ARMC5 Variants in PRKAR1A-Mutated Patients Modify Cortisol Levels and Cushing’S Syndrome

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Endocrine-Related A G Maria et al. ARMC5 variants in PRKAR1A 27:9 509–517 Cancer mutated patients RESEARCH ARMC5 variants in PRKAR1A-mutated patients modify cortisol levels and Cushing’s syndrome

Andrea Gutierrez Maria1, Christina Tatsi1,2, Annabel Berthon1, Ludivine Drougat1, Nikolaos Settas1, Fady Hannah-Shmouni 1, Jerome Bertherat3, Fabio R Faucz 1 and Constantine A Stratakis1,2

1Section on Endocrinology & Genetics (SEGEN), National Institutes of Health (NIH), Bethesda, Maryland, USA 2Pediatric Endocrinology Inter-Institute Training Program, Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland, USA 3Service d’Endocrinologie, Hôpital Cochin, APHP, Institut Cochin, INSERM U1016, Université de Paris, Paris, France

Correspondence should be addressed to A G Maria: [email protected]

Abstract

Mutations in the protein kinase A (PKA) regulatory subunit type 1A (PRKAR1A) and Key Words armadillo repeat-containing 5 (ARMC5) genes cause Cushing‘s syndrome (CS) due to ff cortisol primary pigmented nodular adrenocortical disease (PPNAD) and primary bilateral ff adrenocortical hyperplasia macronodular adrenocortical hyperplasia (PBMAH), respectively. Between the two genes, ff PRKAR1A gene ARMC5 is highly polymorphic with several variants in the population, whereas PRKAR1A ff ARMC5 gene has very little, if any, non-pathogenic variation in its coding sequence. We tested the hypothesis that ARMC5 variants may affect the clinical presentation of PPNAD and CS among patients with PRKAR1A mutations. In this study, 91 patients with PPNAD due to PRKAR1A mutations were tested for abnormal cortisol secretion or CS and for ARMC5 sequence variants. Abnormal cortisol secretion was present in 71 of 74 patients with ARMC5 variants, whereas 11 of 17 patients negative for ARMC5 variants did not have hypercortisolemia. The presence of ARMC5 variants was a statistically strong predictor of CS among patients with PRKAR1A mutations (P < 0.001). Among patients with CS due to PPNAD, ARMC5 variants were associated with lower cortisol levels at baseline (P = 0.04) and after high dose dexamethasone administration (P = 0.02). The ARMC5 p.I170V variant increased ARMC5 protein accumulation in vitro and decreased viability of NCI-H295 cells (but not HEK 293T cells). PPNAD tissues with ARMC5 variants showed stronger ARMC5 protein expression than those that carried a normal ARMC5 sequence. Taken together, our results suggest that ARMC5 variants among patients with PPNAD due to PRKAR1A defects may play the role of a genetic modifier for the presence and severity of hypercortisolemia. Endocrine-Related Cancer (2020) 27, 509–517

Introduction

Cyclic adenosine monophosphate (cAMP)-dependent and RIIβ) and four C-subunits (Cα, Cβ, and Cγ, and PRKX); protein kinase or PKA mediates most cAMP-signaling in each are coded by their own genes (PRKAR1A, PRKAR1B, the adrenal cortex through its regulatory (R) and catalytic PRKAR2A, PRKAR2B, PRKACA, PRKACB, PRKACG, and (C) subunits (Almeida & Stratakis 2011, de Joussineau et al. PRKAX, respectively) (Almeida & Stratakis 2011). RIα, Cα 2012). There are four different R-subunits (RIα, RIβ, RIIα and Cβ defects have been implicated in corticotrophin

-20-0273 Endocrine-Related A G Maria et al. ARMC5 variants in PRKAR1A 27:9 510 Cancer mutated patients

