Giampaolo Trivellin

Characterization of aryl hydrocarbon receptor interacting protein (AIP) mutations in familial isolated pituitary adenoma families

Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15–40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and β‐galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6±11.2 years) than AIP mutation‐negative patients (40.4±14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A‐pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two‐hybrid assay of protein–protein interaction of all missense variants showed variable disruption of AIP‐phosphodiesterase‐4A5 binding. In summary, exonic, promoter, splice‐site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants. Hum Mutat 31:1–11, 2010.

Somatostatin analogs modulate AIP in somatotroph adenomas: the role of the ZAC1 pathway.

CONTEXT Somatotroph adenomas harboring aryl hydrocarbon receptor interacting protein (AIP) mutations respond less well to somatostatin analogs, suggesting that the effects of somatostatin analogs may be mediated by AIP. OBJECTIVE The objective of the investigation was to study the involvement of AIP in the mechanism of effect of somatostatin analogs. DESIGN In the human study, a 16-wk somatostatin analog pretreatment compared with no pretreatment. In the in vitro cell line study, the effect of somatostatin analog treatment or small interfering RNA (siRNA)/plasmid transfection were studied. SETTING The study was conducted at a university hospital. PATIENTS Thirty-nine sporadic and 10 familial acromegaly patients participated in the study. INTERVENTION Interventions included preoperative lanreotide treatment and pituitary surgery. OUTCOME For the human study, GH and IGF-I levels, AIP, and somatostatin receptor staining were measured. For the cell line, AIP and ZAC1 (zinc finger regulator of apoptosis and cell cycle arrest) expression, metabolic activity, and clone formation were measured. RESULTS Lanreotide pretreatment reduced GH and IGF-I levels and tumor volume (all P < 0.0001). AIP immunostaining was stronger in the lanreotide-pretreated group vs. the surgery-only group (P < 0.001). After lanreotide pretreatment, the AIP score correlated to IGF-I changes in females (R = 0.68, P < 0.05). Somatostatin receptor staining was not reduced in samples with AIP mutations. In GH3 cells, 1 nm octreotide increased AIP mRNA and protein (both P < 0.01) and ZAC1 mRNA expression (P < 0.05). Overexpression of wild-type (but not mutant) AIP increased ZAC1 mRNA expression, whereas AIP siRNA knockdown reduced ZAC1 mRNA (both P < 0.05). The siRNA-mediated knockdown of AIP led to an increased metabolic activity and clonogenic ability of GH3 cells compared with cells transfected with a nontargeting control (both P < 0.001). CONCLUSION These results suggest that AIP may play a role in the mechanism of action of somatostatin analogs via ZAC1 in sporadic somatotroph tumors and may explain their lack of effectiveness in patients with AIP mutations.

AIP and its interacting partners

Germline mutations in the aryl hydrocarbon receptor-interacting protein gene (AIP) predispose to young-onset pituitary tumours, most often to GH- or prolactin-secreting adenomas, and most of these patients belong to familial isolated pituitary adenoma families. The molecular pathway initiated by the loss-of-function AIP mutations leading to pituitary tumour formation is unknown. AIP, a co-chaperone of heat-shock protein 90 and various nuclear receptors, belongs to the family of tetratricopeptide repeat (TPR)-containing proteins. It has three antiparallel α-helix motifs (TPR domains) that mediate the interaction of AIP with most of its partners. In this review, we summarise the known interactions of AIP described so far. The identification of AIP partners and the understanding of how AIP interacts with these proteins might help to explain the specific phenotype of the families with heterozygous AIP mutations, to gain deeper insight into the pathological process of pituitary tumour formation and to identify novel drug targets.

