Nuclear Receptor NR1H3 in Familial Multiple Sclerosis

Abstract

Multiple sclerosis (MS) is an inflammatory disease characterized by myelin loss and neuronal dysfunction. Despite the aggregation observed in some families, pathogenic mutations have remained elusive. In this study, we describe the identification of NR1H3 p.Arg415Gln in seven MS patients from two multi-incident families presenting severe and progressive disease, with an average age at onset of 34 years. Additionally, association analysis of common variants in NR1H3 identified rs2279238 conferring a 1.35-fold increased risk of developing progressive MS. The p.Arg415Gln position is highly conserved in orthologs and paralogs, and disrupts NR1H3 heterodimerization and transcriptional activation of target genes. Protein expression analysis revealed that mutant NR1H3 (LXRA) alters gene expression profiles, suggesting a disruption in transcriptional regulation as one of the mechanisms underlying MS pathogenesis. Our study indicates that pharmacological activation of LXRA or its targets may lead to effective treatments for the highly debilitating and currently untreatable progressive phase of MS.

Figure 1

Simplified pedigree for families presenting the NR1H3 p.Arg415Gln mutation. Males are represented by squares and females by circles, the proband is arrowed and a diagonal line indicates deceased subjects. Patients diagnosed with MS have black filled symbols and mutation carriers of unknown phenotype have grey filled symbols. Both families are of Caucasian descent. Heterozygote mutation carriers (M) with corresponding age at onset of disease and wild-type (wt) genotypes are indicated. An asterisk indicates an inferred mutation carrier. NA; not available.

Figure 2

NR1H3 p.Arg415Gln conservation in orthologs and human paralogs. Protein homologs were aligned via ClustalO. Amino acid position for NR1H3 p.Arg415Gln is highlighted in black. Protein homologs with amino acid positions differing from those of the human NR1H3 sequence are indicated in gray. RefSeq accession numbers is provided for orthologs, and gene, protein and RefSeq accession numbers for paralogs. An arrow indicates the exclusion of two or three amino acids from Caenorhabditis elegans at these positions.

Figure 3

Structural and functional analysis of NR1H3. A) Crystal structure of NR1H3 (LXRA) and NR2B1 (RXRA) heterodimer showing the highly conserved arginine residues in their corresponding ligand binding domain was generated with PyMol (PDB ID: 2ACL). Inserts provide a detailed view of the LXRA wild-type (Arg) and mutated (Gln) protein structure. B) FLAG-LXRA wild type (WT) and p.Arg415Gln mutant (R415Q) were co-expressed with RXRA-mycHis (+) or empty vector pcDNA4-myc-his-A (V) in HEK cells with or without 10μM LXRA agonist T0901317 overnight. RXRA was pulled down from the lysates with nickel-charged magnetic beads. Cell lysates and precipitates were immunoblotted with FLAG or Myc antibodies. C) Transcriptional activation of control (pGL-pl) and LXR response element-containing plasmids (LXRE) after co-transfection with empty vector, wild-type (WT) or mutant LXRA (R415Q). Firefly/Renilla luciferase activity (mean ± standard error) for the different constructs, with and without the presence of 10μM of a LXRA agonist (T0901317) normalized to pGL-pl under the same conditions are provided. Statistically significant Fisher’s Least Significant Difference (LSD) post hoc values are provided. r.u., relative units.