(ACTH)-independent, adrenocortical Cushing‘s syndrome may also lead to CS of varying degrees of severity: PPNAD (CS) (Stratakis 2014, Espiard et al. 2018, Hannah-Shmouni can be insidious or lead to early onset, pediatric CS (Sarlis & Stratakis 2020). PRKAR1A mutations cause Carney et al. 1997, Pereira et al. 2010, Lowe et al. 2017). Like complex (CNC), a rare multiple neoplasia syndrome CTNNB1 (Tadjine et al. 2008) and PDE11A (Libé et al. 2011) inherited in an autosomal dominant manner (Kirschner defects in the past, in this study, we tested the hypothesis et al. 2000, Horvath et al. 2010), manifesting with primary that ARMC5 variants may play a role in the adrenocortical pigmented nodular adrenocortical disease (PPNAD), as expression of a PRKAR1A-inactivating mutation, causative well as (rarely) cortisol-producing adenomas (CPA) causing of PPNAD and CS. The data suggest that ARMC5 may have CS (Bertherat et al. 2003, 2009). The importance of the a role in modifying cortisol secretion in PPNAD. role of cAMP-signaling for adrenocortical function and regulation of cortisol secretion was further enhanced by the discovery of cAMP-phosphodiesterase 11 A (PDE11A) defects in association with PPNAD and PPNAD-like lesions Materials and methods and CS (Horvath et al. 2006). Clinical and DNA studies cAMP-signaling defects and abnormal PKA activity have also been implicated in another form of adrenocortical Patients were evaluated at the NIH Clinical Center CS, primary bilateral macronodular adrenocortical between 1995 and 2020 under protocol 95-CH-0059. hyperplasia (PBMAH) (Bourdeau et al. 2006, Almeida et al. Ethics approval was granted from the Institutional Review 2011, 2012). In both PPNAD and PBMAH, regardless of Boards of the Eunice Kennedy Shriver National Institute the underlying primary defect, other genes or pathways of Child Health and Human Development (NICHD) appear to play a modifier role in the clinical phenotype (until 2010) and the National Institute of Diabetes and of the affected patients and/or the onset and severity of Digestive and Kidney Diseases (NIDDK) (2010 to present), hypercortisolemia and CS. For example, somatic CTNNB1 NIH (Clinical Trial Registration no. NCT00001452). For mutations have been described in patients with PRKAR1A all patients, informed consents were obtained; assents defects that present with a CPA in addition to PPNAD were signed by minors and consents obtained from their and severe CS (Tadjine et al. 2008). Furthermore, PDE11A parents, as appropriate. variants may increase the severity of CS in PPNAD (Libé A total of 91 patients with CNC and PPNAD who were et al. 2011) and participate in the pathogenesis of other evaluated for hypercortisolemia and CS and were older adrenocortical lesions, from PBMAH to CPAs, and possibly than 20 years of age at the time of the last follow-up or had adrenocortical cancer (Libé et al. 2008, Vezzosi et al. 2012). already developed CS were included in the study. Patients Mutations of the armadillo repeat-containing 5 were initially examined for clinical features of CNC per (ARMC5) gene are the main causative genetic defect of established guidelines (Stratakis & Raygada 1993). The PBMAH (Assié et al. 2013, Faucz et al. 2014), although biochemical diagnosis of hypercortisolemia relied upon others exist too, from GNAS1 to MEN1 and GIPR (Hsiao elevations in 24-h urinary-free cortisol (UFC) above et al. 2009, Lecoq et al. 2017). In addition to PDE11A (Libé the upper cutoffs of reference intervals; loss of diurnal et al. 2008, Vezzosi et al. 2012), it has long been suspected circadian rhythm in midnight salivary cortisol; and lack that additional genes or pathways (Bourdeau et al. 2004, of suppression or a paradoxical response of serum cortisol Bimpaki et al. 2010) may modify the phenotype of CS following the overnight high dose dexamethasone among patients with PBMAH that can be very variable, suppression test, as detailed elsewhere (Stratakis & from asymptomatic to cyclical and, less frequently, severe Raygada 1993, Stratakis et al. 1999, Tirosh et al. 2016). CS (Hsiao et al. 2009). Yet, little is known about the newly ACTH-independent hypercortisolemia was ascertained identified ARMC5 gene and its possible interactions through ACTH measurement, second-tier biochemical or regulators. testing and radiographic imaging. Tumor samples were The highly polymorphic ARMC5 gene in the general obtained from patients, as previously described (Bertherat population is widely expressed in human tissues, et al. 2003, Horvath et al. 2006). particularly in the adrenal cortex (Berthon et al. 2017a). DNA was extracted from peripheral blood and fresh- Conversely, PRKAR1A is also ubiquitously expressed but, frozen tissues, as previously described (Kirschner et al. unlike ARMC5, does not have frequent coding variants 2000, Bertherat et al. 2009, Horvath et al. 2010, Libé et al. in the general population (Bertherat et al. 2009, Horvath 2011). Sequencing for PRKAR1A and ARMC5 genes was et al. 2010). Hypercortisolemia due to PRKAR1A defects obtained, as described elsewhere (Kirschner et al. 2000,

