Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis

Abstract

Familial tumoral calcinosis (FTC; OMIM 211900) is a severe autosomal recessive metabolic disorder that manifests with hyperphosphatemia and massive calcium deposits in the skin and subcutaneous tissues. Using linkage analysis, we mapped the gene underlying FTC to 2q24-q31. This region includes the gene GALNT3, which encodes a glycosyltransferase responsible for initiating mucin-type O-glycosylation. Sequence analysis of GALNT3 identified biallelic deleterious mutations in all individuals with FTC, suggesting that defective post-translational modification underlies the disease.We assessed 12 individuals with FTC from two large kindreds of Druze and African-American origin (Fig. 1a) that have been extensively described 1,2 . All affected individuals reported recurrent painful, calcified subcutaneous masses of up to 1 kg (Fig. 1b), often resulting in secondary infection and incapacitating mutilation. Three individuals developed deep periarticular tumors (Fig. 1b), and one succumbed to the disease. All affected individuals had hyperphosphatemia (family 1, 6.2-8.5 mg dl -1 ; family 2, 5.2-6.6 mg dl -1 ) but normal levels of calcium, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3.With informed consent of all participants, we obtained DNA samples and carried out a genome-wide scan using 362 microsatellite markers (Research Genetics) in family 1. Consanguinity in this kindred allowed us to apply homozygosity mapping to identify in all affected individuals a 15-Mb segment identical by descent, flanked by D2S142 and D2S2284/D2S2177 on 2q24-q31 (Fig. 1). We obtained a maximum multipoint lod score of 6.7 (HOMOZ 3 ). Multipoint linkage analysis in family 2 using seven markers in this critical region further reduced the interval to 3 Mb flanked by D2S111 and D2S1776 (Fig. 1) and yielded a maximum multipoint lod score of 3.4 (GeneHunter 4 ).Using Mapviewer, we identified 11 genes in the 3-Mb region associated with FTC. Of these, B3GALT1, SCN7A, SCN9A, SCN1A and STK39 have roles in neural or neuroendocrine tissues; the functions of TAIP-2, CMYA3, FLJ11457, LOC90643 and LASS6 are mostly unknown. The last positional candidate gene, GALNT3, encodes the UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (ppGaNTase-T3; ref. 5). ppGaNTase-T3 belongs to a large family of Golgi-associated biosynthetic enzymes that transfer GalNac from the sugar donor UDP-GalNac to serine and threonine residues and are thereby responsible for initiating O-glycan synthesis, a prevalent form of post-translational modification 6 . RT-PCR analysis showed strong expression of GALNT3 in the skin and kidneys, two tissues of functional relevance to the pathogenesis of FTC 1,2 (Fig. 2a). Using balanced primer pairs, we screened PCR amplicons of all ten coding exons and conserved splice sites of GALNT3 for pathogenic mutations in the genomic DNA of affected individuals (primer pairs and PCR conditions are available on request). Members of the Druze family carried a homozygous G→A transition at position 1524+1 (from the ATG ...

Hyperphosphatemic familial tumoral calcinosis caused by a mutation in GALNT3 in a European kindred

“…The existence of various trade routes between Africa and the Middle East during many centuries has been invoked to explain the peculiar geographical distribution of FTC. However, different HFTC-causing mutations have been identified in African-American and Middle Eastern patients (Topaz et al 2004). In the present report, we describe the identification of a novel GALNT3 mutation in a patient of European extraction with features typical of HFTC, demonstrating the existence of GALNT3 mutations outside the Middle Eastern and African populations.…”

“…It is often associated with hyperphosphatemia (Smack et al 1996), and is then termed hyperphosphatemic familial tumoral calcinosis (HFTC; MIM211900). HFTC has been shown to result from loss-of-function mutations in at least two genes: GAL-NT3 coding for UDP-N-acetyl-alpha-D-galactosamine: polypeptide n-acetylgalactosaminyltransferase 3 (ppGalNacT3) (Topaz et al 2004;Ichikawa et al 2005), a glycosyltransferase, which initiates O-glycosylation (Ten Hagen et al 2003);and FGF23 (Benet-Page`s et al 2005;Larsson et al 2005;Araya et al 2005;Chefetz et al 2005) coding for fibroblast growth factor 23 (FGF23), a potent phosphaturic protein (Berndt et al 2005).…”

Mechanism of FGF23 processing in fibrous dysplasia

“…(12) FTC and HHS can result from homozygous mutations in the O-linked glycosyl transferase N-acetylgalactosaminyltransferase 3 (ppGalNAcT3), a member of the galactosaminyl transferase family that is known to transfer N-acetylgalactomine residues to specific serine/threonine residues. (13) It has been shown that mutations in ppGalNAcT3 lead to a loss of FGF23 glycosylation and enhanced cleavage of FGF23, resulting in very low levels of iFGF23 and correspondingly high cFGF23 levels. (14) On the other hand, patients with ADHR who carry mutations in FGF23 within the conserved furin cleavage motif, 176 RHTR 179 , (15,16) have elevated blood iFGF23 levels.…”

Biological activity of FGF-23 fragments

“…It is interesting to note that patients with tumoral calcinosis have elevated concentrations of FGF-23 measured by an assay that detects carboxyl terminal fragments of FGF-23 [17,29]. In contrast, these patients have normal or low-normal concentrations of intact FGF-23.…”

“…Mutations in the gene alter the furin proconvertase recognition site (176RHTR179) in the protein such that the arginine residue at 176 is replaced by glutamine (R176Q) or the arginine residue at amino acid 179 is replaced by a glutamine (R179Q) or tryptophan residue (R179W). In contrast, individuals with tumoral calcinosis have elevated serum phosphate levels and elevated serum levels of the FGF-23 carboxyl terminal fragments [17,29]. Shimada et al have tested the bioactivity of a biosynthetic carboxyl terminal fragment of FGF-23 (aa 180-251) and an N-terminal fragment of FGF-23 and have shown that in contrast to the phosphaturic action of the full-length protein, these peptides fragments are biologically inactive 24 hours after their administration intraperitoneally to mice [11] at a dose of 5 g per mouse every 12 hours.…”