There is increasing recognition that pituitary adenomas may occur in a familial setting, and a number of families have been identified to have familial isolated pituitary adenoma (FIPA), without features of the MEN1 syndrome, Carney complex, or other known familial disorders. Heterozygote germline mutations were identified in a gene encoding AIP (aryl-hydrocarbon receptor interacting protein) in some FIPA families. We have characterised our unique large collection of 140 families in terms of clinical presentation, age of onset, penetrance, genetic mutations, existence of other co-morbidities, responsiveness to somatostatin analogue therapy and histological characteristics.
FIPA is an autosomal dominant disease with a heterogeneous genetic background. A third of our families had a missense, nonsense, frameshift, splice-site, large deletion, and the first described promoter AIP-mutation. We found striking differences in the clinical phenotype of AIP mutation positive and AIP mutation negative FIPA families. Patients with AIP mutations were characterised by early onset, often aggressive disease, and showed a predominance of somatotroph and lactotroph adenomas, but occasionally other types of pituitary tumours. The penetrance of the disease in the AIP mutant families was 3035%, while in AIP mutation-negative families it was lower. In addition, we studied over thirty patients with childhood-onset pituitary adenomas and AIP mutations can be identified in this cohort as well. Typically patients with AIP mutations respond less well to somatostatin analogue treatment and we further explored the mechanism of this lack of response.
We studied AIP immunohistochemical staining in sporadic somatotroph adenomas from patients pretreated with a somatostatin analogue before surgery and compared them to matched patients with no pre-treatment. We found increased AIP expression in the group pretreated with a somatostatin analogue. Similar upregulation of AIP expression both at mRNA and protein levels were observed in a rat pituitary cell line cell (GH3 cells) in vitro. AIP has been shown to bind the tumour suppressor gene ZAC1 and somatostatin analogues are known to upregulate ZAC1 expression. Therefore, we speculated that AIP and ZAC1 might be involved in the somatostatin pathway. Over-expression of wild-type AIP increased ZAC1 mRNA expression in GH3 cells, while knock-out of AIP using siRNA resulted in a reduction of ZAC1 levels. These data suggest that i) ZAC1 is part of the AIP pathway and ii) AIP may play a role in the mechanism of action of somatostatin analogues, which would explain the lack of effect in AIP mutation-positive FIPA families.
Our in vitro data support the previous suggestion that AIP is a tumour suppressor gene. Over-expression of wild-type AIP reduced cell proliferation compared with the empty vector control, while mutant AIP had no or reduced ability to block cell proliferation. On the other hand, siRNA knockdown of endogenous AIP increased cell proliferation compared to a non-coding siRNA control. In addition, using DAPI staining and caspase-assays, we showed that AIP over-expression increased apoptosis which can be inhibited by a caspase inhibitor.
In addition to truncation mutations, which are likely to be disease-causing abnormalities, numerous variants have been described with missense or silent changes leading to no alteration in the amino-acid sequence. The functional characterisation of these AIP variants is important to clarify their pathogenic role and allow appropriate counselling of families. We have studied 13 AIP variants including missense as well as promoter (using a luciferase assay) and splice-site variants (using patient cDNA and a minigene approach). We have found that missense changes disrupting the conserved amino-acids of the molecule tetratricopeptide domain reduced the proteinprotein interaction between AIP and its phosphodiesterase partner PDE4A5. Exonic splice mutations led to a truncated transcript or reduced AIP expression. Our promoter mutation caused decreased basal and stimulated promoter activity. In addition we have, for the first time, characterised the regulation of the AIP promoter and found that it is stimulated by the cAMPPKA signalling pathway.
We have identified an AIP mutation in several families clustering to the same geographical area. To establish if these families have a common ancestor we studied 14 microsatellites around the AIP gene and found a 2.68Mb common area. Using the coalescent theory, which is a retrospective model of gene genealogy, we identified the time when the common ancestor was alive.
We have identified AIP protein in the secretory vesicles of GH-secreting normal and tumour cells with electron-microscopy and showed that human adenoma samples studied ex vivo release AIP protein. In collaboration, we have prepared synthetic AIP protein and set up a time-resolved fluorescence sandwich immunoassay to measure AIP levels in human serum. AIP is present in the circulation at 1.322 nmol/l range in normal volunteers.
Finally, seventy percent of patients with FIPA do not have an AIP mutation, suggesting another gene or genes is/are involved in the pathogenesis of this condition. We have recently carried out a genome-wide analysis which has identified a novel locus for another candidate gene involved in this condition; finer scale mapping is underway.
In conclusion, detailed characterisation of FIPA patients, their mutations and the pathway involved in AIP-related tumorigenesis together with the increasing recognition of a genetic background of familial and early-onset pituitary adenoma cases indicate that these findings are likely to have considerable significance to practising clinical endocrinologists.