The clinical characteristics and pathogenic variants of primary pigmented nodular adrenocortical disease in 210 patients: a systematic review.

Aims: Primary pigmented nodular adrenocortical disease (PPNAD), as a rare kind of Cushing's syndrome, is frequently misdiagnosed. To get a better understanding of the disease, we analyzed the clinical characteristics and pathogenic variants of PPNAD. Methods: Databases were searched, and the pathogenic variants and clinical manifestations of patients were summarized from the relevant articles. Results: A total of 210 patients in 86 articles were enrolled with a median age of 22 and a female-to-male ratio of 2:1. Sixty-six (31.43%) patients were combined with Carney complex (CNC) and 94.29% were combined with osteoporosis/osteopenia. Among 151 patients who underwent genetic testing, 87.42% (132/151) had pathogenic variants. Six gene mutations (PRKAR1A, PDE11A, PRKACA, CTNNB1, PDE8B, and ARMC5) were detected in the patients. The most common mutation was PKAR1A, accounting for 79.47% (120/151). There was a significant correlation between PRKAR1A pathogenic variant and spotty skin pigmentation in CNC concurrent with PPNAD (p < 0.05). Among pregnant patients with PPNAD, those without surgical treatment and with bilateral adrenalectomy suffered from a high-risk perinatal period. However, patients with unilateral adrenalectomy presented a safe perinatal period. Conclusions: For young patients with Cushing's syndrome, especially female patients with spotty skin pigmentation and osteoporosis/osteopenia, PPNAD should be considered. Unilateral adrenal resection may be considered as an option for women with fertility needs. In view of the difficulty of PPNAD diagnosis, genetic testing before surgery might be a reasonable option. Patients with PPNAD with spotty skin pigmentation should consider the PRKAR1A pathogenic variant and pay attention to CNC. Systematic review registration: https://www.crd.york.ac.uk/prospero, identifier CRD42023416988.

Germline PRKACA amplification-associated primary pigmented nodular adrenocortical disease: a case report and literature review.

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare adrenocorticotropin hormone (ACTH)-independent Cushing's syndrome (CS). Pediatric patients with PPNAD typically have unusual skin lesions and slow growth with unknown causes. We present a case of a female Chinese patient with PPNAD caused by the germline PRKACA gene copy number gain of chromosome 19. The patient initially presented with kidney stones, short stature, and obesity. After further testing, it was discovered that the patient had diabetes, mild hypertension, low bone mass, a low ACTH level, and hypercortisolemia, and neither the low-dose or high-dose dexamethasone suppression test was able to inhibit hematuric cortisol, which paradoxically increased. PPNAD was pathologically diagnosed after unilateral adrenalectomy. Chromosome microarrays and whole exon sequencing analyses of the peripheral blood, as well as testing of sectioned adrenal tissue, showed a rise in the copy number of the duplication-containing PRKACA gene on chromosome 19p13.13p13.12, a de novo but not heritable gene defect that causes disease. The clinical signs and symptoms supported the diagnosis of Carney complex (CNC). One significant mechanism of CNC pathogenesis may be the rise in germline PRKACA copy number of chromosome 19. When assessing PPNAD patients for CNC, the possibility of PRKACA gene amplification should be considered. The effect of PRKACA gene amplification on the clinical manifestations of CNC needs to be confirmed by more cases.

Adrenal hyperplasias in childhood: An update.

Pediatric adrenocortical hyperplasias are rare; they usually present with Cushing syndrome (CS); of them, isolated micronodular adrenal disease and its variant, primary pigmented adrenocortical disease are the most commonly encountered. Most cases are due to defects in the cyclic AMP/protein kinase A (cAMP/PKA) pathway, although a few cases remain without an identified genetic defect. Another cause of adrenal hyperplasia in childhood is congenital adrenal hyperplasia, a group of autosomal recessive disorders that affect steroidogenic enzymes in the adrenal cortex. Clinical presentation varies and depends on the extent of the underlying enzymatic defect. The most common form is due to 21-hydroxylase deficiency; it accounts for more than 90% of the cases. In this article, we discuss the genetic etiology of adrenal hyperplasias in childhood.

Carney Complex Complicated with Primary Pigmented Nodular Adrenocortical Disease without Cushing's Syndrome Recurrence for Five Years after Unilateral Adrenalectomy.

We herein report a case of Carney complex (CNC) complicated with primary pigmented nodular adrenocortical disease (PPNAD) after unilateral adrenalectomy. A 44-year-old woman was admitted to our hospital for PPNAD surgery. She had previously undergone surgery for cardiac myxoma and had a PRKAR1A mutation with no family history of CNC. She had Cushing's signs, but her metabolic abnormalities were mild. Adrenal insufficiency due to poor medication adherence was a concern, so she underwent unilateral adrenalectomy. Cushing's signs improved postoperatively and without recurrence for five years. Treatment plans for PPNAD should be determined based on the patient's condition, medication adherence, and wishes.

