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. Author manuscript; available in PMC: 2010 Sep 9.
Published in final edited form as: Hum Mutat. 2010 Apr;31(4):369–379. doi: 10.1002/humu.21178

Mutations and Polymorphisms in the Gene Encoding Regulatory Subunit Type 1-alpha of Protein Kinase A (PRKAR1A): An Update

Anélia Horvath 1, Jérôme Bertherat 2,3,4, Lionel Groussin 2,3,4, Marine Guillaud-Bataille 2,5, Kitman Tsang 1, Laure Cazabat 2, Rosella Libe 2,3, Elaine Remmers 6, Fernande René-Corail 2,3, Fabio Rueda Faucz 1,7, Eric Clauser 2,5, Alain Calender 8, Xavier Bertagna 2,3,4, J Aidan Carney 10, Constantine A Stratakis 1,*
PMCID: PMC2936101  NIHMSID: NIHMS231175  PMID: 20358582

Abstract

PRKAR1A encodes the regulatory subunit type 1-alpha (RIα), of the main mediator of the cAMP effects in the eukaryotic cells – cAMP dependant Protein Kinase A (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified. The majority of them are subject to non-sense mediated mRNA decay (NMD), leading to PRKAR1A haploinsufficiency and, as a result, activated cAMP signaling in the affected tissues. In recent years it became apparent that CNC may be caused not only by RIα haploinsufficiency, but also by the expression of altered PRKAR1A protein, as proven by analysis of the relatively small proportion of expressed mutations in the gene, consisting of aminoacid substitutions and in-frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered rather than missing protein has been analyzed – frame-shifts in the 3′end of the coding sequence that shift the stop codon downstream of the regular one and result in the expression of longer than the wild type protein. The PRKAR1A mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation negative CNC patients are characterized by significant phenotypic heterogeneity. In this paper, we present a comprehensive analysis of all 117 known to date pathogenic PRKAR1A sequence variations, and discuss their molecular context and clinical relevance.

Keywords: PRKAR1A, PKA, mutations, polymorphisms, Carney complex

Introduction

Cyclic AMP (cAMP) dependent Protein Kinase A (PKA, EC 2.7.1.37) is the major mediator of the cAMP effects in the eukaryotic cells. In its inactive state, the PKA holoenzyme is a tetramer comprised of dimer of two regulatory subunits bound to two catalytic subunits (Kinderman, et al., 2006; Tasken, et al., 1997). Upon elevated cellular cAMP concentrations, two molecules of cAMP bind each of the regulatory subunits, which leads to the dissociation of the tetramer into regulatory dimers and two free catalytic subunits (Tasken, et al., 1997). The free catalytic subunits represent active serine threonine kinases which in turn phosphorylate series of cellular targets that regulate downstream effector enzymes, ion channels, and transcription of specific genes that mediate cell growth and differentiation (Shabb, 2001).

In the human, 4 different regulatory (PRKAR1A, PRKAR1B, PRKAR2A, and PRKAR2B) and 3 catalytic subunits (PRKACA; PRKACB, PRKACG) have been identified so far. It is now assumed that the multiple combinations of homo- or heterodimers of regulatory subunits with associated catalytic subunits may, at least in part, account for the specificity of the transduction of the signal generated by numerous different hormones, neurotransmitters, and other signaling through one sole second messenger, cAMP.

The only PKA subunit in which mutations have been found to lead to human disease is PRKAR1A: inactivating PRKAR1A mutations cause the multiple neoplasia and lentiginosis syndrome Carney complex (CNC) (Casey, et al., 1998; Kirschner, et al., 2000a). CNC (MIM# 160980) is an autosomal dominant disorder associated with skin lesions, cardiac and other myxomas, and different types of endocrine tumors including primary pigmented nodular adrenocortical disease (PPNAD), growth hormone–secreting pituitary tumors, gonadal and thyroid neoplasias; rarely, malignant psammomatous melanotic schwannomas (PMS) may also be present (Carney, 1995; Carney, et al., 1986; Stratakis, et al., 1998). The vast majority of the reported PRKAR1A mutations lead to premature stop codons and subsequent degradation of the mutant mRNAs species by NMD (Kirschner, et al., 2000b). Inactivating PRKAR1A mutations lead to excess PKA signaling in affected tissues (Robinson-White, et al., 2006a).

PRKAR1A is located on chromosome 17q23-q24 and extends to a total genomic length of approximately 21 kB. The gene is composed of 11 exons, 10 of which coding, with a coding region of 1143 bp starting from exon 2. The RIα protein consists of 384 AA organized in a dimerization/docking domain at the amino-terminal, followed by a PKA inhibitor site, two tandem binding domains for cAMP at the carboxyl terminus (cAMP:A and cAMP:B), and a linker region that contains the main docking site for the C subunit (Zawadzki and Taylor, 2004). Until recently, it was widely accepted that, in contrast to RIIα, which typically is docked at discrete cellular locations through A Kinase Anchoring Proteins (AKAPs), RIα tends to diffuse in the cytoplasm (Kinderman, et al., 2006; Skalhegg, et al., 1994; Wong and Scott, 2004). In the past several years, however, evidence has accumulated that RIα can be dynamically recruited as well localized at certain compartments of the cell such as the plasma membrane, mitochondria, cytoskeleton, and centrosomes, and AKAPs capable of binding it have been described (Huang, et al., 1997a; Huang, et al., 1997b).

PRKAR1A orthologues have been characterized in many species, and high level of preservation can be tracked back to invertebrates (Aedes aegipty, 73% homology) and protozoa (Ciona intestinalis, 63% homology). The high degree of amino acid sequence conservation underlines the significance of PRKAR1A, the functional “receptor” for cAMP. Indeed, both cloning and gene knockout studies on the individual regulatory subunits have confirmed the essential role of PRKAR1A in maintaining cAMP control of PKA activity through compensatory mechanisms assuring balanced regulation of the free catalytic subunits (Amieux and McKnight, 2002).

