Somatic Activating Mutations in GNAQ and GNA11 Are Associated with Congenital Hemangioma
2016; Elsevier BV; Volume: 98; Issue: 4 Linguagem: Inglês
10.1016/j.ajhg.2016.03.009
ISSN1537-6605
AutoresUgur M. Ayturk, Javier Couto, Steven Hann, John B. Mulliken, Kaitlin L. Williams, August Yue Huang, Steven J. Fishman, Theonia K. Boyd, Harry P. Kozakewich, Joyce Bischoff, Arin K. Greene, Matthew L. Warman,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoCongenital hemangioma is a rare vascular tumor that forms in utero. Postnatally, the tumor either involutes quickly (i.e., rapidly involuting congenital hemangioma [RICH]) or partially regresses and stabilizes (i.e., non-involuting congenital hemangioma [NICH]). We hypothesized that congenital hemangiomas arise due to somatic mutation and performed massively parallel mRNA sequencing on affected tissue from eight participants. We identified mutually exclusive, mosaic missense mutations that alter glutamine at amino acid 209 (Gln209) in GNAQ or GNA11 in all tested samples, at variant allele frequencies (VAF) ranging from 3% to 33%. We verified the presence of the mutations in genomic DNA using a combination of molecular inversion probe sequencing (MIP-seq) and digital droplet PCR (ddPCR). The Gln209 GNAQ and GNA11 missense variants we identified are common in uveal melanoma and have been shown to constitutively activate MAPK and/or YAP signaling. When we screened additional archival formalin-fixed paraffin-embedded (FFPE) congenital cutaneous and hepatic hemangiomas, 4/8 had GNAQ or GNA11 Gln209 variants. The same GNAQ or GNA11 mutation is found in both NICH and RICH, so other factors must account for these tumors' different postnatal behaviors. Congenital hemangioma is a rare vascular tumor that forms in utero. Postnatally, the tumor either involutes quickly (i.e., rapidly involuting congenital hemangioma [RICH]) or partially regresses and stabilizes (i.e., non-involuting congenital hemangioma [NICH]). We hypothesized that congenital hemangiomas arise due to somatic mutation and performed massively parallel mRNA sequencing on affected tissue from eight participants. We identified mutually exclusive, mosaic missense mutations that alter glutamine at amino acid 209 (Gln209) in GNAQ or GNA11 in all tested samples, at variant allele frequencies (VAF) ranging from 3% to 33%. We verified the presence of the mutations in genomic DNA using a combination of molecular inversion probe sequencing (MIP-seq) and digital droplet PCR (ddPCR). The Gln209 GNAQ and GNA11 missense variants we identified are common in uveal melanoma and have been shown to constitutively activate MAPK and/or YAP signaling. When we screened additional archival formalin-fixed paraffin-embedded (FFPE) congenital cutaneous and hepatic hemangiomas, 4/8 had GNAQ or GNA11 Gln209 variants. The same GNAQ or GNA11 mutation is found in both NICH and RICH, so other factors must account for these tumors' different postnatal behaviors. Congenital hemangiomas are rare vascular tumors that are present at birth. They are different from common infantile hemangiomas that rapidly enlarge postnatally and immunostain for the cell surface marker GLUT1.1North P.E. Waner M. Mizeracki A. Mihm Jr., M.C. GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas.Hum. Pathol. 2000; 31: 11-22Abstract Full Text PDF PubMed Scopus (651) Google Scholar In contrast, congenital hemangiomas are GLUT1 negative and display one of two clinical patterns: "rapidly involuting congenital hemangioma" (RICH)2Berenguer B. Mulliken J.B. Enjolras O. Boon L.M. Wassef M. Josset P. Burrows P.E. Perez-Atayde A.R. Kozakewich H.P. Rapidly involuting congenital hemangioma: clinical and histopathologic features.Pediatr. Dev. Pathol. 2003; 6: 495-510Crossref PubMed Scopus (264) Google Scholar, 3Boon L.M. Enjolras O. Mulliken J.B. Congenital hemangioma: evidence of accelerated involution.J. Pediatr. 