 patients with one or two melanoma cases in family history, six CMM patients from DNS families, five families with DNS and a group of CMM patients with a spectrum of other types of cancers in family history.The latter group included: five -BRCA2, probably one Lynch syndrome and one Li-Fraumeni syndrome.Moreover, in 14 families also lung, stomach, breast, colon and liver cancer were reported.
Blood samples taken from 100 healthy blood donors served as a control.
Sequencing analysis.Bands showing different mobility shift were cut out from the gel, eluted into water and amplified.Products of the reamplification were purified and served as templates in the cycle sequencing procedure using the fmol DNA sequencing system (Promega, WI, U.S.A.) and end labeled amplimers as sequencing primers.
Methylation assay.Genomic DNA was modified using 3 M sodium bisulfite with 10 mM hydroquinone according to a previously described method (Herman et al., 1996).Modified DNA was purified with the Wizard purification resin (Promega, WI, U.S.A.).The reaction was terminated by NaOH treatment, followed by ethanol precipitation.The isolated DNA served as template in PCR reaction with primers and reaction conditions as described previously (Herman et al., 1996).PCR products were analyzed by electrophoresis in 2% agarose gel.The accuracy of the procedure was confirmed by using known methylated p16 promoter region DNA template as a control of all steps.

RESULTS
PCR-SSCP analysis and sequencing identified the following genetic alterations: six substitutions in codon 58 CGA/TGA (Arg/ Stop), one G/T substitution in codon 69 Glu/Stop in the WM902b cell line, 16 substitutions GAC/GAT in codon 84 (Asp/Asp), 6 substitutions CGA/TGA in codon 148 (Arg/Thr), 14 substitutions G/C in 3¢UTR and four double changes (two GAC/GAT in codon 84 and 3¢UTR; two polymorphisms in codon 148 and 3¢UTR) (Fig. 1).Methylated CpG islands in the promoter region of CDKN2A were not found.The mutation identified in codon 58 (Arg/Stop) was found only in DNA isolated from melanoma tissue samples obtained from CMM patients without family history.The somatic character of this change was confirmed by analysis of DNA from blood samples derived from the same patients.
The polymorphism in codon 148 Arg/Thr was found in six CMM cases both in blood and tissue samples.The most frequently seen substitution was found in codon 84 GAC/GAT (Asp/Asp), both in tissue and blood samples.In 12 cases the genetic alteration appeared in CMM patients without family history, and in one DNS family but only in four healthy persons (one analyzed CMM member of the family did not have this genetic change).The nucleotide substitution in 3¢UTR was also frequently found (18 cases).The G/C change was localized six nucleotides from the last codon.The substitution was found in eight CMM pa-tients: six of them were sporadic and two reported stomach, breast and lung cancer in the family.Healthy persons with the 3¢UTR substitution were members of the families with one reported melanoma case.Moreover, in four individuals double genetic changes were identified: G/C in 3¢UTR and GAC/GAT in codon 84 (two cases), G/C in 3¢UTR and Ala/Thr in codon 148 (two cases).The above alternations were seen in two CMM patients and their healthy children.The frequency of the genetic alterations found was verified on DNA isolated from 100 healthy blood donors.All the detected genetic changes in the p16 gene are summarized in Table 1.

DISCUSSION
The analysis of the CDKN2A gene mutations in Polish population demonstrated: (i) no germline mutations; (ii) a relatively high num- Original studies on melanoma cell lines suggested an important role of mutated p16 in arising and progression of melanoma.Later studies demonstrated that CDKN2A is rather not the first target in the development of CMM.Studies of p16 mRNA and p16 protein expression in different stages of melanoma progression demonstrated a gradual loss of p16 expression during malignant transformation of melanocytes (Morita et al., 1998).Moreover, the loss of p16 protein expression may be involved in the acquisition of metastatic phenotype of melanoma (Reed et al., 1995).In our studies, five cell lines derived from melanoma at various stages of progression, such as horizontal phase, radial phase and metastases were analyzed.Only in one cell line (derived from metastatic tumor) one CDKN2A mutation was found.Such mutation in codon 69 was described earlier and is considered as typical for cell lines (Smith-Sorensen & Hoving, 1996).
