Abstract
Context
Radiological characterization of adrenal size in primary bilateral macronodular adrenocortical hyperplasia (PBMAH) has not been previously investigated.
Objective
We hypothesized that volumetric modeling of adrenal gland size may correlate with biochemical disease severity in patients with PBMAH. Secondary analysis of patients with concurrent primary aldosteronism (PA) was performed.
Design
A retrospective cross-sectional analysis of 44 patients with PBMAH was conducted from 2000 to 2019.
Setting
Tertiary care clinical research center.
Patients
Patients were diagnosed with PBMAH based upon clinical, genetic, radiographic and biochemical characteristics.
Intervention
Clinical, biochemical, and genetic data were obtained. Computed tomography scans were used to create volumetric models by manually contouring both adrenal glands in each slice using Vitrea Core Fx v6.3 software (Vital Images, Minnetonka, Minnesota).
Main Outcome and Measures
17-hydroxycorticosteroids (17-OHS), ARMC5 genetics, and aldosterone-to-renin ratio (ARR) were retrospectively obtained. Pearson test was used for correlation analysis of biochemical data with adrenal volume.
Results
A cohort of 44 patients with PBMAH was evaluated, with a mean age (±SD) of 53 ± 11.53. Eight patients met the diagnostic criteria for PA, of whom 6 (75%) were Black. In the Black cohort, total adrenal volumes positively correlated with midnight cortisol (R = 0.76, P = 0.028), urinary free cortisol (R = 0.70, P = 0.035), and 17-OHS (R = 0.87, P = 0.0045), with a more pronounced correlation with left adrenal volume alone. 17-OHS concentration positively correlated with total, left, and right adrenal volume in patients harboring pathogenic variants in ARMC5 (R = 0.72, P = 0.018; R = 0.65, P = 0.042; and R = 0.73, P = 0.016, respectively).
Conclusions
Volumetric modeling of adrenal gland size may associate with biochemical severity in patients with PBMAH, with particular utility in Black patients.
Keywords: steroids, Cushing syndrome, PBMAH, ARMC5, adrenal hyperplasia, volumetric
Primary bilateral macronodular adrenocortical hyperplasia (PBMAH), previously referred to as massive macronodular adrenocortical hyperplasia (MMAD) and adrenocorticotropic hormone (ACTH)-independent macronodular adrenal hyperplasia (AIMAH), is a rare cause of ACTH-independent Cushing syndrome, primarily in older adults [1]. PBMAH typically manifests with incidental bilateral adrenal nodules, with or without subclinical, overt or cyclic hypercortisolemia, or less commonly mineralocorticoid and/or androgen/estrogen excess [2-9]. Characteristic appearance of the adrenal glands includes bilateral macronodules (≥1cm) with internodular hyperplasia or atrophy.
The pathogenesis of PBMAH is diverse and multifaceted. Expression of aberrant or ectopic G protein-coupled receptors (GPCR), dysregulation of cAMP/PKA signaling, along with germline and somatic pathogenic variants in MEN1, GNAS, PDE8B, PDE11A, and PRKACA, are among some of the molecular mechanisms that contribute to disease pathophysiology [1, 10, 11]. Most recently, we and others identified inactivating pathogenic variants in the armadillo repeat containing 5 (ARMC5; OMIM 615549; 16p11.2) gene, a putative tumor suppressor, in up to half of all the cases; this has now been confirmed internationally and across all ethnicities [3, 12]. Thus, it appears that PBMAH is not a sporadic disease in most individuals [3, 6, 12-15]; instead the disorder is frequently inherited in an autosomal dominant manner, with incomplete penetrance. Although the function of ARMC5 remains unknown, the gene has essential functions in embryological development as well as adrenal and T-cell biology [16, 17]. Additionally, ARMC5 is involved in the regulation of steroidogenic enzymes, including glucocorticoid synthesis [3, 16, 17]. While clinical presentation is variable, even among family members harboring the same disease-causing variant, several studies have reported an association of pathogenic variants in ARMC5 with larger elevations in serum glucocorticoids or mineralocorticoids [18, 19]. Moreover, nodules harboring pathogenic variants in ARMC5 have been associated with greater proliferation potential compared to those without pathogenic variants [17].
