Clinical, Molecular, and Functional Characterization of CLCN1 Mutations in Three Families with Recessive Myotonia Congenita

Abstract

Myotonia congenita (MC) is an inherited muscle disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. Both recessive (Becker's disease) or dominant (Thomsen's disease) MC are caused by mutations in the CLCN1 gene encoding the voltage-dependent chloride ClC-1 channel, which is quite exclusively expressed in skeletal muscle. More than 200 CLCN1 mutations have been associated with MC. We provide herein a detailed clinical, molecular, and functional evaluation of four patients with recessive MC belonging to three different families. Four CLCN1 variants were identified, three of which have never been characterized. The c.244A>G (p.T82A) and c.1357C>T (p.R453W) variants were each associated in compound heterozygosity with c.568GG>TC (p.G190S), for which pathogenicity is already known. The new c.809G>T (p.G270V) variant was found in the homozygous state. Patch-clamp studies of ClC-1 mutants expressed in tsA201 cells confirmed the pathogenicity of p.G270V, which greatly shifts the voltage dependence of channel activation toward positive potentials. Conversely, the mechanisms by which p.T82A and p.R453W cause the disease remained elusive, as the mutated channels behave similarly to WT. The results also suggest that p.G190S does not exert dominant-negative effects on other mutated ClC-1 subunits. Moreover, we performed a RT-PCR quantification of selected ion channels transcripts in muscle biopsies of two patients. The results suggest gene expression alteration of sodium and potassium channel subunits in myotonic muscles; if confirmed, such analysis may pave the way toward a better understanding of disease phenotype and a possible identification of new therapeutic options.

Fig. 1

Pictures of family 1 brother showing generalized muscle hypertrophy at shoulder girdle and lower limbs

Fig. 2

Hematoxylin and eosin (H&E) stain and ATPase stain of Vastus Lateralis muscle biopsies from family 1 brother (a, b) and family 3 proband (c, d) suggest muscle fiber size variability and type 2 fibers predominance

Fig. 3

Chloride currents generated by wild-type hClC-1 channels and MC ClC-1 variants in high intracellular chloride condition. a Typical chloride currents recorded in HEK293 cells transfected with wild-type, p.T82A, or p.R453W hClC-1 variants. Cells are held at 0 mV, and 400-ms voltage pulses are applied from −200 to +120 mV in 10-mV intervals every 3 s. For clarity, only current traces obtained every 20 mV are shown. b Voltage pulses are applied from −120 to +200 mV to elicit chloride currents in HEK293 cells expressing p.G270V hClC-1 variant. c The instantaneous currents are measured at the beginning of test voltage pulses, normalized with respect to cell capacitance (pA/pF), and reported as a function of voltage. Each point is the mean ± SEM from 11 to 13 cells. Similar current density and strong inward rectification are observed for WT, p.T82A, and p.R453W channels. The relationship for p.G270V channels is linear. d Steady state currents are measured at the end of test voltage pulses and reported as mean current density ±SEM in function of voltage. Again, relationships for WT, p.T82A, and p.R453W channels are very similar, whereas current density and rectification are different for p.G270V. e The voltage dependence of activation is determined by plotting the apparent open probability (Po), calculated from tail currents measured at −105 mV, as a function of test voltage pulses. The relationships obtained from averaged data are fitted with a Boltzmann equation, and fit parameters are reported in Table 1. The activation curves for WT, p.T82A, and p.R453W are superimposed, whereas p.G270V channels displayed voltage dependence greatly shifted toward positive voltages

Fig. 4

Chloride currents generated by p.G190S and coexpressed p.G190S and p.T82A mutants. a Chloride currents are recorded in HEK293 cells using high intracellular chloride condition. The cells are held at 0 mV, and voltage pulses are applied from −200 to +200 mV in 10-mV intervals every 3 s. For clarity, only current traces obtained every 20 mV are shown. b Typical chloride currents recorded in HEK293 cells transfected with 5 µg of p.G190S mutant. c Typical chloride currents recorded in HEK293 cells cotransfected with 5 µg of p.G190S and 5 µg of p.T82A. d The instantaneous currents are measured at the beginning of test voltage pulses, normalized with respect to cell capacitance (pA/pF), and reported as a function of voltage. Each point is the mean ± SEM from 9 to 12 cells. The mean I–V curves are shown for p.T82A, p.G190S, and coexpressed p.G190S + p.T82A. In yellow are reported the algebraic sum of current densities calculated for p.G190S and p.T82A expressed alone. The yellow points are quite superimposed to the I–V relationship for coexpressed p.G190S + p.T82A. e Steady state currents are measured at the end of test voltage pulses and reported as mean current density ±SEM in function of voltage. Again, the yellow points are quite superimposed to the I–V relationship for p.G190S + p.T82A coexpression. f The voltage dependence of activation is determined by plotting the apparent open probability (Po), calculated from tail currents measured at −105 mV, as a function of test voltage pulses. The relationships obtained from averaged data are fitted with a Boltzmann equation, and fit parameters are reported in Table 1. The activation curves for coexpressed p.T82A and p.G190S are intermediate between the relationships for p.G190S and p.T82A expressed alone (Color figure online)

Fig. 5

Quantitative gene expression of selected ion channels in biopsies of MC and control individuals. The transcript level is normalized with respect to ACTB housekeeping gene encoding β-actin. Each bar represents the mean ± SEM of triplicate measures in each bioptic sample. The Vastus Lateralis muscle biopsies are from the index myotonic patients of family 3 carrying p.G270V in homozygosis and of family 1 carrying p.T82A and p.G190S in compound heterozygosis. They are compared to biopsies of two control individuals