AIP ja CDKN1B/p27Kip1 geenien rooli endokriinisissä kasvaimissa

Identification of genes predisposing to tumor syndromes has raised general awareness of tumorigenesis. Genetic testing of tumor susceptibility genes aids the recognition of individuals at increased risk of tumors. Identification of novel predisposing genes enables further studies concerning the classification of potential associated tumors and the definition of target patient group. Pituitary adenomas are common, benign neoplasms accounting for approximately 15% of all intracranial tumors. Accurate incidence estimation is challenging since a great portion of these adenomas are small and asymptomatic. Clinically relevant adenomas, that cause symptoms due to the expansion of the cell mass or the over-secretion of normally produced hormones, occur in approximately one of 1 000 individuals. Although the majority of pituitary adenomas are sporadic, a minority occur as components of familial syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 syndrome is caused by germ-line mutations in the MEN1 gene, whereas most of the CNC patients carry the mutated protein kinase A (PKA) regulatory subunit-1-α (PRKAR1A) gene. Recently, other conditions predisposing to endocrine tumors have been identified: Pituitary Adenoma Predisposition (PAP) and MEN type 4 (MEN4). PAP was originally identified in a genetically homogeneous Finnish population. In a population based cohort from Northern Finland, aryl hydrocarbon receptor-interacting protein (AIP) gene mutations were found in 16% of all patients diagnosed with growth hormone (GH) producing pituitary adenoma, and in 40% of the subset of patients who were diagnosed under the age of 35 years. Since AIP mutations were originally described in a defined, homogeneous population from Northern Finland, it was relevant to study whether mutations also occur in more heterogeneous populations. In patient cohorts with different ethnic origins and variable clinical phenotypes, germ-line AIP mutations were detectable at low frequencies (range 0.8-7.4%). AIP mutation-positive patients were often diagnosed with a GH-producing adenoma at a young age, and usually had no family history of endocrine tumors. The low frequency of AIP mutations in randomly selected patients, and the lack of any family history of pituitary adenomas create a challenge for the identification of PAP patients. Our preliminary study suggests that AIP immunohistochemistry may serve as a pre-screening tool to distinguish between the AIP mutation-negative and the mutation-positive tumors. Tumors of various endocrine glands are components of MEN1 and CNC syndromes. Somatic MEN1 and PRKAR1A mutations in sporadic pituitary adenomas are rare, but occur in some of the other tumors related to these syndromes. The role of AIP mutations in endocrine neoplasia was studied and our results indicated that somatic AIP mutations are rare or non-existent in sporadic tumors of endocrine glands (0 of 111). Furthermore, germ-line AIP mutations in prolactin producing adenomas (2 of 9) confirmed the role of this pituitary tumor type in the PAP phenotype. Thyroid disorders are common in the general population, and the majority of them are sporadic. Interestingly, it has been suggested that thyroid disorders might be more common in PAP families. For this reason we studied germ-line AIP mutations in 93 index cases from familial non-medullary thyroid cancer (NMTC) families. The underlying gene or genes for familial NMTC have not been identified yet. None of the patients had any potentially pathogenic AIP mutation. This suggests that AIP is unlikely to play a role in familial NMTCs. A novel multiple endocrine syndrome was originally described in rats with phenotypic features of human MEN type 1 and 2. Germ-line mutations of cyclin-dependent kinase inhibitor 1B (CDKN1B also known as p27Kip1) gene were reported later in these rats and a germ-line mutation was also identified in one human family with MEN1-like phenotype (later named MEN4). To confirm the importance of this gene’s mutations in humans, we performed a mutation screening in MEN-like patients and in patients with pituitary adenoma. Our results indicate that CDKN1B/p27Kip1 mutations appear in a small portion of MEN1-like patients (one of 36), and that such mutations are rare or non-existent in both familial (0 of 19) and sporadic pituitary adenoma patients (0 of 50). In conclusion, this work strengthens the tumor susceptibility role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia. Clarifying the PAP phenotype facilitates the identification of potential AIP mutation carriers. Genetic counseling can be offered to the relatives and follow-up