Since the identification of PRKAR1A as the gene causing CNC, more than a hundred different pathogenic mutations have been reported, and their molecular and functional analysis has been investigated in several major studies (Bertherat, et al., 2009; Greene, et al., 2008; Groussin, et al., 2002; Kirschner, et al., 2000a; Veugelers, et al., 2004). Herein we review all described to date PRKAR1A sequence variations, including 17 that have not been published before, in an attempt for better understanding of their molecular context and clinical relevance.

Mutation spectrum

To date, a total of 117 different PRKAR1A mutations have been identified in 387 unrelated families of various ethnic origin; they are summarized in Table 1. The molecular changes involve single base substitutions and small (up to 15bp) deletions, insertions or combined rearrangements that are spread along the whole open reading frame of the gene; in addition, several relatively large deletions are reported (Blyth, et al., 2008; Horvath, et al., 2008a). A schematic representation of the PRKAR1A mutations’ type and location is shown on Figure 1.

Table 1.

List of known mutations in the PRKAR1A (sorted by their position along the gene).

# Position DNA Change Protein/RNA Change Type Effect on the protein Phenotype Reference
1 exon 1B c.-97G>A Pseudo ATG frame-shift NMD, no protein CNC (Groussin, et al., 2002)
2 exon 2 c.1A>G p.M1V missense abolished initiation CNC (Kirschner, et al., 2000b)
3 exon 2 c.18delC p.A7PfsX122 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
4 exon 2 c.26G>A p.S9N missense altered protein CNC (Greene, et al., 2008)
5 exon 2 c.43_58del16 p.L15SfsX109 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
6 exon 2 c.46C>T p.R16X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
7 exon 2 c.52_53delTG p.C18X nonsense NMD, no protein CNC (Veugelers, et al., 2004)
8 exon 2 c.54_55dupTG p.E19VfsX111 frame-shift NMD, no protein CNC This study
9 exon 2 c.63_64delCGinsGA p.Y21X nonsense NMD, no protein CNC (Almeida, et al., 2008)
10 exon 2 c.69_70duplGA p.K24RfsX106 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
11 exon 2 c.75_78dupTAAC p.l27X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
12 exon 2 c.82C>T p.Q28X nonsense NMD, no protein CNC (Kirschner, et al., 2000b)
13 exon 2 c.85_95del11 p.A29RfsX12 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
14 exon 2 c.96_97insT p.D33X nonsense NMD, no protein CNC This study
15 exon 2 c.101_105del5 p.S34CfsX9 frame-shift NMD, no protein CNC (Kirschner, et al., 2000b)
16 exon 2 c.109C>T p.Q37X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
17 exon 2 c.124C>T p.R42X nonsense NMD, no protein CNC (Kirschner, et al., 2000b)
18 exon 2 c.140delT p.M46RfsX82 frame-shift NMD, no protein CNC (Imai, et al., 2005)
19 exon 2 c.177G>A retains part of intr 2 splice NMD, no protein CNC (Bertherat, et al., 2009)
20 intron 2 c.177+1 g>a retains part of intr 2 splice NMD, no protein CNC (Bertherat, et al., 2009)
21 intron 2 c.178–2 a>g skips ex 3 splice shorter prot. (324AA) CNC (Kirschner, et al., 2000b)
22 intron 2 c.178_348del171 p.E60_K116del In-frame del shorter prot. (328AA) CNC (Horvath, et al., 2008)
23 exon 3 c.187A>T p.K63X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
24 exon 3 c.190C>T p.Q64X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
25 exon 3 c.205C>T p.Q69X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
26 exon 3 c.206A>G p.Q69R missense altered protein CNC This study
27 exon 3 c.220_221delCG p.R74YfsX7 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
28 exon 3 c.220C>T p.R74C missense altered protein CNC (Veugelers, et al., 2004)
29 exon 3 c.242delA p.E81GfsX48 frame-shift NMD, no protein CNC This study
30 exon 3 c.267delA p.V90WfsX39 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
31 exon 3 c.279_282delTAGG p.R94GfsX34 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
32 exon 3 c.286C>T p.R96X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
33 exon 3 c.289C>T p.R97X nonsense NMD, no protein CNC (Veugelers, et al., 2004)
34 exon 3 c.316_317delAC p.T106GfsX9 frame-shift NMD, no protein CNC (Skamrov, et al., 2003)
35 exon 3 c.319G>T p.Q107X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
36 exon 3 c.340delG p.V114LfsX15 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
37 Inton 3 c.348+1 g>c retains intr 3 splice NMD, no protein CNC (Kirschner, et al., 2000b)
38 exon 4 c.350_353delTTAT p.V117DfsX11 frame-shift NMD, no protein CNC (Kirschner, et al., 2000b)
39 exon 4 c.353_365del13 p.I118TfsX6 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
40 exon 4 c.407_408delTG p.V136AfsX6 frame-shift NMD, no protein CNC (Mabuchi, et al., 2005)
41 exon 4 c.408_412delGCTGT p.L137FfsX4 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
42 exon 4 c.418_419delCA p.H140SfsX1 frame-shift NMD, no protein CNC This study
43 exon 4 c.438A>T p.R146S missense altered protein CNC (Greene, et al., 2008)
44 exon 4 c.439A>G p.S147G missense altered protein CNC (Bertherat, et al., 2009)
45 exon 4 c.440G>A p.S147N missense, splice altered protein CNC (Gu, et al., 2004)
46 intron 4 c.440+1g>a retains part of intr 4 splice NMD, no protein CNC This study
47 intron 4 c.440+1_440+2insg retains part of intr 4 splice NMD, no protein CNC (Bertherat, et al., 2009)
48 intron 4 c.440+2t>c retains part of intr 4 splice NMD, no protein CNC (Bertherat, et al., 2009)
49 intron 4 c.440+4del g retains part of intr 4 splice NMD, no protein CNC (Bertherat, et al., 2009)
50 exon 5 c.463delT p.S155RfsX10 frame-shift NMD, no protein CNC This study
51 exon 5 c.466delG p.V156SfsX9 frame-shift NMD, no protein CNC (Vargas-Alarcon, et al., 2008)
52 exon 5 c.491_492delTG p.V164DfsX5 frame-shift NMD, no protein CNC (Kirschner, et al., 2000a)
53 exon 5 c.496C>T p.Q166X nonsense NMD, no protein CNC This study