1996; 128: 329-335Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar or "non-involuting congenital hemangioma" (NICH).4Enjolras O. Mulliken J.B. Boon L.M. Wassef M. Kozakewich H.P. Burrows P.E. Noninvoluting congenital hemangioma: a rare cutaneous vascular anomaly.Plast. Reconstr. Surg. 2001; 107: 1647-1654Crossref PubMed Scopus (265) Google Scholar RICH can be detected prenatally (as early as 12 weeks gestation) and presents in a newborn as a raised, gray-violaceous solitary cutaneous tumor with fine telangiectasias, ectatic veins, and a pale halo (Figure 1). RICH demonstrates fast-flow and can be associated with congestive cardiac failure and transient low-grade thrombocytopenia. If there are not overwhelming complications from heart failure or hemorrhagic ulceration, the tumor rapidly regresses by 6–14 months, leaving a patch of subcutaneous atrophy, dilated veins, and persistent fast-flow. RICH is also well documented to occur in the liver (Figure 1), where it spontaneously regresses just as in skin.5Fishman S.J. Burrows P.E. Treatment of visceral vascular tumors.in: Mulliken J.B. Burrows P.E. Fishman S.J. Mulliken and Young's Vascular Anomalies: Hemangiomas and Malformations. Oxford University Press, 2013: 242-245Crossref Google Scholar The second category of congenital hemangioma (NICH) presents as a well-circumscribed, plaque-like tumor with a purple-pink hue, pale rim, coarse telangiectasia, and fast-flow (Figure 1). It remains unchanged throughout childhood; however, there are uncommon examples of growth and expansion in adolescence.6Mulliken J.B. Diagnosis and natural history of hemangiomas.in: Mulliken J.B. Burrows P.E. Fishman S.J. Mulliken and Young's Vascular Anomalies: Hemangiomas and Malformations. Oxford University Press, 2013: 69-110Crossref Google Scholar In some cases RICH can cease regressing and transform into NICH.7Mulliken J.B. Enjolras O. Congenital hemangiomas and infantile hemangioma: missing links.J. Am. Acad. Dermatol. 2004; 50: 875-882Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 8Nasseri E. Piram M. McCuaig C.C. Kokta V. Dubois J. Powell J. Partially involuting congenital hemangiomas: a report of 8 cases and review of the literature.J. Am. Acad. Dermatol. 2014; 70: 75-79Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar There are histopathological similarities between RICH and NICH (Figure 1) and between congenital hemangioma and placental chorangioma, suggesting that chorangioma might be the placental counterpart of cutaneous and intrahepatic congenital hemangioma.9Mulliken J.B. Bischoff J. Kozakewich H.P. Multifocal rapidly involuting congenital hemangioma: a link to chorangioma.Am. J. Med. Genet. A. 2007; 143A: 3038-3046Crossref PubMed Scopus (24) Google Scholar We hypothesized that somatic mutations initiate the formation of congenital hemangiomas and searched for these mutations by performing massively parallel sequencing on fresh frozen specimens. The Committee on Clinical Investigation of Boston Children's Hospital approved this study. Congenital hemangioma samples were collected during a clinically indicated procedure, and all participants provided written consent. Tissues were immediately flash-frozen or placed in RNAlater (Thermo Fisher Scientific) and stored at −80°C until further processing. The original intent was to perform RNA-seq looking for coding sequence mutations or genetic alterations that might produce chimeric or miss-spliced transcripts and to perform whole-exome sequencing (WES) looking for coding sequence mutations in genes that were not highly expressed in affected tissue. RNA-seq libraries were prepared via standard Illumina TruSeq protocols and sequenced on one flowcell of an Illumina HiSeq 2500 system. We did not perform WES because we were able to successfully identify mutations with RNA-seq. We generated 32–47 million 100-bp paired-end reads per RNA-seq library. Following the Genome Analysis Toolkit (GATK) best practices workflow,10Van der Auwera G.A. Carneiro M.O. Hartl C. Poplin R. Del Angel G. Levy-Moonshine A. Jordan T. Shakir K. Roazen D. Thibault J. et al.From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline.Curr. Protoc. Bioinformatics. 2013; 11 (1, 33)PubMed Google Scholar we mapped each set of reads to the reference human genome (GRCh37) with STAR aligner,11Dobin A. Davis C.A. Schlesinger F. Drenkow J. Zaleski C. Jha S. Batut P. Chaisson M. Gingeras T.R. STAR: ultrafast universal RNA-seq aligner.Bioinformatics. 