Accepted Manuscript A novel inherited mutation in PRKAR1A abrogates preRNA splicing in a Carney complex family Yunpeng Sun, MD, Xia Chen, PhD, Jingnan Sun, MD, PhD, Xue Wen, MD, Xuguang Liu, MD, PhD, Yanli Zhang, MD, Andrew R. Hoffman, MD, Ji-Fan Hu, MD, PhD, Yongsheng Gao, PhD PII:
S0828-282X(15)00400-6
DOI:
10.1016/j.cjca.2015.05.018
Reference:
CJCA 1695
To appear in:
Canadian Journal of Cardiology
Received Date: 16 February 2015 Revised Date:
18 April 2015
Accepted Date: 5 May 2015
Please cite this article as: Sun Y, Chen X, Sun J, Wen X, Liu X, Zhang Y, Hoffman AR, Hu J-F, Gao Y, A novel inherited mutation in PRKAR1A abrogates preRNA splicing in a Carney complex family, Canadian Journal of Cardiology (2015), doi: 10.1016/j.cjca.2015.05.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A novel inherited mutation in PRKAR1A abrogates preRNA splicing in a Carney complex family Yunpeng Sun, MD,a,b Xia Chen, PhDa# , Jingnan Sun, MD, PhD,c Xue Wen, MD,c
MD, PhD,c,e# Yongsheng Gao, PhDb# a
Department of Pharmacology, College of Basic Medical Sciences, Changchun, Jilin
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130061, China b
Department of Cardiac Surgery, First Hospital of Jilin University, Changchun, Jilin
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130061, China c
Cancer and Stem Cell Center, First Hospital of Jilin University, Changchun, Jilin
130061, China d
Department of Ultrasonic Cardiogram, First Hospital of Jilin University, Changchun,
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Jilin 130061, China
Stanford University Medical School, VA Palo Alto Health Care System, Palo Alto,
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CA 94304, USA
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e
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Xuguang Liu, MD, PhD,b Yanli Zhang, MD,d Andrew R. Hoffman, MD,e Ji-Fan Hu,
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# Correspondences to: Ji-Fan Hu, M.D., Ph.D., Palo Alto Veterans Institute for Research, Palo Alto, CA 94304, USA, Tel: 650-493-5000, x63175, Fax: 650-725-7085, E-mail:
[email protected]; or Xia Chen, M.D., Department of Pharmacology, College of Basic Medical Sciences, Jilin University, Changchun, Jilin 130061, P.R. China, e-mail:
[email protected], or Yongsheng Gao, M.D., Department of Cardiac Surgery, Jilin University, Changchun, Jilin 130061, P.R. China, e-mail:
[email protected].
Running title: PRKAR1A splicing mutation in Carney complex
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ACCEPTED MANUSCRIPT Brief summary
The aim of this study was to identify genetic abnormalities in a Chinese Carney
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complex family. Using whole-exome sequencing, we discovered a heterozygous mutation at the splicing acceptor site of PRKAR1A exon 6. This splicing mutation causes early termination of transcription, resulting in PRKAR1A haploinsufficiency in
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the subjects with Carney complex in the family.
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ACCEPTED MANUSCRIPT ABSTRACT Background: Carney complex (CNC) is an autosomal dominant inherited disease, characterized by spotty skin pigmentation, cardiac and cutaneous myxomas, and
protein kinase A regulatory subunit 1 (PRKAR1A) gene.
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endocrine overactivity. We report a Chinese CNC family with a novel mutation in the
PRKAR1A in two members of the CNC family.
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Methods: Target-exome sequencing was performed to identify the mutation of
Results: The proband was a young man with typical CNC, including pigmentation,
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cutaneous myxomas, cardiac myxoma, Sertoli cell tumor of his left testis, and multiple hypoechoic thyroid nodules. His mother also had CNC with skin pigmentation, cutaneous myxomas, and a cardiac myxoma. Target-exome capture analysis revealed that both the proband and the mother carried a novel heterozygous
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mutation in the exon 6 splicing donor site of PRKAR1A. Sequencing analysis of
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myxoma mRNA revealed that the mutation abrogated exon 6 preRNA splicing, leading to a frameshift starting at Valine 185 and premature translation termination in
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intron 6. The truncated enzyme lacks the functional cAMP binding domain at the
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C-terminus, causing PRKAR1A haploinsufficiency. Conclusion: This study reports a novel splicing mutation in the PRKAR1A gene that adds to the genetic heterogeneity of CNC.
