Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene

Abstract

Human disorders of phosphate (Pi) handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. We present the identification of an inactivating mutation in the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR) with phosphaturia by positional cloning. ENPP1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and previously described inactivating mutations in this gene were shown to cause aberrant ectopic calcification disorders, whereas no aberrant calcifications were present in our patients. Our surprising result suggests a different pathway involved in the generation of ARHR and possible additional functions for ENPP1.

Figure 1

Identification of the Y901S Mutation at the Evolutionarily Conserved Y901 Amino Acid (A) Haplotype analysis based on microsatellite markers from 6q revealed a founder haplotype (black bar) for which the patient is homozygous in the critical region harboring the ENPP1 gene. The numbers of the patients correspond to those present in Table 1. (B) Sequence of the corresponding c.A2722C mutation resulting in Y901S. Patients were homozygous for the mutation, parents and siblings carrying the founder haplotype were heterozygous, and the healthy siblings without the founder haplotype were normal. (C) Evolutionary conservation of the replaced amino acid Y901. Multiple sequence alignment was carried out by ClustalW. S represents the mutation. Asterisk (^∗), identical; colon (:), strictly conserved.

Figure 2

The Effect of the Y901S Mutation and the 779P SNP (A) ENPP1 activity in intact COS7 cells transfected with the full-length WT human ENPP1 cDNA (WT), the c.A2722C (Y901S) mutated cDNA (Mut), and the 779P variation (SNP) in pSVT7 expression vectors. Values are expressed as the ratio of NPP activity to time, normalized by total protein concentration and ENPP1 protein levels to correct for transfection efficiency. Untransfected COS7 cells treated in the same way as others served as negative controls, and their background activity was subtracted from the WT, Mut, and SNP activities. ENPP1 levels were determined by immunoblotting and normalized to β-actin. Values are means ± SEM of five independent experiments, each consisting of triplicates of all samples (n = 15). WT: 1.633 ± 0.083. Mut: 0.065 ± 0.302. SNP: 2.775 ± 0.282. ^∗p = 0.006. ^∗∗p = 0.0002 (two-tailed Student's t test, α level was 0.05; data were tested for normality by the Shapiro-Wilk test). (B) Representative immunoblot of the ENPP1 protein of WT, Mut, SNP, and negative control. All visible bands resulting from the transfection were used for the normalization to ENPP1 protein levels. (C) Immunofluorescence staining of COS7 cells transfected with WT and Mut constructs, showing similar membrane localization. Staining performed with the 3E8 mouse monoclonal antibody and secondary antibody Alexa 488 goat anti-mouse IgG. Representative figures of four independent experiments, taken with the Zeiss LSM Axiovert 100 laser scanning microscope. WT scaling (resolution): X, 0.29 μm; Y, 0.29 μm; Z, 0.37 μm. Mut scaling (resolution): X, 0.29 μm; Y, 0.29 μm; Z, 0.32 μm. (D) ENPP1 activity in lysed COS7 cells transfected with WT, Mut, and SNP constructs. Cells were mechanically lysed by passing through a 21G syringe needle until no pellet was visible. Activity is calculated and represented as in (A). Values are means ± SEM of five independent experiments, each consisting of triplicates of all samples (n = 15). WT: 1.653 ± 0.073. Mut: 0.059 ± 0.328. SNP: 2.869 ± 0.307. ^∗p = 0.0067. ^∗∗p = 0.0004 (Student's t test).