Association Study of Exon Variants in the NF-κB and TGFβ Pathways Identifies CD40 as a Modifier of Duchenne Muscular Dystrophy

Abstract

The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor β [TGFβ]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8 × 10^-6). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFβ pathways. The minor allele at rs1883832, in the 5'-untranslated region of CD40, was associated with earlier LoA (p = 3.5 × 10^-5). This allele diminishes the expression of CD40, a co-stimulatory molecule for T cell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD.

Figure 1

Manhattan Plot of Exome Chip Associations with Age at Loss of Ambulation Additive genotype p values of the Cox proportional-hazards model, with glucocorticoid treatment as a covariate, are shown for 27,025 Exome Chip SNPs with MAF > 0.05. SNPs within, or <10,000 kb upstream or downstream of, prioritized genes in the NF-κB and TGFβ pathways are highlighted in green. The red horizontal line (p = 1.8 × 10⁻⁶) refers to Bonferroni correction for 27,025 SNPs, and the blue line (p = 1.1 × 10⁻⁴) refers to Bonferroni correction for 438 SNPs within prioritized genes. Top p values are annotated with corresponding gene names (also see Table 1).

Figure 2

Kaplan-Meier Plots of Age at Loss of Ambulation by CD40 rs1883832 Genotype Kaplan-Meier curves are shown (additive and dominant genotype models) in the CINRG Exome Chip cohort and validation cohorts. Abbreviations are as follows: add, additive inheritance model; dom, dominant inheritance model. The overall validation cohort is the sum of CINRG validation, Bio-NMD, Padova, and UDP cohorts.