Key words: Carney complex, PRKAR1A gene, cardiac myxoma, splicing mutation, premature termination
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ACCEPTED MANUSCRIPT INSTRUCTIONS Carney complex (CNC) is a rare autosomal dominant inherited disease characterized by spotty skin pigmentation, cardiac and cutaneous myxomas, and endocrine
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overactivity1-5. The syndrome was first described in 1985 by Carney et al6. Cardiac myxoma is the most common cause of death in CNC patients. The diagnostic criteria
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of the syndrome, have recently been modified 7.
Mutation of the cAMP-dependent protein kinase regulatory subunit type 1A
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(PRKAR1A) gene has been identified as a cause of CNC8. More than 1000 patients with CNC have been reported, of whom approximately 70% are familial cases. PRKAR1A mutation is found in >70% of CNC patients, and >100 different mutations have been reported throughout the coding region of PRKAR1A9, 10. Horvath et al
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reviewed all the known PRKAR1A mutations10 and established an online database
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(http://PRKAR1A.nichd.nih.gov). However, little is known about the role of the
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PRKAR1A mutation in the development of CNC in Asian populations.
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In this communication, we report a Chinese CNC family characterized by cardiac and cutaneous myxomas, skin pigmentation, and multiple hypoechoic thyroid nodules. Using target-exome capture, we identified a novel mutation in the PRKAR1A gene in this family.
Subjects and methods The protocol for this study was prospectively reviewed and approved by the Human Medical Ethical Review Committee at Jilin University First Hospital, and informed consent was obtained from each member of the family.
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The Proband The patient was a 20-year-old man who was admitted to the hospital for removal of a
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right ventricular myxoma and a testicular tumor. A Sertoli cell tumor of his left testis had been surgically removed 10 years prior to admission. On physical examination, extensive pigmentation was observed on his face, especially on his upper lips
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(Fig.S1A). Cutaneous myxomas (0.5cm in diameter) were located on both ears (Fig.S2). The right testis was enlarged. Echocardiography revealed an intracardiac
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tumor in the right ventricle affecting the tricuspid valve (Fig.S3A). Thyroid ultrasonography detected multiple small hypoechoic thyroid nodules, and the patient was euthyroid.
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While the growth hormone (GH) level was somewhat elevated at 3.6 ng/ml, the
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patient did not have an acromegalic appearance. No sellar abnormalities were seen on a pituitary CT-scan. An abdominal CT scan revealed a slightly enlarged left adrenal
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gland. Based on clinical manifestations and family history, the patient was diagnosed
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with CNC and the cardiac myxoma was removed. The myxoma originated in a papillary muscle and was adherent to the anterior tricuspid valve leaflet chordae (Fig.S3B).
The mother with CNC The mother was 48-years-old. A left atrium myxoma located at the root of posterior mitral valve leaflet had been resected 20 years ago. She suffered a cerebral infarction due to recurrent left atrium myxoma two years ago, and underwent another cardiac operation to remove the mass one month after the stroke. She also had a history of
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ACCEPTED MANUSCRIPT recurrent cutaneous myxomas of her left nipple. She had multiple areas of pigmentation on her face (Fig.S1B). Thyroid and pituitary function were both normal.
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Thyroid ultrasonography detected multiple small hypoechoic thyroid nodules.
Target-exome capture sequencing
Target-exome capture was used to screen for mutations related to CNC as previously
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described11-13. Briefly, genomic DNA was extracted from peripheral blood using QIAamp Blood Midi Kit (Qiagen, CA). After shearing, the DNA was ligated to
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adapters and amplified using standard Illumina protocols to construct the indexed Illumina libraries. The amplified DNA was then captured with MyGenostics GenCap Whole Exome Enrichment System (MyGenostics Inc, China). Sequencing was performed on a HiSeq 2000 sequencer. The sequencing depth was 340X, and the
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coverage of the target exome was 98.8% (Fig.S4).
The low-quality reads and the 3’ and 5’ adapters were filtered by Trim Galore. The
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filtered reads with sequencing quality > 20 and read length > 80 were aligned to the
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reference genome by the Burrows-Wheeler Aligner (BWA)14. Duplicated reads were removed using Sequence Alignment/Map tools (SAM)11. SNPs were identified by UnifiedGenotyper of the GATK program with dbSNP (v138), using hg19 as the reference genome. InDels were identified by IndelRealigner of GATK with and filtered through a 1000 genome database (http://www.1000genomes.org/). Genetic variants were annotated across the genome by ANNOVAR for mutation (missense, nonsense or frameshift mutations) and location (exonic, intronic or intergenic region)11.
