De novo variants in LRRC8C resulting in constitutive channel activation cause a human multisystem disorder

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  Figure 1. Genetic and clinical features of the two cases with LRRC8C variants. (A) Pedigree of patient P1 (Family 1, II.2), of Italian descent, and of patient P2 (Family 2, II.1), a Canadian individual whose parents are from Chile and Canada. The monoallelic de novo LRRC8C variants found in the patients are indicated below. (B, C) Facial appearance of P1 (at age 2 year) (B) and P2 (at age 2 years and 10 months) (C) showing short palpebral fissures, a small nose, and smooth facial features. Note the resemblance between the two unrelated individuals. (D, E) Prominent telangiectasia on the trunk (P1) and thigh (P2). (F, G) Radiographs of P1 showing thin diaphyses of the radius and ulna (F) and of the tibia and fibula (G) as well as marked metaphyseal fraying at the distal radius and ulna, distal femur and proximal tibia compatible with metaphyseal dysplasia. See statement in the disclosure and competing interest section at the end of the manuscript for parental consent to publishing these images. Source data are available online for this figure.

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  Figure 2. Characteristics of LRRC8C and of the variants identified. (A) Schematic representation of the main LRRC8C transcript and its protein product. Both LRRC8Ctrunc (p.Leu400IlefssTer8) and LRRC8CV390L (p.Val390Leu), depicted by black arrowheads, fall into a region of the protein that is bereft of loss-of-function (pLoF, red track) or missense (orange track) variants in normal controls, as reported in the gnomAD database of normal controls, likely representing a sensitive site for DNA changes to result in pathogenic variants, as also shown by the MetaDome track (red, highest intolerance to changes; blue, lowest intolerance to changes). NTC N-terminal coil, TM transmembrane domain, EC extracellular loop, IC intracellular loop, LRRD leucine-rich repeat domain. (B) LRRC8C topology with secondary structure element of the pore PD indicated and the LRRD approximated as blue arc. (C) Conservation of the CTH2 and CTH3 region domains, with respect to the amino acid residues impacted by LRRC8Ctrunc and LRRC8CV390L, in human paralogues of LRRC8C (hLRRC8x) and orthologues from mouse (mLrrc8c) and zebrafish (zLrrc8c). LRRC8C is not present outside of bony vertebrates. (D) Model of the protein…

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  Figure 3. Structural properties of LRRC8C disease mutants. (A) Size exclusion profiles of detergent-solubilized constructs of human LRRC8CWT and the two disease constructs LRRC8Ctrunc and LRRC8CV390L, each containing a C-terminal fusion to GFP. The elution of extracted proteins was detected via their GFP-fluorescence. (B, C) Structure of a homomeric hLRRC8Ctrunc construct. The cryo-EM densities at 7.3 Å prior to the application of symmetry (B) and at 4.9 Å after the application of C7 symmetry (C) are shown superimposed on a model of the PD of the heptameric protein viewed from within the membrane (top) and from the extracellular side (bottom, left). The density of the filter at 3.4 Å, obtained after masking, is shown on the bottom, right (C). Although the ESD is well-defined, the largest parts of the TM and the CSD are absent. (D) Cryo-EM density of a homomeric structure of hLRRC8CV390L at 4.4 Å processed without application of symmetry (C1). (E) Cryo-EM density of hLRRC8CWT at 3.7 Å processed without application of symmetry (C1). (D, E) Left, view from within the membrane contoured at 6.5 σ. Right, view from the extracellular side at 8.5 σ with model shown as ribbon and sidechains of the…

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  Figure EV1. Features of the hLRRC8C mutant V390L. Comparison of the highest resolved cryo-EM maps (contoured at 8σ) obtained from the hLRRC8CV390L and hLRRC8CWT datasets. (A) Cryo-EM map of hLRRC8CV390L (top) and hLRRC8CWT (bottom) superimposed on indicated subunits. (B) hLRRC8CV390L (left) and hLRRC8CWT (right) densities viewed from the cytoplasm. (A, B) Parts of protein subunits that are defined in the density are shown as ribbons, subunit numbering is as in Fig. 3D, E.

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  Figure 4. Heteromeric assembly and function of LRRC8C disease variants. (A) Strategy for the investigation of heteromerization of LRRC8C disease variants. LRRC8C constructs carry an SBP tag for affinity purification and LRRC8A contains a fusion to GFP for detection. (B) Results from a single-step affinity purification. Left, Coomassie-stained SDS-PAGE gel showing both subunits. The LRRC8A-GFP and full-length LRRC8C constructs run at a similar molecular weight around 100 kDa (*), the LRRC8Ctrunc construct (<) at 50 kDa. Right, detection of in-gel fluorescence of GFP-tagged LRRC8A subunits demonstrates its pulldown by different LRRC8C constructs with similar efficiency. LRRC8AGFP refers to cells that were only transfected with LRRC8A-GFP as negative control. (C) SEC chromatogram of purified heteromeric LRRC8A/C channels containing WT and disease constructs of LRRC8C. Inset shows blowup of the peak region. (D–F) Current densities (at 100 mV) of LRRC8−/− cells transfected with constructs coding for hLRRC8A and either hLRRC8CWT or disease mutants assayed by patch-clamp electrophysiology in the whole-cell configuration. Currents were periodically recorded by a ramp protocol. Perfusion with…

