Molecular diagnosis of pituitary predisposition caused by aryl hydrocarbon receptor-interacting protein gene mutations

Marianthi Georgitsia, Anniina Raitilaa, Auli Karhua, Karoliina Tuppurainenb, Markus J. Ma¨ kinenb, Outi Vierimaac, Ralf Paschked, Wolfgang Saegere, Rob B. van der Luijtf, Timo Saneg, Mercedes Robledoh, Ernesto De Menisi, Robert J. Weilj, Anna Wasikk, Grzegorz Zielinskil, Olga Lucewiczm, Jan Lubinskik,m, Virpi Launonena, Pia Vahteristoa, and Lauri A. Aaltonena,n

aDepartment of Medical Genetics, Molecular and Cancer Biology Research Program, University of Helsinki, P.O. Box 63, 00014, Helsinki, Finland; bDepartment of Pathology, University of Oulu, P.O. Box 5000, 90014, Oulu, Finland; cDepartment of Clinical Genetics, Oulu University Hospital, P.O. Box 60, 90029, Oulu, Finland; dMedical Department III, Leipzig University, Ph-Rosenthal-Street 27, 04103 Leipzig, Germany; eInstitute of Pathology, Marienkrankenhaus, Alfredstrasse 9, 22087 Hamburg, Germany; fDepartment of Medical Genetics, University Medical Centre Utrecht, P.O. Box 85090, 3508 GA, Utrecht, The Netherlands; gDepartment of , Helsinki University Central Hospital, P.O. Box 340, 00029, Helsinki, Finland; hHereditary Endocrine Cancer Group, Human Cancer Genetics Programme, Spanish National Cancer Center (CNIO), Melchor Ferna´ndez Almagro 3, 28029 Madrid, Spain; iDepartment of Internal Medicine, General Hospital, Piazza Ospedale 1, 31100 Treviso, Italy; jBrain Tumor Institute and Department of Neurosurgery, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195; kDepartment of Cell Biology, Nencki Institute of Experimental Biology, Pasteura 3, 02093, Warsaw, Poland; lDepartment of Neurosurgery, Military Institute of the Health Services, Szaserow 128, 00909, Warsaw, Poland; and mDepartment of Genetics and Pathology, International Hereditary Cancer Center, Pomeranian Medical University, Polabska 4, 70115, Szczecin, Poland

Communicated by Bert Vogelstein, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, January 2, 2007 (received for review November 28, 2006) Pituitary are common of the multiple endocrine neoplasia type 1 (MEN1) and Carney com- . Germ-line mutations in the aryl hydrocarbon receptor-inter- plex (CNC) (4, 5). Recent data suggest that a genetic predispo- acting protein (AIP) gene cause pituitary adenoma predisposition sition to pituitary tumors is less rare than thought and that genes (PAP), a recent discovery based on genetic studies in Northern Finland. other than those for MEN1 and CNC are also involved (5–7). In this population, a founder mutation explained a significant pro- Recently, we showed that germ-line mutations of the aryl portion of all cases. Typically, PAP patients were of a hydrocarbon receptor-interacting protein (AIP) gene cause pi- young age at diagnosis but did not display a strong family history of tuitary adenoma predisposition (PAP) (8). A nonsense muta- pituitary adenomas. To evaluate the role of AIP in pituitary adenoma tion, p.Q14X, was found in members of two Finnish families. The susceptibility in other populations and to gain insight into patient mutation segregated perfectly with the GH phenotype and was selection for molecular screening of the condition, we investigated also present in three patients. In addition, a the possible contribution of AIP mutations in pituitary tumorigenesis nonsense mutation, p.R304X, was found in two Italian siblings in patients from Europe and the United States. A total of 460 patients with GH-secreting adenomas. In a population-based series from were investigated by AIP sequencing: young acromegaly patients, Northern Finland, AIP mutations accounted for 16% of all unselected acromegaly patients, unselected pituitary adenoma pa- patients diagnosed with pituitary adenomas secreting GH and tients, and endocrine neoplasia-predisposition patients who were for 40% of patients younger than 35 years of age. Typically, PAP negative for MEN1 mutations. Nine AIP mutations were identified. patients were of a young age at disease onset and did not display Because many of the patients displayed no family history of pituitary a strong family history of pituitary adenomas. Loss of the normal adenomas, detection of the condition appears challenging. Feasibility allele was detected in eight of eight pituitary adenomas; AIP is of AIP (IHC) as a prescreening tool was tested likely to act as a tumor suppressor gene (8). in 50 adenomas: 12 AIP mutation-positive versus 38 mutation-nega- AIP encodes a protein of 330 aa. The protein contains an tive pituitary tumors. AIP IHC staining levels proved to be a useful FKBP-homology domain, and three tetratricopeptide (TPR) predictor of AIP status, with 75% sensitivity and 95% specificity for repeats. AIP forms interactions with the aryl hydrocarbon germ-line mutations. AIP contributes to PAP in all studied popula- receptor (AHR, also known as dioxin receptor), two HSP90 ␣ tions. AIP IHC, followed by genetic counseling and possible AIP proteins, PDE4A5, PPAR , and survivin (9–12). mutation analysis in IHC-negative cases, a procedure similar to the In our first study, for gene identification purposes, we focused diagnostics of the Lynch syndrome, appears feasible in identification on a defined, homogeneous population (8). To gain insight into of PAP. clinical features and approaches to diagnose the condition, it was relevant to examine the contribution of germ-line AIP mutations immunohistochemistry ͉ growth /prolacting–secreting adenomas ͉ in other patient materials as well. Here we sequenced the whole acromegaly AIP coding region in a large, heterogeneous collection of 460

