MOLECULAR GENETICS OF ADRENOCORTICAL TUMORS AND RELATED DISORDERS
, Head, Section on Endocrinology and Genetics
Maria Nesterova, PhD, Staff Scientist
Sosipatros Boikos, MD, Visiting Fellow
Christopher Giatzakis, PhD, Visiting Fellow
Anelia Horvath, PhD, Visiting Fellow
Michael Muchow, PhD, Postdoctoral Fellow
Andrew Bauer, MD, Guest Researcher1
Audrey Robinson-White, PhD, Contract Researcher
Maya Lodish, MD, Clinical Associate
Somya Verma, MD, Clinical Associate
Linda Kotz, RN, Clinical Assistant, Nursing Coordinator
Elizabeth Levine, BS, Student Fellow
Yianna Patronas, BA, MS, Student Fellow
Kit-Man Tsang, BS, Student Fellow
Alfredo Gueron, MD, Special Volunteer
Hui-Pin Hsiao, MD, Special Volunteer2
Virginia Kamvissi, MD, Special Volunteer
Maria Lopez-Iglesias, MD, Special Volunteer
Carney complex genetics
We have collected families with CNC and related syndromes from a number of collaborating institutions worldwide. Through genetic linkage analysis, we identified loci harboring genes for CNC—a genetically heterogeneous condition—on chromosomes 2 (2p16) and 17 (17q22-24); we are currently investigating other possible loci for the condition. With the application of state-of-the-art molecular cytogenetic techniques, we are investigating the participation of these genomic loci in the expression of CNC and, for the cloning of the CNC-associated sequences from this region, have constructed a comprehensive genetic and physical map of the 2p16 chromosomal region. Studies in cultured primary tumor cell lines (established from our patients) identified a region of genomic amplification in CNC tumors in the center of the map. The PRKAR1A gene on 17q22-24, the gene responsible for CNC in most cases of the disease, appears to undergo loss of heterozygosity in at least some CNC tumors. PRKAR1A is also the main regulatory subunit of protein kinase A (PKA), a central signaling pathway for many cellular functions and hormonal responses. We are extending our studies to more patients with CNC for genotype-phenotype correlations, which will likely shed light on the complex biochemical and molecular pathways regulated by PRKAR1A and PKA. We expect to identify new genes through ongoing genome-wide searches for patients and families that do not carry PRKAR1A mutations.
Boikos SA, Stratakis CA. Carney complex: pathology and molecular genetics. Neuroendocrinology 2006;83:189-99.
Groussin L, Horvath A, Jullian E, Boikos S, Rene-Corail F, Lefebvre H, Cephise-Velayoudom FL, Vantyghem MC, Chanson P, Conte-Devolx B, Lucas M, Gentil A, Malchoff CD, Tissier F, Carney JA, Bertagna X, Stratakis CA, Bertherat J. A PRKAR1A mutation associated with primary pigmented nodular adrenocortical disease in 12 kindreds. J Clin Endocrinol Metab 2006;91:1943-9.
Stratakis CA. Adrenocortical tumors, primary pigmented adrenocortical disease (PPNAD)/Carney complex, and other bilateral hyperplasias: the NIH studies. Horm Metab Res 2007;39:467-73.
Toydemir RM, Chen H, Proud VK, Martin R, van Bokhoven H, Hamel BC, Tuerlings JH, Stratakis CA, Jorde LB, Bamshad MJ. Trismus-pseudocamptodactyly syndrome is caused by recurrent mutation of MYH8. Am J Med Genet A 2006;140:2387-93.
Wieacker P, Stratakis CA, Horvath A, Klose S, Nickel I, Muschke P. Male infertility as a component of Carney complex. Andrologia 2007;39:196-7.