2013; 29: 15-21Crossref PubMed Scopus (19220) Google Scholar removed PCR duplicates with Picard, realigned the reads around small insertions and deletions, and recalibrated the base quality scores with GATK.12McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. Garimella K. Altshuler D. Gabriel S. Daly M. DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14799) Google Scholar We then compiled single-nucleotide variant lists with Samtools13Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31615) Google Scholar and identified positions with a minimum of 20× read depth, 3× variant read depth, and 10% variant allele frequency using VarScan.14Koboldt D.C. Zhang Q. Larson D.E. Shen D. McLellan M.D. Lin L. Miller C.A. Mardis E.R. Ding L. Wilson R.K. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing.Genome Res. 2012; 22: 568-576Crossref PubMed Scopus (2945) Google Scholar We removed variants with greater than 40% variant allele frequency, assuming that these changes most likely represent germline variants rather than somatic changes. We further filtered the variants by removing those previously reported in the Exome Variant Server (ESP6500), 1000 Genomes Project, Exome Aggregation Consortium (v.0.3), and dbSNP as a non-clinical entry (build 138). We annotated the remaining variants with ANNOVAR.15Wang K. Li M. Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7883) Google Scholar A total of 43 genes exhibited protein sequence altering variants in ≥3 of 8 specimens (Figure S1). We narrowed this list further by comparing these variants to those found in RNA-seq data from unrelated vascular lesions (arteriovenous malformation [n = 3] and common infantile hemangioma [n = 1]). A total of 12 genes contained a variant found only in the congenital hemangioma samples (Table 1). The variants found in the other RNA-seq datasets were assumed to be associated with RNA editing or with errors in sequencing, mapping, or annotation. Close inspection of variants in the 12 remaining genes for potential mismapping, sequencing errors, and lack of evolutionary conservation left only variants in GNAQ (MIM: 600998) as likely true-positive somatic mutations (Tables 2 and S1). GNAQ encodes guanine nucleotide binding protein G(q) alpha, a subunit within a complex that hydrolyzes the intracellular messenger GTP to GDP. GNAQ shares 90% protein sequence similarity with GNA11 (MIM: 139313). Somatic missense mutations that alter codon 209 in GNAQ (GenBank: NP_002063.2) and GNA11 (GenBank: NP_002058.2) have been reported in >80% of uveal melanomas.16Van Raamsdonk C.D. Bezrookove V. Green G. Bauer J. Gaugler L. O'Brien J.M. Simpson E.M. Barsh G.S. Bastian B.C. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.Nature. 2009; 457: 599-602Crossref PubMed Scopus (1142) Google Scholar, 17Van Raamsdonk C.D. Griewank K.G. Crosby M.B. Garrido M.C. Vemula S. Wiesner T. Obenauf A.C. Wackernagel W. Green G. Bouvier N. et al.Mutations in GNA11 in uveal melanoma.N. Engl. J. Med. 2010; 363: 2191-2199Crossref PubMed Scopus (1071) Google Scholar A different somatic mutation in GNAQ (GRCh37; GenBank: NM_002072.4, NP_002063.2; c.548G>A [p.Arg183Gln]) occurs in isolated capillary malformations and in individuals with Sturge-Weber syndrome (MIM: 185300).18Shirley M.D. Tang H. Gallione C.J. Baugher J.D. Frelin L.P. Cohen B. North P.E. Marchuk D.A. Comi A.M. Pevsner J. Sturge-Weber syndrome and port-wine stains caused by somatic mutation in GNAQ.N. Engl. J. Med. 2013; 368: 1971-1979Crossref PubMed Scopus (641) Google Scholar Stringent filtering revealed evidence for a GNAQ missense mutation in three of eight samples (Figure S1). When we reanalyzed with less stringent filtering, we found that six of eight samples had a somatic GNAQ mutation and the remaining two samples had a somatic GNA11 mutation (Table 2).Table 1Variant Filtering Strategy Employed in the Analysis of RNA-Seq DataParticipant12345678AverageMin 20× read depth, 3× var read depth, 10% var allele freq56,33149,04740,44038,34837,67232,47339,38026,90040,074Known variants filtered24,33318,45412,67112,89311,75610,20611,2369,57013,890Coding sequence variants279208234253241276261348263Nonsynonymous variants172120144138134155164221156Variants without strand bias133839410599114110167113Variants with T)2/6 (33%)–101/1,853 (5%)0/1,499 (0%)2NICH9 yearsmaleneckfrozen tissueGNAQ p.Gln209Pro (c.626A>C)1/10 (10%)–392/6,061 (6%)0/2,215 (0%)3NICH14 yearsfemaletemplefrozen tissueGNA11 p.Gln209Leu (c.626A>T)15/124 (12%)13/211 (6%)84/1,042 (8%)0/2,498 (0%)4NICH5 yearsmalenosefrozen tissueGNAQ p.Gln209Leu (c.626A>T)3/15 (20%)–235/3,233 (7%)0/2,359 (0%)5NICH7 yearsmalenosefrozen