of the mutation carriers can be organized, hence an earlier diagnosis is feasible.Kasvainten muodostumiselle altistavien geenivirheiden tunnistaminen mahdollistaa riskihenkilöiden varhaisdiagnostiikan. Alkuperäisen alttiusgeenilöydöksen jälkeen voidaan tutkia muun muassa mille kasvaimille ko. geenin virheet altistaa sekä minkälaisista potilasryhmistä ko. geenin virheitä on mahdollista löytää. Aivolisäkeadenoomat ovat hyvänlaatuisia kasvaimia, jotka ovat yleisiä normaalissa väestössä. Valtaosa näistä adenoomista eivät aiheuta oireita, koska ovat pieniä ja hormonaalisesti toimimattomia. Aivolisäkeadenoomia, joissa kasvaimen massan kasvu ja/tai hormonien liikaeritys johtaa kliinisiin oireisiin, esiintyy noin yhdellä henkilöllä tuhannesta. Suurin osa aivolisäkeadenoomista muodostuvat sattumalta, mutta osa tapauksista on perinnöllisiä. Tunnistettuja perinnöllisiä oireyhtymiä ovat esimerkiksi multippeli endokriininen neoplasia tyypin 1 (MEN1) syndrooma sekä Carneyn kompleksi (CNC). Äskettäin julkaistiin kaksi uutta aivolisäkeadenoomille altistavaa oireyhtymää: aivolisäke adenooma alttius (pituitary adenoma predisposition, PAP) sekä multippeli endokriininen neoplasia tyypin 4 (MEN4) syndrooma. Väitöskirjatutkimukseni tarkoituksena on selventää näille uusille oireyhtymille altistavien geenien roolia endokriinisissä kasvaimissa. PAP johtuu ituradan mutaatiosta AIP (aryl hydrocarbon receptor-interacting protein) geenissä. Alttiusgeenin tunnistaneessa tutkimuksessa havaittiin ko. geenin olevan virheellinen 16 %:lla pohjoissuomalaisista potilaista, joilla oli kasvuhormonia erittävä aivolisäkeadenooma. AIP mutaatio oli 40 % niistä potilaista, jotka oli diagnosoitu alle 35-vuotiaina. Väitöskirjan ensimmäisessä osatyössä tutkittiin AIP geenin osuutta erilaisissa aivolisäkeadenooma potilasnäytteissä. Tuloksissa havaittiin, että verrattuna pohjoissuomalaisista saatuihin tuloksiin, AIP mutaatioita esiintyi vähemmän (0.8-7.4 %) niin eurooppalaisissa kuin yhdysvaltalaisissa aivolisäkeadenooma potilaissa. Tutkimuksen avulla varmennettiin tyypillinen AIP mutaatiopositiivisen potilaan fenotyyppi: nuori potilas, jolla on yleensä kasvuhormonia erittävä aivolisäkeadenomaa ja jolla ei välttämättä ole sukutaustaa endokriinisistä kasvaimista. Lisäksi mutaatiopositiivisten tunnistamiseksi todettiin immunohistokemiallisen analyysin olevan mahdollista, mutta menetelmän optimointi vaatii lisätutkimuksia. MEN1 ja CNC oireyhtymissä esiintyy kasvaimia aivolisäkkeen lisäksi myös muissa endokriinisissä kudoksissa. Somaattiset mutaatiot näille oireyhtymille altistavissa geeneissä ovat harvinaisia satunnaisissa aivolisäkeadenoomissa, mutta niitä esiintyy muissa oireyhtymille tyypillisissä satunnaisissa endokriinisissä kasvaimissa. Toisessa osatyössä tutkittiin somaattisia AIP mutaatioita erilaisessa satunnaisissa endokriinisissä kasvaimista. Tuloksien mukaan AIP somaattisia mutaatioita ei ole tai ne ovat harvinaisia (0/111). Kuitenkin tutkimuksessa löydettiin ituradan AIP mutaatio kahdelta potilaalta (2/9), joiden prolaktiinia erittävät aivolisäkeadenoomat oli diagnosoitu nuorella iällä. Löydös vahvisti siten tämän kasvaintyypin kuuluvan PAP fenotyyppiin. Kilpirauhasen kasvaimet, joista suurin osa muodostuu satunnaisesti, ovat yleisiä väestössä. Perinnöllinen ei-medullaarinen kilpirauhasen syöpä (familial non-medullary thyroid cancer, NMTC) on tunnistettu oireyhtymä, jonka geneettistä taustaa ei vielä ole löydetty. Koska kilpirauhasen kasvaimia esiintyy myös PAP perheissä, kolmannessa osatyössä tutkittiin altistavatko AIP mutaatiot perinnölliselle NMTC:lle. Tuloksien mukaan AIP ei näyttäisi osallistuvan ko. oireyhtymään, sillä ituradan mutaatiota ei löydetty. CDKN1B (cyclin-dependent kinase inhibitor 1B, myös nimeltään p27Kip1) geenin mutaatio tunnistettiin MEN1 ja MEN2 fenotyypin kaltaisesta rottalinjasta ja lisäksi MEN1-kaltaisen fenotyypin perheestä. Tämä oireyhtymä nimettiin myöhemmin MEN4:ksi. Neljännessä osatyössä tutkittiin CDKN1B/p27Kip1 geenin osuutta potilasnäytteistä, joilla oli joko MEN1-kaltainen fenotyyppi tai aivolisäkeadenooma. Tutkimuksessa tunnistettiin toinen mutaatio MEN1-kaltaisen fenotyypin potilaasta (1/36) ja siten vahvistettiin ko. geenin rooli endokriinisessä oireyhtymässä. Kuitenkin ko. geenin mutaatiot ovat harvinaisia tai niitä ei ole perinnöllisillä (0/19) tai satunnaisilla (0/50) aivolisäkeadenooma potilailla. Väitöskirjatyö vahvistaa AIP and CDKN1B/p27Kip1 roolia endokriinissä kasvaimissa. PAP fenotyypin selventäminen mahdollistaa virheellisen AIP geenin kantajien tunnistamisen. Näiden potilaiden sukulaisille voidaan tarjota geenitestausta, jolloin seurannan avulla voidaan diagnoosin tekemistä sekä hoitojen aloittamista aikaistaa

