Clinical Validity Scoring Notes and points | GnomAD pLOF - LOEUF score of 0.61, moderately constrained for LOF. Not constrained for missense. There are many different transcripts. GENETIC EVIDENCE (total 3.5 points): HGMD: NM_001025366.3 c.655G>T p.E219* (exon 2)- absent gnomAD. NMD+, PMID: 30232381 - reported as Glu39* in proband TOF241 with TOF, RAA, bicuspid pulmonic valve; short stature, obesity; learning difficulties; depression and/or anxiety; stillborn offspring. 1 POINT (COMMON) c.454C>T p.R152* (exon 1) - absent gnomAD, NMD +, PMID: 20420808 - in patient 2 with Left ventricular outflow tract obstruction BAV, AVS, CoA,PDA, ascending aorta dilation. Also inherited from unaffected family members, and they only sequenced VEGFA, so would decrease points for both. 0.5 POINT c.19_22dupGACA p.(Thr8Argfs*78 (exon 1) - variant in 0.04% (23/39188) East Asian chr gAD v4. PMID: 20420808 - Present in patient with coarctation of the aorta, VSD and PDA. High MAF, also inherited from unaffected family members, and they only sequenced VEGFA, will not score points for this variant. c.998G>A p.R333Q (exon 6) - 7/44788 East Asian chr. REVEL score is not in gAD. PMID: 20420808 - Found in one proband with coarctation of the aorta and vsd, but variant inherited from unaffected family members, and they only sequenced this gene. Deduct points. 0.5 POINT c.973C>T p.R325* (exon 6) - 0.002% (35/1156678) European alleles gnomAD. PMID: 22067973 - found in a patient with isolated tricuspid aortic valve stenosis, but inherited from unaffected parent, VEGFA only gene sequenced. Deduct points. 0.5 POINT Case/Control: Griffin 2009 PMID: 19308252 - 771 congenital cardiovascular malformation cases (included 595 with tetralogy of Fallot) and performed a case-control analysis investigating the common promoter variants previously reported. No significant effect of the three VEGF promoter variants was found. 93 TOF patients were tested for rare variants and one were found. Sample size was powered at 90% to find at least 1 case if 5% of TOF cases were caused by VEGF. Internal cases: E3740147955 - ENSP00000361125: p.His362IlefsTer39 aka NM_001025366.3(VEGFA):c.1079del p.gly360Alafs*41. Variant is located in a region that is not expressed in most transcripts, and has a really low pext score (0) in all tissues and in cardiac tissues in gnomad v2. Proband with eczema, laryngomalacia, patent foramen ovale, right aortic arch w/ mirror image branching, tricuspid regurgitation, vasuclar ring, double aortic artch and atelectasis. In addition, this variant has a somewhat high maf (0.07%) 67/74948 alleles in AFrican’African American with 3 HOM in gAD v4. Due to uncertain expression of this part of the protein and high maf, not scoring for now. E3730537808 - NM_001025366.3(VEGFA):c.735C>A p.(Tyr245*) (exon 3) - this variant is absent gnomad v2, expressed in all available transcripts. Patient phenotype - small for GA, abnormal heart morphology, atrial septal defect, patent ductus arteriosus, biscuspid aortic valve, total anomalous pulmonary venous return, abnormal morphology of the great vessels. PEXT score for this exon is good. Adding 1 point. EXPERIMENTAL EVIDENCE (total 2.6 points) Jin 2017 PMID: 29037991 - zebrafish vegfaa mutants. Vegfaa -/- embryos are embryonic lethal and display a complete loss of the dorsal aorta (DA) and expansion of the cardinal vein. Homozygous model, not replicating haploinsufficiency seen in humans, downgrade. 0.5pt animal model Ferrara 1006 PMID: 8602242 - Loss of a single VEGF allele is lethal in the mouse embryo betweens days 11 and 12. Angiogenesis and blood-island formation were impaired, resulting in several developmental anomalies. VEGF+/- embryonic stem cells were used to gemerate chimeric mice. Observed a defect in blood supply of the yolk sac in several embryos at E9.5-11.5 (Fig. 3a). The VEGF+I- embryos exhibited several anomalies23 (Fig. 3). In the cranial region, the branchial arches were poorly developed and unsegmented. The forelimb buds were positioned at their earlier caudal location and were unsegmented. Also, the forebrain region appeared significantly underdeveloped (Fig. 3b,g). In the heart region, the common atrium and primitive ventricle were developmentally delayed23, the dorsal aortae were rudimentary, and the thickness of the ventricular wall was markedly decreased (Fig. 3b, c,g, h). Embryonic lethal heterozygous in mice, not fully replicating human phenotype. Downgrade. 0.5 points animal model Stalmans 2003 PubMed: 12539040 - absence of the Vegf164 isoform caused birth defects in mice similar to those found in del22q11 patients. Mice have 3 isoforms that differ in # of residues (120, 164, and 188). Compared with wild-type, Vegf +/120 or Vegf 164/164 neonates, severe DGS-like vessel malformations were detected in approximately 50% of Vegf 120/120 and Vegf 188/188 neonates (n = 26–56; P < 0.05). Defects include cardiac outlfow defects, tetralogy of fallot, persistent truncus arteriosus, hypoplasia of the pulmonary trunk. Other congenital birth defects included cleft palate, hypoplastic thyrmus. Vascular density was significantly reduced in multiple tissues tested (mandible, thymus, interventricular septum) in Vegf120/120 mice. Expression of Tbx1 was reduced in Vegf120/120 mice. Knockdown of zebrafish vegf in tbx1 knockdown zebrafish resulted in higher % of aortic arch artery defects (fig 5). 