Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome
2019; Elsevier BV; Volume: 104; Issue: 6 Linguagem: Inglês
10.1016/j.ajhg.2019.04.013
ISSN1537-6605
AutoresYline Capri, Elisabetta Flex, Oliver H.F. Krumbach, Giovanna Carpentieri, Serena Cecchetti, Christina Lißewski, Soheila Rezaei Adariani, Denny Schanze, Julia Brinkmann, Juliette Piard, Francesca Pantaleoni, Francesca Romana Lepri, Elaine Goh, Karen Chong, Elliot Stieglitz, Julia Meyer, Alma Kuechler, Nuria C. Bramswig, Stephanie Sacharow, Marion Strullu, Yoann Vial, Cédric Vignal, George Kensah, Goran Čuturilo, Neda S. Kazemein Jasemi, Radovan Dvorský, Kristin G. Monaghan, Lisa M. Vincent, Hélène Cavé, Alain Verloès, Mohammad Reza Ahmadian, Marco Tartaglia, Martin Zenker,
Tópico(s)RNA modifications and cancer
ResumoAberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%–20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations. Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%–20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations. Noonan syndrome (NS [MIM: PS163950]) is one of the most common monogenic disorders affecting development and growth.1Roberts A.E. Allanson J.E. Tartaglia M. Gelb B.D. Noonan syndrome.Lancet. 2013; 381: 333-342Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar The phenotype of NS comprises a distinctive facies (e.g., hypertelorism, downslanting palpebral fissures, ptosis, and low-set/posteriorly rotated ears), cardiac abnormalities (a wide spectrum of congenital heart defects and cardiomyopathy), postnatally reduced growth, skeletal defects (chest and spine), cryptorchidism, bleeding diathesis, as well as variable neurocognitive impairment and predisposition to malignancies,1Roberts A.E. Allanson J.E. Tartaglia M. Gelb B.D. Noonan syndrome.Lancet. 2013; 381: 333-342Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 2Tartaglia M. Gelb B.D. Zenker M. Noonan syndrome and clinically related disorders.Best Pract. Res. Clin. Endocrinol. Metab. 2011; 25: 161-179Crossref PubMed Scopus (282) Google Scholar most commonly juvenile myelomonocytic leukemia (JMML [MIM: 607785]).3Strullu M. Caye A. Lachenaud J. Cassinat B. 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Elucidation of MRAS-mediated Noonan syndrome with cardiac hypertrophy.JCI Insight. 2017; 2: e91225Crossref PubMed Scopus (56) Google Scholar Although the causal link between mutations in a subset of these genes and the disorder still remains to be confirmed,4Grant A.R. Cushman B.J. Cavé H. Dillon M.W. Gelb B.D. Gripp K.W. Lee J.A. Mason-Suares H. Rauen K.A. Tartaglia M. et al.Assessing the gene-disease association of 19 genes with the RASopathies using the ClinGen gene curation framework.Hum. Mutat. 2018; 39: 1485-1493Crossref PubMed Scopus (48) Google Scholar the accumulated molecular evidence strongly supports the view that NS is caused by upregulated intracellular traffic through the RAS-mitogen-activated protein kinase (MAPK) signaling pathway.21Tartaglia M. Gelb B.D. Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.Ann. N Y Acad. Sci. 2010; 1214: 99-121Crossref PubMed Scopus (154) Google Scholar, 22Rauen K.A. The RASopathies.Annu. Rev. Genomics Hum. Genet. 2013; 14: 355-369Crossref PubMed Scopus (542) Google Scholar Other disorders clinically related to NS (e.g., cardio-facio-cutaneous syndrome [MIM: PS115150], Costello syndrome [MIM: 218040], neurofibromatosis type 1 [MIM: 162200], Legius syndrome [MIM: 611431], Mazzanti syndrome [MIM: 607721], and Noonan syndrome with multiple lentigines [MIM: PS151100]) are also caused by mutations in genes encoding key proteins of the RAS-MAPK signaling backbone or upstream regulators (i.e., CBL, HRAS, KRAS, NF1, SPRED1, SHOC2, BRAF, MAP2K1, and MAP2K2).21Tartaglia M. Gelb B.D. Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.Ann. N Y Acad. Sci. 2010; 1214: 99-121Crossref PubMed Scopus (154) Google Scholar, 22Rauen K.A. The RASopathies.Annu. Rev. Genomics Hum. Genet. 