Clinical and molecular genetics of Carney complex

Carney complex (CNC) is a rare multiple familial neoplasia syndrome that is characterized by multiple types of skin tumors and pigmented lesions, endocrine neoplasms, myxomas and schwannomas and is inherited in an autosomal dominant manner. Clinical and pathologic diagnostic criteria are well established. Over 100 pathogenic variants in the regulatory subunit type 1A (RI-A) of the cAMP-dependant proteine kinase (PRKAR1A) have been detected in approximately 60% of CNC patients, most leading to R1A haploinsufficiency. Other CNC-causing genes remain to be identified. Recent studies provided some genotype-phenotype correlations in CNC patients carrying PRKAR1A-inactivating mutations, which provide useful information for genetic counseling and/or prognosis; however, CNC remains a disease with significant clinical heterogeneity. Recent mouse and in vitro studies have shed light into how R1A haploinsufficiency causes tumors. PRKAR1A defects appear to be weak tumorigenic signals for most tissues; Wnt signaling activation and cell cycle dysregulation appear to be important mediators of the tumorigenic effect of a defective R1A.

Introduction

Carney complex (CNC) is a rather unique, relatively rare autosomal dominant multiple endocrine neoplasia syndrome first described by Dr. J. Aidan Carney in 1985 as ‘the complex of myxomas, spotty pigmentation, and endocrine overeactivity’. Most patients previously diagnosed with having the syndromes known as LAMB (lentigines, atrial myxoma, myxoid neurofibroma and ephelide) or NAME (navi, atrial myxoma, blue nevi) would now be more appropriately described as CNC.1, 2 To this date, over 500 patients of diverse ethnicity from all continents have been registered by the NIH-Mayo clinic (USA) and the Cochin Hospital (France); 43% are males and 57% are females; approximately 70% are familial cases.³

The diagnosis of CNC is made if two or more major manifestations of the syndrome are present (Table 1).*3, *4, *5, 6 These must be confirmed by histology, biochemical testing, and imaging. However, one can also make the diagnosis when only one of the major criteria is present if the patient is a carrier of a known inactivating mutation of PRKAR1A.⁷ Additionally, a considerable number of clinical and biochemical manifestations listed in Table 1 are suggestive, but not diagnostic, of CNC.

Pigmented skin lesions are reported in the majority of CNC patients (over 80%). They constitute one of the three major criteria of CNC and are diagnostically very important because, easily recognizable and occurring early in life, they may lead to early detection of a potentially life threatening disease. The most common skin lesions are lentigines (they are present in 70–75% of cases); these are hamartomatous melanocytic lesions clinically similar to, but histologically different from freckles (ephelides). Morphologically, lentigines are flat, poorly circumcised, brown-to-black macules usually measuring less than 0.5 cm in diameter. The appearance of lentigines may vary in different ethnic groups. For example, in African-Americans, lentigines may be slightly raised, dark papules, similar to nevi. Histologically, lentigines show hyper-pigmentation of the basal cell layer associated to melanocytic hyperplasia and hypertrophy. In contrast, the pigmentation in common freckles is the result of increased melanin production, without, usually, any melanocytic hyperplasia.8, *9 Even though lentigines may be the first sign of CNC at birth, they usually do not acquire their typical intensity and characteristic distribution (lips, conjunctiva and inner and outer canthi; vaginal and penile mucosa) until the late prepubertal and early peripubertal period.*4, *10 CNC-related lentigines tend to fade after the fourth decade of life, in contrast to common age- or sun-related skin macules.*4, 11

The second most frequent skin manifestations in CNC patients are blue nevi, and in particular, epithelioid blue nevi. These are small (usually < 5 mm), circular or star-shaped, blue to black marks which are variably distributed (often on the face, trunk, limbs) and uncommon in the general population.*9, *10

Cutaneous myxomas are the third most frequent skin manifestation in CNC patients (30–55% of studied patients).*4, *10 They are preferably located on the eyelids, ears and nipples but can also be seen in other areas. Cutaneous myxomas are asymptomatic, small (rarely bigger than 1 cm in diameter), opalescent or dark pink papules which can be diagnosed early in life (mean age 18 yr). In the majority of patients (>70%), cutaneous myxomas show multiple locations and have a tendency to recur. Histologically they are characterized by their location in the dermis or exceptionally in the sub-cutaneous tissues, they are well circumscribed, hypo-cellular with an abundant myxoid stroma, prominent capillaries, and occasional lobulation, especially in larger lesions. The frequency of cutaneous myxomas is probably underestimated; they are often confused with common “skin tags” and other overgrowths (collagenomas, fibromas). Early diagnosis is essential since it is estimated that almost 80% of CNC patients with a life-threatening cardiac myxoma had presented earlier in life with cutaneous myxoma.*3, *4, *10 Thus, when there is clinical suspicion, one should not hesitate to perform a histological examination of a lesion.