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Bertherat et al. 2009, Horvath et al. 2010, Assié et al. 2013, Analysis of protein expression in vitro Faucz et al. 2014). The coding and the flanking intronic HEK 293T and NCI-H295 cells were seeded into six-well sequences were sequenced for both genes. plates at a density of 3 × 105 and 5 × 105 cells per well, respectively. After 24 h of incubation, HEK 293T cells In silico modeling were transfected with Lipofectamine 2000 (11668030, Invitrogen) using Opti-MEM I Reduced Serum Medium Germline variants in ARMC5 were evaluated by (31985-070, Gibco) and NCI-H295 cells were transfected MutationTaster (http://www.mutationtaster.org), with Effectene Transfection Reagent (301425, Qiagen) Polymorphism Phenotyping v2 algorithm tool following manufacturer’s protocol. Five hundred (PolyPhen-2) (http://genetics.bwh.harvard.edu/pph2), nanograms of each vector were used for the transfection and SIFT (Sorting Tolerant From Intolerant) algorithm and the empty pCMV6-Entry vector was used as a (http://sift.jcvi.org) to predict the possible impact of the negative control. After 24 h of transfection, cells were amino acid substitution on the structure and function of washed with PBS and resuspended in 50 μL of ice-cold a human protein, as we have described elsewhere (Assié lysis buffer (Tris–HCl 10 mM, pH 7.5, NaCl 150 mM, et al. 2013). EDTA 1 mM, EGTA 1 mM, SDS 0.1%, Nonidet P-40 1%) containing a cocktail of protease and phosphatase inhibitors (PPC1010, Sigma-Aldrich). The collected cells DNA constructs, cell culture, and ARMC5 were incubated for 30 min on ice and centrifuged for 15 expression studies min at 4°C, 15,000 g. The total protein concentration The human ARMC5 WT (NM_001105247.1) coding of the supernatant was determined by Pierce™ BCA sequence was cloned into the pCMV6-Entry vector with Protein Assay (23227, Thermo Scientific), following the C-terminal Myc-DDK tag (RC226267, Origene, Rockville, manufacturer’s protocol. After quantification of protein MD, USA). The p.Ile170Val variant was introduced into extracts, 50 μg of total proteins of each sample were the human ARMC5 WT template using the QuikChange separated by electrophoresis in 10% polyacrylamide gel Lightning Site-directed Mutagenesis Kit (210518-5, under denaturing conditions (SDS-PAGE). Proteins were Agilent Technologies, Santa Clara, CA, USA), following transferred to a nitrocellulose membrane (1620115, Bio- the manufacturer’s protocol. The following primers were Rad) and Western Blot was performed using antibodies used for mutagenesis: against DDK diluted 1:1000 (NB600-345, Novus Biologicals, USA), and GAPDH diluted 1:2000 (SC-32233, ARMC5-Ile170Val MUT_F: GCCGTTCGGTTCTGGACG Santa Cruz Biotechnology). Fluorescent secondary CTGTCTGTCTTCATG antibodies (827-08364 IRDye 800CW Goat anti-Mouse ARMC5-Ile170Val MUT_R: CATGAAGACAGACAGCGT and 926-68073 IRDye 680RD Donkey anti-Rabbit, LiCor, CCAGAACCGAACGGC. USA) were diluted 1:20,000 and Odyssey CLx Imaging System (Licor, USA) was used to acquire the signal of the bands. Densitometric quantification was performed Cell culture using ImageJ software and a ratio between the intensity of ARMC5 and GAPDH bands was calculated. Human Embryonic Kidney 293T cells (HEK 293T) were grown in Dulbecco’s modified Eagle’s medium (DMEM, 10313, Gibco) supplemented with 10% fetal bovine Cell viability studies serum (100-106, Gemini Bio Products, USA) and 1% antibiotic (Penicillin–Streptomycin – 15140-148, Gibco). HEK 293T and NCI-H295 cells were seeded into 96-well NCI-H295 cells were grown in Dulbecco’s Modified Eagle plates at a density of 5 × 103 and 1 × 104 cells per well, Medium: Nutrient Mixture F-12 (DMEM/F-12, A4192002, respectively. Cells were transfected as described previously Gibco), supplemented with 2% fetal bovine serum for 2, 24 and 48 h. In the end of incubation time, MTT (100-106, Gemini Bio Products), 1% antibiotic (Penicillin– (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Streptomycin – 15140-148, Gibco) and 5 μg/mL insulin, salt) was added to a final concentration of 0.5 mg/mL into 5 μg/mL transferrin and 5 ng/mL sodium selenite (1% ITS the culture media and incubated for 3 h at 37°C. Media – 41400045, Thermo Fisher). Cells were incubated in a was removed and 200 μL of isopropanol containing acetic humidified atmosphere at 37°C with 5% CO2. acid 0.04 M was added into the wells to solubilize the

