6.15 Carney’s complex

Carney’s complex (CNC) is an autosomal dominant disorder, which was described in 1985 as ‘the complex of myxomas, spotty pigmentation, and endocrine overactivity’ in 40 patients (1). Since then, more than 500 index cases have been reported, resulting in better definition of the disease and the establishment of diagnostic criteria (2, 3). As implied from the initial description, CNC is not only a multiple neoplasia syndrome, but also causes a variety of pigmented lesions of the skin and mucosae. (4) Several patients described in earlier years under the acronyms NAME (nevi, atrial myxomas, and ephelides) and LAMB (lentigines, atrial myxomas, and blue nevi) probably had CNC (5, 6). Thus, lentigines, blue nevi, café-au-lait spots, and cutaneous tumours, such as myxomas, fibromas, and others, are major features of the disease (4, 7–10).

The clinical characteristics of CNC have been reviewed and are presented in Box 6.15.1 (2, 9). A definite diagnosis of CNC is given if two or more major manifestations are present (4, 9, 11, 12). A number of related manifestations may accompany or suggest the presence of CNC but are not considered diagnostic of the disease (Box 6.15.1). Cutaneous manifestations constitute three of the major disease manifestations: (1) spotty skin pigmentation with a typical distribution (lips, conjunctiva, and inner or outer canthi, genital mucosa); (2) cutaneous or mucosal myxoma; and (3) blue nevi (multiple) or epithelioid blue nevus. Suggestive or associated with CNC findings but not diagnostic are: (1) intense freckling (without darkly pigmented spots or typical distribution); (2) multiple blue nevi of common type; (3) café-au-lait spots or other ‘birthmarks’; and (4) multiple skin tags or other skin lesions, including lipomas and angiofibromas.

The relationship between the cutaneous and noncutaneous manifestations of CNC appears to be an essential clue to the molecular aetiology of the disease. According to the latest reports, more than half of CNC patients present with both characteristic dermatological and endocrine signs; however, a significant number of patients present with skin lesions that are only ‘suggestive’ and not characteristic of CNC (9). A recent classification based on both dermatological and endocrine markers has subgrouped CNC patients as: multisymptomatic (with extensive endocrine and skin signs); intermediate (with few dermatological and endocrine manifestations); and, paucisymptomatic (with isolated primary pigmented nodular adrenocortical disease (PPNAD) alone and no cutaneous signs) (9).

Skin lesions are consistently reported in the majority of the CNC patients (above 80%), the most common being lentigines (in 70–75% of cases). Other pigmented lesions, most frequently blue nevi and café-au-lait spots, with or without lentigines, are seen in approximately 50% of CNC patients. The effort to systemize the knowledge on the cutaneous lesions in CNC patients is driven by their high diagnostic value—presented early in life and easily recognizable, the skin manifestations are an early sign that directs dermatologists’ attention towards underlying endocrine or other pathology. In an attempt to outline the most specific and sensitive skin abnormalities in CNC, several research groups have published exhaustive analyses that add to an improved diagnostic and preventive approach (9, 10, 13). The major challenge appears to be in distinguishing the disease-associated prominent lesions from the more common non-CNC-specific, age- or sun-related skin alterations.

Characteristic CNC pigmented skin lesions are shown in Fig. 6.15.1. Lentigo is a hamartomatous melanocytic lesion, clinically similar but histologically different from freckles (14). Morphologically, lentigines are flat, poorly circumscribed, brown-to-black macules, usually less than 0.5 cm in diameter, but these may differ in different ethnic groups. In African-Americans, for example, lentigines may be slightly raised, dark papules, similar to nevi (14). In contrast to the common freckles, on histological examination lentigines show basal cell layer hyperpigmentation associated with an increased number of melanocytes (hyperplasia), the majority of which appear hypertrophic. This distinguishes them from freckles (ephelides), which present with a regular number of melanocytes and are pigmented as a result of melanin disposition in the surrounding keratinocytes.

