Mutations in the Protein Kinase a R1α Regulatory Subunit Cause Familial Cardiac Myxomas and Carney Complex

Amendment history: Corrigendum (January 2001) Mutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complex

Mairead Casey, … , Cynthia C. Morton, Craig T. Basson

J Clin Invest. 2000;106(5):R31-R38. https://doi.org/10.1172/JCI10841.

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Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1α gene encoding the R1α regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1α frameshift mutations in three unrelated families result in haploinsufficiency of R1α and cause Carney complex. We did not detect any truncated R1α protein encoded by mutant PRKAR1α. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1α deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1α alleles and that wild-type R1α protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1α to R2β regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1α haploinsufficiency on PKA activity and the role […]

Find the latest version: https://jci.me/10841/pdf α Mutations in the protein kinase A R1 Online ﬁrst regulatory subunit cause familial cardiac PUBLICATION myxomas and Carney complex

Mairead Casey,1,2 Carl J. Vaughan,1 Jie He,1 Cathy J. Hatcher,1,2 Jordan M. Winter,1 Stanislawa Weremowicz,3 Kate Montgomery,4 Raju Kucherlapati,4 Cynthia C. Morton,3,5 and Craig T. Basson1,2

1Molecular Cardiology Laboratory, Cardiology Division, Department of Medicine, Weill Medical College of Cornell University, New York, New York, USA 2Department of Cell Biology, Weill Medical College of Cornell University, New York, New York, USA 3Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA 4Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York, USA 5Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Boston, Massachusetts, USA Address correspondence to: Craig T. Basson, Molecular Cardiology Laboratory, Cardiology Division, Weill Medical College of Cornell University, 525 E. 68th Street, New York, New York 10021, USA. Phone: (212) 746-2201; Fax: (212) 746-2222; E-mail: [email protected].

Mairead Casey and Carl J. Vaughan contributed equally to this work.

Received for publication July 21, 2000, and accepted July 28, 2000.

Cardiac myxomas are benign mesenchymal tumors that can present as compo- nant cardiac myxomas are associated nents of the human autosomal dominant disorder Carney complex. Syndromic with spotty pigmentation of the skin cardiac myxomas are associated with spotty pigmentation of the skin and and nonneoplastic hyperfunctioning endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to endocrine states, e.g., primary pigment- chromosome 17q24. We now demonstrate that the PRKAR1α gene encoding the ed nodular adrenocortical hyperplasia. R1α regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to Individuals affected with Carney com- this chromosome 17q24 locus. Furthermore, we show that PRKAR1α frameshift plex may have extracardiac (most often mutations in three unrelated families result in haploinsufficiency of R1α and cutaneous) myxomas, as well as a vari- cause Carney complex. We did not detect any truncated R1α protein encoded ety of other benign neoplasms includ- by mutant PRKAR1α. Although cardiac tumorigenesis may require a second ing schwannomas, pituitary adenomas, somatic mutation, DNA and protein analyses of an atrial myxoma resected from thyroid adenomas, and breast fibroade- a Carney complex patient with a PRKAR1α deletion revealed that the myxoma nomas. Cardiac myxomas of Carney cells retain both the wild-type and the mutant PRKAR1α alleles and that wild- complex are histologically indistin- type R1α protein is stably expressed. However, in this atrial myxoma, we did guishable from more common spo- observe a reversal of the ratio of R1α to R2β regulatory subunit protein, which radic cardiac myxomas and, like the lat- may contribute to tumorigenesis. Further investigation will elucidate the cell- ter, most often arise in the left atrium at specific effects of PRKAR1α haploinsufficiency on PKA activity and the role of the fossa ovalis (7). However, unlike PKA in cardiac growth and differentiation. sporadic cardiac myxomas, which most This article may have been published online in advance of the print edition. The date of public- often occur as isolated single lesions in ation is available from the JCI website, http://www.jci.org. J. Clin. Invest. 106:R31–R38 (2000). middle-aged women and which are usually amenable to surgical resection, syndromic cardiac myxomas exhibit no Introduction and rheumatologic symptoms attrib- age or sex preference and may present Cardiac myxomas are benign neo- uted (4) to myxoma-mediated produc- as multiple concurrent lesions in any plasms that occur in 7 per 10,000 indi- tion of IL-6. Seven percent of cardiac cardiac chamber. Affected individuals viduals (1). These slowly proliferating myxomas (1) are components of a may have multiple recurrences at any lesions arise from subendocardial familial autosomal dominant syn- cardiac location despite adequate sur- pluripotent primitive mesenchymal drome (Figure 1) that has been variably gical margins (7). cells, which can differentiate within referred to as LAMB (lentigines, atrial Clinical evaluation and genetic link- myxomas along a variety of lineages myxoma, mucocutaneous myxoma, age analysis of families affected by Car- including epithelial, hematopoietic, blue nevi) and NAME (nevi, atrial myx- ney complex (8, 9) suggested two and muscular (2, 3). Morbidity and oma, myxoid neurofibromata, ephel- human chromosomal loci for disease mortality from cardiac myxomas are ides) and more recently as Carney com- genes: chromosome 2p16 and chro- the result of embolic stroke, heart fail- plex (OMIM #160980, ref. 5; and ref. 6). mosome 17q24. Linkage to the chro- ure due to intracardiac obstruction, In Carney complex, autosomal domi- mosome 17q24 locus (CAR) was

