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Numerous molecules, including the vascular endothelial growthfactor (VEGF), have been implicated in the assembly of a primi-tive vascular network1. It is unclear, however, which moleculescontrol the subsequent remodeling into a functional vasculature.The pharyngeal arch arteries undergo complex remodeling intothe adult circuit of large thoracic vessels. Life-threatening defectsof pharyngeal arch artery patterning can exist in isolation, butoften occur in combination with craniofacial, thymic andparathyroid birth defects as part of the DiGeorge/velo-cardio-facial syndrome (DGS/VCFS), one of the most common congeni-tal syndromes, affecting 1 in 4,000 infants2–6. In 8–28% of cases,the disease is inherited7, but the prevalence of familial DGS/VCFSis increasing due to improved treatment, often enabling patients

to survive until reproductive age8. Consequently, there is a grow-ing need for genetic counseling and prenatal screening7.

Most DGS/VCFS patients have an approximately 3 millionbase pair chromosomal deletion of 22q11 (del22q11). Becausethe deletion is hemizygous, haploinsufficiency of one or moregenes on 22q11 has been suggested to be responsible for thissyndrome2,3. Studies of mouse genetics have identified the tran-scription factor gene Tbx1 as a candidate for the main clinicalfindings of the disorder2,9–11. The basis for the disorder in humansremains questionable, as an isolated mutation of TBX1 has notbeen documented in the rare DGS/VCFS patients in whom ahemizygous deletion of 22q11 could not be detected12. Similarlyperplexing is the extreme phenotypic variability among

VEGF: A modifier of the del22q11 (DiGeorge) syndrome?

INGEBORG STALMANS1, DIETHER LAMBRECHTS1, FREDERIK DE SMET1, SANDRA JANSEN1, JIAN WANG5, SUNIT MAITY1,6, PAIGE KNEER5, MAREN VON DER OHE6, ANN SWILLEN2,

CHRISTA MAES3, MARC GEWILLIG4, DANIEL G.M. MOLIN7, PETER HELLINGS1, THURID BOETEL6,MAARTIN HAARDT6, VEERLE COMPERNOLLE1, MIEKE DEWERCHIN1, STEPHANE PLAISANCE1,

ROBERT VLIETINCK2,8, BEVERLY EMANUEL9, ADRIANA C. GITTENBERGER-DE GROOT7, PETER SCAMBLER10, BERNICE MORROW11, DEBORAH A. DRISCOL9,

LIEVE MOONS1, CAMILA V. ESGUERRA1,6, GEERT CARMELIET3, ANNETT BEHN-KRAPPA6, KOEN DEVRIENDT2, DÉSIRÉ COLLEN1, SIMON J. CONWAY5 & PETER CARMELIET1

1The Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, and2The Center for Human Genetics, 3Laboratory of Experimental Medicine and Endocrinology and

4Department of Pediatrics, Katholieke Universiteit Leuven, Leuven, Belgium5Institute of Molecular Medicine & Genetics, Medical College of Georgia, Augusta, Georgia, USA

6Mermaid Pharmaceuticals GmbH, Falkenried 88 (CiM), Hamburg, Germany7Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands

8Department of Population Genetics, Genomics & Bioinformatics, University Maastricht, Maastricht, The Netherlands

9Division of Reproductive Genetics, Department of Obstetrics & Gynecology, Division of Human Genetics & Molecular Biology, Department of Pediatrics,

University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA10Molecular Medicine Unit, Institute of Child Health, London, UK

11Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York, USAI.S. and D.L. contributed equally to this study.

Correspondence should be addressed to S.J.C. and P.C.; email: sconway@mail.mcg.edu orpeter.carmeliet@med.kuleuven.ac.be

Published online 21 January 2003; doi:10.1038/nm819

Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacialand life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome islikely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involve-ment of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birthdefects in mice, reminiscent of those found in del22q11 patients. The close correlation of birthand vascular defects indicated that vascular dysgenesis may pathogenetically contribute to thebirth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf 164-deficient em-bryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced bytbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplo-type was associated with an increased risk for cardiovascular birth defects in del22q11 individu-als. These genetic data in mouse, fish and human indicate that VEGF is a modifier ofcardiovascular birth defects in the del22q11 syndrome.

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del22q11 patients. Haploinsufficiency or polymorphisms of ad-ditional genes in the 22q11 region or polymorphisms in genesthat might act in the genetic pathway of TBX1 may be responsi-ble for the variable expressivity of the disorder, but few candi-dates have been identified thus far13. We show here that VEGF isa candidate modifier of the cardiovascular syndrome phenotype.

Mice expressing a single Vegf isoformAt least three Vegf isoforms exist (Vegf120, Vegf164 and Vegf188; thenumber of residues is given in superscript). They differ in bind-ing heparin-rich matrix components and receptors; although allisoforms bind Flk1 (fetal liver kinase-1 or VEGF receptor-2) andFlt1 (fms-like tyrosine kinase-1 or VEGF receptor-1), only Vegf164

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o pFig. 1 Aortic arch and conotruncal defects in Vegf120/120 and Vegf188/188 em-bryos and neonates. The left side of the mouse is on the right side of the pic-ture in all panels, except in k and l. a–j, Micrographs of the thoracic vessels(above) are complemented with a scheme (below). a and b, Normal aorticarch configuration in Vegf +/+ newborn. c and d, Interruption of the aortic archin E14.5 Vegf 120/120 embryo, caused by abnormal regression of the left 4th archartery. The blood flows from the heart into the carotid artery, and via an ab-normally persisting carotid duct to the descending left aorta. e and f, Vegf 188/188

neonate (oblique view): the blood flows through the right-bending aortic archand persistent right dorsal aorta into the left-sided descending aorta (the 4tharch artery is interrupted). The left subclavian artery and the ductus connect tothe left dorsal aorta. A vascular ring around the trachea and esophagus (thelatter is removed for clarity) is formed by the right-bending aorta, the rightdorsal aorta, the left dorsal aorta, the ductus arteriosus and the pulmonarytrunk. g and h, E14.5 Vegf 120/120 embryo, showing a ‘duplication’ of the aorticarch segment between the left carotid and subclavian artery, resulting from apersistent left carotid duct. The ductus arteriosus has been partially removedduring preparation (g), but is in place in (h). i and j, Vegf 188/188 neonate: dou-ble-sided aortic arch with a right-sided ductus arteriosus, caused by abnormalpersistence of the right dorsal aorta, abnormal regression of the left 6th archartery and persistence of the right 6th arch artery. k and l, Ink injection in E10.5embryos to visualize the pharyngeal arch arteries (right lateral view). In aVegf +/+ embryo (k), the 3rd, 4th and 6th pharyngeal arch arteries (indicated by 3,4, 6) and dorsal aorta (DA) have a normal size and shape, whereas in theVegf 188/188 embryo (l), the right dorsal aorta is enlarged and dominant. In addi-tion, the right 4th arch artery, which normally persists in wild-type embryos, is