The biochemical diagnosis of PBMAH currently relies on the criteria used to establish the diagnosis of Cushing syndrome (CS) as described by The Endocrine Society Clinical Practice Guidelines [20]. Importantly, analysis of 24-hour urinary 17-hydroxycorticosteroids (17-OHS) as a screening test for PBMAH is a sensitive marker of disease, although it lacks specificity and availability by most commercial testing sites [2]. In addition to biochemical evidence, the diagnosis of PBMAH requires the identification of bilateral adrenal lesions using computed tomography (CT). In one study, PBMAH patients with pathogenic variants in ARMC5 had a greater total adrenal size and total adrenal weight after surgical resection when compared with their ARMC5 negative counterparts [12]. However, accurate radiological characterization of adrenal volume in PBMAH and its putative correlation to disease severity and race have not been previously investigated. Volumetric contouring from CT has been informative in several adrenal conditions, including primary pigmented nodular adrenocortical disease (PPNAD) and congenital adrenal hyperplasia (CAH) [21, 22]. In this study, we hypothesized that volumetric modeling of adrenal gland size may correlate with biochemical disease severity in subjects with PBMAH.
1. Materials and Methods
A. Clinical and biochemical evaluation of patients with PBMAH
Subjects with PBMAH were evaluated at the National Institutes of Health (NIH) Clinical Center for CS, ranging from subclinical to overt disease. Clinical, biochemical, and radiographic characteristics were assessed. Biochemical diagnosis of hypercortisolemia and adrenal CS was made according to The Endocrine Society Clinical Practice Guidelines for the diagnosis of CS [20]. Subjects were initially examined for clinical features of CS, with subsequent biochemical confirmation when 2 or more of the following screening tests were consistent with hypercortisolemia: 24-hour urinary free cortisol (UFC), midnight serum cortisol, and the low-dose dexamethasone suppression test [20]. ACTH-independent CS was ascertained through ACTH measurement, second-tier biochemical testing, and radiographic imaging [20]. The biochemical diagnosis of PBMAH also relied on elevations in 17-OHS, as detailed elsewhere [2]. In cases where patients were treated surgically, a histological diagnosis of PBMAH was performed by an experienced pathologist.
A stepwise case confirmation and subtype classification for primary aldosteronism (PA) was performed in all included patients [23]. Plasma aldosterone concentration (PAC) (ng/dL), 24-hour urine aldosterone (UA, μg/24hr) and plasma renin activity (PRA) (ng/mL ∙ h) was obtained in all patients. Aldosterone-to-renin ratio (ARR) was reported in conventional units (ng/dL per ng/mL ∙ h), with an adopted cutoff value of ≥15 for a positive case detection for PA. Potassium was normalized in all patients and liberal salt intake prior to biochemical evaluation was encouraged. Medications that may lead to false-positive or false-negative ARR results during case detection and confirmation were all discontinued at least 2 to 4 weeks prior to enrollment. Antihypertensive therapy with minimal effect on ARR was achieved with one or more of the following oral agents: α-blocker (doxazosin or terazosin), calcium channel blocker (verapamil), and/or vasodilator (hydralazine). Case confirmation was ascertained with the saline suppression test and oral salt-loading tests with 24-hour UA.
B. Volumetric modeling of adrenal glands
Criteria for radiographic diagnosis included bilateral macronodules (≥1cm) with internodular atrophy. Non-contrast CT scans were used to create volumetric models by manually contouring both adrenal glands, in each slice, using Vitrea Core Fx v6.3 software (Vital Images, Minnetonka, Minnesota) (Fig. 1). Ethics approval was granted from the Institutional Review Boards of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) (until 2010) and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (2010 to present), NIH (Clinical Trial Registration no. NCT00005927).
Figure 1.
Axial non-contrast CT image of the abdomen. Three-dimensional volumetric contouring is represented by the yellow dotted lines delineating each adrenal gland.
