A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis

Abstract

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Herein we report a homozygous missense mutation (H193R) in the KLOTHO (KL) gene of a 13-year-old girl who presented with severe tumoral calcinosis with dural and carotid artery calcifications. This patient exhibited defects in mineral ion homeostasis with marked hyperphosphatemia and hypercalcemia as well as elevated serum levels of parathyroid hormone and FGF23. Mapping of H193R mutation onto the crystal structure of myrosinase, a plant homolog of KL, revealed that this histidine residue was at the base of the deep catalytic cleft and mutation of this histidine to arginine should destabilize the putative glycosidase domain (KL1) of KL, thereby attenuating production of membrane-bound and secreted KL. Indeed, compared with wild-type KL, expression and secretion of H193R KL were markedly reduced in vitro, resulting in diminished ability of FGF23 to signal via its cognate FGF receptors. Taken together, our findings provide what we believe to be the first evidence that loss-of-function mutations in human KL impair FGF23 bioactivity, underscoring the essential role of KL in FGF23-mediated phosphate and vitamin D homeostasis in humans.

Figure 1. Radiographic imaging of the patient carrying the KL H193R mutation.

(A) Intracranial calcification and short bulbous tooth roots. (B) Sclerosis in the left hand. (C) Head CT demonstrating midline areas of dural calcification and posterior fossa calcifications (arrows). (D) Plain radiograph of ankle with Achilles tendon calcifications (arrow).

Figure 2. Mutational and structural analyses of the KL H193R mutation.

(A) Mutational analysis of the KL gene. DNA sequencing of the KL exons revealed an A-to-G transition in exon 1 (boxes). Left, normal control; right, patient. c.578A>G, homozygous A-to-G transition. (B) Sequence alignment of the region encompassing the mutated histidine residue of KL and related family-1 glycosidases. LPH, lactase phlorizinhydrolase; MYR, myrosinase. Residue numbers are in parentheses to the left of the alignment. For myrosinase, secondary structure elements are denoted below the sequence, and the locations and lengths of the secondary structure elements are indicated by boxes in the sequence. H, α helix; β, β strand; g, g helix. A dash in the sequence represents a gap introduced to optimize the alignment. The histidine residue, which is mutated in KL of the patient presented in this study, is colored red. Note that this residue is conserved among family-1 glycosidases and their orthologs. (C) Ribbon representation of the crystal structure of myrosinase (Protein Data Bank [PDB] ID, 1E6S) and surface representation of catalytic site residues. White mustard myrosinase is a plant homolog of human KL. A close-up view of the catalytic cavity with bound substrate (gluco-hydroximolactam) is shown on the right, and to orient the reader, a view of the whole structure is shown on the left. Note that histidine 141 of the catalytic site is homologous to H193 of human KL.

Figure 3. The KL mutation affects the total KL protein expression.

(A) The KL mutation significantly reduces expression and secretion of KL. Wild-type and H193R KL isoforms were transiently expressed in HEK293 cells. Proteins in culture media (M) and cell lysates (L) were analyzed by immunoblotting with anti-V5 (KL) and β-actin antibodies. Only 1/20 volume of wild-type–secreted KL media (M_1/20; compared with H193R KL) was loaded on the gel. mKL, membrane-bound KL; sKL, secreted KL. (B) Temperature rescue of membrane KL expression. Cells stably expressing either wild-type or H193R membrane-bound KL were seeded equally and maintained in culture at either 37°C or 28.5°C. Expression of wild-type KL was comparable at the 2 temperatures tested, whereas increased amounts of fully glycosylated mutant KL protein was produced at the lower temperature in 2 independent experiments. All data are representative of at least 3 independent experiments.

Figure 4. The KL mutation impairs KL-dependent FGF23 signaling.

HEK293 cells were transiently transfected with vector alone (BLK), wild-type, or mutant (MUT) membrane-bound KL. The cells were treated for 30 minutes with 100 ng/ml FGF23 or vehicle. Relative EGR1 mRNA expression was determined by real-time quantitative RT-PCR using β-actin as the internal standard (n = 6–8). Results are presented as fold change (mean ± SEM) compared to unstimulated cells. *P < 0.01, compared with BLK; ^†P < 0.01, compared with the same plasmid transfection without FGF23 treatment; ^‡P < 0.01, compared with WT+FGF23.

Figure 5. The KL mutation impairs the ability of membrane-bound KL to form a ternary complex with FGF23 and FGFR1c.

HEK293 cells overexpressing wild-type or mutant mKL were transfected with FGFR1c and treated with FGF23. KL or FGF23 were immunoprecipitated, and the immunoprecipitates were analyzed for coprecipitated proteins by immunoblotting with the antibodies indicated.