Correlation of Phenotype-Genotype and Protein Structure in RYR1-Related Myopathy

Abstract

Introduction: Next generation sequencing results in an explosive identification of rare variants of RYR1, making the correlation between phenotype and genotype complicated. We analyzed the data of 33 patients with RYR1-related myopathy, attempting to elucidate correlations between phenotype, genotype, and protein structure of RyR1.

Methods: Clinical, histopathologic, and genetic data were evaluated, and variants were mapped to the cryo-EM RyR1 structure. The three-dimensional structure of the variant on RyR1 was analyzed.

Results: The clinical spectrum was highly variable regardless of the mode of inheritance. Recessive variations were associated with more severe feeding problems and respiratory insufficiency in infancy (p < 0.05). Forty pathogenic and likely pathogenic variations were identified, and 14 of them were novel. Missense was the most common variation type regardless of inheritance mode. Arginine (15/45) was the most frequently involved residue. All but one dominant variation clustered in Pore forming and pVSD domains, while recessive variations enriched in Bsol (7/25) and SPRYs (6/25) domains. Analysis of the spatial structure of variants showed that dominant variants may impact RyR1 mainly by breaking down hydrogen or electrovalent bonds (10/21); recessive variants located in different domains may impact the function of RyR1 through different pathways. Variants located in RyR1 coupling sites (PY1&2 and the outermost of Bsol) may cause the most severe clinical manifestation.

Conclusion: Clinical diversity of RYR1-related myopathy was impacted by the inheritance mode, variation type, and variant location. Dominant and recessive variants have different sensitive domains impacting the function of RyR1 through different pathways.

Keywords: RYR1-related myopathy; cohort study; genotype; phenotype; protein structure.

Figure 1

Distribution of variations across the RYR1 coding region.

Figure 2

Variants mapping on the rabbit RyR1 monomers, presented with residues in rabbit matched to human variants. a-b, Structure of RyR1 tetramer from the membrane plane (A) and cytosolic plane (B); (C,D) Structure of a single RyR1 monomer (C) and with 90-degree rotation (D). Each domain was assigned a unique color. (E) Dominant RyR1 variants mapped on RyR1 tetramer with one monomer highlighted in color; (F–H) Dominant RyR1 variants on a single monomer (F), with 90-degree (G) and 180-degree rotation (H). (I) Recessive RyR1 variants mapped on RyR1 tetramer with one monomer highlighted in color; (J–L) Recessive RyR1 variants on a single monomer (J), with 90-degree (K) and 180-degree rotation (L). Each variant is represented by a colored sphere. Color coding for clinical severity was shown at the right bottom: mild 1-5 = green; moderate 6-10 = blue; severe >10= red. Arabic number before each residue designated the corresponding case number. Addictive number following case number “-1,” “-2” showing different recessive variants in the same case.

Figure 3

Three-dimensional structure of variants on rabbit RyR1 monomers, presented with residues in rabbit matched to human variants. (A) R4860 located on the U-motif of the S5-S6 loop, connecting with residues bring negative charges enriched in the U-motif; (B) view of the interactions among the three subunits of NTD. R221 on NTD-B connected with E397 on NSol; (C) T4979 located on the binding site of ATP, interfering with the combination between ATP and RyR1; (D) R2241 located on the binding site of calmodulin, interfering with the binding of Calmodulin to RyR1; (E) Gly3191 and Leu932 located on PY1&2 and the outermost part of Bsol, respectively, the presumed coupling sites among RyR1s; (F) the enlarged view of PY1&2, L931 located in the center of the domain, interacting with surrounding hydrophobic residues.