Abstract
Ryanodine receptor ion channels (RyR1s) release Ca2+ ions from the sarcoplasmic reticulum to regulate skeletal muscle contraction. By whole-exome sequencing, we identified the heterozygous RYR1 variant c.14767_14772del resulting in the in-frame deletion p.(Phe4923_Phe4924del) in two brothers with a lethal form of the fetal akinesia deformation syndrome (FADS). The two deleted phenylalanines (RyR1-Δ4923FF4924) are located in the S6 pore-lining helix of RyR1. Clinical features in one of the two siblings included severe hypotonia, thin ribs, swallowing inability, and respiratory insufficiency that caused early death. Functional consequences of the RyR1-Δ4923FF4924 variant were determined using recombinant 2,200-kDa homotetrameric and heterotetrameric RyR1 channel complexes that were expressed in HEK293 cells and characterized by cellular, electrophysiological, and computational methods. Cellular Ca2+ release in response to caffeine indicated that the homotetrameric variant formed caffeine-sensitive Ca2+ conducting channels in HEK293 cells. In contrast, the homotetrameric channel complex was not activated by Ca2+ and did not conduct Ca2+ based on single-channel measurements. The computational analysis suggested decreased protein stability and loss of salt bridge interactions between RyR1-R4944 and RyR1-D4938, increasing the electrostatic interaction energy of Ca2+ in a region 20 Å from the mutant site. Co-expression of wild-type and mutant RyR1s resulted in Ca2+-dependent channel activities that displayed intermediate Ca2+ conductances and suggested maintenance of a reduced Ca2+ release in the two patients. Our findings reveal that the RYR1 pore variant p.(Phe4923_Phe4924del) attenuates the flow of Ca2+ through heterotetrameric channels, but alone was not sufficient to cause FADS, indicating additional genetic factors to be involved.