Immunohistochemistry (IHC) The germline variants in ARMC5 that were identified among patients with PPNAD carriers of PRKAR1A IHC was performed on serial sections from paraffin- mutations are listed in Table 1. They have all been embedded adrenal tissue from selected patients. Slides described previously and most have been predicted to be were deparaffinized in Histoclear (HS-202, National benign by all in silico analyses. Diagnostics, USA) and rehydrated through ethanol Among the 91 patients who met the eligibility criteria gradient. Epitope retrieval was done in Vector Antigen for the study (PRKAR1A mutation status confirmed and Retrieval Solution (H3300, Vector Labs, USA) at 95°C. adequate screening for hypercortisolemia or CS), there ARMC5 antibody (NBP1-94024, Novus, USA) was diluted were 74 patients who had an ARMC5 variant; among 1:50 and sections were incubated overnight at 4°C. them, 71 (96%) had CS and only 3 (4%) did not have Detection of the primary antibody was performed using CS. Among those who did not have an ARMC5 variant an anti-rabbit antibody coupled to biotin (111-065-144, (n 17), 6 (35%) had CS and 11 (65%) did not have CS. Jackson ImmunoResearch Laboratory) followed by = This clear excess of PPNAD patients with an ARMC5 streptavidin-HRP amplification (016-030-084, Jackson variant that had CS was highly statistically significant ImmunoResearch Laboratory). HRP activity was detected (P < 0.001). There were no specificARMC5 variants that with 3,3′-diaminobenzidinetetrahydrochloride (DAB) were reliably statistically overrepresented among patients (SK-4105, Vector Labs). Negative controls were not used with CS, given the small numbers and the rarity of some since the tissue itself showed positive and negative of the variants. Data are shown in Supplementary Table 1 ARMC5 staining. Representative pictures of the staining (see section on supplementary materials given at the end were taken in an inverted light microscope at 5× and 10× of this article). As expected from its population frequency objective lenses. Quantification of DAB positive staining (gnomAD database), the p.A705V variant was the most was performed using ImageJ software and optical density frequent; it was even more frequent among patients with (OD) was calculated as follows: OD = log(maximum CS, but the difference was not significant P( = 0.11). intensity/mean intensity).

Clinical data analysis for patients with CS and ARMC5 Statistics variant carriers vs non-carriers

Categorical data are presented as percentage and Collective clinical data for patients diagnosed with CS proportions were analyzed using χ2 test or Fisher exact are presented in Table 2. There were no differences in test as appropriate. Continuous data were checked for the median age of diagnosis of CS between patients with normality based on histogram distribution and the no ARMC5 variants and those with a variant (18.9 vs Shapiro–Wilk statistical test. Based on the previous results, 19.4, respectively) or in mean BMI Z-score (1.5 vs 1.4, continuous data normally distributed are presented as respectively). However, patients with ARMC5 variants mean (s.d.) and were compared between groups using had consistently lower cortisol levels than those without: Student’s t-test. Continuous data not normally distributed median midnight cortisol levels (7.7 vs 17.6 µg/dL, are presented as median (inter-quartile range: 25th–75th respectively, P = 0.06), median UFC levels adjusted for the percentile) and were compared between groups using upper limit of normal (ULN) (0.4-fold increase vs 2.34- the Wilcoxon rank-signed test. Hazard risk analysis fold increase, respectively, P = 0.01), and median cortisol for diagnosis of CS based on ARMC5 genotype status levels in response to high dose dexamethasone adjusting was performed using Cox proportional hazard and for baseline cortisol (12.2 vs 25.8 µg/dL, respectively, is presented as hazard ratio (HR, 95% CIs). Statistical P = 0.02) were lower. These differences did not depend on analysis of clinical data was performed in R. Data related the presence of particular variants when we analyzed the to functional assays are presented as the mean (s.e.m.) and data per the most frequent ARMC5 sequence alterations. P-value was obtained by two-way ANOVA followed by No difference in the prevalence of disorders of glucose Tukey’s multiple comparisons test carried out using the metabolism or elevated blood pressure were observed software GraphPadPrism 6 (GraphPad®). between patients with and without ARMC5 variants.