tissueGNAQ p.Gln209Leu (c.626A>T)7/51 (14%)–61/759 (8%)–6NICH2.5 yearsmaleneckfrozen tissueGNA11 p.Gln209Leu (c.626A>T)3/103 (3%)–15/633 (2%)–7RICH3 yearsmalelower extremityfrozen tissueGNAQ p.Gln209His (c.627A>C)7/30 (23%)–––8RICH1 weekmaleorbital areafrozen tissueGNAQ p.Gln209Leu (c.626A>T)6/31 (19%)–––9NICH12 yearsmalelower extremityFFPE tissueGNAQ p.Gln209Pro (c.626A>C)–1139/19,657 (6%)125/1,089 (11%)–10NICH14 monthsmalelower extremityFFPE tissueNA–3/1,383 (< 1%)0/640 (0%)–11RICH3 monthsmalelower extremityFFPE tissueGNAQ p.Gln209Leu (c.626A>T)–-26/717 (4%)–12NICH3 yearsmaleearFFPE tissueNA–1/400 (< 1%)-–13RICH2 weeksfemaleupper extremityFFPE tissueNA–2/772 (< 1%)0/516 (0%)–14NICH2 yearsmalelower extremityFFPE tissueNA–2/179 (1%)0/152 (0%)–15RICH2 weeksmalelower extremityFFPE tissueGNA11 p.Gln209Leu (c.626A>T)––27/2,576 (1%)–16RICH4 monthsmaleliverFFPE tissueGNA11 p.Gln209Leu (c.626A>T)––52/537 (10%)–17CHORneonatalfemaleplacentaFFPE tissueNA–0/850 (0%)––18CHORneonatalfemaleplacentaFFPE tissueNA–3/1,855 (< 1%)––19CHORneonatalfemaleplacentaFFPE tissueNA–3/1,875 (< 1%)––20CHORneonatalfemaleplacentaFFPE tissueNA–7/2,233 (< 1%)––RNA and DNA (MIP-seq) columns indicate read depth, and DNA (ddPCR) column indicates number of droplets. The rate of variant/total alleles is also depicted in the aforementioned columns. For samples that were not associated with a mutation (indicated with NA), available MIP-seq and ddPCR results with the lowest power (in terms of read depth and number of droplets) are depicted. Control DNA samples were retrieved from blood or saliva. Abbreviations are as follows: NICH, non-involuting congenital hemangioma; RICH, rapidly involuting congential hemangioma; CHOR, chorangioma. Dash (–) indicates that the assay was not performed. Open table in a new tab RNA and DNA (MIP-seq) columns indicate read depth, and DNA (ddPCR) column indicates number of droplets. The rate of variant/total alleles is also depicted in the aforementioned columns. For samples that were not associated with a mutation (indicated with NA), available MIP-seq and ddPCR results with the lowest power (in terms of read depth and number of droplets) are depicted. Control DNA samples were retrieved from blood or saliva. Abbreviations are as follows: NICH, non-involuting congenital hemangioma; RICH, rapidly involuting congential hemangioma; CHOR, chorangioma. Dash (–) indicates that the assay was not performed. We confirmed that the mutations were real by testing DNA from six of eight samples via an orthologous method, digital droplet PCR (ddPCR), and/or molecular inversion probe sequencing (MIP-seq) (Table 2 and Figure 2). We performed ddPCR (Table S2) as previously described19Couto J.A. Vivero M.P. Kozakewich H.P. Taghinia A.H. Mulliken J.B. Warman M.L. Greene A.K. A somatic MAP3K3 mutation is associated with verrucous venous malformation.Am. J. Hum. Genet. 2015; 96: 480-486Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar and considered variants present at frequencies ≥1% to represent true positives. We also verified that the mutations we identified are somatic by testing control DNA (extracted from blood or saliva) of four participants via ddPCR (Table 2). For MIP-seq, we enriched the genomic DNA samples for the protein-coding sequences of GNAQ and GNA11 by hybridization to probes (Table S3) containing an 8-nucleotide barcode that uniquely identifies individual MIPs. MIP capture and sequencing were performed as previously described.20Luks V.L. Kamitaki N. Vivero M.P. Uller W. Rab R. Bovée J.V. Rialon K.L. Guevara C.J. Alomari A.I. Greene A.K. et al.Lymphatic and other vascular malformative/overgrowth disorders are caused by somatic mutations in PIK3CA.J. Pediatr. 