Mutation of SGK3, a Novel Regulator of Renal Phosphate Transport, Causes Autosomal Dominant Hypophosphatemic Rickets.

CONTEXT: Hypophosphatemic rickets (HR) is a group of rare hereditary renal phosphate wasting disorders caused by mutations in PHEX, FGF23, DMP1, ENPP1, CLCN5, SLC9A3R1, SLC34A1, or SLC34A3. OBJECTIVE: A large kindred with 5 HR patients was recruited with dominant inheritance. The study was undertaken to investigate underlying genetic defects in HR patients. DESIGN: Patients and their family members were initially analyzed for PHEX and FGF23 mutations using polymerase chain reaction sequencing and copy number analysis. Exome sequencing was subsequently performed to identify novel candidate genes. RESULTS: PHEX and FGF23 mutations were not detected in the patients. No copy number variation was observed in the genome using CytoScan HD array analysis. Mutations in DMP1, ENPP1, CLCN5, SLC9A3R1, SLC34A1, or SLC34A3 were also not found by exome sequencing. A novel c.979-96 T>A mutation in the SGK3 gene was found to be strictly segregated in a heterozygous pattern in patients and was not present in normal family members. The mutation is located 1 bp downstream of a highly conserved adenosine branch point, resulted in exon 13 skipping and in-frame deletion of 29 amino acids, which is part of the protein kinase domain and contains a Thr-320 phosphorylation site that is required for its activation. Protein tertiary structure modelling showed significant structural change in the protein kinase domain following the deletion. CONCLUSIONS: The c.979-96 T>A splice mutation in the SGK3 gene causes exon 13 skipping and deletion of 29 amino acids in the protein kinase domain. The SGK3 mutation may cause autosomal dominant HR.

Micro-CT assessment of dental mineralization defects indicative of vitamin D deficiency in two 17th-19th century Dutch communities.

OBJECTIVES: This study investigates vitamin D deficiency patterns in individuals from birth to the beginning of adolescence. Microscopic computed tomography (micro-CT) evaluation of interglobular dentine (IGD) in teeth provides information on the age of disease onset and the number of deficient periods per individual, which will increase our understanding of factors influencing vitamin D deficiency prevalence, including sociocultural practices and latitude. MATERIALS AND METHODS: Beemster and Hattem, two Dutch 17th-19th century communities, yielded relatively high prevalences of rickets (15-24%) and residual rickets (15-24%). From the affected individuals, a subsample of 20 teeth were selected for micro-CT scanning. Thin sections were made of 17 teeth, consisting of 6 teeth with and 11 teeth without observable IGD on micro-CT that were included for method comparison. RESULTS: About 19 out of 29 (65.5%) individuals (one tooth was deemed unobservable) presented with IGD on micro-CT. Eight of the 11 (72.7%) individuals without IGD on micro-CT demonstrated histologically visible IGD. In 40.7% (11/27) of the affected individuals (combined micro-CT and histology results), vitamin D deficiency was recurrent, and in four individuals, some episodes occurred at approximately annual intervals suggesting vitamin D deficiency was seasonal. In three individuals, IGD occurred in the dentine formed around birth, suggesting maternal vitamin D deficiency. DISCUSSION: Micro-CT analysis of IGD is found to be a valuable non-destructive method that can improve our understanding of the influence of sociocultural practices and latitude on disease development within age and sex groups in past communities.

Analysis of patterning in the occurrence of skeletal lesions used as indicators of vitamin D deficiency in subadult and adult skeletal remains.

Paleopathological investigations of conditions linked to vitamin D deficiency have increased in the last twenty years, and a suite of skeletal lesions has been established to aid in the diagnosis of vitamin D deficiency disease in subadults and adults. This paper analyzes the occurrence of these lesions in a large skeletal series comprising 3541 Roman period individuals (1st-6th century AD). Sixteen lesions reported in rickets in subadults, and 13 associated with residual rickets and osteomalacia in adults, were analyzed. Among subadults, there were clear associations among post-cranial lesions. Porotic cranial changes were associated with each other, but not with post-cranial lesions. A range of conditions could have produced the cranial lesions. There was a general paucity of correlations between indicators found in adults, and the difficulty in recording bending deformities was clear. Pseudofractures appear to provide a useful means of investigating osteomalacia in adults. In general, a simple algorithmic approach using presence or absence of lesions is unlikely to provide an adequate means of diagnosing vitamin D deficiency in paleopathology. Knowledge and consideration of the underlying physiological mechanisms involved in lesion formation, combined with individual judgement, will be required to differentially diagnose cases.