ituitary adenomas are common, benign, monoclonal neo- Author contributions: M.G. and A.R. contributed equally to this work; M.G., A.R., A.K., O.V., Pplasms of the anterior . They account for V.L., P.V., and L.A.A. designed research; M.G., A.R., K.T., and M.J.M. performed research; Ϸ15% of intracranial tumors (1). Approximately two-thirds R.P., W.S., R.B.v.d.L., T.S., M.R., E.D.M., R.J.W., A.W., G.Z., O.L., and J.L. contributed new reagents/analytic tools; M.G., A.R., A.K., K.T., M.J.M., V.L., P.V., and L.A.A. analyzed data; produce pituitary in excess; among these, and M.G., A.R., A.K., E.D.M., R.J.W., V.L., P.V., and L.A.A. wrote the paper. MEDICAL SCIENCES (PRL)- and (GH)-oversecreting adenomas are The authors declare no conflict of interest. the most common. GH-secreting adenomas cause acromegaly Abbreviations: AHR, aryl hydrocarbon receptor; AIP, aryl hydrocarbon receptor-interacting and . Less common are adrenocorticotropin hormone protein; GH, growth hormone; IHC, immunohistochemistry; MEN1, multiple endocrine (ACTH)-secreting adenomas, causing Cushing’s disease. The neoplasia type 1; PAP, pituitary adenoma predisposition; PRL, prolactin. remaining one-third of pituitary adenomas is endocrinologically Data deposition: The sequences reported in this paper have been deposited in the GenBank silent, known as nonfunctioning pituitary adenomas, and cause database (accession nos. EF203234–EF203240). symptoms or signs due to tumor growth (1–3). Pituitary adeno- nTo whom correspondence should be addressed. E-mail: lauri.aaltonen@helsinki.fi. mas are components of rare, well established syndromes, such as © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700004104 PNAS ͉ March 6, 2007 ͉ vol. 104 ͉ no. 10 ͉ 4101–4105 Downloaded by guest on September 25, 2021 Table 1. AIP mutations identified in pituitary adenoma patients from the European and North American populations Normal No of AIP patients with allele Age at Family history AIP mutation lost in diagnosis, of pituitary Patients Mutation* Fragment (%) Clinical data tumor Sex years adenoma Controls

