HMGCS1 variants cause rigid spine syndrome amenable to mevalonic acid treatment in an animal model

Abstract

Rigid spine syndrome is a rare childhood-onset myopathy characterized by slowly progressive or non-progressive scoliosis, neck and spine contractures, hypotonia and respiratory insufficiency. Biallelic variants in SELENON account for most cases of rigid spine syndrome, however, the underlying genetic cause in some patients remains unexplained. We used exome and genome sequencing to investigate the genetic basis of rigid spine syndrome in patients without a genetic diagnosis. In five patients from four unrelated families, we identified biallelic variants in HMGCS1 (3-hydroxy-3-methylglutaryl-coenzyme A synthase). These included six missense variants and one frameshift variant distributed throughout HMGCS1. All patients presented with spinal rigidity primarily affecting the cervical and dorso-lumbar regions, scoliosis and respiratory insufficiency. Creatine kinase levels were variably elevated. The clinical course worsened with intercurrent disease or certain drugs in some patients; one patient died from respiratory failure following infection. Muscle biopsies revealed irregularities in oxidative enzyme staining with occasional internal nuclei and rimmed vacuoles. HMGCS1 encodes a critical enzyme of the mevalonate pathway and has not yet been associated with disease. Notably, biallelic hypomorphic variants in downstream enzymes including HMGCR and GGPS1 are associated with muscular dystrophy resembling our cohort's presentation. Analyses of recombinant human HMGCS1 protein and four variants (p.S447P, p.Q29L, p.M70T, p.C268S) showed that all mutants maintained their dimerization state. Three of the four mutants exhibited reduced thermal stability, and two mutants showed subtle changes in enzymatic activity compared to the wildtype. Hmgcs1 mutant zebrafish displayed severe early defects, including immobility at 2 days and death by Day 3 post-fertilisation and were rescued by HMGCS1 mRNA. We demonstrate that the four variants tested (S447P, Q29L, M70T and C268S) have reduced function compared to wild-type HMGCS1 in zebrafish rescue assays. Additionally, we demonstrate the potential for mevalonic acid supplementation to reduce phenotypic severity in mutant zebrafish. Overall, our analyses suggest that these missense variants in HMGCS1 act through a hypomorphic mechanism. Here, we report an additional component of the mevalonate pathway associated with disease and suggest biallelic variants in HMGCS1 should be considered in patients presenting with an unresolved rigid spine myopathy phenotype. Additionally, we highlight mevalonoic acid supplementation as a potential treatment for patients with HMGCS1-related disease.

Keywords: HMGCS1; enzymopathy; mevalonate pathway; neuromuscular disease; rigid spine myopathy.

Figure 1

HMGCS1-myopathy cohort. (A) Pedigrees of the four families with biallelic variants in HMGCS1 segregating with rigid spine syndrome. (B) Rigid spine presentation demonstrated in images of Patients P1 and P2 (Family SPA1), P3 (Family JPN1) and P4 (Family ITA1). Features include spinal rigidity affecting the cervical and dorso-lumbar regions, limited neck flexion, scoliosis, scapular winging and hypotrophy.

Figure 2

Muscle MRI and pathology associated with HMGCS1-related myopathy. (A) Muscle MRI performed for Patient P1 (Family SPA1) at 23 years of age [A(i–iii)] and Patient P4 (Family ITA1) at 36 years of age [A(iv–vi)]. Variation in T₁ signal indicative of fatty replacement in the posterior muscles of the cervical paraspinal muscles (stars) [A(i)], the posterior compartment of the thigh (arrows) [A(ii, iv and v)] and the posterior compartment of the leg (arrows) [A(vi)]. Evidence of oedema in the posterior compartment of the thighs in Patient P1 shown by STIR (arrows) [A(iii)]. Vastus intermedius is also affected to a lesser extent (arrow heads). (B) Light microscopy staining of muscle biopsies from Patients P1 (Family SPA1) at age 13 years (top), P3 (Family JPN1) at age 5 years and at 20 years (middle) and P4 (Family ITA1) at 4 years (bottom). Haematoxylin and eosin (H&E) and Gömöri trichrome (GT) staining show occasional internal nuclei and rimmed vacuoles in some patients. Succinate dehydrogenase (SDH) staining of SPA1 and nicotinamide adenine dinucleotide (NADH) staining of Families JPN1 and ITA1 patient biopsies reveal irregularities in staining including moth-eaten appearances and core-like regions (arrows). Scale bars = 20 μm or 50 μm, as indicated. yo = years old.