Other CNC-related skin manifestations include: café-au-lait spots (typically less pigmented than those seen in Mc-Cune Albright syndrome) or depigmented lesions that can also be present at birth or develop during childhood; melanocytic and atypical nevi, and the so called Spitz nevus.

Cardiac myxomas, which can appear as early as in infancy, are responsible for over 50% of the disease-specific mortality of CNC patients. Early detection and regular screening for cardiac myxomas by echocardiography is essential, as these tumors can lead to sudden death by embolism, strokes, or cardiac failure. Cardiac myxomas are the most common, clinically significant non-cutaneous lesions in CNC patients. Compared to non-CNC-related, sporadic myxomas that are more common in older women and almost always are solitary and affect the left atrium, CNC-associated cardiac myxomas have no predilection for age, gender, and cardiac location; they are also often multiple, tend to recur frequently, and grow rather aggressively.*3, *5

Pigmented nodular adrenocortical disease (PPNAD) (Fig. 3) is the most frequent endocrine manifestation in CNC patients. Adrenocorticotropic hormone-independent Cushing syndrome (CS), is present in 25–30% of CNC patients.¹² PPNAD is named after the macroscopic appearance of the adrenal glands that is characterized by small, cortisol-producing, pigmented micro-nodules (less than 1 cm in diameter) of the adrenal cortex. Diagnosis of CS due to PPNAD is often difficult because hypercortisolism can develop progressively over years and may be periodic: cyclical and atypical CS is more often the rule, rather than the exception, among patients with CNC.*3, 12 Diagnosis is established using the 6-day Liddle test: patients with PPNAD show a “paradoxical” increase in their 24-hour urinary free cortisol and/or 17-hydroxysteroids that is progressive and reaches its peak on the second day of the high-dose dexamethasone administration.¹³ Occasionally, patients with chronic, atypical CS will present with an asthenic rather than obese body habitus; these patients have for many years normal or near-normal 24-hour urinary free cortisol production but consistently abnormal cortisol diurnal rhythm.¹⁴

The true incidence of PPNAD among patients with CNC is probably under-estimated biochemical testing with a dexamethasone stimulation test has been shown to detect additional patients with sub-clinical, atypical, or periodic Cushing’s syndrome.¹³ Furthermore, histological evidence of PPNAD has been found in almost every patient with CNC who underwent an autopsy. Pathological investigation reveals that adrenal glands from patients with PPNAD are usually normal in size and weight; it is for this reason, that one out of three patients has essentially normal-appearing adrenal glands on computed tomography (CT-scan). The remaining patients may have visible round micronodules (smaller than 1 cm in diameter) or, rarely, macronodules (larger than 1 cm) within the background of hyperplasia.

PPNAD has a bimodal age of distribution. Most cases are diagnosed during the second and third decade of life; however, a substantial proportion of patients present during early childhood (2-10 years).³

Growth hormone (GH) secreting pituitary hyperplasia and adenomas leading to the biochemical hallmarks of acromegaly, with elevation of insulin-like growth factor 1 (IGF 1) and GH levels, and subtle hyperprolactinemia, can be present in up to 75% of CNC patients. However, clinical acromegaly due to a rapidly growing adenoma remains rare and may be seen in only 10–20% of the patients. These GH-producing adenomas do not appear until the third decade of life. Biochemical acromegaly is often unmasked by abnormal results of an oral glucose tolerance test or paradoxical responses to thyrotropin-releasing hormone administration.¹⁵