PRKAR1A, protein kinase A activity, and endocrine and other tumor development
We are investigating the functional and genetic consequences of PRKAR1A mutations in cell lines established from CNC patients and their tumors. We measure both cAMP and PKA activity in the cell lines, along with expression of the other subunits of the PKA tetramer. In addition, to explore more about PRKAR1A’s role as a general tumor suppressor, we are seeking mutations of the gene in sporadic endocrine and non-endocrine tumors (thyroid adenomas and carcinomas, adrenocortical adenomas and carcinomas, ovarian carcinomas, melanomas and other benign and malignant pigmented lesions, and myxomas in the heart and other sites); investigators within the NIH and around the world are providing needed specimens on a collaborative basis.
Horvath A, Mathyakina L, Vong Q, Baxendale V, Pang AL, Chan WY, Stratakis CA. Serial analysis of gene expression in adrenocortical hyperplasia caused by a germline PRKAR1A mutation. J Clin Endocrinol Metab 2006;91:584-96.
Nesterova MV, Johnson N, Cheadle C, Bates SE, Mani S, Stratakis CA, Kahn I, Gupta RK, Cho-Chung YS. Autoantibody cancer biomarker: extracellular protein kinase A. Cancer Res 2006;66:8971-4.
Robinson-White A, Meoli E, Stergiopoulos S, Horvath A, Boikos S, Bossis I, Stratakis CA. PRKAR1A mutations and protein kinase A interactions with other signaling pathways in the adrenal cortex. J Clin Endocrinol Metab 2006;91:2380-8.
Shi Z, Henwood MJ, Bannerman P, Batista D, Horvath A, Guttenberg M, Stratakis CA, Grimberg A. Primary pigmented nodular adrenocortical disease reveals insulin-like growth factor binding protein-2 regulation by protein kinase A. Growth Horm IGF Res 2007;17:113-21.
Zembowicz A, Knoepp SM, Bei T, Stergiopoulos S, Eng C, Mihm MC, Stratakis CA. Loss of expression of protein kinase A regulatory subunit 1a in pigmented epithelioid melanocytoma but not in melanoma or other melanocytic lesions. Am J Surg Pathol 2007;31:1764-75.
Prkar1a+/− and antisense (AS) Prkar1a transgenic animal models
Since the discovery of PRKAR1A’s involvement in CNC, Lawrence Kirschner, while a member of our laboratory and in collaboration with Heiner Westphal, developed a Prkar1a KO floxed by a lox-P system to generate first a novel Prkar1a+/− and then knockouts of the Prkar1a gene in a tissue-specific manner after crossing this new mouse model with mice that express the cre protein in, respectively, the adrenal cortex, anterior lobe of the pituitary, and thyroid gland. The heterozygote mouse developed several tumors reminiscent of the human disease. Ongoing crosses with mice such as the transgenic GHRH-expressing mouse attempted to identify tissue-specific effects (in the case of the GHRH-expressing mouse, the pituitary) or specific signaling events (such as involvement of the p53 and Rb proteins in prkara1a-related tumorigenesis). In addition to the model described above, we created a transgenic mouse carrying an anti-sense transgene for exon 2 of the mouse Prkar1a gene (X2AS) under the control of a regulatable promoter. Similar to human CNC tumors, tissues from mice with the X2AS transgene showed higher cAMP-stimulated kinase activity. The mice had several CNC-compatible histologic and clinical changes, including obesity attributed to subclinical Cushing syndrome.
Kirschner LS, Kusewitt DF, Matyakhina L, Towns WH, Carney JA, Westphal H, Stratakis CA. A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissue. Cancer Res 2005;65:4506-14.
PRKAR1A, the cell cycle, chromosomal stability, mitogen-activated protein kinases (MAPK), and other signaling pathways
We aim to identify PRKAR1A-interacting mitogenic and other growth-signaling pathways in cell lines expressing PRKAR1A constructs and/or mutations. Several genes that regulate PKA function and increase cAMP-dependent proliferation and related signals may be altered in the process of endocrine tumorigenesis initiated by a mutant PRKAR1A, a gene with important functions in both the cell cycle and chromosomal stability. Recently, we found an interaction with the mTOR pathway in both human and mouse cells with altered PKA function.
