Genetic modifiers of ambulation in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study

Abstract

Objective: We studied the effects of LTBP4 and SPP1 polymorphisms on age at loss of ambulation (LoA) in a multiethnic Duchenne muscular dystrophy (DMD) cohort.

Methods: We genotyped SPP1 rs28357094 and LTBP4 haplotype in 283 of 340 participants in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS). Median ages at LoA were compared by Kaplan-Meier analysis and log-rank test. We controlled polymorphism analyses for concurrent effects of glucocorticoid corticosteroid (GC) treatment (time-varying Cox regression) and for population stratification (multidimensional scaling of genome-wide markers).

Results: Hispanic and South Asian participants (n = 18, 41) lost ambulation 2.7 and 2 years earlier than Caucasian subjects (p = 0.003, <0.001). The TG/GG genotype at SPP1 rs28357094 was associated to 1.2-year-earlier median LoA (p = 0.048). This difference was greater (1.9 years, p = 0.038) in GC-treated participants, whereas no difference was observed in untreated subjects. Cox regression confirmed a significant effect of SPP1 genotype in GC-treated participants (hazard ratio = 1.61, p = 0.016). LTBP4 genotype showed a direction of association with age at LoA as previously reported, but it was not statistically significant. After controlling for population stratification, we confirmed a strong effect of LTBP4 genotype in Caucasians (2.4 years, p = 0.024). Median age at LoA with the protective LTBP4 genotype in this cohort was 15.0 years, 16.0 for those who were treated with GC.

Interpretation: SPP1 rs28357094 acts as a pharmacodynamic biomarker of GC response, and LTBP4 haplotype modifies age at LoA in the CINRG-DNHS cohort. Adjustment for GC treatment and population stratification appears crucial in assessing genetic modifiers in DMD.

Figure 1

Flow diagram of analysis plan and population grouping. Subgroups included in different analyses are shown, starting from the top with the whole Duchenne Natural History Study (DNHS) cohort, and in subsequent steps excluding patients with no available DNA for genotyping; subjects with no available genome-wide markers for multidimensional scaling analysis for population stratification; and subjects leading to population stratification. Thick-border boxes indicate groups selected for specific analyses. CINRG = Cooperative International Neuromuscular Research Group; GC = glucocorticoid corticosteroid; LoA = loss of ambulation; MDS = multidimensional scaling; SNP = single nucleotide polymorphism.

Figure 2

Kaplan–Meier plots of age at loss of ambulation grouped by SPP1 rs28357094 genotype. (A) All patients genotyped for SPP1 rs28357094, including all races and ethnicities (n = 279), grouped 2 ways by rs28357094 genotype (black line = TT; gray line = TG/GG). (B) All patients genotyped for SPP1 rs28357094, including all races and ethnicities (n = 279), grouped 4 ways by rs28357094 genotype (black lines = TT; gray lines = TG/GG) and GC treatment (continuous lines = at least 1 year while ambulatory; dashed lines = <1 year or untreated). (C) Caucasian cohort controlled for population stratification and genotyped for SPP1 rs28357094 (n = 116), grouped 2 ways by rs28357094 genotype (black line = TT; gray line = TG/GG). (D) Caucasian cohort controlled for population stratification and genotyped for SPP1 rs28357094 (n = 116), grouped 4 ways by rs28357094 genotype (black lines = TT; gray lines = TG/GG) and GC treatment (continuous lines = at least 1 year while ambulatory; dashed lines = <1 year or untreated).

Figure 3

Kaplan–Meier plots of age at loss of ambulation grouped by LTBP4 rs10880 genotype. (A) All patients genotyped for LTBP4 rs10880, including all races and ethnicities (n = 274), grouped 2 ways by rs10880 genotype (black line = TT; gray line = CC/CT). (B) All patients genotyped for LTBP4 rs10880, including all races and ethnicities (n = 274), grouped 4 ways by rs10880 genotype (black line = TT; gray line = CC/CT) and GC treatment (continuous lines = at least 1 year while ambulatory; dashed lines = <1 year or untreated). (C) Caucasian cohort controlled for population stratification and genotyped for LTBP4 rs10880 (n = 115), grouped 2 ways by rs10880 genotype (black line = TT; gray line = CC/CT). (D) Caucasian cohort controlled for population stratification and genotyped for LTBP4 rs10880 (n = 115), grouped 4 ways by rs10880 genotype (black line = TT; gray line = CC/CTy) and GC treatment (continuous lines = at least 1 year while ambulatory; dashed lines = <1 year or untreated).

Figure 4

Cartesian plot of multidimensional scaling analysis of genome-wide marker population stratification. Values of the 2 highest ranking components are shown (1st on the x-axis and 2nd on the y-axis). Shape and color of the markers indicate self-identified ethnicity. Participants self-identifying as non-Hispanic Caucasian, indicated by x-shaped markers, form a cluster with low values of the first component (< −0.0025, vertical cutoff line). Forty-five participants with other self-identified races and ethnicities are mostly positioned right of the cutoff line: African American (filled circles), Asian (filled squares), Hispanic Caucasian (empty triangles), Hispanic (filled triangles), and Other (empty circles). Twelve participants self-identifying as non-Hispanic Caucasians appear as outliers, whereas 3 participants self-identifying as Hispanic Caucasian or Other cluster together with non-Hispanic Caucasians and are included in subsequent analyses.