Abstract
Inherited disorders characterized by motor neuron loss and muscle weakness are genetically heterogeneous. The recent identification of mutations in the gene encoding transient receptor potential vanilloid 4 (TRPV4) in distal spinal muscular atrophy (dSMA) prompted us to screen for TRPV4 mutations in a small group of children with compatible phenotype. In a girl with dSMA and vocal cord paralysis, we detected a new variant (p.P97R) localized in the cytosolic N-terminus of the TRPV4 protein, upstream of the ankyrin-repeat domain, where the great majority of disease-associated mutations reside. In another child with congenital dSMA, in this case associated with bone abnormalities, we detected a previously reported mutation (p.R232C). Functional analysis of the novel p.P97R mutation in a heterologous system demonstrated a loss-of-function mechanism. Protein localization studies in muscle, skin, and cultured skin fibroblasts from both patients showed normal protein expression. No TRPV4 mutations were detected in four children with dSMA without bone or vocal cord involvement. Adding to the clinical and molecular heterogeneity of TRPV4-associated diseases, our results suggest that molecular testing of the TRPV4 gene is warranted in cases of congenital dSMA with bone abnormalities and vocal cord paralysis.
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Acknowledgments
We thank Mrs. Catherine Wrenn for her valuable help in the English writing. This study was supported in part by the Italian Ministry of Health, Regione Toscana grant RR5/09-RT (to CB) and Fondazione Telethon (Grant GUP08005 to CB).
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The authors declare that all the experiments and clinical studies comply with the current laws of the country in which they were performed.
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Supplementary Figure 1
Immunofuorescence (IF) panel of muscle and skin sections from a control and patient 1 to evaluate the expression of the TRPV4 gene product. In muscle sections from both patient and control, TRPV4 is diffusely but modestly expressed in the cytosol (A, C). In skin sections (B, D) TRPV4 shows intense binding on keratinocyte layer, endothelium and nerve endings, as described [11, 22]. No significant differences can be observed between patient and control. (JPEG 83 kb)
Supplementary Figure 2
Expression of the TRPV4 gene product in cultured skin fibroblasts from control, patients 1 and 2. TRPV4 has intense staining in the nucleus as described by the antibody manufacturer. No differences can be seen between the two patients in respect to control. (JPEG 51 kb)
Supplementary Figure 3
Structural representation of the N-terminus of the TRPV4 protein is presented. (A) Ribbon representation of one of the structural models obtained for the N-terminal region (Met1-Thr399) of TRPV4. The structural prediction was performed using the protein structure prediction server Robetta [24, 25]. The first 130 N-terminal residues, without a detectable PDB homolog, were modeled using the Rosetta de novo approach [26], while comparative models for the ankyrin-repeat domain (ARD) were built using the PDB structure of the chicken TRPV4 ARD as template [3]. The ARD with six putative repeat units, the proline-rich domain and the N-terminal 1–131 domain are depicted in red, orange and green, respectively. Residues Pro97 (in blue) and Arg232 (in cyan) are shown. (B) Modeling of the NH2-terminus region (residues 1–470) with I-TASSER server (http://zhang.bioinform-atics.ku.edu/I-TASSER/). Visualization and molecular graphic of the final model was rendered using Chimera (http://www.cgl.ucsf.edu/chimera/). The ribbon representation shows the six ankyrin repeats (ANK; blue), the proline-rich domain in yellow and the P97 and R232 residues, indicated by arrows with dotted lines. (JPEG 44 kb)
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Fiorillo, C., Moro, F., Brisca, G. et al. TRPV4 mutations in children with congenital distal spinal muscular atrophy. Neurogenetics 13, 195–203 (2012). https://doi.org/10.1007/s10048-012-0328-7
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DOI: https://doi.org/10.1007/s10048-012-0328-7