Somatic PDGFRB Activating Variants in Fusiform Cerebral Aneurysms
2019; Elsevier BV; Volume: 104; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2019.03.014
ISSN1537-6605
AutoresYigit Karasozen, Joshua W. Osbun, Carolina A. Parada, Tina Busald, Philip D. Tatman, Luis F. Gonzalez‐Cuyar, Christopher J. Hale, Diana Alcantara, Mark O’Driscoll, William B. Dobyns, Mitzi L. Murray, Louis J. Kim, Peter H. Byers, Michael O. Dorschner, Manuel Ferreira,
Tópico(s)Fibroblast Growth Factor Research
ResumoThe role of somatic genetic variants in the pathogenesis of intracranial-aneurysm formation is unknown. We identified a 23-year-old man with progressive, right-sided intracranial aneurysms, ipsilateral to an impressive cutaneous phenotype. The index individual underwent a series of genetic evaluations for known connective-tissue disorders, but the evaluations were unrevealing. Paired-sample exome sequencing between blood and fibroblasts derived from the diseased areas detected a single novel variant predicted to cause a p.Tyr562Cys (g.149505130T>C [GRCh37/hg19]; c.1685A>G) change within the platelet-derived growth factor receptor β gene (PDGFRB), a juxtamembrane-coding region. Variant-allele fractions ranged from 18.75% to 53.33% within histologically abnormal tissue, suggesting post-zygotic or somatic mosaicism. In an independent cohort of aneurysm specimens, we detected somatic-activating PDGFRB variants in the juxtamembrane domain or the kinase activation loop in 4/6 fusiform aneurysms (and 0/38 saccular aneurysms; Fisher's exact test, p < 0.001). PDGFRB-variant, but not wild-type, patient cells were found to have overactive auto-phosphorylation with downstream activation of ERK, SRC, and AKT. The expression of discovered variants demonstrated non-ligand-dependent auto-phosphorylation, responsive to the kinase inhibitor sunitinib. Somatic gain-of-function variants in PDGFRB are a novel mechanism in the pathophysiology of fusiform cerebral aneurysms and suggest a potential role for targeted therapy with kinase inhibitors. The role of somatic genetic variants in the pathogenesis of intracranial-aneurysm formation is unknown. We identified a 23-year-old man with progressive, right-sided intracranial aneurysms, ipsilateral to an impressive cutaneous phenotype. The index individual underwent a series of genetic evaluations for known connective-tissue disorders, but the evaluations were unrevealing. Paired-sample exome sequencing between blood and fibroblasts derived from the diseased areas detected a single novel variant predicted to cause a p.Tyr562Cys (g.149505130T>C [GRCh37/hg19]; c.1685A>G) change within the platelet-derived growth factor receptor β gene (PDGFRB), a juxtamembrane-coding region. Variant-allele fractions ranged from 18.75% to 53.33% within histologically abnormal tissue, suggesting post-zygotic or somatic mosaicism. In an independent cohort of aneurysm specimens, we detected somatic-activating PDGFRB variants in the juxtamembrane domain or the kinase activation loop in 4/6 fusiform aneurysms (and 0/38 saccular aneurysms; Fisher's exact test, p < 0.001). PDGFRB-variant, but not wild-type, patient cells were found to have overactive auto-phosphorylation with downstream activation of ERK, SRC, and AKT. The expression of discovered variants demonstrated non-ligand-dependent auto-phosphorylation, responsive to the kinase inhibitor sunitinib. Somatic gain-of-function variants in PDGFRB are a novel mechanism in the pathophysiology of fusiform cerebral aneurysms and suggest a potential role for targeted therapy with kinase inhibitors. Intracranial aneurysms occur in approximately 2% of the population and have a rupture risk of 6 per 100,000 individual-years.1Brown Jr., R.D. Broderick J.P. Unruptured intracranial aneurysms: Epidemiology, natural history, management options, and familial screening.Lancet Neurol. 2014; 13: 393-404Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 2Rinkel G.J. Djibuti M. Algra A. van Gijn J. Prevalence and risk of rupture of intracranial aneurysms: A systematic review.Stroke. 1998; 29: 251-256Crossref PubMed Scopus (1169) Google Scholar There are two types of aneurysms, the more common saccular type (90%–95%) and the fusiform type (4%–8%).1Brown Jr., R.D. Broderick J.P. Unruptured intracranial aneurysms: Epidemiology, natural history, management options, and familial screening.Lancet Neurol. 