Molecular Analysis of the Cyclic AMP-Dependent Protein Kinase A (PKA) Regulatory Subunit 1A (PRKAR1A) Gene in Patients with Carney Complex and Primary Pigmented Nodular Adrenocortical Disease (PPNAD) Reveals Novel Mutations and Clues For Pathophysiology: Augmented PKA Signaling is Associated with Adrenal Tumorigenesis in PPNAD
2002; Elsevier BV; Volume: 71; Issue: 6 Linguagem: Inglês
10.1086/344579
ISSN1537-6605
AutoresLionel Groussin, Lawrence S. Kirschner, Caroline Vincent-Dejean, Karine Perlemoine, Eric Jullian, Brigitte Delemer, Sabina Zacharieva, Duarte Pignatelli, J. Aidan Carney, J P Luton, Xavier Bertagna, Constantine A. Stratakis, Jérôme Bertherat,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoWe studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G→T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC. We studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G→T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC. Carney complex (CNC [MIM 160980]) is a familial multiple neoplasia syndrome transmitted as an autosomal dominant trait (Carney et al. Carney et al., 1986Carney JA Hruska LS Beauchamp GD Gordon H Dominant inheritance of the complex of myxomas, spotty pigmentation, and endocrine overactivity.Mayo Clin Proc. 1986; 61: 165-172Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). CNC was initially described as the association of myxomas, spotty skin pigmentation, and endocrine overactivity (Carney et al. 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Primary pigmented nodular adrenocortical disease (PPNAD), a rare cause of ACTH-independent Cushing syndrome, is the main endocrine manifestation of CNC. PPNAD is observed in one-fourth of patients with CNC (Stratakis et al. Stratakis et al., 1999Stratakis CA Sarlis N Kirschner LS Carney JA Doppman JL Nieman LK Chrousos GP Papanicolaou DA Paradoxical response to dexamethasone in the diagnosis of primary pigmented nodular adrenocortical disease.Ann Intern Med. 1999; 131: 585-591Crossref PubMed Scopus (171) Google Scholar, Stratakis et al., 2001Stratakis CA Kirschner LS Carney JA Clinical and molecular features of the Carney complex: diagnostic criteria and recommendations for patient evaluation.J Clin Endocrinol Metab. 2001; 86: 4041-4046Crossref PubMed Scopus (513) Google Scholar). Approximately half of the cases of CNC are familial (Stratakis et al. Stratakis et al., 2001Stratakis CA Kirschner LS Carney JA Clinical and molecular features of the Carney complex: diagnostic criteria and recommendations for patient evaluation.J Clin Endocrinol Metab. 2001; 86: 4041-4046Crossref PubMed Scopus (513) Google Scholar). Putative genetic loci have been identified by linkage analysis at chromosome 2p16 and 17q22-24 (Stratakis et al. Stratakis et al., 1996Stratakis CA Carney JA Lin JP Papanicolaou DA Karl M Kastner DL Pras E Chrousos GP Carney complex, a familial multiple neoplasia and lentiginosis syndrome: analysis of 11 kindreds and linkage to the short arm of chromosome 2.J Clin Invest. 1996; 97: 699-705Crossref PubMed Scopus (358) Google Scholar; Casey et al. 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Casey et al., 2000Casey M Vaughan CJ He J Hatcher CJ Winter JM Weremowicz S Montgomery K Kucherlapati R Morton CC Basson CT Mutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complex.J Clin Invest. 2000; 106: R31-R38Crossref PubMed Scopus (220) Google Scholar; Kirschner et al. Kirschner et al., 2000bKirschner LS Sandrini F Monbo J Lin JP Carney JA Stratakis CA Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex.Hum Mol Genet. 2000b; 9: 3037-3046Crossref PubMed Scopus (343) Google Scholar). PRKAR1A encodes the type 1α regulatory subunit of cAMP-dependent protein kinase A (PKA). Overall, inactivating mutations of this gene have been observed in ∼41% of CNC kindreds (Kirschner et al. Kirschner et al., 2000bKirschner LS Sandrini F Monbo J Lin JP Carney JA Stratakis CA Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex.Hum Mol Genet. 