Variable frequencies of p16 germline mutations have been reported in different collections of melanoma families in Sweden, Aus-tralia, Great Britain, Israel, U.S.A. and other countries (Hashemi et al., 2001;Holland et al., 1995;MacKie et al., 1998;Yakobson et al., 2000;Goldstein et al., 2000).It is also known that the incidence of other cancers, such as pancreatic adenocarcinoma, is higher in CMM families with the 358delG mutation (Holuska & Hodi, 1998).On the other hand, CDKN2A mutations occurred rarely and did not play an important role in genetic melanoma predisposition (Tsao et al., 2000).Very low number of genetic changes identified in melanoma cases with reported other cancers or one/two melanoma cases in family history showed that CDKN2A is not a target for mutational changes in such groups of patients (Shennan et al., 2000).In our CMM patients with either sporadic CMM or with one/two melanoma incidents, or other cancer cases in the family no CDKN2A gene germline mutations were found.Unfortunately, there were no classical FMM families, thus the lack of germline mutations need not necessarily be representative of the Polish population.
In contrast, in sporadic CMM a relatively high number of genetic alterations was found.In six out of 55 (10.9%) tissue samples studied the somatic mutation CGA/TGA creating a stop codon was identified.In addition several other polymorphisms were seen.They included a silent change in codon 84, Arg/Thr substitution in codon 148 and G/C substitution in 3¢UTR.The substitution identified in codon 148 is the most frequently reported polymorphisms in the CDKN2A gene.However, more frequent in our studies is the polymorphism identified in 3¢UTR.Such polymorphism is regarded as common in melanoma (Kumar et al., 2001).The most frequently identified substitution was GAC/GAT in codon 84.The silent mutation, which has not been described before, was found in 11% of sporadic CMM.Surprisingly, it was detected in one healthy control only.
In over 90% of DNS and CMM/DNS families CDNK2A polymorphisms were seen.In 2/3 of CMM/DNS families the codon 84 silent mutation was observed.Interestingly, in one of the CMM/DNS families all the members except the CMM patient demonstrated the C/G substitution in 3¢UTR.This further underlines the genetic diversity between DNS and CMM (Puig et al., 1997).
The discovery of the p14/ARF gene may radically change our understanding of genetic alterations linked to the CDKN2A gene so far.Studies of the alternative exon 1 beta demonstrated a 16 bp insertion specifically altering p14/ARF, which in consequence fails to stabilize p53 and arrest growth of a p53 expressing melanoma cell line.This 60ins16 mutation was also found in an individual with multiple primary melanomas (Rizos et al., 2001b).However, most of the mutations appear in the common exon 2. In fact more than 40% of all mutations described were localized there and affected both the p16 and p14/ARF genes.Nevertheless, a clear distinction between p16 and p14/ARF mutations and their function exists since studies of five definitive mutations inactivating the p16 gene demonstrated correct p14/ARF transcripts in melanoma patients (Stott et al., 1998).In other studies three of seven CDKN2A/ARF mutations tested altered the subcellular distribution of p14/ARF and diminished the ability of p14 to activate the p53 pathway.All these facts indicate that p14/ARF might also be involved in melanoma predisposition (Rizos et al., 2001a).
Accordingly, the substitution C/T in codon 84 of p16/CDKN2A (silent) corresponds to a change in codon 99 of p14/ARF-substitution CGC/TGC (Arg/Cys).Thus, the functional target of the above nucleotide alteration might be rather p14/ARF than p16.However, the biological consequences of the alteration have to be confirmed by functional analysis of this p14/ARF variant.