2014; 13: 393-404Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 2Rinkel G.J. Djibuti M. Algra A. van Gijn J. Prevalence and risk of rupture of intracranial aneurysms: A systematic review.Stroke. 1998; 29: 251-256Crossref PubMed Scopus (1169) Google Scholar Saccular aneurysms are abnormal arterial outpouchings at branch points and have histological loss of the media and intima. Fusiform aneurysms are circumferential abnormal arterial dilatation with histological medial and intimal hyperplasia. The size, location in the cerebrovascular tree, and type of aneurysm all influence the natural history of this disease.1Brown Jr., R.D. Broderick J.P. Unruptured intracranial aneurysms: Epidemiology, natural history, management options, and familial screening.Lancet Neurol. 2014; 13: 393-404Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 2Rinkel G.J. Djibuti M. Algra A. van Gijn J. Prevalence and risk of rupture of intracranial aneurysms: A systematic review.Stroke. 1998; 29: 251-256Crossref PubMed Scopus (1169) Google Scholar Abundant evidence supports a genetic component to the etiology of intracranial aneurysms.1Brown Jr., R.D. Broderick J.P. Unruptured intracranial aneurysms: Epidemiology, natural history, management options, and familial screening.Lancet Neurol. 2014; 13: 393-404Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 2Rinkel G.J. Djibuti M. Algra A. van Gijn J. 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Genetics of intracranial aneurysms.Lancet Neurol. 2005; 4: 179-189Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 4Bor A.S. Rinkel G.J. van Norden J. Wermer M.J. Long-term, serial screening for intracranial aneurysms in individuals with a family history of aneurysmal subarachnoid haemorrhage: A cohort study.Lancet Neurol. 2014; 13: 385-392Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 5Mackey J. Brown R.D. Sauerbeck L. Hornung R. Moomaw C.J. Koller D.L. Foroud T. Deka R. Woo D. Kleindorfer D. et al.Affected twins in the familial intracranial aneurysm study.Cerebrovasc. Dis. 2015; 39: 82-86Crossref PubMed Scopus (11) Google Scholar Several genetic syndromes are associated with intracranial aneurysms, and they confer increased risk compared to the risk for the general population.1Brown Jr., R.D. Broderick J.P. Unruptured intracranial aneurysms: Epidemiology, natural history, management options, and familial screening.Lancet Neurol. 2014; 13: 393-404Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 2Rinkel G.J. Djibuti M. Algra A. van Gijn J. Prevalence and risk of rupture of intracranial aneurysms: A systematic review.Stroke. 1998; 29: 251-256Crossref PubMed Scopus (1169) Google Scholar, 3Ruigrok Y.M. Rinkel G.J. Wijmenga C. Genetics of intracranial aneurysms.Lancet Neurol. 2005; 4: 179-189Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar Studies in mono- and dizygotic twins also suggest both genetic and environmental contributions.5Mackey J. Brown R.D. Sauerbeck L. Hornung R. Moomaw C.J. Koller D.L. Foroud T. Deka R. Woo D. Kleindorfer D. et al.Affected twins in the familial intracranial aneurysm study.Cerebrovasc. Dis. 2015; 39: 82-86Crossref PubMed Scopus (11) Google Scholar Established environmental risk factors, which might somatically alter coding regions of the genome, include cigarette smoking and hypertension.1Brown Jr., R.D. Broderick J.P. Unruptured intracranial aneurysms: Epidemiology, natural history, management options, and familial screening.Lancet Neurol. 2014; 13: 393-404Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 2Rinkel G.J. Djibuti M. Algra A. van Gijn J. Prevalence and risk of rupture of intracranial aneurysms: A systematic review.Stroke. 1998; 29: 251-256Crossref PubMed Scopus (1169) Google Scholar The role of post-zygotic variants of genes that function in critical intracellular signaling pathways has been established for several types of overgrowth syndromes8Lindhurst M.J. Sapp J.C. Teer J.K. Johnston J.J. Finn E.M. Peters K. Turner J. Cannons J.L. Bick D. 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Liang M.G. Bischoff J. Warman M.L. Greene A.K. A somatic GNA11 mutation is associated with extremity capillary malformation and overgrowth.Angiogenesis. 