Young acromegaly Germany c.66-71delAGGAGA Exon 1 1 of 27 (3.7) Acromegaly- Yes M 20 Yes 0of532 GHoma (acromegaly) c.878-879AG3GT (p.E293G) and Exon 6 1of 27 (3.7) Acromegaly- Yes F 29† NA 0 of 255 c.8803891delCTGGACCCAGCC GHoma Finland c.40C3T (p.Q14X ) Exon 1 2 of 36 (5.5) Acromegaly- NA M 36 No 0 of 532 GHoma — — — Acromegaly- NA F 41 No 0 of 532 GHoma Unselected acromegaly Italy — — 0 of 71 — — — — — — Unselected pituitary adenoma U.S. IVS2-1G3C Intron 2 1 of 113 (0.9) Acromegaly- NA M 20 No 0 of 202 GHoma c.824-825insA Exon 6 1 of 113 (0.9) GHoma Yes M 8 No 0 of 201 Poland c.911G3A (p.R304Q) Exon 6 1 of 122 (0.8) Cushing’s NA NA 26 No 0 of 255 disease– ACTHoma MEN1-negative Spain c.542delT Exon 4 1 of 55 (1.8) Acromegaly– NA M 18 Yes 0of203 GHoma (acromegaly) The Netherlands c.896C3T (p.A299V) Exon 6 1 of 36 (2.8) Acromegaly– NA F 16 No 0 of 255 GHoma

ACTHoma, ACTH-secreting adenoma; F, female; GHoma, GH-secreting adenoma; M, male; NA, not available. *Only putative pathogenic changes are depicted. †Age at time of operation; age at time of diagnosis is not known.

pituitary adenoma patients and patients from families with seen in controls and was associated with a phenotype strongly MEN1 features and who were derived from different popula- suggestive of PAP, it was presumed pathogenic. Other changes tions in Europe and the United States. In addition, because the were presumed to be neutral. It is clear that this subdivision is genetic evidence suggests that many AIP-associated pituitary preliminary and should be interpreted with some caution. The adenomas are null for AIP protein, we tested whether negative results are reviewed and the details of the missense changes are staining in AIP immunohistochemistry (IHC) in pituitary ade- depicted below. nomas would be a useful marker for PAP. Young acromegaly patients. In the German samples, two AIP mutations were identified (2 of 27, 7.4%; see Table 1). In Results addition, a heterozygous intronic change, IVS1-18C3T, was Mutation Analysis. Nine presumably pathogenic mutations were identified in one sample but was not predicted to have an effect identified. These mutations, and the features of the respective on splicing as tested in silico. This same intronic change was also ´ patients, are depicted in Table 1 and Fig. 1. It is typically detected in 1 of 107 Centre d’Etude du Polymorphism Humain challenging to robustly evaluate the nature of missense changes controls (1%) and 1 of 96 Caucasian U.K. controls (1%), in hereditary predisposition. Here, if a missense change was not suggesting that the variant is a polymorphism. From the Helsinki University Central Hospital (HUCH) patient cohort, 36 Finnish patients were analyzed. The Finnish germ-line founder mutation was identified in two (5.5%; see Table 1) patients. Unselected acromegaly patients. Among the 71 Italian sporadic acromegaly patients, one heterozygous germ-line missense change was found: p.R16H (c.47G3A, resulting in the substi- tution of arginine at position 16 by histidine). Loss-of- heterozygosity analysis from this individual’s pituitary tumor tissue was negative. p.R16H was absent in 181 Caucasian U.K., 52 Italian, and 209 Finnish controls. The change was found in 1 Fig. 1. Diagram of AIP displaying the presumably pathogenic mutations of 90 healthy German controls (1%), 1 unselected pituitary identified in this study. The locations of the FKBP-homology region and the adenoma patient from the United States, and in 3 Polish three tetratricopeptide repeats (TPRs) are indicated by colored boxes. AHR unselected pituitary adenoma patients (see below), suggesting and HSP90 interaction regions are indicated by black lines. that this change may be a neutral polymorphism.

4102 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700004104 Georgitsi et al. Downloaded by guest on September 25, 2021 Fig. 2. AIP IHC. (A) AIP immunoreaction is observed in both the cytoplasm and the nucleus in normal adenohypophysis. (B) AIP expression in AIP-proficient adenoma. (C and D) AIP-deficient adenomas from two acromegaly patients bearing the p.Q14X mutation. There is complete loss of immunoreaction in adenoma cells, whereas AIP heterozygous peripheral blood leucocytes display positive immunoreaction, indicated by black arrows.