Figure 3

Localization of HMGCS1 variants within the gene and protein and their impact on HMGCS1 expression and function. (A) Schematic representations of the HMGCS1 gene (top) and protein (bottom) linear architectures. The HMGCS1 gene includes untranslated regions (empty boxes), coding exons (filled boxes) and introns (horizontal line). Variants identified in patients with rigid spine syndrome are labelled, including both missense (circle) and truncating (triangle) variants. The variants are coloured according to the families they have been identified in (red: Family JPN1; blue: Family USA1; green: Family ITA1; orange: Family SPA1). Letters ‘A’, ‘B’ and ‘s’ within the protein structure (bottom) represent residues of the active site (E95, C129, H264), co-enzyme A binding site (N167, S221, K269, K273) and salt bridge (D119, E121, R194, D208, K239, K461 and H462), respectively. Diagram not to scale. (B) Conservation of the six missense variant positions in various species and in HMGCS2; the mitochondrial paralogue. Asterisks indicate complete conservation of residues in the analysed species. (C) RNA-sequencing fold-change in HMGCS1 expression in Patient P3 (Family JPN1) compared to three unaffected controls (left). Error bars correspond to standard deviation. RNA-seq reads indicate virtually monoallelic expression of HMGCS1 c.86A>T, p.(Q29L) suggesting the c.344_345del, p.(S115Wfs*12) variant undergoes nonsense mediated decay (right). (D) Visualization of the HMGCS1 dimer (PDB: 2P8U) using PyMOL. HMGCS1 chain A (right) represented by a ribbon and chain B (left) shown as a cartoon. Ac-CoA represented as dark grey spheres. Variant positions (represented by sticks) coloured by family. Close-up representation showing the minimum distance between S447 and Q29 is ∼4.3 Å (dashed box).

Figure 4

HMGCS1 is expressed in various tissues including patient skeletal muscle. (A) Quantitative real-time PCR analysis using cDNA from healthy human cell lines and tissues. Expression data from human embryonic kidney cells (HEK293FT cell line), fibroblasts, myoblasts, myotubes at D2, D4, D6, D8 of differentiation, cortex, skeletal muscle controls from in vitro contractile testing and fetal muscle. Transcript levels were normalized to the geometric mean of EEF2 and TBP using the delta Ct method. Data presented as mean ± standard error of the mean (n = 2–7 biological). (B) Graphical presentation of HMGCS1 peptides detected by quantitative mass spectrometry from myoblasts (MB) and myotubes at Day 2 (D2) and Day 8 (D8) of differentiation. (C–F) Western blotting for HMGCS1 (top) in (C) primary human (Cook Myosite) myoblasts and myotubes from Days 0–12 of differentiation, in (D) control human tissue and cell lines, and in (E) Patient P1 (Family SPA1) skeletal muscle alongside healthy skeletal muscle controls. Recombinant wild-type HMGCS1 (Rec. HMGCS1) used as a control for antibody specificity. (F) Western blotting for HMGCS1 in mouse extensor digitorum longus (EDL) and soleus muscles (top). Western blots for GAPDH and gel-stained myosin heavy chain (MHC) bands are shown to demonstrate comparable loading of total and muscle proteins.

Figure 5

Thermal shift and circular dichroism analyses of recombinant wild-type and mutant HMGCS1. (A) Thermal shift assay first derivative fluorescence curves measured for HMGCS1 wild-type and four mutants (12.5 μg) over increasing temperature. The minimum point of the first derivative curves was estimated as the protein melting temperature (T_m). Thermal shift assays were conducted three independent times using four replicates per assay. The data plotted in (B) represent the T_m averages from each assay (n = 3) and are presented as mean ± standard error of the mean. Significance was determined by an ordinary one-way ANOVA followed by Dunnett’s multiple comparison test. (C–F) Circular dichroic spectra scans (195–260 nm) of wild-type and four mutant HMGCS1 (0.1 mg/ml) measured at (C) 15°C, (D) 25°C, (E) 55°C and (F) 95°C in buffer containing 10 mM sodium phosphate buffer pH 7.4, (including 1% of original protein buffer: 150 mM NaCl, 50 mM Tris and 10% glycerol).

