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A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with adrenocortical hyperplasia

Abstract

Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signaling has been associated with PRKAR1A or GNAS mutations and leads to adrenocortical tumors and Cushing syndrome1,2,3,4,5,6,7. We investigated the genetic source of Cushing syndrome in individuals with adrenocortical hyperplasia that was not caused by known defects. We performed genome-wide SNP genotyping, including the adrenocortical tumor DNA. The region with the highest probability to harbor a susceptibility gene by loss of heterozygosity (LOH) and other analyses was 2q31–2q35. We identified mutations disrupting the expression of the PDE11A isoform-4 gene (PDE11A) in three kindreds. Tumor tissues showed 2q31–2q35 LOH, decreased protein expression and high cyclic nucleotide levels and cAMP-responsive element binding protein (CREB) phosphorylation. PDE11A codes for a dual-specificity PDE that is expressed in adrenal cortex and is partially inhibited by tadalafil and other PDE inhibitors8,9; its germline inactivation is associated with adrenocortical hyperplasia, suggesting another means by which dysregulation of cAMP signaling causes endocrine tumors.

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Figure 1: Micronodular BAH, identification of the 2q31–2q35 locus by LOH studies and PDE11A gene mapping and allelic losses.
Figure 2: The chromosome 2q PDE11A locus, PDE11A4 expression alterations in patients with adrenocortical hyperplasia, and mutations in the PDE11A gene in four patients with the disease.
Figure 3: Immunohistochemistry of human adrenal glands using a polyclonal antibody to PDE11A reported in ref. 11.
Figure 4: Effects of PDE11A mutations in cyclic nucleotide levels and CREB phosphorylation.

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Acknowledgements

This work is dedicated to our patients and their families. It was supported by US NIH intramural project Z01-HD-000642-04 to C.A.S. and, in part, by Groupement d'Intérêt Scientifique-Institut National de la Santé et de la Recherche Médicale Institut des Maladies Rares and the Plan Hospitalier de Recherche Clinique (AOM 02068) to the Comete Network. We thank S. Libutti and R. Alexander (National Cancer Institute (NCI), NIH) for expert surgical care on the individuals described in this study. We thank the nursing and other support staff of NICHD, NIH on the former 8W and 9W, and current 1NW and 5NW wards of the National Institutes of Health Warren Grant Magnuson Clinical Center for their support of our research studies and their help in the management of patients with adrenal tumors. We also thank D. Gunther (University of Washington, Seattle) and W.W. de Herder (Department of Internal Medicine, Endocrinology, Erasmus Medical Center, Rotterdam, the Netherlands) and the many other clinicians who have sent us samples from their patients. DNA samples from France were screened in that country for PRKAR1A mutations by E. Clauser, Unité d'Oncogénétique, CHU Cochin, Paris, and by E. Jullian, Institut Cochin, INSERM U567, Paris, to whom we are grateful. We thank C. Wayman (Discovery Biology, Pfizer Global Research and Development, Sandwich, Kent, UK) and J. Beavo (Department of Pharmacology, University of Washington, Seattle) for insightful advice in the field of PDE11A and their collaboration on the Pde11a−/− mouse. We thank I. Aksentijevich and E. Remmers (National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH), B. Brooks (National Eye Institute, NIH) and F. Porter (NICHD, NIH) for providing us with control DNA samples; K. Calis and F. Pucino (Pharmacy Department, NIH Clinical Center) for checking the PDE inhibitors toxicity database; and M. Abu-Asab and M. Tsokos (Laboratory of Pathology, NCI, NIH) for expert assistance with electron miscroscopy of adrenocortical specimens. We also thank V. Manganiello (National Heart, Lung and Blood Institute, NIH) and A. Spiegel (National Institute of Diabetes and Digestive and Kidney Diseases, NIH) for discussions on phosphodiesterases and cAMP signaling. We thank W.-Y. Chan's laboratory and staff (NICHD, NIH) for accommodating our increased sequencing needs and P. Soni for assisting with sequencing analysis. Finally, we thank C.A. Bondy and O.M. Rennert (NICHD, NIH) for editing our manuscript and for their continuing support of our studies.

