Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Dis Model Mech
2014 Jan 01;71:119-28. doi: 10.1242/dmm.012500.
Show Gene links
Show Anatomy links
A missense mutation accelerating the gating of the lysosomal Cl-/H+-exchanger ClC-7/Ostm1 causes osteopetrosis with gingival hamartomas in cattle.
Sartelet A, Stauber T, Coppieters W, Ludwig CF, Fasquelle C, Druet T, Zhang Z, Ahariz N, Cambisano N, Jentsch TJ, Charlier C.
???displayArticle.abstract???
Chloride-proton exchange by the lysosomal anion transporter ClC-7/Ostm1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or Ostm1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/Ostm1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC-7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/Ostm1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/Ostm1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/Ostm1 in lysosomal function and bone resorption.
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).
Adzhubei,
A method and server for predicting damaging missense mutations.
2010, Pubmed
Adzhubei,
A method and server for predicting damaging missense mutations.
2010,
Pubmed Barvencik,
CLCN7 and TCIRG1 mutations differentially affect bone matrix mineralization in osteopetrotic individuals.
2014,
Pubmed Browning,
Rapid and accurate haplotype phasing and missing-data inference for whole-genome association studies by use of localized haplotype clustering.
2007,
Pubmed Chalhoub,
Grey-lethal mutation induces severe malignant autosomal recessive osteopetrosis in mouse and human.
2003,
Pubmed Charlier,
Highly effective SNP-based association mapping and management of recessive defects in livestock.
2008,
Pubmed Charlier,
A deletion in the bovine FANCI gene compromises fertility by causing fetal death and brachyspina.
2012,
Pubmed Coury,
SLC4A2-mediated Cl-/HCO3- exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts.
2013,
Pubmed Drost,
Complications during gestation in the cow.
2007,
Pubmed Druet,
A hidden markov model combining linkage and linkage disequilibrium information for haplotype reconstruction and quantitative trait locus fine mapping.
2010,
Pubmed Fasquelle,
Balancing selection of a frame-shift mutation in the MRC2 gene accounts for the outbreak of the Crooked Tail Syndrome in Belgian Blue Cattle.
2009,
Pubmed Feng,
Structure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle.
2010,
Pubmed Friedrich,
Mutational analysis demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane currents.
1999,
Pubmed
,
Xenbase Grobet,
A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle.
1997,
Pubmed Jentsch,
CLC chloride channels and transporters: from genes to protein structure, pathology and physiology.
2008,
Pubmed Kasper,
Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration.
2005,
Pubmed Koivusalo,
In situ measurement of the electrical potential across the lysosomal membrane using FRET.
2011,
Pubmed Kornak,
Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.
2001,
Pubmed Lange,
ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function.
2006,
Pubmed Leisle,
ClC-7 is a slowly voltage-gated 2Cl(-)/1H(+)-exchanger and requires Ostm1 for transport activity.
2011,
Pubmed Li,
Fast and accurate short read alignment with Burrows-Wheeler transform.
2009,
Pubmed Li,
Biophysical properties of ClC-3 differentiate it from swelling-activated chloride channels in Chinese hamster ovary-K1 cells.
2000,
Pubmed Li,
The Sequence Alignment/Map format and SAMtools.
2009,
Pubmed Ludwig,
Common gating of both CLC transporter subunits underlies voltage-dependent activation of the 2Cl-/1H+ exchanger ClC-7/Ostm1.
2013,
Pubmed
,
Xenbase Lupski,
Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy.
2010,
Pubmed Luzzi,
Malignant infantile osteopetrosis: dental effects in paediatric patients. Case reports.
2006,
Pubmed Marchler-Bauer,
CDD: a Conserved Domain Database for the functional annotation of proteins.
2011,
Pubmed McKenna,
The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.
2010,
Pubmed Meyers,
A deletion mutation in bovine SLC4A2 is associated with osteopetrosis in Red Angus cattle.
2010,
Pubmed Neagoe,
The late endosomal ClC-6 mediates proton/chloride countertransport in heterologous plasma membrane expression.
2010,
Pubmed
,
Xenbase Ng,
Targeted capture and massively parallel sequencing of 12 human exomes.
2009,
Pubmed Pressey,
Distinct neuropathologic phenotypes after disrupting the chloride transport proteins ClC-6 or ClC-7/Ostm1.
2010,
Pubmed Robinson,
Integrative genomics viewer.
2011,
Pubmed Sartelet,
Allelic heterogeneity of Crooked Tail Syndrome: result of balancing selection?
2012,
Pubmed Sartelet,
A splice site variant in the bovine RNF11 gene compromises growth and regulation of the inflammatory response.
2012,
Pubmed Schrider,
Pervasive multinucleotide mutational events in eukaryotes.
2011,
Pubmed Schulz,
The G215R mutation in the Cl-/H+-antiporter ClC-7 found in ADO II osteopetrosis does not abolish function but causes a severe trafficking defect.
2010,
Pubmed Sim,
SIFT web server: predicting effects of amino acid substitutions on proteins.
2012,
Pubmed Stauber,
Sorting motifs of the endosomal/lysosomal CLC chloride transporters.
2010,
Pubmed
,
Xenbase Stauber,
Cell biology and physiology of CLC chloride channels and transporters.
2012,
Pubmed Stauber,
Chloride in vesicular trafficking and function.
2013,
Pubmed Steinberg,
A cation counterflux supports lysosomal acidification.
2010,
Pubmed Steward,
Neurological aspects of osteopetrosis.
2003,
Pubmed Sui,
Rapid gene identification in a Chinese osteopetrosis family by whole exome sequencing.
2013,
Pubmed Teitelbaum,
Bone resorption by osteoclasts.
2000,
Pubmed Tolar,
Osteopetrosis.
2004,
Pubmed Volpi,
Targeted next-generation sequencing appoints c16orf57 as clericuzio-type poikiloderma with neutropenia gene.
2010,
Pubmed Wartosch,
Lysosomal degradation of endocytosed proteins depends on the chloride transport protein ClC-7.
2009,
Pubmed Weinert,
Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal Cl- accumulation.
2010,
Pubmed Wu,
HCO3-/Cl- anion exchanger SLC4A2 is required for proper osteoclast differentiation and function.
2008,
Pubmed Xue,
Report of two Chinese patients suffering from CLCN7-related osteopetrosis and root dysplasia.
2012,
Pubmed Zhang,
Ancestral haplotype-based association mapping with generalized linear mixed models accounting for stratification.
2012,
Pubmed