Mavrakis M, Lippincott-Schwartz J, Stratakis CA, Bossis I. Depletion of type IA regulatory subunit (RI) of protein kinase A (PKA) in mammalian cells and tissues activates mTOR and causes autophagic deficiency. Hum Mol Genet 2006;15:2962-71.
Mavrakis M, Lippincott-Schwartz J, Stratakis CA, Bossis I. mTOR kinase and the regulatory subunit of protein kinase A (PRKAR1A) spatially and functionally interact during autophagosome maturation. Autophagy 2007;3:151-3.
Robinson-White AJ, Leitner WW, Aleem E, Kaldis P, Bossis I, Stratakis CA. PRKAR1A inactivation leads to increased proliferation and decreased apoptosis in human B lymphocytes. Cancer Res 2006;66:10603-12.
Phosphodiesterase (PDE) genes in endocrine and other tumors
In patients without CNC or PRKAR1A mutations but with bilateral adrenal tumors such as those seen in CNC, we found inactivating mutations of the PDE11A gene (Figure 5.9), which regulates PKA in the normal physiologic state. Phosphodiesterase 11A is a member of a 22-gene family of proteins that break down cyclic nucleotides controlling PKA. PDE11A appears to act as a tumor suppressor; thus, when its action is abolished, tumors develop, as observed in pediatric and adult patients with bilateral adrenal tumors. This discovery provides the first evidence of a mutated PDE in a genetic disorder predisposing to tumors. Recent data indicate that PDE11A sequence changes may be present in the general population. The finding that genetic alterations of such a major biochemical pathway may be associated with tumors in humans raises the reasonable hope that drugs that modify PKA and/or PDE activity will eventually be developed for use in both CNC and general patients with other, non-genetic adrenal and perhaps other endocrine tumors.
Figure 5.9
Loss of the PRKAR1A gene locus in adrenal tumors from patients with massive macronodular adrenocortical disease (MMAD). Interphase FISH with BACs from the 2p16 (A, B) and 17q22-24 (C) regions in AIMAH tissues. (A) Dual color FISH with BACs 400-P-14 (R) and 514-O-11 (G) from the 2p16 region shows allelic losses of both BACs in adrenal tissue from patient 4. (B) Adrenal tissue cells from patient 6 after hybridization with BAC 400-P-14 and α-satellite probe specific for chromosome 2 show an allelic deletion of BAC400-p-14 (one G signal). The presence of one R signal in two of three cells suggests that the whole chromosome 2 loss is frequent. (C) FISH with BAC321G-8–containing PRKARIA shows an allelic loss of the 17q22-24 region in adrenal tissue cells of patient 2.
Horvath A, Boikos S, Giatzakis C, Robinson-White A, Groussin L, Griffin KJ, Stein E, Levine E, Delimpasi G, Hsiao HP, Keil M, Heyerdahl S, Matyakhina L, Libe R, Fratticci A, Kirschner LS, Cramer K, Gaillard RC, Bertagna X, Carney JA, Bertherat J, Bossis I, Stratakis CA. A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with adrenocortical hyperplasia. Nat Genet 2006;38:794-800.
Horvath A, Giatzakis C, Robinson-White A, Boikos S, Levine E, Griffin K, Stein E, Kamvissi V, Soni P, Bossis I, de Herder W, Carney JA, Bertherat J, Gregersen PK, Remmers EF, Stratakis CA. Adrenal hyperplasia and adenomas are associated with inhibition of phosphodiesterase 11A in carriers of PDE11A sequence variants that are frequent in the population. Cancer Res 2006;66:11571-5.
Genetic investigations on other adrenocortical diseases and tumors
Our project aims (1) to use general and pathway-specific microarrays on a variety of adrenocortical tumors, including single adenomas and massive macronodular adrenocortical disease (MMAD), to identify genes with important functions in adrenal oncogenetics; (2) to examine specific candidate genes for their roles in adrenocortical tumors and development; and (3) to identify additional genes with a role in inherited adrenocortical and related diseases, such as Allgrove syndrome.
