p16 (INK4(a), MTS-1) gene polymorphism and methylation status in human pituitary tumours

OBJECTIVE The p16 gene, which encodes a physiological inhibitor of the cycl in D-CDK4 complex, is now considered as an important tumour-suppressor gene in a variety of tumours. A marked reduction of its expression has been rep orted to occur without significant genetic alterations in human pituitary a denomas, although rare point mutations of uncertain functional significance have been described. On the other hand, pi6 gene silencing due to hypermet hylation has been reported in several human primary tumours. The aim of thi s study was to further investigate the pathogenetic events leading to p16 g ene inactivation in pituitary tumours. DESIGN To screen a european series of human pituitary tumours for p16 gene alterations and possible gene hypermethylation. PATIENTS A representative series of 31 human pituitary tumours-30 macroaden omas, including a MEN-I non-secreting pituitary adenoma and a non-MEN-1 fam ilial giant ON-secreting adenoma, and one FSH-secreting pituitary carcinoma -was studied. METHODS Polymerase chain reaction/single strand conformation polymorphism ( PCR-SSCP) analysis was used to screen for p16 gene alterations in all cases . Direct sequencing of POE-products was obtained by the di-deoxynucleotide method where suspected abnormalities of the PCR-SSCP analysis were observed . In 24 samples, a methylation-specific PCR assay (MSP-PCR) was used to det ermine p16 gene methylation status. RESULTS Two sporadic cases of pituitary adenomas had a similar single A to G base substitution leading to an heterozygous Ala140Thr p16 polymorphism, which has not previously been described in such tumours, but is known to be functionally silent, No other pig abnormality could be suspected from PCR- SSCP analysis in this series. In contrast, the presence of methylated-speci fic PCR products was observed in 20/24 cases (83.3%). CONCLUSIONS This study confirms that p16 gene mutations are not involved in the pathogenesis of human pituitary tumours, although polymorphisms can be demonstrated, depending on the population considered, In contrast, the hig h incidence of hypermethylation of the p16 gene suggests that such an alter ation occurs early in pituitary tumours, and may play a role in pituitary t umorigenesis.