To date, a total of 117 different PRKAR1A
mutations have been identified in 387 unrelated families of various ethnic origin; they are summarized in . 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 .
List of known mutations in the PRKAR1A (sorted by their position along the gene).
Figure 1 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, (more ...)
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 . 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.
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.
The expressed missense substitutions in PRKAR1A
are listed in . 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.
In silico modeling and in vitro analysis of the effect of PRKAR1A missense substitutions on the protein function
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 ). 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, ).
Separate attention requires the missense substitution affecting the first coding aminoacid - M1V. In silico
modeling predicted very severe effect on the protein function (See ). 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.
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
Twenty-eight splice variants are identified to date in the close proximity of the exon-intron junctions of PRKAR1A (). 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 ). 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 ). 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.
In silico modeling of the effect of PRKAR1A splice variants
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
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.