Bourdeau I, Matyakhina L, Stergiopoulos SG, Sandrini F, Boikos S, Stratakis CA. 17q22-24 chromosomal losses and alterations of protein kinase A (PKA) subunits expression and activity in ACTH-independent macronodular adrenal hyperplasia (AIMAH). J Clin Endocrinol Metab 2006;91:3626-32.
Keegan CE, Hutz JE, Krause AS, Koehler K, Metherell LA, Boikos S, Stergiopoulos S, Clark AJ, Stratakis CA, Huebner A, Hammer GD. Novel polymorphisms and lack of mutations in the ACD gene in patients with ACTH resistance syndromes. Clin Endocrinol (Oxford) 2007;67:168-74.
Genetic investigations on pituitary tumors, other endocrine neoplasias, and related syndromes
Additional work in our laboratory—conducted in collaboration with investigators at the NIH and elsewhere—focuses on elucidating the genetics of CNC- and adrenal-related endocrine tumors, including childhood adrenocortical cancer, testicular tumors, and thyroid and pituitary masses related or unrelated to PRKAR1A mutations. As part of this work, we have described novel genetic abnormalities in thyroid and other endocrine tumors and are identifying the genetic defects in patients with related syndromes (the lentigenoses, i.e., Peutz-Jeghers syndrome and others, the Carney Triad).
Bilodeau S, Vallette-Kasic S, Gauthier Y, Figarella-Branger D, Brue T, Berthelet F, Lacroix A, Batista D, Stratakis C, Hanson J, Meij B, Drouin J. Role of Brg1 and HDAC2 in GR trans-repression of the pituitary POMC gene and misexpression in Cushing disease. Genes Dev 2006;20:2871-86.
Bowden SA, Sotos JF, Stratakis CA, Weil RJ. Successful treatment of an invasive growth hormone-secreting pituitary macroadenoma in an 8-year-old boy. J Pediatr Endocrinol Metab 2007;20:643-7.
Nandagopal R, Vortmeyer A, Oldfield EH, Keil MF, Stratakis CA. Cushing syndrome due to a pituitary corticotropinoma in a child with tuberous sclerosis: an association or a coincidence? Clin Endocrinol (Oxford) 2007;67:639-41.
Genetic investigations on other endocrine neoplasias and related syndromes; hereditary paragangliomas and related conditions
Carried out collaboratively with other investigators at the NIH and elsewhere and with an international consortium organized by our laboratory, additional work in our laboratory concerns the genetics of a rare syndrome that predisposes to adrenal and other tumors—the Carney Triad—and related conditions (Figure 5.10). In the course of this work, we identified a new syndrome known as the paraganglioma and gastrointestinal stromal tumor syndrome; we found mutations in the genes encoding succinate dehydrogenase (SDH) subunits B, C, and D (Figure 5.11).
Figure 5.10
Typical tumors in patients with the paraganglioma and gastrointestinal stromal tumor (GIST) syndrome. (a) Several GISTs protrude from the anterior wall of stomach of a patient CTRS35; two show surface bosselation. (b) Hematoxylin and eosin staining of one of the stomach GISTs from the CTRS35 patient and staining with (c) cKIT, (d) CD-34, (e) actin, (f) desmin, and (g) vimentin.
Figure 5.11
Families with the dyad of "paraganglioma and gastric stromal sarcoma"; the identified mutations in succinate dehydrogenase subunit genes (SDHB, SDHC, and SDHD) (panels a, b, d, e, g, and j) and associated tumor alterations. (e) Mitochondrial respiratory chain function studies in the gastrointestinal stromal tumor (GIST) of a patient. (f) More than 75 percent of the cells from the CTRS8 patient’s GIST showed only one SDHC signal (bright spot); in this panel, four of the five cells show one signal of the 1q-located SHDC gene.
