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Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization of a new in-frame deletion.

ABSTRACT: BACKGROUND:Point mutations of the PRKAR1A gene are a genetic cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD), but in 30% of the patients no mutation is detected. OBJECTIVE:Set up a routine-based technique for systematic detection of large deletions or duplications of this gene and functionally characterize these mutations. METHODS:Multiplex ligation-dependent probe amplification (MLPA) of the 12 exons of the PRKAR1A gene was validated and used to detect large rearrangements in 13 typical CNC and 39 confirmed or putative PPNAD without any mutations of the gene. An in-frame deletion was characterized by western blot and bioluminescence resonant energy transfer technique for its interaction with the catalytic subunit. RESULTS:MLPA allowed identification of exons 3-6 deletion in three patients of a family with typical CNC. The truncated protein is expressed, but rapidly degraded, and does not interact with the protein kinase A catalytic subunit. CONCLUSIONS:MLPA is a powerful technique that may be used following the lack of mutations detected by direct sequencing in patients with bona fide CNC or PPNAD. We report here one such new deletion, as an example. However, these gene defects are not a frequent cause of CNC or PPNAD.

SUBMITTER: Bataille MG

PROVIDER: S-EPMC4733623 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization of a new in-frame deletion.

Bataille M Guillaud MG Rhayem Y Y Sousa S B SB Libé R R Dambrun M M Chevalier C C Nigou M M Auzan C C North M O MO Sa J J Gomes L L Salpea P P Horvath A A Stratakis C A CA Hamzaoui N N Bertherat J J Clauser E E

European journal of endocrinology 20131129 1

<h4>Background</h4>Point mutations of the PRKAR1A gene are a genetic cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD), but in 30% of the patients no mutation is detected.<h4>Objective</h4>Set up a routine-based technique for systematic detection of large deletions or duplications of this gene and functionally characterize these mutations.<h4>Methods</h4>Multiplex ligation-dependent probe amplification (MLPA) of the 12 exons of the PRKAR1A gene was valida ...[more]

PMID: 24144965

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Project description:BACKGROUND:Carney complex (CNC) is a multiple endocrine neoplasia syndrome due to inactivating mutations in the PRKAR1A gene that codes for type I? regulatory (RI?) subunit of protein kinase A. Most PRKAR1A mutations are subject to nonsense mRNA decay (NMD) and, thus, lead to haploinsufficiency. METHODS AND SETTING:Patient phenotyping for CNC features and DNA, RNA, protein, and transfection studies were carried out at a research center. RESULTS:We describe in unrelated kindreds with CNC four naturally occurring PRKAR1A mutations (1055del4, 1067del4ins5, 1076delTTins13, and 1142del4) that are predicted to escape NMD because they are located in the last coding exon of the gene. The phenotype of CNC was not different from that in other patients with the condition, although the number of patients was small. Each of the mutations caused a frameshift that led to a new stop codon into the 3' untranslated open reading frame, predicting an elongated protein that, however, was absent in patient-derived cells. After site-directed mutagenesis, in vitro transcription, and cell-free translation experiments, the expected size mutant proteins were present. However, when the mutant constructs were transfected in adrenal (NCI-295), testicular (N-TERA), and embryonic (HEK293) cells and despite the presence of the mutant mRNA, Western blot analysis indicated that there were no longer proteins. The subsequent application of proteasome inhibitors to cells transfected with the mutant constructs led to the detection of the aberrant proteins, although a compound that affects protein folding had no effect. The wild-type protein was also decreased in both patient-derived cells and/or tissues as well as in the in vitro systems used in this study. CONCLUSIONS:This was the first demonstration of proteasomal degradation of RI? protein variants leading to PRKAR1A haploinsufficiency and CNC, adding protein surveillance to NMD in the cellular mechanisms overseeing RI? synthesis. In agreement with the molecular data, CNC patients bearing PRKAR1A defects that extend the open reading frame did not have a different phenotype, although this has to be confirmed in a larger number of patients.

Project description:ContextMost tumors in Carney complex (CNC) are benign, including primary pigmented nodular adrenocortical disease (PPNAD), the main endocrine tumor in CNC. Adrenocortical cancer (AC) has never been observed in the syndrome. Herein, we describe a large Azorean family with CNC caused by a point mutation in the PRKAR1A gene coding for type 1-α (RIα) regulatory subunit of the cAMP-dependent protein kinase A, in which the index patient presented with AC.ObjectiveWe studied the genotype-phenotype correlation in CNC.Design and settingWe reported on case series and in vitro testing of the PRKAR1A mutation in a tertiary care referral center.PatientsTwenty-two members of a family were investigated for Cushing syndrome and other CNC components; their DNA was sequenced for PRKAR1A mutations.ResultsCushing syndrome due to PPNAD occurred in four patients, including the proposita who presented with AC and three who had Cushing syndrome and/or PPNAD. Lentigines were found in six additional patients who did not have PPNAD. A base substitution (c.439A>G/p.S147G) in PRKAR1A was identified in the proposita, in the three others with PPNAD, in the proposita's twin daughters who had lentigines but no evidence of hypercortisolism, and in five other family members, including one without lentigines or evidence of hypercortisolism. Unlike in other RIα defects, loss of heterozygosity was not observed in AC. The S147G mutation was compared to other expressed PRKAR1A mutations; it led to decreased cAMP and catalytic subunit binding by RIα and increased protein kinase A activity in vitro.ConclusionsIn a large family with CNC, one amino acid substitution caused a spectrum of adrenal disease that ranged from lack of manifestations to cancer. PPNAD and AC were the only manifestations of CNC in these patients, in addition to lentigines. These data have implications for counseling patients with CNC and are significant in documenting the first case of AC in the context of PPNAD.

Dataset Information

Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization of a new in-frame deletion.

Publications

Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization of a new in-frame deletion.

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