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ACCEPTED MANUSCRIPT Gene and cDNA sequencing Gene sequencing was performed for the mother, father, proband, and brother. DNA was extracted from peripheral blood. DNA samples were amplified by polymerase
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chain reaction (PCR) with forward primer 5’-TTCATTTTGTGGCAGCCTGA-3’ and reverse primer 5’-CCACCACTCCAGCAAAGTTC-3’. The amplified products were sequenced using a Big Dye Terminator Sequencing Kit (Applied Bio systems, Foster
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City, CA) on an automated sequencer ABI Prism 3100.
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Exon splicing analysis
To determine if the PRKAR1A mutation altered mRNA splicing, total RNA was extracted from tumor biopsy samples and was reverse transcribed into cDNA as previously described15-17. Unspliced mRNA from the mutated allele was amplified a
set
of
primers
SJ028
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with
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5’-AATGGCCGCTTTAGCCAAAGCC-3’ and
(located SJ029
in
(located
exon in
intron
6): 6):
5’-GTCTCGGTCGATGCCCCACAAT-3’. The PCR products were cloned using a
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pJet cloning kit and sequenced16.
RESULTS
Clinical manifestation in the CNC family The family pedigree analysis indicated that only the proband and the mother suffered from CNC (Fig.1A). The proband had a cardiac myxoma, skin pigmentation, cutaneous myxomas, a Sertoli cell tumour of his left testis, and multiple hypoechoic nodules in the thyroid. Similarly, his mother had cardiac myxomas, pigmentation, and cutaneous myxomas. Pathological examination confirmed that the cardiac tumors
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ACCEPTED MANUSCRIPT from both the proband and the mother were typical cardiac myxomas (Fig.1B). Aspiration biopsy on the right testis of the proband showed Sertoli cell tumor, confirmed by immunohistochemical examination (vimentin+, calretinin+ and
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inhinbin+)(Fig.1C).
Target-exome capture identifies a novel mutation in PRKAR1A
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We performed whole-exome capture sequencing with a Solexa HiSeq 2000 sequencer (Fig.2A). Using GATK IndelRealigner program, we identified a total of three Indel
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mutations in exonic DNAs of NF1, PRKAR1A, and RECQL4 in the proband (Fig.2B). A “G” insertion was detected in exon 55 of NF1, a gene that causes Neurofibromatosis type I (von Recklinghausen disease). By alignment, we identified a two-base substitution mutation (CG>TT) in exon 6 of the PRKAR1A gene (Fig.2C).
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This mutation has not been reported in either the PRKAR1A mutation database
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(http://PRKAR1A.nichd.nih.gov) or in the HGMDpro database. In addition, we also identified a “G” deletion in intron 15 of RECQL1, a RecQ DNA helicase family gene
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that participates in DNA repair and recombination pathways in cell cycle replication.
Gene sequencing links the mutation with CNC We then focused our interest on PRKAR1A as it is frequently mutated in CNC. Figure 3A shows the alignment of exome sequencing data with a typical heterozygous pattern of the mutation in PRKAR1A. In the mutated allele of PRKAR1A exon 6, the normal “CG” was replaced by “TT” and there was also a “G” deletion.
To correlate the identified mutation with CNC, we sequenced the PRKAR1A exon 6 mutation region for all family members (Fig.3B). The proband and his mother had the
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ACCEPTED MANUSCRIPT identical mutation in chr17 region 66521094-66521101, with the CG>TT replacement at chr17 66521094-66521094, a deletion of G at chr1766521100, and a T>C mutation at chr17 66521101. Neither the father nor the older brother carried these mutations.
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The presence of the mutations correlated with the clinical manifestation of CNC in the
The mutation abrogates mRNA splicing in exon 6
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family.
As the identified mutation was located in the splicing donor site of PRKAR1A exon 6,
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it was expected that mRNA splicing would be altered (Fig.4A). We extracted total RNA from tumor tissues. After cDNA synthesis, the unspliced mRNA was amplified by a pair of PCR primers that are located in exon 6 and intron 6, respectively (Fig.4B, top panel). As expected, we detected the presence of unspliced mRNA only in the
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CNC patients (Fig.4B, bottom panel). Sequencing of the PCR product confirmed the
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presence of a frameshift at Valine 185. After continuous translation of an additional 9 amino acids, protein coding was terminated at a TGA stop codon located in intron 6.
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As a result, this splicing mutation led to the loss of translation from exons 7-11 coding
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sequences (Fig.4C).
SNPs that carry amino acid mutations Using target-exome capture sequencing, we also identified SNPs that were uniquely present in the proband, but not in normal populations by filtering through dbSNP, HAPMAP, and 1000 genome databases. With GATK UnifiedGenotyper program, we identified a total of 130 SNPs in the proband’s DNA that alter amino acid coding. Tables 1-3 list the SNP mutations based on gene functions.