abnormally locally narrowed (arrowhead) in this embryo. The caudal part ofthe left dorsal aorta regressed (not shown). A, aorta; AS, aortic sac; B, brachio-cephalic trunk; C, carotid artery; CD, carotid duct; D, ductus arteriosus; DA,dorsal aorta; P, pulmonary trunk; S, subclavian artery. Colors in the schematicoverview correspond to their embryonic origin: gray, aortic sac; turquoise, 3rd

arch artery; purple, 4th arch artery; pink, dorsal aorta β-segment; dark pink,dorsal aorta α-segment; olive green, 7th intersegmental artery, subclavianartery; light green, 6th arch artery, ductus arteriosus; brown, descending aorta;light blue, pulmonary arteries; orange, dorsal aorta γ-segment, carotid duct.m–p, LacZ staining of E10.5 Vegf +/+ (m, o) and Vegf 188/188 (n, p) embryos har-boring a TieLacZ transgene. The left and right 4th and 6th arch arteries (indi-cated by ‘4’ and ‘6’) and dorsal aortae have a comparable size in Vegf +/+

embryos, whereas in Vegf 188/188 embryos, the left 4th and 6th arch arteries andthe left dorsal aorta are smaller; the right dorsal aorta is enlarged (which wouldlead to a right-sided aortic arch and ductus). Scale bar, 100 µm in m–p.

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binds neuropilin-1 (Nrp1)14. To study their role, we previouslygenerated knock-in mice, expressing a single isoform15,16. Miceexpressing only the Vegf 164 variant (Vegf 164/164) were normal15. Incontrast, half of Vegf 188/188 embryos died between embryonic day(E) 9.5 and E13.5, whereas the remaining mice survived for pro-longed periods after birth15. Half of Vegf 120/120 mice died in theperinatal period due to congenital birth defects (group A),whereas the remaining mice succumbed within 2 weeks afterbirth, in part due to myocardial ischemia (group B)16.

Vascular malformations in Vegf 120/120 and Vegf 188/188 miceVegf120/120 neonates of group A became cyanotic immediatelyafter birth. To determine whether they had cardiovascular birthdefects, we injected a dye to visualize the large thoracic arteries.Compared with wild-type, Vegf +/120 or Vegf 164/164 neonates, severeDGS-like vessel malformations were detected in approximately50% of Vegf 120/120 and Vegf 188/188 neonates (n = 26–56; P < 0.05).Similar aortic arch malformations were detected in Vegf 120/120 andVegf 188/188 embryos, with the former suffering additionally fatalcardiac defects. E14.5 embryos and neonates had remodeling de-

fects of the 4th, 6th and, less frequently, 3rd pharyngeal arch arter-ies (Supplementary Table 1 and Fig. 1 online). Malformations ofthe 4th arch artery included a type-B left 4th aortic arch interrup-tion (Fig. 1c–f), a persistence of the cartoid duct segment be-tween the left carotid and subclavian artery (Fig. 1c,d and g,h), adouble aortic arch (Fig. 1i and j), a right 4th arch interruption to-gether with an aberrant subclavian artery (resulting in a retro-esophageal right subclavian artery, aberrantly originating fromthe descending aorta ‘arteria lusoria’) and a right-sided aorticarch and descending aorta (Supplementary Fig. 1 online). Thetype-B interruption of the aortic arch could be associated withpersistence of the ductus caroticus (resulting in a ‘cervical aorticarch’; Fig. 1c and d) or persistence of the right distal dorsal aortaleading, in combination with a left ductus arteriosus, to a vascu-lar ‘ring’ (Fig. 1e and f). Evidence of abnormal development ofthe 6th arch artery was highlighted by hypoplasia of the pul-monary trunk (Fig. 2i) or malformations of the ductus arteriosusleading to an absent or right-sided (Fig. 1i and j) ductus.Malformations of the 3rd arch artery included a double left orright carotid artery. Aortico-pulmonary collateral arteries were

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Fig. 2 Cardiac outflow defects in Vegf 120/120 mice. The left side of themouse is on the right side of the picture in all panels. a–c, Hematoxylin andeosin (H&E) staining, revealing a normal configuration of the aorta (A; a)and pulmonary trunk (P; b) in Vegf +/+ neonates. Panel c displays a schematicillustration. d–f, Vegf 120/120 neonate: Tetralogy of Fallot characterized byventricular septal defect (VSD; d), overriding aorta (A; d) and hypoplasticpulmonary trunk (P; e). Note the characteristic subvalvular stenosis of theright ventricle (arrowheads). The arrow in e indicates the pulmonary valve.Panel f schematically illustrates the cardiac defect. g and h, Vegf 120/120 new-

born: persistent truncus arteriosus (PTA; g). Panel h shows a schematic illus-tration. i, Vegf 120/120 newborn: isolated hypoplasia of the pulmonary trunk(P); the aorta (A) is normal. j and k, Thrombomodulin staining, revealingthat the vascular density in the interventricular septum is normal in a Vegf +/+

newborn (j), but significantly diminished in a sick Vegf 120/120 newborn thatdies at birth due to cardiovascular malformations (group A; k). Note alsothe increasing size and irregularity of the vessels. LV, left ventricle; LA, leftatrium; RV, right ventricle; RA, right atrium. The mice in d, e, i were injectedwith ink. Scale bar, 200 µm in a, b, d, e, g, i; and 25 µm in j, k.

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also observed. Malformations of the 4th arch artery were also ob-served in group B Vegf 120/120 mice.