C. Genetic analysis of germline variants in ARMC5
DNA was extracted from peripheral blood leukocytes as previously described [3, 18]. The complete ARMC5-coding and intronic sequence of all 4 ARMC5 isoforms expressed in the adrenal glands (ARMC5-001 [NM_001288767], ARMC5-002 [NM_024742], ARMC5-003 [NM_001301820], and ARMC5-201 [NM_001105247]) was amplified using the conditions previously described [18]. Each polymerase chain reaction product was amplified using BigDye Terminator V3.1 (Life Technologies), purified using ZR DNA Sequencing Clean-up Kit (Zymo Research), and analyzed by classical bidirectional Sanger sequencing. For the variations in nomenclature, the most frequent isoform in the literature (NM_001105247.1) was used. The germline variants in ARMC5 predicted to be damaging by in silico analysis were inferred to alter the level or biochemical function of the gene or gene product. Based on Varsome data (https://varsome.com, visualized on November 2019), 9 different predictions software packages were used: MutationTaster, FATHMM, MetaSVM, MetalR, phyloP20way, LRT, MutationAssessor, SIFT, and Provean. Since the function of ARMC5 remains unknown, missense variants that represented common variants in the general population or were predicted to be nondamaging by in silico analysis were excluded.
D. Statistical analysis
Statistical analyses were performed using R Studio (Version 1.2.5001, RStudio Inc.) and on SPSS 20.0 software (IBM Corp, USA). Results are presented as mean and 95% confidence interval (CI) unless otherwise indicated. Categorical variables were analyzed using the chi-square test and the Fisher exact test was used when the sample size was ≤5. The Mann-Whitney U test was used for continuous variables. Pearson test was used for correlation analysis, unless mentioned otherwise. A 2-tailed P value of <0.05 was used to define statistically significant differences.
2. Results
A. Clinical and biochemical characteristics of patients with PBMAH
Forty-four patients with PBMAH (13 males, 30%) were evaluated and divided into 3 groups: overall, Black, and White (Table 1). For the purposes of this study, the term Black incorporates individuals who self-identify as Black, African American, or the Caribbean diaspora. The mean age of the subjects was 53 years with a standard deviation (SD) of 11.53 years at the time of presentation. Ten subjects (23%) were Black and 32 (73%) were White. In the overall cohort, the midnight serum cortisol concentration, UFC, and 17-OHS were 8.37 μg/dL (6.84-9.89), 54.73 μg/24-hour (35.69-73.77), and 9.34 mg/24-hour (7.90-10.78), respectively; mean adrenal volumes were 45.18 mL (37.49-52.87) for total, 26.96 mL (20.92-33.0) for the left, and 18.72 mL (15.41-22.03) for the right. Bilateral adrenal nodules were comparable in sizes across all groups.
Table 1.
Clinical and Biochemical Characteristics of Patients With PBMAH
| Overall | Black | White | P value (Black vs White) | |
|---|---|---|---|---|
| n (%) | 44a | 10 (23) | 32 (73) | <0.001 |