0.5 points animal model; not scoring full points due to differences in the isoforms, particularly compared to human (MANE select ENST00000672860 is 395 residues). Rykiel 2021 PMID: 33620155 - increased VEGF applied to quail embryos shown to lead to impaired heart tube elongation accompanied by diameter expansion. VEGF treatment increased the rate of cardiac malformations in surviving embryos. Shows some potential impact of VEGF and congenital heart defects, but applying endogenous VEGF is not replicating the LOF model proposed in humans (based on the variants reported above). Increasing VEGF, not replicating the haploinsufficiency seen in humans, Downgraded and scored only 0.1 points, Non-human model organism 0.1 points Carmeliet 1996 PMID: 8602241 - Targeted inactivation of one or both VEGF allele in embryos that were generated by aggregation of embryonic stem cells with tetrapoid embryos. The dorsal aorta was poorly developed in VEGF+/- tetraploid embryonic stem cell dereived embryos. endothelial cells lined a much smaller lumen than normal, especially in the anterior part of the embryo (Fig. 2b, c). At 8.5 d.p.c. the dorsal aorta was missing over its entire length in VEGF-/- embryos. However, no VEGF+/- offspring were obtained (embryonic lethal). VEGF -/- were also embryonic lethal. Fewer endothelial cells lined the smaller lumen of the dorsal aorta in the anterior part of F1 VEGF+/- than VEGF+/+ embryos. Ink injection to view the dorsal aorta and head vessels showed no connection between the heart and the vessel system in VEGF- 1- T-ES cell-derived embryos (Fig. 3c, d). Yolk sac of F1 VEGF +/- embryos displayed an irregular plexus of small vessels but no larger collecting vessels (Fig. 3f, i). 0.5 point non-human model organism. PMID: 28230770 - Zebrafish have duplicate copies of vegfa (vegfaa and vegfab). Used morpholino oligonucleotides to knockdown transcription levels. Gross morphology and heart development was almost normal w/ 10ng doses of vegfab MO, therefore hypothesized that vegfaa dominantly functions in vegfa signaling. In vegfaa morpholinos, observed defects in vescular development, shrunken ventricle and atrium, and abnormal heart looping. Dose-dependence was shown with increasing MO doses resulting in higher % of cardiac defects. vegfaa mutants showed improperly leftward-looped hearts and the tube extends aberrantly and develops a narrow but longer heart tube. Downgrading points; genetic structure in zebrafish, having two duplicate copies makes scoring in this model more difficult to say it is truly replicating the phenotype scored in humans. 0.5 points non-human model organism.
No points awarded: Hiratsuka 2005 PMID: 15601856 - VEGF-A is part of the vascular endothelial growth factor signaling system, which is likely involved in the migration of hemangioblast precursor cells in the fetal trunk during early embryogenesis. This study helps support that VEGF-A is important for VEGF signaling during embryogenesis, but isn’t specific to cardiac defects, and it isn’t clear that VEGR is known to be associated w/ cardiac defects, therefore did not score any points. In mouse embryos, using RT-PCR and IHC to assess expression, found that VEGF-A is dominant in the anterior portion of the embryo, whereas VEGFR1 and 2 are expressed in the posterior (fig 2 and 3). VEGFA guides VEGFR cells in vitro - whole embryo cultures were treated with different concentrations of VEGFA in different corners of the cell culture wells, and VEGFA at 40ng/ml was most effective (fig 4). VEGFR-positive cells collected from embryos were placed on cover glass and placed next to a spot on the cover glass where cells rom the anterior or posterior region of the embryos had been seeded. VEGFR-positive cells migrated to cells obtained from the anterior portion, and this migration was eliminated by neutralizing anti-mouse VEGFA antibody. VEGFR-positive cells did not migrate toward cells obtained from the posterior region of embryos. (Fig 5). VEGF-A heterozygous (+/−) mice showed embryonic lethality due to multiple defects in vascular structure formation (6, 13
Carmeliet 1999 PMID: 10229225 - same authors as Stalmans 2003 PubMed: 12539040. VEGF120/120 mice (ie those lacking VEGF164 and VEGF184 had impaired postnatal myocardial angiogenesis, resulting in ischemic cardiomyopathy characterized by reduced contractility and sarcomere breakdown, but normal stores of high-energy phosphates Giordano 2001 PubMed: 11331753 - cardiomyocyte-specific knockout mouse had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to b-adrenergic stimulation
Ootaki 2003 PMID: 12735573 - Children with cyanotic congenital heart disease were found to have significantly elevated VEGF levels compared with those with acyanotic heart disease, especially those with a single ventricle associated w/ asplenia syndrome. However a commentary brought up methodological concerns that could have caused bias; serum VEGF concentrations may reflect blood platelet degranulation rather than synthesis by peripheral tissues PMID: 15511516. Scoring no points.
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