PMC9263075_01
Heterozygous STIM1 mutation in a patient with myopathy. (A) The sequence of the PCR products of genomic DNA from the patient's leukocytes is shown. g.4095848T>C (c.1477T>C, p.L303P). This mutation has not been previously reported. (B) Multiple alignment of the sequences from humans and 6 other species, showing that L303 is a conserved amino acid in 7 analysed species. (C) The left panel shows the structure of human CC1-IH STIM1. The top right panel shows the interactions between 2 alpha helices of the CC1-IH region of STIM1. Blue is Q314 and light blue is E318 of one alpha helix, and L303 and R304 are the red and yellow residues, respectively, of the other alpha helix. The bottom right panel shows the L303P mutation. The figure was produced using Swiss-PdbViewer. (D) Mutation significance cut-off () of STIM1 L303P mutation. (E) Immunoblot analysis of STIM1 protein from the patient's (P1) peripheral blood mononuclear cells (PBMCs) and from 2 healthy donors (C1 and C2). We employed GAPDH as a loading control. The panels illustrate the results from a single experiment, representative of 3. (F) Calcium flux analysis in the PBMCs of P1 and 3 healthy donors (C1, C2, and C3) in response to ionomycin. The panels illustrate the results from a single experiment, representative of 3. (G) Calcium flux geometric mean (Geo Mean) is represented for C1, C2, and C3. +- SD and P1 in non-stimulated PBMCs (NS) or ionomycin-stimulated PBMCs (Iono). p < 0.05 (*).
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This mutation affects leucine at position 303, generating a missense mutation by a proline (c.1477T>C, p.L303P, transcript ID ENST00000300737.4), a previously unreported variant, which we validated by Sanger sequencing of genomic DNA from peripheral leukocytes ( Figure 1A ). Alignment of the human STIM1 protein sequence with sequences from the 7 animal species in which STIM1 has been sequenced showed L303 to be highly conserved throughout evolution ( Figure 1B ). The L303P mutation modifies the position of the lateral chain, affecting the proper conformation of the CC1-IH region of STIM1 ( Figure 1C ). A mutation significance cut-off study () predicted this variant as likely to be damaging ( Figure 1D ). We then assessed STIM1 protein levels in the patient's peripheral blood mononuclear cells (PBMCs), detecting normal STIM1 protein levels compared with healthy donors ( Figure 1D ). Higher calcium flux in response to ionomycin was detected in the patient compared with the 3 healthy donors ( Figures 1E-G ), confirming that the L303P mutation generates a GOF in PBMCs that affects calcium homeostasis.