2013; 14: 355-369Crossref PubMed Scopus (542) Google Scholar In all these related conditions, termed RASopathies, increased signaling through RAS and the MAPK cascade can result from upregulated activity of RAS proteins, enhanced function of upstream signal transducers (e.g., proteins positively controlling RAS function) or downstream RAS effectors, as well as from the inefficient signaling switch-off by feedback mechanisms (e.g., neurofibromin and CBL loss of function). More recently, the use of whole-exome sequencing (WES) has allowed the discovery of RASopathy-associated genes encoding signal transducers or modulators that do not belong to the canonical RAS-MAPK pathway, but when functionally perturbed, are predicted to impact RAS signaling by still poorly characterized circuits.20Higgins E.M. Bos J.M. Mason-Suares H. Tester D.J. Ackerman J.P. MacRae C.A. Sol-Church K. Gripp K.W. Urrutia R. Ackerman M.J. Elucidation of MRAS-mediated Noonan syndrome with cardiac hypertrophy.JCI Insight. 2017; 2: e91225Crossref PubMed Scopus (56) Google Scholar, 23Flex E. Jaiswal M. Pantaleoni F. Martinelli S. Strullu M. Fansa E.K. Caye A. De Luca A. Lepri F. Dvorsky R. et al.Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis.Hum. Mol. Genet. 2014; 23: 4315-4327Crossref PubMed Scopus (100) Google Scholar, 24Gripp K.W. Aldinger K.A. Bennett J.T. Baker L. Tusi J. Powell-Hamilton N. Stabley D. Sol-Church K. Timms A.E. Dobyns W.B. A novel rasopathy caused by recurrent de novo missense mutations in PPP1CB closely resembles Noonan syndrome with loose anagen hair.Am. J. Med. Genet. A. 2016; 170: 2237-2247Crossref PubMed Scopus (90) Google Scholar, 25Martinelli S. Krumbach O.H.F. Pantaleoni F. Coppola S. Amin E. Pannone L. Nouri K. Farina L. Dvorsky R. 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Evers C. et al.Genotype and phenotype in patients with Noonan syndrome and a RIT1 mutation.Genet. Med. 2016; 18: 1226-1234Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 12Cirstea I.C. Kutsche K. Dvorsky R. Gremer L. Carta C. Horn D. Roberts A.E. Lepri F. Merbitz-Zahradnik T. König R. et al.A restricted spectrum of NRAS mutations causes Noonan syndrome.Nat. Genet. 2010; 42: 27-29Crossref PubMed Scopus (239) Google Scholar, 13Schubbert S. Zenker M. Rowe S.L. Böll S. Klein C. Bollag G. van der Burgt I. Musante L. Kalscheuer V. Wehner L.-E. et al.Germline KRAS mutations cause Noonan syndrome.Nat. Genet. 2006; 38: 331-336Crossref PubMed Scopus (600) Google Scholar, 14Zenker M. Lehmann K. Schulz A.L. Barth H. Hansmann D. Koenig R. Korinthenberg R. Kreiss-Nachtsheim M. Meinecke P. Morlot S. et al.Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations.J. Med. Genet. 2007; 44: 131-135Crossref PubMed Scopus (161) Google Scholar, 20Higgins E.M. Bos J.M. Mason-Suares H. Tester D.J. Ackerman J.P. MacRae C.A. Sol-Church K. Gripp K.W. Urrutia R. Ackerman M.J. Elucidation of MRAS-mediated Noonan syndrome with cardiac hypertrophy.JCI Insight. 2017; 2: e91225Crossref PubMed Scopus (56) Google Scholar, 23Flex E. Jaiswal M. Pantaleoni F. Martinelli S. Strullu M. Fansa E.K. Caye A. De Luca A. Lepri F. Dvorsky R. et al.Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis.Hum. Mol. Genet. 2014; 23: 4315-4327Crossref PubMed Scopus (100) Google Scholar, 24Gripp K.W. Aldinger K.A. Bennett J.T. Baker L. Tusi J. Powell-Hamilton N. Stabley D. Sol-Church K. Timms A.E. Dobyns W.B. A novel rasopathy caused by recurrent de novo missense mutations in PPP1CB closely resembles Noonan syndrome with loose anagen hair.Am. J. Med. Genet. A. 2016; 170: 2237-2247Crossref PubMed Scopus (90) Google Scholar, 25Martinelli S. Krumbach O.H.F. Pantaleoni F. Coppola S. Amin E. Pannone L. Nouri K. Farina L. Dvorsky R. Lepri F. et al.University of Washington Center for Mendelian GenomicsFunctional Dysregulation of CDC42 Causes Diverse Developmental Phenotypes.Am. J. Hum. Genet. 