Macronodular Adrenal Hyperplasia due to Mutations in an Armadillo Repeat Containing 5 (ARMC5) Gene: A Clinical and Genetic Investigation

CONTEXT Inactivating germline mutations of the probable tumor suppressor gene, armadillo repeat containing 5 (ARMC5), have recently been identified as a genetic cause of macronodular adrenal hyperplasia (MAH). OBJECTIVE We searched for ARMC5 mutations in a large cohort of patients with MAH. The clinical phenotype of patients with and without ARMC5 mutations was compared. METHODS Blood DNA from 34 MAH patients was genotyped using Sanger sequencing. Diurnal serum cortisol measurements, plasma ACTH levels, urinary steroids, 6-day Liddles test, adrenal computed tomography, and weight of adrenal glands at adrenalectomy were assessed. RESULTS Germline ARMC5 mutations were found in 15 of 34 patients (44.1%). In silico analysis of the mutations indicated that seven (20.6%) predicted major implications for gene function. Late-night cortisol levels were higher in patients with ARMC5-damaging mutations compared with those without and/or with nonpathogenic mutations (14.5 ± 5.6 vs 6.7 ± 4.3, P < .001). All patients carrying a pathogenic ARMC5 mutation had clinical Cushings syndrome (seven of seven, 100%) compared with 14 of 27 (52%) of those without or with mutations that were predicted to be benign (P = .029). Repeated-measures analysis showed overall higher urinary 17-hydroxycorticosteroids and free cortisol values in the patients with ARMC5-damaging mutations during the 6-day Liddles test (P = .0002). CONCLUSIONS ARMC5 mutations are implicated in clinically severe Cushings syndrome associated with MAH. Knowledge of a patients ARMC5 status has important clinical implications for the diagnosis of Cushings syndrome and genetic counseling of patients and their families.

A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27KIP1 expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5′UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF–encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patients pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27KIP1 expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.

X-linked acrogigantism syndrome: clinical profile and therapeutic responses.

X-linked acrogigantism (X-LAG) is a new syndrome of pituitary gigantism, caused by microduplications on chromosome Xq26.3, encompassing the gene GPR101, which is highly upregulated in pituitary tumors. We conducted this study to explore the clinical, radiological, and hormonal phenotype and responses to therapy in patients with X-LAG syndrome. The study included 18 patients (13 sporadic) with X-LAG and microduplication of chromosome Xq26.3. All sporadic cases had unique duplications and the inheritance pattern in two families was dominant, with all Xq26.3 duplication carriers being affected. Patients began to grow rapidly as early as 2-3 months of age (median 12 months). At diagnosis (median delay 27 months), patients had a median height and weight standard deviation scores (SDS) of >+3.9 SDS. Apart from the increased overall body size, the children had acromegalic symptoms including acral enlargement and facial coarsening. More than a third of cases had increased appetite. Patients had marked hypersecretion of GH/IGF1 and usually prolactin, due to a pituitary macroadenoma or hyperplasia. Primary neurosurgical control was achieved with extensive anterior pituitary resection, but postoperative hypopituitarism was frequent. Control with somatostatin analogs was not readily achieved despite moderate to high levels of expression of somatostatin receptor subtype-2 in tumor tissue. Postoperative use of adjuvant pegvisomant resulted in control of IGF1 in all five cases where it was employed. X-LAG is a new infant-onset gigantism syndrome that has a severe clinical phenotype leading to challenging disease management.

Clinical and genetic characterization of pituitary gigantism: an international collaborative study in 208 patients

Despite being a classical growth disorder, pituitary gigantism has not been studied previously in a standardized way. We performed a retrospective, multicenter, international study to characterize a large series of pituitary gigantism patients. We included 208 patients (163 males; 78.4%) with growth hormone excess and a current/previous abnormal growth velocity for age or final height >2 s.d. above country normal means. The median onset of rapid growth was 13 years and occurred significantly earlier in females than in males; pituitary adenomas were diagnosed earlier in females than males (15.8 vs 21.5 years respectively). Adenomas were ≥10 mm (i.e., macroadenomas) in 84%, of which extrasellar extension occurred in 77% and invasion in 54%. GH/IGF1 control was achieved in 39% during long-term follow-up. Final height was greater in younger onset patients, with larger tumors and higher GH levels. Later disease control was associated with a greater difference from mid-parental height (r=0.23, P=0.02). AIP mutations occurred in 29%; microduplication at Xq26.3 - X-linked acrogigantism (X-LAG) - occurred in two familial isolated pituitary adenoma kindreds and in ten sporadic patients. Tumor size was not different in X-LAG, AIP mutated and genetically negative patient groups. AIP-mutated and X-LAG patients were significantly younger at onset and diagnosis, but disease control was worse in genetically negative cases. Pituitary gigantism patients are characterized by male predominance and large tumors that are difficult to control. Treatment delay increases final height and symptom burden. AIP mutations and X-LAG explain many cases, but no genetic etiology is seen in >50% of cases.