The Journal of Clinical Investigation | Volume 106 R31 observed in five families affected by These oligonucleotides were also used ATAGTG-3′ Carney complex (8). We have now to amplify a GNAS13 gene segment Exon 5: 5′-GACAGTCTGGGGTCTTTAAT- refined the CAR interval and used a from human genomic DNA. This TCTA-3′ / 5′-TCAAAGAGGAAAACAAACT- positional cloning strategy to demon- product was random hexamer–radio- TCAAT-3′ strate that familial cardiac myxomas labeled and used as a probe to screen Exon 6: 5′-TTTCTTTAATTTGGAATATGC- and Carney complex are caused by membrane arrays of the Roswell Park TTC-3′ / 5′-ATCTGACATACAAGGGATG- mutations in the gene (PRKAR1α) Cancer Institute human genomic BAC TAATG-3′ encoding the R1α regulatory subunit library. BAC and PAC DNA was pre- Exon 7: 5′-TTTTTAAAACAAAGTTCAGGA- of cAMP-dependent protein kinase A pared with the QIAGEN Large Con- TTG-3′ / 5′-CTAAATCACACTCTCAAACA- (PKA) on chromosome 17q24. We fur- struct kit (QIAGEN Inc.) and was CCAT-3′ ther propose that PRKAR1α acts as a sequenced directly with oligonu- Exon 8: 5′-ATTATTCCATAGCATTATGTG- tumor suppressor gene to regulate cell cleotides randomly designed from GTG-3′ / 5′-AGTCACAGAGGAAATAACT- proliferation within the human heart. GNAS13 coding sequence on an ABI GTGAA-3′ 377 sequencer (Perkin-Elmer Inc.). Exon 9: 5′-GGCTATTTGTTGAATCTCTT- Methods Primers were designed as follows from TAT-3′ / 5′-TGAGTTCTTTACCTCTAAAA- Clinical evaluation. Informed consent intronic sequence flanking exons 2, 3, TTCAA-3′ was obtained from all participants in and 4: Exon 10: 5′-TTGTTTAGCTTTTTGG GAT- accordance with the Weill Medical Exon 2: 5′-GAGTCAGTTCGCTGGTTCC-3′ TTTA-3′ / 5’GGAGAAGACAAAA TATGGA- College of Cornell University Com- / 5′-TGCCCTTAACCCCGGCCCCATTC-3′ AGAC-3′ mittee on Human Rights in Research. Exon 3: 5′-AAGGTTTGTCTAGGTTAATT- Exon 11: 5′-TATTGTCTTCTTTCTCA- All family members were evaluated by C-3′ / 5′-TAGCCTTTCAGTCTGTTCCTC- GAAGTGC-3′ / 5′-GTGCAATAAAAGCAA- a thorough history and physical exam CA-3′ CTTTCAATA-3′ without knowledge of genotype status. Exon 4: 5′-GAGTCAGTTCGCTGGTTCC-3′ Exons 1 and 2 were amplified by RT- If there was any evidence of dermato- / 5′-TGCCCTTAACCCCGGCCCCATTC-3′ PCR from patient lymphocyte or lym- logic, cardiac, or endocrine disease Exon 5 was amplified in two overlapping phoblast RNA samples with the suggestive of Carney complex, patients segments using primer pairs: Access RT-PCR kit (Promega Corp., were further evaluated by electrocar- 5′-AGGCATGACCACCATGTCTGACCA-3′ Madison, Wisconsin, USA) and diography, transthoracic echocardiog- / 5′-GATTGGTCAGTCGATCTTCCA-3′ 5′- primers: raphy, and serum chemistry. GTTTCGACAGTGTGACATCAATAC-3′ / 5′-GCTGGGAGCAAAGCGCTGAGGGA-3′ Genetic analyses. Peripheral blood was 5′-TCTAATTCTGGTTGTAAACTGCTA-3′ / 5′-TGTAGACCTCAGCGCTGATAGC-3′ obtained from each family member, Each exon was PCR-amplified with Amplified