2017; 20: 303-306Crossref PubMed Scopus (82) Google Scholar, 16Nikolaev S.I. Vetiska S. Bonilla X. Boudreau E. Jauhiainen S. Rezai Jahromi B. Khyzha N. DiStefano P.V. Suutarinen S. Kiehl T.R. et al.Somatic activating KRAS mutations in arteriovenous malformations of the brain.N. Engl. J. Med. 2018; 378: 250-261Crossref PubMed Scopus (229) Google Scholar but the role of somatic genetic alterations or mosaicism in intracranial aneurysms remains unknown. The index individual was first treated for a dissecting fusiform paraclinoid internal carotid artery aneurysm at nine years of age. All individuals' data and specimen collection were reviewed and approved by the University of Washington institutional review board and human subjects division. The individual was noted to have an impressive ipsilateral cutaneous phenotype. 14 years later, he developed a giant dissecting fusiform aneurysm of the right vertebral artery, which was previously normal according to angiography (Figures 1A–C and S1). He had apparently normal cognition and no neurological deficits or other birth defects. No other abnormalities (including intracranial calcifications) were found on brain, cardiovascular, or peripheral vascular imaging. He later developed both radial and coronary artery aneurysms (Figures S1J and S1K), but never an aortic aneurysm or dissection, beneath his dermal phenotype. His family history was negative. The left neurovascular tree remained normal (Figures S1C and S1E). Detailed phenotype information for this individual is shown in Figure S1. He underwent a series of operations for treatment of the giant, rapidly growing fusiform vertebral aneurysm. DNA was extracted from multiple vascular and perivascular tissue samples (Figure 1K) obtained during surgery. Initial variant discovery was carried out via paired-sample exome sequencing to an average depth of ∼150× between blood and fibroblasts derived from the diseased areas (>99% of the exome was covered for all samples). Exome sequencing was performed on blood and abnormal tissue with a customized exome-capture probe set that is built upon the xGen Exome Research Panel v1.0 (IDT) backbone and from the UW Medicine Center for Precision Diagnostics. Initial variant discovery was carried out via a comparison between blood and diseased-area cultured fibroblast exomes sequenced to an average depth of ∼150× on the Illumina HiSeq 2500 platform. Subsequent exome sequencing was performed on other diseased specimens and healthy radial artery (Figures 1K and 1L) to an average depth of at least 40× (>99% of the exome). Resulting reads were aligned with the Burrows-Wheeler Alignment Tool BWA-MEM (v0.7.5) according to the Broad Institute's Genome Analysis Toolkit (GATK) best practices. Somatic variants were identified via MuTect (v1.1.7) with default parameters. Our analysis detected a single novel variant within the platelet-derived growth factor receptor β gene (PDGFRB) juxtamembrane-coding region (p.Tyr562Cys [g.149505130T>C (GRCh37/hg19); c.1685A>G]). Variant allele fractions ranged from 18.75% to 53.33% within histologically abnormal tissue (Figures 1D–1L). No other somatic variants were found. The highest allele fractions were found in a specimen from an occipital artery aneurysm (Figures 1D–1I and S2). This PDGFRB variant was not found in DNA isolated from blood or the histologically normal, left-sided, radial artery (Figures 1J–1L), confirming post-zygotic or somatic mosaicism. PDGFRB encodes a conserved transmembrane receptor tyrosine kinase involved in diverse signaling processes during embryonal development.17Lindahl P. Johansson B.R. Levéen P. Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.Science. 1997; 277: 242-245Crossref PubMed Scopus (1743) Google Scholar, 18Boucher P. Gotthardt M. Li W.-P. Anderson R.G. Herz J. LRP: Role in vascular wall integrity and protection from atherosclerosis.Science. 2003; 300: 329-332Crossref PubMed Scopus (467) Google Scholar, 19Daneman R. Zhou L. Kebede A.A. Barres B.A. Pericytes are required for blood-brain barrier integrity during embryogenesis.Nature. 2010; 468: 562-566Crossref PubMed Scopus (1363) Google Scholar, 20He C. Medley S.C. Hu T. Hinsdale M.E. Lupu F. Virmani R. Olson L.E. PDGFRβ signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis.Nat. Commun. 