On hazard risk assessment analysis of diagnosis of CS based on ARMC5 genotype status, patients with ARMC5 variants tended to be diagnosed with CS earlier (Fig. 1A); however, the number of patients without ARMC5

rs number rs151069962 rs201768837 rs201280100 rs35923277 rs377221865 rs35461188 rs141923065 rs142376949 rs61732352 rs201720272 rs55800131 rs11150624 rs9926717 variants was small compared to those with a variant (HR: 1.93 (0.83–4.52), P = 0.13) and no statistical significant differences were observed. When we examined individual variants with a sufficient number of events, it appeared 4.95 2.5 2.53 4.15 3.5199 3.5499 4.67 0.4519 7.38 3.43 7.2699 0.666 11.3999 − − − −

(GERP RS) that the higher risk was identified in patients with a − Conservation single ARMC5 variant (Fig. 1B), the p.I170V (HR: 3.67 (0.84–15.96), P = 0.08). Although patients with the

c p.I170V variant were also carriers for the common p.A705V variant, when we assessed only at the p.A705V variant compared to patients with no ARMC5 variant, the Tolerated Tolerated NA Tolerated NA Tolerated Tolerated Tolerated NA NA NA Damaging NA Pathogenicity (SIFT) risk was lower (HR: 1.77 (0.74–4.28), P = 0.20). Indeed, the four patients who presented with both CS due to PPNAD and p.I170V ARMC5 variant developed severe CS at an

prediction for pathogenicity are listed the variants found. earlier age, requiring bilateral adrenalectomy. in silico Polymorphism Polymorphism Disease causing Polymorphism Disease causing Polymorphism Polymorphism Polymorphism Disease causing Disease causing Polymorphism Polymorphism Polymorphism Pathogenicity (mutation taster) In vitro expression and proliferation studies

When a flag-tagged construct bearing the p.I170V ARMC5 variant was expressed in HEK 293T and NCI-H295 cells, it led to higher expression of the protein in both cell types, especially in adrenocortical NCI-H295 cells (Fig. 2A and Benign Benign Benign Benign Benign Benign Benign Benign Benign Uncertain Benign Benign Benign Pathogenicity (varsome) B). Moreover, MTT assay were performed to determine the consequences of ARMC5 p.I170V variant on the cell Predicted as damaging in only 1 out of 11 prediction tools.

c viability of both cell lines. Our results show that, after

2.02 0.68 1.35 2.7 0.68 0.68 0.68 2.7 0.68 0.68 6.08 48 h of transfection, the viability of the HEK 293T cells 53.38 14.19 + Cushing’s b (Fig. 2C) was not affected, whereas the NCI-H295 cells

CNC overexpressing WT or mutant ARMC5 I170V variant had decreased viability compared to the empty vector; the a p.I170V ARMC5 variant decreased the viability of the 1.65 0.55 1.1 2.19 0.55 0.55 0.55 2.19 0.55 0.55 4.94 CNC 45.05 18.13 NCI-H295 cells significantly more than the WT, suggesting

a possible role of this variant in protein expression (Fig. 2D). (%) 4.84 0.31 0.83 3.64 0.03 0.0287 0.317 2.12 0.487 0.01569 6.55 MAF gnomAD 40.6 29.5 Immunohistochemistry (IHC) 74 patients with 148 alleles examined; b IHC showed that immunostaining for ARMC5 was prediction. stronger among adrenal tumor samples that carried an Protein

p.F14Y p.A110T p.R146 p.I170V p.L303 p.G323A p.Q408R p.P507L p.A547 p.L580 p.L614 p.A705V N/A ARMC5 variant. Moreover, ARMC5 expression was shown in silico to be widely distributed in samples of patients that had ARMC5 variants compared to samples of patients that did not have ARMC5 variants (Fig. 3). variants and c.41T>A c.328G>A c.438G>A c.508A>G c.909G>C c.968G>C c.1223A>G c.1520C>T c.1641G>A c.1740C>T c.1842C>G c.2114C>T c.475+58A>G c.DNA 12.3 to 6.17, with 6.17 being the most conserved; GERP RS, typical score. It quantifies position-specific constraint in terms of rejected substitutions (RS) by estimating actual −

ARMC5 Discussion variants found in germline DNA of PPNAD individuals with Cushing’s syndrome. Minor allele frequency (MAF) and The present study shows that germline variants in 91 patients with a total of 182 alleles examined;

Table 1 Intronic g.1978A>G ARMC5 a GERP, genomic evolutionary rate profiling (GERP) is a conservation score calculated by quantifying substitution deficits across multiple alignments of orthologues using the genomes 35 mammals. It ranges from number of substitutions at that site and subtracting it from the expected assuming neutrality; NA, not applicable. ARMC5 among patients with PRKAR1A defects and https://erc.bioscientifica.com © 2020 Society for Endocrinology https://doi.org/10.1530/ERC-20-0273 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/02/2021 05:03:31PM via free access Endocrine-Related A G Maria et al. ARMC5 variants in PRKAR1A 27:9 514 Cancer mutated patients

Table 2 Clinical profile of patients with Cushing‘s syndrome.