Large-cell calcifying Sertoli cell tumors (LCCSCT) (Fig. 4) is a very common, almost always benign tumor (only one case of malignancy has been reported in a 62-year-old man) in male CNC patients which appear in the first decade of life and are most often diagnosed during routine testicular ultrasound when micro-calcifications are found. The earliest detection of LCCSCT (by ultrasonography) was made in a two year old boy. Only rarely, LCCSCTs in CNC (as in Peutz-Jeghers syndrome) may have endocrine consequences and lead to gynecomastia (due to increased P-450 aromatase expression) and precocious puberty. LCCSCT may impair fertility through obstruction of semiferous tubules or inappropriate hormone production or aromatization. Leydig cell tumors and adrenal rests have also been reported in CNC patients but only with the concomitant presence of LCCSCT.*3, 7 Testicular adrenal rests in these patients are also affected by PPNAD and can lead to recurrent Cushing syndrome after adrenalectomy in patients with CNC and PPNAD.

Thyroid gland disease in CNC is a spectrum spanning from follicular hyperplasia and nodular disease to carcinoma. Up to 75% of CNC patients may have multiple thyroid nodules, detected as small, hypo echoic lesions on ultrasonography.¹⁶ Thyroid nodules often appear within the first ten years of life. On biopsy, follicular adenoma is the most common finding, whereas thyroid cancer, follicular or papillary, may develop in up to 10% of CNC patients with preexisting thyroid pathology.⁷

Ovarian cysts are often found on ultrasonography as multiple hypoechoic lesions. Although usually clinically insignificant, they may occasionally progress to ovarian carcinoma.17, 18

PMS was present in 8% of CNC patients studied by Bertherat and al; and four patients died of metastatic PMS.¹⁰ PMS may occur anywhere in the peripheral nervous system, but it is most frequently found in the gastrointestinal tract (esophagus and stomach) and the paraspinal sympathetic chain. Schwannomas in CNC are characterized by their heavy pigmentation (melanin), frequent calcifications, and multicentricity. CNC is the only genetic condition other than the neurofibromatosis syndromes and isolated familial schwannomatosis that includes schwannomas as a manifestation; however, PMS is unlike any of the schwannomas seen in these other conditions.*3, 19, 20

Osteochondromyxoma is one of the latest components of the disorder to be described.²¹ It usually affects the long bones and rarely the facial skeleton and spine. These tumors become apparent early in life and are often confused to osteochondromas or osteosarcomas.

Genetic linkage analysis identified two independent loci for CNC, CNC1 located on chromosome 17p22–24 and CNC2 located on chromosome 2p16.*22, *23 The gene responsible for CNC at locus 2p16 remains unknown. In most cases, CNC is caused by inactivating mutations in the regulatory subunit type 1 alpha gene (PRKAR1A) located at 17q22–24 which encodes the most widely expressed of the protein kinase A (PKA) regulatory subunits.³ Thus, PRKAR1A is a key component of the cAMP signaling pathway. PRKAR1A’s genomic region is approximately 21 kb-long and the open reading frame contains 11 exons that code for a protein that totals 384 amino acids. The peptide consists of a dimerisation/docking domain and two cAMP binding domains (A and B) that are essential for its function within the PKA tetramer (see below).

To date, over 100 disease-causing pathogenic sequence variants, spread all over the coding length of the gene, have been identified. These coding variants show no preference for a particular genomic region or exon. A recent extensive update by Horvath and al., reviewed all the known PRKAR1A mutations²⁴; an online database is available at http://prkar1a.nichd.nih.gov. The majority of CNC-causing PRKAR1A mutations are base substitutions, small deletions and insertions or rearrangements.³ Rarely, large PRKAR1A deletions can occur.²⁵.

Over 70% of CNC patients with a classical phenotype show a PKAR1A mutation which leads to a premature stop codon and subsequently non-sense mediated mRNA decay (NMD)*3, *10 and, thus, PRKAR1A haploinsufficiency. The mutations that escape NMD are significantly less frequent and lead to the expression of an abnormal and defective PRKAR1A protein.25, *26, 27, 28 The in vitro effect of a certain number of these expressed mutations has been evaluated and confirmed their pathogenic potential.27, 29