products were sequenced and lymphoblastoid lines were estab- the appropriate primer pair from in both directions on an ABI 377 auto- lished by transformation with the human genomic DNA samples with mated sequencer using BigDye Termi- Epstein-Barr virus (8). Genomic DNA reaction conditions as follows: [94°C × nator Cycle Sequencing reagents and RNA were isolated from peripher- 20 s, 57°C × 30 s, 72°C × 45 s] × 35 (Perkin-Elmer Inc.). Two novel STRs, al lymphocytes, lymphoblasts, or cycles. In all cases except exon 2 ampli- an AG28 dinucleotide repeat and an tumor cells with QIAamp columns fication, standard PCR reagents were ATT11 trinucleotide repeat, were noted (QIAGEN Inc., Valencia, California, used with AmpliTaq Gold (Perkin- in the PRKAR1α 5´ untranslated USA). Polymorphic short tandem Elmer Inc.). Exon 2 was amplified with region and 21 kb 3′ to the gene, respec- repeats (STRs) were amplified by PCR the QIAGEN Taq DNA Polymerase Kit tively. PCR primers used to amplify with published nucleotide primer (QIAGEN Inc.). Amplified products using these repeats for genotyping sequences (10), analyzed on denatur- were sequenced in both directions on studies were: ing polyacrylamide gels (8), and visu- an ABI 377 automated sequencer using AG: 5′-CTGCAGGAAGAGGGAGAAATGG- alized by autoradiography or on an BigDye Terminator Cycle Sequencing GAA-3′ / 5′-AGAAGTTAAGTGACTTGCC- ABI 377 sequencer (Perkin-Elmer Inc., reagents (Perkin-Elmer Inc.). CATGGC-3′ Norwalk, Connecticut, USA). Cytoge- PRKAR1α sequence analysis. Oligonu- ATT: 5′-TTTCAGCCTCAGTATCTTTATT- netic analysis of tumor and lympho- cleotides (as below) were designed based TG-3′ / 5′-ACCTAAAGTCTTTCTGCCAGT- cyte samples was performed as previ- on intronic sequence flanking each TAT-3′ ously described (11). PRKAR1α exon (3–11) and were used to Western blot analysis. Lymphoblast and Cloning and sequence analysis of PCR-amplify them from human genom- tumor samples were solubilized in 2% GNAS13. Oligonucleotides (5′- ic DNA samples. PCR conditions were SDS. Equivalent protein concentra- CTATTCTGCATGACAACCTGAAGC-3′ / [94°C × 20 s, 57°C × 30 s, 72°C × 45 s] × tions of lysate were electrophoresed on 5′-TCTAATTCTGGTTGTAAACTGCTA-3′) 35 cycles with standard PCR reagents 10% acrylamide denaturing gels and corresponding to the 3′ portion of the and AmpliTaq Gold (Perkin-Elmer Inc.). transferred to PVDF membrane. West- GNAS13 gene (12), including untrans- Exon 3: 5′-GAATTGGTGTTTTCCTCTTA- ern blotting was performed with anti- lated sequence, were used to screen, by ACTT-3′ / 5′-TATGATTCATTCATCAAAG- actin (Sigma Chemical Co., St. Louis, PCR, arrayed pools of the Roswell Park GAGAC-3′ Missouri, USA) and with primary Cancer Institute (Buffalo, New York, Exon 4: 5′-AATGTTTTTGGTTTATGGAA- mAb’s to the R1α and R2β PKA sub- USA) human genomic PAC library. TTGT-3′ / 5′-CACACCCTTACTTGAAAA- units (Becton Dickinson Transduction