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ACCEPTED MANUSCRIPT DISCUSSION Carney complex (CNC) is a rare autosomal dominant disease characterized by spotty skin pigmentation, cardiac and cutaneous myxomas, and endocrine overactivity. The
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syndrome was found to be responsible for many endocrine tumors in addition to myxomas. In this Chinese CNC family, the proband had a cardiac myxoma, skin pigmentation on his upper lips, a cutaneous myxoma on his ear, and multiple
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cutaneous myxomas as well as skin pigmentation.
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hypoechoic thyroid nodules. Similarly, the mother also exhibited cardiac and
Mutation of the PRKAR1A gene has been shown to cause CNC8. By target exome capture sequencing, we identified a novel mutation in a CNC kindred in PRKAR1A exon 6 that includes replacement of CG with TT at chr17 66521094-66521095, a
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deletion of G at chr1766521100, and a T>C transition at chr17 66521101.
Interestingly, the identified mutation is located exactly in the splicing donor site of
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PRKAR1A exon 6. The mutation not only leads to a frameshift starting at Valine 185,
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but also abrogates the splicing of intron 6. As a result, RNA polymerase II reads through the intron 6 sequence and terminates immediately following the translation of an additional nine amino acids from intron 6 (Fig.4). Consequently, the mutated PRKAR1A protein would only have 183 amino acids, 198 amino acids short of the normal protein. This truncated enzyme subunit lacks most of the C-terminal cAMP binding domain. This mutation would then lead to PRKAR1A haploinsufficiency18 and CNC in this family.
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ACCEPTED MANUSCRIPT PRKAR1A encodes the type 1A regulatory (R) subunit of protein kinase A (PKA) or cAMP-dependent protein kinase. The inactive PKA enzyme consists of a tetramer of two homo- or heterodimers of R subunits (PRKAR1A, PRKAR1B, PRKAR2A, and
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PRKAR2B) and a homodimer of two catalytic (C) subunits (from a choice of four molecules: PRKACA, PRKACB, PRKACG, and PRKX). Activation of PKA occurs upon the binding of two molecules of cAMP to each R subunit19, followed by the
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dissociation of the holoenzyme and the release of the active C subunit, which in turn phosphorylates a series of downstream cellular target genes20. PRKAR1A
cAMP-stimulated kinase activity8.
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haploinsufficiency leads to an increase in total (and not only PKA-specific)
Two mechanisms have been suggested to explain the increment in total cAMP
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signaling activity in CNC. First, PRKAR1A haploinsufficiency leads to a higher
subunits
that
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intracellular C:R subunit ratio, thereby increasing the availability of free catalytic phosphorylate
downstream
targets.
Second,
PRKAR1A
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haploinsufficiency leads an upregulation of other components of the PKA tetramer,
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including both type I (PRKAR1B) and type II (PRKAR2A or PRKAR2B) subunits, in a tissue-dependent manner21. These regulatory subunits may lack the ability to regulate the catalytic subunit as effectively as PRKAR1A, especially in the presence of high cAMP levels. PRKAR1A has been reported to act as a tumor suppressor. The mTOR pathway was activated by PKA to increase cell survival in adrenocortical cells and follicular thyroid cancer cells, which is correlated with hyperphosphorylation of BAD22, 23, a very distant BCL2 family member.
It should be noted that we cannot completely exclude the possibility that some of the
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ACCEPTED MANUSCRIPT SNPs and InDels from this study may represent rare genetic variations. Our sequencing data were quality controlled and filtered through the 1000 genomes project database. The database contains the genomes of 1,092 individuals from 14 populations,
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constructed using a combination of low-coverage whole-genome and exome sequencing. In addition, we have also searched the Exome Aggregation Consortium (ExAC) database (URL: http://exac.broadinstitute.org). There are a total of 782 SNPs
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and InDels of PRKAR1A recorded in the ExAC database. However, none of them covers the exon 6 InDel mutation that was identified in this study. Thus, we believe
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that the exon 6 InDel is a new mutation associated with the CNC in this family.
In summary, we have described a Chinese CNC kindred with typical clinical features including cardiac myxomas, cutaneous myxomas, skin pigmentation, and multiple
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hypoechoic thyroid nodules associated with a novel mutation in PRKAR1A exon 6.
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The mutation abrogates exon 6 preRNA splicing, leading to a frameshift starting at Valine 185 and premature termination of translation within intron 6. The truncated
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PRKAR1A enzyme lacks the cAMP binding domain, leading to PRKAR1A
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haploinsufficiency in the CNC patients.