Lentiginosis is one of the manifestations of CNC that can occur early; lentigines usually acquire their typical intensity and distribution during the peripubertal period (9, 10, 15). They typically involve the centrofacial area, including the vermilion border of the lips, and the conjunctiva, especially the lacrimal caruncle and the conjunctival semilunar fold; intraoral pigmented spots have also been reported (16). In contrast to age-related skin lesions, CNC-associated lentigines tend to fade after the fourth decade of life, but may be detectable as late as the eighth decade (9, 15).

The next very common skin manifestation in CNC is a lesion known as blue nevus, which is infrequent in the general population. Blue nevi can be seen as small (usually <5 mm), blue to black-coloured marks with a circular or star-shaped appearance. Their distribution is variable; most often they occur on the face, trunk, and limbs, and less frequently on the hands or feet.

An interesting subtype of blue nevus, which is exceedingly rare as a sporadic lesion in the general population but is sometimes seen in patients with CNC, is the epithelioid blue nevus (17). Epithelioid blue nevus usually presents with intensive pigmentation and poorly circumscribed proliferative regions containing two cell types: heavily pigmented globular and fusiform cells; and lightly pigmented, polygonal spindle melanocytes with a single prominent nucleolus. In contrast to blue nevi, epithelioid blue nevi display no dermal fibrosis (18). After comprehensive comparative analysis, and based on the fact the epithelioid blue nevi have also been reported in patients with none of the other features of CNC, epithelioid blue nevi are not considered pathognomonic for CNC but simply associated with the disease (9, 18).

Blue nevi and lentigines in CNC are often accompanied by café-au-lait spots, which are otherwise rarely present as an isolated skin manifestation of CNC. Like lentigines, café-au-lait spots can be present at birth. In general, café-au-lait spots in CNC are less intensely pigmented than those seen in McCune–Albright syndrome and they are more similar to those seen in the neurofibromatosis syndromes.

The third most common skin manifestation of CNC—cutaneous myxoma—is reported in between 30 and 55% of the studied patients (4, 9, 10). Cutaneous myxomas rarely exceed 1 cm in diameter and often affect the eyelids, ears, and nipples, but may also be seen on other areas of the face, ears, trunk, and perineum. They usually appear as asymptomatic, sessile, small, opalescent, or dark pink papules and large, finger-like, pedunculated lesions. They are typically diagnosed early in life, most often during the teenage years (mean age, 18 years). In the majority of patients (>70%) cutaneous myxomas show multiple appearance and a tendency to recur. The frequency of myxoma may be underestimated because of the sometimes difficult clinical diagnosis; therefore histological examination is strongly recommended when in doubt. Histopathologically, myxomas are characterized by a location in the dermis or, occasionally, more superficially in the subcutaneous tissues, sharp circumscription (sometimes encapsulation), relative hypocellularity with abundant myxoid stroma, prominent capillaries, lobulation (larger lesions), and occasional presence of an epithelial component. It is estimated that approximately 80% of CNC patients with life-threatening cardiac myxoma present with cutaneous myxoma earlier in life; therefore, cutaneous myxoma can serve as good marker for the disease with high prognostic significance (4, 9, 10).

Other CNC-related skin abnormalities include melanocytic and atypical nevi, and the so-called Spitz nevus. Occasionally, depigmented lesions can be present at birth or, more often, develop in early childhood. These manifestations, although usually not considered specific, may be suggestive for the disease or may accompany other CNC signs of importance for the diagnosis.

Most cases of CNC are caused by inactivating mutations in the gene encoding one of the subunits of the protein kinase A (PKA) tetrameric enzyme, namely regulatory subunit type 1 α (PRKAR1A), located at 17q22–24 (4). Although a second locus (2p16) has been implicated, sequencing of the region in the linked families did not reveal alterations in other coding sequences (19).

PRKAR1A extends to a total genomic length of approximately 21 kb and consists of 11 exons, encoding a total of 381 amino acids, with a dimerization/ docking domain, and two cAMP binding domains, A and B. Since the identification of PRKAR1A mutations in CNC, more than 100 disease-causing pathogenic sequence changes have been reported; they are spread over the entire coding sequence of the gene, without a notable preference for a region or exon. Structurally, the vast majority of the mutations consist of base substitutions, small deletions, and insertions or combined rearrangements, involving up to 15 bp (4); although rare, large PRKAR1A deletions have been reported (20).