Until recently, no genotype-phenotype correlations had been found for the stop codon mutations that lead to PRKAR1A haploinsufficiency. This is because most of the PRKAR1A mutations in CNC patients are family-or patient-specific and only three mutations have been identified in more than three kindreds: c.82t, c.491–492delTG in exon 5 and c.709-7del6 in intron 7.*3, *26 Recently, Bertherat and al. reported a genotype-phenotype study in 353 CNC patients of which 73% were positive for a PRKAR1A mutation.¹⁰ PRKAR1A mutations were observed in 80% of the familial cases compared to 37% of the sporadic CNC patients. The overall penetrance of CNC in PRKAR1A-mutated patients was 97.5%. Patients with PRKAR1A mutations more frequently had myxomas, skin lesions, thyroid and gonadal tumors and these clinical manifestations appeared at an earlier age compared to CNC patients that did not have PRKAR1A mutations or deletions. Acromegaly, cardiac myxoma, lentigines and PMS were more often associated with exonic mutations. Other findings of this study included (1) the hot-spot c.491–492del TG mutation was more significantly associated with cardiac myxoma, lentigines and thyroid tumors when compared to all other PRKAR1A mutations; and (2) mutations that escaped NMD were associated with a higher total number of CNC manifestations. An ongoing study, by the same group, is evaluating the clinical implications of the co-occurrence of phosphodiesterase 11A (PDE11A) mutations in CNC patients with PRKAR1A mutations. The PDE11A locus was identified by a genome-wide SNP genotyping study in individuals with adrenocortical hyperplasia leading to Cushing’s syndrome that was not caused by known genetic defects.³⁰ More CNC-causing genes remain to be identified as over 60% and 25% of respectively sporadic and familial cases are negative for PRKAR1A mutations.

In its inactive form, cyclic AMP (cAMP)-dependent PKA is a holotetramer composed of two regulatory (R) and two catalytic (C) homodimers. The tetramer is dissociated into two regulatory and two free enzymaticaly active catalytic subunits when intra-cellular cAMP levels increase and two molecules of cAMP bind to each of the regulatory subunits.31, 32 In turn, free catalytic subunits phosphorylate a variety of cellular target substrate proteins, that regulate a wide range of cellular processes: transcription, metabolism, cell cycle progression and apoptosis.33, 34 So far, four different regulatory subunits (PRKAR1A, PRKAR1B, PRKAR2A, and PRKAR2B) and four catalytic subunits (PRKACA, PRKACB, PRKACG and PRKX) have been identified. It is the PRKAR1A gene coding for the PKA type I-A regulatory subunit (R-Iα) that is mutated in CNC and PRKAR1A is the only PKA subunit in which mutations have been found to lead to human disease.

PRKAR1A haploinsufficiency leads to excess cellular cAMP signaling in affected tissues.*23, 35 Loss of R-Iα leads to an increase in total (but not only PKA-specific) cAMP-stimulated kinase activity.*23, 36 Two mechanisms are suggested to explain the increase in total cAMP signaling: 1. R-Iα haploinsufficiency leads to a higher intracellular C to R subunit ratio, and thus, increased availability of free catalytic subunits that phosphorylate downstream targets. 2. R-Iα haploinsufficiency leads an upregulation of other components of the PKA tetramer including both type I (PRKAR1B) and type II (PRKAR2A or PRKAR2B) subunits, in a tissue-dependent manner¹⁵; these other regulatory subunits may not be able to control the catalytic subunit as effectively as PRKAR1A, at least not in the presence of high cAMP levels. Data on animal models of prkara1a downregulation or haploinsufficiency are consistent with these mechanisms.37, 38, 39

The role of R1α has been explored in several different cancer tissues and cell lines, including colorectal, breast, renal, and ovarian cancer.40, 41, 42, 43, 44, 45, 46 The CNC data suggest that PRKAR1A is a tumor suppressor gene, as tumors from CNC patients frequently carry both the germline mutation and LOH of the 17q22–24 PRKAR1A locus. However, a few CNC tumors did not show PRKAR1A LOH*23, 36 and, in certain tissues, loss of heterozygocity is probably only one factor leading to tumerigenesis. Other yet unidentified genetic abnormalities contribute to neoplastic transformation in CNC.