ACKNOWLEDGMENTS This work was supported by California Institute of Regenerative Medicine (CIRM) grant (RT2-01942), Jilin International Collaboration Grant (#20120720), the National Natural Science Foundation of China grant (#81272294, #31430021) to J.F.H; the National Natural Science Foundation of China grant (#81071920, #81372835) and Jilin Science and Technique Program grant (11GG003) to W.L.; and the grant of Key Project of Chinese Ministry of Education (#311015) to C.J. and the Medical Research
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ACCEPTED MANUSCRIPT Service of the Department of Veterans Affairs to A.R.H.;
The authors report no conflicts of interest.
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14. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754-1760. 15. Sun J, Li W, Sun Y, et al. A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies. Nucleic Acids Res. 2014;42:9588-9601. 16. Wang H, Li W, Guo R, et al. An intragenic long noncoding RNA interacts epigenetically with the RUNX1 promoter and enhancer chromatin DNA in hematopoietic malignancies. Int J Cancer. 2014;135:2783-2794. 17. Zhang S, Zhong B, Chen M, et al. Epigenetic reprogramming reverses the malignant epigenotype of the MMP/TIMP axis genes in tumor cells. Int J Cancer. 2014;134:1583-1594. 18. Patronas Y, Horvath A, Greene E, et al. In vitro studies of novel PRKAR1A mutants that extend the predicted RIalpha protein sequence into the 3'-untranslated open reading frame: proteasomal degradation leads to RIalpha haploinsufficiency and Carney complex. The Journal of clinical endocrinology and metabolism. 2012;97:E496-502. 19. Bruystens JG, Wu J, Fortezzo A, Kornev AP, Blumenthal DK, Taylor SS. PKA RIalpha homodimer structure reveals an intermolecular interface with implications for cooperative cAMP binding and Carney complex disease. Structure. 2014;22:59-69. 20. Bossis I, Stratakis CA. Minireview: PRKAR1A: normal and abnormal functions. Endocrinology. 2004;145:5452-5458. 21. Stergiopoulos SG, Stratakis CA. Human tumors associated with Carney complex and germline PRKAR1A mutations: a protein kinase A disease! FEBS letters. 2003;546:59-64. 22. Pringle DR, Vasko VV, Yu L, et al. Follicular thyroid cancers demonstrate dual activation of PKA and mTOR as modeled by thyroid-specific deletion of Prkar1a and Pten in mice. The Journal of clinical endocrinology and metabolism. 2014;99:E804-812. 23. de Joussineau C, Sahut-Barnola I, Tissier F, et al. mTOR pathway is activated by PKA in adrenocortical cells and participates in vivo to apoptosis resistance in primary pigmented nodular adrenocortical disease (PPNAD). Human molecular genetics. 2014;23:5418-5428.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS
Figure 1. Pedigree of the Carney Complex Family.
noted by an arrow.
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A. The CNC pedigree. NN: normal, NM: heterozygous mutation. Proband is
staining, magnification ×400.
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B. Pathological findings of cardiac myxoma in the proband and the mother. HE
C. Pathological and immunohistochemical staining of Sertoli cell tumor from
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right testis aspiration biopsy. A: HE staining; B-D: immunohistochemical staining of calretinin, inhibin, and vimentin, respectively. Magnification x100.
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Figure 2. InDel mutations identified by exome capture sequencing.
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A. Diagram of exome capture sequencing. Library was constructed by shearing genomic DNA into short fragments. Exons of target genes were captured by
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biotinylated oligonucleotide probes and amplified for Hyseq2000 sequencing.
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B. Three InDel mutations identified by exome capture sequencing. C. Comparison of the InDel mutation-containing allele with the normal allele. For NF1, there is a “G” insertion in exon 55. For PRKAR1A, the ttGAT-c mutation from the normal CGGATGT sequence is located in the exon 6 splicing donor region. For RECQL4, a “G” deletion is detected in exon 16 splicing acceptor region.
Figure 3. The PRKAR1A exon 6 mutation is correlated with CNC.
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ACCEPTED MANUSCRIPT A. Alignment of sequencing data of the PRKAR1A exon 6 in the proband. B. Correlation of the PRKAR1A exon 6 mutation with the CNC cases. Both CNC cases in the family (the proband and the mother) carried the same PRKAR1A
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exon 6 mutation, while the healthy father and the healthy brother did not carry
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the mutation.
Figure 4. The mutation abrogates the splicing of PRKAR1A exon 6.
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A. Mutation of the PRKAR1A exon 6 splicing donor site. With the mutation of CG>TT, G deletion, and T>C in the exon6-intron 6 donor site, it is expected that the splicing will be abrogated.