To assess when the pharyngeal arch vascular malformationsoccurred, Vegf-variant mice were intercrossed with mice express-ing β-galactosidase under the control of the endothelial-specificTie1-promoter (Tie1LacZ mice). Initial formation of the embryonicvasculature was indistinguishable in all genotypes at E8.5.However, in 19% of E9.5 Vegf 188/188 embryos (n = 21), the firstthree pharyngeal arches were hypoplastic and the arch arteriesappeared as primitive capillary networks, often with an irregularsmall lumen and signs of regression—the vascular defects beingso severe that they caused circulatory collapse. In the remainingfraction of Vegf 188/188 embryos and in all other genotypes, the

pharyngeal arch arteries developed normally byE9.5. While the 3rd, 4th and 6th pharyngeal arch ar-teries were present by E10–10.5 in Vegf 120/120 andVegf 188/188 embryos, subsequent malformationsincluded hypoplasia, an irregular lumen size andsigns of regression in vessels that should nor-mally persist (Fig. 1k, l and m–p). A dominantright dorsal aorta was observed in approximately10% and 20% of Vegf 120/120 and Vegf 188/188 em-bryos, respectively (Fig. 1m–p). Thus, Vegf iso-forms are redundant for the initial formation ofarch arteries, but have distinct functions for theirsubsequent patterning.

Cardiac defects in Vegf 120/120 and Vegf 188/188 miceThe initial cardiogenesis at E9.0 was indistin-guishable in all genotypes. However, subsequent

formation of the interventricular septum and septation of thecardiac outflow tract into the aorta and pulmonary trunk wereabnormal in Vegf 120/120 and Vegf 188/188 embryos at E13.5–14.5. As aresult, a wide spectrum of congenital cardiac outflow tractanomalies, characteristically observed in DGS/VCFS patients2–6,was observed in Vegf 120/120 neonates of group A: Tetralogy ofFallot (Fig. 2d–f), persistent truncus arteriosus (Fig. 2g and h), hy-poplasia of the pulmonary trunk (Fig. 2i) or isolated ventricularseptum defect (VSD; data not shown). Such cardiac defects werenever observed in Vegf +/+ neonates (Fig. 2a–c) or in group BVegf 120/120 mice, which explains why they escaped the neonatallethality. Similar cardiac defects were observed in Vegf 188/188 em-bryos with circulatory collapse at E13.5, whereas Vegf 188/188 em-

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Fig. 3 Craniofacial, thymic and parathyroid defects inVegf 120/120 neonates. a–d, Alizarin red and Alcian bluestaining of bone and cartilage, revealing a normally fusedpalate in a Vegf +/+ neonate (a) and a cleft palate (indi-cated by white dashed line in b) in a Vegf 120/120 neonate.Compared with a Vegf +/+ neonate (c), the mandible is dis-proportionally smaller in a Vegf 120/120 neonate (d). The lineindicates the naso-otic distance, used for the measure-ments. Note also the aplasia of the incisors in a Vegf 120/120

neonate (arrowhead in d). e and f, CD31 staining, reveal-ing the lower vascular density in the trabecular region ofthe mandible in a Vegf 120/120 neonate (f) than in a Vegf +/+

neonate (e). g, Compared with a Vegf +/+ neonate (left),the thymus is hypoplastic in a Vegf 120/120 neonate (right).h, H&E staining of a section through the neck of aVegf 120/120 neonate, revealing a normally positioned thy-roid (TH) but an ectopic thymus (T). Note also that theparathyroid (PTH) is not normally associated with thethyroid, but ectopically with the thymus. Distances 1 and2, used for measurement of the ratio, are indicated. i andj, H&E staining of a section through the neck of a Vegf +/+

(i) and a Vegf 120/120 (j) neonate. In the Vegf +/+ mouse, theparathyroid is normally associated with the thyroidglands, which lay symmetrically around the trachea (TR).In the Vegf 120/120 mouse, an ectopically located thymus,lateral to the thyroid glands, compresses the trachea.Note also the complete absence of parathyroid glands.The border of the thyroid gland is indicated by a dashedline. E, esophagus. k and l, Immunostaining for thrombo-modulin, revealing numerous small capillaries in aVegf +/+

thymus (k) and a reduced vessel density in a Vegf 120/120

thymus (l). Note also the presence of abnormally dilatedvessels in the Vegf 120/120 thymus (l). Scale bar, 25 µm in e,f, k, l; 100 µm in h; and 200 µm in i, j.

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bryos that survived mid-gestation had less severe defects (smallVSD or subtle defects in cardiac outflow tract alignment).

Craniofacial and thymic anomalies in Vegf 164-deficient miceVegf 120/120 neonates also had the typical DGS-like craniofacial,thymic and parathyroid anomalies2–6: 43% lacked incisors and74% had a cleft palate (n = 19; P < 0.05; Fig. 3a–d). Cranial su-tures between their frontal and parietal bones failed to closeproperly and their jaws were shorter (mandibular to naso-oticlength ratio: 0.58 ± 0.01 in Vegf +/+ neonates versus 0.50 ± 0.06 inVegf 120/120 mice; n = 5; P < 0.05). No defects were detected in theother genotypes. However, the first three pharyngeal arches,which give rise to the craniofacial structures, were hypoplastic inthe severely affected Vegf 188/188 E9.5 embryos. In 5 of 12 Vegf 120/120

mice, the thymus was either hypoplastic (Fig. 3g), absent, or ec-topically located in the neck region (Fig. 3h and j). Thymic de-velopment was normal in other genotypes (Fig. 3i). The fractionof circulating lymphocytes was under-represented in Vegf 120/120

neonates: 59 ± 7% in four Vegf +/+ mice; 34 ± 6% in five Vegf 120/120

mice (P < 0.05); 47 ± 5% in four Vegf 164/164 mice; and 50 ± 5% infour Vegf188/188 mice (P = NS). Parathyroid glands were absent in 4of 12 Vegf 120/120 neonates (Fig. 3j) and, when present, they wereassociated with an ectopic thymic lobe (Fig. 3h). Parathyroidgland defects were not observed in the other genotypes (Fig. 3i).Plasma calcium levels at birth were comparable in all genotypes(see Supplementary Note online).

