Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization
M Guillaud Bataille1,2, Y Rhayem1,2, S B Sousa3,4
1Département de Biologie Hormonale, Hôpital Cochin, Assistance Publique - Hôpitaux de Paris, 27 Rue du Faubourg Saint Jacques, 75014 Paris, France.
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.
Insights
Multiplex ligation-dependent probe amplification (MLPA) detects large PRKAR1A gene deletions in Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD) patients. This method identified a novel deletion causing protein degradation and loss of function.
Area of Science:
- Genetics
- Molecular Biology
- Endocrinology
Background:
- Point mutations in the PRKAR1A gene are a known cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD).
- Approximately 30% of patients with these conditions do not have detectable mutations via standard sequencing.
- This suggests other genetic alterations, such as large deletions or duplications, may be involved.
Purpose of the Study:
- To establish a reliable method for detecting large deletions or duplications in the PRKAR1A gene.
- To functionally characterize any identified large genomic rearrangements.
Main Methods:
- Multiplex ligation-dependent probe amplification (MLPA) was employed to analyze all 12 exons of the PRKAR1A gene.
- The technique was validated and applied to patients with CNC and PPNAD who lacked detectable mutations.
- Functional analysis of a identified deletion involved western blotting and bioluminescence resonant energy transfer (BRET) assays.
Main Results:
- MLPA successfully identified a deletion spanning exons 3-6 in three family members with typical CNC.
- The resulting truncated PRKAR1A protein was expressed but rapidly degraded.
- The aberrant protein failed to interact with the catalytic subunit of protein kinase A.
Conclusions:
- Multiplex ligation-dependent probe amplification (MLPA) is an effective tool for detecting large PRKAR1A gene rearrangements in patients with suspected CNC or PPNAD when sequencing is negative.
- A novel deletion causing protein instability and loss of function was identified and characterized.
- These types of large gene defects, while significant, are not the most common cause of CNC or PPNAD.
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