A Gene-Based Classification of Primary Adrenocortical Hyperplasias.

Primary or adrenocorticotropin-independent adrenocortical tumors and hyperplasias represent a heterogeneous group of adrenocortical neoplasms that arise from various genetic defects, either in isolation or familial. The traditional classification as adenomas, hyperplasias, and carcinomas is non-specific. The recent identification of various germline and somatic genes in the development of primary adrenocortical hyperplasias has provided important new insights into the molecular pathogenesis of adrenal diseases. In this new era of personalized care and genetics, a gene-based classification that is more specific is required to assist in the understanding of their disease processes, hormonal functionality and signaling pathways. Additionally, a gene-based classification carries implications for treatment, genetic counseling and screening of asymptomatic family members. In this review, we discuss the genetics of benign adrenocorticotropin-independent adrenocortical hyperplasias, and propose a new gene-based classification system and diagnostic algorithm that may aid the clinician in prioritizing genetic testing, screening and counseling of affected, at risk individuals and their relatives.

Update of Genetic and Molecular Causes of Adrenocortical Hyperplasias Causing Cushing Syndrome.

Bilateral hyperplasias of the adrenal cortex are rare causes of chronic endogenous hypercortisolemia also called Cushing syndrome. These hyperplasias have been classified in two categories based on the adrenal nodule size: the micronodular types include Primary Pigmented Nodular Adrenocortical Disease (PPNAD) and isolated Micronodular Adrenal Disease (iMAD) and the macronodular also named Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH). This review discusses the genetic and molecular causes of these different forms of hyperplasia that involve mutations and dysregulation of various regulators of the cAMP/protein kinase A (PKA) pathway. PKA signaling is the main pathway controlling cortisol secretion in adrenocortical cells under ACTH stimulation. Although mutations of the regulatory subunit R1α of PKA (PRKAR1A) is the main cause of familial and sporadic PPNAD, inactivation of two cAMP-binding phosphodiesterases (PDE11A and PDE8B) are associated with iMAD even if they are also found in PPNAD and PBMAH cases. Interestingly, PBMAH that is observed in multiple familial syndrome such as APC, menin, fumarate hydratase genes, has initially been associated with the aberrant expression of G-protein coupled receptors (GPCR) leading to an activation of cAMP/PKA pathway. However, more recently, the discovery of germline mutations in Armadillo repeat containing protein 5 (ARMC5) gene in 25-50% of PBMAH patients highlights its importance in the development of PBMAH. The potential relationship between ARMC5 mutations and aberrant GPCR expression is discussed as well as the potential other causes of PBMAH.

Primary pigmented nodular adrenocortical disease (PPNAD): single centre experience.

Background Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of Cushing's syndrome (CS) in childhood. We describe a case series of patients presenting at our centre along with a review of the literature. Methods A retrospective analysis of six index cases and one family were done for demographic features, hormonal profile, imaging findings, genetic mutation status, histopathologic findings and follow-up details. Diagnosis was based on biochemistry and confirmed with histopathology and or genetic mutation. All patients had suppressed 8 am adrenocorticotropic hormone (ACTH) (<10 pg/mL) despite evidence of hypercortisolism. Results The mean age in our cohort was 8.2 years (range 15 months to 20 years). All patients presented with overt CS, including one patient with cyclic Cushing's. Three patients had additional features of Carney complex (CNC). Imaging did not reveal any obvious mass lesions on computed tomography (CT), the classical beaded appearance was present in only two of the patients. Mutation analysis was positive in three patients. Five patients underwent bilateral adrenalectomy and had features of PPNAD on histopathology. Conclusions PPNAD is a rare cause of ACTH-independent CS in childhood and may signal underlying CNC. Patients with younger age of onset with overt CS may still have a mutation in the PRKAR1A gene and warrant genetic testing.

Familial Forms of Cushing Syndrome in Primary Pigmented Nodular Adrenocortical Disease Presenting with Short Stature and Insidious Symptoms: A Clinical Series.

Cushing syndrome (CS) is a rare disease in children, frequently associated with subtle or periodic symptoms that may delay its diagnosis. Weight gain and growth failure, the hallmarks of hypercortisolism in pediatrics, may be inconsistent, especially in ACTH-independent forms of CS. Primary pigmented nodular adrenocortical disease (PPNAD) is the rarest form of ACTH-independent CS, and can be associated with endocrine and nonendocrine tumors, forming the Carney complex (CNC). Recently, phenotype/genotype correlations have been described with particular forms of CNC where PPNAD is isolated or associated only with skin lesions. We present four familial series of CS due to isolated PPNAD, and compare them to available data from the literature. We discuss the clinical and molecular findings, and underline challenges in diagnosing PPNAD in childhood.

PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease.

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD.

A Novel Mutation in the type Iα Regulatory Subunit of Protein Kinase A (PRKAR1A) in a Cushing's Syndrome Patient with Primary Pigmented Nodular Adrenocortical Disease.

A 40-year-old man presented with Cushing's syndrome due to bilateral adrenal hyperplasia with multiple nodules. Computed tomography scan results were atypical demonstrating an enlargement of the bilateral adrenal glands harboring multiple small nodules, but the lesion was clinically diagnosed to be primary pigmented nodular adrenocortical disease (PPNAD) based on both endocrinological test results and his family history. We performed bilateral adrenalectomy and confirmed the diagnosis histologically. An analysis of the patient and his mother's genomic DNA identified a novel mutation in the type Iα regulatory subunit of protein kinase A (PRKAR1A) gene; p.E17X (c.49G>T). This confirmed the diagnosis of PPNAD which is associated with Carney Complex.

ARMC5 Mutations in a Large Cohort of Primary Macronodular Adrenal Hyperplasia: Clinical and Functional Consequences.

CONTEXT: Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of primary adrenal Cushing's syndrome (CS). ARMC5 germline mutations have been identified recently in PBMAH. OBJECTIVE: To determine the prevalence of ARMC5 mutations and analyze genotype-phenotype correlation in a large cohort of unrelated PBMAH patients with subclinical or clinical CS. PATIENTS AND METHODS: ARMC5 was sequenced in 98 unrelated PBMAH index cases. PBMAH was identified by bilateral adrenal nodular enlargement on computed tomography scan. The effect on apoptosis of ARMC5 missense mutants was tested in H295R and HeLa cells. Clinical and hormonal data were collected including midnight and urinary free cortisol levels, ACTH, androgens, renin/aldosterone ratio, cortisol after overnight dexamethasone suppression test, cortisol and 17-hydroxyprogesterone after ACTH 1-24 stimulation and illegitimate receptor responses. Computed tomography and histological reports were analyzed. RESULTS: ARMC5-damaging mutations were identified in 24 patients (26%). The missense mutants and the p.F700del deletion were unable to induce apoptosis in both H295R and HeLa cell lines, unlike the wild-type gene. ARMC5-mutated patients showed an overt CS more frequently, compared to wild-type patients: lower ACTH, higher midnight plasma cortisol, urinary free cortisol, and cortisol after dexamethasone suppression test (P = .003, .019, .006, and <.001, respectively). Adrenals of patients with mutations were bigger and had a higher number of nodules (P = .001 and <.001, respectively). CONCLUSIONS: ARMC5 germline mutations are common in PBMAH. Index cases of mutation carriers show a more severe hypercortisolism and larger adrenals. ARMC5 genotyping may help to identify clinical forms of PBMAH better and may also allow earlier diagnosis of this disease.

Molecular and clinical evidence for an ARMC5 tumor syndrome: concurrent inactivating germline and somatic mutations are associated with both primary macronodular adrenal hyperplasia and meningioma.

CONTEXT: Primary macronodular adrenal hyperplasia (PMAH) is a rare cause of Cushing's syndrome, which may present in the context of different familial multitumor syndromes. Heterozygous inactivating germline mutations of armadillo repeat containing 5 (ARMC5) have very recently been described as cause for sporadic PMAH. Whether this genetic condition also causes familial PMAH in association with other neoplasias is unclear. OBJECTIVE: The aim of the present study was to delineate the molecular cause in a large family with PMAH and other neoplasias. PATIENTS AND METHODS: Whole-genome sequencing and comprehensive clinical and biochemical phenotyping was performed in members of a PMAH affected family. Nodules derived from adrenal surgery and pancreatic and meningeal tumor tissue were analyzed for accompanying somatic mutations in the identified target genes. RESULTS: PMAH presenting either as overt or subclinical Cushing's syndrome was accompanied by a heterozygous germline mutation in ARMC5 (p.A110fs*9) located on chromosome 16. Analysis of tumor tissue showed different somatic ARMC5 mutations in adrenal nodules supporting a second hit hypothesis with inactivation of a tumor suppressor gene. A damaging somatic ARMC5 mutation was also found in a concomitant meningioma (p.R502fs) but not in a pancreatic tumor, suggesting biallelic inactivation of ARMC5 as causal also for the intracranial meningioma. CONCLUSIONS: Our analysis further confirms inherited inactivating ARMC5 mutations as a cause of familial PMAH and suggests an additional role for the development of concomitant intracranial meningiomas.