Unselected pituitary adenoma patients. AIP mutation analysis per- of 12) lacked both cytoplasmic and nuclear immunoreactivity formed in 113 unselected pituitary adenoma patients from the against AIP (Fisher’s Exact test, P ϭ 0.000004). In tumor tissues, United States revealed two germ-line mutations (2 of 113, 1.8%; see leukocytes served as internal positive controls (Fig. 2 C and D). Table 1) and two heterozygous missense changes that are likely to AIP IHC had 75% sensitivity and 95% specificity for truncating be polymorphisms. A heterozygous c.906G3A, resulting in the AIP germ-line mutations. silent p.V302V change in exon 6, was found in three cases. When p.V302V was tested in silico, the prediction programs showed no Discussion significant effect on splicing. None of the three patients had a family In our original study, we evaluated the contribution of AIP in a history of pituitary adenomas. Loss-of-heterozygosity analysis was population-based material of acromegaly patients, diagnosed possible from tumor DNA samples of 2/3 individuals, and showed between 1980 and 1999 in the Oulu region of Northern Finland. retention of the wild type allele. p.V302V was not detected in 109 In this isolated population, two germ-line AIP mutations Centre d’E´tude du Polymorphism Humain, 94 Caucasian U.K., and (p.Q14X and IVS3-1G3A) accounted for 16% of all patients 52 Italian controls. Finally, the previously seen missense change diagnosed with pituitary adenomas secreting GH and for 40% of p.R16H in exon 1, was found in one individual: a 78 year-old woman patients that were younger than 35 years at the age of diagnosis of Polish descent. Tumor DNA sequence did not show loss of (8). In the current study, we examined the role of AIP in more heterozygosity. heterogeneous patient groups to provide clues to clinical and In 122 unselected pituitary adenoma patients from Poland, molecular identification of PAP. three different germ-line heterozygous missense changes were The analysis of 71 Italian acromegaly patients systematically detected, of which one was considered disease associated (1 of collected from the Treviso region revealed no significant find- 122, 0.8%; Table 1). In addition, the previously detected p.R16H ings, but two siblings (2 of 73, 2.7%) belonging to this same change was seen also in three Polish individuals all diagnosed sample collection were shown to display a truncating AIP with Cushing’s disease. A heterozygous c.696G3C, resulting in mutation in our previous study (8). the silent p.P232P change in exon 5, was found in one patient In both of the two patient sets with acromegaly that presented with Cushing’s disease. This change did not have any predicted at a young age, the Helsinki and Leipzig regions, two PAP effect on splicing as tested in silico. p.P232P was absent in 108 patients were detected; altogether, 4 of 63 (6.3%) patients Centre d’E´tude du Polymorphism Humain or 95 Caucasian U.K. had PAP. controls. Two putative AIP mutations were found among unselected Patients counseled and examined for MEN1 with negative genetic testing pituitary adenoma cases from the United States. IVS2-1G3C results. In the 55 Spanish samples analyzed, one AIP mutation was splice site mutation was detected in a patient diagnosed with

identified (1 of 55, 1.8%; see Table 1). Likewise, of the 36 Dutch acromegaly at the age of 20 years. c.824–825insA insertion, also MEDICAL SCIENCES samples, an AIP germ-line mutation was identified in one causing a premature stop codon, was seen in a patient diagnosed specimen (1 of 36, 2.8%; see Table 1). with GH-secreting adenoma at the age of 8 years. These two cases account for 1.8% of the unselected pituitary adenoma AIP Immunohistochemical Staining. AIP immunoreaction was ob- cases in this series. The number of acromegaly patients in the served in both the cytoplasm and the nucleus in normal adeno- sample set was only 13. Although the numbers are very small, it hypophysis (Fig. 2A). Most AIP mutation-negative adenomas (36 is noteworthy that the contribution of PAP in acromegaly in this of 38) had preserved cytoplasmic and nuclear immunoreaction consecutive U.S. series was similar to that seen in Northern against AIP (Fig. 2B), whereas most AIP-deficient adenomas (9 Finland (2 of 13, 15%).