Expression of Vegf and Nrp1Hotspots of Vegf expression were detected at all sites, commonlyaffected in del22q11 individuals. For example, Vegf was highlyexpressed at E10 in the endoderm of the 4th pharyngeal arch,overlaying the developing 4th arch artery (Fig. 4a). At E10.5, a

Vegf hotspot in the endoderm between the 4th and the develop-ing 6th arch preceded remodeling of the 6th arch artery (Fig. 4b).Vegf was also highly expressed in the aortic sac, the cardiac out-flow tract, the ventricular septum, the maxillary and mandibularprominences, the future midline of the palate and the thymus. Asimilar expression pattern for Vegf was found in all genotypes,indicating that the three Vegf variants were co-expressed in theprecursors of the affected structures. The Vegf 164-specific Nrp1 re-ceptor was co-expressed with Vegf at all of these sites, althoughin different cell types. Nrp1 was expressed in endothelial cellslining the pharyngeal arch arteries (Fig. 4c) and, at a lower level,in pharyngeal endoderm, in periendothelial cells and in the sur-rounding neural crest-derived mesenchyme at E10.5 (Fig. 4c).Nrp1 was also expressed in the aortic sac (E10.5) and outflowtract (E12.5), in the mandibular and maxillary prominences(E10.5), in the palate, tooth bud, tongue and mandible (E13.5),and in the neural crest-derived capsule and perivascular tissue ofthe thymus (E13.5). Notably, Nrp1 expression was prominent inthe right 4th pharyngeal arch artery (which often persists inVegf 120/120 embryos), whereas it was undetectable in the left 4th

pharyngeal arch artery (which aberrantly regressed in these em-bryos; Fig. 4d), indicating that downregulated expression of Nrp1preceded vascular regression.

Birth defects correlate with vascular defectsBecause birth defects in Vegf 120/120 and Vegf 188/188 mice did notseem to be attributable to primary neural crest abnormalities(data not shown), we investigated whether they might be relatedto vascular defects. Compared with wild-type mice, the vasculardensity in the mandible was significantly reduced in Vegf 120/120

mice (vessels per mm2, 500 ± 27 in eight Vegf +/+ versus 310 ± 7 infive Vegf 120/120; P < 0.05; Fig. 3e and f). These vessels were abnor-

Table 1 Association of VEGF variations with cardiovascular defects in del22q11S

Genotypes del22q11S cases with Healthy controlsalleles CV defects

Number (frequency) Number (frequency) P value Crude odds ratio Logistic odds ratio P value(n = 58) (n = 316) (95% CI) (95% CI)

–2578C→ACC 12 (0.21) 97 (0.31)CA 28 (0.48) 157 (0.50)AA 18 (0.31) 62 (0.20) 0.096a 2.3 (1.1–5.2) 2.4 (1.1–5.4) 0.032c

C 52 (0.45) 351 (0.56)A 64 (0.55) 281 (0.44) 0.033b 1.5 (1.0–2.3)

–1154G→AGG 16 (0.28) 152 (0.48)AG 32 (0.55) 132 (0.42)AA 10 (0.17) 32 (0.10) 0.012a 3.0 (1.2–7.1) 2.9 (1.2–7.1) 0.016c

G 64 (0.55) 436 (0.69)A 52 (0.45) 196 (0.31) 0.004b 1.8 (1.2–2.7)

–634G→CGG 30 (0.52) 147 (0.47) 0.40a 2.3 (0.7–8.0) 2.3 (0.7–8.0) 0.19c

GC 25 (0.43) 135 (0.43)CC 3 (0.05) 34 (0.11)

G 85 (0.73) 429 (0.68) 0.25b 1.3 (0.8–2.0)C 31 (0.27) 203 (0.32)

Distribution of genotypes and alleles (frequencies between brackets) for the indicated SNPs in del22q11S cases with cardiovascular defects and in healthy controls. P values werecalculated by comparing del22q11S cases with cardiovascular defects to the healthy controls using the c2 contingency test: a2 d.f.; b1 d.f. Odds ratios (95% confidence intervalsbetween brackets) were calculated relative to the –2578CC, –1154GG or –634CC genotypes or to the –2578C, –1154G or –634C allele. cP values of odds ratios, derived from lo-gistic regression, were calculated after adjustment for gender.

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mally dilated and tortuous and likely contributed to impairedtissue perfusion. In the thymus, the vessel density and total vas-cular volume were also reduced in Vegf 120/120 mice (vessels permm2 and total vascular area per thymic area, 590 ± 121 and 7.6 ±1.2% in Vegf +/+ versus 230 ± 45 and 4.0 ± 0.3% in Vegf 120/120, re-spectively; n = 5; P < 0.05; Fig. 3k and l). In addition, vasculardensities in the interventricular septum were lower in group AVegf 120/120 neonates with cardiac anomalies than in healthy wild-type neonates (vessels per mm2, 1,220 ± 86 in five Vegf +/+ miceversus 560 ± 62 in five Vegf 120/120 mice; P < 0.05; Fig. 2j and k), andtheir vessels were grossly abnormal and dilated (mean vesselarea, 100 ± 11 µm2 in five Vegf +/+ mice versus 165 ± 23 µm2 in fiveVegf 120/120 mice; P < 0.05; Fig. 2j and k). In contrast, capillary den-sity and size in the septum were less affected in group B Vegf 120/120

mice (900 ± 140 vessels per mm2; n = 3; P < 0.05 versus Vegf +/+

mice and 125 ± 21 µm2, P = NS) and were normal in survivingVegf 188/188 neonates15, which lacked cardiac outflow defects. Thus,vascular defects in Vegf 120/120 mice were present in all organs thatsubsequently developed birth defects. The correlation betweenthe severity of the vascular defects and the birth defects indicatesthat the two processes are linked.

Reduced expression of Tbx1 in Vegf 120/120 miceTo evaluate whether Vegf interacted with Tbx1 in pharyngealarch development, we quantified its expression in a microdis-sected region containing the arches in E10.5 Vegf 120/120 embryos.By RT-PCR, 340 ± 80 copies of Tbx1 mRNA per 100 copies of HprtmRNA were detected in wild-type embryos. Tbx1 transcript lev-els in Vegf 120/+ and Vegf 120/120 embryos were only 77 ± 15% and 34± 9% of those in wild-type embryos, respectively (n = 10; P = 0.06and P < 0.0001). Tbx1 transcript levels in Vegf 164/164 embryos were116 ± 9% of those found in wild-type embryos (n = 6; P = NS). Insitu hybridization analysis further confirmed that Tbx1 expres-

sion was reduced in the pharyngeal arches in Vegf 120/120, but notin wild-type or Vegf 164/164, embryos (Fig. 4e and f).