Presentation No ARMC5 variant ARMC5 variant present Overall P-valueb Age at diagnosis, yearsa 18.9 (17.5–21.5), n = 6 19.4 (13.6–34.1), n = 59 19.4 (13.6–33.5) 0.73 Serum midnight cortisol, µg/dLa 17.6 (14.4–20.9), n = 5 7.7 (4.3–14.9), n = 47 8.2 (4.7–16.1) 0.06 Serum morning cortisol, µg/dLa 20.9 (15.5–21.4), n = 5 12.1 (9.2–16.6), n = 48 12.5 (10.1–18.6) 0.04 24-h UFC corrected for ULN, fold changea 2.34 (1.8–3.3), n = 4 0.38 (0.1–0.5), n = 33 0.44 (0.15–0.91) 0.01 Serum cortisol after low dose DST, µg/dLa 26 (17.5–36.7), n = 3 10.4 (6.8–16.5), n = 24 12.1 (7.1–17.4) 0.26 Serum cortisol after high dose DST, µg/dLa 25.8 (21.4–32.2), n = 4 12.2 (7.4–16.7), n = 30 13.4 (7.5–19.3) 0.02 Weight Z-score at diagnosisa 0.8 (−1.1–1.5), n = 5 1.5 (0.6–2.0), n = 49 1.3 (0.5–2.0) 0.33 BMI Z-score at diagnosis 1.5 (1.1), n = 5 1.4 (1.0), n = 49 1.4 (1.0) 0.86 Fasting glucose, mg/dLa 89 (81.3–93.3), n = 4 85 (80–92), n = 33 87 (80–92) 0.87 Fasting insulin, IU/La 8.7, n = 1 9.1 (6.9–14.2), n = 29 9 (7–13.8) 0.86 Diagnosis of prediabetes or diabetes, n (%) Yes: 1 (50) Yes: 5 (18.5) Yes: 6 (20.7) 0.38 No: 1 (50) No: 22 (81.5) No: 23 (79.3) Diagnosis of pre-hypertension or Yes: 4 (80) Yes: 32 (66.7) Yes: 36 (68) 0.99 hypertension, n (%) No: 1 (20) No:16 (33.3) No: 17 (32)

BSA, body surface area; DST, dexamethasone suppression test; n, indicates the number of available data for each variable; UFC, urinary-free cortisol. Bold indicates statistical significance. Clinical and biochemical data of patients with Cushing‘s syndrome included in the study overall and according to ARMC5 genotype. Values represent the median (min–max). aNot normally distributed data; bComparison between patients with and without ARMC5 variant.

PPNAD may play the role of a modifier of the phenotype of hypercortisolemia and/or CS – patients with ARMC5 variants had hypercortisolemia and/or CS more frequently than those without any ARMC5 sequence variants. This is consistent with our initial hypothesis when we started this investigation. On the other hand, patients with ARMC5 variants had overall lower cortisol levels than those without (Table 2). At first glance, these two sets of data are contradictory. Yet, we know that ARMC5 in vitro decreased steroidogenesis in NCI-H295 adrenocortical cells (Assié et al. 2013, Espiard et al. 2015) and that the Armc5 haploinsufficient mouse showed decreased corticosterone secretion before it reverted to increased glucocorticoids and a PBMAH-like phenotype (Espiard et al. 2015). The function of ARMC5 is unknown, but the normal protein, when overexpressed in adrenocortical cell cultures (NCI-H295), appears to increase apoptosis (Assié et al. 2013, Espiard et al. 2015, Berthon et al. 2017a). In our experiments, normal ARMC5 decreased the viability of NCI-H295 cells (Fig. 2). Interestingly, this effect of ARMC5 appeared to be cell specific, as it was not observed in non-adrenal HEK 293T cells. In the same experiments, the p.I170V ARMC5 variant, found in four of our patients with PPNAD and severe CS, decreased the viability of the H295R cells significantly more than the WT (Fig. 2D). It is noteworthy that the p.I170V Figure 1 variant increased the presence of the ARMC5 protein in Event plot of the time of CS diagnosis. (A) Age of PPNAD patients at CS both HEK 293T and NCI-H295 cells (Fig. 2A and B) but diagnosis based on the presence of any variant in the ARMC5 gene. (B) Age of PPNAD patients at CS diagnosis based on the presence of the affected viability only in the latter. Corroborating this p.I170V ARMC5 variant compared to patients with no ARMC5 variants. data, ARMC5 expression was also increased in adrenal