Georgitsi et al. PNAS ͉ March 6, 2007 ͉ vol. 104 ͉ no. 10 ͉ 4103 Downloaded by guest on September 25, 2021 p.R304Q change was seen in one Polish unselected pituitary 34 to 120 cases per million (20). Although these observations adenoma patient (1 of 122, 1%). If pathogenic, this AIP mutation suggest that Ϸ1,000 new cases will be diagnosed annually in the would be previously unrecognized in Cushing’s disease. We have United States, the insidious nature of acromegaly and the frequent detected this variant in an Italian patient with acromegaly (M.G., delays in diagnosis in this disease have led to estimates that A.R., A.K., E.D.M., V.L., P.V., and L.A.A., unpublished data) GH-secreting pituitary adenomas are present but undiagnosed in as but in none of the healthy controls, which strongly supports the many as 20,000 persons in the Unites States alone (21). Because the notion that the change is pathogenic. Interestingly, p.R304Q majority of patients present with a macroadenoma, and younger locates on the AHR-binding region (13, 14) and possibly affects patients frequently have larger, more invasive tumors with poorer the interaction of AIP with AHR. outcomes, the potential for prolonged biochemical remission with Approximately 10% of cases clinically suggestive of MEN1 do any single modality is diminished (22, 23). In patients with more not seem to harbor germ-line MEN1 mutations (4, 15–17). Thus, advanced disease, successful therapy eliminates or resolves all we examined whether AIP is involved in such cases of unex- manifestations of the disease in a minority, and diminished quality- plained endocrine neoplasia susceptibility. Two mutation- of-life persists in the majority (24). Overall morbidity and mortality, positive cases were found (2 of 91, 2%). The phenotypes of these related primarily to chronic cardiovascular disease, is a function of two patients were in line with the above findings; both the biochemical control: risk of mortality may be as high as 3.5-fold Spanish and the Dutch patient were diagnosed with acromegaly greater in patients with persistent disease compared with those in at an early age: 18 and 16 years, respectively. The Spanish patient remission (22, 23, 25–28). Although molecular diagnosis of PAP in also had a positive family history, with two maternal uncles being unselected pituitary adenomas would be first performed in a diagnosed with acromegaly. research setting, thousands of paraffin-embedded somatotropi- These data firmly confirm that AIP is directly implicated in the noma samples in the United States alone are available for pre- molecular pathogenesis of pituitary tumors, particularly of the screening of PAP by AIP IHC, pending consent from the patients. GH/PRL lineage. In the future, it will be of interest to examine Although current management of patients with pituitary adenomas what the contribution of de novo mutations in PAP is. In the does not seem to be influenced by diagnosed AIP mutation current work, the relevant additional samples were not available. positivity, offering genetic counseling and predictive testing to The prevalence of AIP germ-line mutations varies in different family members provides a powerful tool for prevention of mor- clinical settings. In unselected sporadic pituitary tumors, the bidity in at-risk individuals. The great majority of AIP-related overall prevalence seems to be low: 2 of 113 and 1 of 122 from tumors are GH- and/or PRL-secreting adenomas, and the clinical our U.S. and Poland cases, respectively. Also, none of the diagnosis of acromegaly at onset is difficult. Therefore, we suggest previously reported p.Q14X, p.R304X, and IVS3-1G3A mu- that asymptomatic relatives testing positive for an AIP mutation tations were found in a recently published U.S. series (18). On should undergo annual PRL and IGF1 monitoring (29), as sug- the contrary, AIP mutations are enriched in patients of a very gested for MEN1 carriers (30). Finally, we highlight the particular young age at onset and/or a positive family history of acromegaly, importance of AIP analyses in patients with a positive family history although some of the PAP patients display neither of these for acromegaly or with early onset of the tumor. features. Selection of patients for genetic counseling and possi- ble genetic testing for PAP appeared challenging. Materials and Methods To help simplify selection, we tested AIP IHC in 50 pituitary Study Subjects. The study was approved by the appropriate ethics adenomas as a tool for molecular screening for PAP. Because many review committees. Appropriate informed consent was obtained mutations are truncating and because the condition is typically from all subjects. associated with loss of the wild-type allele in tumors, a strategy Young acromegaly patients. DNA extracted from paraffin- similar to that used to screen patients for hereditary nonpolyposis embedded tumor tissue was available from 27 patients with colorectal cancer or Lynch syndrome (19) appeared attractive: IHC acromegaly from the German pituitary tumor register, Institute screening of tumors for loss of the predisposition gene product, of Pathology, Marienkrankenhaus Hamburg. The search was genetic counseling, and possible germ-line mutation testing in cases conducted for entries during the last 3 years for patients younger displaying negative IHC for AIP, followed by cascade screening in than 40 years old at the time of surgery. family members to identify individuals at risk. Because pituitary DNA derived from blood was available from 36 Finnish acro- adenomas are examined immunohistochemically as a routine di- megaly patients who were Ͻ45 years old and originally diagnosed agnostic practice, this screening method appears to be feasible. and treated at the Department of Endocrinology, Helsinki Uni- Indeed, we found that negative AIP IHC staining is a strong versity Central Hospital (HUCH). This cohort represented 57.1% predictor of PAP. Two cases with a negative AIP mutation analysis of all young (Ͻ45 years) acromegaly patients diagnosed at HUCH but a negative IHC result may have had occult (such as deletion not between the years 1980–2005. detected by sequencing) germ-line mutations or somatic loss of Unselected acromegaly patients. Blood-extracted DNA samples from AIP, although technical problems are also a possible explanation. 71 unselected Italian acromegaly patients who were referred to The individuals had no family history of endocrine tumors but were Treviso General Hospital were available. Age at diagnosis ranged 40 and 47 years of age, and, thus, PAP is possible. Similarly, in the between 23 and 90 years, with a mean age of 45 years. three mutation-positive cases displaying positive AIP IHC, the Unselected pituitary adenoma patients. Altogether, 113 samples col- unexpected IHC finding could have been due to technical difficul- lected consecutively from patients undergoing resection of a pitu- ties such as unspecific staining or missense-type second hits en- itary tumor at the Cleveland Clinic were analyzed. DNA was abling production of nonfunctional yet immunoreactive AIP pro- isolated from either blood or tumor tissue. Age at diagnosis ranged tein. It is likely that, with greater experience with this approach, the between 8 and 87 years, with a mean age of 52 years. Of these 113 ability to screen for AIP alterations by IHC will further improve. patients, all underwent biochemical and immunohistochemically Although the cost of DNA sequencing will be reduced dramatically confirmed diagnoses: 13 with Cushing’s disease, 13 with acromegaly in the near future, IHC screening will remain useful, because direct due to GH-secreting adenomas, 11 with hyperprolactinemia due to DNA testing for AIP germ-line mutations would require prior PRL-secreting adenomas. The remaining 76 patients had a non- genetic counseling. Genetic counseling demands resources and functioning pituitary adenoma. None of the patients had a family needs to be reserved for those pituitary adenoma cases that display history of pituitary tumors. features of hereditary susceptibility. Blood-extracted DNA samples from 122 unselected Polish pitu- The annual incidence of newly diagnosed cases of acromegaly itary adenoma patients were collected at the International Hered- ranges from 2.8 to Ͼ4 per million, with prevalences ranging from itary Cancer Center, Pomeranian Medical University, in Szczecin,

