Genetic analysis and natural history of Charcot-Marie-Tooth disease CMTX1 due to GJB1 variants

Abstract

Charcot-Marie-Tooth disease (CMT) due to GJB1 variants (CMTX1) is the second most common form of CMT. It is an X-linked disorder characterized by progressive sensory and motor neuropathy with males affected more severely than females. Many reported GJB1 variants remain classified as variants of uncertain significance (VUS). In this large, international, multicentre study we prospectively collected demographic, clinical and genetic data on patients with CMT associated with GJB1 variants. Pathogenicity for each variant was defined using adapted American College of Medical Genetics criteria. Baseline and longitudinal analyses were conducted to study genotype-phenotype correlations, to calculate longitudinal change using the CMT Examination Score (CMTES), to compare males versus females, and pathogenic/likely pathogenic (P/LP) variants versus VUS. We present 387 patients from 295 families harbouring 154 variants in GJB1. Of these, 319 patients (82.4%) were deemed to have P/LP variants, 65 had VUS (16.8%) and three benign variants (0.8%; excluded from analysis); an increased proportion of patients with P/LP variants compared with using ClinVar's classification (74.6%). Male patients (166/319, 52.0%, P/LP only) were more severely affected at baseline. Baseline measures in patients with P/LP variants and VUS showed no significant differences, and regression analysis suggested the disease groups were near identical at baseline. Genotype-phenotype analysis suggested c.-17G>A produces the most severe phenotype of the five most common variants, and missense variants in the intracellular domain are less severe than other domains. Progression of disease was seen with increasing CMTES over time up to 8 years follow-up. Standard response mean (SRM), a measure of outcome responsiveness, peaked at 3 years with moderate responsiveness [change in CMTES (ΔCMTES) = 1.3 ± 2.6, P = 0.00016, SRM = 0.50]. Males and females progressed similarly up to 8 years, but baseline regression analysis suggested that over a longer period, females progress more slowly. Progression was most pronounced for mild phenotypes (CMTES = 0-7; 3-year ΔCMTES = 2.3 ± 2.5, P = 0.001, SRM = 0.90). Enhanced variant interpretation has yielded an increased proportion of GJB1 variants classified as P/LP and will aid future variant interpretation in this gene. Baseline and longitudinal analysis of this large cohort of CMTX1 patients describes the natural history of the disease including the rate of progression; CMTES showed moderate responsiveness for the whole group at 3 years and higher responsiveness for the mild group at 3, 4 and 5 years. These results have implications for patient selection for upcoming clinical trials.

Keywords: ACGS; ACMG; CMT1X; Cx32; connexin 32.

Figure 1

Schematic of Cx32 amino acid sequence demonstrating 146 individual coding variants described in this study (A) and mean age-adjusted baseline CMTES for pathogenic/likely pathogenic missense variants by structural domain of the Cx32 (B). (A) Pathogenic/likely pathogenic variants are depicted in red, variant of uncertain significance (VUS) in yellow and benign variants in green. Variant types are delineated as shown in the key. There are 10 frameshift, 10 nonsense, 120 missense, five in-frame deletions/insertions, and one stop-lost; the 10 non-coding variants are not shown. The variants for the two families harbouring two variants each, are here depicted separately. (B) Error bars indicate 95% confidence interval. NTerm = N-terminus, amino acids (AA) 0–20; TM1 = first transmembrane domain, AA 21–41; EC1 = first extracellular domain, AA 42–72; TM2 = second transmembrane domain, AA 73–97; IC = intracellular cytoplasmic domain, AA 98–128; TM3 = third transmembrane domain, AA 129–157; EC2 = second extracellular domain, AA 158–185; TM4 = fourth transmembrane domain, AA 186–215. There were no pathogenic/likely pathogenic missense variants in the C terminal domain. *Significance at <0.05, ** < 0.01, *** < 0.001. Analysis of covariance (ANCOVA) used with post hoc P-values adjusted for Bonferroni correction.

Figure 2

Disease progression over time for pathogenic/likely pathogenic variants (males and females combined). (A) Mean change in CMTES and CMTES-R [Charcot-Marie-Tooth Examination Score (Rasch-modified)] from baseline. (B) Mean change in CMTES from baseline stratified by baseline disease severity. Error bars indicate 95% confidence intervals. Baseline CMTES stratification: mild = 0–7 (blue), moderate = 8–14 (orange), severe = ≥15 (grey). Patient numbers at each time point in the figures are found in Tables 3 and 4.

Figure 3

Scatter plot showing correlation between (A) baseline CMTES and age for pathogenic/likely pathogenic variants and VUS, and (B) baseline CMTES and age by sex for all pathogenic/likely pathogenic variants. Each point represents an individual patient. In A, the lines of best fit for simple linear regression model are plotted in red [pathogenic/likely pathogenic variants (P/LP), n = 305, Spearman’s ρ = 0.362, P = 7.3 ×10⁻¹¹] and blue (VUS, N = 61, ρ = 0.380, P = 0.003). The near identical lines suggest there is no difference clinically between pathogenic/likely pathogenic and VUS at baseline and that disease progression is indistinguishable between the groups. In B, the lines of best fit for simple linear regression model are plotted in black (all variants, n = 305, Spearman’s ρ = 0.362, P = 7.3 ×10⁻¹¹), blue (male, n = 155, ρ = 0.577, P = 3.9 ×10⁻¹⁵) and red (female, n = 150, ρ = 0.273, P = 0.001). The divergent regression lines for male and female disease progression suggest a slower disease progression in females over time. All correlations are significant at the 0.01 level. CMTES = Charcot-Marie-Tooth Examination Score; VUS = variant of uncertain significance.