Figure 2 Functional study of ARMC5 p.I170V variant. Western blot assay of ARMC5 in (A) HEK 293T cells and (B) NCI-H295 cells transfected with plasmids expressing WT or mutated p.I170V ARMC5. GAPDH was used as an internal loading control. Data are expressed as fold of change from relative expression of ARMC5/GAPDH ± s.e.m.; **P = 0.0098, ****P < 0.0001. Blots shown are representative of two independent experiments. MTT assay of (C) HEK 293T cells and (D) NCI-H295 cells after transfection with plasmids expressing WT or mutated p.I170V ARMC5. Measurements were performed 2, 24 and 48 h after transfection. Data represent values of two independent experiments ± s.e.m.; *P < 0.05, ****P < 0.0001. tissues of PPNAD patients with CS that carried the presence of ARMC5 may be undesirable under physiologic p.I170V variant compared to patients that did not carry adrenocortical function (Cavalcante et al. 2020). any ARMC5 variants (Fig. 3). The possible interaction between ARMC5 and PKA How does one reconcile these data? We would have to remains a matter of speculation too, although it is suggested speculate, given the absence of information on the exact by the data in this paper. ARMC5 may be implicated in function of ARMC5 in the adrenal cortex; we may rely the regulation of adrenocortical beta-catenin (CTNNB1), on speculation on how ARMC5 mediates tumorigenicity. a protein with which it shares a structure and early One assumption is that, under physiological conditions, developmental adrenal expression (Berthon et al. 2017a,b) ARMC5 is a negative regulator of adrenocortical and one that is involved in PRKAR1A’s tumorigenicity proliferation and glucocorticoid secretion (Berthon et al. (Tadjine et al. 2008, Almeida & Stratakis 2011). CTNNB1 2017b, Hu et al. 2017); its deficiency and/or dysfunction mutations were found in the somatic state in CPAs that may lead to compensatory proliferation and eventually developed in patients with PPNAD and severe forms of CS overgrowth and tumor formation along with excess (Tadjine et al. 2008). This was the first demonstration that steroid hormone secretion (Assié et al. 2013, Espiard other genetic events can modify PRKAR1A’s phenotypic et al. 2015, Berthon et al. 2017b). Recent data indicate effects. PDE11A variants were also shown to lead more that cullin 3 targets ARMC5 for ubiquitination and frequently to the development of CS among patients with degradation, a finding that may suggest that increased PPNAD (Libé et al. 2011).

Figure 3 ARMC5 expression in adrenal of patients with PRKAR1A mutation. (A) Immunohistochemistry staining of ARMC5 in adrenal glands of patients who carry PRKAR1A mutation, have Cushing‘s syndrome (CS) and underwent adrenalectomy. Representative pictures were taken in an inverted light microscope at 5× and 10× objective lenses. (B) Quantification of DAB ARMC5 positive staining using ImageJ software. P* = 0.0193.