2000b; 9: 3037-3046Crossref PubMed Scopus (343) Google Scholar). All of the PRKAR1A defects reported so far are functionally null mutations. All sequence changes were predicted to cause premature stop codons, with the exception of one mutation that altered the transcriptional start site (at the ATG codon) (Kirschner et al. Kirschner et al., 2000bKirschner LS Sandrini F Monbo J Lin JP Carney JA Stratakis CA Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex.Hum Mol Genet. 2000b; 9: 3037-3046Crossref PubMed Scopus (343) Google Scholar). It was then demonstrated that mutant mRNAs bearing a premature stop codon were unstable, as a result of nonsense-mediated mRNA decay (NMD) (Kirschner et al. Kirschner et al., 2000bKirschner LS Sandrini F Monbo J Lin JP Carney JA Stratakis CA Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex.Hum Mol Genet. 2000b; 9: 3037-3046Crossref PubMed Scopus (343) Google Scholar). It is interesting that PRKAR1A seems to function as a classic tumor-suppressor gene in tumors from CNC patients as demonstrated by loss of the normal allele in CNC lesions (Kirschner et al. Kirschner et al., 2000aKirschner LS Carney JA Pack SD Taymans SE Giatzakis C Cho YS Cho-Chung YS Stratakis CA Mutations of the gene encoding the protein kinase A type I-α regulatory subunit in patients with the Carney complex.Nat Genet. 2000a; 26: 89-92Crossref PubMed Scopus (835) Google Scholar). Loss of the normal PRKAR1A protein and NMD of the mutant allele in these studies suggested that oncogenesis in CNC tumors was due to the complete absence of a functional PRKAR1A. In the present study, 11 kindreds with CNC, in which almost all index cases were referred for Cushing syndrome caused by PPNAD, were investigated for PRKAR1A germline mutations. Family members were evaluated by a thorough history and physical exam. Affection status was determined on the basis of the diagnostic criteria proposed recently (Stratakis et al. Stratakis et al., 2001Stratakis CA Kirschner LS Carney JA Clinical and molecular features of the Carney complex: diagnostic criteria and recommendations for patient evaluation.J Clin Endocrinol Metab. 2001; 86: 4041-4046Crossref PubMed Scopus (513) Google Scholar) and as reported by Groussin et al. (Groussin et al., 2002Groussin L Jullian E Perlemoine K Leheup B Luton JP Bertagna X Bertherat J Mutations of the PRKAR1A gene in Cushing's syndrome due to sporadic primary pigmented nodular adrenocortical disease (PPNAD).J Clin Endocrinol Metab. 2002; 87: 4324-4329Crossref PubMed Scopus (141) Google Scholar). Clinical data of all the index cases are shown in table 1.Table 1Clinical Manifestations among the Index CasesFamilyLentiginesHeart MyxomaPPNADEndocrine ManifestationsOtherCNC07++CNC03++CNC09++GH-PRL pituitary-producing adenomaEthmoidal osteochondromyxoma; PMS; blue nevus; breast ductal adenomaCNC04+++Toxic multinodular goiter; ovarian cystBenign hepatoma; PMS; breast ductal adenoma; splenic lipoma; skin myxoma; mesenteric myxoma; pelvic leiomyoma; pancreatic acinar adenocarcinomaCNC08+Ovarian cystBlue nevusCNC06+++CNC02++Calcifications on testicular ultrasonographyBlue nevusCNC01+++Thyroid tumor AcromegalyBenign hepatomaCNC05++CNC10++CNC11+Ovarian cystPMS; breast ductal adenomaNote.—Abbreviations:+=present; GH=growth hormone; PRL=prolactin; PMS=psammomatous melanotic schwannoma. Open table in a new tab Note.— Abbreviations:+=present; GH=growth hormone; PRL=prolactin; PMS=psammomatous melanotic schwannoma. DNA was extracted as reported elsewhere (Groussin et al. Groussin et al., 2002Groussin L Jullian E Perlemoine K Leheup B Luton JP Bertagna X Bertherat J Mutations of the PRKAR1A gene in Cushing's syndrome due to sporadic primary pigmented nodular adrenocortical disease (PPNAD).J Clin Endocrinol Metab. 