| Age, years (mean ± SD) | 53 ± 11.53 | 50 ± 13.11 | 55 ± 10.50 | 0.25 |
| Sex (male [%]) | 13 (30) | 6 (60) | 6 (19) | 0.034 |
| BMI (mean ± SD) | 34.74 ± 7.46 | 35.23 ± 6.41 | 34.45 ± 7.70 | 0.77 |
| Total adrenal volume (mL) | 45.18 (37.49-52.87) | 38.69 (23.30–54.09) | 45.94 (36.96-54.93) | 0.42 |
| Left adrenal volume (mL) | 26.96 (20.92-33.0) | 25.57 (14.05-37.08) | 26.27 (19.12-33.41) | 0.92 |
| Right adrenal volume (mL) | 18.72 (15.41-22.03) | 16.63 (7.49-25.78) | 19.38 (15.59-23.17) | 0.50 |
| Left adrenal mass (cm) | 2.36 (1.96-2.75) | 2.18 (1.44-2.92) | 2.39 (1.88-2.90) | 0.67 |
| Right adrenal mass (cm) | 2.39 (2.02-2.77) | 1.73 (0.90-2.56) | 2.59 (2.15-3.03) | 0.056 |
| Serum ACTH (pg/mL) | 9.73 (6.75-12.71) | 14.71 (5.29-24.13) | 8.62 (5.44-11.81) | 0.10 |
| Midnight serum cortisol (µg/dL) | 8.37 (6.84-9.89) | 8.82 (4.27-13.38) | 8.08 (6.43-9.74) | 0.69 |
| UFC (µg/24-hour) | 54.73 (35.69-73.77) | 80.08 (-3.19-163.35) | 41.88 (32.45-51.32) | 0.086 |
| Urinary 17-OHS (mg/24-hour) | 9.34 (7.90-10.78) | 8.23 (5.41-11.06) | 9.15 (7.48-10.81) | 0.59 |
| HbA1c (%) | 6.35 (5.96-6.75) | 6.17 (5.44-6.91) | 6.36 (5.86-6.86) | 0.68 |
| Plasma aldosterone concentration (ng/dL) | 17.35 (8.28-26.42) | 39.81 (4.90-74.72) | 10.13 (5.95-14.31) | 0.0040 |
| Urine aldosterone (µg/24-hour) | 17.36 (11.99-22.74) | 25.38 (4.10-46.65) | 14.93 (10.55-19.30) | 0.093 |
| Serum potassium (mmol/L) | 3.88 (3.75-4.01) | 3.69 (3.43-3.95) | 3.93 (3.77-4.08) | 0.12 |
| PRA (ng/mL ∙ h) | 1.27 (0.80-1.73) | 0.82 (0.58-1.06) | 1.27 (0.73-1.80) | 0.31 |
| ARR (ng/dL per ng/mL ∙ h) | 23.43 (8.03-38.84) | 65.04 (6.09-123.99) | 9.52 (6.68-12.36) | 0.0010 |
| Elevated ARR (n [%]) | 8 (18) | 6 (60) | 2 (7) | 0.0030 |
Continuous variables are presented as mean (95% CI) unless indicated otherwise.
a Includes 2 Asian patients.
Abbreviations: ACTH, adrenocorticotropic hormone; ARR, aldosterone-to-renin ratio; BMI, body mass index; PRA, plasma renin activity; UFC, urinary free cortisol.
Black patients had significantly higher PAC (39.81 [4.90-74.72] vs 10.13 ng/dL [5.95–14.31], P = 0.0040, Black vs White, respectively) and ARR (65.04 [6.09-123.99] vs 9.52 [6.68-12.36], P = 0.0010) compared with White patients with a similar trend for 24-hour UA levels (25.38 [4.10-46.65] vs 14.93 µg/24-hour [10.55-19.30], P = 0.093, Table 1). Black patients without pathogenic variants in ARMC5 were also found to have increased PAC and ARR compared with White patients (52.48 [−8.39 to 113.36] vs 11.12 [5.87-16.36] ng/dL, P = 0.0030, and 86.64 [−15.70 to 188.98] vs 10.39 [6.79-13.99], P = 0.001 Black vs White, PAC and ARR, respectively, Table 2). Limited sample size prevented a sufficient comparison of PAC and ARR levels between White and Black patients with ARMC5 pathogenic variants (data not shown). Interestingly, Black patients with a pathogenic variant in ARMC5 presented at a younger age when compared with White patients with an ARMC5 defect (39 ± 6.14 [SD] vs 56 ± 13.09 [SD], P = 0.042, Black vs White, respectively).
Table 2.