Mice with Inactivation of Aryl Hydrocarbon Receptor-Interacting Protein (Aip) Display Complete Penetrance of Pituitary Adenomas with Aberrant ARNT Expression

Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been shown to predispose to pituitary adenoma predisposition, a condition characterized by growth hormone (GH)-secreting pituitary tumors. To study AIP-mediated tumorigenesis, we generated an Aip mouse model. Heterozygous mice developed normally but were prone to pituitary adenomas, in particular to those secreting GH. A complete loss of AIP was detected in these lesions, and full penetrance was reached at the age of 15 months. No excess of any other tumor type was found. Ki-67 analysis indicated that Aip-deficient tumors have higher proliferation rates compared with Aip-proficient tumors, suggesting a more aggressive disease. Similar to human AIP-deficient pituitary adenomas, immunohistochemical studies showed that expression of aryl hydrocarbon receptor nuclear translocator 1 or 2 (ARNT or ARNT2) protein was lost in the mouse tumors, suggesting that mechanisms of AIP-related tumorigenesis involve aberrant ARNT function. The Aip+/− mouse appears to be an excellent model for the respective human disease phenotype. This model constitutes a tool to further study AIP-associated pituitary tumorigenesis and may be potentially valuable in efforts to develop therapeutic strategies to treat pituitary adenomas

Pituitary adenoma predisposition caused by germline mutations in the AIP gene

Pituitary adenomas are common in the general population, and understanding their molecular basis is of great interest. Combining chip-based technologies with genealogy data, we identified germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene in individuals with pituitary adenoma predisposition (PAP). AIP acts in cytoplasmic retention of the latent form of the aryl hydrocarbon receptor and also has other functions. In a population-based series from Northern Finland, two AIP mutations account for 16% of all patients diagnosed with pituitary adenomas secreting growth hormone and for 40% of the subset of patients who were diagnosed when they were younger than 35 years of age. Typically, PAP patients do not display a strong family history of pituitary adenoma; thus, AIP is an example of a low-penetrance tumor susceptibility gene

The Expression of AIP-Related Molecules in Elucidation of Cellular Pathways in Pituitary Adenomas