4104 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700004104 Georgitsi et al. Downloaded by guest on September 25, 2021 Poland. Of these, 74 patients were diagnosed with Cushing’s negative adenomas included 32 somatotropinomas, five prolacti- disease, 30 with acromegaly, and 18 with pituitary adenomas of nomas, and one GH- and PRL-negative adenoma. various types. Age of onset ranged between 8 and 67 years, with a mean age of 39 years. Age of onset was not known for 21 cases. Mutation Analysis. Mutation analysis was performed by direct Patients counseled and examined for MEN1 with negative genetic testing sequencing of genomic DNA. The whole coding region of AIP was results. Blood-extracted DNA from 36 Dutch patients was analyzed. sequenced, as well as flanking intronic sequences and 5Ј and 3Ј Patients had been referred to the DNA Diagnostics Laboratory untranslated regions. PCR protocols and primer sequences have (Department of Medical Genetics, University Medical Centre been described by Vierimaa et al. (8) and are available on request. Utrecht, The Netherlands) for MEN1 molecular diagnostics, during DNA sequencing was performed using Big Dye 3.1 termination the period of 2004–2006. Patients suspected for MEN1 were chemistry on an ABI3730 DNA sequencer (Applied Biosystems, defined as those with at least three of the following five lesions: Foster City, CA). hyperparathyroidism/parathyroid tumors, pancreatic endocrine tu- In Silico Analysis. The potential effects on splicing of the detected mors, pituitary adenomas, tumors, and/or neuroen- intronic and silent changes were predicted in silico by computa- docrine carcinoid tumors. The patients fulfilled the following Ͻ tional methods by using NetGene2, Alternative Splice Site Predic- criteria: young age at onset ( 35 years) of any of the five MEN1- tor (ASSP), and SpliceScan programs (31–34). related lesions and/or multiple MEN1-related lesions in a single organ or two distinct organs, and at least one first-degree relative AIP IHC. Five-micrometer-thick sections were cut from the paraffin in whom at least one target organ was affected. Age at tumor blocks. After deparaffinization and rehydration, sections were diagnosis ranged between 15 and 81 years, with a mean age of 50 pretreated in either 0.01 M citrate (pH 6.0) buffer in a microwave years. oven at 800 W for 2 min and at 300 W for 10 min or in 0.01 M Another set consisted of individuals suspected for MEN1 and Tris-EDTA (pH 6.0) buffer in a microwave oven at 800 W for 2 min referred to the Spanish National Cancer Center during the period and at 300 W for 15 min. AIP was detected in tumors by using AIP of 1997–2006. Blood-extracted DNA samples from 55 unselected antibody (AIP SP5213P; Acris Antibodies, Hiddenhausen, Ger- and consecutive MEN1-negative patients were available for AIP many) at a 1:4,000 dilution for 30 min. Positive antibody reaction mutation analysis. Age at diagnosis ranged between 12 and 78 years, was detected with diaminobenzidine (DAKO, Copenhagen, Den- with a mean age of 50 years. Information was not available for two mark) with hematoxylin counterstain. patients. We thank all patients for their valuable help. We are grateful to R. Lehtonen Control Samples. DNA from unrelated, anonymous, individuals was for help with the in silico analysis; S. Marttinen, I. L. Svedberg, I. Vuoristo, ´ P. Hannuksela, M. Aho, and R. Vuento for their excellent technical used as control samples: 110 Caucasian Centre d’Etude du Poly- assistance; P. Ellonen for providing sequencing facilities and service; and morphism Humain individuals, 288 Caucasians from the U.K. D. K. Luedecke (University of Hamburg, Germany) for providing material (Human Random Control DNA Panels, Porton Down, Salisbury, and clinical data. This study was supported by Academy of Finland Grants Wiltshire, U.K.), 209 Finns, 90 Germans, and 52 Italians. 213183 (to V.L.) and 212901 (to P.V.), the Center of Excellence in Translational Genome-Scale Biology, the Sigrid Juse´lius Foundation, the IHC Samples. AIP protein expression was analyzed in 50 pituitary Cancer Society of Finland, Association for International Cancer Research adenomas. Twelve tumors were from AIP mutation-positive indi- Grant 05-001 (to A.K.), a Jalmari and Rauha Ahokas Foundation research 3 grant (to M.G.), a Bodossaki Foundation postgraduate scholarship (to viduals (nine cases with p.Q14X, and one case with IVS2-1G C, M.G.), the Melvin Burkhardt Chair in Neurosurgical , and the c.824–825insA, and IVS3-1G3A, respectively) including 10 so- Karen Colina Wilson Research Endowment within the matotropinomas and two . Thirty-eight mutation- Institute at the Cleveland Clinic Foundation.