Characteristics of PBMAH Patients Without Pathogenic Variants in ARMC5
| Total wild-type ARMC5 | Black | White | P value (Black vs White) | |
|---|---|---|---|---|
| n (%) | 33a | 6 (18) | 26 (79) | <0.001 |
| Age, years (mean ± SD) | 54 ± 10.85 | 58 ± 10.50 | 55 ± 10.11 | 0.50 |
| Sex (male [%]) | 8 (24) | 4 (67) | 4 (15) | 0.036 |
| BMI (mean ± SD) | 34.53 ± 6.40 | 37.25 ± 6.75 | 34.17 ± 6.28 | 0.29 |
| Total adrenal volume (mL) | 43.47 (35.29-51.65) | 44.06 (26.41-61.71) | 44.07 (34.17-53.96) | 1.00 |
| Left adrenal volume (mL) | 24.24 (17.87-30.61) | 28.14 (16.86-39.42) | 23.80 (16.01-31.60) | 0.62 |
| Right adrenal volume (mL) | 19.83 (15.87-23.80) | 21.30 (6.31-36.29) | 19.88 (15.55-24.21) | 0.79 |
| Left adrenal mass (cm) | 2.34 (1.86-2.81) | 2.32 (1.52-3.11) | 2.34 (1.74-2.95) | 0.97 |
| Right adrenal mass (cm) | 2.58 (2.15-3.02) | 2.27 (1.09-3.44) | 2.67 (2.16-3.18) | 0.48 |
| Serum ACTH (pg/mL) | 10.67 (6.84-14.51) | 18.72 (4.58-32.85) | 9.03 (5.12-12.95) | 0.049 |
| Midnight serum cortisol (µg/dL) | 8.11 (6.44-9.79) | 8.58 (3.79-13.38) | 7.66 (5.82-9.51) | 0.66 |
| UFC (µg/24-hour) | 44.93 (34.51-55.35) | 48.30 (21.03-75.57) | 39.77 (31.49-48.04) | 0.39 |
| Urinary 17-OHS (mg/24-hour) | 8.92 (7.37-10.47) | 7.92 (4.70-11.13) | 8.74 (7.03-10.45) | 0.66 |
| HbA1c (%) | 6.40 (5.95-6.84) | 6.27 (5.20-7.33) | 6.44 (5.89-6.99) | 0.73 |
| Plasma aldosterone concentration (ng/dL) | 19.61 (7.60-31.63) | 52.48 (−8.39 to 113.36) | 11.12 (5.87-16.36) | 0.003 |
| Urine aldosterone (µg/24-hour) | 19.26 (12.95-25.57) | 29.41 (−2.06 to 60.87) | 16.59 (11.62-21.56) | 0.088 |
| Serum potassium (mmol/L) | 3.90 (3.75-4.06) | 3.65 (3.25-4.05) | 3.97 (3.79-4.15) | 0.11 |
| PRA (ng/mL ∙ h) | 1.40 (0.75-2.04) | 0.73 (0.48-0.99) | 1.34 (0.66-2.03) | 0.34 |
| ARR (ng/dL per ng/mL ∙ h) | 26.41 (5.76-47.06) | 86.64 (−15.70 to 188.98) | 10.39 (6.79-13.99) | 0.001 |
| Elevated ARR (n [%]) | 6 (21) | 4 (67) | 2 (10) | 0.016 |
Continuous variables are presented as mean (95% CI) unless indicated otherwise.
a Includes 1 patient of Asian ancestry.
Abbreviations: ACTH, adrenocorticotropic hormone; ARR, aldosterone-to-renin ratio; BMI, body mass index; PRA, plasma renin activity; UFC, urinary free cortisol.
Of 39 patients with available biochemical data, 8 (21%) met the criteria of an ARR of ≥15 (86.39 [19.08-153.69] vs 7.19 [5.60-8.77] P < 0.001, PA vs without PA, respectively) [24]. All patients with PA were male, 6 (75%) were Black, and 2 (25%) harbored pathogenic variants in ARMC5. Right adrenal mass was significantly less in patients with PA compared to those without PA (1.39 [0.69-2.09] vs 2.74 [2.29-3.19] cm, P = 0.005, respectively).
B. Biochemical parameters correlated with adrenal volumetrics in patients with PBMAH
In the overall cohort, total adrenal volume positively correlated with 17-OHS (R = 0.38, P = 0.014, Table 3). When analyzing left and right adrenal volumes separately, we found a more pronounced correlation between left adrenal volumes and 17-OHS (R = 0.43, P = 0.0048) and a significant correlation between UFC and left adrenal volume (R = 0.36, 0.018). A trend toward a significant correlation was also observed with midnight serum cortisol and glycated hemoglobin A1c (HbA1c) levels and left adrenal volume (R = 0.28, P = 0.075; and R = 0.33, P = 0.078, respectively), while right adrenal volume did not show correlations with biochemical parameters [24]. All other biochemical parameters did not significantly correlate with adrenal volume.
Table 3.