Mutations in PRKAR1A are seen in more than 70% of the patients with classical CNC and, in the majority of these cases, they lead to complete inactivation of one of the PRKAR1A alleles as a result of premature stop codon generation and subsequent nonsense-mediated mRNA decay (NMD) (4, 10). In its inactive form, PKA is a tetramer composed of two regulatory and two catalytic subunits (21). The decreased cellular concentration of regulatory subunits results in a balance shift between the formation and the disassembly of the PKA tetramer, towards the release of the catalytic subunits. The free catalytic subunits, which are active serine–threonine kinases, further phosphorylate a series of targets that regulate downstream effectors and transcription of specific genes, mediating cell growth and differentiation (22). Thus, functionally, the mechanism by which PRKAR1A haploinsufficiency causes CNC is through excess cellular cAMP signalling in affected tissues (23). CNC lesions frequently show loss-of-heterozygosity, suggesting a tumour-suppressor function for PRKAR1A (4, 3).

Although significantly less frequent, mutations that escape NMD and lead to the expression of an abnormal, defective PRKAR1A protein have been reported (20, 24, 25). These expressed mutations may lead to a characteristic phenotype that reflects the location and the type of the genetic change. Examples include a germline in-frame deletion of exon 3 which results in severe expression of the majority of the CNC manifestations—a phenotype illustrating the importance of exon 3 in linking the dimerization/docking and the first cAMP binding domain (20). In contrast, another in-frame variant—a splice-site deletion that eliminates exon 7—is seen associated mostly with lentiginosis and the adrenal component of CNC, PPNAD. Just as lentiginosis is the most common nonendocrine CNC manifestation, PPNAD is the most frequently observed endocrine tumour of the disease. Thus, the presence of only two features of CNC, the most common ones, with this splice-site variant is consistent with the anticipation of a milder phenotype associated with certain splice mutations, due to their incomplete penetrance at the mRNA level, (i.e. not all DNA molecules harbouring the splice variant result in mRNA species lacking exon 7) (24–26).

Apart from the above mentioned, expressed mutant PRKAR1A isoforms, several other expressed isoforms that result from single amino acid substitutions have been reported (25, 26). Detailed in vitro analysis of their effects on protein function have revealed important PRKAR1A domain features (26, 27). The six naturally occurring missense substitutions examined by this study (Ser9Asn, Arg74Cys, Arg146Ser, Asp183Tyr, Ala213Asp, Gly289Trp) are spread over all the functional domains of the protein. Although, as mentioned before, the low number of individuals affected by each of these mutations prevented detailed phenotype–genotype analysis, these studies support the previous suggestion that the alteration of PRKAR1A function alone (and not only its complete loss) is sufficient to increase PKA activity, leading to CNC.

Until recently, no genotype–phenotype correlations had been found for the different stop codon mutations, which are expected to uniformly lead to lack of the PRKAR1A mutant allele’s protein product in cells. This was because most of the mutations were identified in single patients only and only two (c.491–492delTG/p.Val164fsX4, and c.709(−7–2) del6(TTTTTA)) had been seen in more than three kindreds (4, 24). The first study to explore all PRKAR1A mutations found to date against all CNC phenotypes was recently completed; 353 individuals, 258 of whom (73%) were positive for a PRKAR1A mutation, were studied (10). Several features that distinguish PRKAR1A mutation carriers from mutation-negative CNC patients were identified; the former presented more frequently and earlier in life with pigmented skin lesions, myxomas, thyroid, and gonadal tumours. In addition, essential correlations between certain genetic defects and the severity and type of CNC manifestations were found. Bertherat et al. (10) outlined subgroups of patients; the first group presented with isolated PPNAD, in some cases accompanied with lentiginosis. In this group the following tendencies were observed: (1) patients diagnosed before 8 years of age were rarely carriers of PRKAR1A mutations; and (2) most of the patients with isolated PPNAD and the presence of PRKAR1A mutation were carriers of either the c.709(−7–2) del6(TTTTTA) mutation (p <0.0001) or the c.1A>G/p.Met1Val substitution affecting the initiation codon of the protein. These observations were in line with previously published reports (4, 24) and both mutations are rather rare. Although the molecular mechanism of the Met1Val substitution is not completely clear, it is the only mutation that alters the protein initiation site, and may, in theory, result in alternative initiation (28); c.709(−7–2) del6(TTTTTA) is a splice variant that is expected to result in an exon skip, frame shift, and premature stop codon generation. However, since it does not affect the two immediate nucleotides on either site of the splice junction, it is expected to lead to splicing in less than 100% of the molecules that harbour it, and thus, presumably, to lead to a milder phenotype. The fact that a milder phenotype involves only the adrenal and skin is suggestive of their high sensitivity to changes in PKA activity.

