Correlation of phenotype with genotype and protein structure in RYR1-related disorders

Abstract

Variants in the skeletal muscle ryanodine receptor 1 gene (RYR1) result in a spectrum of RYR1-related disorders. Presentation during infancy is typical and ranges from delayed motor milestones and proximal muscle weakness to severe respiratory impairment and ophthalmoplegia. We aimed to elucidate correlations between genotype, protein structure and clinical phenotype in this rare disease population. Genetic and clinical data from 47 affected individuals were analyzed and variants mapped to the cryo-EM RyR1 structure. Comparisons of clinical severity, motor and respiratory function and symptomatology were made according to the mode of inheritance and affected RyR1 structural domain(s). Overall, 49 RYR1 variants were identified in 47 cases (dominant/de novo, n = 35; recessive, n = 12). Three variants were previously unreported. In recessive cases, facial weakness, neonatal hypotonia, ophthalmoplegia/paresis, ptosis, and scapular winging were more frequently observed than in dominant/de novo cases (all, p < 0.05). Both dominant/de novo and recessive cases exhibited core myopathy histopathology. Clinically severe cases were typically recessive or had variants localized to the RyR1 cytosolic shell domain. Motor deficits were most apparent in the MFM-32 standing and transfers dimension, [median (IQR) 85.4 (18.8)% of maximum score] and recessive cases exhibited significantly greater overall motor function impairment compared to dominant/de novo cases [79.7 (18.8)% vs. 87.5 (17.7)% of maximum score, p = 0.03]. Variant mapping revealed patterns of clinical severity across RyR1 domains, including a structural plane of interest within the RyR1 cytosolic shell, in which 84% of variants affected the bridging solenoid. We have corroborated genotype-phenotype correlations and identified RyR1 regions that may be especially sensitive to structural modification.

Keywords: Genotype-phenotype; Myopathy; Neuromuscular disease; RyR1; Structure-function.

Fig. 1

Distribution of variants across the RYR1 coding region including MH/CCD hot spots. Numbers within green and red boxes correspond to the affected amino acid residue

Fig. 2

Median (IQR) MFM-32 scores (a) and pulmonary function test values (b) by mode of inheritance. a Except for standing and transfers, participants with an AR mode of inheritance achieved a significantly lower median (IQR) percentage of maximum MFM-32 score across all other MFM-32 domains, when compared to AD/DN cases [standing and transfers, 71.8 (33.3)% vs. 59.0 (27.6)% p = 0.078; axial and proximal motor function, 100.0 (19.1)% vs. 97.2 (16.0)% p = 0.017; distal motor function, 95.2 (4.8)% vs. 92.9 (8.3)% p = 0.046; total score, 87.5 (17.7)% vs. 79.7 (18.8)% p = 0.037]. There was no difference in pulmonary function parameters when compared by mode of inheritance (all, p > 0.05), (^∘ and * denote outliers)

Fig. 3

Bar charts of clinical symptom frequency by mode of inheritance (a) and affected RyR1 domain(s) (b) expressed as a percentage of cases. Statistically significant differences were as follows for AD/DN versus AR categories, respectively; facial weakness 69% versus 100%, p = 0.026; neonatal hypotonia 6% versus 33%, p = 0.034; ophthalmoplegia/paresis 0% versus 42%, p < 0.001; ptosis 6% versus 42%, p = 0.003; and scapular winging 31% versus 67%, p = 0.032. When symptom frequency was compared by affected RyR1 domain(s), the only statistically significant difference, after adjustment for multiple comparisons, was in ophthalmoplegia/paresis between CS versus CAC, 25% versus 0%, p = 0.005 and both domains versus CAC, 33% versus 0%, p = 0.001. Differences in symptom frequency for all other symptoms, by mode of inheritance and affected RyR1 structural domain(s), were not significant, p > 0.05

