In this study, we identified what we believe to be the first disease-causing mutation in KL, H193R, in a patient presenting with severe tumoral calcinosis manifested by marked hyperphosphatemia with ectopic and vascular calcifications. The mutation drastically decreased protein expression of the membrane-bound and secreted forms of KL, which led to reduced FGF23-KL-FGFR1c complex formation on the cell surface and, ultimately, to impaired FGF23 signaling. Although substitution of H193 with arginine affects the tertiary fold of KL1 domain in KL, we cannot rule out the possibility that defective enzymatic activity of KL is implicated in impaired FGF23 signaling. Regardless, our data clearly show that KL dysfunction due to H193R mutation leads to a clinical phenotype reflecting impaired biological activity of FGF23.
Kl was originally described as a gene involved in aging, as Kl-deficient mice exhibited shortened lifespan and multiple aging-related phenotypes (13
), whereas overexpression of the Kl gene in mice extended lifespan (24
).Importantly, the phenotype of Kl
-deficient mice closely parallels that of Fgf23
-null mice (16
), indicating that KL and FGF23 have functions in the same metabolic pathway. Similar to mice deficient in Kl
, our patient developed hypercalcemia, hyperphosphatemia, and increased 1,25(OH)2
D levels, resulting in ectopic calcifications and osteopenia. In contrast to Kl
-deficient mice, however, our patient did not have a phenotype suggestive of abnormal or early aging. In addition, there was also no clinical evidence of emphysema, skin atrophy, delayed puberty (hypogonadotropic hypogonadism), or neural degeneration as was observed in the Kl
-deficient mice. Likewise, tumoral calcinosis patients with FGF23
mutations do not show signs of premature aging, although early dementia, most likely from cerebral calcifications, may occur (25
). These observations, together with our present findings, support that KL likely serves as a factor controlling mineral ion homeostasis rather than aging. However, there are potential explanations for a lack of aging features in our patient. The KL
mutation found in our patient did not result in complete loss of KL expression or secretion. Therefore, the patient retained partial KL function, as indicated in Figure . Possibly due to this partial activity of KL, the patient had not developed features of premature aging, although we cannot rule out that she may develop such features over time. It is also possible that humans are able to partially compensate for deficient KL function in a manner that lessens the effect on phosphate metabolism or other metabolic pathways, such that the phenotype may vary in severity and in details from the mouse model. Finally, this study demonstrated that the KL
mutation impaired FGF23 signaling but it may not have affected other less well characterized functions of KL, which could be more relevant to premature aging.
Under normal physiological circumstances, hyperphosphatemia and FGF23 inhibit renal 1,25(OH)2
D synthesis, thereby leading to low 1,25(OH)2
D levels. However, compromised FGF23 signaling due to mutations in any of the 3 genes involved in tumoral calcinosis (FGF23
, and now KL
) disrupts this negative feedback mechanism, resulting in increased serum 1,25(OH)2
D concentrations and hyperphosphatemia (1
). In response to severe hyperphosphatemia and increased 1,25(OH)2
D concentrations, our patient had appropriately elevated intact (functional) and C-terminal FGF23 levels. However, FGF23 signaling in our patient was severely compromised as a result of the diminished ability of mutant KL to form a ternary FGF23-KL-FGFR1c complex (Figure ). In contrast, tumoral calcinosis patients with inactivating mutations in FGF23
have highly elevated C-terminal fragments but low or undetectable levels of intact (active) FGF23 (1
). In these patients, FGF23 production is increased to compensate for hyperphosphatemia, but the FGF23 protein is unstable and readily cleaved by intracellular furin-like convertases, thereby preventing the necessary inhibition of renal phosphate reabsorption.
The disease severity of tumoral calcinosis is quite variable, ranging from solely eyelid calcifications (20
) to massive periarticular calcifications (8
). Even considering the phenotypic variability of this disorder, our patient appeared to be more severely affected than previously reported cases. In this regard, the brain calcifications described herein have only been reported in 2 other patients with GALNT3
mutations (in the choroid plexus [ref. 5
] and dura [refs. 6
]). Furthermore, our patient had clinical features such as osteopenia and hypercalcemia that are seen in Kl
-deficient mice (13
) but have not been reported in previous tumoral calcinosis cases. Interestingly, both our patient and the Kl
-deficient mouse have a combination of osteopenic and hyperostotic bones (13
), similar to the hyperostotic lesions seen in a variant of tumoral calcinosis, hyperostosis-hyperphosphatemia syndrome. These differing clinical phenotypes most likely result from the different functional roles of the mutated genes.
Despite biochemical similarities such as high circulating levels of Fgf23 and hyperphosphatemia (12
), there exist several biochemical differences between the Kl
-deficient mice and our patient. First, although Kl
-deficient mice have increased urinary calcium excretion (29
), our patient did not have hypercalciuria. Second, Kl
-deficient mice have abnormal regulation of insulin/IGF-1 signaling, resulting in hypoglycemia and increased insulin sensitivity. In contrast, our patient had normal fasting glucose and insulin concentrations. Third, PTH levels are low in the Kl
-deficient mice when compared with wild-type mice (15
) and similarly are predominantly low or normal in tumoral calcinosis cases that result from FGF23
mutations. However, our patient had hyperparathyroidism at presentation and subsequently underwent subtotal parathyroidectomy of hyperplastic glands. The reason for the hyperparathyroidism was unclear, as elevated serum phosphate is a stimulus for PTH but high calcium and 1,25(OH)2
D inhibit PTH production. Since KL is expressed in the parathyroid glands (30
), KL may have a direct role in the regulation of PTH in humans. Alternatively, this may be the result of tertiary hyperparathyroidism caused by persistent hyperphosphatemia. The lack of hyperparathyroidism in other tumoral calcinosis patients implies that this may be a feature limited to this particular patient. Further study of additional human cases will be required to clarify whether hyperparathyroidism is a unique response of our patient or a feature inherent to the disorder in humans.
In conclusion, we demonstrated that a homozygous loss-of-function KL mutation in a human resulted in severe tumoral calcinosis, not early aging as previously proposed. Despite the elevated circulating FGF23 concentrations, the H193R mutation diminished the ability of KL to function as a cofactor necessary for FGF23 signaling through FGFRs, leading to hyperphosphatemia, increased 1,25(OH)2D concentrations, and subsequent ectopic calcifications. The fact that a mutation in KL was found in a patient with tumoral calcinosis demonstrates that, in addition to FGF23 and GALNT3, KL should now be considered in the molecular diagnosis of tumoral calcinosis.