B. Detection of the unspliced mRNA from the mutated allele. Top panel:
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Location of PCR primers. Arrows: primer orientation. Bottom panel: PCR of
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unspliced mRNA. After removal of residual genomic DNA by DNase I, mRNA was converted into cDNA and amplified with a primer located in exon
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6 and the second primer located in intron 6. Note the detection of the mutated
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unspliced mRNA only in the proband and the mother. C. The CNC mutation prematurely terminates the coding of PRKAR1A protein. Unspliced PCR products were cloned and sequenced. Sequencing confirmed that the PRKAR1A protein was truncated without amino acids coded in exons 7-11 (mNRA sequence NM_001278433).
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ACCEPTED MANUSCRIPT Table 1. Oncogene and growth factor SNP genotypes with altered amino acids (target exome sequencing)
Location Access ID
SNP Mutation*
ALK
chr2
4587C>G (D1529E)1 4472A>G (K1491R)1 4381A>G (I1461V)1 3375C>A (G1125G)1 4C>A (P2T)2 2076T>C (A692A)2 474C>T (N158N)2 1562G>A (R521K)2 1887T>A (T629T)2 344T>C (M115T)2 465G>A (P155P)2 1621A>C (M541L)2 453T>C (F151F)2 3912C>T (D1304D)2 4071G>A (A1357A)2 1566G>A (Q522Q)1 1701A>G (P567P)1 900C>T (H300H)2 3944C>T (P1315L)2 135A>G (A45A)2 1296A>G (A432A)1 561T>C (N187N)2 759A>G (L253L)2 1815G>C (E605D)2
NM_004304
NM_004329 NM_004360 NM_201284
EPCAM HSD3B2 KIT LMO1 MET
chr2 chr1 chr4 chr11 chr7
NM_002354 NM_000198 NM_000222 NM_002315 NM_001127500
NTRK1 PDGFRA PRF1 PTCH1 RET
chr1 chr4 chr10 chr9 chr10
NM_001007792 NM_006206 NM_005041 NM_000264 NM_020975
TSHR
chr14
NM_000369
ZNF276
chr16
NM_001113525
2709T>C (T903T)1 2982C>T (D994D)2
2586G>C (L862L)2 156C>G (A52A)2 4146G>A (P1382P)2
3222T>C (D1074D)1 1686C>T (A562A)2 2307G>T (L769L)2 2181G>C (E727D)2
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BMPR1A chr10 CDH1 chr16 EGFR chr7
3036G>A (T1012T)2 2535T>C (G845G)2 1500A>G (Q500Q)2 702T>A (P234P)1
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Gene
* Alterations in SNP genotypes and amino acids (parenthesis) 1 homozygous, 2 heterozygous
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ACCEPTED MANUSCRIPT Table 2. Tumor suppressor SNP genotypes with altered amino acids in coding sequence (target exome sequencing)
Access ID
SNP Mutation*
APC
chr5
NM_000038
AXIN2
chr17
NM_004655
BARD1
chr2
NM_001282543
BRCA1
chr17
NM_007294
BRCA2
chr13
NM_000059
BRIP1
chr17
NM_032043
EXT1 FANCA
chr8 chr16
NM_000127 NM_000135
FANCE FANCF HNF1A
chr6 chr11 chr12
NM_021922 NM_022725 NM_000545
1458T>C (Y486Y)2 1635G>A (A545A)2 4479G>A (T1493T)2 5034G>A (G1678G)2 1386C>T (P462P)2 1365A>G (P455P)2 1462G>A (V488M)2 1461T>C (H487H)2 1077G>C (R359S)2 4837A>G (S1613G)2 4308T>C (S1436S)2 3548A>G (K1183R)2 3113A>G (E1038G)2 3807T>C (V1269V)2 4563A>G (L1521L)2 6513G>C (V2171V)2 3411T>C (Y1137Y)2 2755T>C (S919P)2 1761G>A (E587E)2 2426G>A (G809D)2 1501G>A (G501S)2 387A>C (P129P)2 96C>T (R32R)2 1375C>T (L459L)2 1460G>A (S487N)2 1636A>G (T546A)2 1314T>C (H438H)2 702G>A (L234L)2 1676A>G (Q559R)2 1623T>G (D541E)2 18C>A (A6A)2 98C>G (P33R)2 2829C>T (A943A)2 1335A>G (E445E)2 4349C>G (P1450R)2