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Authors and Affiliations

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Contributions

C.A.S.: overall design and planning of the project, clinical evaluation of patients, analysis of the genome-wide genotyping, selection of PDE11A4 as a candidate gene, overall supervision and organization of experiments, presentation of results, design of figures and writing the manuscript. C.A.S., J.A.C. and J.B. are principal investigators in the International Carney Complex Consortium. C.A.S. is a Senior Investigator at NICHD, which provided most of the funding for this project under an intramural NIH grant to C.A.S. A.H.: participation in specimen collection, database construction, analysis of the genome-wide genotyping, selection of candidate genes, design and optimization of the amplification and sequencing protocols, sequencing analysis, identification of pathogenic and polymorphic genetic variations, participation in organization of working processes, participation in the presentation of the results and editing of the paper. S.B.: participation in specimen collection, database construction sequencing analysis, FISH analysis; PDE11A4 protein expression evaluation (protein blot analysis, immunohistochemistry). C.G.: participation in the design of the project, expression of the PDE11A isoforms in different tissues, in vitro functional analysis of the effect of the pathogenic genetic variants, participation in the analysis of the cAMP and cGMP activity data, analysis for LOH (SSCP and microsatellite analysis), participation in the presentation of the results and editing of the paper. A.R.-W.: in vitro cAMP and cGMP assays and production of other functional data. L.G.: sequencing analysis and specimen collection; co-investigator in the consortium. K.J.G.: PDE11A4 expression analysis, mouse tissue analysis, editing the paper. E.S.: PDE11A4 expression analysis and in vitro assays. E.L., G.D. and H.P.H.: sequencing analysis. M.K.: clinical specimen collection and evaluation of patients. S.H: PDE11A4 expression analysis (protein blots, cDNA, mRNA). L.M.: FISH analysis of adrenocortical tumor specimens. R.L. and A.F.: sequencing analysis. L.S.K.: clinical specimen collection, mouse data analysis, editing the manuscript. K.C.: genome-wide genotyping, statistical evaluation of the data. R.C.G.: clinical specimen collection, patient evaluation. X.B.: clinical specimen collection, patient evaluation; co-investigator in the consortium. J.A.C.: design and planning of the project, clinical specimen collection, patient evaluation, review of all histopathology, editing of the manuscript. J.B.: design and planning of the project,, clinical specimen collection, patient evaluation, editing of the manuscript. I.B.: in vitro functional analysis of the effect of the pathogenic genetic variants, analysis of the cAMP and cGMP activity data, analysis for LOH (SSCP and microsatellite analysis), PDE11A protein expression evaluation (protein blot analysis), participation in the organization of most experiments and the presentation of results, editing of the manuscript.

Corresponding author

Correspondence to Constantine A Stratakis.

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Competing interests

C.A.G. has filed a patent application (no. 601761,446, filed 24 January 2006) in which he is named as the primary inventor of the PDE11A gene in endocrine tumors.

K.C. is co-owner of Sapio Sciences, Inc., the company that analyzed the genotyping data in this project.

Supplementary information

Supplementary Fig. 1

De novo mutation and clinical data from family CAR36. (PDF 87 kb)

Supplementary Fig. 2

LOH analysis. (PDF 63 kb)

Supplementary Fig. 3

Expression of other PDEs in the adrenal cortex. (PDF 68 kb)

Supplementary Table 1

SNPs with significant LOH from chromosome 2q. (PDF 29 kb)

Supplementary Table 2

All primers. (PDF 31 kb)

Supplementary Table 3

All sequence changes and number of controls. (PDF 16 kb)

Supplementary Table 4

Novel benign PDE11A4 polymorphisms. (PDF 96 kb)

Supplementary Methods (PDF 1219 kb)

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Horvath, A., Boikos, S., Giatzakis, C. et al. A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with adrenocortical hyperplasia. Nat Genet 38, 794–800 (2006). https://doi.org/10.1038/ng1809

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