2015; 6: 7770Crossref PubMed Scopus (81) Google Scholar, 21Olson L.E. Soriano P. PDGFRβ signaling regulates mural cell plasticity and inhibits fat development.Dev. Cell. 2011; 20: 815-826Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar PDGFRB is normally expressed in several cell types, including pericytes and vascular smooth muscle cells, and has an essential role in vascular progenitor cell signaling.19Daneman R. Zhou L. Kebede A.A. Barres B.A. Pericytes are required for blood-brain barrier integrity during embryogenesis.Nature. 2010; 468: 562-566Crossref PubMed Scopus (1363) Google Scholar, 20He C. Medley S.C. Hu T. Hinsdale M.E. Lupu F. Virmani R. Olson L.E. PDGFRβ signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis.Nat. Commun. 2015; 6: 7770Crossref PubMed Scopus (81) Google Scholar, 21Olson L.E. Soriano P. PDGFRβ signaling regulates mural cell plasticity and inhibits fat development.Dev. Cell. 2011; 20: 815-826Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar On the basis of the findings in the individual described above, we performed targeted sequencing of PDGFRB in a validation cohort of 50 aneurysm and arterial walls (Table S1). The validation cohort was sequenced similarly to the exome sequencing performed on the index individual with the exception that a custom capture-probe set (IDT) was used rather than the full exome. Variants were batch-identified across the cohort with the Platypus variant caller (v0.8.1), which used a minimum variant-allele fraction of 2%, a minimum coverage of 5 reads, and a minimum posterior probability of 0 (no variant reads), allowing more inclusive initial analysis. Germline variants and sequencing artifacts were further filtered out with an in-house script. All somatic variants were analyzed with IGV (v2.3.71) and functionally annotated with Oncotator (v1.9.3.0). In three additional sporadic individual cases, targeted sequencing revealed four variants: a juxtamembrane domain variant predicted to result in a four aa in-frame deletion (p.Tyr562_Arg565del) in exon 12, and two additional variants (p.Asp850Tyr and p.Arg849_Lys860delinsHisAlaGlyLeuGluLeuHisLeuGln) in the activation loop of the kinase domain in exon 18 (Figure 2A). The latter variant was comprised of two deletions located in cis, and these deletions together are predicted to result in a complex, in-frame insertion-deletion (Figure S4). Variants were only found in fusiform aneurysms (3/5, 60%), which were radiographically and histologically similar to the aneurysms found in our index individual (Figures 2B–F and S3). All saccular aneurysms had wild-type PDGFRB. Exome sequencing was performed on aneurysm walls and control tissues from all three PDGFRB-variant sporadic fusiform aneurysms (see the Supplemental Data). All aneurysm samples were sequenced to at least a depth of 175× (>99% of exome), and control samples, with the exception of lymph node DNA from the VAL-44 individual, were sequenced to a depth of at least 90× (>99% of exome) average coverage. Additional sequencing was added to the PDGFRB variant region of the lymph node DNA (non-aneurysm control DNA) with the custom capture-probe set in order to study a germline contribution for VAL-44. The aneurysm exome of VAL-44 was analyzed on its own. For every available control tissue, complete pairs were analyzed with a variant via FreeBayes (v1.0.2), Strelka2 (v2.0.17), VarDict (v1.5.1), and VarScan2 (v2.4.3), and the output was filtered with an in-house script and confirmed with manual inspection on IGV. For the VAL-44 aneurysm without a good-quality control, variant calling was done with FreeBayes, Platypus, and VarDict, and the output was filtered with an in-house script and confirmed with manual inspection on IGV. Exome sequencing of aneurysm and normal tissue DNA revealed only recurrent PDGFRB variants (Figure S4 and Table S3), suggesting a causal role in the formation of sporadic fusiform aneurysms. DNA was available from blood and/or unaffected healthy tissue, allowing for the exploration of the germline contribution of the variant in all cases (Table S3). The skewed PDGFRB allele fractions, in sporadic fusiform aneurysms, ranged from 5.6 to 21.4% (Figure 2A), also consistent with post-zygotic, somatic variants. These results were also confirmed by next-generation sequencing after independent primer-pair amplification across the variant locations, resulting in similar allele fractions (Tables S2 and S3). Two of the PDGFRB missense variants observed (p.Tyr562Cys and p.Asp850Val) have recently been found in sporadic myofibromas.22Agaimy A. Bieg M. Michal M. Geddert H. Märkl B. Seitz J. Moskalev E.A. Schlesner M. Metzler M. Hartmann A. et al.Recurrent somatic PDGFRB mutations in sporadic infantile/solitary adult myofibromas but not in angioleiomyomas and myopericytomas.Am. J. Surg. Pathol. 