Delineation of the clinical, molecular and cellular aspects of novel JAM3 mutations underlying the autosomal recessive hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts

Abstract

We have recently shown that the hemorrhagic destruction of the brain, subependymal, calcification, and congenital cataracts is caused by biallelic mutations in the gene encoding junctional adhesion molecule 3 (JAM3) protein. Affected members from three new families underwent detailed clinical examination including imaging of the brain. Affected individuals presented with a distinctive phenotype comprising hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. All patients had a catastrophic clinical course resulting in death. Sequencing the coding exons of JAM3 revealed three novel homozygous mutations: c.2T>G (p.M1R), c.346G>A (p.E116K), and c.656G>A (p.C219Y). The p.M1R mutation affects the start codon and therefore is predicted to impair protein synthesis. Cellular studies showed that the p.C219Y mutation resulted in a significant retention of the mutated protein in the endoplasmic reticulum, suggesting a trafficking defect. The p.E116K mutant traffics normally to the plasma membrane as the wild-type and may have lost its function due to the lack of interaction with an interacting partner. Our data further support the importance of JAM3 in the development and function of the vascular system and the brain.

Figure 1. Pedigree Drawings of the studied Families

Three families with different ethnic origin were recruited in this study. Family 1 is Turkish, family 2 is Afghani, while family 3 is Moroccan. Filled symbols denote affected individuals with the syndrome of hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Sequenced individuals are indicated with an asterisk.

Figure 2. Clinical data

A-1) Facial picture of the affected individual in Family 1 shows bilateral nuclear cataracts. A-2) Head CT image of the patient in Family 1 reveals multifocal intraparenchymal hemorrhages (arrows) and subependymal calcification (arrowheads). B) T2-weighted axial brain MRI of Patient 1 in Family 2 reveals multifocal intraparenchymal hemorrhages of varying ages (arrows) as well as intraventricular hemorrhage (asterisks). C-1) Facial picture of the affected individual in family 3 shows bilateral cataracts, enlarged frontal part of the cranium and prominent scalp veins. C-2) Head CT of the patient in family 3 shows multifocal intraparenchymal hemorrhages (arrows) and subependymal calcification (arrowheads). Diffusely hypodensity of brain parenchyma is also noted, suggesting severe edema. C-3) T2-weighted coronal MRI image of the same patient shows intraventricular hemorrhage with associated enlargement of the ventricles.

Figure 3. p.C219Y mutation disrupts normal intracellular JAM3 protein localization

HeLa Cells transiently transfected with C-terminal Flag-tagged JAM3-pCMV5 vector constructs wild type (WT), A–F; p.E116K, G–L; and p.C219Y, M–R) and stained with anti-Flag Ab (red). WT vector localizes to the plasma membrane (PM) with the hRas-GFP-tagged marker (A–C) and not with the endoplasmic reticulum (ER) marker calnexin (green; D–F). JAM3–p.E116K–Flag construct localization was similar to the WT with hRas-GFP-tagged marker (G–I) and the calnexin (J–L). P.C219Y construct do not localize at the PM like hRas-GFP-tagged marker (M–O) and appears to be accumulated or clustered within the ER With calnexin (P–R).