2018; 102: 309-320Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 26Young L.C. Hartig N. Boned Del Río I. Sari S. Ringham-Terry B. Wainwright J.R. Jones G.G. McCormick F. Rodriguez-Viciana P. SHOC2-MRAS-PP1 complex positively regulates RAF activity and contributes to Noonan syndrome pathogenesis.Proc. Natl. Acad. Sci. USA. 2018; 115: E10576-E10585Crossref PubMed Scopus (38) Google Scholar, 30Aoki Y. Niihori T. Kawame H. Kurosawa K. Ohashi H. Tanaka Y. Filocamo M. Kato K. Suzuki Y. Kure S. Matsubara Y. Germline mutations in HRAS proto-oncogene cause Costello syndrome.Nat. Genet. 2005; 37: 1038-1040Crossref PubMed Scopus (525) Google Scholar Missense mutations in these genes affect a small number of highly conserved amino acid residues that lead to overactivation of these proteins by decreasing/impairing their GTPase activity in response to GTPase-activating proteins (GAPs), increasing guanine nucleotide exchange factor (GEF)-independent GDP release, altering binding properties to effectors, or a combination of these mechanisms.31Gremer L. Gilsbach B. Ahmadian M.R. Wittinghofer A. Fluoride complexes of oncogenic Ras mutants to study the Ras-RasGap interaction.Biol. Chem. 2008; 389: 1163-1171Crossref PubMed Scopus (38) Google Scholar Notably, while these germline mutations may affect the same residues that are generally mutated in cancer, multiple lines of evidence indicate that RASopathy-causing changes are generally less activating than their respective cancer-associated somatic lesions.21Tartaglia M. Gelb B.D. Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.Ann. N Y Acad. Sci. 2010; 1214: 99-121Crossref PubMed Scopus (154) Google Scholar Despite the large number of genes implicated in NS and related phenotypes, about 10%–20% of affected individuals with a convincing clinical diagnosis of NS do not have mutations in currently known RASopathy-associated genes, indicating that other unidentified genes contribute to this disorder. Through the use of complementary approaches based on "functional candidacy" (parallel sequencing of selected gene panels containing functionally related candidate genes) or WES, we identified RRAS2 (MIM: 600098; GenBank: NM_012250.5) as a gene implicated in NS. We provide structural, biochemical, and functional data to support the causal link between RRAS2 mutations and NS, outline the mechanisms by which mutations perturb RRAS2 function, and characterize the clinical phenotype associated with these gene lesions. Subjects from six unrelated families were included in the study. Clinical data and DNA samples were collected from the participating families after written informed consent was obtained. DNA samples were stored and used under research projects approved by the Review Boards of the participating institutions. Because of a suspected RASopathy, subjects 1, 2, 3-III-1, and 5 were referred for diagnostic genetic testing by sequencing of an "extended" panel of RASopathy-associated genes designed to include a set of candidate disease genes selected in the frame of the NSEuroNet Consortium, while subjects 4 and 6 were analyzed by WES (Supplemental Subjects and Methods). In five cases, the RRAS2 variant (c.68G>T [p.Gly23Val], c.65_73dup [p.Gly22_Gly24dup], c.70_78dup [p.Gly24_Gly26dup], c.208G>A [p.Ala70Thr], c.215A>T [p.Gln72Leu]) arose de novo (i.e., it was not identified in parental blood DNA samples). In family 3, mutation scan in one affected family member (3-III-1) identified the heterozygous c.208G>A missense change, and subsequent co-segregation analysis confirmed the occurrence of the variant in three similarly affected relatives. All variants were validated by Sanger sequencing. In all cases, no other candidate variant was identified, further supporting the clinical relevance of this finding. In subject 4, the RRAS2 variant was detected in both amniocyte and peripheral blood DNA, at 44% and 46% of reads, respectively, indicating the heterozygous mutation was present in the germline of the subject. The