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene predispose to the development of pituitary adenomas. Here, we characterized AIP mutation positive (AIPmut+) and AIP mutation negative (AIPmut−) pituitary adenomas by immunohistochemistry. The expressions of the AIP-related proteins aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), cyclin-dependent kinase inhibitor 1B encoding p27(Kip1), and hypoxia-inducible factor 1-α were examined in 14 AIPmut+ and 53 AIPmut− pituitary adenomas to detect possible expression differences. In addition, the expression of CD34, an endothelial and hematopoietic stem cell marker, was analyzed. We found ARNT to be less frequently expressed in AIPmut+ pituitary adenomas (P = 0.001), suggesting that AIP regulates the ARNT levels. AIP small interfering RNA-treated HeLa, HEK293, or Aip-null mouse embryonic fibroblast cells did not show lowered expression of ARNT. Instead, in the pituitary adenoma cell line GH3, Aip silencing caused a partial reduction of Arnt and a clear increase in cell proliferation. We also observed a trend for increased expression of nuclear AHR in AIPmut+ samples, although the difference was not statistically significant (P = 0.06). The expressions of p27(Kip1), hypoxia-inducible factor 1-α, or CD34 did not differ between tumor types. The present study shows that the expression of ARNT protein is significantly reduced in AIPmut+ tumors. We suggest that the down-regulation of ARNT may be connected to an imbalance in AHR/ARNT complex formation arising from aberrant cAMP signaling

Candidate driver genes in microsatellite-unstable colorectal cancer

Defects in the mismatch repair system lead to microsatellite instability (MSI), a feature observed in similar to 15% of all colorectal cancers (CRCs). Microsatellite mutations that drive tumourigenesis, typically inactivation of tumour suppressors, are selected for and are frequently detected in MSI cancers. Here, we evaluated somatic mutations in microsatellite repeats of 790 genes chosen based on reduced expression in MSI CRC and existence of a coding mononucleotide repeat of 6 similar to 10 bp in length. All the repeats were initially sequenced in 30 primary MSI CRC samples and whenever frameshift mutations were identified in &gt;20%, additional 70 samples were sequenced. To distinguish driver mutations from passengers, we similarly analyzed the occurrence of frameshift mutations in 121 intronic control repeats and utilized a statistical regression model to determine cut-off mutation frequencies for repeats of all types (A/T and C/G, 610 bp). Along with several know target genes, including TGFBR2, ACVR2, and MSH3, six novel candidate driver genes emerged that harbored significantly more mutations than identical control repeats. The mutation frequencies in 100 MSI CRC samples were 51% in G8 of GLYR1, 47% in T9 of ABCC5, 43% in G8 of WDTC1, 33% in A8 of ROCK1, 30% in T8 of OR51E2, and 28% in A8 of TCEB3. Immunohistochemical staining of GLYR1 revealed defective protein expression in tumors carrying biallelic mutations, supporting a loss of function hypothesis. This is a large scale, unbiased effort to identify genes that when mutated are likely to contribute to MSI CRC development.</p

Germline CDKN1B/p27(Kip1) mutation in multiple endocrine neoplasia

Context: Germline mutations in the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome, but in up to 20-25% of clinical MEN1 cases, no MEN1 mutations can be found. Recently, a germline mutation in the CDKN1B gene, encoding p27(Kip1), was reported in one suspected MEN1 family with two acromegalic patients. Objective: Our objective was to evaluate the role of CDKN1B/p27(Kip1) in human tumor predisposition in patients clinically suspected of MEN1 but testing negative for MEN1 germline mutation as well as in familial and sporadic acromegaly/pituitary adenoma patients. Design: Genomic DNA was analyzed for germline mutations in the CDKN1B/p27(Kip1) gene by PCR amplification and direct sequencing. Setting: The study was conducted at nonprofit academic research and medical centers. Patients: Thirty-six Dutch and one German suspected MEN1 patient, who previously tested negative for germline MEN1 gene mutations, were analyzed. In addition, 19 familial and 50 sporadic acromegaly/pituitary adenoma patients from Europe and the United States were included in the study. Main Outcome Measures: We analyzed germline CDKN1B/p27(Kip1) mutations in individuals with pituitary adenoma and MEN1-like features. Results: A heterozygous 19-bp duplication (c.59_77dup19) leading to a truncated protein product was identified in one Dutch patient with suspected MEN1 phenotype, pituitary adenoma, carcinoid tumor, and hyperparathyroidism (one of 36, 2.8%). No mutations were detected in either familial or sporadic acromegaly/pituitary adenoma patients. Conclusions: Our results support the previous finding that germline CDKN1B/p27(Kip1) mutations predispose to a human MEN1-like condition. However, such mutations appear uncommon in suspected MEN1 cases and rare or nonexistent in familial or sporadic acromegaly/pituitary adenoma patients