2002; 87: 4324-4329Crossref PubMed Scopus (141) Google Scholar), and the 12 exons and the flanking intronic sequences of the PRKAR1A gene were separately PCR amplified using the primers and the conditions described elsewhere for exons 1A, 1B, 2, and 7 (Kirschner et al. Kirschner et al., 2000bKirschner LS Sandrini F Monbo J Lin JP Carney JA Stratakis CA Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex.Hum Mol Genet. 2000b; 9: 3037-3046Crossref PubMed Scopus (343) Google Scholar) and the oligonucleotides listed in table 2 for exons 3, 4A, 4B, 5, 6, 8, 9, and 10. Sequencing was performed as reported elsewhere (Groussin et al. Groussin et al., 2002Groussin L Jullian E Perlemoine K Leheup B Luton JP Bertagna X Bertherat J Mutations of the PRKAR1A gene in Cushing's syndrome due to sporadic primary pigmented nodular adrenocortical disease (PPNAD).J Clin Endocrinol Metab. 2002; 87: 4324-4329Crossref PubMed Scopus (141) Google Scholar). Ethnically matched controls (N=90) and the CEPH collection of DNA samples (N=100) were tested for PRKAR1A pathogenic disease-causing mutations; none was found, although some of the common polymorphisms of this gene were present (data not shown).Table 2Novel PRKAR1A Oligonucleotide Sequences for Mutation DetectionExon and DirectionSequenceExon 3 Sense:5′- GAATTGGTGTTTTCCTCTTAACTT-3′ Antisense:5′-TATGATTCATTCATCAAAGGAGAC-3′Exon 4A Sense:5'-AATGTTTTTGGTTTATGGAATTGT-3′ Antisense:5′-CACACCCTTACTTGAAAAATAGTG-3′Exon 4B Sense:5′-GACAGTCTGGGGTCTTTAATTCTA-3′ Antisense:5′-TCAAAGAGGAAAACAAACTTCAAT-3′Exon 5 Sense:5′-TTTCTTTAATTTGGAATATGCTTC-3′ Antisense:5′-ATCTGACATACAAGGGATGTAATG-3′Exon 6 Sense:5′-TTTTTAAAACAAAGTTCAGGATTG-3′ Antisense:5′-CTAAATCACACTCTCAAACACCAT-3′Exon 8 Sense:5′-GGCTATTTGGTTGAATCTCTTTAT-3′ Antisense:5′-TGAGTTCTTTACCTCTAAAATTCAA-3′Exon 9 Sense:5′-TTGTTTAGCTTTTTGGTGATTTTA-3′ Antisense:5′-GGAGAAGACAAAATTATGGAAGAC-3′Exon 10 Sense:5′-TATTGTCTTCTTTCTCAGAAGTGC-3′ Antisense:5′-GTGCAATAAAAGCAACTTTCAATA-3′ Open table in a new tab To prepare total cellular protein extracts, cultured lymphocytes were harvested and pelleted. Tumor tissues were obtained as reported elsewhere (Groussin et al. Groussin et al., 2000Groussin L Massias J Bertagna X Bertherat J Loss of expression of the ubiquitous transcription factor cAMP response element-binding protein (CREB) and compensatory overexpression of the activator CREMτ in the human adrenocortical cancer cell line H295R.J Clin Endocrinol Metab. 2000; 85: 345-354Crossref PubMed Scopus (55) Google Scholar). Protein assays were performed using the protein assay kit (Bio-Rad Laboratories). Equivalent protein concentrations were resolved by electrophoresis on 10% SDS-polyacrylamide gel and were transferred to nitrocellulose sheets. Western blotting was performed with primary mouse antibody to the RIα subunit (1/250; Becton Dickinson Transduction Laboratories). For detection of the first antibody with a goat anti-mouse IgG antibody (1/5000; Santa Cruz; sc-2005), chemiluminescence was used. For loss of heterozygosity (LOH) analysis, DNA from different tumors of the proband from family CNC04 was analyzed along with a paired DNA sample from peripheral blood. Seven microsatellite markers located on 17q22-24 were used: centromere-D17S807, D17S1882, D17S1813, PRKAR1A(CA)n, D17S795, D17S789, and D17S840-telomere. The sequences of the primers and genomic order of their loci were derived from the publicly available genomic databases (Genome Database; Whitehead Institute MIT, Center for Genome Research Web site). Markers were PCR amplified with end labeled 32P-radiolabeled oligonucleotide primers, and 30 cycles were performed (95°C for 30 s, 51°C for 40 s, 72°C for 40 s), followed by a final 5-min extension at 72°C. Aliquots of amplified DNA were mixed with an equal volume of loading buffer, were denatured at 94°C for 5 min, and were electrophoresed on a 6% polyacrylamide gel. Gels were dried and placed on Kodak X-Omat films. Marker PRKAR1A(CA)n PCR fragments were analyzed by electrophoresis through 4% polyacrylamide gel and were visualized with ultraviolet light after staining with