2015; 166 (1048–54.e1, 5)Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar Raw reads were mapped to the reference human genome sequence (GRCh37) with BWA; PCR duplicates were removed with FastqMcf and Picard. The minimum read depths for GNAQ c.626A>T and GNA11 c.626A>T (GRCh37, GenBank: NM_002067.4) were 2,032× and 179×, respectively. We considered true positive somatic variants to have allele frequencies >2%. Using a combination of the aforementioned ddPCR and MIP-seq assays, we also analyzed eight archival formalin-fixed paraffin-embedded (FFPE) congenital hemangiomas and four chorangioma samples. Four of the hemangiomas contained a likely somatic GNAQ or GNA11 mutation, whereas no such mutations were found in the chorangiomas (Table 2). Our findings expand the spectrum of genes and alleles that are somatically altered in congenital vascular anomalies (Figure S2).18Shirley M.D. Tang H. Gallione C.J. Baugher J.D. Frelin L.P. Cohen B. North P.E. Marchuk D.A. Comi A.M. Pevsner J. Sturge-Weber syndrome and port-wine stains caused by somatic mutation in GNAQ.N. Engl. J. Med. 2013; 368: 1971-1979Crossref PubMed Scopus (641) Google Scholar, 19Couto J.A. Vivero M.P. Kozakewich H.P. Taghinia A.H. Mulliken J.B. Warman M.L. Greene A.K. A somatic MAP3K3 mutation is associated with verrucous venous malformation.Am. J. Hum. 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Activating PIK3CA alleles and lymphangiogenic phenotype of lymphatic endothelial cells isolated from lymphatic malformations.Hum. Mol. Genet. 2015; 24: 926-938Crossref PubMed Scopus (89) Google Scholar To determine which features within a congenital hemangioma sample are enriched for mutant cells, we performed laser capture microdissection on a FFPE tissue section from participant 4. We observed a mutant allele frequency of 13% in the vascular-enriched lobules within the lesion, whereas the remaining tissues exhibit less than 2% mutant allele frequency (Figure 3). We were unable to identify strongly suggestive GNAQ or GNA11 mutations in 4/16 congenital hemangiomas, leading us to conclude that the frequency of mutated cells in some specimens was either below our threshold of detection or that congenital hemangiomas exhibit further locus heterogeneity. Although our sample size for chorangiomas is small, our data indicate that chorangioma is probably not the placental counterpart of congenital hemangiomas associated with mutations altering Gln209 in GNAQ or GNA11. In contrast to an earlier study in which we detected somatic mutations in PIK3CA with WES but not with RNA-seq,23Kurek K.C. Luks V.L. Ayturk U.M. Alomari A.I. Fishman S.J. Spencer S.A. Mulliken J.B. Bowen M.E. Yamamoto G.L. Kozakewich H.P. Warman M.L. Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome.Am. J. Hum. Genet. 2012; 90: 1108-1115Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar RNA-seq was useful in this study. For WES, the sensitivity of detecting somatic mutations is dependent on depth of coverage and frequency of mutant cells (Figure S3). Interestingly, when we compared the relative frequencies of the GNAQ and GNA11 mutant allele at the RNA and DNA levels in the affected tissue specimens for which we had paired RNA and DNA, the mutant allele frequency was always higher in the RNA (Table 2). This latter finding is compatible with mutation-containing cells within the affected tissue expressing GNAQ or GNA11 at a greater level relative to other cells. Thus, in addition to the advantages of using RNA-seq to find mutations that produce abnormal splicing or chimeric transcripts, RNA-seq can be advantageous for detecting somatic coding sequence mutations. We are grateful for the contributions of the participants to this study. This work was supported by NIH grants R01-AR064231 (to M.L.W.), R01-HL096384 (to J.B.), and R21-HD081004 (to A.K.G.). Download .pdf (.45 MB) Help with pdf files Document S1. Figures S1–S3 and Tables S1–S3 The URLs for data presented herein are as follows:1000 Genomes, http://browser.1000genomes.orgANNOVAR, http://annovar.openbioinformatics.org/en/latest/Burrows-Wheeler Aligner, http://bio-bwa.sourceforge.net/dbSNP, build 138, http://www.ncbi.nlm.nih.gov/projects/SNP/ExAC Browser, http://exac.broadinstitute.org/Fastq-MCF, https://code.google.com/p/ea-utils/wiki/FastqMcfGATK, https://www.broadinstitute.org/gatk/NHLBI Exome Sequencing Project (ESP) Exome Variant Server, http://evs.gs.washington.edu/EVS/OMIM, http://www.omim.org/Picard, http://broadinstitute.github.io/picard/RefSeq, http://www.ncbi.nlm.nih.gov/RefSeqsamtools, https://github.com/samtools/STAR Aligner, https://github.com/alexdobin/STAR/releasesVarScan, http://varscan.sourceforge.net/ Somatic Activating Mutations in GNAQ and GNA11 Are Associated with Congenital HemangiomaAyturk et al.The American Journal of Human GeneticsJune 02, 2016In Brief(The American Journal of Human Genetics 98, 789–795; April 7, 2016) Full-Text PDF Open Archive
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