Bi-allelic variants in HMGCR cause an autosomal-recessive progressive limb-girdle muscular dystrophy

Abstract

Statins are a mainstay intervention for cardiovascular disease prevention, yet their use can cause rare severe myopathy. HMG-CoA reductase, an essential enzyme in the mevalonate pathway, is the target of statins. We identified nine individuals from five unrelated families with unexplained limb-girdle like muscular dystrophy and bi-allelic variants in HMGCR via clinical and research exome sequencing. The clinical features resembled other genetic causes of muscular dystrophy with incidental high CPK levels (>1,000 U/L), proximal muscle weakness, variable age of onset, and progression leading to impaired ambulation. Muscle biopsies in most affected individuals showed non-specific dystrophic changes with non-diagnostic immunohistochemistry. Molecular modeling analyses revealed variants to be destabilizing and affecting protein oligomerization. Protein activity studies using three variants (p.Asp623Asn, p.Tyr792Cys, and p.Arg443Gln) identified in affected individuals confirmed decreased enzymatic activity and reduced protein stability. In summary, we showed that individuals with bi-allelic amorphic (i.e., null and/or hypomorphic) variants in HMGCR display phenotypes that resemble non-genetic causes of myopathy involving this reductase. This study expands our knowledge regarding the mechanisms leading to muscular dystrophy through dysregulation of the mevalonate pathway, autoimmune myopathy, and statin-induced myopathy.

Keywords: HMG-CoA reducatase; HMGCR; autoimmune myopathy; limb-girdle like muscular dystrophy; mevalonate pathway; rare genetic disease; statin-induced myopathy.

Graphical abstract

Figure 1

Bi-allelic variants in HMGCR in five affected families results in an autosomal-recessive progressive limb-girdle muscular dystrophy (A) Five families with nine affected individuals and bi-allelic variant segregation. Filled symbols indicate affected individuals. Parents of consanguineous family are fourth cousins. (B) Spatial distribution of muscle weakness in six affected individuals (age during examination) utilizing MRC muscle strength scores generated by MuscleViz denoting: 0, paralysis; 1, trace or minute muscle contraction; 2, muscle movement is possible without gravity; 3, muscle movement is possible against gravity; 4, muscle strength is reduced, but movement against resistance is possible; and 5, preserved normal strength. (C) T1 axial MRI images (from family 5 II:I, at 31 years of age) show striking replacement of almost all muscles of the upper leg (top image) with fibroadipose tissue (signal artifact in right upper leg due to metallic implant). In the lower leg (bottom image), there is increased signaling in the soleus and gastrocnemius muscles with relative sparing of the anterior muscles (tibialis anterior, extensor hallucis longus, and extensor digitorum longus) and lateral muscles (peroneus longus and brevis). (D) Histopathologic findings from family 2 II:1 (top row) includes mild variation in myofiber size on H&E (left), increased subsarcolemmal oxidative enzyme activity on NADH stain (middle), and increased subsarcolemmal mitochondria on electron microscopy (right). Findings from family 2 II:2 (bottom row) includes dystrophic myopathic changes with marked variation in myofiber size with atrophic and hypertrophic myofibers, degenerating fibers with phagocytosis, endomysial fibrosis, and many rimmed vacuoles with basophilic granule deposition on H&E (left). Gomori trichrome (middle) highlights the endomysial fibrosis and stains the rimmed vacuoles stain red. Electron microscopy shows myofibrillar disruption and vacuoles containing lysosomal debris (right). (E) Ultrastructural findings of moderately increased subsarcolemmal mitochondria of the tibialis anterior muscle from family 5 II:I at 35 years of age.

Figure 2

Pathogenic variants in HMGCR cluster in linker and catalytic domains in highly conserved residues (A) Schematic representation of HMGCR transcript map and location of variants identified in cohort. Compound heterozygous variant combinations per family and affected probands are colored the same. (B) HMGCR domains are highlighted and reveal clustering of variants in the catalytic region and linker domain with a constraint plot by MetaDome. SSD, sterol-sensing domain. (C) Ortholog alignment of HMG-CoA reductase residues altered by missense or in-frame deletion variants are highlighted with a red rectangle.

Figure 3

3D molecular structure of HMGCR reveals likely molecular mechanisms (A) Each monomer of the tetramer is colored distinctly to illustrate the dimer-of-dimers architecture. Each dimer of the tetramer is a domain-swapped structure where the linker of one monomer is adjacent to the catalytic center of another (see Figure S1 for more details). (B) Three monomers have been colored white and one monomer remains colored as in (A). Sites of case variants are colored gray in three monomers and colored in one monomer with the WT amino acid labeled. (C and D) Arg443 is within the linker region, which is partially resolved in crystallographic experiments. Residues within the linker that are resolved participate in a hydrogen bond network with Arg443 (C) and moderately stabilizing effects on residue Ile467 (D). (E and F) Ser508 is a position directly at the monomer-monomer interface, and (F) Arg515 is at the monomer-monomer interface in the domain-swapped region (see Figure S1). Arg515 is flanked by negatively charged residues and may dynamically form inter- and intra-monomer interactions. (G) Leu546 is at the end of a small loop between the two beta strands of the domain-swapped sheet. It is surrounded by other hydrophobic residues and between the side chains of the adjacent alpha helix. (H) Asp623 is at the base of a loop that connects a beta sheet to an alpha helix, and its side chain forms multiple specific contacts with residues from both secondary structures. (I) Tyr792 is directly part of the cross-dimer interface of the tetramer. PTM, post translational modification.

Figure 4

Enzymatic activity, size, and stability of purified recombinant HMGCR (A and B) Enzymatic activity of the protein measured as a reduction in absorbance at 340 nm. The activity was quantified relative to WT HMGCR with all three variants showing significant reductions in activity compared to WT protein. Error bars are mean ± SD. (C) Fluorescence curves from thermal shift assays over increasing temperature from three missense variants constructs and WT. Thermal shift assay averaged across four replicates show the three missense variants cause significant reductions in stability compared to WT protein Tm estimates. (D) Estimation of the protein size using the S200 increase column. Two of the variant proteins, p.Asp623Asn and p.Tyr792Cys, eluted at the same volume as WT, while p.Arg443Gln eluted earlier. Acrylamide gels are depicted and stained with Coomassie blue showing only the p.Arg443Gln variant was different in size compared with WT with an approximately 20% increase in size. ^∗∗p value 0.0041, ^∗∗∗0.0002, and ^∗∗∗∗<0.0001.