The clinical characteristics of CNC have been recently reviewed and are also presented in Table 1 (Boikos and Stratakis, 2006; Mateus et al., 2008). A definite diagnosis of CNC is given if two or more major manifestations are present (Bertherat, 2006; Mateus et al., 2008; Sandrini and Stratakis, 2003; Stratakis et al., 2001). A number of related manifestations may accompany or suggest the presence of CNC, but are not considered diagnostic of the disease (Table 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.

Characteristic for CNC pigmented skin lesions are presented on Figure 1. Lentigo is a hamartomatous melanocytic lesion clinically similar but histologically different from the freckles (Stratakis, 2000). Morphologically, lentigines are flat, poorly circumscribed, brown-to-black macules usually less than 0.5 cm in diameter, but may differ in different ethnic groups. In African-Americans, for example, lentigines may be slightly raised, dark papules, similar to nevi (Stratakis, 2000). In contrast to the common freckles, on histologic 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 increased melanin production and 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 (Bertherat et al., 2009; Mateus et al., 2008; Young et al., 1989). 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 (Carney, 1995). In contrast to the 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 (Mateus et al., 2008; Young et al., 1989).

The next very common skin manifestation in CNC is a lesion known as blue nevus that is infrequent in the general population. Blue nevi can be seen as small (usually <5 mm), blue to black–colored marks with 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 that is exceedingly rare as a sporadic lesion in the general population but is sometimes seen in patients with CNC is the epithelioid blue nevus (EBN) (Zembowicz et al., 2004). EBN 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 the other blue nevi, EBN display no dermal fibrosis (Carney and Ferreiro, 1996). After comprehensive comparative analysis, and, based on the fact the EBN have also been reported in patients with none of the other features of CNC, EBN are not considered pathognomonic for CNC, but simply associated with the disease (Carney and Ferreiro, 1996; Mateus et al., 2008).

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 (Bertherat et al., 2009; Mateus et al., 2008; Stratakis et al., 2001). 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 teen years (mean age, 18 yr). In the majority of the 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 the CNC patients with the life threatening cardiac myxoma present with cutaneous myxoma earlier in life; therefore, cutaneous myxoma can serve as a good disease marker with high prognostic significance (Bertherat et al., 2009; Mateus et al., 2008; Stratakis et al., 2001).

PRKAR1A extends to a total genomic length of approximately 21 kb and consists of 11 exons, encoding a total of 381 AA, assembled in a dimerisation/docking domain, and two cAMP binding domains – A and B. Since the identification of PRKAR1A mutations in CNC, more than 80 disease-causing pathogenic sequence changes have been reported; they are spread all over the coding length 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 (Stratakis et al., 2001); although rare, large PRKAR1A deletions have been reported (Horvath et al., 2008a).

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 non-sense mediated mRNA decay (NMD) (Bertherat et al., 2009; Stratakis et al., 2001). In its inactive form, PKA is a tetramer composed of two regulatory and two catalytic subunits (Tasken et al., 1997). The decreased cellular concentration of regulatory subunits results in balance shift between the formation and the disassembly of the PKA tetramer, toward 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 enzymes, ion channels, and activate the transcription of specific genes mediating the cell growth and differentiation (Shabb, 2001). Thus, functionally, the mechanism by which PRKAR1A haploinsufficiency causes CNC is through excess cellular cAMP signaling in affected tissues (Robinson-White et al., 2006). CNC lesions frequently show loss-of-heterozygosity (LOH) suggesting a tumor-suppressor function for PRKAR1A (Boikos and Stratakis, 2007; Stratakis et al., 2001).

Although significantly less frequent, mutations that escape NMD and lead to the expression of an abnormal, defective PRKAR1A protein have been reported (Groussin et al., 2006; Horvath et al., 2008a; Meoli et al., 2008;Veugelers et al., 2004). These expressed mutations may lead to characteristic phenotype that reflects the location and the type of the genetic change. Examples include a germline ‘in frame’ deletion of exon 3 that results in severe expression of the majority of the CNC manifestations – a phenotype illustrating the importance of exon 3 in linking the dimerisation/docking and the first cAMP binding domain (Horvath et al., 2008a). 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, primary pigmented nodular adrenocortical disease (PPNAD). Like lentiginosis is the most common non-endocrine CNC manifestation, PPNAD is the most frequently observed endocrine feature of the disease. Thus, the presence of only two features of CNC, and just the common ones, with the described 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 (etc. not 100% the DNA molecules harboring the splice variant result in mRNA species lacking exon 7) (Greene et al., 2008; Groussin et al., 2006; Veugelers et al., 2004).

Apart from the above mentioned expressed mutant PRKAR1A isoforms, several other expressed isoforms that result from single aminoacid substitutions have been reported (Greene et al., 2008; Veugelers et al., 2004). Detailed in vitro analysis of their effects on the protein function revealed important PRKAR1A domain features (Greene et al., 2008; Horvath et al., 2008b). The six naturally occurring missense substitutions examined by this study (Ser9Asn, Arg74Cys, Arg146Ser, Asp183Tyr, Ala213Asp, Gly289Trp) are spread all over the functional domains of the protein. Although, as mentioned before, the restricted number of affected individuals by each of these mutations prevented detailed phenotype-genotype analysis, these studies supported the previous suggestion that the alteration of PRKAR1A function alone (not only its complete loss) is sufficient for increasing PKA activity leading to CNC.