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  Figure EV2. Functional properties of hLRRC8C mutants in buffer set Δsalt. (A) Representative current traces and current–voltage relationship of an LRRC8−/− cell transfected with constructs coding for hLRRC8A and hLRRC8CWT after swelling of cells in buffer set Δsalt showing the expected features of an LRRC8A/C channel. (B, C) Representative current traces and current–voltage relationships of an LRRC8−/− cell transfected with constructs coding for hLRRC8A and hLRRC8CV390L under isotonic (B) and hypertonic conditions (C). (D, E) Representative current traces and current–voltage relationships of an LRRC8−/− cell transfected with constructs coding for hLRRC8A and hLRRC8Ctrunc under isotonic (D) and hypertonic conditions (E). The similar general properties of currents obtained under isotonic and hypotonic conditions with respect to the negative reversal potential, the pronounced outward rectification and the inactivation at strongly positive voltages show characteristics of VRACs composed of LRRC8A and C-subunits. (A–E) Currents were recorded by patch-clamp electrophysiology in the whole-cell configuration upon perfusion of cells with either isotonic (B, D) or hypotonic buffer (A, C, E, described…

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  Figure EV3. Functional properties of hLRRC8C mutants in buffer set Δmannitol. (A, B) Mean current density (at 100 mV) of LRRC8A-/- cells (n = 10) (A) and HEK293 cells (n = 8) (B) recorded in buffer set Δmannitol. (C) Representative current traces and current–voltage relationship of LRRC8-/- cells transfected with constructs coding for hLRRC8A and hLRRC8CWT after swelling of cells in buffer set Δmannitol not leading to channel activation. (D) Mean current density (at 100 mV) of hLRRC8A/CWT channels (n = 12) recorded in buffer set Δmannitol. (E) Representative current traces and current–voltage relationship of LRRC8-/- cells transfected with constructs coding for hLRRC8A and hLRRC8CV390L after swelling of cells in buffer set Δmannitol. (F) Mean current density (at 100 mV) of hLRRC8A/CV390L (n = 11) recorded in buffer set Δmannitol. (G) Representative current traces and current–voltage relationship of LRRC8-/- cells transfected with constructs coding for hLRRC8A and hLRRC8Ctrunc after swelling of cells in buffer set Δmannitol. (H) Mean current density (at 100 mV) of hLRRC8A/Ctrunc channels (n = 10) recorded in buffer set Δmannitol. (A–H) Currents were recorded by patch-clamp electrophysiology…

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  Figure EV4. Biochemical and functional characterization of the constructs LRRC8Ct379 and LRRC8Ct419. (A) Models of truncated LRRC8 constructs. A ribbon representation of LRRC8Ctrunc is shown left as reference. The gray box indicates the region of interest in different constructs shown to the right: Center left, LRRC8Ct379, the region on the C-terminus of LRRC8Ctrunc (380–400) that is absent in this construct is highlighted in orange; Center right, LRRC8Ct419, the sequence extension compared to LRRC8Ctrunc (residues 401–419) is highlighted in green; Right, equivalent region of the LRRC8A mutant found in éburiffé mice (which is truncated after residue 442), with the extension compared to LRRC8Ctrunc highlighted in orange. (B) Fluorescence size exclusion chromatogram of GFP-fusions of the constructs LRRC8Ct379 and LRRC8Ct419 after extraction in detergent. Asterisk corresponds to the volume of the heptameric channel. While no oligomer is detected in case of the shorter LRRC8Ct379, the longer LRRC8Ct419 elutes at a similar volume as LRRC8Ctrunc. (C) Mean current density (at 100 mV) of LRRC8-/- cells transfected with constructs coding for LRRC8A and either LRRC8Ct419 (n = 5) or LRRC8Ct419 (n = 8)…

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  Figure 5. Phenotype of human LRRC8C variants. (A) Distribution of variants identified in human LRRC8C and their phenotypical consequence. Positions of variants are mapped on the LRRC8C subunit and shown as spheres. Variants leading to a premature termination of the protein without clinical phenotype (as ascertained in gnomAD, see text) are colored in green, the two variants discussed in this study with strong gain-of-function phenotype in red and violet. (B) Schematic depiction of the effect of disease variants in LRRC8C with pronounced gain-of-function phenotype on channel activity. Two subunits of a VRAC heteromer are shown, where the two LRRC8C disease mutants increase the flexibility of the subunit, leading to channel opening already under isotonic conditions.