Biochemical Parameters Significantly Correlated with Total and Left Adrenal Volume in Patients with PBMAH
| Cohort | Biochemical Parameter | Total Adrenal Volume | Left Adrenal Volume | Right Adrenal Volume | |||
|---|---|---|---|---|---|---|---|
| R | P value | R | P value | R | P value | ||
| Overall | Urinary 17-OHS (mg/24-hour) | 0.38 | 0.014 | 0.43 | 0.0048 | 0.074 | 0.64 |
| UFC (µg/24-hour) | 0.28 | 0.072 | 0.36 | 0.018 | -0.034 | 0.83 | |
| Black | Urinary 17-OHS (mg/24-hour) | 0.87 | 0.0045 | 0.86 | 0.0050 | 0.24 | 0.54 |
| UFC (µg/24-hour) | 0.70 | 0.035 | 0.81 | 0.0080 | 0.00046 | 1.0 | |
| Midnight serum cortisol (µg/dL) | 0.76 | 0.028 | 0.85 | 0.0070 | 0.17 | 0.67 | |
| Pathogenic ARMC5 | Urinary 17-OHS (mg/24-hour) | 0.72 | 0.018 | 0.65 | 0.042 | 0.73 | 0.016 |
Correlation coefficients in bold are statistically significant.
Abbreviations: 17-OHS, 17-hydroxycorticosteroid; UFC, urinary free cortisol.
Subgroup analysis by race demonstrated significant positive correlations between total adrenal volume and 17-OHS (R = 0.87, P = 0.0045), UFC (R = 0.70, P = 0.035), and midnight cortisol concentration (R = 0.76, P = 0.028) among Black patients (Table 3). Additionally, left adrenal volume showed a strong positive correlation with 17-OHS (R = 0.86, P = 0.0050), UFC (R = 0.81, P = 0.0080), and midnight cortisol concentration (R = 0.85, P = 0.0070), while similar to the overall cohort, right adrenal volume did not show correlations with biochemical parameters [24]. Among White patients, total adrenal volumes trended toward a significant positive correlation with 17-OHS, while other biochemical parameters showed no correlation (data not shown).
C. Germline variants in ARMC5
We sequenced all subjects, including 3 families with 2 affected members. Genotype c.1736_1739delGCCT (p.Cys579SerfsTer50; rs1387565854) was only counted once in the analysis (ADT 85.01 and ADT 85.06, Table 4 [25]). Eleven (25%) subjects were heterozygous for pathogenic variants in ARMC5 that were predicted to be damaging by at least 5 out of 9 in silico tools. Genotype analysis among patients with pathogenic variants in ARMC5 demonstrated a positive correlation between total adrenal volume and 17-OHS levels (R = 0.72, P = 0.018), with similar correlations when analyzing separately left (R = 0.65, P = 0.042) and right adrenal volumes (R = 0.73, P = 0.016) (Table 3). Other biochemical parameters did not significantly correlate with total, left, or right adrenal volume [24].
Table 4.
In Silico Prediction of Pathogenic Variants in ARMC5 in Patients With PBMAH
| ID | DNA change | Protein change | SNP ID | Domains | In silico modelinga | Allele frequency (%)b |
|---|---|---|---|---|---|---|
| ADT36.01 | c.170dupG | p.Ile58Asnfs*45 | rs951869246 | Armadillo Repeat-Containing-Related | Likely pathogenic | N/A |
| ADT125.02 | c.944G > A | p.R315Q | rs1415974570 | Armadillo Repeat-Containing-Related | Likely Pathogenic | 0.0004 |
| ADT306.03 | c.1084C > T | p.R362W | rs1385397608 | arm_5/ARM repeat | Likely pathogenic | 0.0032 |
| ADT202.03 | c.1090C > T | p.R364* | rs1386368908 | Armadillo | Likely Pathogenic | N/A |
| ADT241.02 | c.1223A > G | p.Q408R | rs141923065 | Armadillo-like helical | Likely pathogenic | 0.313 |
| ADT307.03 | c.1223A > G | p.Q408R | rs141923065 | Armadillo-like helical | Likely pathogenic | 0.313 |
| ADT310.03 | c.1223A > G | p.Q408R | rs141923065 | Armadillo-like helical | Likely pathogenic | 0.313 |
| ADT85.01 | c.1736_1739delGCCTc | p.Cys579Serfs*50 | rs1387565854 | Armadillo-like helical | Likely pathogenic | N/A |
| ADT85.06 | c.1736_1739delGCCTc | p.Cys579Serfs*50 | rs1387565854 | Armadillo-like helical | Likely pathogenic | N/A |
| ADT150.02 | c.1777C > T | p.R593W | rs587777662 | Armadillo-like helical | Likely Pathogenic | 0.0004 |
| ADT130.01 | c.1928C > T | p.T643M | rs370836071 | Armadillo-type fold | Likely pathogenic | 0.0037 |
| ADT91.13 | c.2477C > A | p.P826H | no ID | BTB/POZ domain | Likely pathogenic | N/A |
Analyses were made based on the NM_001105247.1 ARMC5 isoform.
