Dominant mutations in ITPR3 cause Charcot-Marie-Tooth disease

Abstract

Objective: ITPR3, encoding inositol 1,4,5-trisphosphate receptor type 3, was previously reported as a potential candidate disease gene for Charcot-Marie-Tooth neuropathy. Here, we present genetic and functional evidence that ITPR3 is a Charcot-Marie-Tooth disease gene.

Methods: Whole-exome sequencing of four affected individuals in an autosomal dominant family and one individual who was the only affected individual in his family was used to identify disease-causing variants. Skin fibroblasts from two individuals of the autosomal dominant family were analyzed functionally by western blotting, quantitative reverse transcription PCR, and Ca²⁺ imaging.

Results: Affected individuals in the autosomal dominant family had onset of symmetrical neuropathy with demyelinating and secondary axonal features at around age 30, showing signs of gradual progression with severe distal leg weakness and hand involvement in the proband at age 64. Exome sequencing identified a heterozygous ITPR3 p.Val615Met variant segregating with the disease. The individual who was the only affected in his family had disease onset at age 4 with demyelinating neuropathy. His condition was progressive, leading to severe muscle atrophy below knees and atrophy of proximal leg and hand muscles by age 16. Trio exome sequencing identified a de novo ITPR3 variant p.Arg2524Cys. Altered Ca²⁺ -transients in p.Val615Met patient fibroblasts suggested that the variant has a dominant-negative effect on inositol 1,4,5-trisphosphate receptor type 3 function.

Interpretation: Together with two previously identified variants, our report adds further evidence that ITPR3 is a disease-causing gene for CMT and indicates altered Ca²⁺ homeostasis in disease pathogenesis.

Figure 1

Clinical features and sequencing. Photographed at age 64, the index patient P1 had distal muscle atrophy (A). In the family, his father (P4) and one of his two brothers (P3) had been similarly affected (B). Moreover his daughter (P2) had had foot deformities since an early age and was noted to have pes cavus when examined at age 35 (C). In sural nerve biopsy of P1, a clear hypertrophic neuropathy with prominent onion bulbs was seen (D, plastic section toluidine blue staining, scale bar 25 µm). Muscle biopsy from tibialis anterior muscle of P1 showed prominent small group atrophy, fiber type grouping and secondary myopathic change (E, frozen section HE‐staining, scale bar 125 µm). Sanger sequencing confirmed the presence of ITPR3 c.1843G> A (p.Val615Met) in all affected individuals, shown are P1 and P2 chromatograms (F), and it was absent in the unaffected brother.

Figure 2

Position and conservation of IP₃R3 mutations. The p.Val615Met and the p.Arg2524Cys mutations affect conserved stretch of amino acids (A). The p.Val615Met mutation lies adjacent to the cytoplasmic surface of IP₃R3 and in proximity to the IP₃ binding site, while the p.Arg2524Cys mutation lies in the channel pore (B‐D), as predicted based on the previously published model of the tetramer ²³ . The amino acids 610‐620 and 2520‐2530 are highlighted in red (B and C). Key domains of IP₃R3 channel and IP₃‐molecule at its binding site are highlighted in the figure (D). ARM1‐3 = armadillo repeat domains 1‐3, BTF1‐2 = β‐trefoil domains 1‐2, CLD = center linker domain, JD = juxtamembrane domain, TMD = transmembrane domain.

Figure 3

Fibroblast protein levels in Finnish family patients. Western blots of fibroblast lysates showed that the levels of IP₃R1 were not changed between patient and control fibroblasts (A, B). In P2 fibroblasts, the levels of IP₃R2 and IP₃R3 were significantly decreased compared with the controls, whereas the levels in P1 fibroblasts were unchanged (A, B). qPCR showed that the mRNA level of ITPR3 was increased in P1 but not in P2 fibroblasts (C). Finally, siRNA knockdown of ITPR3 in control fibroblasts led to clear reduction in the protein level of IP₃R3 by western blot, while the levels of IP₃R1 and IP₃R2 were unchanged (D) KD = ITPR3 siRNA knockdown, NT = non‐targeting siRNA, wt = non‐treated. Data points marked in the figures were excluded from the data‐analysis (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

Ca²⁺ flux analysis in patient and control fibroblasts. The Ca²⁺ flux measurements were performed with two different methods, using cell‐permeant Fluo‐4 AM and Fura‐2 AM fluorescent Ca²⁺ indicators. In the first method (A and B), we used fluorescent microscopy to monitor Ca²⁺ in single cells, and 80 μmol/L ATP as GPCR agonist to evoke Ca²⁺ release. In each case, representative Ca²⁺ response curves after addition of ATP are shown. Grey thin lines are recordings from single cells whereas the thick line represents the averaged trace for all the cells in the given experiment. (A) siRNA of ITPR3 led to increased average time to peak as compared with cells that were untreated (wt) or treated with non‐targeting (NT) siRNA (n = 4 individual experiments). KD = ITPR3 siRNA knockdown, NT = non‐targeting siRNA, wt = non‐treated. (B) In response to ATP, P1 fibroblasts had decreased area under curve (AUC), while P2 cells had increased time to peak compared with unrelated controls (P1 n = 20, P2 n = 14 and three controls n = 17‐22 individual experiments). In the second method (C–E), we measured Ca²⁺ in single wells of a 96‐well plate, using 10 μmol/L ionomycin, 10 μmol/L thapsigargin or 50 nmol/L bradykinin to evoke responses, and compared patient cells to one control line performing five independent experiments in each setting. All stimuli (added after 90 sec, 2nd dotted line) were added in the presence of EGTA (added after 30 sec, 1st dotted line). (C) Traces showed a decrease in ionomycin‐induced Ca²⁺‐transients for both patients compared to the healthy control, with a significant decrease in peak amplitude for P2. (D) In response to SERCA inhibitor thapsigargin, patient fibroblasts did not display statistically significant decrease in Ca²⁺ ER store content compared to the healthy control. (E) In response to bradykinin, we observed a significant decrease in the peak amplitude of the response in P2 fibroblasts. All results are presented as mean ± SEM of independent experiments and statistical comparisons performed with one‐way ANOVA. (*P < 0.05, **P < 0.01, ***P < 0.001).