Figure 6

hmgcs1^−/− zebrafish have severe mitochondrial abnormalities and an early lethal phenotype. (A) Brightfield images of wild-type and hmgcs1^−/− embryos at 48 h post-fertilization (hpf). hmgcs1^−/− embryos have developmental delay, reduced pigmentation and blood pooling in the brain. (B) Quantification of movement to a touch-evoke assay. hmgcs1^−/− embryos lack a movement response to touch, unlike wild-type and heterozygous siblings. Injection of mRNA encoding human HMGCS1 rescues the movement defects in hmgcs1^−/− embryos (Student’s t-test). Twenty-four injected embryos were used for each assay. (C) Survival assay on the progeny of a hmgcs1^+/− in-cross. In control injected fish only 79% (n = 19) survived until Day 4 and no hmgcs1^−/− fish were detected at this time, as indicated in brackets, demonstrating that loss of Hmgcs1 results in early embryo lethality. Injection of HMGCS1 mRNA rescues the lethality in hmgcs1^−/− embryos, with 100% (n = 24) of injected fish surviving until Day 4, and subsequent genotyping indicating 33% (n = 8) of the fish were hmgcs1^−/−. Twenty-four embryos from the hmgcs1^+/− in-cross were used for each assay. (D) There is a significant reduction in the survival of embryos resulting from a hmgcs1^+/− in-cross injected with HMGCS1 variant mRNA compared to injection of wild-type mRNA. P-values are listed next to the figure legend. Statistics were performed in SPSS using a generalized linear model. (E) Scanning electron microscopy images of longitudinal muscle sections at 48 hpf, demonstrating mitochondria associated with skeletal muscle. hmgcs1^−/− embryos have an increased proportion of mitochondria with detached membranes. Magenta arrowheads indicate mitochondria, cyan arrowheads indicate myofibres. Scale bar = 1 μm. Three samples were analysed for each genotype. (F) Mitochondrial density is not affected in hmgcs1^−/− embryo skeletal muscle (Student’s t-test). (G) The proportion of mitochondria with detached membranes is higher in hmgcs1^−/− embryos compared to wild-type (Fisher’s exact test). Statistics were performed in Prism 9 (GraphPad). Statistically significant differences are indicated on the graphs by the P-value.

Figure 7

Mevalonic acid treatment improves hmgcs1^−/− zebrafish survival and reduces phenotypic severity. (A) Survival analysis of hmgcs1^−/− zebrafish embryos treated with 50, 500 and 1000 μM mevalonic acid, E3 control and vehicle control (ethanol) until 6 days post-fertilization (dpf). Control hmgcs1^−/− embryos do not survive past 2 dpf. Treatment with 50 μM mevalonic acid significantly extends survival until 3 dpf, whereas 500 and 1000 μM treatments extended survival until 6 dpf, at which point the experiment was ceased. Statistically significant differences are indicated on the graphs by the P-value next to the figure legend. (B) Phenotypic severity of hmgcs1^−/− embryos rescued with mevalonic acid treatment at 6 dpf. Treatment with 1000 μM mevalonic acid further reduces the phenotypic severity of hmgcs1 loss-of-function compared to 500 μM. (C) Swimming activity in hmgcs1^−/− embryos at 6 dpf treated with mevalonic acid. hmgcs1^−/− embryos treated with 1000 μM mevalonic acid (n = 66) swam significantly further than embryos treated with the 500 μM treatment (n = 40). Rep = biological replicate. Statistics were performed in SPSS using a linear mixed model. (D) Transmission electron microscopy images of longitudinal muscle sections of vehicle control and 1000 μM mevalonic acid treated embryos at 48 hpf, demonstrating mitochondria associated with skeletal muscle. hmgcs1^−/− embryos have an increased proportion of mitochondria with detached membranes. Magenta arrowheads indicate mitochondria, cyan arrowheads indicate myofibres. Scale bar = 1 μm. Three samples were analysed for each genotype. (E) Mitochondrial density is not affected by mevalonic acid treatment. (F) The percentage of mitochondria with detached membranes is significantly reduced in mevalonic acid treated hmgcs1^−/− embryos compared to vehicle-treated controls. Statistics were performed in in SPSS using a two-way ANOVA. For E and F, the graphs show the estimated means and 95% confidence intervals together with the raw data.