Association of VEGF variations with del22q11 patientsEven though the gene VEGF does not map to the del22q11 re-gion, the mouse phenotypic findings raised the question ofwhether VEGF might act in the genetic pathway of a critical genethat is hemizygously deleted in del22q11 patients. No VEGF se-quence variations were detected in the exons or intron/exonboundaries in 40 non-deleted subjects with DGS/VCFS. We there-fore examined whether variations of the –2578C→A, –1154G→Aand –634G→C single-nucleotide polymorphisms (SNPs) in thepromoter or 5′ untranslated region (5′-UTR) of VEGF conferred anincreased risk for specific birth defects, as these SNPs were previ-ously documented to downregulate VEGF expression17,18. Wegenotyped a Flemish population of 91 del22q11 cases (47male/44 female) and 316 unrelated healthy controls with compa-rable distribution of gender, geography and ethnicity (165male/151 female). None of the polymorphisms in the controlpopulation deviated from the Hardy-Weinberg equilibrium.When comparing controls with all cases, we found no association(Supplementary Table 2 online). However, genotype and allelefrequencies of the –1154G→A polymorphism differed signifi-cantly between del22q11 cases with cardiovascular (CV) defectsand healthy controls (χ2 = 8.81, P = 0.012, 2 degrees of freedom(d.f.) for genotypes and χ2 = 8.44, P = 0.004, 1 d.f. for alleles; Table 1). The risk for CV defects associated with the homozygousA/A genotype increased 3.0-fold (95% CI = 1.2–7.1). Analysis bylogistic regression to adjust for gender yielded similar results(Table 1). When studying the –2578C→A polymorphism, we alsofound a significant association of the variant A allele (χ2 = 4.52, P= 0.033; Table 1) and a 2.4-fold increased risk associated with theA/A genotype after correction for gender by logistic regression

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Fig. 4 Expression of Vegf, Nrp1 and Tbx1 ana-lyzed by in situ hybridization. The left side of themouse is on the right side of the picture in allpanels. The 4th and 6th pharyngeal arch arteriesare indicated by ‘4’ and ‘6’. All panels displayVegf +/+ embryos, except the Vegf 120/120 embryo ind and f. a and b, Hotspot expression of Vegf inthe pharyngeal arch endoderm lining the 4th

arch (arrowheads in a) at E10, and in the pouchbetween the 4th and the developing 6th arch (ar-rowheads in b) at E10.5, overlaying the pharyn-geal arch arteries. Vegf expression isspatio-temporally correlated and coincidentwith the remodeling of the pharyngeal arch ar-teries. The 6th arch artery (yellow dashed line;‘6’) buds off from the dorsal aorta (DA; whitedashed line). CV, cardinal vein. c, Nrp1 isstrongly expressed in the endothelial cells liningthe 4th pharyngeal arch artery (arrowheads) andis expressed at a lower level in neural crest-de-rived periendothelial cells, arch core and endo-derm. d, E10.5 Vegf 120/120 embryo: Nrp1 isexpressed in the dorsal aorta (DA), aortic sac(AS) and in the right 4th arch artery, but down-regulated in the left 4th arch artery (arrow-heads). e and f, Expression of Tbx1 in the 4th

arch is higher in a wild-type (e) than a Vegf 120/120

(f) embryo at E10.5. Tbx1 was expressed in thepharyngeal endoderm (F) and in the head mes-enchyme surrounding the dorsal aorta.

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(95% CI = 1.1–5.4; P = 0.032; Table 1). The –634G allele alsotended to be more common in del22q11 cases with CV defects(0.73 versus 0.68 in controls; Table 1).

We also determined haplotypes, because the susceptibility tocomplex diseases is often attributable to multiple polymor-phisms, whose collective effects are more readily predicted by de-termining the full haplotype information. Haplotypes of the–2578, –1154 and –634 loci were inferred by the maximum likeli-hood method and their frequencies compared between cases andcontrols (Supplementary Table 3 online). When analyzing haplo-types at two loci, the –2578A/–1154A and –1154A/–634G haplo-types were more common in del22q11 cases with CV defects thanin controls (0.45 versus 0.29, P = 0.003 for –2578A/–1154A and0.45 versus 0.31; P = 0.025 for –1154A/–634G). Haplotype analysisincluding all three loci revealed that the –2578A/–1154A/–634Ghaplotype was more prevalent in del22q11 cases with CV defectsthan in controls (0.45 in cases versus 0.29 in controls, P = 0.008),identifying this haplotype as the ‘at-risk’ haplotype. These obser-vations thus revealed a critical role for the –1154A allele in deter-mining the susceptibility for CV defects in del22q11 patients, butdo not exclude the possibility that additional SNPs may also beassociated. Calculation of the population-attributable risk re-vealed that up to 14% of cases in the study population would not

have occurred, had the ‘at-risk’ –2578A/–1154A/–634G haplotypenot been present.

To examine whether the association was not attributable topopulation stratification (type I error), we compared the –1154Aallele and –2578A/–1154A/–634G haplotype distributions inFlemish del22q11 subjects with CV defects (n = 58) to thosewithout CV defects (n = 33). Even despite the small number ofavailable del22q11 subjects without CV defects, we still found asignificant association of the ‘at-risk’ –1154A allele with CV de-fects. Indeed, of the 33 del22q11 individuals without CV defects,58% were G/G, 30% G/A and 12% A/A, compared with 28%G/G, 55% G/A and 17% A/A in the 58 del22q11 subjects with CVdefects (χ2 = 8.1, P = 0.017, 2 d.f.). Both the variant –1154A alleleand the –2578A/–1154A/–634G haplotype were more prevalentin del22q11 subjects with than without CV defects: 0.45 in casesversus 0.27 in controls for both the A allele (χ2 = 5.48, P = 0.019,1 d.f.) and haplotype (P = 0.037). These results indicate that ourobservations are not an artifact of admixture or unrecognizedsubstructure in the Flemish (control) population.

vegf modifies del22q11 syndrome in zebrafishWe used the zebrafish model to obtain additional evidence thatvegf and tbx1 genetically interacted and to provide functional

a b c d e

g h i

j k l

Fig. 5 Knockdown of zebrafish vegf aggravates aortic arch artery (AAA)patterning defects in tbx1 knockdown zebrafish. a and b, Whole mount insitu hybridization of zebrafish tbx1, revealing prominent expression in thepharyngeal arches at 28 (a) and 48 (b) h post fertilization (hpf; anterior tothe left, dorsal view). c and d, Whole mount in situ hybridization for ze-brafish tie1, revealing a normal pattern of the AAAs in zebrafish embryos (3 dpf) after injection of buffer. e, Diagram, schematically representing thethree-dimensional organization of the AAA system (fromhttp://mgchd1.nichd.nih.gov:8000/zfatlas). f, Incidence of the AAA pat-terning defects, determined by calculating the percentage of embryos af-fected with AAA malformations (average of at least three experiments) afterinjection of the indicated concentration (µM) of zebrafish vegf or tbx1 mor-

pholino oligomers. g–l, All panels display an anterior to the left, lateral viewexcept k, which shows an anterior to the right, lateral view. Injection ofmorpholinos against zebrafish vegf (80 µM; g) or tbx1 (200 µM; h) did notaffect AAA patterning, whereas injection of tbx1 morpholino (300 µM; i)disturbed AAA patterning, most often resulting in the disappearance of the6th AAA. When co-injected, vegf (80 µM) plus tbx1 (200 µM) morpholinosseverely impaired AAA patterning and, often, only one or two irregular, dis-organized and discontinuous AAAs persisted (j). Often, the AAA defectswere more severe at either the right (k) or left (l) side of the embryo whenco-injected with vegf (80 µM) plus tbx1 (200 µM) morpholinos. The preciseAAA-type could not be unambiguously identified in the latter group. Panelsj and k, l are from two different embryos.