The second group of CNC patients that was suggested to have a particular genotype–phenotype correlation comprised individuals with myxomas (affecting all locations—skin, heart, and breast), PMS, thyroid tumours, and large-cell calcifying Sertoli cell tumours (LCCSCT). In these patients, PRKAR1A mutations were seen substantially more often. Related to this is the recognition that certain tumours present at a significantly younger age in PRKAR1A mutation carriers: cardiac myxomas (p = 0.02), thyroid tumours (p = 0.03), and LCCSCTs (p = 0.04) (10). Another finding in these patients was that mutations that escaped NMD and led to an alternate, usually shorter, protein were associated with an overall higher total number of CNC manifestations (p = 0.04).

In terms of pigmented skin lesions in CNC, two important correlations have been observed: (1) lentigines (as well as PMS, acromegaly, and cardiac myxomas), were seen significantly more often in CNC patients with exonic PRKAR1A mutations, compared to those with intronic ones (p = 0.04); and (2) lentigines (as well as cardiac myxoma and thyroid tumours) were significantly associated with the hot spot c.491–492delTG mutation compared to all other PRKAR1A defects (p = 0.03). These data add greatly to the understanding of the molecular mechanisms of the involvement of PRKAR1A in endocrine and other tumourigenesis and, thus, for genetic counselling and prognosis in CNC families.

Interestingly, a 2.3-Mb deletion in chromosome band 17q24.2–q24.3, which involved PRKAR1A together with another 13 genes, resulted in a number of clinical features, including posterior laryngeal cleft, growth restriction, microcephaly, and moderate mental retardation. The only CNC manifestation was numerous freckles and lentigines at a young age (29); the authors called the observed phenotype ‘CNC plus’.

To date, the molecular causes underlying the formation of pigmented skin lesions in CNC are not fully understood. A possible mechanism involves the PKA-mediated activation of pathways downstream of the melanocortin receptors (MCRs), which form a subfamily of the G protein-coupled receptors (GPCRs) and regulate a wide variety of processes, including skin pigmentation (30–32). The melanocortin 1 receptor (MC1R) is expressed preferentially in epidermal melanocytes and is known to be the key regulator of mammalian pigmentation (31, 33). MC1R is stimulated by the proopiomelanocortin-derived melanocyte-
stimulating hormone and ACTH and, in turn, activates the rate-limiting enzyme in melanin synthesis, tyrosinase. As a GPCR, MC1R is positively coupled with adenylate cyclase, and its actions are mainly mediated by PKA, in coordination with other signalling molecules involving protein kinase C (PKC) and MAPKs (34–36).

CNC shares clinical features and molecular pathways with several other familial lentiginosis syndromes, such as McCune–Albright syndrome (OMIM #174800), Peutz–Jeghers (OMIM #175200), LEOPARD (OMIM #151100), Noonan’s (OMIM #163950), Cowden’s disease (OMIM #158350), and Bannayan–Ruvalcaba–Riley syndrome (OMIM #153480). In all of these conditions skin lesions accompany underlying endocrine and/or other abnormalities, which, as in CNC, are considered an important diagnostic sign.