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal α-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

Mutations of the aryl hydrocarbon receptor interacting protein (AIP) have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h) mutations, R304* (nonsense mutation), R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE) of TOMM20.

Prevalence of AIP mutations in a large series of sporadic Italian acromegalic patients and evaluation of CDKN1B status in acromegalic patients with multiple endocrine neoplasia

BACKGROUND Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene and the p27(KIP1) encoding gene CDKN1B have been associated with two well-defined hereditary conditions, familial isolated pituitary adenoma (FIPA) and multiple endocrine neoplasia type 4 (MEN4). Somatotropinomas are present in most AIP mutated FIPA kindreds, as well as in two-thirds of MEN4 patients who carry pituitary tumors. METHODS Germline DNA samples of 131 Italian sporadic acromegalic patients including 38 individuals with multiple tumors, and of six FIPA families (four homogeneous for prolactinomas and two heterogeneous with prolactin/nonfunctioning pituitary adenomas) were collected in a multicentric collaborative study. The prevalence of AIP and CDKN1B gene point mutations and copy number variations were evaluated. RESULTS Two novel (IVS3+1G>A and c.871G>A) and one previously described (c.911G>A) AIP mutations were detected in four apparently sporadic cases (3.1%) with relatively high age at diagnosis (49+/-18, range 30-67). No mutations/rearrangements were detected in FIPA families. The highly conserved c.871G>A substitution was detected in a patient who also carried a MEN1 mutation suggesting that she is a double heterozygote. The possible pathogenic effect on AIP splicing of the silent substitution c.144G>A found in another patient was ruled out using a minigene-based approach. CDKN1B mutations/rearrangements were neither identified in patients with multiple neoplasia nor in FIPA families. CONCLUSION AIP is mutated in about 3% of apparently sporadic acromegalic patients. The relatively high age at diagnosis, as well as its sporadic presentation, suggests that these patients are carriers of mutations with reduced pathogenicity. p27(KIP1) is unlikely to represent the common unifying nonendocrine etiology for acromegaly and cancer.

Somatic mosaicism underlies X-linked acrogigantism syndrome in sporadic male subjects

Somatic mosaicism has been implicated as a causative mechanism in a number of genetic and genomic disorders. X-linked acrogigantism (XLAG) syndrome is a recently characterized genomic form of pediatric gigantism due to aggressive pituitary tumors that is caused by submicroscopic chromosome Xq26.3 duplications that include GPR101 We studied XLAG syndrome patients (n= 18) to determine if somatic mosaicism contributed to the genomic pathophysiology. Eighteen subjects with XLAG syndrome caused by Xq26.3 duplications were identified using high-definition array comparative genomic hybridization (HD-aCGH). We noted that males with XLAG had a decreased log2ratio (LR) compared with expected values, suggesting potential mosaicism, whereas females showed no such decrease. Compared with familial male XLAG cases, sporadic males had more marked evidence for mosaicism, with levels of Xq26.3 duplication between 16.1 and 53.8%. These characteristics were replicated using a novel, personalized breakpoint junction-specific quantification droplet digital polymerase chain reaction (ddPCR) technique. Using a separate ddPCR technique, we studied the feasibility of identifying XLAG syndrome cases in a distinct patient population of 64 unrelated subjects with acromegaly/gigantism, and identified one female gigantism patient who had had increased copy number variation (CNV) threshold for GPR101 that was subsequently diagnosed as having XLAG syndrome on HD-aCGH. Employing a combination of HD-aCGH and novel ddPCR approaches, we have demonstrated, for the first time, that XLAG syndrome can be caused by variable degrees of somatic mosaicism for duplications at chromosome Xq26.3. Somatic mosaicism was shown to occur in sporadic males but not in females with XLAG syndrome, although the clinical characteristics of the disease were similarly severe in both sexes.