Abbreviations: N/A, not available; SNP, single nucleotide polymorphism.
a In silico modeling was performed based on Varsome (varsome.com)
bAllele frequency is based on gnomAD total frequency (gnomAD.com)
cGenotype c.1736_1739delGCCT was only accounted once in the analysis
Reference: Quang D, Chen Y, Xie X. DANN: a deep learning approach for annotating the pathogenicity of genetic variants. Bioinformatics. 2015;31(5):761-763.
3. Discussion
PBMAH is a heterogenous disease manifesting as a clinical and biochemical spectrum, ranging from incidentally identified asymptomatic bilateral adrenal nodules to overt or severe ACTH-independent CS [2-5, 12, 13, 15]. Previously, volumetric contouring from CT has been used to assess disease outcomes in other bilateral adrenal pathologies, including PPNAD [21]. In this study, correlation between adrenal gland volumetrics and biochemical severity of PBMAH was investigated using volumetric contouring from CT. Our results indicate in the overall cohort, total adrenal volume positively correlated with 17-OHS. When analyzing left adrenal volume alone, a more pronounced correlation was observed for 17-OHS, and UFC significantly correlated with left adrenal volume. Interestingly, in the Black cohort, 3 biochemical parameters, 17-OHS, UFC, and midnight serum cortisol, all positively correlated with total and left adrenal volumes, despite the smaller sample size. The prevalence of ARMC5 in our overall cohort was consistent with other published estimates [3, 12, 13, 15], and our genotype analysis demonstrated a positive correlation between 17-OHS and total, left, and right adrenal volumes in PBMAH patients harboring pathogenic variants in ARMC5. Ultimately, adrenal volume positively correlated with biochemical parameters, but future analyses, including regression models to evaluate a possible causal relationship, are required to affirm the utility of this modality in PBMAH.
Volumetric measurement of the adrenal glands is a reproducible modality with low interobserver variability [26]. Most studies report adrenal size (mm) using CT or MRI, whereas adrenal weight is only reported if tumor samples are available [6, 13, 27]. Adrenal volume data is limited to patients who have undergone adrenalectomy, which in some cases represents a limited fraction of the total cohort [27]. Interestingly, a greater proportion of left adrenalectomies are performed in PBMAH, likely due to larger adrenal size on radiography [27], and as a result of larger adrenal volumes rather than nodular size. This is supported by findings from Schneller et al, who determined that mean left adrenal volume was larger than right adrenal volume in patients with normal radiographic findings of the adrenal glands, independent of sex [26]. We found larger left adrenal volumes and a more pronounced correlation with biochemical parameters, including 17-OHS. Further investigation using adrenal venous sampling or fluorodeoxyglucose–positron emission tomography (FDG-PET) to discern left and right adrenal biochemical activity will be required to affirm this observation. Ultimately, volumetric contouring of the adrenal glands may provide a supplemental tool for calculation of disease severity, which may aid with patient counseling including surgical decisions surrounding unilateral adrenalectomy.
Additionally, 8 patients who met the diagnostic criteria for PA were identified. Interestingly, 6 of these patients were Black, which likely accounts for the significant elevation of PAC and ARR in the overall, and ARMC5 wild-type Black cohorts compared with the respective White cohorts. Previously, Zilbermint et al reported that all patients harboring pathogenic ARMC5 variants in their PA cohort were Black [19]. We did not report significant differences in PAC or ARR between the White and Black cohorts with pathogenic ARMC5 variants, although 50% of the Black cohort had an elevated ARR. Further analysis with a larger cohort is required to evaluate the relationship between pathogenic ARMC5 variants and PA in Black patients.