54 exon 5 c.499C>T* p.Q167X nonsense NMD, no protein UDC (Sandrini, et al., 2002)
55 exon 5 c.502G>A p.G168S missense, splice altered protein CNC (Bertherat, et al., 2009)
56 intron 5 c.502+1g>t retains part of intr 5 splice NMD, no protein CNC (Groussin, et al., 2002)
57 intron 5 c.502+1g>a retains part of intr 5 splice NMD, no protein CNC (Gennari, et al., 2008)
58 exon 6 c.528_531delGATTins11 p.I177MfsX13 frame-shift NMD, no protein CNC (Veugelers, et al., 2004)
59 exon 6 c.531_(549+32)del51 p.I177MfsX1 frame-shift, splice NMD, no protein CNC (Bertherat, et al., 2009)
60 exon 6 c.545dupC p.D182GfsX5 frame-shift NMD, no protein CNC (Veugelers, et al., 2004)
61 exon 6 c.547G>T p.D183Y missense altered protein CNC (Greene, et al., 2008)
62 intron 6 c.550(-9-2)del8 Skip ex 7 splice shorter prot. (328AA) CNC (Kirschner, et al., 2000b)
63 exon 7 c.550G>A p.V184I missense, splice altered protein CNC (Bertherat, et al., 2009)
64 exon 7 c.558_559dupTA p.N187IfsX20 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
65 exon 7 c.566_567delAAinsCAC p.E189AfsX44 frame-shift NMD, no protein CNC (Kirschner, et al., 2000b)
66 exon 7 c.569G>A p.W190X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
67 exon 7 c.570G>A p.W190X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
68 exon 7 c.587_588insGG p.G198EfsX10 frame-shift NMD, no protein CNC (Veugelers, et al., 2004)
69 exon 7 c.597delC p.F200LfsX6 frame-shift NMD, no protein CNC (Sasaki, et al., 2008)
70 exon 7 c.619delT p.Y207MfsX15 frame-shift NMD, no protein CNC (Veugelers, et al., 2004)
71 exon 7 c.623delG p.G208EfsX14 frame-shift NMD, no protein CNC (Casey, et al., 2000)
72 exon 7 c.624dupA p.T209NfsX24 frame-shift NMD, no protein CNC (Veugelers, et al., 2004)
73 exon 7 c.638C>A* p.A213D missense altered protein ODM (Perdigao, et al., 2005)
74 exon 7 c.652_653delAA p.K218DfsX14 frame-shift NMD, no protein CNC This study
75 exon 7 c.658_659delAA p.N220CfsX12 frame-shift NMD, no protein CNC (Veugelers, et al., 2004)
76 exon 7 c.664A>T* p.L222X nonsense NMD, no protein ACA (Bertherat, et al., 2003)
77 exon 7 c.669_670delGT p.W224GfsX8 frame-shift NMD, no protein CNC This study
78 exon 7 c.671G>A p.W224X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
79 exon 7 c.672G>A p.W224X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
80 exon 7 c.679delG p.D227TfsX14 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
81 exon 7 c.682C>T p.R228X nonsense NMD, no protein CNC (Kirschner, et al., 2000b)
82 exon 7 c.687delT* p.D229EfsX12 frame-shift NMD, no protein ODM (Perdigao, et al., 2005)
83 exon 7 c.693dupT p.R232X nonsense NMD, no protein CNC (Kirschner, et al., 2000b)
84 intron 7 c.708+1g>t Skip ex 7 splice shorter prot. (328AA) CNC (Groussin, et al., 2002)
85 intron 7 c.709(-5-107)del103 skips ex 8 splice NMD, no protein CNC (Bertherat, et al., 2009)
86 intron 7 c.709(-7-2)del6 skips ex 8 splice NMD, no protein CNC** (Groussin, et al., 2002)
87 intron 7 c.709-1g>c skips ex 8 splice NMD, no protein CNC (Bertherat, et al., 2009)
88 exon 8 c.711_712dupAA p.S238KfsX4 frame-shift NMD, no protein CNC (Kirschner, et al., 2000b)
89 exon 8 c.718dupC p.L240PfsX8 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
90 exon 8 c.725dupA p.R243AfsX5 frame-shift NMD, no protein CNC (Groussin, et al., 2002)
91 exon 8 c.728_740del13 p.K244NfsX9 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
92 exon 8 c.738T>G p.Y246X nonsense NMD, no protein CNC (Bertherat, et al., 2009)
93 exon 8 c.758_759delTC p.S254YfsX15 frame-shift NMD, no protein CNC (Casey, et al., 2000)
94 exon 8 c.763_764delAT p.I255PfsX14 frame-shift NMD, no protein CNC (Groussin, et al., 2002)
95 exon 8 c.764_768delTTTTA p.I255RfsX12 frame-shift NMD, no protein CNC This study
96 exon 8 c.769G>A p.E257K missense, splice NMD, no protein CNC This study
97 intron 8 c.769+5g>c Retains part of intr 8 splice NMD, no protein CNC (Bertherat, et al., 2009)
98 exon 9 c.786_787delGGinsCT p.W262CfsX2 frame-shift NMD, no protein CNC (Kirschner, et al., 2000a)
99 exon 9 c.804dupT p.D269X nonsense NMD, no protein CNC (Veugelers, et al., 2004)
100 exon 9 c.812dupT p.L271FfsX7 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
101 exon 9 c.845_846insA p.V283CfsX9 frame-shift NMD, no protein CNC (Bertherat, et al., 2009)
102 exon 9 c.865G>T p.G289W missense altered protein CNC (Greene, et al., 2008)
103 intron 9 c.891+3a>g Retains part of intr 9 splice skips ex 10 CNC (Kirschner, et al., 2000a)
104 intron 9 c.892-2a>g skips ex 10 splice skips ex 10 CNC This study
105 exon 10 c.910C>T p.Q304X nonsense NMD, no protein CNC (Veugelers, et al., 2004)
106 exon 10 c.920C>G p.S307X nonsense NMD, no protein CNC This study
107 exon 10 c.951delA p.R317SfsX14 frame-shift NMD, no protein CNC (Veugelers, et al., 2004)
108 exon 10 c.963_985del23* p.D322TfsX20 frame-shift NMD, no protein ACA (Bertherat, et al., 2003)
109 inton 10 c.973+1g>a Retains part of intr 10 splice NMD, no protein CNC This study
110 exon 11 c.974-1g>a* skips ex 11 splice NMD, no protein ACA (Bertherat, et al., 2003)
111 exon 11 c.1055_1058delGACC p.R352LfsX88 frame-shift elongated prot. (438AA) CNC (Bertherat, et al., 2009)
112 exon 11 c.1067_1070delAACGins5 p.E356GfsX71 frame-shift elongated prot. (425AA) CNC (Bertherat, et al., 2009)
113 exon 11 c.1076_1077delTTins13 p.L359QfsX86 frame-shift elongated prot. (443AA) CNC (Bertherat, et al., 2009)
114 exon 11 c.1083delA p.C362AfsX79 frame-shift elongated prot. (439AA) CNC This study
115 exon 11 c.1131_1132delTG p.S378TfsX48 frame-shift elongated prot. (425AA) CNC (Bertherat, et al., 2009)
116 exon 11 c.1142_1145delTCTG p.V381EfsX59 frame-shift predicted elongated prot. (439AA) CNC (Bertherat, et al., 2009)
117 promoter? c.(-10,752_6,876)del3,876 Before ex 1 deletion of part of the promoter decreased prot. expression CNC (Horvath, et al., 2008)
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*

