Abstract
Ectopic calcification (EC), which is the pathological deposition of calcium and phosphate in extra-skeletal tissues, may be associated with hypercalcaemic and hyperphosphataemic disorders, or it may occur in the absence of metabolic abnormalities. In addition, EC may be inherited as part of several monogenic disorders and studies of these have provided valuable insights into the metabolic pathways regulating mineral metabolism. For example, studies of tumoural calcinosis, a disorder characterised by hyperphosphataemia and progressive EC, have revealed mutations of fibroblast growth factor 23 (FGF23), polypeptide N-acetyl galactosaminyltransferase 3 (GALNT3) and klotho (KL), which are all part of a phosphate-regulating pathway. However, such studies in humans are limited by the lack of available large families with EC, and to facilitate such studies we assessed the progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for EC. This identified two mutants with autosomal recessive forms of EC, and reduced lifespan, designated Ecalc1 and Ecalc2. Genetic mapping localized the Ecalc1 and Ecalc2 loci to a 11.0 Mb region on chromosome 5 that contained the klotho gene (Kl), and DNA sequence analysis identified nonsense (Gln203Stop) and missense (Ile604Asn) Kl mutations in Ecalc1 and Ecalc2 mice, respectively. The Gln203Stop mutation, located in KL1 domain, was severely hypomorphic and led to a 17-fold reduction of renal Kl expression. The Ile604Asn mutation, located in KL2 domain, was predicted to impair klotho protein stability and in vitro expression studies in COS-7 cells revealed endoplasmic reticulum retention of the Ile604Asn mutant. Further phenotype studies undertaken in Ecalc1 (kl203X/203X) mice demonstrated elevations in plasma concentrations of phosphate, FGF23 and 1,25-dihydroxyvitamin D. Thus, two allelic variants of Kl that develop EC and represent mouse models for tumoural calcinosis have been established.
Highlights
Ectopic calcification (EC) is characterized by the pathological deposition of calcium and phosphate in extra-skeletal tissues, and represents a major cause of adverse cardiovascular outcomes and mortality [1]
Studies have highlighted the central role of pyrophosphate as a mineralization regulator, as germline mutations of the ectonucleotide pyrophosphatase/ phosphodiesterase 1 (ENPP1) gene, which encodes an enzyme mediating the generation of extracellular pyrophosphate, lead to idiopathic infantile arterial calcification [4], whilst germline mutations of the ANKH gene, which encodes a transmembrane protein involved in pyrophosphate transport, may lead to chondrocalcinosis [5]
Our study describes two EC mouse mutants, designated kl203X and kl604N, which harbour germline coding-region nonsense and missense klotho gene (Kl) mutations, respectively, that were induced by ENU
Summary
Ectopic calcification (EC) is characterized by the pathological deposition of calcium and phosphate in extra-skeletal tissues, and represents a major cause of adverse cardiovascular outcomes and mortality [1]. Metastatic EC is associated with metabolic abnormalities and arises from sustained elevations in circulating calcium and/or phosphate concentrations, which lead to widespread mineral deposition that affects arterial vessels, kidneys, articular cartilage and peri-articular soft tissues [2], and occurs frequently in major chronic diseases such as chronic renal failure [3]. Studies of tumoural calcinosis (TC), an autosomal recessive disorder characterized by the progressive deposition of calcium phosphate crystals in peri-articular and other soft tissues [6], have revealed hyperphosphataemia to be a major promoter of ectopic calcification and delineated a hormonal mechanism regulating circulating phosphate concentrations [6, 7]. ENU mouse mutants, which can be associated with loss-of-function, hypomorphic, hypermorphic kl203X/203X mice kl/kl mice
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