R32 The Journal of Clinical Investigation | Volume 106 Laboratories, Lexington, Kentucky, cause McCune-Albright syndrome, 5 exons with an open reading frame USA). Antigenic epitopes in the PKA which shares several phenotypic fea- beginning in exon 2. Exons 2–5 regulatory subunits are in the NH2-ter- tures with Carney complex, including (GNAS13 coding sequence) were minal hinge region (13), and positive abnormal skin pigmentation and amplified from patient DNA samples controls supplied by Becton Dickinson endocrinopathy (5), but Gsα muta- and subjected to automated sequenc- Transduction Laboratories were rat tions are not found in Carney complex ing. Although a GGC7 trinucleotide cerebrum lysate (R1α) and human aor- patients (14). Given the homologies STR within the 5′ untranslated tic endothelial lysate (R2β). Bound pri- between Gα13 and Gsα and the chro- sequence in exon 2 of GNAS13 (3 bp mary antibody was detected by ECL mosomal location of the GNAS13 5′ to the translation start site) was (Amersham Pharmacia Biotech, Piscat- gene, we searched for mutations in the found to be polymorphic, no muta- away, New Jersey, USA). Densitometry GNAS13 gene in five unrelated Carney tions were founding within the was performed with a Fluor-S imaging complex patients. GNAS13 coding sequence. system and MultiAnalyst software We screened the Roswell Park Can- Identification of PRKAR1α mutations. (Bio-Rad Laboratories Inc., Hercules, cer Institute BAC and PAC human Interrogation of the Human Genome California, USA). An equivalent genomic DNA libraries and identified Project databases further revealed that amount of total cellular protein for six BAC (472J20, 484A6, 583F2, the PRKAR1α gene (accession no. lymphoblast samples was loaded on 489G5, 657B12, 752A23) and four NM002374) encoding the R1α regula- each gel, based on actin expression. As PAC (704J11556, 704K19308, tory subunit of cAMP-dependent PKA estimated relative to actin expression, 704A09613, 704A10613) clones for was also included within the CAR the amount of myxoma lysate cellular GNAS13. Comparison of BAC/PAC locus minimal interval (Figure 2). protein loaded was 10% of lymphoblast sequences with GNAS13 cDNA PRKAR1α had previously been lysate cellular protein. sequence established the genomic mapped (as the Tissue-Specific Extin- structure of the human GNAS13 gene: guisher 1 [TSE1] locus) to chromo- Results Genetic and physical mapping of the CAR locus. Our previous analyses demonstrated that the Carney complex disease gene is located in the 17-cM interval between D17S807 and D17S785. Analy- sis of high-density genetic and physical maps (10, 12) of this locus (Figure 2) permitted the fine ordering of ten additional microsatellite polymorphisms (Figure 2, Table 1) within this interval, which were used to genotype families YA and YB. Haplotype analysis revealed recombination events between the Car- ney complex disease gene and D17S1882 and D17S929 at the centromeric and telomeric ends, respectively, of the CAR locus. Thus, these genetic analyses refined the CAR locus and established a minimal genetic interval of about 12 cM between D17S1882 and D17S929 for the CAR locus. Exclusion of GNAS13. Computerized analysis of the Human Genome Pro- ject/GenBank database (12) revealed that the D17S2016 sequence-tagged site (STS) was included within the centromeric portion of the CAR locus. BLAST analysis of this STS demonstrated that the sequence correspond- Figure 1 ed to the coding sequence of the Pedigree of family YF and cardiac myxoma in a proband. The subject number and disease GNAS13 gene (GenBank accession no. status of each family member analyzed are indicated. Squares denote male family members, and circles females. Affected and unaffected individuals are represented by closed and open L22075) encoding the GTP-binding α symbols, respectively. Carney complex is transmitted in an autosomal dominant fashion. protein, G 13. Mutations in another Transesophageal echocardiography of individual I-1 revealed an unusual posterior mass GTP-binding protein involved in intra- (arrow) in the right atrium (RA) below the eustachian valve (EV). Pathologic analysis at sur- cellular signal transduction, Gsα, gery revealed that the mass was a cardiac myxoma.