somatic mutation (the presence of the mutation was ruled out in lymphocytes)

**

In the carriers of mutation c.709(-7-2)del6 and c.63_64delCGinsGA CNC phenotype was restricted to PPNAD, and, in some cases, skin manifestations

***

CNC+ phenotype is defined by the authors as „posterior laryngeal cleft and the presence of numerous freckles and lentigines, growth restriction, microcephaly and moderate mental retardation“.

CNC – Carney complex; ACA – adenocortical adenoma; UDC – undiferentiated thyroid carcinoma; ODM – odontogenic myxoma; NMD – Non-sense mediated m-RNA decay

Figure 1.

Figure 1

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Schematic presentation of the type and the approximate location of the 117 identified to date mutations in PRKAR1A; a large deletion eliminating the whole PRKAR1A together with 13 more genes (2.3Mb del 17q24.2-q24.3) is also shown. The docking/dimerisation, the linker and the two cAMP functional domains (from left to right) of the protein are denoted with ovals.

The mutations in PRKAR1A are spread along the whole coding sequence, without significant preference for an exon or a domain. Most of them are unique – they are identified in single families only (Bertherat, et al., 2009). To date, only 3 mutations have been found in more than three unrelated pedigrees: c.82C>T, c.491_492delTG and c.709(-7-2)del6; these mutations have been seen in kindreds with different racial and ethnic background, suggesting that they are likely to result from more than one independent mutation events.

A graph representing the relationship between the molecular type of the PRKAR1A mutations’ and their functional effect is shown on Figure 2. Relatively small proportion (20/117, 17.1%) of the unique mutations result in the expression of an altered protein; these group convenes the 11 missense substitutions, 6 frameshift mutations affecting the last exon of the gene and thus escaping NMD, one in-frame deletion that eliminates exon 3 in frame, and one splice variant that leads to exon 7 skipping. The vast majority of mutations (97/117, 82.9%) result in premature stop codon (PSC) generation caused by nonsense and frameshift changes upstream of the last gene exon; the mutant mRNAs are degraded through NMD. In this second group fall also the majority of the splice variants – disrupted donor or acceptor sequences lead to exon skip and/or retaining of part of the intron, and, thus, direct or by frame-shift incorporation of PSC. The different mutations’ type functional effect and relation to the phenotype are discussed below.

Figure 2.

Figure 2

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Quantitative schematic representation of the expressed vs NMD mutations in PRKAR1A.

Mutations generating premature stop codon

As mentioned, PSC are caused by nonsense, frame shift and splice variants located before the last exon of PRKAR1A. The expression of the shortened protein is abolished by the NMD, as shown by our experiments (Kirschner, et al., 2000b); thus, the overall effect of this type of mutation is a 50% reduction in cellular PRKAR1A levels. The resulting higher proportion of catalytic to regulatory subunits causes increased signaling and activation of the downstream cellular processes such as proliferation and differentiation, (Robinson-White, et al., 2006b). Hence, NMD mutations cause disease in the affected tissues through a mechanism of haploinsufficiency. This type of mutations is found in the majority of the CNC patients and the resulting phenotypes vary in a very wide range both as number of manifestation and severity of their expression. The factors to affect this variability, besides age, gender and environmental agents are likely to include other genetic determinants with modifying effects. The later is supported by family clustering of particular sets of manifestations as well as our recent studies showing influence of genetic variants in other cAMP-related molecules on the severity of the CNC phenotype (manuscript in preparation). Although the uniqueness of the most of the mutations limits genotype/phenotype analysis, it is now accepted that phenotype/genotype correlation is rare among carriers of NMD mutations in PRKAR1A - they all share haploinsufficiency at the protein level.