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confirmation that reduced vegf expression increases the risk ofpharyngeal arch vessel malformations. The zebrafish ortho-logues of tbx1 (Fig. 5a and b) and vegf (ref. 19 and data notshown) were expressed in the pharyngeal arches after 1 day postfertilization (dpf). As the 3rd to 6th aortic arch artery (AAA) de-velop only by 2.5 dpf, their patterning was analyzed at 3 dpf bywhole mount Tie-1 staining (Fig. 5c and d). The 3rd and 4th AAAarise as direct branches from the ventral aorta, whereas the 5th

and 6th AAA have a common trunk from the ventral aorta anddrain to the midline dorsal aorta by means of an independentroute (Fig. 5e). Because the phenotype in knockdown zebrafishembryos is dependent on the dose of the morpholino oligomerinjected, we counted the fraction of embryos with AAA defects(Fig. 5f). The AAAs developed normally in all embryos after in-jection with buffer or a solution containing 60, 80, 100 or 120µM zebrafish vegf morpholino (Fig. 5f and g). Microinjection ofzebrafish tbx1 morpholinos did not affect aortic arch artery de-velopment at 50, 100, 150 or 200 µM (Fig. 5f and h), but, at 300µM, the 6th pharyngeal arch artery and the common trunk(draining the 6th AAA to the midline dorsal aorta) were irregular,discontinuous or even absent in approximately 90% of 3 dpf em-bryos (Fig. 5f and i). Similar AAA defects have been documentedin the van Gogh (vgo) zebrafish20, a mutant that lacks functionalzebrafish tbx1 (T. Piotrowski, pers. comm.). To analyze whetherzebrafish vegf and tbx1 genetically interacted, vegf and tbx1 mor-pholinos were co-injected. A low concentration of each mor-pholino was chosen, which, when injected separately, onlyminimally affected AAA development (60, 80 or 100 µM for ze-brafish vegf morpholino; 200 µM for zebrafish tbx1 morpholino).After co-injection of zebrafish vegf (60 µM) and tbx1 (200 µM)morpholinos, AAA development was normal in all embryos (Fig.5f). Co-injection of zebrafish tbx1 morpholino (200 µM) with in-creasing zebrafish vegf morpholino (80–100 µM) dose-depen-dently impaired AAA development, resulting in more frequentand severe defects. For example, at 200 µM zebrafish tbx1 plus 80or 100 µM zebrafish vegf morpholinos, the AAAs were irregular,thin, discontinuous and disorganized or even absent in 30 ± 3%and 38 ± 1% of embryos, respectively (Fig. 5f and j). Often, vas-cular defects were more severe on either the right or left side,only one or two AAAs were detectable and the vessel drainingthe caudal AAAs was absent (Fig. 5k and l). Thus, reduced vegflevels increased the risk for AAA and pharyngeal cartilage (datanot shown) defects in a tbx1-knockdown fish model.

DiscussionVEGF is well established to play a critical role in angiogenesis,but has not been previously implicated in birth defects. The fol-lowing evidence suggests that VEGF modifies the expressivity ofthe del22q11 syndrome, in particular the cardiovascular anom-alies: (i) the birth defects of Vegf 120/120 and Vegf 188/188 mice arereminiscent of those seen in del22q11 patients2,3,5,6 and in micewith a disrupted syntenic region of human chromosome 22q11(refs. 9–11)—the cardiac birth defects in Vegf 120/120 mice beingeven more prominent than in previous del22q11 mouse mod-els9–11; (ii) expression of Vegf spatio-temporally correlates withaortic arch remodeling during pharyngeal development,whereas expression of Nrp1 is downregulated in vessels that ab-normally regress in Vegf 120/120 mice; (iii) like tbx1 (ref. 2), the ef-fects of vegf are dependent on gene dosage, as exemplified bythe graded vascular defects after progressive vegf knockdown inzebrafish embryos and by the haploinsufficient vascular defectsin mice lacking a single Vegf allele21; (iv) Vegf regulates expres-

sion of Tbx1 in the pharyngeal apparatus at the time of its pat-terning; (v) knockdown of vegf enhances the penetrance of thepharyngeal arch artery and cartilage defects in a tbx1-knock-down zebrafish; and (vi) a VEGF haplotype, documented to re-duce VEGF expression (unpublished data and refs. 17,18), isassociated with an increased risk for cardiovascular defects indel22q11 individuals. Thus, the Vegf variant–specific mouse ge-netic data reveal that Vegf is capable of influencing pharyngealarch patterning, the zebrafish genetic data underscore that re-duced vegf levels predispose to aortic pharyngeal arch defects,and the human genetic data suggest that VEGF is involved in thehuman del22q11 syndrome. We therefore propose the followinginterpretation of the mouse, fish and human genetic data: Vegf ,and in particular Vegf 164, is critical for normal pharyngeal archdevelopment, and reduced or absent Vegf164 levels increase therisk of pharyngeal arch birth defects—this is true for mice andfish and, by extrapolation, therefore is also likely applicable tohumans. Any allelic VEGF promoter variation, lowering the ex-pression of VEGF, will also lower the expression of the VEGF165

(the human ortholog of the mouse Vegf164 isoform) variant.Thus, if VEGF165 levels in human del22q11 embryos fall below acritical threshold required for proper pharyngeal arch develop-ment, the risk of birth defects may increase. The zebrafishknockdown experiments support a model whereby vegf thresh-old levels are critical for tbx1-dependent pharyngeal arch devel-opment. This model presumes, however, that del22q11 embryoscarrying an at-risk haplotype have lower VEGF levels—a pre-sumption that cannot be easily tested in the early human fetusand therefore remains speculative.