ethidium bromide. A PCR-cloning method was used to construct both wild-type and exon 6–skipping mutant expression constructs. Total RNA was extracted from peripheral blood leukocytes of family CNC04 proband using RNABle (Eurobio). cDNA was synthesized by Moloney murine leukemia virus-reverse transcriptase (Invitrogen). RIα cDNA from lymphocytes was amplified using the primers 5′-GAG CAA AGC GCT GAG GGA GCT C-3′ (sense) and 5′-AAG CAT GGA TTG GGG AGA GGA G-3′ (antisense). The reaction performed with Expand Long Template PCR System (Roche) consisted of 2 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 58°C, and 2 min at 72°C. The PCR fragments corresponding to the full-length wild-type RIα cDNA and the natural mutant with the exon 6 skipping were electrophoresed on agarose gel and were purified by using a QIA quick gel extraction kit (Qiagen). These were introduced into the pGEM-T easy vector by using a TA cloning kit (Promega). The two constructs were verified by sequencing before a second PCR amplification was performed using Pwo DNA Polymerase (Roche). Each sample was incubated successively at 94°C for 15 s, 63°C for 30 s, 72°C for 1 min, for a total of 30 cycles, followed by a final extension at 72°C for 7 min. The following specific oligonucleotide primers were used: HA-RIα sense primer containing a HindIII site with the hemagglutinin HA sequence (underlined): 5′-CCT CCA AGC TTG CCA CCA TGG CTT ACC CAT ACG ACG TCC CAG ACT ACG CTG AGT CTG GCA GTA CCG CCG CC-3′ or nonHA-RIα sense primer: 5′-CCT CCA AGC TTG AGA ACC ATG GAG TCT GGC-3′; a common antisense primer containing a Xho site: 5′-CCG TTC TCG AGT CAG ACA GAC AGT GAC ACA AAA CT-3′. The four products were purified by gel electrophoresis, were digested with HindIII and Xho, and were cloned into the HindIII/Xho sites of the pREP4 expression vector (Invitrogen) to create HA-RIα-WT, HA-RIα-Δ184–236, RIα-WT, and RIα-Δ184–236. All constructs were sequenced prior to their use in expression studies. Lymphocytic cell culture and cycloheximide treatment were performed as reported elsewhere (Kirschner et al. Kirschner et al., 2000bKirschner LS Sandrini F Monbo J Lin JP Carney JA Stratakis CA Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex.Hum Mol Genet. 2000b; 9: 3037-3046Crossref PubMed Scopus (343) Google Scholar). COS7 cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% FCS, glutamine, and gentamycine. The cells were plated in six-well dishes and were transfected 24 h later by using Lipofectamine Plus (Invitrogen) following the protocol provided. We used the luciferase reporter pSS-CRE-LUC containing a sequence of the rat somatostatin gene −71–53 (including the CRE site) inserted in the 5′-region of the luciferase gene. The reporter gene RSV-βGal (Bertherat et al. Bertherat et al., 1995Bertherat J Chanson P Montminy M The cyclic adenosine 3′,5′-monophosphate-responsive factor CREB is constitutively activated in human somatotroph adenomas.Mol Endocrinol. 1995; 9: 777-783PubMed Google Scholar) was used as an internal control for transfection efficiency. Cells were cotransfected with 0.2 μg of pSS-CRE-LUC, 0.25 μg of RSV-βGal, and 1.5 μg of plasmid expressing HA-RIα-WT or molar equivalent of HA-RIα-Δ184–236 and empty pREP4 plasmids. Six hours prior to harvesting, half of the transfected dishes were incubated in a mixture of 10−5 M forskolin (Sigma) and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma). Cells were harvested 24 h after transfection and were subjected to lysis; luciferase activity was then assayed for and was normalized to β-galactosidase activity. All transfection experiments were performed in triplicate and were repeated five times; the results are expressed as the means. Statistical significance was assessed by a Student's t test (StatView 5.0., SAS Institute). Control immunoblotting analysis was performed to confirm that HA-RIα constructs had equivalent levels of expression. Total lysates from empty