2017; 41: 195-203Crossref PubMed Scopus (58) Google Scholar, 23Arts F.A. Sciot R. Brichard B. Renard M. de Rocca Serra A. Dachy G. Noël L.A. Velghe A.I. Galant C. Debiec-Rychter M. et al.PDGFRB gain-of-function mutations in sporadic infantile myofibromatosis.Hum. Mol. Genet. 2017; 26: 1801-1810Crossref PubMed Scopus (58) Google Scholar None of the variants we detected in intracranial aneurysms was seen in >120,000 normal genomes (from dbSNP, 1000 Genomes, NHLBI-EVS, and the gnomAD databases); this finding supports their pathogenicity and suggests that they might be embryonic lethal. These variants altered conserved regions and were predicted to be protein-altering and pathogenic by PolyPhen2, SIFT, and MutationTaster (Figure S5). In fusiform aneurysms, missense variants and in-frame deletions occurred in either the ArgTyrGluIleArg motif of the juxtamembrane region or the adjacent AspPheGly motif in the activation loop (Figures 3A–C and S5) of PDGFRB. Disruption of juxtamembrane region auto-inhibitory sites causes constitutive activation.24Dibb N.J. Dilworth S.M. Mol C.D. Switching on kinases: Oncogenic activation of BRAF and the PDGFR family.Nat. Rev. Cancer. 2004; 4: 718-727Crossref PubMed Scopus (156) Google Scholar, 27Irusta P.M. Luo Y. Bakht O. Lai C.C. Smith S.O. DiMaio D. Definition of an inhibitory juxtamembrane WW-like domain in the platelet-derived growth factor β receptor.J. Biol. Chem. 2002; 277: 38627-38634Crossref PubMed Scopus (48) Google Scholar All four variants occur in known homologous PDGFRA and KIT "hot spots" within the juxtamembrane or the activation loop of the kinase domains.24Dibb N.J. Dilworth S.M. Mol C.D. Switching on kinases: Oncogenic activation of BRAF and the PDGFR family.Nat. Rev. Cancer. 2004; 4: 718-727Crossref PubMed Scopus (156) Google Scholar, 25Corless C.L. Schroeder A. Griffith D. Town A. McGreevey L. Harrell P. Shiraga S. Bainbridge T. Morich J. Heinrich M.C. PDGFRA mutations in gastrointestinal stromal tumors: Frequency, spectrum and in vitro sensitivity to imatinib.J. Clin. Oncol. 2005; 23: 5357-5364Crossref PubMed Scopus (676) Google Scholar The conserved residues are found in all tyrosine protein kinases, and the analogous residues (Tyr555 and Tyr552) in the PDGFRA and KIT kinases are somatically altered in cancers24Dibb N.J. Dilworth S.M. Mol C.D. Switching on kinases: Oncogenic activation of BRAF and the PDGFR family.Nat. Rev. Cancer. 2004; 4: 718-727Crossref PubMed Scopus (156) Google Scholar, 25Corless C.L. Schroeder A. Griffith D. Town A. McGreevey L. Harrell P. Shiraga S. Bainbridge T. Morich J. Heinrich M.C. PDGFRA mutations in gastrointestinal stromal tumors: Frequency, spectrum and in vitro sensitivity to imatinib.J. Clin. Oncol. 2005; 23: 5357-5364Crossref PubMed Scopus (676) Google Scholar (Figure 3B). The aneurysm alterations in PDGFRB are predicted to result in p.Tyr562Cys and p.Tyr562_Arg565del. Aligned amino-acid sequences of the activation loops of human KIT, PDGFRA, and PDGFRB start and end with residues AspPheGly and AlaProGlu. Within the kinase loop, the two alterations that are known to be important for autoregulation24Dibb N.J. Dilworth S.M. Mol C.D. Switching on kinases: Oncogenic activation of BRAF and the PDGFR family.Nat. Rev. Cancer. 2004; 4: 718-727Crossref PubMed Scopus (156) Google Scholar, 25Corless C.L. Schroeder A. Griffith D. Town A. McGreevey L. Harrell P. Shiraga S. Bainbridge T. Morich J. Heinrich M.C. PDGFRA mutations in gastrointestinal stromal tumors: Frequency, spectrum and in vitro sensitivity to imatinib.J. Clin. Oncol. 