Bi-allelic variants in the ESAM tight-junction gene cause a neurodevelopmental disorder associated with fetal intracranial hemorrhage

Abstract

The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs^∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies."

Keywords: ESAM; blood-brain barrier; epilepsy; exome sequencing; global developmental delay; intellectual disability; intracranial hemorrhage; neurodevelopmental disorders; pregnancy loss; retinopathy; tight junctions.

Graphical abstract

Figure 1

Neuroimaging abnormalities in individuals with homozygous ESAM variants Individual 1: T1-weighted (a and b) images showing various parenchymal hemorrhagic area in different evolution phases, located at the parietal-occipital area. T2-weighted and multi-echo gradient-recalled echo (GRE) T2-weighted images (c and d) showing blood occupying the ventricular spaces (hemoventricle). T1-weighted MRI (e and f) and FLAIR sequences (g) highlight a dysmorphic corpus callosum (e) and poromalacic evolution of the hemorrhagic areas with ventricular dilatation (f and g) and diffuse subependymal microhemorrhage foci. The orange arrow in (g) indicates ventricular derivation. Individual 4: MRI images at 2 days after birth: 3DT2 sagittal (a), 3DT2 coronal MPR (b), 3DT1 axial (c), 3D TOF (d). Bilateral grade III germinal matrix - intraventricular hemorrhage (blue arrows) with supratentorial posthemorrhagic hydrocephalus. Bilateral parenchymal hematomas with associated subpial hemorrhages (orange arrows) at different stages and parietal encephalomalacia secondary to prior bleeding with residual clot inside (red stars). No evident vascular anomalies in MRI angiography (3D TOF sequence) were found. Individual 5 (sibling of individual 4): MRI images at 7 days after birth: 3DT2 sagittal (a), 3DT2 coronal MPR (b), 3DT1 axial (c), 3D TOF (d). Microcephaly with simplified gyral pattern and severe hypoplastic corpus callosum (blue arrow). Left parietal subcortical white matter hematoma with subpial hemorrhage (red stars) and encephalomalacia. Right frontal (orange arrow) and multiple bilateral periventricular hemorrhages. MRI angiography (3D TOF sequence) shows slight narrowing of the proximal anterior and middle cerebral arteries (green arrows). Individual 6: MRI images at 5 days old. T1- (a) and T2- (b) weighted images showing massive dilatation of lateral ventricles with global cerebral parenchymal destruction. Diffuse meningeal and intraventricular hemorrhage with intraventricular clotting. Focal destruction of the septum pellucidum. Individual 8: axial T2-weighted images showed hyperintensity and volume loss in the bilateral periventricular white matter, frontotemporal atrophy, dilatation in the lateral ventricles (a and b) and hypoplasia of the corpus callosum (c) (orange arrows). Bilateral periventricular calcifications were observed on cranial CT (d), as indicated by the arrow. Individual 9: Head CT (axial) showing bilateral subependymal (a) and basal ganglia/thalamus calcifications (b) (orange arrows), suggestive of a previous bleeding. Dilation of the lateral ventricles and their straight shape suggest agenesis of the corpus callosum, as confirmed in the sagittal multiplanar reformation (c). Individual 11: Axial T2-weighted true fast sequence (a and b), axial T2 HASTE sequence (c), and axial T1-weighted sequence (d). The fetal brain MRI shows hydrocephalus, intraventricular hemorrhage, diffuse intraparenchymal, and periventricular hemorrhages with calcifications (hypointense signals on T1-weighted images). Individual 13: Brain MRI at 37 weeks of gestation. Axial T2 (a and b) showing severe irregular ventriculomegaly with increased extra-axial spaces and significant decrease in gray and white matter volume. Orange arrows indicate an abnormal Sylvian fissure development in (a) and an asymmetric gyral pattern suggestive of dysgyria/polymicrogyria in (b). Small periventricular cysts are indicated by red stars. Sagittal T2 (c) highlighting corpus callosum dysgenesis with absence of rostrum (arrow). Coronal T2 (d): hypointense lesions in the temporal parenchyma (indicated by the arrow) protruding into the temporal horn of the right ventricle. GRE T2 sequences (e) demonstrate susceptibility artifacts consistent with brain calcification (arrow).

Figure 2

Family pedigrees and genetic findings Bi-allelic ESAM variants are shown in thirteen affected individuals, including four fetuses, from eight unrelated families. The c.115del (p.Arg39Glyfs^∗33) frameshift variant was identified in six individuals from families A, B, E, and F, originating from the same geographic area in Turkey (southeastern Anatolia). The c.287del (p.Pro96Leufs^∗33) frameshift variant was detected in two affected siblings from Spain (family C). The c.35T>A (p.Leu12^∗) nonsense variant was identified in a family of Algerian descent (family D). The c.451+1G>A splice site variant was identified in two independent families of Arab Bedouin descent (families G and H). +/+, +/−, and −/− represents homozygosity for the wild-type allele, heterozygosity, and homozygosity for the mutant allele, respectively. An asterisk beneath an individual indicates that ES was performed. Arrowheads indicate the probands. TOP, termination of pregnancy; SB, stillbirth; SAB, spontaneous abortion; wks, weeks of gestation.

Figure 3

In vitro matrigel tubulogenesis assay (A) Endothelial colony-forming cells (ECFCs) isolated from individual 1 showed significantly decreased tubulogenesis compared to the ECFCs obtained from a healthy control (HC) and parents of individual 1. (B and C) Quantification of tubulogenesis was performed considering the number of branch points (B) and the tube length (C). Data shown (±SD) are from 3 independent experiments. ^∗∗p < 0.01. Scale bar: 200 mm. More technical details are provided in the supplemental note.

Figure 4

ESAM immunohistochemistry and hematoxylin and eosin staining in the damaged brain tissue (white matter) of individual 10 (A and B) A capillary with ESAM-negative endothelial cells in the proband (A) compared to a control sample (B), as indicated by the blue arrows. Magnification: 40 HPF. (C) Periventricular leukomalacia in individual 10: the white matter close to the lateral ventricle (V) shows multiple areas of calcification (blue arrow). Hematoxylin and eosin staining, magnification 4 HPF. (D) Calcified areas showing elongated sticks and round bodies as a result of encrusted (“ferruginated”) neurons and their axons with mineral salts. Hematoxylin and eosin staining, magnification 20 HPF. More technical details are provided in the supplemental note.