vector, HA-RIα-WT, or HA-RIα-Δ184–236 COS transfected cells were used in Western blotting, which then was evaluated by chemiluminescence using anti-HA antibody (Santa Cruz, sc-805). Sequencing of the 12 coding exons of the genomic DNA from the index cases revealed a PRKAR1A mutation in the heterozygotic state in 9 of the 11 kindreds (details are given in table 3 and figs. 1, 2, and 3). Seven of these mutations have not been previously reported. Four of them occurred de novo (fig. 1). The 578 delTG and the exon 8 IVS + 3 A→G mutations were reported elsewhere (Kirshner et al. Kirschner et al., 2000bKirschner LS Sandrini F Monbo J Lin JP Carney JA Stratakis CA Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex.Hum Mol Genet. 2000b; 9: 3037-3046Crossref PubMed Scopus (343) Google Scholar). The 578 delTG mutation is the most frequent CNC mutation found to occur de novo in more than eight kindreds so far; founder effect has been excluded in most of these kindreds by extensive genotyping of chromosome 17 markers when relatives were available (data not shown).Table 3PRKAR1A Mutations in Nine Families with CNCFamilyMutationEffect on RIαCNC07Exon 1B 12G→AAdditional out-of-frame ATG initiation codon within a consensus sequence for translation initiation; could abolish translation of the wild-type proteinCNC03Exon 4B IVS +1G→AExon skippingCNC09Exon 4B 578 delTGFrameshift after codon 163; stop codon after 4 missense residuesCNC04Exon 6 IVS +1G→TExon skippingCNC08Exon 7 IVS del (−7→−2)Exon skippingCNC06810 ins A (exon 7)Frameshift after codon 241; stop codon after 6 missense residuesCNC02815 del 13 bp (exon 7)Frameshift after codon 243; stop codon after 9 missense residuesCNC01850 del AT (exon 7)Frameshift after codon 255; stop codon after 13 missense residuesCNC05Exon 8 IVS +3A→GActivation of a cryptic splice site Open table in a new tab Figure 2A heterozygous G→A transversion in exon 1B gives rise to a novel ATG translation initiation codon. A, Pedigree of family CNC07. The proband is indicated by the arrow. B, Sequencing of PRKAR1A exon 1B revealed a heterozygous mutation that creates an upstream, out-of-frame ATG codon in the RIα1B mRNA within a consensus sequence for translation initiation. This AUG codon is classified as a strong start site on the basis of the A and G, respectively, at position −3 and +4 (Kozak Kozak, 1997Kozak M Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6.Embo J. 1997; 16: 2482-2492Crossref PubMed Scopus (407) Google Scholar). According to the scanning model of eukaryotic translation, the novel ATG should initiate translation of a truncated protein and decrease translation from the wild-type start codon (Kozak Kozak, 1999Kozak M Initiation of translation in prokaryotes and eukaryotes.Gene. 1999; 234: 187-208Crossref PubMed Scopus (1079) Google Scholar), as administrated for CDKN2A gene (Liu et al. Liu et al., 1999Liu L Dilworth D Gao L Monzon J Summers A Lassam N Hogg D Mutation of the CDKN2A 5′ UTR creates an aberrant initiation codon and predisposes to melanoma.Nat Genet. 1999; 21: 128-132Crossref PubMed Scopus (208) Google Scholar). The location of mutant ATG codon is shown with a horizontal band. The mutation is indicated by an arrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3An exon 6 splice-site mutation leads to an exon 6 skipping in family CNC04. A, Pedigree of family CNC04. The subjects who were studied in each generation are numbered. The question mark (?) represents an individual of unknown affection status. The proband (II.1) is indicated by the arrow. She presented with a severe form of Carney complex and died of a pancreatic adenocarcinoma with rapidly growing liver metastasis. Her unaffected mother was also investigated for the presence of the mutation. Only the proband and her affected son were heterozygous for the intron 6 splice mutation. The proband's sequence trace of the sense strand showed a G→T transversion in the splice donor site of intron 