Identification of Serum Interleukin 6 Levels as a Disease Severity Biomarker in Facioscapulohumeral Muscular Dystrophy

Abstract

Background: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common myopathies in adults, displaying a progressive, frequently asymmetric involvement of a typical muscles' pattern. FSHD is associated with epigenetic derepression of the polymorphic D4Z4 repeat on chromosome 4q, leading to DUX4 retrogene toxic expression in skeletal muscles. Identifying biomarkers that correlate with disease severity would facilitate clinical management and assess potential FSHD therapeutics' efficacy.

Objectives: This study purpose was to analyze serum cytokines to identify potential biomarkers in a large cohort of adult patients with FSHD.

Methods: We retrospectively measured the levels of 20 pro-inflammatory and regulatory cytokines in sera from 100 genetically confirmed adult FSHD1 patients. Associations between cytokine concentrations and various clinical scores were investigated. We then measured serum and muscle interleukin 6 (IL-6) levels in a validated FSHD-like mouse model, ranging in severity and DUX4 expression.

Results: IL-6 was identified as the only cytokine with a concentration correlating with several clinical severity and functional scores, including Clinical Severity Score, Manual Muscle Testing sum score, Brooke and Vignos scores. Further, FSHD patients displayed overall IL-6 levels more than twice high as control, and patients with milder phenotypes exhibited lower IL-6 serum concentration than those with severe muscular weakness. Lastly, an FSHD-like mouse model analysis confirmed that IL-6 levels positively correlate with disease severity and DUX4 expression.

Conclusions: Serum IL-6, therefore, shows promise as a serum biomarker of FSHD severity in a large cohort of FSHD1 adult patients.

Keywords: Facioscapulohumeral muscular dystrophy (FSHD); Interleukin-6; cytokines; disease biomarkers.

Fig. 1

Correlations between serum cytokine levels and clinical scores in FSHD1. Heat maps of Spearman’s correlation coefficient (Spearman R) and False Discovery Rate (FDR) using Benjamini and Hochberg method for cytokines and clinical scores in all patients (a.), males (b.), and females (c.).

Fig. 2

Correlations between serum IL-6 levels and clinical scores in FSHD1. Scatter plot representations of serum IL-6 levels versus MMT sum score (a.), Brooke score (b.), Vignos score (c.), clinical severity score (CSS) (d.), and age-corrected CSS (e.) in all patients, males, and females. Spearman R coefficients and p-values are indicated.

Fig. 3

Comparison of serum IL-6 levels with clinical severity in FSHD1 and with control. a. Scatter plot representation of serum IL-6 levels in the different groups according to clinical severity. A significant difference in serum IL-6 levels was obtained between patients with a CCS≤6 and or > 6 in the overall population (p < 0.0001^****), in males (p = 0.0016^**) and in females (p = 0.015^*). b. Scatter plot representation of serum IL-6 levels in the control and FSHD1 patient. A significant difference in serum IL-6 levels was obtained between control and patients in the overall population (p = 0.006^**), in males (p = 0.027^*), and in females (p = 0.011^*).

Fig. 4

FSHD-like mouse model: IL-6 levels in the serum and muscle. ACTA1-MCM/ + with TMX (control), ACTA1-MCM, FLExD/ + no TMX (mild) and ACTA1-MCM, FLExD/ + 7 days post-TMX (Severe D7) mice were assessed for muscle function and IL-6 levels. Ex vivo muscle physiology assessment of extensor digitorum longus muscle in each group of mice (a., b.): Control, blue (n = 9), Mild, green (n = 10), and Severe D7, red (n = 11). Mice were assessed for maximum tetanus (a.), and cross-sectional muscle area normalized specific tetanus (b.). IL-6 levels in the serum (c.) and gastrocnemius muscle (d.) in each group of mice: Control, blue (n = 6 in serum, n = 10 in muscle), Mild, green (n = 8 in serum, n = 10 in muscle), and Severe D7 (n = 6 in serum, n = 10 in muscle). Red dots in panels c and d depict corresponding samples used for serum and muscle analysis. Orange dots in panel d represent samples exclusively used for muscle analysis. Significance: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.