Sartelet A , Stauber T , Coppieters W , Ludwig CF , Fasquelle C , Druet T , Zhang Z , Ahariz N , Cambisano N , Jentsch TJ , Charlier C .

	Fig. 1. Clinical features of congenital hamartomas in affected Belgian Blue calves. (A) Premature stillborn calf exhibiting gingival hamartoma, abnormal skull shape, hydrops and hepatomegaly. Inset shows livers from mutant (left) and wild-type (right) calves. (B) Sagittal section of a case head revealing a hamartoma within the inferior jaw. (C) Dead case presenting a voluminous hamartoma; note teeth inclusion (arrow). (D) Alive case with abnormal skull shape accompanied by a protruding tongue.
	Fig. 2. Genome-wide association mapping for the hamartoma disease locus. (A) Manhattan plot for case/control GWAS presenting a unique genome-wide significant signal on chromosome 25; the 29 autosomes are alternately labeled in gray or black. inset shows a typical hamartoma case. (B) Cases genotypes for 1256 chromosome 25 SNP; homozygous genotypes are shown in yellow or white, heterozygous genotypes in red; the centromeric 1.15-Mb homozygosity region, identical by state among all cases, is highlighted in red.
	Fig. 3. Missense mutations in the second CBS domain of the ClC-7 protein. (A) Sequence traces of CLCN7 exon 23 for wild-type (top) and mutant (bottom) calves; triangles pinpoint three nucleotide substitutions with the corresponding Y750Q amino acid mutation in red. (B) X-ray structure of CmCLC displaying the localization of the mutated amino acid (red spheres in either subunit of the dimer) according to the published alignment between CmCLC and ClC-7 (Feng et al., 2010). The transmembrane core-forming parts are depicted in gray, the cytosolic domains CBS1 in yellow and CBS2 in green, using darker colors for one subunit. (C) ClC-7 CBS2 domain alignment, from mammals to fish, showing its conservation through evolution with the Y750Q mutation in red.
	Fig. 4. Severe osteopetrotic phenotype exhibited by Y750Q homozygous calves. (A) Dorsoventral radiographs of extended hind legs of a one-week-old homozygous mutant calf (MUT, right) and an age-matched Belgian Blue control calf (WT, left) are presented. X-rays were performed using a digital radiograph system [Vertix Vet (150 kV/600 mA), Siemens, Germany] with 75 kV and 100 mA as technical parameters. The bone marrow cavity is clearly visible within metatarsus of the wild-type calf but absent from the mutant corresponding bone (red arrows). The mutant calf also exhibits a tibia fracture (white arrow) probably consecutive to the acknowledged increased fragility of osteopetrotic long bones. (B) Fresh transversal (left) and sagittal (right) sections of long bones (tibia) of age-matched mutant (MUT) and control (WT) calves showing an absence of central bone marrow cavity for the mutant.
	Fig. 5. Lysosomal storage in a homozygous ClC-7(Y750Q) calf. Lysates (60 μg protein per lane) of cerebellum from a control calf (WT) and from a calf homozygous for the Y750Q mutation (MUT) were analyzed by immunoblot against the indicated proteins. Immunoblotting for α-tubulin served as loading control. (A) Levels of subunit c of the mitochondrial ATP synthase were increased in the mutated calf. (B) The animals displayed no differences in the levels of the preprocathepsin D (immature) or the intermediate and mature forms of cathepsin D. (C) The autophagic marker LC3-II was increased in the mutated calf whereas overall LC3 levels were unchanged.
	Fig. 6. Protein levels of ClC-7 and Ostm1. Membrane protein-enriched lysates (80 μg per lane) of kidney from a control calf (WT) and a calf homozygous for the Y750Q mutation (MUT), as well as from a Clcn7−/− mouse (KO) and its wild-type littermate (WT) were analyzed by western blot for ClC-7 (A) and Ostm1 (with an antibody directed against a C-terminal epitope that also recognizes the proteolytically processed transmembrane fragment) (B). Immunoblotting for α-tubulin served as loading control. Lack of ClC-7 signal in lysate from the Clcn7−/− mouse shows the specificity of the antibody. Ostm1, which migrates with an apparently higher molecular weight in the bovine samples, exists predominantly in its proteolytically processed form (~30 kDa). The different sizes could be due to species-specific differences in glycosylation.
	Fig. 7. Correct subcellular targeting of ClC-7/Ostm1 upon heterologous expression. (A) After transient transfection with rat ClC-7, either wild-type (WT) or the Y744Q mutant (MUT), HeLa cells were immunolabeled for ClC-7 (green in overlay) and the late endosomal/lysosomal marker protein LAMP-1 (red); blue in overlay indicates DAPI staining of nuclei. (B) HeLa cells co-transfected with rat ClC-7 (either WT or MUT) and mOstm1-GFP (green in overlay), or with Ostm1-GFP alone (bottom panel) were immunolabeled for ClC-7 (red) and the late endosomal/lysosomal marker protein LAMP-1 (blue).
	Fig. 8. Accelerated gating of ClC-7/Ostm1 by the disease-causing mutation. Xenopus oocytes coexpressing Ostm1 and partially plasma membrane-localized hClC-7PM, either without further mutation (‘WT′) or carrying the Y746Q mutation (MUT), were recorded in a two-electrode voltage clamp. (A) Current traces (representative for three batches of oocytes) recorded with the clamp protocol shown on the right (holding potential −30 mV, subsequent 2-second test pulses between −80 mV and 80 mV in 20-mV intervals, each followed by a 0.5-second deactivation pulse at −80 mV) are shown as superimposition for ‘WT′ and MUT. Activation (black arrows, quantified in C) and relaxation kinetics (white arrows) of the currents were accelerated by the Y746Q mutation. Scales for the time and intensity of the current traces are shown on the right (the clamp protocol is not shown in scale). (B) Mean currents after 2 seconds were normalized to the current at +80 mV and plotted as function of voltage. Values are the mean of 13 (‘WT′) and 18 (MUT) oocytes from three batches of oocytes, small error bars (s.e.m.) are hidden behind the symbol. (C) Rate constants of current activation were determined by a single exponential fit of the current trace during the first 250 milliseconds of depolarization to 80 mV for each of the measured oocytes. Thick lines in data point clouds indicate the arithmetic mean, and thin lines the s.e.m. (P<10−6 calculated by t-test between ‘WT′ and MUT).

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