6. B, Migration, on a 1% agarose gel, of RT-PCR products from transformed lymphocytes and PPNAD from family CNC04's proband. After gel extraction and purification, RT-PCR products were directly sequenced. Top, Schematic representation of the gene organization of coding exons giving rise to the mature RIα mRNA. Regions that encode functional domains are denoted. The localization of the primers used for the RT-PCR is indicated with arrows. Bottom, Schematic representation of exon 6 skipping mRNA. C, RT-PCR products from transformed lymphocytes from patient carrying the exon 6 IVS del(−9→−2) mutation. No change is obvious after 4 h of treatment with 100 μg/ml cycloheximide (X) compared to the vehicle (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The mutation found in one kindred (CNC04) was a G→T transversion in the 5′ splice-donor site of intron 6. The proband had died of a severe form of CNC; her severely affected son also had the mutation, but her unaffected mother did not (fig. 3A). This unique mutation was predicted to lead to exon skipping; however, the sequence change was in frame, because exon 6 contains 53 triplet codons, making it unlikely that NMD was ongoing (fig. 3B). In fact, the mutant mRNA was present in the proband's peripheral lymphocytes before and after treatment with cycloheximide (fig. 3C), consistent with an exon 6 skipping mutation, exon 6 IVS del(−9→−2), shown elsewhere (Kirschner et al. Kirschner et al., 2000bKirschner LS Sandrini F Monbo J Lin JP Carney JA Stratakis CA Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the Carney complex.Hum Mol Genet. 2000b; 9: 3037-3046Crossref PubMed Scopus (343) Google Scholar). Exon 6 is located in the region encoding for cAMP-binding domain A and gives rise to 53 amino acids (184–236) of the human RIα subunit. To detect shortened RIα forms (RIα-Δ184–236), we performed western-blot analysis of cell lysates from peripheral lymphocytes from six CNC patients with known mutations of the PRKAR1A gene, including the proband of family CNC04 and three control subjects (fig. 4A). Only the patient with the exon 6 mRNA skipping mutation showed the shortened RIα form. We then studied five different tumors from this patient (adrenal nodules [left and right PPNAD], a pancreatic adenocarcinoma, schwannoma, and hepatoma); the deleted form of RIα was found in all tumors (fig. 4B). It is interesting that only the pancreatic adenocarcinoma demonstrated loss of expression of the RIα wild-type protein. To understand the mechanism underlying this observation, we performed LOH analysis using peripheral blood DNA and DNA from the tumors examined above. Seven markers located around the PRKAR1A gene were studied, including the intragenic dinucleotide repeat. Consistent with the western blotting data, only the malignant tumor showed LOH for all informative markers, including the intragenic one (fig. 4C). To investigate the consequences of the deleted form of the RIα protein at the transcriptional level, transient transfections were performed under the assumption that changes in expression of cAMP-responsive genes reflect PKA activity, as shown elsewhere (Gonzalez and Montminy Gonzalez and Montminy, 1989Gonzalez GA Montminy MR Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (1996) Google Scholar). We created HA-expression vectors for the wild-type RIα protein (RIα-WT) and the deleted mutant (RIα-Δ184–236). Forskolin, an adenylyl-cyclase activator and IBMX, a phosphodiesterase inhibitor, were used as stimulants of the cAMP signaling pathway. We then examined the effects of the wild-type RIα and RIα-Δ184–236 on the activity of a luciferase reporter gene under the control of the somatostatin promoter containing a cAMP-responsive element (CRE), a binding site for ATF/CREB transcription factors. As shown in figure 5A, RIα-Δ184–236 showed higher transcriptional activity of the CRE-somatostatin gene t
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