1. Heaney AP, Melmed S (2004) Nat Rev Cancer 4:285–295. 20. Stewart PM (2004) Eur J Endocrinol 151:431–432. 2. Melmed S (2003) J Clin Invest 112:1603–1618. 21. Melmed S (2006) in Endocrinology, eds DeGroot LJ, Jameson JL (Elsevier 3. Arafah BM, Nasrallah MP (2001) Endocr Relat Cancer 8:287–305. Saunders, Philadelphia), pp 411–428. 4. Daly AF, Jaffrain-Rea ML, Beckers A (2005) Horm Metab Res 37:347–354. 22. Drange MR, Fram NR, Herman-Bonert V, Melmed S (2000) J Clin Endocrinol 5. Daly AF, Jaffrain-Rea ML, Ciccarelli A, Valdes-Socin H, Rohmer V, Tam- Metab 85:168–174. burrano G, Borson-Chazot C, Estour B, Ciccarelli E, Brue T, et al. (2006) J Clin 23. Besser GM, Burman P, Daly AF (2005) Eur J Endocrinol 153:187–193. Endocrinol Metab 91:3316–3323. 24. Biermasz NR, Pereira AM, Smit JW, Romijn JA, Roelfsema F (2005) J Clin 6. Frohman LA, Eguchi K (2004) Growth Horm IGF Res 14(Suppl A):90–96. Endocrinol Metab 90:2731–2739. 7. Pellegata NS, Quintanilla-Martinez L, Siggelkow H, Samson E, Bink K, Hofler H, 25. Holdaway IM, Rajasoorya RC, Gamble GD (2004) J Clin Endocrinol Metab Fend F, Graw J, Atkinson MJ (2006) Proc Natl Acad Sci USA 103:15558–15563. 89:667–674. 8. Vierimaa O, Georgitsi M, Lehtonen R, Vahteristo P, Kokko A, Raitila A, 26. Colao A, Ferone D, Marzullo P, Lombardi G (2004) Endocr Rev 25:102– Tuppurainen K, Ebeling TM, Salmela PI, Paschke R, et al. (2006) Science 152. 312:1228–1230. 27. Kauppinen-Makelin R, Sane T, Reunanen A, Valimaki MJ, Niskanen L, 9. Carver LA, Bradfield CA (1997) J Biol Chem 272:11452–11456. Markkanen H, Loyttyniemi E, Ebeling T, Jaatinen P, Laine H, et al. (2005) 10. Bolger GB, Peden AH, Steele MR, MacKenzie C, McEwan DG, Wallace DA, J Clin Endocrinol Metab 90:4081–4086. Huston E, Baillie GS, Houslay MD (2003) J Biol Chem 278:33351–33363. 28. Swearingen B, Barker FG, II, Katznelson L, Biller BM, Grinspoon S, Klibanski 11. Sumanasekera WK, Tien ES, Turpey R, Vanden Heuvel JP, Perdew GH (2003) A, Moayeri N, Black PM, Zervas NT (1998) J Clin Endocrinol Metab 83:3419– J Biol Chem 278:4467–4473. 3426. 12. Kang BH, Altieri DC (2006) J Biol Chem 281:24721–24727. 29. Giustina A, Barkan A, Casanueva FF, Cavagnini F, Frohman L, Ho K, 13. Bell DR, Poland A (2000) J Biol Chem 275:36407–36414. Veldhuis J, Wass J, Von Werder K, Melmed S (2000) J Clin Endocrinol Metab 14. Petrulis JR, Perdew GH (2002) Chem Biol Interact 141:25–40. 85:526–529.