Matyakhina L, Bei TA, McWhinney SR, Pasini B, Cameron S, Gunawan B, Stergiopoulos SG, Boikos S, Muchow M, Dutra A, Pak E, Campo E, Cid MC, Gomez F, Gaillard RC, Assie G, Fuzesi L, Baysal BE, Eng C, Carney JA , Stratakis CA. Genetics of Carney triad: recurrent losses at chromosome 1 but lack of germline mutations in genes associated with paragangliomas and gastrointestinal stromal tumors. J Clin Endocrinol Metab 2007, in press.
McWhinney SR, Pasini B, Stratakis CA (from the Carney Triad & Carney-Stratakis Dyad/Syndrome Consortium). Mutations of the genes coding for the succinate dehydrogenase subunit genes in familial gastrointestinal tumors. N Engl J Med 2007;357:1054-6.
Pasini B, Matyakhina L, Bei T, Muchow M, Boikos S, Ferrando B, Carney JA, Stratakis CA. Multiple gastrointestinal stromal tumors caused by platelet-derived growth factor receptor alpha gene mutations: a case associated with a germline V561D defect. J Clin Endocrinol Metab 2007;92:3728-32.
Pasini B, McWhinney SR, Bei T, Matyakhina L, Stergiopoulos S, Muchow M, Boikos SA, Ferrando B, Pacak K, Assie G, Baudin E, Chompret A, Ellison JW, Briere JJ, Rustin P, Gimenez-Roqueplo AP, Eng C, Carney JA, CA Stratakis. Clinical and molecular genetics of patients with the Carney-Stratakis syndrome and germline mutations of the genes coding for the succinate dehydrogenase subunits SDHB, SDHC, and SDHD. Eur J Hum Genet 2007 [E-pub ahead of print].
Perry CG, Young WF Jr, McWhinney SR, Bei T, Stergiopoulos S, Knudson RA, Ketterling RP, Eng C, Stratakis CA, Carney JA. Functioning paraganglioma and gastrointestinal stromal tumor of the jejunum in three women: syndrome or coincidence. Am J Surg Pathol 2006;30:42-9.
Clinical investigations in the diagnosis and treatment of adrenal and pituitary tumors
Patients with adrenal tumors and other types of Cushing syndrome (and, occasionally, other pituitary tumors) come to the NIH Clinical Center for diagnosis and treatment. Ongoing studies are investigating (1) the prevalence of ectopic hormone receptor expression in adrenal adenomas and in MMAD; (2) the diagnostic use of high-sensitivity magnetic resonance imaging for the earlier detection of pituitary tumors; and (3) the diagnosis, management, and postoperative care of children with Cushing disease and other pituitary tumors.
Batista D, Gennari M, Riar J, Chang R, Keil MF, Oldfield EH, Stratakis CA. An assessment of petrosal sinus sampling for localization of pituitary microadenomas in children with Cushing disease. J Clin Endocrinol Metab 2006;91:221-4.
Batista DL, Riar J, Keil M, Stratakis CA. Diagnostic tests for children referred for the investigation of Cushing syndrome. Pediatrics 2007;120:e575-86.
Batista DL, Zhang X, Gejman R, Ansell PJ, Zhou Y, Johnson SA, Swearingen B, Hedley-Whyte ET, Stratakis CA, Klibanski A. The effects of SOM230 on cell proliferation and ACTH secretion in human corticotroph pituitary adenomas. J Clin Endocrinol Metab 2006;91:4482-8.
Hsiao HP, Iglesias ML, Keil MF, Boikos S, Robinson-White A, Stratakis CA. Differences in cortisol levels and body mass index between East Asians and Caucasians with Cushing syndrome: an “East Asian” phenotype for Cushing syndrome. Clin Endocrinol (Oxford) 2007;66:753-55.
Stratakis CA. Cortisol and growth hormone: clinical implications of a complex, dynamic relationship. Pediatr Endocrinol Rev 2006;2:333-8.
