Scapuloperoneal spinal muscular atrophy and CMT2C are allelic disorders caused by alterations in TRPV4

Abstract

Scapuloperoneal spinal muscular atrophy (SPSMA) and hereditary motor and sensory neuropathy type IIC (HMSN IIC, also known as HMSN2C or Charcot-Marie-Tooth disease type 2C (CMT2C)) are phenotypically heterogeneous disorders involving topographically distinct nerves and muscles. We originally described a large New England family of French-Canadian origin with SPSMA and an American family of English and Scottish descent with CMT2C. We mapped SPSMA and CMT2C risk loci to 12q24.1-q24.31 with an overlapping region between the two diseases. Further analysis reduced the CMT2C risk locus to a 4-Mb region. Here we report that SPSMA and CMT2C are allelic disorders caused by mutations in the gene encoding the transient receptor potential cation channel, subfamily V, member 4 (TRPV4). Functional analysis revealed that increased calcium channel activity is a distinct property of both SPSMA- and CMT2C-causing mutant proteins. Our findings link mutations in TRPV4 to altered calcium homeostasis and peripheral neuropathies, implying a pathogenic mechanism and possible options for therapy for these disorders.

Figure 1

Pathology of an individual with SPSMA. (a) Phosphotungstic acid hematoxylin (PTAH) staining of muscle biopsy showing severe muscle fiber–type grouping and atrophy (arrow). (b) Hematoxylin and eosin (H&E) staining of muscle autopsy sample showing severe muscle fiber–type grouping and atrophy (arrow). (c) ATPase staining (pH 9.4) of muscle autopsy samples showing both small type 1 and type 2 fibers and fiber type grouping. (d) Luxol fast blue/H&E staining of spinal cord sections showing a normal number of anterior horn cells. A representative motor neuron in the anterior horn is indicated by an arrow.

Figure 2

TRPV4 mutations in SPSMA and CMT2C pedigrees. (a) A heterozygous mutation, C946T, resulting in R316C, was identified in exon 6 of TRPV4 in the SPSMA family. The wild-type (WT) sequence is shown in the lower panel. (b) All the affected members whose DNA samples were available for sequencing analysis had the R316C substitution. (c) A heterozygous mutation, G806A (leading to the R269H amino acid substitution) in exon 5 of TRPV4 was identified in the CMT2C family. The WT sequence is shown in the lower panel. (d) All the affected members whose DNA samples were available for sequencing analysis had this R269H substitution. (e) Evolutionary conservation of amino acids in the mutated region of TRPV4 in different species. Comparison of human (Homo sapiens) TRPV4 and its orthologs in dog (Canis lupus familiaris), cattle (Bos taurus), mouse (Mus musculus), rat (Rattus norvegicus), chicken (Gallus gallus) and zebrafish (Danio rerio). Amino acids identical to those in human TRPV4 are in black letters; nonidentical ones are denoted in red letters. The positions of the C-terminal amino acids are shown on the right. The mutated amino acids are indicated by arrows on the top. (+) indicates TRPV4 mutation; (−) indicates wild type.

Figure 3

Localization of wild-type and mutant TRPV4 on the plasma membrane. (a–l) Confocal microscopy was performed using HEK293 cells transfected with plasmids pIRES2-ZsGreen1 containing wtTRPV4 (a–d), R269H (e–h) or R316C (i–l). Cells expressing exogenous TRPV4 were labeled by green fluorescent protein (GFP) (c, g, k). TRPV4 is shown by blue (a, e, i) and cadherin by red (b, f, j). Merged images are shown on the right panels (d, h, l). Arrows indicate TRPV4 signals on the plasma membrane. Arrowheads indicate representative cells without significant expression of GFP and TRPV4 (f, h).

Figure 4

Effect of mutations on TRPV4 activity when stimulated with 4αPDD. (a–c) Effect of stimulation with 4αPDD (2 μM) on internal fluorescence ratio in wtTRPV4- (a), R269H- (b) and R316C-transfected (c) HEK293 cells. (d) Application of 4αPDD induced an increase in intracellular calcium ([Ca²⁺]_i). Average increases, basal and maximum values are given. For each condition, n > 11 in at least three independent recordings. *, significant differences when compared with wtTRPV4 (two-tailed Student’s t test, P < 0.001). Error bars, means ± s.e.m.

Figure 5

Effect of mutations on TRPV4 activity when stimulated with hypotonic solution. (a–c) Effect of stimulation with a hypotonic stimulus (HTS) (200 mOsm) on internal fluorescence ratio in wtTRPV4- (a), R269H- (b) and R316C-transfected (c) HEK293 cells. (d) Application of HTS induced an increase in intracellular calcium ([Ca²⁺]_i). Average increases, basal and maximum values are given. For each condition, n > 15 in at least three independent recordings. *, significant differences when compared with wtTRPV4 (two-tailed Student’s t test, P < 0.005). Error bars, means ± s.e.m.

Figure 6

Whole-cell recordings of TRPV4 currents from transfected HEK293 cells. (a, b) Voltage clamp recordings of the currents elicited by a slow voltage ramp (from -100 to 100 mV, 600 ms) in control conditions (black traces) and in the presence of 20 μM ruthenium red (red traces) in cells expressing wtTRPV4 (a) or R269H (b) channels. (c, d) Ruthenium red–sensitive current (obtained by digital subtraction) in cells expressing wtTRPV4 (c) and R269H (d). (e) Plot summarizing the amount of current (normalized to capacitance) recorded at +100 mV in control conditions (black bars) and in the presence of ruthenium red (red bars) in cells expressing the wild-type and mutated channels. (f) Plot summarizing the size of the ruthenium red–sensitive current in cells expressing the two channel types. (g) The ruthenium red–sensitive current was transformed into conductance. The conductance of R269H-expressing cells was ~6 times larger (0.35 ± 0.5 in wild type and 2.1 ± 0.6 in the mutant, 9 and 6 cells, respectively, P < 0.01). Error bars, means ± s.e.m.