clinical data of the affected subjects from the six families are shown in Table 1, facial features of four affected individuals as well as the pedigree of family 3 are presented in Figure 1, and a detailed clinical history is provided in the Supplemental Note. Taken together, the identified RRAS2 variants included three different nucleotide substitutions predicting missense changes of highly conserved amino acid residues (Gly23, Ala70, and Gln72) among RRAS2 orthologs and paralogs (Figure S1). Alterations to the corresponding positions in other GTPases of the RAS superfamily have already been reported to cause RASopathies or to contribute to oncogenesis (Table S1). In the remaining cases, we identified two small in-frame duplications (p.Gly22_Gly24dup, p.Gly24_Gly26dup) affecting the well-established mutational hotspot of RAS proteins (Figure 2A). Of note, p.Gly22_Gly24dup had previously been reported as somatic event in an uterine leiomyosarcoma specimen,32Huang Y. Saez R. Chao L. Santos E. Aaronson S.A. Chan A.M. A novel insertional mutation in the TC21 gene activates its transforming activity in a human leiomyosarcoma cell line.Oncogene. 1995; 11: 1255-1260PubMed Google Scholar and other similar, but not identical, small in-frame duplications affecting these residues have also been reported in association with different cancers in the Catalogue of Somatic Mutations in Cancer (COSMIC database). The two small in-frame duplications and c.68G>T (p.Gly23Val) and c.215A>T (p.Gln72Leu) substitutions were absent from general population databases, while the c.208G>A (p.Ala70Thr) change had previously been reported in two subjects in gnomAD (heterozygous state, frequency < 0.00001) (Table S2). Multiple in silico prediction algorithms uniformly rated these changes as deleterious/pathogenic (Table S2).Table 1Clinical Features and Genotype of Individuals with RRAS2 VariantsSubject 1Subject 2Family 3Subject 4Subject 5Subject 63-II-13-II-23-III-13-III-2OriginAlgerianSri LankaGermanIndianSerbianSouth American/ AshkenaziGenderMMFFFMMFMAge at last visit7 y 11 m12 y 2 m32 y40 y7 y 1 m1 y 7 m2 weeks8 y 10 m22 m (last measurement 18 m)RRAS2 variantc.65_73dup (p.Gly22_Gly24dup)c.68G>T (p.Gly23Val)c.208G>A (p.Ala70Thr)c.208G>A (p.Ala70Thr)c.208G>A (p.Ala70Thr)c.208G>A (p.Ala70Thr)c.215A>T (p.Gln72Leu)c.208G>A (p.Ala70Thr)c.70_78dup (p.Gly24_Gly26dup)Inheritancede novode novopresumed paternalpresumed paternalmaternalmaternalde novode novode novoPrenatal featuresNE, PHPHNANANANNE, fetal ventriculo-megaly and cardiac abnormalitiesNEPH, LGABirth measurements: weight, length, OFC (weeks GA)3,730 g, 50.5 cm, 37 cm (35)3,180 g, 46.5 cm, 35 cm (35)NA3,740 g, 51 cm, 36 cm3,110 g, 48 cm, 36 cm (39)2,440 g, 48 cm, 32 cm (35)2,400 g (33)NA3,600 g, 51 cm, 38 cm (35)Feeding difficultiesPFPF, TFNANAPFNNANNHeight at last examination125.5 cm (+0.3 SD)139.5 (−1.5 SD) 85 cm (−3.3 SD)aBefore onset of growth hormone treatment at age 3 y 6 m.160 cm (−1.3 SD)170 cm (+0.3 SD)108 cm (−3.0 SD)78 cm (−1.8 SD)NA122 cm (−2.1 SD)84.5 cm (+0.5 SD)Weight27.5 kg (+0.5 SD)32.5 kg (−1.4 SD)NA59 kg (+0.1 SD)18.6 kg (−1.8 SD)11 kg (−0.4 SD)NA22 kg (−1.9 SD)12.5 kg (+0.7 SD)OFC54 cm (+1.2 SD)57 cm (+2.5 SD)52.5 cm (−2.2 SD)55.5 cm (+0.2 SD)52 cm (+0.4 SD)49 cm (+0.2 SD)NA52.5 cm (+0.2 SD)54.5 cm (+5.0 SD)CryptorchidismNNNANANANhypoplastic scrotumNANCongenital heart defectSVAoSVSDVSDNNNTOFAVSD, multiple VSDsNLymphatic anomaliesNNNNNNNNNFacial anomaliestypical NStypical NSsuggestive NSvery mild in adulthoodtypical NStypical NSmultiple anomaliessuggestive NStypical NSDevelopmentNmild MD, mild LDNNmild MD, mild LDNNANmild global delayNeurologyNChiari malformationNNNNnon-obstructive hydrocephalusNmild ventriculomegaly, hypotoniaSkeletalNNNNNN11 rib pairs, proximally placed thumb, spinal canal stenosispectus excavatumNHematology & oncologyNlymphopeniaNNNNthrombocytopeniaNNSkin and hairglabellar heamangiomaNNNatopic dermatitis,NNNglabellar