The Journal of Clinical Investigation | Volume 106 R33 some 17q23-q24 by somatic cell sequence analysis. Two probands were nucleotides 576–577 at Thr163 was hybrid analysis (15, 16). Additionally, from families that had been linked to present in all affected individuals but we noted that the 155-kb human chromosome 17q24 (families YA and not in unaffected individuals in family genomic BAC clone RP11-62F10 YB), and the others (YE, YF, and SY2) YF with a resultant frameshift leading (accession no. AC005799.1) contained were from families that were too small to a premature stop 6 codons later. sequences corresponding to both to definitively establish linkage to any Mutations (∆FSterGly208 in family PRKAR1α and the anonymous STR chromosomal locus. Clinical features YA, ∆FSterVal253 in family YB, D17S789, which exhibited no recombi- of families YA and YB have been previ- ∆FSterThr163 in family YF) were con- nation with the Carney complex dis- ously described (8). Probands from the firmed by sense and antisense sequence ease gene in the families that we had other three families all exhibit spotty analysis and by gel electrophoresis of studied. Therefore, the PRKAR1α pigmentation of the skin and have had PCR products to demonstrate the dif- chromosomal location, the known cardiac myxomas. ferent lengths of the alleles. None of role of PKA in signal transduction and We observed heterozygous PRKAR1α these sequence variants was observed cell growth, and the ubiquitous expres- gene mutations (Figures 3 and 4) in in 100 normal unrelated chromo- sion pattern of the R1α subunit three Carney complex probands, somes. We also observed a single base (16–18) all suggested that PRKAR1α including the two probands from chro- pair thymine insertion in a poly-T tract was a candidate disease gene for Car- mosome 17q24–linked families (YA in intron 3 that is 9 nucleotides 5′ to ney complex. and YB). Bidirectional sequence analy- the exon 4 splice acceptor site. This The coding sequence of PRKAR1α is sis demonstrated the presence of a 1-bp insertion was a common polymor- 1.146 kb in length. Comparison of deletion (G) of nucleotide 710 at phism that was present in 50% of indi- the genomic sequence derived from Gly208 in affected individuals but not viduals genotyped. BAC RP1162F10 with PRKAR1α unaffected individuals in family YA PRKAR1α analysis in a cardiac myxo- cDNA sequence elucidated the with a consequent frameshift and pre- ma. The frameshift PRKAR1α muta- intron-exon boundaries and showed mature stop 13 codons later. A 2-bp tions observed in families YA, YB, and that the gene was comprised of 11 deletion (TC) of nucleotides 845–846 YF would all yield truncated protein exons (Figure 3), not 10 as previously at Val253 was observed in affected indi- products lacking the cAMP-binding reported (19). However, no novel viduals but not unaffected individuals domains (Figure 3) if the mutant alle- genomic sequence was available in the from family YB with a consequent les were transcribed and translated. Human Genome Project database frameshift and premature stop 15 Alternatively, these mutant alleles corresponding to exons 1 and 2 of the codons later. A 2-bp deletion (TG) of might be null alleles due to degrada- PRKAR1α gene to compare with the previously described genomic structure (accession no. Y07641). Analysis of the genomic sequence of a PRKAR1α pseudogene contained within BAC clone RP4-621B10 (ref. 19; accession no. AL13883), which maps to chromosome 1 and begins with sequence 5′ to the open reading frame, raised the possibility that the proposed boundary (19) between exons 1 and 2 might be imprecise. To analyze the coding sequence of the PRKAR1α gene for mutations that might cause Carney complex, we designed PCR primers from the intron sequence flanking exons 3–11 and amplified these exons from human genomic DNA samples. Because of ambiguity about the genomic organi- zation of PRKAR1α exons 1 and 2, we designed oligonucleotides based on coding sequence from exon 3 and on 5′ Figure 2 untranslated sequence within exon 1 Ideogram of chromosome 17 with Giemsa banding pattern showing localization of the Car- ney complex locus and the PRKAR1α gene. Positive bands are black and the pericentromeric to amplify, via RT-PCR, exons 1 and 2 region is gray. Numbers indicate cytogenetic designations for bands. The genetic map loca- from lymphocyte/lymphoblast RNA tions of polymorphic loci analyzed from 17q are given. One centimorgan (cM) is indicated. samples. PRKAR1α exons 1–11 ampli- Linkage data suggested that the gene responsible for Carney complex is located in the 12-cM fied from five Carney complex unre- interval between D17S1882 and D17S929 (CAR). Both GNAS13 and PRKAR1α map cytoge- lated probands were then subjected to netically to chromosome 17q23-q24.

