Thomas O. Carpenter

SLC34A3 Mutations in Patients with Hereditary Hypophosphatemic Rickets with Hypercalciuria Predict a Key Role for the Sodium-Phosphate Cotransporter NaPi-IIc in Maintaining Phosphate Homeostasis

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare disorder of autosomal recessive inheritance that was first described in a large consanguineous Bedouin kindred. HHRH is characterized by the presence of hypophosphatemia secondary to renal phosphate wasting, radiographic and/or histological evidence of rickets, limb deformities, muscle weakness, and bone pain. HHRH is distinct from other forms of hypophosphatemic rickets in that affected individuals present with hypercalciuria due to increased serum 1,25-dihydroxyvitamin D levels and increased intestinal calcium absorption. We performed a genomewide linkage scan combined with homozygosity mapping, using genomic DNA from a large consanguineous Bedouin kindred that included 10 patients who received the diagnosis of HHRH. The disease mapped to a 1.6-Mbp region on chromosome 9q34, which contains SLC34A3, the gene encoding the renal sodium-phosphate cotransporter NaP(i)-IIc. Nucleotide sequence analysis revealed a homozygous single-nucleotide deletion (c.228delC) in this candidate gene in all individuals affected by HHRH. This mutation is predicted to truncate the NaP(i)-IIc protein in the first membrane-spanning domain and thus likely results in a complete loss of function of this protein in individuals homozygous for c.228delC. In addition, compound heterozygous missense and deletion mutations were found in three additional unrelated HHRH kindreds, which supports the conclusion that this disease is caused by SLC34A3 mutations affecting both alleles. Individuals of the investigated kindreds who were heterozygous for a SLC34A3 mutation frequently showed hypercalciuria, often in association with mild hypophosphatemia and/or elevations in 1,25-dihydroxyvitamin D levels. We conclude that NaP(i)-IIc has a key role in the regulation of phosphate homeostasis.

Multisystem study of 20 older adults with Williams syndrome

To address the natural history of Williams syndrome (WS), we performed multisystem assessments on 20 adults with WS over 30 years of age and documented a high frequency of problems in multiple organ systems. The most striking and consistent findings were: abnormal body habitus; mild–moderate high frequency sensorineural hearing loss; cardiovascular disease and hypertension; gastrointestinal symptoms including diverticular disease; diabetes and abnormal glucose tolerance on standard oral glucose tolerance testing; subclinical hypothyroidism; decreased bone mineral density on DEXA scanning; and a high frequency of psychiatric symptoms, most notably anxiety, often requiring multimodal therapy. Review of brain MRI scans did not demonstrate consistent pathology. The adults in our cohort were not living independently and the vast majority were not competitively employed. Our preliminary findings raise concern about the occurrence of mild accelerated aging, which may additionally complicate the long‐term natural history of older adults with WS. We provide monitoring guidelines to assist in the comprehensive care of adults with WS.

A clinician's guide to X-linked hypophosphatemia

X‐linked hypophosphatemia (XLH) is the prototypic disorder of renal phosphate wasting, and the most common form of heritable rickets. Physicians, patients, and support groups have all expressed concerns about the dearth of information about this disease and the lack of treatment guidelines, which frequently lead to missed diagnoses or mismanagement. This perspective addresses the recommendation by conferees for the dissemination of concise and accessible treatment guidelines for clinicians arising from the Advances in Rare Bone Diseases Scientific Conference held at the NIH in October 2008. We briefly review the clinical and pathophysiologic features of the disorder and offer this guide in response to the conference recommendation, based on our collective accumulated experience in the management of this complex disorder.

A translocation causing increased alpha-klotho level results in hypophosphatemic rickets and hyperparathyroidism.

Phosphate homeostasis is central to diverse physiologic processes including energy homeostasis, formation of lipid bilayers, and bone formation. Reduced phosphate levels due to excessive renal loss cause hypophosphatemic rickets, a disease characterized by prominent bone defects; conversely, hyperphosphatemia, a major complication of renal failure, is accompanied by parathyroid hyperplasia, hyperparathyroidism, and osteodystrophy. Here, we define a syndrome featuring both hypophosphatemic rickets and hyperparathyroidism due to parathyroid hyperplasia as well as other skeletal abnormalities. We show that this disease is due to a de novo translocation with a breakpoint adjacent to α-Klotho, which encodes a β-glucuronidase, and is implicated in aging and regulation of FGF signaling. Plasma α-Klotho levels and β-glucuronidase activity are markedly increased in the affected patient; unexpectedly, the circulating FGF23 level is also markedly elevated. These findings suggest that the elevated α-Klotho level mimics aspects of the normal response to hyperphosphatemia and implicate α-Klotho in the selective regulation of phosphate levels and in the regulation of parathyroid mass and function; they also have implications for the pathogenesis and treatment of renal osteodystrophy in patients with kidney failure.

