p16 (INK4a, MTS-1) gene polymorphism and methylation status in human pituitary tumors

OBJECTIVE: The p16 gene, which encodes a physiological inhibitor of the cyclin 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 reported to occur without significant genetic alterations in human pituitary adenomas, although rare point mutations of uncertain functional significance have been described. On the other hand, p16 gene silencing due to hypermethylation has been reported in several human primary tumours. The aim of this study was to further investigate the pathogenetic events leading to p16 gene 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 macroadenomas, including a MEN-1 non-secreting pituitary adenoma and a non-MEN-1 familial giant GH-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 PCR-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 determine 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 p16 abnormality could be suspected from PCR-SSCP analysis in this series. In contrast, the presence of methylated-specific 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 high incidence of hypermethylation of the p16 gene suggests that such an alteration occurs early in pituitary tumours, and may play a role in pituitary tumorigenesis.