R34 The Journal of Clinical Investigation | Volume 106 in the R1α:R2β ratio secondary to decreased R1α expression consistent with PRKAR1α haploinsufficiency. However, in the tumor sample there was a reversal in the R1α:R2β ratio that may relate to further decreased R1α and/or increased R2β (Figure 5).

Discussion Figure 3 We have demonstrated that mutations Three frameshift mutations in PRKAR1α that cause Carney complex. A schematic represen- in the PRKAR1α gene encoding the tation of PRKAR1α cDNA shows exons 1–11. Regions that encode functional domains R1α regulatory subunit cause familial (dimerization domain, antigenic sites, hinge/pseudophosphorylation site, cAMP-binding cardiac myxomas in autosomal domi- α α domains A and B) of R1 are denoted (13). Location of the PRKAR1 deletion mutations nant Carney complex. Individuals at ∆ ∆ ∆ FSterGly208 in family YA, FSterVal253 in family YB, and FSterThr163 in family YF are risk for cardiac myxomas who are shown. Mutations are denoted by ∆FSter to indicate a deletion with resultant frameshift and affected by Carney complex develop premature stop codon, and by the first amino acid residue affected by the deletion. disease based on PRKAR1α haploinsufficiency secondary to heterozygous tion of the mutant mRNA via non- deleted mutant alleles are present in constitutional frameshift mutation of sense-mediated decay (20). To distin- this tumor. Finally, to date, we have PRKAR1α that results in a nonex- guish between these possibilities, not detected any additional sequence pressed allele. Moreover, PRKAR1α expression of the R1α protein was variants in PRKAR1α amplified from acts as a tumor suppressor gene in the determined with specific antibody to this atrial myxoma. Densitometry of heart and other tissues, presumably by the NH2-terminus of R1α by Western Western blot analysis (Figure 5) of the regulation of PKA activity. PRKAR1α blot (Figure 5) of lymphoblasts derived R2β regulatory subunit in the same function as a tumor suppressor gene is from a normal individual, lym- samples (normal lymphoblasts, Car- consistent with findings (21) demon- phoblasts derived from an affected ney complex lymphoblasts, and atrial strating that PRKAR1α accounts for individual (family YA, IV-1 with the myxoma) analyzed for R1α protein tissue-specific inhibition of gene tran- Gly208 deletion/frameshift mutation expression demonstrated that R2β scription at the TSE1 locus on chro- described above), and a left atrial myx- expression was similar in normal and mosome 17q24. Haploinsufficiency of oma from this affected individual. affected lymphocytes, with a decrease another kinase, STK11, followed by Only full-length R1α protein was present in all three samples without any evidence of truncated product. Den- Table 1 sitometry showed that the amount of Haplotype analysis of individuals in families affected by Carney complex R1α protein in Carney complex lymphocytes was 60% less than that in normal lymphocytes, consistent with haploinsufficiency of PRKAR1α in Carney complex. Full-length R1α was also present in the atrial myxoma sample, suggesting that the wild-type allele is preserved in the tumor. Cytogenetic analysis of this atrial myxoma revealed a normal 46,XX karyotype and failed to demonstrate any abnormalities at chromosome 17q23-24. Genotyping with STR D17S789, which is closely genetically and physically linked to the PRKAR1α gene as described above, as well as with an ATT trinucleotide repeat 21 kb 3′ to the PRKAR1α coding sequence demonstrated no loss of heterozygosity at this locus. (Genotyp- ing with an AG dinucleotide repeat in Designations of individuals who are affected by Carney complex are in bold. Microsatellites from chro- the 5′ untranslated PRKAR1α mosome 17q are ordered from centromere to telomere. Recombination events between Carney complex sequence was uninformative.) Se- disease gene and microsatellites are shown in black. Individuals who are concordant are shown in white, 1α and uninformative analyses are shown in gray. The boxed region shows the minimal chromosome 17q24 quence analysis of PRKAR exon 7 interval (12 cM between D17S1882 and D17S929) for the Carney complex disease gene in which there is shows that both the wild-type and no evidence of recombination.