2005; 23: 5357-5364Crossref PubMed Scopus (676) Google Scholar, 28Looman C. Sun T. Yu Y. Zieba A. Ahgren A. Feinstein R. Forsberg H. Hellberg C. Heldin C.H. Zhang X.Q. et al.An activating mutation in the PDGF receptor-beta causes abnormal morphology in the mouse placenta.Int. J. Dev. Biol. 2007; 51: 361-370Crossref PubMed Scopus (12) Google Scholar, 29Chiara F. Goumans M.J. Forsberg H. Ahgrén A. Rasola A. Aspenström P. Wernstedt C. Hellberg C. Heldin C.H. Heuchel R. A gain of function mutation in the activation loop of platelet-derived growth factor β-receptor deregulates its kinase activity.J. Biol. Chem. 2004; 279: 42516-42527Crossref PubMed Scopus (24) Google Scholar were p.Asp850Tyr and an in-frame deletion and insertion spanning this region (Figure 3C). These data suggest that variants found in cerebral aneurysms act via gain-of-function mechanisms. Deep, targeted sequencing of the genes coding for the kinases KRAS, PDGFRA, BRAF, TGFBR1, and TGFBR2 identified no variants in the cohort of 50 aneurysms, consistent with an etiology specific to PDGFRB. Several heterozygous germline or mosaic gain-of-function variants in PDGFRB result in infantile myofibromatosis (IM; MIM: 228550) or sporadic myofibromas, and single, heterozygous germline variants were found in the rare Kosaki overgrowth (MIM: 616592) and Penttinen syndromes (MIM: 601812) (Figures 3 and Table S2). Several other heterozygous, germline loss-of-function variants cause primary familial brain calcification (PFBC; MIM: 615007). None of the above phenotypes were found in our four aneurysm-affected individuals (Table S2). Genome copy-number alterations, including chromosome 5 deletions encompassing PDGFRB, have been associated with developmental delay but not with aneurysm development in affected individuals.26Coe B.P. Witherspoon K. Rosenfeld J.A. van Bon B.W. Vulto-van Silfhout A.T. Bosco P. Friend K.L. Baker C. Buono S. Vissers L.E. et al.Refining analyses of copy number variation identifies specific genes associated with developmental delay.Nat. Genet. 2014; 46: 1063-1071Crossref PubMed Scopus (391) Google Scholar Mice deficient in Pdgfb or Pdgfrb die from multiple developmental defects including hemorrhages due to a lack of pericytes and vascular smooth muscle cells in blood vessels.17Lindahl P. Johansson B.R. Levéen P. Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.Science. 1997; 277: 242-245Crossref PubMed Scopus (1743) Google Scholar, 18Boucher P. Gotthardt M. Li W.-P. Anderson R.G. Herz J. LRP: Role in vascular wall integrity and protection from atherosclerosis.Science. 2003; 300: 329-332Crossref PubMed Scopus (467) Google Scholar, 19Daneman R. Zhou L. Kebede A.A. Barres B.A. Pericytes are required for blood-brain barrier integrity during embryogenesis.Nature. 2010; 468: 562-566Crossref PubMed Scopus (1363) Google Scholar, 20He C. Medley S.C. Hu T. Hinsdale M.E. Lupu F. Virmani R. Olson L.E. PDGFRβ signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis.Nat. Commun. 2015; 6: 7770Crossref PubMed Scopus (81) Google Scholar Mutation that activate Pdgfrb in mice cause vascular smooth muscle cell de-differentiation, hyperplasia, and increased extracellular-matrix synthesis.20He C. Medley S.C. Hu T. Hinsdale M.E. Lupu F. Virmani R. Olson L.E. PDGFRβ signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis.Nat. Commun. 2015; 6: 7770Crossref PubMed Scopus (81) Google Scholar The wide range of phenotypes suggests a complexity of PDGFRB function and downstream signaling that is likely a result of cell lineage and developmental-timing-specific expression. To study the functional status of PDGFRB variants, we performed assays by using cells collected from skin punches of the index individual (healthy and affected regions), and we performed site-directed mutagenesis by using the QuickChange II Site-Directed Mutagenesis Kit (Catalog #200518, Agilent Technologies). For details, see the Supplemental Data. Ligand binding induces PDGFRB dimerization and activates autophosphorylation in trans. Phosphorylation on multiple tyrosine residues creates docking sites for signaling proteins, including phosphatidylinositol-3 kinase (PI3K), AKT, STAT transcription factors, and phospholipase Cγ (PLCγ).27Irusta P.M. Luo Y. Bakht O. Lai C.C. Smith S.O. DiMaio D. Definition of an inhibitory juxtamembrane WW-like domain in the platelet-derived growth factor β receptor.J. Biol. Chem. 2002; 277: 38627-38634Crossref PubMed Scopus (48) Google Scholar, 28Looman C. Sun T. Yu Y. Zieba A. Ahgren A. Feinstein R. Forsberg H. Hellberg C. Heldin C.H. 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