Expressed mutations

The expressed missense substitutions in PRKAR1A are listed in Table 2. While the pathogenic effect of the NMD mutations is almost certain to destroy the PRKAR1A function, the expressed mutations that lead to altered protein clearly require estimation of their pathogenic potential. Eight of these mutations – six aminoacid substitutions (S9N, R74C, R146S, D183Y, A213D and G289W), and the two in-frame deletions of exons 3 and 7 - have been subject of detailed investigation of their effect in vitro (Greene, et al., 2008; Meoli, et al., 2008). The expressed mutations were spread over all functional PRKAR1A domains and every one of them led to variable extends of decrease of the cAMP binding and increase in the cAMP-specific PKA activation (Greene, et al., 2008; Meoli, et al., 2008). As expected, the highest impact on the protein function was measured for the in-frame deletion that eliminates exon 3, and, respectively, the primary binding site for the catalytic subunit (Greene, et al., 2008); this mutation leads to significant elevation in the PKA activity even in the absence of cAMP. Similar, although milder effect on the protein is caused by the R146S, which disrupts the secondary binding site for the catalytic subunit (residues 138-148 and 232-247 within the cAMP binding domain A (Greene, et al., 2008). Expressed mutations affecting the cAMP binding domains (A183Y, A213D and G289W), are shown, in line with their position, to significantly decrease the cAMP binding; the proposed mechanism of action is through conformational changes that alter the enzymatic function. Conformational changes are also proposed to take place for the last two of the studied expressed mutations: S9N, located in the dimerisation/docking domain, and R74C, which is positioned in the linker region, outside any known functional sequence.

Table 2.

In silico modeling and in vitro analysis of the effect of PRKAR1A missense substitutions on the protein function

# DNA Change Protein Change Domain In silico modeling In vitro analysis Inter-species alignment
prediction score* cAMP binding** cAMPspecific PKA activation** Mus musculus Gallus gallus Anolis carolinensis Xenopus laevis Danio regio Plasmodium falciparum
1 c.1A>G p.M1V Initiation codon Probably damaging 2.055 0.31*** 2.46*** M M M M M M
2 c.26G>A p.S9N Dimerization/docking Benign 0.243 0.28*** 2.14** S S S S S -
3 c.206A>G p.Q69R Linker region Probably damaging 2.013 na na Q Q Q Q Q -
4 c.220C>T p.R74C Linker region Possibly damaging 1.686 0.69*** 2.77 R R R R R -
5 c.438A>T p.R146S cAMP binding A Benign 0.733 0.49*** 2.12*** R R R R R F
6 c.439A>G p.S147G cAMP binding A Benign 1.033 0.84*** 2.16*** S S S S S E
7 c.440G>A p.S147N cAMP binding A Benign 0.145 na na S S S S S E
8 c.502G>A p.G168S cAMP binding A Probably damaging 2.250 na na G G G G G G
9 c.547G>T p.D183Y cAMP binding A Probably damaging 2.521 0.24*** 1.63 D D D D D E
10 c.550G>A p.V184I cAMP binding A Benign 0.052 na na V V V V V I
11 c.638C>A p.A213D cAMP binding A Probably damaging 2.507 0.17*** 2.06 A A A A A A
12 c.865G>T p.G289W cAMP binding B Probably damaging 2.592 0.43*** 2.92*** G G G G G G
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*

greater score indicates higher probability to impair the protein function; the factors taken into account for the calculation of the score are

(1) difference in the thermo-physical properties of the wt and mutant protein, and

(2) evolutionary preservation of the residue in the corresponding position

**

both cAMP binding and PKA activity changes in response to cAMP are presented as ratio to the wt PRKAR1A

***

P<0.05

Notably, substantial correlation between the in vitro studies and the in silico modeling of the effect of the expressed missense substitutions on the protein function, along with the homology data, was observed (See Table 2). In silico prediction of the effect of the missense substitutions on the protein function (http://coot.embl.de/PolyPhen/) has estimated highest potential to impact the protein function generally for mutations residing in the cAMP binding domains (G168S, D183Y, A213D and G289D, Table 2).

Separate attention requires the missense substitution affecting the first coding aminoacid - M1V. In silico modeling predicted very severe effect on the protein function (See Table 2). Consistent with the above, the in vitro studies have shown significant effect of the mutation on both cAMP binding and cAMP specific PKA activation. However, although the mutant mRNA is found to be expressed in equal to the wt levels in carriers of the mutation, it is not clear if the mutant protein is expressed and stable in vivo (Kirschner, et al., 2000b). The possibility of alternate expression from a surrogate initiation site has been explored – an in-frame ATG in the context of a relatively good match for Kozak sequence is located 141 bp downstream from the original initiation codon; however, Western blot did not detect shorter protein forms.

Analysis of the phenotype characteristics in the carriers of expressed PRKAR1A mutations revealed severe CNC phenotype in the carrier of the in-frame deletion of ex3 and the c.708 +1G>T mutation, expressed both as number of manifestations and severity of their expression. Apart from this finding, no genotype-phenotype connection could be characterized within this relatively small group, which is expected taken the limited number of affected patients and the variable number CNC manifestations. Nevertheless, the finding of expressed PRKAR1A mutant variations that cause disease related changes in vitro, confirms that altered PRKAR1A function, not only haploinsufficiency, is enough to lead to the disease phenotype.