The Journal of Clinical Investigation | Volume 106 R35 conformational change and dissoci- ates from the catalytic subunits; the free catalytic subunits are enzymati- cally active. Activity of cAMP-dependent PKA can either inhibit or stimulate cell proliferation depending on the cell type (18, 26–28). PKA activity in any given cell type is dependent not only upon the concentration of free catalytic subunit but also upon the cell type–specific ratio of regulatory sub- Figure 4 units (3). R1:R2 is modulated not only Mutational analysis of PRKAR1α in Carney complex families YA, YB, and YF. Automated at the gene expression level (18, 19, 29) sequence analyses of wild-type and mutant exons 7, 8, and 5 amplified from normal and but at the protein level by a wide array affected individuals in families YA, YB, and YF, respectively, are shown. Sequence of the sense of different cell type–specific binding strand is shown for family YA, and the antisense strand for families YB and YF. A heterozy- proteins for the regulatory subunits (A gous 1-bp deletion is noted in family YA, and 2-bp deletions in families YB and YF (arrows; kinase–anchoring proteins, or AKAPs) deleted bases are underlined). These mutations all result in a frameshift with consequent (26–31). Changes in one regulatory premature stop codons. subunit concentration may incom- pletely compensate for changes in another. For instance, knockout of somatic mutation, causes the pheno- the NF1 tumor suppressor gene as well R1β or R2β in mice leads to a partial typically related Peutz-Jeghers syn- as p53 for tumorigenesis (24, 25). increase in R1α, but overall PKA activ- drome, characterized by benign neo- Loss of the R1α regulatory subunit ity is still decreased (26, 29). McKnight plasms and spotty pigmentation of promotes cell proliferation and and colleagues (26, 29) have hypothe- the skin (22). Similarly, constitutional growth of benign tumors in multiple sized that maintenance of adequate mutation of PRKAR1α followed by tissues. The cell biological conse- R1α is a critical intracellular protective somatic mutation of PRKAR1α or quences of the genetic abnormalities mechanism against unregulated cat- other melanocyte genes likely accounts in R1α are likely mediated via changes alytic subunit activity. for the spotty skin pigmentation in in PKA activity. The PKA holoenzyme Hyperendocrine states, benign Carney complex. We have not observed is a tetramer comprised of two catalyt- endocrine neoplasms (e.g., pituitary a second acquired somatic mutation ic subunits and two regulatory sub- and thyroid adenomas), and the cuta- of the PRKAR1α wild-type allele in the units. Genes for four regulatory sub- neous spotty pigmentation of the skin tumor analyzed here, but a “two-hit” units have been identified — R1α, R1β, seen in Carney complex are all likely mechanism of tumorigenesis (23) R2α, and R2β (3, 18) — which differ in related to increased PKA activity. Ele- remains possible for other syndromic their tissue-specific expression pat- vated PKA activity can be caused by and nonsyndromic cardiac myxomas. terns (3, 26). In its tetrameric form, increased free active catalytic subunit Although our Western blot analyses PKA is inactive. Upon binding of in the absence of the reg- detected no mutant, truncated R1α cAMP to the regulatory subunits, the ulatory/inhibitory R1α regulatory protein in a cardiac myxoma, failure to regulatory subunit dimer undergoes a subunit. For instance, constitutive recognize truncated protein could be due to low levels of protein or to conformational abnormalities with consequent loss of antigenicity. Thus, a dominant-negative contribution of a mutant protein remains a formal possibility. Our findings do show that if a second “hit” is necessary for cardiac tumorigenesis, the acquired mutation is not required to occur in PRKAR1α. Figure 5 The diverse signal transduction pro- Western blot analysis of R1α and R2β in normal lymphoblasts, Carney complex lym- teins that interact with R1α and phoblasts, and Carney complex cardiac myxoma. (a) Positive controls for antibodies to R1α cAMP-dependent PKA, as well as other and R2β were electrophoresed and Western blotted as described in Methods. (b, c) Protein PKA subunits, are candidates to be lysates of lymphoblasts from (b) a normal individual and (c) an individual affected by Car- ney complex (family YA, IV-1) were electrophoresed and analyzed. Densitometry revealed studied for somatic mutation in these similar levels of R2β protein in the two samples but a 60% decrease in R1α levels. (d) R1α tumors. Such a two-gene model of and R2β were analyzed in lysate from a left atrial myxoma resected from individual IV-1 in tumorigenesis is similar to that family YA. Densitometry was used to determine R1α and R2β expression in all samples, and observed in murine neurofibromatosis the ratio of R1α:R2β was calculated. A reversal of the R1α:R2β ratio is observed in the tumor models, which require null alleles in sample compared with affected and unaffected lymphocytes.