M AN U
D
TE
EP
AC C
MEN1
chr11
NM_130801
NF1 PALB2 RNASEL SDHB TP53 TSC1
chr17 chr16 chr1 chr1 chr17 chr9
NM_000267 NM_024675 NM_021133 NM_003000 NM_001276760 NM_000368
TSC2
chr16
NM_000548
5268T>G (S1756S)2 5465T>A (V1822D)1 5880G>A (P1960P)2
RI PT
Location
148C>T (P50S)2
SC
Gene
996G>C (T332T)2 70C>T (P24S)2
2612C>T (P871L)2 2311T>C (L771L)2 2082C>T (S694S)2 7397T>C (V2466A)1 8525G>A (R2842H)2 2637A>G (E879E)1 1652C>A (A551E)2 796A>G (T266A)1
1720A>G (S574G)1 1269C>T (D423D)2 2034G>A (P678P)2
965T>C (M322T)2
* SNP genotypes and altered amino acids (parenthesis). 1 homozygous mutation, 2 heterozygous mutation
18
ACCEPTED MANUSCRIPT
Location
Access ID
SNP Mutation*
ATM BIVMERCC5 DDB2 ERCC2 MLH3
chr11 chr13
NM_000051 NM_000123
chr11 chr19 chr14
NM_000107 NM_001130867 NM_001040108
MSH2 MSH6 MUTYH NBN
chr2 chr2 chr1 chr8
NM_001258281 NM_001281493 NM_001048171 NM_002485
5948A>G (N1983S)1 138T>C (H46H)2 3157G>C (R1053G)1 378T>C (T126T)1 396A>C (R132R)2 4335A>G (Q1445Q)2 2531C>T (P844L)2 970C>T (L324F)2 2400T>A (T800T)2 1389G>C (T463T)2 2016A>G (P672P)2 1197T>C (D399D)1
3238G>C (R1080G)1 3310G>C (D1104H)2
2476A>G (N826D)1
972G>C (Q324H)2 553G>C (E185Q)2 102G>A (L34L)2
AC C
EP
TE
D
M AN U
SC
Gene
RI PT
Table 3. DNA repair gene SNP genotypes with altered amino acids (target exome sequencing)
19
ACCEPTED MANUSCRIPT NM_000535
RECQL4
chr8
NM_004260
WRN
chr8
NM_000553
XPC
chr3
NM_001145769
2570G>C (G857A)2 2466T>C (L822L)2 1621A>G (K541E)2 3127T>C (L1043L)1 3014G>A (R1005Q)1 801G>C (E267D)1 513C>T (C171C)1 2361G>T (L787L)2 3222G>T (L1074F)2 2704C>A (Q902K)2 1770T>A (A590A)1
1454C>A (T485K)2 780C>G (S260S)2 288C>T (A96A)2 738C>T (S246S)1 274T>C (S92P)1
RI PT
chr7
3893G>T (G1298V)2 4099T>C (C1367R)2 46C>G (L16V)2
SC
PMS2
AC C
EP
TE
D
M AN U
*: Changes in SNP genotypes and amino acids (parenthesis). 1 homozygous mutation, 2: heterozygous mutation
20
ACCEPTED MANUSCRIPT
1
RI PT
A. Pedigree of the Carney Complex Family 2
I
1
NM
SC
NN
2
NN
NM
The mother
EP
Calretinin
AC C
C. Testis sertoli cell tumor HE staining
TE
D
B. Cardiac myxomas (×400) Proband
M AN U
II
Inhinbin
Vimentin
Figure 1. Pedigree of the Carney Complex Family
RI PT SC
A. Whole exome sequencing Genomic DNAs Library construction
M AN U
Library DNA fragments
Biotin
Hybridization Biotin-streptavidin capture
Biotin-probe
Streptavidin capture
Probe-captured exon DNAs
B. InDel analyses 5HI%DVH 0XW%DVH KRPKHW
1) 35.$5$ 5(&4/
* *
*
KHW KHW KRP
1RUPDO )UHTXHQF\
C. Gene mutations
H[RQ
F'1$&KDQJHV 0XW5DWLR 'HSWK 8QLT,'
H[RQ H[RQ H[RQ
FGXS* FGHO* FGHO&G
EP
*HQH
TE
SNP/InDel analyses
D
Hyseq2000 sequencing
6FULSWV
FKU 10B FKU 10B FKU 10B
AC C
-
ACCEPTED MANUSCRIPT
JWWWWJJWWWWDDWJJFWWJWJJFJJWWWJFDJJDFFJWWWWFDDDJ ([RQ JWWWWJJWWWWDDWJJFWWJWJJFJJWWWJFDJJ*DFFJWWWWFDDDJ 0XWDWLRQDOOHOH ([RQ 35.$5$ 1RUPDODOOHOH FDDJJDJDJDFJJDW JWDDJDWWWJFDFWWWDJ JWFWDWJWWDDFDDW ([RQ ,QWURQ ([RQ 0XWDWLRQDOOHOH FDDJJDJDJD77JDW & DDJDWWWJFDFWWWDJ JWFWDWJWWDDFDDW ([RQ ,QWURQ ([RQ 5(&4/ 1RUPDODOOHOH JDFDWJFFJJDJJDJJ JWJDJJDDFWJFFFFFDJ FFDWFJDJDFWWWJF ([RQ ,QWURQ ([RQ 0XWDWLRQDOOHOH JDFDWJFFJJDJJDJJ JWJDJJDDFWJFFFFFD FFDWFJDJDFWWWJF ([RQ ([RQ ,QWURQ 1)