Frameshift mutations leading to elongated PRKAR1A protein

Six of the frameshift mutations (c.1055_1058delGACC, c.1067_1070delAACGins5, c.1076_1077delTTins13, c.1083delA, c.1131_1132delTG, and c.1142_1145delTCTG) are located in the last exon of PRKAR1A and are predicted to escape NMD. Each of the mutations causes a frame shift that abolishes the wild type termination codon and generates a new one further downstream. Compared to the wild type PRKAR1A, the predicted mutant proteins are longer and carry a different 3′ AA sequence, resulting from codon rearrangement. The functional significance of the elongated proteins versus the wild-type R1α is subject of another report under submission.

Large deletions

Several relatively large deletions in the region of PRKAR1A have been described so far (Blyth, et al., 2008; Horvath, et al., 2008a), including the above discussed expressed deletion that eliminates exon 3 in frame (c.178_348del171/p.E60_K116del). This mutation has been shown to result to the in vivo expression of shorter PRKAR1A protein lacking part of the linker region connecting the dimerisation/docking domain and the first cAMP binding domain; the deleted region includes the binding site for the catalytic subunit/inhibitor (Horvath, et al., 2008a). Only one patient was found to carry this mutation, and he presented with very severe phenotype, both as number of CNC manifestation and level of their expression (Horvath, et al., 2008a). In vitro studies with expression vectors harboring PRKAR1A ORF lacking exon 3 have shown extreme effect on the protein function (Greene, et al., 2008). The second identified deletion was located in the upstream regulatory region of PRKAR1A and did not affect the ORF of the gene; this mutation is expected to lead to decreased PRKAR1A mRNA levels but no other effects on the protein; the molecular phenotype is predicted to be PRKAR1A haploinsufficiency, consistent with the majority of PRKAR1A mutations causing CNC. The third noteworthy deletion encompasses significantly larger genomic region from 17q24.2-q24.3 and eliminates, along with PRKAR1A, 13 more genes (Blyth, et al., 2008). This mutation was identified as a de novo genomic rearrangement in a 12 years old patient presenting with posterior laryngeal cleft, moderate growth and mental retardation, microcephaly, and, as the only CNC related manifestation - multiple freckles and lentigines. However, other CNC manifestations can be underrepresented due to the severity of the main phenotype, or, more likely may occur later in the development. Although larger rearrangements involving PRKAR1A region are reported in the literature, in most of the cases they led to extremely severe phenotype and premature death that prevented comprehensive analysis of relatively mild CNC manifestation with later onset (Bridge, et al., 1985; Levin, et al., 1995; Olney, et al., 1999).

Splice mutations

Twenty-eight splice variants are identified to date in the close proximity of the exon-intron junctions of PRKAR1A (Table 3). As mentioned, the vast majority of them lead to a frame-shift and subsequent incorporation of PMS. Notably, for the most of the splice mutations (23, 82%), the in silico modeling predicted complete abrogation of the junction formation; in 2 of them accompanied by a shift of the predicted junction with 1 base (see Table 3). For the remaining five variants (c440+4delG, c.502G>A, c.550G>A, c.763delAT, and 891+3A>G) a significant decrease in the probability score to form junction compared to the wt was estimated (see Table 3). It is noteworthy that three of these last variants affected directly exonic sequences and their effect on the splice-site was complemented by a change in the approximate coding sequence, which additionally increased their pathogenic impact. Combined with the family data that show segregation of these variants with the CNC phenotype, all described 28 splice changes were classified as pathogenic.

Table 3.

In silico modeling of the effect of PRKAR1A splice variants

# DNA Change Exon affected Spliceport WT Score Spliceport mutation Score
1 c.177G>A Incorporates intron2 0.679639 0.00
2 c.177+1g>a Incorporates intron2 0.679639 0.00
3 c.178–2a>g Skip exon 2 0.894923 0.00
5 c.348 +1 g>c Incorporates intron3 1.14417 0.00
6 c.438A>T Incorporates intron4 0.820566 0.00
7 c.439A>G Incorporates intron4 0.820566 0.00
8 c.440G>A Incorporates intron4 0.820566 0.00
9 c.440+ 1 insG Incorporates intron4 0.820566 0.268882 (+1 base)
10 c.440+ 4del g Incorporates intron4 0.820566 0.142182
11 c.440+2 t>c Incorporates intron4 0.820566 0.00
13 c.502 +1g>t Incorporates intron5 1.69211 0.00
14 c.502+1g>a Incorporates intron5 1.69211 0.00
15 c.502G>A Incorporates intron5 1.69211 0.705629
17 c.550(-8-2) del 7 Skip exon 7 0.802186 0.00
18 c.550G>A Skip exon 7 (+ −) 0.802186 0.478702
19 c.708 +1g>t Skip exon 7 1.74672 0.00
20 c.709 (-7-2)del6(ttttta) Skip exon 8 0.502791 0.0
21 c.709 (-5-107)del103 Skip exon 8 0.502791 0.0
22 c.709-1G>C Skip exon 8 0.502791 0.0
24 c.763delAT Incorporates intron8 1.02912 0.778435
25 c.769+5g>c Incorporates intron8 1.02912 0.00
26 c.891+3A>G Incorporates intron9 1.41361 0.158643
27 c.892-2 a>g Skip exon 10 1.64415 0.0
28 c.974-1g>a Skip exon 11 1.2552 0.112813 (+1 base)
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Interestingly, some splice-site alleles may express both mutant and wild type mRNA molecules in a proportion depending on the type of the mutation and its location relevant to the junction. Thus, the relative decrease of the cellular wt PRKAR1A for these mutations is expected to be lower, and, accordingly, to lead to a milder phenotype. This assumption is in line with a recent analysis of a “mild” splice variant (c.709(-7-2)del6), which is one of the very few mutations where incomplete penetrance of PRKAR1A mutation is seen, and, in the affected individuals, only PPNAD and lentiginosis – the most common disease manifestations - have been diagnosed (Groussin, et al., 2006).

