Germline CDKN2A/ARF alterations in human melanoma
Approximately 10% of cases of human cutaneous malignant melanoma (CMM) have been estimated to occur in individuals with a familial predisposition, frequently in association with dysplastic nevus syndrome (DNS). The genetics of familial melanoma is complex and heterogeneous. To date only two melanoma predisposing genes have been identified. The CDKN2A/ARF locus on human chromosome 9p21 encodes two distinct cell cycle regulatory proteins, p16 and p14ARF. Germline alterations in the CDKN2A gene have been detected in approximately 20% of CMM families. Germline mutations have also been reported in the CDK4 gene on chromosome 12q15, in a few families.
The primary aim of this thesis was to investigate alterations in the CDKN2A/ARF locus among individuals belonging to Swedish families with melanoma heredity and to study their biological consequences.
The relationships between germline CDKN2A mutations, melanoma and DNS phenotype were studied in five Swedish melanoma families with the mutations: a proline to leucine substitution (P48L), and an arginine insertion (113insR). We found significant correlations between CDKN2A mutations and CMM/DNS. Our results are consistent with the hypothesis that germline CDKN2A mutations and DNS both contribute to melanoma predisposition and may lead to early onset melanoma when present in the same individual.
The 113insR germline mutation was detected in 17 Swedish melanoma families. Haplotype analysis, using microsatellite markers in the 9p21 region, showed that these families share a common allele at markers close to CDKN2A, suggesting that 113insR is a founder mutation. Statistical analysis of meiotic recombination events in the 9p21 region indicated that the mutation arose approximately 2000 years ago.
We screened 80 individuals with multiple primary melanomas (MPM) for germline CDKN2A mutations. Mutations were detected in 11 % of these patients and the majority of them had a family history of melanoma. A novel 24 base pair deletion, which included codons 62-69 (delta-62- 69) was detected in one individual belonging to a family with melanoma heredity. CDKN2A mutation screening of individuals with MPM may thus identify high-risk families.
We confirmed the role of the P48L and A62-69 CDKN2A germline mutations in the development of melanoma tumors by demonstrating that the mutant p 16 proteins are functionally abnormal and do not bind to CDK4 or CDK6.
Due to overlapping open reading frames in exon 2 of CDKN2A, the p16-delta-62-69 also causes an in frame deletion of residues 77-84 in p14ARF. Our studies in cultured tumor cells showed that partial functional loss in p14ARF-delta-77-84 may complement the defective p16-delta-62-69 mutant and may contribute to melanoma development in patients carrying the 24 bp deletion in CDKN2A.
The high rate of CDKN2A inactivation in human cancer encouraged us to address the mechanisms and frequency of Cdkn2a/b gene loss during immortalization of Ras-transformed rat embryo fibroblast clones. We found homozygous deletions of Cdkn2a/b in all established cell lines studied. The mechanisms for loss of heterozygosity (LOH) were mitotic non-disjunction, deletion/rearrangements and, rarely, mitotic recombination. The frequency of Cdkn2a/b gene deletions was estimated to be approximately 2 x 10-8 /cell/generation.
List of scientific papers
I. Hashemi J, Linder S, Platz A, Hansson J (1999). Melanoma development in relation to non-functional p16/INK4A protein and dysplastic naevus syndrome in Swedish melanoma kindreds. Melanoma Res. 9(1): 21-30.
https://pubmed.ncbi.nlm.nih.gov/10338331
II. Hashemi J, Bendahl PO, Sandberg T, Platz A, Linder S, Stierner U, Olsson H, Ingvar C, Hansson J, Borg A (2001). Haplotype analysis and age estimation of the 113insR CDKN2A founder mutation in Swedish melanoma families. Genes Chromosomes Cancer. 31(2): 107-16.
https://pubmed.ncbi.nlm.nih.gov/11319798
III. Hashemi J, Platz A, Ueno T, Stierner U, Ringborg U, Hansson J (2000). CDKN2A germ-line mutations in individuals with multiple cutaneous melanomas. Cancer Res. 60(24): 6864-7.
https://pubmed.ncbi.nlm.nih.gov/11156381
IV. Hashemi J, Lindstrom MS, Asker CE, Platz A, Hansson J, Wiman K (2002). A melanoma-predisposing germline CDKN2A mutation with functional significance for both p16 and p14ARF. Cancer Letters.
V. Zhou JN, Hashemi J, Helou K, Zhang A, Rohme D, Zetterberg A, Levan G, Linder S (1998). Analysis of mechanisms and frequency of CDKN2A/B gene loss during progression of RAS-transformed rat embryo fibroblast clones. Somat Cell Mol Genet. 24(6): 327-39.
https://pubmed.ncbi.nlm.nih.gov/10763411
History
Defence date
2002-03-22Department
- Department of Oncology-Pathology
Publication year
2002Thesis type
- Doctoral thesis
ISBN-10
91-7349-148-9Number of supporting papers
5Language
- eng