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Aryl Hydrocarbon Receptor Interacting Protein Variants in Sporadic Pituitary Adenomas

Cedars-Sinai Research Institute, David Geffen School of Medicine at University of California, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 2015, Los Angeles, California 90048. E-mail: Melmed{at}cshs.org.

	Abstract

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Context: Proximal pathogenesis of pituitary tumors remains largely unclear. Recently, three heterozygous germline mutations were reported in the aryl hydrocarbon receptor interacting protein (AIP) gene in Finnish and Italian families with pituitary tumor predisposition and in Finnish patients harboring sporadic pituitary tumors.

Objective: The objectives of this study were to examine the frequency of the three AIP germline mutations in U.S. patients harboring sporadic pituitary tumors and to correlate clinical features of pituitary tumors with these mutations, if they exist in these patients.

Design: Genomic DNA was extracted from lymphoblastoid cell lines established from patients with sporadic pituitary tumors. Three segments of the AIP gene that contain the reported mutation sites for Q14X, IVS3–1G>A, and R304X were amplified by PCR and sequenced.

Setting: The study was conducted in a private nonprofit academic medical center.

Patients: The subjects were 66 consecutive patients (including 52 with acromegaly or prolactinoma) participating in a pituitary tumor database who consented to genetic study.

Main Outcome Measure(s): The main outcome measure was the prevalence of these specific germline mutations in affected individuals.

Results: AIP mutations were not detected in the 66 patients. A synonymous polymorphism was found in a single patient with acromegaly.

Conclusions: The three specific AIP germline mutations do not play an important role in pathogenesis of sporadic pituitary tumors in U.S. patients.

	Introduction

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PITUITARY ADENOMAS COMPRISE up to 15% of intracranial tumors and are the most common cause of hyperpituitarism and hypopituitarism in adults (1). These mostly benign tumors arise from pituitary cell types the hormonal secretory products of which determine the biochemical and clinical phenotype. Virtually all pituitary adenomas are monoclonal, arising from intrinsic somatic mutations leading to dysregulated cell proliferation including loss of heterozygosity, cell cycle dysregulation, or gene activation (2, 3). Multiple endocrine neoplasia type 1 (MEN1), familial acromegaly, and Carney Complex are well recognized but rare inherited disorders that all include pituitary tumors (1). Genetic abnormalities are associated with oncogene activation or inactivation of tumor suppressor genes (4, 5). Three activating oncogenes, gsp, ccnd1, and PTTG, as well as growth factors, have been implicated in pituitary tumor pathogenesis (4). The importance of tumor suppressor gene inactivation in pituitary tumorigenesis is also evident both in humans and mice: menin inactivation causes MEN1 in humans and Rb deletion results in pituitary tumors in mice (5, 6, 7).

A germline nonsense mutation (Q14X) of low penetrance in the aryl hydrocarbon receptor interacting protein (AIP) gene was recently identified in members of several Finnish families with a unique pituitary adenoma predisposition (mostly somatotropinoma and prolactinoma) (8). Another AIP germline mutation (R304X) was found in an Italian family with acromegaly (8). In a pooled group of Finnish population-based acromegaly patients, four of 51 patients possess the Q14X mutation, and one of 51 subjects harbored an IVS3–1G>A mutation, which interferes with the exon 4 splice acceptor site of the AIP gene (8). Loss of heterozygosity was detected in all five somatotropinomas, two prolactinomas, and one mixed tumor derived from patients with AIP germline mutations, suggesting that AIP may behave as a tumor suppressor gene. In the same report, however, AIP gene mutations were not detected in several hundred nonaffected individuals or in a German and a Turkish family with familial acromegaly (8). To determine whether AIP germline mutations contribute to sporadic pituitary adenomas in an ethnically diverse patient population in the United States, we examined genomic DNA derived from transformed lymphocytes for the three specific AIP mutations in 66 patients.

	Subjects and Methods

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Subjects

Patients diagnosed with pituitary tumors who were participating in the Cedars-Sinai Pituitary Database (9) underwent biochemical, imaging, and clinical evaluation, and peripheral blood was drawn for white blood cell isolation after informed consent was obtained. Sixty-six such patients gave consent for genetic studies, with 52 of 66 patients harboring acromegaly or prolactinoma. The study was approved by the Institutional Review Board at Cedars-Sinai Medical Center.

Methods

Peripheral blood lymphocytes were transformed with Epstein-Barr virus and grown to a density of 1–2 x 10⁶ cells/ml, and cell pellets were frozen at –70 C. DNA was extracted from cell pellets with DNeasy Tissue Kit from Qiagen (Valencia, CA). Genomic DNA sequences containing potential mutation sites were amplified by PCR. To amplify a 355-bp sequence harboring Q14X, primers 5'-TGG CGC TAG CTC GGA AGC TGC-3' and 5'-ATT CAG CCC AAT CAG CGT CGA G-3' were used. To amplify a 350-bp sequence harboring IVS3–1G>A, primers 5'-TGC TGG GCA CAC AGG AGA TGT G-3' and 5'-TCT GCA GGT TCT TGA GGC AGG CA-3' were used. For a 335-bp sequence harboring R304X, primers 5'-CCT CAT GCC CTT GCA TGC CCA C-3' and 5'-ACA CAG AAG CAT GAC GCA GCA CG-3' were used. PuReTag Ready-to-Go PCR beads (Amersham BioSciences, Piscataway, NJ) were used for PCR. PCR conditions: 95 C for denaturing, 58 C for annealing, 72 C for polymerization. PCR products were purified and sequenced in both directions using the PCR primers. Sequencing results were both machine read and manually read independently by two of the authors.

