Abstract 2239: Macronodular adrenal hyperplasia due to mutations in ARMC5: New mutations in humans and modeling in zebrafish
Authors: Faucz Fabio R. , Zilbermint Mihail , Assié Guillaume , Lodish Maya B. , Szarek Eva , Trivellin Giampaolo , Berthon Annabel , Sinaii Ninet , Libé Rossella , Espiard Stéphanie , Drougat Ludivine , Ragazzon Bruno , Feldman Benjamin , Bertherat Jerome , Stratakis Constantine A. 
Published: May 2015

Abstract
Inactivating germline mutations of the probable tumor suppressor gene Armadillo Repeat Containing 5 (ARMC5) have recently been identified (N Engl J Med. 2013;369:2105-14) as a genetic cause of ACTH-independent macronodular adrenal hyperplasia (AIMAH) . We assessed for the presence of ARMC5 mutations in a cohort of patients with AIMAH. Blood DNA from 34 AIMAH patients was genotyped using Sanger sequencing. Germline ARMC5 mutations were found in 15 out of 34 patients (44.12%). In silico analysis of the mutations indicated that 7 (20.6%) predicted major implications for gene expression. All patients carrying ARMC5 mutation, that were predicted to be pathogenic, had clinical Cushing's Syndrome (7/7, 100%) compared to 14/27 (52%) of those without or with mutations predicted as benign (p=0.029). ARMC5 mutations are implicated in clinically severe Cushing's syndrome associated with AIMAH. Knowledge of a patient's ARMC5 status and the pathway related with this gene has important clinical implications for the diagnosis of some diseases, including Cushing's syndrome and for genetic counseling of patients and their families. To further investigate the function of ARMC5 we are investigating its function using the zebrafish model. We knocked down the zebrafish ortholog, armc5, with an antisense morpholino oligonucleotide (MO). Zebrafish embryos injected with 15 pg of our armc5 MO display phenotypes at mid- through late stages of embryogenesis; these are: hyperactive spontaneous movement at 36 hours post fertilization (hpf) and irregularities in the notochord and a “curly down” body curvature at 48 hpf. Whole mount in situ hybridization (WISH) of MO-injected embryos at an earlier stage (10 hpf/ late gastrula) reveals reductions in a marker of paraxial mesoderm and a foreshortening of labeled chordamesoderm. Intriguingly, reduction of the same paraxial mesoderm marker causes cysts to form in the embryonic zebrafish kidney and several zebrafish models of polycystic kidney disease display a curly down body shape. RNA-Seq of Armc5 MO-injected embryos reveals enrichment in genes normally expressed in the musculature and liver among the cohort of upregulated genes. Based on these preliminary findings, we hypothesize that armc5 acts to repress one or more components involved in the differentiation of muscle and liver and possibly formation of embryonic kidney cysts. Ongoing studies will examine embryonic kidney cyst incidence in armc5 MO-injected embryos and validate selected RNA-seq findings by WISH and by assessment of the armc5 overexpression phenotype in zebrafish.
Citation Format: Fabio R. Faucz, Mihail Zilbermint, Guillaume Assié, Maya B. Lodish, Eva Szarek, Giampaolo Trivellin, Annabel Berthon, Ninet Sinaii, Rossella Libé, Stéphanie Espiard, Ludivine Drougat, Bruno Ragazzon, Benjamin Feldman, Jerome Bertherat, Constantine A. Stratakis. Macronodular adrenal hyperplasia due to mutations in ARMC5: New mutations in humans and modeling in zebrafish. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2239. doi:10.1158/1538-7445.AM2014-2239