A limitation of this study includes its retrospective References nature which led unavoidably to missing data. In addition, we identified a small number of patients Almeida MQ, Azevedo MF, Xekouki P, Bimpaki EI, Horvath A, Collins MT, Karaviti LP, Jeha GS, Bhattacharyya N, Cheadle C, et al. without ARMC5 variants, which resulted in a small 2012 Activation of cyclic AMP signaling leads to different pathway number of patients in the group comparisons. Although alterations in lesions of the adrenal cortex caused by germline we present statistical analyses of the results, the reader PRKAR1A defects versus those due to somatic GNAS mutations. Journal of Clinical Endocrinology and Metabolism 97 E687–E693. should critically review the data, and we acknowledge (https://doi.org/10.1210/jc.2011-3000) that final conclusions are difficult to be drawn. However, Almeida MQ, Harran M, Bimpaki EI, Hsiao HP, Horvath A, Cheadle C, to our knowledge, this remains the largest study on the Watkins T, Nesterova M & Stratakis CA 2011 Integrated genomic analysis of nodular tissue in macronodular adrenocortical investigation of ARMC5 variants in patients with CNC. hyperplasia: progression of tumorigenesis in a disorder associated Additionally, it should be mentioned that patients with multiple benign lesions. Journal of Clinical Endocrinology and with CNC undergo regular testing for CS given their Metabolism 96 E728–E738. (https://doi.org/10.1210/jc.2010-2420) Almeida MQ & Stratakis CA 2011 How does cAMP/protein kinase A predisposition for PPNAD, which may, however, differ signaling lead to tumors in the adrenal cortex and other tissues? depending on their family and personal preferences or Molecular and Cellular Endocrinology 336 162–168. (https://doi. whether they are the first probands diagnosed with the org/10.1016/j.mce.2010.11.018) Assié G, Libé R, Espiard S, Rizk-Rabin M, Guimier A, Luscap W, disease in their family or not. Furthermore, although Barreau O, Lefèvre L, Sibony M, Guignat L, et al. 2013 ARMC5 the screening protocol used at our institute has mutations in macronodular adrenal hyperplasia with Cushing’s remained the same over the last years, we acknowledge syndrome. New England Journal of Medicine 369 2105–2114. (https:// doi.org/10.1056/NEJMoa1304603) that our screening testing may differ from other Bertherat J, Groussin L, Sandrini F, Matyakhina L, Bei T, Stergiopoulos S, hospitals. Thus, the timing of diagnosis and markers Papageorgiou T, Bourdeau I, Kirschner LS, Vincent-Dejean C, et al. of CS may not be reliable factors as is the presence or 2003 Molecular and functional analysis of PRKAR1A and its locus (17q22-24) in sporadic adrenocortical tumors: 17q losses, somatic absence of CS. mutations, and protein kinase A expression and activity. Cancer In conclusion, this study shows that germline Research 63 5308–5319. variants in ARMC5 were found more frequent Bertherat J, Horvath A, Groussin L, Grabar S, Boikos S, Cazabat L, Libe R, René-Corail F, Stergiopoulos S, Bourdeau I, et al. 2009 among patients with PRKAR1A mutations and Mutations in regulatory subunit type 1A of cyclic adenosine hypercortisolemia and/or CS than those that never 5′-monophosphate-dependent protein kinase (PRKAR1A): phenotype developed high cortisol levels. The data suggest that analysis in 353 patients and 80 different genotypes. Journal of Clinical Endocrinology and Metabolism 94 2085–2091. (https://doi. ARMC5 can be involved in regulating PKA signaling org/10.1210/jc.2008-2333) and cortisol secretion in a state of PRKAR1A deficiency, Berthon A, Faucz F, Bertherat J & Stratakis CA 2017a Analysis of ARMC5 although the way the two molecular pathways interact expression in human tissues. Molecular and Cellular Endocrinology 441 140–145. (https://doi.org/10.1016/j.mce.2016.08.018) remains unknown. Berthon A, Faucz FR, Espiard S, Drougat L, Bertherat J & Stratakis CA 2017b Age-dependent effects of Armc5 haploinsufficiency on adrenocortical function. Human Molecular Genetics 26 3495–3507. (https://doi.org/10.1093/hmg/ddx235) Bimpaki EI, Iliopoulos D, Moraitis A & Stratakis CA 2010 MicroRNA Supplementary materials signature in massive macronodular adrenocortical disease and This is linked to the online version of the paper at https://doi.org/10.1530/ implications for adrenocortical tumourigenesis. Clinical Endocrinology ERC-20-0273. 72 744–751. (https://doi.org/10.1111/j.1365-2265.2009.03725.x) Bourdeau I, Antonini SR, Lacroix A, Kirschner LS, Matyakhina L, Lorang D, Libutti SK & Stratakis CA 2004 Gene array analysis of macronodular adrenal hyperplasia confirms clinical heterogeneity Declaration of interest and identifies several candidate genes as molecular mediators. C A S holds patents on the function of the PRKAR1A, PDE11A, and GPR101 Oncogene 23 1575–1585. (https://doi.org/10.1038/sj.onc.1207277) genes and related issues. His laboratory has also received research Bourdeau I, Matyakhina L, Stergiopoulos SG, Sandrini F, Boikos S & funding on the GPR101 gene, abnormal growth hormone secretion and its Stratakis CA 2006 17q22-24 chromosomal losses and alterations of treatment by Pfizer, Inc. F R F holds a patent on theGPR101 gene and/or its protein kinase A subunit expression and activity in function. 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Received in final form 27 June 2020 Accepted 2 July 2020 Accepted Manuscript published online 7 July 2020