Randomized trial of the anti-FGF23 antibody KRN23 in X-linked hypophosphatemia

BACKGROUND X-linked hypophosphatemia (XLH) is the most common heritable form of rickets and osteomalacia. XLH-associated mutations in phosphate-regulating endopeptidase (PHEX) result in elevated serum FGF23, decreased renal phosphate reabsorption, and low serum concentrations of phosphate (inorganic phosphorus, Pi) and 1,25-dihydroxyvitamin D [1,25(OH)2D]. KRN23 is a human anti-FGF23 antibody developed as a potential treatment for XLH. Here, we have assessed the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of KRN23 following a single i.v. or s.c. dose of KRN23 in adults with XLH. METHODS Thirty-eight XLH patients were randomized to receive a single dose of KRN23 (0.003-0.3 mg/kg i.v. or 0.1-1 mg/kg s.c.) or placebo. PK, PD, immunogenicity, safety, and tolerability were assessed for up to 50 days. RESULTS KRN23 significantly increased the maximum renal tubular threshold for phosphate reabsorption (TmP/GFR), serum Pi, and 1,25(OH)2D compared with that of placebo (P<0.01). The maximum serum Pi concentration occurred later following s.c. dosing (8-15 days) compared with that seen with i.v. dosing (0.5-4 days). The effect duration was dose related and persisted longer in patients who received s.c. administration. Changes from baseline in TmP/GFR, serum Pi, and serum 1,25(OH)2D correlated with serum KRN23 concentrations. The mean t1/2 of KRN23 was 8-12 days after i.v. administration and 13-19 days after s.c. administration. Patients did not exhibit increased nephrocalcinosis or develop hypercalciuria, hypercalcemia, anti-KRN23 antibodies, or elevated serum parathyroid hormone (PTH) or creatinine. CONCLUSION KRN23 increased TmP/GFR, serum Pi, and serum 1,25(OH)2D. The positive effect of KR23 on serum Pi and its favorable safety profile suggest utility for KRN23 in XLH patients. Trial registration. Clinicaltrials.gov NCT00830674. Funding. Kyowa Hakko Kirin Pharma, Inc.

Genetic Defect in CYP24A1, the Vitamin D 24-Hydroxylase Gene, in a Patient with Severe Infantile Hypercalcemia

CONTEXT Idiopathic infantile hypercalcemia (IIH) is a disorder the genetic etiology and physiological basis of which are not well understood. OBJECTIVE The objective of the study was to describe the underlying physiology and genetic cause of hypercalcemia in an infant with severe IIH and to extend these genetic findings into an additional cohort of children with IIH. DESIGN This was an inpatient study of a single patient with consanguineous parents at an academic medical center with follow-up in a specialty clinic cohort. PATIENTS The patient population was one patient with severe IIH for gene discovery and physiological testing and 27 patients with idiopathic infantile hypercalcemia in the replication cohort. INTERVENTIONS Interventions included a calcium isotopic absorption study as well as homozygosity mapping and whole-exome sequencing in a single patient followed up by gene sequencing in replication cohort. MAIN OUTCOME MEASURE Fractional absorption of calcium and genetic variants causing hypercalcemia were measured. RESULTS Intestinal calcium absorption was extremely elevated (∼90%). A rare homozygous deletion in the CYP24A1 gene was found, leading to the loss of a single highly conserved amino acid. In vivo functional studies confirmed decreased 24-hydroxylase activity because the subject had undetectable levels of 24,25-dihydroxyvitamin D. No coding variants in CYP24A1 were found in the 27 additional patients with IIH. CONCLUSIONS Our study confirms that CYP24A1 plays a causal role in some but not all cases of IIH via markedly increased intestinal absorption of calcium, suggesting that genetic diagnosis could be helpful in a subset of IIH patients. This case demonstrates the power of an unbiased, genome-wide approach accompanied by informative physiological studies to provide new insights into human biology.

Exome sequencing reveals FAM20c mutations associated with fibroblast growth factor 23–related hypophosphatemia, dental anomalies, and ectopic calcification

Fibroblast growth factor 23 (FGF23) plays a crucial role in renal phosphate regulation, exemplified by the causal role of PHEX and DMP1 mutations in X‐linked hypophosphatemic rickets and autosomal recessive rickets type 1, respectively. Using whole exome sequencing we identified compound heterozygous mutations in family with sequence similarity 20, member C (FAM20C) in two siblings referred for hypophosphatemia and severe dental demineralization disease. FAM20C mutations were not found in other undiagnosed probands of a national Norwegian population of familial hypophosphatemia. Our results demonstrate that mutations in FAM20C provide a putative new mechanism in human subjects leading to dysregulated FGF23 levels, hypophosphatemia, hyperphosphaturia, dental anomalies, intracerebral calcifications and osteosclerosis of the long bones in the absence of rickets.

Treatment of X-linked hypophosphatemia with calcitriol and phosphate increases circulating fibroblast growth factor 23 concentrations.