Polymorphisms

Four nonpathogenic unique synonymous substitutions in the third base of the codon have been described so far in PRKAR1A: c.87G>A/p.A29A, c.204G>A/p.L68L, c.318G>C/p.T106T, and c.492G>A/p.V164V. From them, the first one has been seen in at least five unrelated kindreds, and the remaining three are detected in single families only. Multiple intronic variants in close proximity of the junctions have been described, from them the most frequent being c.349-5dup5 (detected in approximately 15% of the studied alleles), followed by c.769-24A>G and c.891-34G>T (in approximately 12% and 10% of the studied alleles, respectively). However, the extend to which different research groups analyze the exon flanking sequences widely vary and the frequency of these variants may therefore be significantly underreported; furthermore, both missense variants and splice changes may be as well underreported. All these polymorphic variants are seen in healthy control individuals and in both CNC families, and in the later, they do not show segregation with the disease status.

Mutation detection rate of PRKAR1A mutations in CNC

Since the first description of the disease, numerous CNC patients have been reported from all ethnic groups and presenting with various number, combination and severity of manifestations. The most recently re-evaluated diagnostic criteria for CNC are listed in Table 4; a definite diagnosis is given when two or more major manifestations are present. Soon after the identification of PRKAR1A and the initial patient screenings, the mutation detection rate was estimated to approximately 50% (Figure 3, A). After a decade of comprehensive phenotype analysis and re-evaluation of the diagnostic criteria, it is now assumed that mutations in PRKAR1A are responsible for the disease in more than 60% of the CNC patients (Figure 2B). The largest to date study of 185 families, detects PRKAR1A mutations in 114 (62%) (Bertherat, et al., 2009). Interestingly, this percent rises up to 80 among CNC patients presenting with Cushing syndrome due to PPNAD (Cazabat, et al., 2007). Among the mutation carriers, Cushing syndrome due to PPNAD is seen in 70% of the female patients before 45 years, but in only 45% of the male patients, likely reflecting the generally higher frequency of Cushing syndrome in females (Grumbach, et al., 2003). These numbers, however, may represent an underestimate of the true incidence of Cushing syndrome, since autopsy of CNC patients detects PPNAD in almost 100% of the cases (Stratakis, et al., 2001).

Table 4.

Diagnostic criteria for CNC

Major Diagnostic criteria for CNC
 1. Spotty skin pigmentation with typical distribution (lips, conjunctiva and inner or outer canthi, vaginal and penile mucosal
 2. Myxoma* (cutaneous and mucosal)
 3. Cardiac myxoma*
 4. Breast myxomatosis* or fat-suppressed magnetic resonance imaging findings suggestive of this diagnosis
 5. PPNAD* or paradoxical positive response of urinary glucocorticosteroid excretion to dexamethasone administration during Liddle’s test
 6. Acromegaly due to GH-producing adenoma*
 7. LCCST* or characteristic calcification on testicular ultrasound
 8. Thyroid carcinoma* or multiple, hypoechoic nodules on thyroid ultrasound in a young patient
 9. Psammomatous melanotic schwannomas*
 10. Blue nevus, epithelioid blue nevus*
 11. Breast ductal adenoma*
 12. Osteochondromyxoma*
Supplementary criteria
 1. Affected first-degree relative
 2. Inactivating mutation of the PRKAR1A gene
Findings suggestive of or possibly associated with CNC, but not diagnostic for the disease
 1. Intense freckling (without darkly pigmented spots or typical distribution)
 2. Blue nevus, common type (if multiple)
 3. Café-au-lait spots or other “birthmarks”
 4. Elevated IGF-I levels, abnormal GTT, or paradoxical GH response to TRH testing in the absence of clinical acromegaly
 5. Cardiomyopathy
 6. Pilonidal sinus
 7. History of Cushing’s syndrome, acromegaly, or sudden death in extended family
 8. Multiple skin tags or other skin lesions; lipomas
 9. Colonic polyps (usually in association with acromegaly)
 10. Hyperprolactinemia (usually mild and almost always combined with clinical or subclinical acromegaly)
 11. Single, benign thyroid nodule in a young patient; multiple thyroid nodules in an older patient (detected on ultrasound)
 12. Family history of carcinoma, in particular of the thyroid, colon, pancreas, and ovary; other multiple benign or malignant tumors
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*

After histological confirmation

Figure 3.

Figure 3

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Mutation detection rate among CNC patients A) soon after the identification of PRKAR1A as a gene responsible for the disease phenotype, and, B) after the most recent analyses. The mutation detection rate increased from ~50 (2000) to above 62% (2009), and, in addition, genetic defect in PDE11A and PDE8B were identified in a phenotypic subgroup of initially recognized as CNC patients.

When opposed to the PRKAR1A mutation carriers, CNC patients with no genetic defect in PRKAR1A presented with disease overall later in life and more frequently in a sporadic fashion suggesting implication of different genetic factors (Bertherat, et al., 2009). Further, several phenotype subgroups could be distinguished among the PRKAR1A mutation negative individuals: with typical CNC manifestations (9%); with PPNAD and lentigines only (11%); with isolated bilateral micronodular hyperplasia (12%); (see Figure 3, B). Recently, a genome wide based study on the last group identified inactivating mutations in two of the human genes encoding phosphodiesterases – PDE11A and PDE8B (Gunther, et al., 2004; Horvath, et al., 2006a; Horvath, et al., 2008b). Another CNC locus on region 2p16 has been suggested by linkage analysis, however, so far no other gene has been identified, and the genetic searches for additional molecular factors continues (Stratakis, et al., 1996; Stratakis, et al., 2001).

