ClinVar

In 7 males and 1 female over 3 generations of a family (family 1) with aortic root aneurysm, either isolated or in association with distal ascending aortic aneurysm, aortic valve stenosis, and/or bicuspid aortic valve (AOVD3; 618496), Gould et al. (2019) identified heterozygosity for a c.2056+1G-T transversion (c.2056+1G-T, NM_019055.5) in intron 12 of the ROBO4 gene. Analysis of cDNA from patient and control fibroblasts showed that the splicing mutation causes skipping of exon 13, resulting in an in-frame transcript encoding a protein missing 36 amino acids from the intracellular domain. The mutation was also present in 2 clinically unaffected female family members, indicating reduced penetrance, but was absent from the ExAC database. Immunofluorescence analysis of patient aorta showed reduced expression in the endothelium compared to control, and there was infiltration of ROBO4(+) cells into the aortic media, which was not seen in controls. Immunostaining for albumin showed substantial penetration into the aortic intima of the patient, whereas it was restricted to the endothelial surface in control aorta. Histologic analyses showed a strong fibroproliferative response in the intima and superficial medial layer of patient aorta, with increased cellularity and accumulation of collagen, as well as decreased elastin content and fragmentation and disarray of elastic fibers in the superficial media. Gould et al. (2019) suggested that ROBO4 variants disrupt endothelial cellular performance and barrier function, contributing to pathologic remodeling of the aortic media. In transiently transfected cultured human aortic endothelial cells, dextran permeability assay showed loss of endothelial barrier function, associated with downregulation of TJP1 (601009) and VE-cadherin (CDH5; 601120); there was also induction of expression of alpha-SMA (ACTC1; 102540) and SNAI1 (604238), with cellular elongation and invasion. The authors also noted a modest decline in BMP (see 112264) and NOTCH (see 190198) activity, with no change in TGF-beta (190180) responses. Mice with Robo4 lacking exon 13 presented an incompletely penetrant complex cardiovascular phenotype that included aortic valve defects and dysfunction as well as aneurysm of the ascending aorta: at 20 weeks of age, 1 (3.2%) of 31 heterozygous males and 4 (11.4%) of 31 homozygous male mice were affected, compared to none of the wildtype males (p less than 0.05).