Recurrent de novo mutations in CLDN5 induce an anion-selective blood–brain barrier and alternating hemiplegia

Abstract

Claudin-5 is the most enriched tight junction protein at the blood brain barrier (BBB). Perturbations in its levels of expression have been observed across numerous neurological and neuropsychiatric conditions; however, pathogenic variants in the coding sequence of the gene have never previously been reported. Here, we report the identification of a novel de novo mutation (c.178G > A) in the CLDN5 gene in two un-related cases of alternating hemiplegia with microcephaly. This mutation (G60R) lies within the first extracellular loop of claudin-5 and based on protein modelling and sequence alignment, we predicted it would modify claudin-5 to become an anion selective junctional component as opposed to a purely barrier forming protein. Generation of stably transfected cell lines expressing wild-type or G60R claudin-5 showed that the tight junctions could still form in the presence of the G60R mutation but that the barrier against small molecules was clearly attenuated and displayed higher Cl- ion permeability and lower Na+ permeability. While this study strongly suggests that CLDN5 associated alternating hemiplegia is a channelopathy, it is also the first study to identify the conversion of the BBB to an anion selective channel mediated by a dominant acting variant in CLDN5.

The Impact of Pathogenic and Artificial Mutations on Claudin-5 Selectivity from Molecular Dynamics Simulations

“…1 , we show a multisequence alignment of a selection of human Cldn proteins. Interestingly, the G60 residue is conserved among all these sequences, with the exception of Cldn8 and Cldn17, and it is located in a region where mutations were predicted to be deleterious (25).…”

“…In lack of experimental information, several structural models of Cldn5 tight junctional assemblies have been proposed, and their investigation, refinement and validation are mandatory steps to unravel the molecular basis of its function. To this goal, the recent discovery of the first Cldn5 variant associated with a neurological condition (G60R)(25) serves as an excellent bench test. The substitution, causing alternating hemiplegia in two unrelated patients, affects an amino acid between the β 3 and β 4 strands on ECL1, and was experimentally demonstrated to promote anion permeability in Cldn5 TJs.…”

“…The first de novo Cldn5 mutation linked to a neurological condition was recently reported (25). The variation, G60R, is located in the ECL1 domain and is associated with two unrelated cases of alternating hemiplegia with microcephaly.…”

The CLDN5 gene at the blood-brain barrier in health and disease

“…Very recently, a pathogenic de novo mutation of CLDN5 was originally identified by our group in two independent patients with alternating hemiplegia of childhood (AHC) [ 159 ]. The mutation in CLDN5 (c.178G > A) is located in the gate and produces a G60R mutant.…”

“…It is still unknown why the BBB in the basal ganglia is highly vulnerable for brain calcification. Patients with CLDN-5 missense mutation also developed brain calcification in the basal ganglia [ 159 , 160 ], but, interestingly, brain calcification in the basal ganglia is not necessarily a common hallmark observed in AHC patients with ATP1A3 mutations or 22q11DS patients. The other gene mutations relating to BBB permeability are occludin ( OCLN ) [ 224 ], JAM-B ( JAM2 ) [ 103 ], JAM-C ( JAM3 ) [ 102 ], PDGF-β ( PDGFB ) [ 225 ], and PDGFR-β ( PDGFBR ) [ 226 ].…”