Mouse models have shed some light into the role of PRKAR1A haploinsufficiency in tumor formation in cAMP-responsive tissues. Prkar1a knock-out (KO) animals died in utero due to failed cardiac morphogenesis.⁴⁷ Heterozygous KO (HetKO) mice developed tumors in cAMP-responsive tissues (non-pigmented schwannomas, bone lesions and thyroid neoplasias) but they lacked some characteristic for CNC lesions, like cardiac and skin myxomas, and pituitary adenomas. However, compared to human CNC tumors, cAMP signaling in HetKO mouse cells was only modestly increased, which may explain the lower susceptibility of the mouse pituitary, heart and skin in tumor formation.³⁹ A different mouse model, which led to significantly higher PRKAR1A downregulation, showed higher cAMP signaling and an overall more severe phenotype, including endocrine manifestations such as hypercorticosteronemia and thyroid cancer, and an overall shorter lifespan.37, 38 Tissue-specific, pituitary and cardiac, complete Prkar1a-KO mice, did develop pituitary adenomas and cardiac lesions that resembled human myxomas, respectively.48, 49

A recent mouse model studied the effect of PRKAR1A haploinsufficiency on tumor formation in the background of other known tumor suppressor gene defects and chemically induced skin papillomas. Interestingly, Prkar1a^+/−Trp53^+/− and Prkar1a^+/−Rb1^+/− double heterozygote mice developed more sarcomas and grew more and larger pituitary and thyroid tumors compared to the single HetKO Trp53^+/− and Rb1^+/− heterozygous mice, respectively. Similarly, HetKO Prkar1a^+/− mice developed more papillomas than wild-type animals after chemical induction.⁵⁰ Thus, Prkar1a haploinsufficiency augmented the previously described phenotype for the respective mouse models without causing any new tumors in other cAMP-responsive or other, tissues. In the same study, Wnt signaling and cell cycle abnormalities were identified by whole-genome transcriptome profiling as the main pathways activated by abnormal cAMP signaling confirming recent data from human studies which identified somatic β-catenin (CTNNB1) mutations in PRKAR1A-haploinsufficient tumors.51, 52 These studies showed that PRKAR1A haploinsufficiency maybe an overall relatively weak tumorigenic signal, unless it is associated with other tumor suppressor gene defects, Wnt signaling activation, and cell cycle dysregulation, mainly an increase in the expression of cyclin D1 and the transcription factor E2F1.⁵⁰

Pediatric patients with CNC should have an echocardiography during the first 6 months of life and annually thereafter. If there is a history of an excised myxoma, echocardiographic evaluation is necessary every six months. For children, growth rate and clinical examination alone are enough to screen for other manifestations of CNC. Although detectable at an early age, most endocrine tumors in CNC become clinically significant during the second decade of life and imaging or biochemical screening in prepubertal children is not considered necessary except for diagnostic purposes (when it is clinically indicated).

For adult and post-pubertal CNC-patients the following annual work-up is recommended: echocardiogram, measurement of urinary free cortisol (possibly supplemented by diurnal cortisol levels and an overnight 1 mg dexamethasone testing), and serum IGF-1 levels. For male patients, annual testicular ultrasound is recommended, if calcifications suggesting LCCSCT were identified on initial work-up. Similarly, thyroid ultrasound should be obtained at initial evaluation and repeated annually as needed (abnormal palpation, history of nodules). In females, ovarian ultrasound is recommended at initial evaluation and repeated if an abnormality was detected. For PPNAD screening, diurnal cortisol levels (23 h30 and 24 h nighttime samples and 7 h30 and 8 h00 morning samples), a dexamethasone stimulation test (protocol and interpretation suggested by Stratakis et al.¹³) and adrenal CT-scans are recommended. Early acromegaly may be detected by obtaining oGGT, in addition to IGF-1 levels and pituitary MRI. These tests may be abnormal in patients with CNC years before a pituitary tumor is visible on MRI (if one is ever detected).⁵³ Brain and spine MRIs should be obtained once at the original evaluation of any adult patient with CNC, and not repeated, unless clinical neurological signs suggest the possibility of a schwanomma (PMS).

Cardiac myxomas need to be removed surgically. The best treatment for PPNAD is bilateral adrenalectomy, although under certain circumstances ketoconazole or mitotane may be used for medical adrenalectomy. GH-producing pituitary adenomas may be removed surgically, but treatment of acromegaly with somatostatin analogues may also be used either as a primary treatment or as an adjuvant to surgery. PMS is the most difficult tumor to treat: these lesions localize in or around nerve roots along the spine, which makes them most of the time inoperable. In addition, 10% of schwannomas are malignant and may metastasize to the lungs, liver or brain.