15. Hai N, Aoki N, Shimatsu A, Mori T, Kosugi S (2000) Clin Endocrinol (Oxford) 30. Brandi ML, Gagel RF, Angeli A, Bilezikian JP, Beck-Peccoz P, Bordi C, MEDICAL SCIENCES 52:509–518. Conte-Devolx B, Falchetti A, Gheri RG, Libroia A, et al. (2001) J Clin 16. Marx SJ (2005) Nat Rev Cancer 5:367–375. Endocrinol Metab 86:5658–5671. 17. Cebrian A, Ruiz-Llorente S, Cascon A, Pollan M, Diez JJ, Pico A, Telleria D, 31. Brunak S, Engelbrecht J, Knudsen S (1991) J Mol Biol 220:49–65. Benitez J, Robledo M (2003) J Med Genet 40:e72. 32. Hebsgaard SM, Korning PG, Tolstrup N, Engelbrecht J, Rouze P, Brunak S 18. Yu R, Bonert V, Saporta I, Raffel LJ, Melmed S (2006) J Clin Endocrinol Metab (1996) Nucleic Acids Res 24:3439–3452. 91:5126–5129. 33. Wang M, Marin A (2006) Gene 366:219–227. 19. Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, 34. Tchourbanov A, Ali HH, Deogun J (2004) Proceedings of the 2004 IEEE Nakagawa H, Sotamaa K, Prior TW, Westman J, et al. (2005) N Engl J Med Computational Systems Bioinformatics Conference (IEEE Comput Soc, Los 352:1851–1860. Alamitos, CA) pp 672–673.

Georgitsi et al. PNAS ͉ March 6, 2007 ͉ vol. 104 ͉ no. 10 ͉ 4105 Downloaded by guest on September 25, 2021