CONTEXT X-Linked hypophosphatemia (XLH) is characterized by renal phosphate wasting, with inappropriately low or normal serum 1,25-dihydroxyvitamin D concentrations causing rickets and osteomalacia. Mutations in PHEX result in increased fibroblast growth factor 23 (FGF23) expression, elevating circulating FGF23 concentrations. Treating XLH with phosphate and calcitriol may further increase FGF23 concentrations, based on in vitro and in vivo models. OBJECTIVE The aim of the study was to investigate whether current standard XLH therapies increase circulating FGF23 concentrations. DESIGN AND SETTING We conducted a prospective observational study of XLH subjects during routine clinical management at two tertiary referral centers. PATIENTS The study included 10 XLH patients (seven children, three adults; age, 2-30 yr) initiating therapy and five XLH patients (age, 18-41 yr) electing not to undergo therapy. INTERVENTION(S) Oral calcitriol and phosphate were administered. MAIN OUTCOME MEASURES We measured circulating intact FGF23 concentrations. RESULTS Baseline circulating FGF23 concentrations were elevated in 14 of 15 subjects, increasing after treatment in most subjects. Follow-up was 14.4 +/- 11.7 months (treatment cohort) and 25 +/- 32 months (nontreatment cohort). FGF23 concentrations increased 132.7 +/- 202.4% from pretreatment to peak during therapy but did not change significantly over time in the nontreatment cohort. FGF23 concentrations were related to phosphate doses (P = 0.04) and nonsignificantly to calcitriol doses (P = 0.06). CONCLUSIONS Treating XLH with phosphate and calcitriol was associated with concurrent increases in circulating FGF23 concentrations, which may diminish therapeutic effect or contribute to complications of therapy. Because it is unknown whether the degree of FGF23 elevation correlates with disease severity in XLH, further study is needed to determine whether adjusting therapy to minimize effects on FGF23 concentration is warranted.

Circulating Levels of Soluble Klotho and FGF23 in X-Linked Hypophosphatemia: Circadian Variance, Effects of Treatment, and Relationship to Parathyroid Status

CONTEXT Circulating fibroblast growth factor (FGF)-23 is variably elevated in individuals with X-linked hypophosphatemia (XLH), and klotho has recently been shown to effect renal phosphate handling, yet limited data are available on circulating FGF23 and klotho in XLH. OBJECTIVE The objective of the study was to characterize circulating FGF23 and klotho in XLH. DESIGN Children and adults with XLH withheld medication for 14 d. Fasting serum FGF23, PTH, klotho, phosphate, and 1,25 dihydroxyvitamin D were obtained. Treated adults were also sampled, and circadian sampling was performed in selected individuals. SETTING The study was conducted at a hospital research unit at an academic medical center. PATIENTS AND OTHER PARTICIPANTS Participants included 23 individuals with XLH and eight controls. INTERVENTIONS There were no interventions. MAIN OUTCOME MEASURES Serum klotho and FGF23 were measured. RESULTS FGF23 was greater in XLH than in controls and greater in treated XLH subjects compared with XLH subjects not receiving medical therapy. Children had higher klotho levels than adults, but values in XLH were similar to controls. A strong positive correlation between FGF23 and PTH was found in XLH subjects, whereas there was no relationship between these variables in controls. Circulating klotho, but not FGF23, has a diurnal pattern. CONCLUSIONS Serum klotho declines with age and demonstrates circadian variation but is normal in XLH. Serum FGF23 is similar in children and adults, is elevated in XLH, further increases with therapy, and demonstrates no diurnal variation. The direct relationship between FGF23 and PTH in subjects with XLH suggests that FGF23 regulation of PTH secretion is aberrant in this disorder.

The expanding family of hypophosphatemic syndromes

Investigation of X-linked hypophosphatemia (XLH) has led to the identification of a novel phosphate-regulating homeostatic system. Initially considered vitamin D-refractory rickets, renal phosphate wasting was identified as the cardinal biochemical feature of XLH and several related disorders. Current therapy employs calcitriol and phosphate, which usually improves, but does not completely heal deformities and short stature. Later complications of XLH include development of osteophytes, entheses, and osteoarthritis. The mutated gene in XLH, PHEX, is expressed in osteocytes, but its role in the pathogenesis of phosphate wasting is poorly understood. Many hypophosphatemic disorders are mediated by FGF23, a unique fibroblast growth factor with endocrine properties. Renal action of FGF23 leads to reduced expression of type II sodium-phosphate co-transporters, as well as reduced expression of CYP27B1, which encodes vitamin D 1α-hydroxylase. FGF23-mediated hypophosphatemia is characterized by inappropriately normal circulating 1,25-dihydroxyvitamin D together with renal phosphate wasting. The FGF23 system serves as a novel mechanism by which the mineralizing skeleton can communicate phosphate supply to the kidney and thereby mediate excretion or conservation of this important skeletal component. Other forms of FGF23-mediated hypophosphatemia represent various aberrations in this axis. Secretion of excess FGF23 (as in tumor-induced osteomalacia), and mutations preventing proteolytic cleavage of FGF23 result in similar clinical features. Other hypophosphatemic disorders are discussed.