1RUPDODOOHOH
Figure 2. Data of InDel analyses from whole exome sequencing
ACCEPTED MANUSCRIPT
A. PRKAR1A exome sequencing
E3 E4 E5 E6
E2
E7
E8
E9 E10 E11
TE
D
M AN U
SC
RI PT
Exons: E1
AC C
Proband
EP
B. Genomic DNA sequencing
Mother
Father
Brother
Figure 3. exome sequencing identifies the mutation of PRKAR1A exon 6
A. PRCART1A exon 6 splicing mutation Intron 6
exon 7
SC
exon 6
RI PT
ACCEPTED MANUSCRIPT
Normal allele:
B. PRCART1A unspliced mRNA 500 bp
M AN U
Mutation allele:
unsplicing cDNA (413 bp)
400 bp 200 bp
β-ACTIN (135 bp)
100 bp paientt
mother
father
brother
D
M
TE
C. PRCART1A premature termination Normal allele:
exon 6
intron 6
AC C
Mutation allele:
exon 7
EP
exon 6
Figure 4. Premature termination mutation in PRCART1A
ACCEPTED MANUSCRIPT
A novel inherited mutation in the PRKAR1A gene that causes protein coding premature truncation in a Carney complex family
AC C
EP
TE
D
M AN U
SC
RI PT
Yunpeng Sun, Jingnan Sun, Jiuwei Cui, Guanjun Wang, Andrew R. Hoffman, Ji-Fan Hu, Wei Li
ACCEPTED MANUSCRIPT
SUPPLEMENTAL FIGURE LEGEND
RI PT
Figure S1. Carney complex. Spotty pigments on the face of the proband and the mother.
SC
Figure S2. Carney complex. Cutaneous myxomas located on the ears of the proband.
Figure S3. Carney complex. Imaging of cardiac myxoma in the proband.
M AN U
A. Echocardiography shows the myxoma located in right ventricle adherent to the anterior tricuspid valve leaflet.
B. The myxoma originated in papillary muscle and adhered to the anterior tricuspid valve
D
leaflet chordae.
TE
Figure S4. SNP and InDel data analyses. The filtered data reads with sequencing quality > 20 and read length > 80 were aligned to the reference genome by the Burrows-Wheeler Aligner
EP
(BWA). InDels were identified by GATK and annotated based on CCDS, 1000 genomes, and dbSNP(c130) databases using hg19 as the reference genome. SNPs available in dbSNP,
AC C
HAPMAP, and 1000 human genome databases were removed from the data set. Those SNPs that affect amino acid coding were considered to be potential disease-associated SNPs.
B. The mother ACCEPTED MANUSCRIPT
AC C
EP
TE
Figure S1. Pigments on the face.
D
M AN U
SC
RI PT
A. Proband
ACCEPTED MANUSCRIPT B. Cutaneous myxomas on the left ear
TE
D
M AN U
SC
RI PT
A. Cutaneous myxomas on the right ear
AC C
EP
Figure S2. The cutaneous myxomas located on Proband’s ears.
ACCEPTED MANUSCRIPT
SC
RI PT
A. Preoperational echocardiography
AC C
EP
TE
D
M AN U
B. Cardiac myxoma
Figure S3. Cardiac myxoma in right ventricle
ACCEPTED MANUSCRIPT
Exome library DNAs
Probe-captured exon DNAs
Hiseq 2000 Sequencing
Sequence Alignment/Map (SAM) tools
SNPs
GATK IndelRealigner
M AN U
GATK UnifiedGenotyper
dbSNP (v138)
InDels
Annotation Insertion Deletion
TE
D
SNP amino acid mutation
EP
Figure S4. Schematic diagram of whole exome capture sequencing
AC C
SC
Burrows -Wheeler Aligner (BWA)
RI PT
Trim-Galore