Until recently, no genotype-phenotype correlations had been found for the different stop codon mutations that 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 and only two [c.491_492delTG/p.Val164fsX4, and c.709(−7-2) del6(ttttta)] had been seen in more than three kindreds (Groussin et al., 2006; Stratakis et al., 2001). The first study to explore all PRKAR1A mutations found to date against all CNC phenotypes was recently completed: 353 individuals, 258 of whom (73%) positive for a PRKAR1A mutation were studied (Bertherat et al., 2009). Several features that distinguished 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 tumors. In addition, essential correlations between certain genetic defects and the severity and type of CNC manifestations were found. Bertherat et al. outlined subgroups of patients: the first group presented with isolated PPNAD, in some cases accompanied with lentiginosis. In these group, the following tendencies were observed: (1) patients diagnosed before 8 yr of age were rarely carriers of PRKAR1A mutation; (2) most of the patients with isolated PPNAD and 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 (Groussin et al., 2006; Stratakis et al., 2001) and both mutations are rather unique. Although the molecular mechanism of the Met1Val is not completely clear, it is the only mutation that alters the protein initiation site, and, may in theory, result in alternative initiation (Kirschner et al., 2000). The splice variant c.709 (−7-2)del6(ttttta) is expected to result in an exon skip, frameshift and premature stop codon generation; however, since it does not affect the two immediate nucleotides on any site of the splice junction, it is expected to take place in less than 100% of the molecules which harbor it, and thus, presumably, to lead to a milder phenotype expression. The fact that a milder phenotype involves only the adrenal and the skin is suggestive of their high sensitivity to changes in PKA activity.

The second group of CNC patients that was suggested to express particular genotype-phenotype correlation was comprised by individuals with myxomas (affecting all locations – skin, heart, and breast), PMS, thyroid tumors, and Large-Cell Calcifying Sertoli Cell Tumor (LCCSCT) – in these patients, PRKAR1A mutations were seen substantially more often. Related to this is the acknowledgment that certain tumors presented at significantly younger age in PRKAR1A mutation carriers: cardiac myxomas (P = 0.02), thyroid tumors (P = 0.03) and LCCSCTs (P = 0.04) (Bertherat et al., 2009). Another finding among 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 tumors) associated significantly with the ‘hot spot’ c.491–492delTG mutation compared to all other PRKAR1A defects (P = 0.03). These data add an inestimable value to the understanding the molecular mechanisms of the involvement of PRKAR1A in endocrine and other tumorigenesis and, thus, for genetic counseling and prognosis in CNC families.

Interestingly, a recently described 2.3 Mb deletion in chromosome band 17q24.2–q24.3 that involved, along with other 13 genes, PRKAR1A, 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 (Blyth et al., 2008); the authors called the observed phenotype ‘CNC plus.’

Probably the closest, at least in terms of a molecular pathway link, to CNC is MAS. Patients with this condition have characteristic lesions that affect predominantly three systems – the skin, the endocrine system and the skeleton. The café-au-lite spots in MAS patients are similar to the ones observed in CNC, but tend to be more intensely pigmented. MAS is caused by post-zygotic activating mutations of the gene encoding the adenylate cyclase stimulating G alpha protein (GNAS1) of the heterotrimeric G protein (Weinstein et al., 1991). 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 GNAS1 are always missense substitutions at the critical 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 (even in the absence of a GPCR ligand).

We have recently 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 (Horvath et al., 2006a,b, 2008c). iMAD patients were initially considered CNC patients, but it soon became clear that iMAD is not the same as PPNAD (Gunther et al., 2004).

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. PJS has been elucidated at the molecular level (Hemminki et al., 1998; Jenne et al., 1998): the disease was first mapped to chromosome 19p13.3 and, soon after that, the gene encoding the serine threonine kinase 11 (STK11 also known as LKB1) was found to be mutated in most patients (Boardman et al., 2000; Jiang et al., 1999; Resta et al., 1998; Wang et al., 1999; Westerman et al., 1999; Ylikorkala et al., 1999). The proposed mechanism of the disease is through elimination of the kinase activity of the STK11/LKB1 tumor suppressor protein.

LEOPARD is an acronym for the manifestations of this syndrome: multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness (Gorlin et al., 1969). LEOPARD is allelic to Noonan syndrome: both diseases are linked to mutations in PTPN11 (12q24), the gene encoding the non-receptor tyrosine phosphatase Shp-2 (Jamieson et al., 1994; Van Der Burgt et al., 1994). The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family, proteins that are known to regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation.

Cowden disease and BRRS share clinical characteristics such as mucocutaneous lesions, hamartomatous polyps of the gastrointestinal tract, and increased risk of developing neoplasms. Both conditions are caused by mutations in the PTEN gene (Liaw et al., 1997; Marsh et al., 1997; Nelen et al., 1996). PTEN is located on 10q23.31 and encodes phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. The gene was recognized as a tumor suppressor gene and has been found mutated in a number of tumors (Yin and Shen, 2008). 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 tumor suppressor effect is expressed by inhibition of AKT/PKB signaling pathway.