hemangiomaOcularNstrabismusNstrabismushyperopia, bilateral ptosisNNANstrabismic amblyopia, esotropiaOther malformations/anomaliesNGH deficiency, GH treatment from age 4 yunilateral duplex kidneyNmultiple allergies, bronchitisNlabyrinth dysplasia, anteriorly placed anusminor hippocampal malformation on brain MRINAbbreviations: AVSD, atrioventricular septal defect; F, female; GA, gestational age; GD, global delay; GH, growth hormone; LD, learning difficulties; LGA, large for gestational age; M, male; m, months; MD, motor delay; N, none/normal; NA, not applicable/not available; NE, nuchal edema; OFC, occipitofrontal head circumference; PF, poor feeding reported; PH, polyhydramnios; SVAoS, supravalvular aortic stenosis; TF, tube feeding (>4 weeks); TOF, Tetralogy of Fallot; y, years.a Before onset of growth hormone treatment at age 3 y 6 m. Open table in a new tab Figure 2RRAS2 Structure and Location and Functional Impact of Noonan Syndrome-Causing VariantsShow full caption(A) Schematic representation of RRAS2 and HRAS proteins. Conserved motifs critical for tight guanine nucleotide binding and hydrolysis, and position of the disease-causing RRAS2 variants are illustrated together with the homologous residues of HRAS. The three residues representing the mutational hotspots of oncogenic HRAS mutations are shown in red.(B) Structural modeling of RRAS2 variants. A structural model of the active GTP-bound RRAS2 protein highlights the relative position of the disease-causing missense or insertion mutations. All RRAS2 mutations affect residues that are located in the nucleotide binding active site region, which contains integral elements involved in GDP/GTP binding, GTP hydrolysis, and interactions with regulators (GEFs and GAPs) and effectors.(C) Biochemical assessment of RRAS2p.Ala70Thr. RRAS2WT and RRAS2p.Ala70Thr proteins were biochemically characterized regarding their nucleotide exchange (left), GTP hydrolysis (middle), and effector binding (right) properties. The nucleotide exchange reaction was measured in the absence (intrinsic) and in the presence of the catalytic RASGEF domain of mouse RASGRF1, while the catalytic activity of the GTPase was assessed in the absence (intrinsic) and in the presence of the p120 RASGAP GAP domain. The RAS-binding and RAS association domains of CRAF and RASSF5 were used to evaluate the binding behavior of the RRAS2p.Ala70Thr mutant to RAS effectors. Overall, the data indicate that the p.Ala70Thr change leads to an accumulation of the protein in its GTP-bound active state, resulting to an increased signaling activity. The missense change, however, is predicted to differentially impact on the diverse downstream signaling pathways.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Abbreviations: AVSD, atrioventricular septal defect; F, female; GA, gestational age; GD, global delay; GH, growth hormone; LD, learning difficulties; LGA, large for gestational age; M, male; m, months; MD, motor delay; N, none/normal; NA, not applicable/not available; NE, nuchal edema; OFC, occipitofrontal head circumference; PF, poor feeding reported; PH, polyhydramnios; SVAoS, supravalvular aortic stenosis; TF, tube feeding (>4 weeks); TOF, Tetralogy of Fallot; y, years. (A) Schematic representation of RRAS2 and HRAS proteins. Conserved motifs critical for tight guanine nucleotide binding and hydrolysis, and position of the disease-causing RRAS2 variants are illustrated together with the homologous residues of HRAS. The three residues representing the mutational hotspots of oncogenic HRAS mutations are shown in red. (B) Structural modeling of RRAS2 variants. A structural model of the active GTP-bound RRAS2 protein highlights the relative position of the disease-causing missense or insertion mutations. All RRAS2 mutations affect residues that are located in the nucleotide binding active site region, which contains integral elements involved i
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