Fig. 4

a–g Variants mapped to the high resolution cryo-EM mammalian (rabbit) RyR1 structure. Letters a–g correspond to specific, affected RyR1 residues. Lettering is consistent between panels. a Topographical image of the RyR1 tetrameric structure with each structural region assigned a unique color. b Topographical image of a single RyR1 monomer with each structural region assigned a unique color. c RyR1 protein structure is shown with a single tetrameric subunit highlighted in teal. Each variant is represented by a sphere which includes the whole side chain of the affected residue. Lettering d–g identify affected RyR1 residues: (A, p.Arg1043Cys/rabbit p.Arg1044; p.Arg975Trp/rabbit p.Arg976), (B, p.Asp708Asn/rabbit p.Asp709), (C, p.Arg1606His/rabbit p.Arg1607), (D, p.Arg530His/rabbit p.Arg531), (E, p.Met485Val/rabbit p.Leu486), (F, p.Arg2163His/rabbit Arg2163), (G, p.Thr2206Met/rabbit Thr2206), (H, p.Arg2224His/rabbit p.Arg2224), (I, p.Cys2233Arg/rabbit p.Cys2233), (J, p.Arg2336His/rabbit p.Arg2336; p.Asn2342Ser/rabbit p.Asn2342; p.Val2354del/rabbit p.Val2354; p.Gly2434Arg/rabbit p.Gly2434), (K, p.Arg109Trp/rabbit p.Arg110), (L, p.Arg2452Trp/rabbit p.Arg2452), (M, p.Tyr3933Cys/rabbit Tyr3934), (N, p.Ser4028Leu/rabbit p.Ser4029), (O, p.Arg3366/rabbit p.Arg3366), (P, p.Arg4737Gln/rabbit p.Arg4736), (Q, p.Thr4709Met/rabbit p.Thr4708), (R, p.Gly4820Arg/rabbit p.Gly4819), (S, p.Asn4575Thr/rabbit p.Asn4574), (T, p.Phe4808Asn/rabbit p.Phe4807), (U, p.Leu4936Arg/rabbit p.Leu4935; Ala4940Thr/rabbit p.Ala4939), (V, p.Met4840Arg/rabbit p.Met4839), (W, p.Arg4893Gln/rabbit p.Arg4892; p.Ala4894Asp/rabbit p.Ala4893; p.Ile4898Thr/rabbit p.Ile4897; p.Phe4921Leu/rabbit p.Phe4920), (X, p.Thr4853Ile/rabbit p.Thr4852), (Y, p.Met4875Val/rabbit p.Met4874; p.Glu4911Lys/rabbit p.Glu4910), (Z, p.Arg4861His/rabbit p.Arg4860). d, e Variants in each RyR1 region are assigned distinct colors, as detailed in a, b. f, g Variant mapping with color coding for clinical severity as follows: mild (clinically mild; 0 = white, 1–2 = green, 3–4 = orange. Clinically severe ≥ 5 = red). Clinical severity scores for each specific variant/participant are provided in Table S3. e RyR1 CS plane of interest with a single monomer highlighted in teal. View is facing the SR from the cytosol and variant coloring is as defined in a, b. Variants are enriched to the Bsol. f RyR1 CS plane of interest with a single monomer in teal. Clinical severity coloring, for each variant, is as defined in c. View is facing the SR from the cytosol. Variants with the greatest clinical severity are localized to the Bsol

Fig. 5

Median (IQR) MFM-32 scores and pulmonary function test values by affected RyR1 domain(s). A trend was observed for participants with only the CS affected, to exhibit lower median (IQR) percentage of maximum MFM-32 scores for each dimension including total score, a. This was deemed statistically significant for comparison of dimension 2 only (axial and proximal motor function), after adjustment for multiple comparisons (only the CS affected, 93.1 (13.2)% versus only the CAC affected 100.0 (1.4)% respectively, p < 0.001). There was no difference in percent predicted maximal effort PFTs according to affected RyR1 domain(s), b. Cases, with variant(s) that affected only the CS, achieved a significantly lower percent predicted SVC when compared to cases with variants that affected only the CAC, after adjustment for multiple comparisons (69.5 ± 17.3% versus 87.0 ± 18.0% respectively, p = 0.03), b (^∘ and * denote outliers)