Penetrance of CNC mutations

The overall penetrance of CNC among PRKAR1A mutations’ carriers is above 95% by the age of 50; only few PRKAR1A carriers are reported who do not meet the diagnostic criteria. Only two mutations so far have been found to result in incomplete penetrance of CNC – the splice variant c.709(-7-2)del6 and the initiation alternating substitution p.M1Vp. Even more interesting is the observation that, when expressed, these two mutations lead to relatively mild CNC phenotype restricted mostly to PPNAD, in some cases accompanied by lentigines. These findings were confirmed in all 12 unrelated families affected by c.709(-7-2)del6 and in the two unrelated kindreds where c.1A>G/p.M1V was seen (Groussin, et al., 2006). The explanation of this mild, and, in some cases, missing phenotype is seek in the light molecular effect of these two mutations – as a splice variant, c.709(-7-2)del6 does not affect 100% of the molecules, and, respectively, leads to lower level of PRKAR1A insufficiency. The molecular effect of M1V substitution is not clear, but the same substitution in the first codon is suggested to be a mild mutation for other genetic disorders such as Smith-Lemli-Opitz syndrome (Witsch-Baumgartner, et al., 2005). Taking into account the particular sensitivity of the adrenal cells to even minor changes in the PKA activity and/or PRKAR1A levels, it is suggested that adrenal will be the first tissue to react on these PRKAR1A mutations.

PRKAR1A in unselected population

Sequencing of the entire coding region of PRKAR1A has not identified alterations in at least 200 unrelated chromosomes. In addition, genotyping of 745 unrelated healthy individuals enrolled in New York Cancer Prospective study (Mitchell, et al., 2004) for 51 unique PRKAR1A mutations (Supplementary Table 1) did not detect the presence of any of them. Thus, PRKAR1A mutations are concluded to be very rare in general population. It is an essential parallel to be made with the gene encoding phosphodiesterase type 11A (PDE11A) – despite the proven association between pathogenic mutations, including null alleles, with adrenal neoplasias, such mutations are seen in general population with lower frequency (Horvath, et al., 2006a; Horvath, et al., 2006b).

Genotype-phenotype correlations

Until recently, genotype-phenotype correlation studies were limited by the high variability of the CNC phenotype, both as number and combinations of manifestations, and the relatively limited series of patients. After more than 20 years of analysis and studying of CNC patients, as well as the fusion of the major databases from the National Institutes of Health, Bethesda, MD, and the Hospital Cochin, Paris, France, more than 380 patients are genotypically characterized and clinically evaluated. An overall phenotypic segregation could be seen when the PRKAR1A mutation carriers were opposed to the mutation negative CNC patients: the former presented more frequently and earlier in life with pigmented skin lesions, myxomas, thyroid and gonadal tumors (Bertherat, et al., 2009). Further, subgroups of patients presenting with particular genotype-phenotype correlation could be outlined. The first such a group was presented by patients with isolated PPNAD, in some cases accompanied with lentiginosis. Among these patients, the following tendencies were observed: (1) patients diagnosed before 8 years 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.M1V substitution affecting the initiation codon of the protein. These observations are in line with the discussed above mild, mainly adrenal or, in some cases absent phenotype associated with these two PRKAR1A mutations.

The second group of CNC patients with particular genotype-phenotype correlation was comprised by individuals with myxomas (affecting all locations - skin, heart, and breast), PMS, thyroid tumors, and 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 the PRKAR1A mutation carriers: cardiac myxomas (p=0.02), thyroid tumors (p=0.03) and LCCSCTs (p=0.04) (Bertherat et al., 2009). Within the group of PRKAR1A carriers the mutations located in exons associated more frequently with lentigines, PMS, acromegaly and cardiac myxomas, compared to the intronic ones (p = 0.04); this observation is in line with the above discussion of a milder phenotype associated with splice variants. And, finally, the “hot spot” c.491-492delTG mutation was most significantly associated with lentigines, cardiac myxoma, and thyroid tumors when opposed to all other PRKAR1A defects combined (p = 0.03).

Diagnostic relevance

Clinical and genetic studies of CNC patients have revealed considerable heterogeneity both in mutational spectrum and in phenotypic variation. Patients with two or more diagnostic criteria from Table 4 or with isolated PPNAD are referred to genetic analysis of PRKAR1A. Once a mutation is identified, genotyping for the particular variant is recommended for all at-risk relatives. The proportion of de novo mutations in PRKAR1A is estimated to be approximately 20% (our unpublished results). Germline mosaicism has not been seen so far and may not be frequent, and thus not of diagnostic concern currently.

Conclusions and future prospects

To date, most PRKAR1A studies are focused on its coding regions and on screening for point mutations and small rearrangements detectable by PCR-based techniques. The identification of large deletions in the gene, and the significant proportion of patients with characteristic CNC phenotype and not detectable PRKAR1A mutations have triggered the application of alternative technologies for detection of non-coding variations and larger re-arrangements that may account for the disease phenotype (our on-going project).

Apart from PRKAR1A, three other genes from the cAMP signaling pathway are known to date to cause related or overlapping to CNC phenotype. Germline mutations in PDE11A and PDE8B associate with iMAD (Horvath, et al., 2006a; Horvath, et al., 2008b). Somatic activation mutations in G-alpha stimulatory subunit of the G-protein coupled receptor (GNAS) are known to result in McCune Albright syndrome – a postzygotic somatic multiple neoplasia characterized by polyostotic fibrous dysplasia (POFD), cafe-au-lait skin pigmentation, and peripheral precocious puberty (Weinstein, et al., 1991). Identification of genetic defects in cAMP-signaling molecules in patients with similar and/or overlapping phenotype underlines the importance of this signaling in the normal functioning of the endocrine and other cells. Identification of genetic defects in cAMP-signaling molecules in patients with similar and/or overlapping with CNC features underscores the importance of this signaling pathway in endocrine and other tumorigenesis; on-going studies aim at characterizing this involvement better.

Supplementary Material

References

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