R36 The Journal of Clinical Investigation | Volume 106 activation of cAMP-dependent PKA factor (37), which is at least partially dependent pathways has already activity produces unregulated growth regulated by PKA-dependent phos- prompted therapeutic modalities for of autonomous hyperfunctioning phorylation. Both the Cα catalytic malignancies (18) and ultimately may human thyrocytes, and mutations in subunit and the R1α regulatory sub- suggest novel therapies to prompt car- Gsα, which yield elevated cAMP levels unit are expressed in the developing diac cell regeneration in the ischemic and elevated PKA activity, cause heart (16–18). Imaizumi-Scherrer et al. and cardiomyopathic heart. growth hormone–secreting pituitary (17) concluded that there were low lev- adenomas and thyroid adenomas (32, els of R1α mRNA throughout the E16 Acknowledgments 33). Thus, loss of the R1α regulatory mouse heart. However, close examina- We are grateful to family members and subunit and increased PKA activity tion of their in situ hybridization data their physicians (including A. Merliss may result in thyroid adenomas as (17) suggests that there may be focal and A. Denio) for their participation in seen in individual I-1 in family YB. increase of R1α expression at the this study, and to B. Batog for techni- Similarly, PKA activity is required for interatrial septum, where most cardiac cal assistance. PACs were provided by tyrosine hydroxylase (TH) activity that myxomas, nonsyndromic and syn- the Resource Center of the German contributes both to catecholamine dromic, arise. Human Genome Project at the Max- synthesis and biosynthesis of melanin We have previously observed (38) Planck-Institut for Molecular Genetics. skin pigmentation (34). PKA deficien- that lipomatous hypertrophy of the The authors appreciate discussion and cy decreases TH activity not only due interatrial septum is common in comments from M. Gershengorn, C. to loss of TH phosphorylation but young adults affected by Carney com- Nathan, and B. Lerman. This work was also because PKA is required for tran- plex. Knockout mice homozygous- supported by an American College of scriptional regulation of TH gene null for the R2β subunit exhibit a par- Cardiology/Merck Research Fellow- expression (34). tial compensatory increase in R1α ship (to C.J. Vaughan), a National In nonendocrine cell types, loss of expression but an overall decrease in Heart, Lung, and Blood Institute PKA activity may be associated with PKA activity, and their phenotype (NHLBI) Minority Postdoctoral Fel- increased cell proliferation (18, 26, 28). includes a loss of adipose tissue via lowship (to C.J. Hatcher), an American For instance, Svenningsen and Kanje increased lipolysis (39). In the atrial Heart Association Student Scholarship (28) demonstrated that cAMP-depend- myxoma studied here, haploinsuffi- in Cardiovascular Disease and Stroke ent PKA inhibits Schwann cell prolif- ciency of R1α combined with tumori- (to J.M. Winter), a National Cancer eration, and pharmacologic inhibition genesis is associated with a reversal of Institute grant (CA78895 to C.C. Mor- of PKA promotes Schwann cell the R1α:R2β ratio. These findings ton), and grants from the Wendy Will growth. Thus, benign schwannomas remain to be confirmed in other Case Cancer Fund, the American Heart seen in Carney complex patients, tumor samples as they become avail- Association (Grant-in-Aid), New York including individual III-10 from fami- able. We hypothesize that haploinsuf- City Affiliate Inc., and NIH/NHLBI ly YA, may be a consequence of ficiency of the R1α subunit in the (R01 HL-61785) to C.T. Basson. decreased Schwann cell PKA activity. heart of Carney complex patients may In these cells, loss of PRKAR1α may lead to decreased PKA activity with 1. Reynen, K. 1995. Cardiac myxomas. N. Engl. J. result in decreased PKA activity consequent partial compensatory Med. 333:1610–1617. 2. Ferrans, V.J., and Roberts, W.C. 1973. Structural through regulatory isoform switching, increase in R2β expression and there- features of cardiac myxomas: histology, histo- as in the atrial myxoma studied here, by induce adipose tissue growth in the chemistry, and electron microscopy. Hum. Pathol. 4:111–146. or through altered intracellular PKA interatrial septum. Furthermore, addi- 3. Burke, A.P., and Virmani, R. 1993. Cardiac myxo- binding and sequestration by tional sporadic mutation of PRKAR1α mas: a clinicopathologic study. Am. J. Clin. Pathol. Schwann cell–specific AKAPs that and/or other genes in the cAMP- 100:671–680. 4. Kanda, T., et al. 1994. Interleukin-6 and cardiac have not yet been identified. dependent signal transduction path- myxoma. Am. J. Cardiol. 74:965–967. The cellular mechanism by which way may contribute to decreased PKA 5. OMIM™: Online Mendelian Inheritance in Man. 1α Center for Medical Genetics, Johns Hopkins Uni- loss of PRKAR modifies PKA activi- activity and altered PKA regulatory versity, Baltimore, Maryland, USA, and National ty in the heart and produces cardiac isoform expression which, in turn, Center for Biotechnology Information, National myxomas remains uncertain. Cardiac ultimately leads to cardiac myxoma Library of Medicine, Bethesda, Maryland, USA. http://www.ncbi.nlm.nih.gov/omim/. myxomas arise from rare pluripotent formation. In fact, the propensity of 6. Carney, J.A., Gordon, H., Carpenter, P.C., Shenoy, subendocardial mesenchymal stem R2β-regulated PKA activity to induce B.V., and Go, V.L. 1985. The complex of myxo- cells (2–3). These unusual cardiac cells cellular differentiation (18) may mas, spotty pigmentation, and endocrine overac- tivity. Medicine. 64:270–283. have not been isolated or cultured (35), explain the wide variety of differenti- 7. Carney, J.A. 1985. Differences between nonfamil- and the effects of PKA on their biology ated cell lineages (2, 3) observed with- ial and familial cardiac myxoma. Am. J. Surg. Pathol. 9:53–55. are unexplored. Rheumatologic symp- in cardiac myxomas. In vitro and in 8. Casey, M., et al. 1998. Identification of a novel toms in patients with cardiac myxo- vivo experimental models that probe genetic locus for familial cardiac myxomas and mas are often attributed to elevated PRKAR1α and other PKA regulatory Carney complex. Circulation. 98:2560–2566. 9. Stratakis, C.A., et al. 1996. Carney complex, a serum IL-6, whose synthesis is PKA- subunit expression in the heart will familial multiple neoplasia and lentiginosis syn- dependent (36). Normal cardiac struc- elucidate PKA-dependent pathways drome: analysis of 11 kindreds and linkage to the short arm of chromosome 2. J. Clin. Invest. ture and function require physiologic for cardiac cell growth and differenti- 97:699–705. expression of the CREB transcription ation. Manipulation of such PKA- 10. Broman, K.W., Murray, J.C., Sheffield, V.C., White,

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