Our data imply a previously unanticipated role for Vegf inpharyngeal arch development. Hotspots of Vegf expression weredetected in the endoderm just before the onset of remodeling ofthe arch arteries. Nrp1, on the other hand, was expressed in(peri-)endothelial cells of arch arteries, but also in the mes-enchyme around the arteries and in the endoderm. In addition,Vegfand Nrp1 were coordinatedly expressed at all other sites thatare commonly affected in del22q11 subjects, indicating thatNrp1 has a role in transmitting instructive Vegf 164 cues. A func-tion for Nrp1 is supported by the observation that absence ofNrp1 causes isolated aortic arch defects22 and that Nrp1 expres-sion in Vegf 120/120 embryos remained elevated in the persistingright 4th arch artery, while it became extinct in the left 4th archartery, which abnormally regressed in these embryos. For a Vegfisoform to provide instructive signals, it should reach the vessel,which is centrally located in the pharyngeal arch. Thus, the Vegfisoform should be diffusible, yet interact with guidance cues inthe extracellular matrix—the Vegf 164 isoform fits all these crite-ria. In contrast, the short Vegf 120 isoform may be too soluble andtherefore diffuse in a chaotic manner or overshoot its vasculartarget, whereas the matrix-bound Vegf 188 isoform may not be re-leased and fails to reach the vessel (Supplementary Fig. 2 online).Localized upregulation of Vegf 164 may provide similar critical sig-nals for nearby Nrp1-expressing neural crest cells or cells derivedfrom the neural crest in the heart, craniofacial skeleton, thymusand parathyroid glands.

The birth defects in Vegf 120/120 mice resemble those seen inTbx1-deficient mice2,9–11. In addition, Tbx1 expression was re-duced in Vegf 120/120 mice and knockdown studies in zebrafish in-dicated that reduced vegf levels aggravated the pharyngeal archdefects in tbx1 knockdown embryos. This suggests that Vegf andTbx1 genetically interact at some level and that some of the de-fects in Vegf 120/120 mice are, at least in part, attributable to re-

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duced Tbx1 levels in the pharyngeal endoderm. How Tbx1 isdownregulated in the absence of the Vegf 164 isoform remains tobe determined. Several arch cell types express Nrp1 and maytherefore respond to Vegf 164 by either directly upregulating Tbx1expression or releasing signals that indirectly affect Tbx1 expres-sion. We cannot exclude an interaction of Vegf/Nrp1 with otherdel22q11 disease candidates, nor do our findings lead to a con-clusion that the role of Vegf in the pharyngeal arch apparatus isrestricted to an induction of Tbx1 expression alone—as evi-denced by the fact that the birth defects in Vegf 120/120 mice resem-ble, but are not phenocopies of, those seen in Tbx1-deficientmice2,9–11.

Surgical ablation and gene-inactivation23,24 studies revealedthat migration and differentiation of neural crest into the pha-ryngeal arches are critical for the generation of a functional pha-ryngeal arch apparatus. However, we did not document a grossimpairment of neural crest migration, accumulation or differen-tiation in vitro or in vivo in Vegf 120/120 and Vegf 188/188 mice, al-though a more subtle functional defect or impairment cannot beexcluded. An alternative hypothesis advocates a primary vascu-lar defect of the pharyngeal arch arteries, whereby insufficientblood perfusion impairs development of the pharyngeal arch de-rivatives25. When vascular dysgenesis also occurs during subse-quent formation of the arch-derived structures, such persistentperfusion imbalance may even further increase the risk for birthdefects. The lower numbers of more dilated capillaries in thethymus, bone and interventricular septum in the heart in groupA Vegf 120/120 mice might indeed have caused such perfusion im-balance and contributed to the more severe pharyngeal arch de-fects in these mice. Capillary densities and birth defects in theseorgans were less pronounced or absent in surviving Vegf 188/188

mice and in group B Vegf 120/120 mice—the severity of the pharyn-geal arch defects thus seems to be related to the degree of vascu-lar impairment. Vascular anomalies have been documented inthe brain, eye, neck, chest, abdomen and extremities indel22q11 patients6,26,27, but also in Vegf 120/120 mice16,28,29. Vasculardysgenesis has been proposed to cause craniofacial anomalies,neuro-psychiatric symptoms, learning difficulties, and malfor-mations of the kidney, intestine, genitals, thyroid glands andfingers26. An imbalance in placental or fetal perfusion inmonozygotic del22q11 twins has also been proposed to explainwhy one of them often has an increased incidence of cardiac30 orother birth defects31,32. The involvement of a prototypic vasculargrowth factor such as VEGF in the morphogenesis of these struc-tures should stimulate future studies to assess whether, to whatextent and how vascular dysgenesis, together with other mecha-nisms, contributes to the del22q11 phenotype.

We did not identify VEGF as a candidate modifier gene of thehuman del22q11 syndrome from a random genome-wide scan,but tested the specific hypothesis, deduced from the mouse andfish genetic evidence, that VEGF might modify the cardiovascu-lar phenotype in del22q11 patients. Thus, the human genetic as-sociation somehow is similar to the mouse and fish genetics.Nonetheless, it is important to notice that the genetic evidencefor a role of VEGF in the human del22q11 syndrome was basedon the comparison of a relatively small population of del22q11cases with CV defects to healthy controls. As genetic associationstudies can be plagued by irreproducibility, in particular when asingle, small population is studied33,34, we replicated the associa-tion by comparing del22q11 cases with and without CV defects.Nevertheless, confirmatory replication studies in additional andlarger study populations, eventually complemented by family-

based association analysis, will be required to conclusively estab-lish that VEGF variations confer an increased risk for CV defectsin del22q11 patients. Performing a meta-analysis using addi-tional SNPs may, however, be a formidable challenge because ofthe limited availability of DNA samples with well-characterizedclinical information. Correlating plasma levels of VEGF withgenotypes may also underscore the association, but the compen-satory (hypoxic) upregulation of VEGF in DGS patients with CVdefects may confound such analysis. On a more general level,such studies will allow validation of the usefulness of a com-bined genetic approach in mice, fish and human to unravel thepathogenesis of human complex traits.