[A pedigree study of a patient with primary pigmented nodular adrenocortical disease and familial gene mutation].

OBJECTIVE: To clarify the clinical features and genetic background of a kindred of primary pigmented nodular adrenocortical disease (PPNAD). METHODS: Detailed clinical characteristics and laboratory test results from a ten-year old girl diagnosed as PPNAD were collected. Seven members of her family were screened for Cushing syndrome and Carney complex, and their blood DNA was extracted and sequenced for PRKAR1A, PDE11A, PDE8B and CTNNB1 mutations with ABI3730. RESULTS: The girl presented with symptoms and signs of hypercortisolism, while no features of Carney complex were observed. Hypercortisolemia, suppressed corticotrophin and high urinary free cortisol level were revealed. Cortisol level could not be suppressed both in high and low dose dexamethasone suppression test. The diagnosis of adrenocorticotrophic hormone (ACTH)-independent Cushing syndrome was established. Image and pathology of adrenal glands were in accordance with PPNAD. Other family members showed no evidence of Cushing syndrome or Carney complex. DNA sequencing showed that the patient harbored a missense mutation, C18G. Her father and younger sister were proved to be carriers of this mutation. CONCLUSION: A Chinese PPNAD family was identified clinically and genetically, and a novel missense mutation of PRKAR1A was found.

A rare case of familial Cushing's syndrome with a common presentation of weight gain due to a mutation of the PRKAR1A gene causing isolated primary pigmented nodular adrenocortical disease.

BACKGROUND: Cushing's syndrome (CS) is uncommon in childhood and adolescence. Variable presentation with subtle symptoms and signs can make diagnosis difficult. CASE REPORT: We report the case of a 17-year-old girl referred for acne and progressive weight gain with an adrenocorticotropic hormone-independent CS. A computed tomography scan of the adrenals showed normal-sized adrenal glands with discrete bilateral shape irregularity. Bilateral adrenalectomy was performed and the histopathological findings were characteristic of primary pigmented nodular adrenocortical disease (PPNAD). Genetic analysis confirmed a germline mutation of the PRKAR1A gene. The same mutation was found in her sister, mother, and maternal grandfather. Endocrine tests showed that the sister of our patient also presented PPNAD requiring bilateral adrenalectomy and a similar histopathological pattern was observed. No other features of Carney complex was found among all affected members of the family. CONCLUSION: It is exceptional for PPNAD to be an isolated phenomenon as well as being revealed by progressive weight gain in adolescence.

mTOR pathway is activated by PKA in adrenocortical cells and participates in vivo to apoptosis resistance in primary pigmented nodular adrenocortical disease (PPNAD).

Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation.

Primary pigmented nodular adrenocortical disease: the original 4 cases revisited after 30 years for follow-up, new investigations, and molecular genetic findings.

The original 4 patients with Cushing syndrome who underwent bilateral adrenalectomy for primary pigmented nodular adrenocortical disease were followed up for an average of 31 years to determine whether they or any of their primary relatives had developed Carney complex or its components. None had. Three of the patients were alive and well; the fourth had died of an unrelated condition. All the adrenal glands contained multiple small, black or brown cortical nodules, up to 4 mm in diameter. The extracapsular extension of the micronodules was limited to the immediate pericapsular adipose tissue and was not considered evidence of low-grade malignancy. Immunocytochemically, the nodules were positive for synaptophysin, inhibin-A, and melan A and negative for vimentin and CD56. Ki-67 antibody stained the cytoplasm of cells in the micronodules but not that of the atrophic cortical cells. The 4 patients had the PRKAR1A deletion that has been associated with the isolated form of primary pigmented nodular adrenocortical disease.

Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization of a new in-frame deletion.

BACKGROUND: Point mutations of the PRKAR1A gene are a genetic cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD), but in 30% of the patients no mutation is detected. OBJECTIVE: Set up a routine-based technique for systematic detection of large deletions or duplications of this gene and functionally characterize these mutations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) of the 12 exons of the PRKAR1A gene was validated and used to detect large rearrangements in 13 typical CNC and 39 confirmed or putative PPNAD without any mutations of the gene. An in-frame deletion was characterized by western blot and bioluminescence resonant energy transfer technique for its interaction with the catalytic subunit. RESULTS: MLPA allowed identification of exons 3-6 deletion in three patients of a family with typical CNC. The truncated protein is expressed, but rapidly degraded, and does not interact with the protein kinase A catalytic subunit. CONCLUSIONS: MLPA is a powerful technique that may be used following the lack of mutations detected by direct sequencing in patients with bona fide CNC or PPNAD. We report here one such new deletion, as an example. However, these gene defects are not a frequent cause of CNC or PPNAD.

Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension.

At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa-like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na(+)/K(+) ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.