Statistics

The sample size justification was based on 95% confidence interval estimation of the success proportion, using the normal approximation method. A success is defined by a patient having any one of the three mutations in the AIP gene. The success proportion is unknown, but is likely to be around 0.10 based on a previous report (8). The sample size of 66 allows 97% chance to detect at least one patient with an AIP mutation if the actual prevalence is 5%, and 74% chance if the actual prevalence is 2%. This actual proportion was estimated with a 95% confidence interval, using the binomial distribution.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Sixty-six (33 female and 33 male) patients from the Pituitary Center Database gave consent for genetic studies (Table 1). They were aged 8–81 yr at the time of pituitary tumor diagnosis, with a mean age of 39 and median age of 37. More than half of the patients had acromegaly (35 of 66), about a quarter of the patients had prolactinoma (17 of 66), nine patients had nonfunctioning tumors, and five patients had Cushing’s disease. This patient profile reflects the status of our Pituitary Center as a tertiary referral center for pituitary tumors with a particular interest in acromegaly. About 75% of patients (49 of 66) harbored macroadenomas, and the remaining 25% (17 of 66) harbored microadenomas. Three of the 66 patients had a sibling with a diagnosed pituitary tumor, and the remainder had no family history of pituitary tumors. Most patients were Caucasians (52 of 66) residing in California, and 14 were minorities or from other locales.

A heterozygous synonymous G to A polymorphism in exon 1 was noted in a 40-yr-old female patient with acromegaly; however, this polymorphism did not change the amino acid residue (glycine) that the gene encodes and is unlikely to be clinically significant.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Although much progress has been made in understanding the genetic basis of familial pituitary tumor syndromes such as MEN1, the pathogenesis of sporadic pituitary tumors remains largely unknown (1). A recent study reports that 10% of Finnish patients with sporadic acromegaly harbor specific AIP germline mutations (Q14X or IVS3–1G>A) (8). Q14X and another mutation, R304X, are also present in patients with familial pituitary tumors (8). Therefore, we tested the prevalence of the three specific AIP germline mutations in our patients with sporadic pituitary tumors.

AIP, also called ARA9, XAP2, or FKBP37, is a 330-amino-acid protein homologous to immunophilin FKBP52 (10). It was first isolated as a binding partner for the hepatitis B virus X protein (11); is one of three proteins that form a cytoplasmic complex with the aryl hydrocarbon receptor (AHR); and helps AHR stabilization, nuclear-cytoplasmic shuttling, and transactivation (10). However, AIP binding to the AHR is not critical for signaling as an AHR mutant that does not interact with AIP appears to function appropriately (12). The AHR is a transcription factor that modulates transcription activities of genes after binding with endogenous ligands or xenobiotics (10). Because ligand-dependent AHR signaling inhibits the cell cycle through interaction with Rb and induction of p27 (13, 14), AIP loss-of-function mutations that abolish AHR inhibition of the cell cycle could result in tumorigenesis.

We did not detect any of the three reported mutations in our 66 patients with sporadic pituitary tumors, and statistical analysis concluded that the chance of any single mutation is no greater than 5.6% in our patient population studied. Our sample size is large enough that the chance of detecting any single mutation is 97% if the true prevalence of any mutation is 5%. Therefore, because we did not detect mutations, the mutation rate appears to be very low in our patient population. Our results are in dramatic contrast to a recent report (8). It is unlikely that our patient sample is biased against detecting the mutations as the only difference between the patients tested and those not tested is that the patients assessed had consented for genetic studies. A possible explanation of the differences in AIP mutation discoveries may be ethnicity; the AIP mutations may be particularly prevalent in Finnish patients, whereas our patients have diverse ethnic backgrounds. Such founder effects among the Finnish population have been reported for a number of disorders (15). The significance of the G to A synonymous polymorphism is not clear.

In summary, we did not find any of the three specific AIP germline mutations in 66 mostly Caucasian patients of diverse ethnic background with sporadic pituitary tumors. We conclude that AIP mutations are unlikely involved in the pathogenesis of pituitary tumors in substantial numbers of non-Finnish patients.

	Footnotes

 
This work was supported by National Institutes of Health Grant CA75979 (to S.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 3, 2006

Abbreviations: AHR, Aryl hydrocarbon receptor; AIP, aryl hydrocarbon receptor interacting protein; MEN1, multiple endocrine neoplasia type 1.

Received August 10, 2006.

Accepted September 25, 2006.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) All Versions of this Article: 91/12/5126    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yu, R. Articles by Melmed, S. Search for Related Content PubMed PubMed Citation Articles by Yu, R. Articles by Melmed, S. Related Collections Endocrine Oncology Neuroendocrinology and Pituitary