MethodsVegf isoform mice. The generation of Vegf isoform–specific mice andstaining methods, neural crest culture, RT-PCR and in situ hybridizationhave been described15,16,29,35,36. Intraventricular ink injections were per-formed using a manually pulled glass capillary. The embryos were fixedovernight in 4% paraformaldehyde, rinsed with PBS, dehydrated in increas-ing concentrations of ethanol and cleared in a mixture of 2:1 benzyl-alco-hol and benzoyl-benzoate. In situ hybridization probes were specific forNrp1 (from nt 358 to 761 in the murine Nrp1 cDNA), Crabp1 (ref. 35), Ap2(ref. 36), Tbx1 (gift from D. Srivastava)37 and Vegf (from nt 85 to 492 in themurine Vegf cDNA; GenBank acc. no. NM009505.1). Housing and proce-dures involving experimental animals were approved by the institutionalanimal care and research advisory committee of the KULeuven, Belgium.

Genetic association studies. Caucasoid individuals (n = 91) of Flemish ori-gin, referred to the Hospital of Leuven (Belgium) for del22q11S, were usedfor genotyping after obtaining informed consent and with approval of thelocal ethical review committee. Criteria for diagnosis of del22q11S includedthe identification of a hemizygous 22q11 chromosomal deletion in patientspresenting a variable combination of craniofacial defects (cleft palate orvelopharyngeal insufficiency and facial dysmorphism), cardiovascular de-fects (n = 58; Tetralogy of Fallot, persistent truncus arteriosus, pulmonarystenosis, membranous ventricular septum defect, abnormal origin of theright subclavian artery, major aortico-pulmonary collateral arteries; overrid-ing, right-bending, interrupted or coarcted aorta), developmental delay (IQdetermination according to the Diagnostic Statistical Manual-IV of theAmerican Psychiatric Association), thymic insufficiency (low lymphocytecount and impaired T-cell function) and/or parathyroid defects (low plasmaparathyroid hormone levels in combination with low baseline hypocal-cemia). The diagnosis of DGS/VCFS individuals without a 22q11 deletionwas based on the presence of two or more major symptoms (such as hy-poparathyroidism, hypoplastic thymus, characteristic cardiovascular de-fects) or 1 major and 2 minor symptoms (including facial dysmorphism,velopharyngeal insufficiency, mental retardation, cleft palate). The controlgroup consisted of 316 unrelated healthy controls of similar age, geogra-phy and ethnicity, randomly sampled in Flanders (median age, 44 years;range, 19–65 years by Kaplan Meier analysis). In addition, 40 non-deletedDGS/VCFS subjects of British origin were analyzed for variations in exonsand intron–exon boundaries of VEGF. DNA of patients and control subjectswas extracted from white blood cells from peripheral blood using theQIAamp Blood Kit (Qiagen, Westburg, Leusden, The Netherlands).

Denaturing high-performance liquid chromatography (dHPLC) on atransgenomic WAVE instrument (Transgenomic Inc., Crewe, UK) was usedto screen for exonic or intronic SNPs (primer sequences available on re-quest). Direct sequencing (Applied Biosystems, PE, Foster City, California)of PCR fragments with aberrant dHPLC patterns was used to confirm thepresence of a variant allele. SNPs in the promoter and 5′-UTR region weregenotyped by pyrosequencing (see Supplementary Methods online).

We tested for Hardy-Weinberg equilibrium using the χ2 contingency test(1 d.f.). For case-control samples, standard genotypic or allelic odds ratioswere calculated relative to the at-risk genotype or allele. P values for differ-ences in the distribution of allelic or genotypic frequencies between casesand controls were determined using a χ2 contingency test with 1 or 2 d.f.,respectively. The population attributable risk (PAR) for alleles or haplotypeswas estimated using the Levin formula: PAR = 100 × [proportion of total

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population exposed × (relative risk for exposed versus nonexposed – 1)] /[(proportion of total population exposed × (relative risk for exposed versusnonexposed – 1)) + 1]. Distributions of haplotype frequencies, estimated bythe maximum-likelihood method using the estimated haplotypes (EH) pro-gram38, were compared using CLUMP (ref. 39). CLUMP generates empiricalP values, based on Monte Carlo simulations by random simulations of10,000 2 × 3 (pairwise haplotypes) or 2 × 4 (three-locus haplotypes) tableswith the same marginal totals as the one under consideration, and bycounting the number of times that a χ2 value associated with the real tablewas achieved. To adjust the P values and odds ratios for gender when com-paring del22q11S patients with and without CV defects, a binary logistic re-gression was performed using the statistical software program SPSSv10.0.

Zebrafish experiments. The supplement (see Supplementary Note online)provides a more complete description of the methods, reporting the main-tenance of wild-type AB/TL zebrafish (Danio rerio) stocks40,41, the cloning ofthe zebrafish tbx1 cDNA, the microinjection of morpholino oligomers, in-cluding positive and negative controls42, whole mount in situ hybridiza-tion43 and the phenotypic analysis. For the latter, at least 30–40 embryoswere analyzed per experiment in 3 independent experiments for alterationsin morphology, rate of blood flow and development of the vasculature.Visualization of the vasculature was achieved by whole-mount in situ hy-bridization for tie1, flk1 and alkaline phosphatase, while the extracellularmatrix associated with the cartilage chondrocytes was stained with Alcianblue. As the phenotype varied according to the dose or combination ofmorpholino oligomer used, the penetrance of the phenotype was scored bycounting the embryos with distinct vascular or cartilage defects.

Supplementary information is available on the Nature Medicine website.

AcknowledgmentsWe thank K. Brepoels, A. Bouché, E. Gils, A. Claeys, B. Hermans, L. Kieckens,W. Man, A. Manderveld, K. Maris, W. Martens, M. Nijs, S. Meynen, T.Vancoetsem, A. Vandenhoeck, K. Vandevelde, C. Van Huylebroeck, P. VanWesemael, B. Vanwetswinkel, S. Wyns, C. Jonas and M. Trautmann forassistance; A. Kasran for performing FACS analyses; D. Anderson for providingthe p75 antiserum; K. Alitalo for providing the Tie1LacZ mice and Christine VanBroeckhoven and Jurgen Del-Favero. I.S. is a Research Assistant and K.D. is aSenior Clinical Investigator of the Fund for Scientific Research-Flanders (FWO,Belgium). D.L. is a Research Assistant of the IWT. This work is furthersupported by grants from the FWO (G012500), the “Research FundK.U.Leuven Belgium” (GOA/2001/09) and the European Union (BMH4-CT98-3380), by a Bristol-Myers-Squibb grant to P.C., by a grant fromInteruniversitary Attraction Poles (Belgian State) and the Belgian Foundationfor Research in Paediatric Cardiology to K.D., by a grant from the British HeartFoundation to P.S., and by NIH grants (HL60714 and HL60104) to S.J.C.

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 9 OCTOBER; ACCEPTED 20 DECEMBER 2002

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