Exome sequencing in multiple sclerosis families identifies 12 candidate genes and nominates biological pathways for the genesis of disease

Abstract

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in 12 genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP12), Wnt signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients.

Fig 1. Flowchart describing the methodology and samples characterized in this study.

Fig 2. Pedigrees for families nominating genes for MS.

Males are represented by squares and females by circles, a diagonal line indicates subjects known to be deceased. Black filled symbol, MS; black filled with gray dot, possible MS; gray filled, unaffected obligate carrier. Heterozygote carriers (M) and wild-type (wt) genotypes are provided. MS patients with inferred genotypes are indicated with an asterisk.

Fig 3. Pathways predicted to be disrupted in multi-incident MS families.

Simplified representation of immunological pathways containing genes harboring disease-causing variants identified in MS patients, highlighting their crosstalk and overlap. Genes of the fibrinolysis and complement cascades are provided in blue, inflammasome activation in orange, Wnt signaling pathways in green, nuclear receptor complexes in purple, and cation channels and exchangers in gray. Genes harboring variants segregating with MS in families are indicated with a ‘MS’ label. NR, nuclear receptor; CH, chemokine; CY, cytokine.

Fig 4. Protein conservation in orthologs and human paralogs.

Organism and RefSeq accession numbers are provided for orthologs and gene name and RefSeq accession numbers for human paralogs, which were obtained from Ensembl release 91. Evolutionarily conserved positions for nominated pathogenic variants are highlighted in black, and the NLRP1 p.Gly578Ser mutation previously identified in MS patients indicated in gray [11].

Fig 5. Functional analysis.

a) Primary structure of single-chain PLAU protein, adapted from Berkenblit et al. 2005 [32]. Kringle domain is provided in gray, with cysteine residues forming disulfide bonds indicated in white. Amino acid substitutions identified in MS families are shown. b) Crystal structure for NLRP12 leucine rich repeat (LRR) sixth domain showing conserved amino acid residues was predicted from NP_653288.1 with I-TASSER (Iterative Threading ASSEmbly Refinement) [181], and the p.Leu972His substitution introduced using PyMol 1.7. c) Western blot showing expression of pro-caspase-1, caspase-1, NLRP12 and β-actin in microglial (BV2) cells transfected with an empty vector (vector), wild-type NLRP12 (WT) or mutant constructs (L475Q or L972H); and d) expression of iNOS, NCOA3 and β-actin in microglial (BV2) cells transfected with an empty vector (vector), wild-type NCOA3 (WT) or NCOA3 p.Cys485 (R485C). Histograms depict mean expression ± standard error. *Tukey’s HSD post hoc p-value < 0.001. r.u. relative units.