Probably the closest, at least in terms of a molecular pathway link, to CNC is McCune–Albright syndrome. Patients with this condition have characteristic lesions that affect predominantly three systems: the skin, the endocrine system, and the skeleton. The café-au-lait spots in McCune–Albright syndrome patients are similar to those observed in CNC, but tend to be more intensely pigmented. McCune–Albright syndrome is caused by postzygotic, activating, somatic mutations of GNAS, located on 20q13, which encodes the adenylate cyclase-stimulating G α protein (Gsa) of the heterotrimeric G protein (37). G proteins couple hormone receptors to adenylyl cyclase and are therefore required for hormone-stimulated cAMP synthesis. Because of the somatic nature of the genetic defect, the presentation of the disease is mosaic and the level of clinical involvement of any tissue is highly variable. The mutations in GNAS are always missense substitutions at the critical sites for the GTPase inactivation (amino acid positions Arg201 and Gln227), and, in contrast to PRKAR1A defects, lead to constant protein activation and prolonged cAMP production.

We have reported another endocrine lesion that is associated with increased tissue levels of cAMP, isolated micronodular adrenocortical hyperplasia (iMAD). In these patients, inactivating mutations in the genes encoding phosphodiesterases types 11A (PDE11A) and 8B (PDE8B) have been reported (38–40). iMAD patients were initially considered CNC patients, but it soon became clear that iMAD is not the same as PPNAD (41).

Peutz–Jeghers syndrome, another autosomal dominant familial lentiginosis syndrome, is characterized by melanocytic macules of the lips, buccal mucosa, and digits, multiple gastrointestinal hamartomatous polyps, and an increased risk of various neoplasms. The lentigines observed in patients with Peutz–Jeghers syndrome shows similar density and distribution to the ones in CNC. Peutz–Jeghers syndrome has been elucidated at the molecular level (42, 43); the disease was first mapped to chromosome 19p13.3 and, soon after, the gene encoding the serine–threonine kinase 11 (STK11 also known as LKB1) was found to be mutated in most patients (44–49). The proposed mechanism of the disease is through elimination of the kinase activity of the STK11/LKB1 tumour suppressor protein.

LEOPARD is an acronym for the manifestations of the syndrome comprising: multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness (50). LEOPARD is allelic to Noonan’s syndrome; both diseases are linked to mutations in PTPN11 (12q24), the gene encoding the nonreceptor tyrosine phosphatase Shp-2 (51, 52). The protein encoded by this gene is a member of the protein tyrosine phosphatase family, proteins that are known to regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation.

Cowden’s disease and Bannayan–Ruvalcaba–Riley syndrome share clinical characteristics, including mucocutaneous lesions, hamartomatous polyps of the gastrointestinal tract, and increased risk of developing neoplasms. Both conditions are caused by mutations in the PTEN gene (53–55). PTEN is located on 10q23.31 and encodes phosphatidylinositol-3, 4, 5-trisphosphate 3-phosphatase. The gene was recognized as a tumour suppressor gene and has been found to be mutated in a number of tumours (56). It contains a tensin-like domain as well as a catalytic domain similar to that of the dual-specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine phosphatases, PTEN preferentially dephosphorylates phosphoinositide substrates. It negatively regulates intracellular levels of phosphatidylinositol-3, 4, 5-trisphosphate in cells and its tumour suppressor effect is expressed by inhibition of the AKT/PKB signalling pathway.

The overlapping clinical manifestations of these syndromes, which are caused by distinct molecular defects, suggest crosstalk between the involved pathways. Indeed, PRKAR1A inactivation leads to phosphorylation of mTOR and ERK1/2 (57, 58), LKB1 is phosphorylated by PKA (59), and PTEN expression is positively regulated by transcription factor Egr-1 in a PKA-dependent manner (60).

Studies on CNC and related syndromes have been supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development, NIH, intramural project Z01-HD-000642–04 (to Dr C. A. Stratakis).