Fig 2: The HMGCR mutation. (A) Sanger sequencing of an HMGCR amplicon of an unaffected individual (V:6), an obligatory carrier (IV:1) and an affected individual (V:13). The mutation causes a glycine to aspartate nonconservative substitution at position 822. (B) Precent identity matrix of HMGCR produced using Clustal-Omega, showing high homology between the human HMGCR and orthologs. (C) Multiple alignment of human HMGCR and orthologs produced using Clustal-Omega, showing that the substituted amino acid is highly conserved throughout evolution. (D) Structural model of HMG CoA-reductase protein in the WT and mutated form, based on 1DQ8. The substitution presumably forms a new H-bond with a distal residue, compromises the helix dipole, and causes electrostatic repulsion. (E) Expression of HMGCR across various tissues, obtained using the GTEx database.

Fig 3: HMGCR mutation impairs protein function. (A and B) Subcellular localization of WT (A) and CRISPR-KI mutant (B) HMGCR in SH-SY5Y cells. Both WT and mutant HMGCR protein (green) are located in clusters in the cytoplasm with some relation to the endoplasmic reticulum (red). (C and D) HMGCR enzymatic activity. The velocity of enzymatic reduction of different concentrations of HMG CoA was measured by spectrophotometric NADPH oxidation assay of the WT (n = 9) and mutant (n = 12) forms of HMGCR. Analyzed using Michaelis–Menten analysis and multiple t-tests. (E–G) Mutant HMGCR shows decreased affinity to pravastatin, a protein inhibitor that binds to the same catalytic pocket as HMG CoA (n = 8). As seen, mutant kinetics are almost identical to the no protein control. PM, plasma membrane; ER, endoplasmic reticulum.

Fig 4: Oral mevalonolactone treatment in human HMGCR-LGMD. (A) Anti-HMGCR autoantibodies titer in patients (n = 6) and healthy controls (n = 10). (B) Plasma mevalonolactone levels of patients V:2 (n = 20 on multiple occasions) and healthy controls (n = 10), normalized to average level of controls. (C) Mevalonolactone levels in peripheral blood of V:2 after an oral dose of 16 mg/kg mevalonolactone, normalized to average level of controls. (D–I) Evaluation of muscle strength of patient V:2 throughout the treatment period by dynamometry (Top) and manual muscle test (MMT; Bottom) by an experienced neurologist. (J–L) Evaluation of distal muscles throughout the treatment by an experienced neurologist. (M) Lung functions throughout the treatment period assessed by spirometry. (N) Muscle strength improvement "heat-map" by dynamometry, precent improvement from weakest state. (O) Pigmentation in the proximal nail fold which appeared occasionally following treatment. I: Gross appearance; II: Dermatoscopic image of the index finger showing pigmentation; and III: Dermatoscopic image of the same index finger several days later, pigmentation has resolved. Statistical analysis using t-test.