Clinical and molecular investigations of other pediatric genetic syndromes
Additional collaborative work in our group investigates pediatric genetic syndromes that are seen in our clinics and wards.
Giri N, Batista DL, Alter BP, Stratakis CA. Endocrine abnormalities in patients with Fanconi anemia. J Clin Endocrinol Metab 2007;92:2624-31.
Mai PL, Korde L, Kramer J, Peters J, Mueller CM, Pfeiffer S, Stratakis CA, Pinto PA, Bratslavsky G, Merino M, Choyke P, Linehan WM, Greene MH. A possible new syndrome with growth-hormone secreting pituitary adenoma, colonic polyposis, lipomatosis, lentigines and renal carcinoma in association with familial testicular germ cell malignancy: a case report. J Med Case Reports (BMC) 2007;1:9-14.
Perl S, Kotz L, Keil M, Patronas NJ, Stratakis CA. Calcified adrenals associated with perinatal adrenal hemorrhage and adrenal insufficiency. J Clin Endocrinol Metab 2007;92:754.
1 Director, Pediatric Endocrinology Program, Walter Reed Army Medical Center, Bethesda, MD
2 former Visiting Scientist
3 Kurt Griffin, MD, PhD, former Staff Fellow, now at University of Arizona
COLLABORATOR
Dalia Batista, MD, Massachusetts General Hospital, Harvard University, Boston, MA
Thalia Bei, PhD, BIOGENOMICA Research Institute, Athens, Greece
Jérôme Bertherat, MD, PhD, Hôpital Cochin, Paris, France
Stephan Bornstein, MD, PhD, Universität Dresden, Dresden, Germany
Isabelle Bourdeau, MD, Université de Montreal, Canada
Brian Brooks, MD, PhD, Ophthalmic Genetics and Clinical Services Branch, NEI, Bethesda, MD
J. Aidan Carney, MD, PhD, Mayo Clinic, Rochester, MN
Wai-Yee Chan, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Yoon S. Cho-Chung, MD, PhD, Basic Research Laboratory, NCI, Bethesda, MD
Adrian Clark, MD, PhD, St. Bartholomew’s Hospital, London, UK
Nickolas Courkoutsakis, MD, PhD, University of Thrace, Alexandroupolis, Greece
Jacques Drouin, PhD, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Canada
Adda Grimberg, MD, Children’s Hospital of Philadelphia, Philadelphia, PA
Gary Hammer, MD, PhD, University of Michigan, Ann Arbor, MI
Friedhelm Hildebrandt, MD, University of Michigan, Ann Arbor, MI
Peter Hornsby, PhD, University of Texas Health Science Center, San Antonio, TX
Meg Keil, RN, PNP, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Lawrence Kirschner, MD, PhD, James Cancer Hospital, Ohio State University, Columbus, OH
Anne Klibanski, MD, Massachusetts General Hospital, Harvard University, Boston, MA
Andre Lacroix, MD, PhD, Centre Hospitalier de l’Université de Montréal, Montreal, Canada
Stephen Libutti, MD, Center for Cancer Research, NCI, Bethesda, MD
Jennifer Lippincott-Schwartz, PhD, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
Stephen Marx, PhD, Surgery Branch, NCI, Bethesda, MD
Ludmila Matyakhina, PhD, Medical Genetics Branch, NHGRI, Bethesda, MD
Nickolas Patronas, MD, Diagnostic Radiology, NIH Clinical Center, Bethesda, MD
Margarita Raygada, PhD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Owen M. Rennert, MD, Program in Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Matthew Ringel, MD, PhD, Ohio State University, Columbus, OH
Michael Stowasser, MD, University of Queensland, Brisbane, Australia
David Torpy, MD, University of Queensland, Brisbane, Australia
Antonis Voutetakis, MD, Gene Therapy and Therapeutics Branch, NIDCR, Bethesda, MD
Heiner Westphal, MD, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
For further information, contact stratakc@cc1.nichd.nih.gov.