Pathogenic variants in ARMC5 account for approximately 20% to 40% of PBMAH cases [6, 15]. In our cohort, 25% of unrelated patients harbored germline pathogenic variants in ARMC5. Several groups have reported adrenal size is significantly larger in patients harboring pathogenic variants in ARMC5 compared with their ARMC5-negative counterparts [13, 15, 18]. Additionally, it has been proposed that patients with this genetic defect manifest with more severe CS, although this conclusion is preliminary. In our study, the only biochemical parameter to correlate with total, left, and right adrenal volumes in patients harboring pathogenic variants in ARMC5 was 17-OHS. Analysis of 17-OHS provides an integrated assessment of the amount of cortisol and its byproducts produced over a 24-hour period. 17-OHS offers a sensitive screening test for PBMAH, although it lacks specificity and availability by most commercial testing sites, and it is cumbersome to perform [2]. Future studies with a larger sample size should be conducted to assess any potential correlation between the presence of pathogenic ARMC5 variants and severity of CS, and across the entire PBMAH spectrum, including those that are incidentally found.
Previous studies by our group and others have aimed to correlate genotypic and phenotypic presentations in PBMAH. Albiger et al were unable to correlate specific pathogenic variants in ARMC5 (frameshift, nonsense, or missense) with age of disease onset, sex, hypercortisolism, or adrenal size [15]. However, the cohort used in this study comprised only White patients. Others have shown elevated midnight cortisol and UFC with decreased ACTH in patients harboring pathogenic variants in ARMC5 when compared with wild-type controls [12, 18]. Kyo et al reported a 71% frequency of ARMC5 pathogenic variants in their Japanese cohort, and additionally found a more severe phenotype in patients harboring pathogenic variants in ARMC5 [28]. Therefore, in conjunction with the apparent correlation of adrenal volume with 17-OHS in patients harboring pathogenic variants in ARMC5 reported in this study, the correlation between ARMC5 status and clinical and biochemical data may be particularly informative in certain racial or ethnic groups. Future studies should compare genotypic and phenotypic data across different races and ethnicities, including analysis of LC/MS-MS steroid profiling, either in plasma or urine, to investigate a possible correlation between pathogenic variants in ARMC5 and disease severity in PBMAH [29].
Limitations of our study include that our subjects had a less common subset of PBMAH comprised of mild to overt hypercortisolism with low normal to suppressed ACTH, rather than incidentally discovered or clinically silent disease. Additionally, our study population was small and not generalizable. Since one radiologist performed manual contouring of the adrenal glands, inter-rater reliability was not assessed. Lastly, we did not perform LC/MS-MS steroid profiling in plasma or urine to further investigate how adrenal volume and ARMC5 status affected other adrenal steroid intermediates.
In conclusion, volumetric contouring of the adrenal glands in conjunction with clinical and biochemical data may predict disease severity in individuals with PBMAH, particularly among Black patients. Further research is necessary to determine if a cause-effect relationship is present between adrenal volume and biochemical severity and to investigate if and why the left adrenal gland appears to be more biochemically active in PBMAH.
Acknowledgments
We would like to thank the patients who participated in the clinical trial.
Financial Support: This work was supported by the research project Z01-HD008920 (C.A.S.) of the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, USA.
Glossary
Abbreviations
- 17-OHS
17-hydroxycorticosteroid
- ACTH
adrenocorticotropic hormone
- ARR
aldosterone-to-renin ratio
- CI
confidence interval
- CS
Cushing syndrome
- CT
computed tomography
- PA
primary aldosteronism
- PAC
plasma aldosterone concentration
- PBMAH
primary bilateral macronodular adrenocortical hyperplasia
- PPNAD
primary pigmented nodular adrenocortical disease
- UA
urine aldosterone
- UFC
urinary free cortisol
Additional Information
Disclosure Summary: C.A.S. holds a patent on the PRKAR1A, PDE11A, and GPR101 genes and/or their function and his laboratory has received research funding from Pfizer Inc. F.R.F holds patent on the GPR101 gene and/or its function. M.Z. received consulting fees from the Guidepoint and GLG. The other authors have nothing to disclose.
Data Availability
The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.