Osteological evidence for juvenile vitamin D deficiency in a 19th century suburban population from Surrey, England.

Vitamin D deficiency rickets was considered endemic in the industrialized cities of 19th century England, but was rarely reported in more rural and suburban areas. The commercial excavation of St. John's Church, Redhill, Surrey, UK provided an opportunity to examine to what extent suburban children were affected by rickets and the factors responsible for its development. Seventy-nine non-adults (0-17 years) from St. John's Church were subjected to macroscopic and radiographic analysis to identify skeletal manifestations of vitamin D deficiency. Rachitic lesions were identified in 14/79 individuals (17.7%) aged from six months to six years. Active cases occurred from six months to two years of age with healed cases observed from three to six years. One seven month old infant also displayed healed lesions. The age-specific pattern of active and healed rickets suggests the population was vulnerable to the seasonal restriction of sunlight hours, with the considerably low vitamin D content of the infant diet unable to provide sufficient amounts to maintain metabolic functions. This research demonstrates that rickets was not simply a disease of industrialization but that a variety of factors contributed to its development in groups previously considered to be low risk.

Cardiac, bone and growth plate manifestations in hypocalcemic infants: revealing the hidden body of the vitamin D deficiency iceberg.

BACKGROUND: Whilst hypocalcemic complications from vitamin D deficiency are considered rare in high-income countries, they are highly prevalent among Black, Asian and Minority Ethnic (BAME) group with darker skin. To date, the extent of osteomalacia in such infants and their family members is unknown. Our aim was to investigate clinical, cardiac and bone histomorphometric characteristics, bone matrix mineralization in affected infants and to test family members for biochemical evidence of osteomalacia. CASE PRESENTATION: Three infants of BAME origin (aged 5-6 months) presented acutely in early-spring with cardiac arrest, respiratory arrest following seizure or severe respiratory distress, with profound hypocalcemia (serum calcium 1.22-1.96 mmol/L). All infants had dark skin and vitamin D supplementation had not been addressed during child surveillance visits. All three had severely dilated left ventricles (z-scores + 4.6 to + 6.5) with reduced ejection fraction (25-30%; normal 55-70), fractional shortening (7 to 15%; normal 29-40) and global hypokinesia, confirming hypocalcemic dilated cardiomyopathy. They all had low serum levels of 25 hydroxyvitamin D (25OHD < 15 nmol/L), and elevated parathyroid hormone (PTH; 219-482 ng/L) and alkaline phosphatase (ALP; 802-1123 IU/L), with undiagnosed rickets on radiographs. One infant died from cardiac arrest. At post-mortem examination, his growth plate showed a widened, irregular zone of hypertrophic chondrocytes. Histomorphometry and backscattered electron microscopy of a trans-iliac bone biopsy sample revealed increased osteoid thickness (+ 262% of normal) and osteoid volume/bone volume (+ 1573%), and extremely low bone mineralization density. Five of the nine tested family members had vitamin D deficiency (25OHD < 30 nmol/L), three had insufficiency (< 50 nmol/L) and 6/9 members had elevated PTH and ALP levels. CONCLUSIONS: The severe, hidden, cardiac and bone pathology described here exposes a failure of public health prevention programs, as complications from vitamin D deficiency are entirely preventable by routine supplementation. The family investigations demonstrate widespread deficiency and undiagnosed osteomalacia in ethnic risk groups and call for protective legislation.

Soluble Klotho causes hypomineralization in Klotho-deficient mice.

The type I transmembrane protein αKlotho (Klotho) serves as a coreceptor for the phosphaturic hormone fibroblast growth factor 23 (FGF23) in kidney, while a truncated form of Klotho (soluble Klotho, sKL) is thought to exhibit multiple activities, including acting as a hormone, but whose mode(s) of action in different organ systems remains to be fully elucidated. FGF23 is expressed primarily in osteoblasts/osteocytes and aberrantly high levels in the circulation acting via signaling through an FGF receptor (FGFR)-Klotho coreceptor complex cause renal phosphate wasting and osteomalacia. We assessed the effects of exogenously added sKL on osteoblasts and bone using Klotho-deficient (kl/kl) mice and cell and organ cultures. sKL induced FGF23 signaling in bone and exacerbated the hypomineralization without exacerbating the hyperphosphatemia, hypercalcemia and hypervitaminosis D in kl/kl mice. The same effects were seen in rodent bone models in vitro, in which we also detected formation of a sKL complex with FGF23-FGFR and decreased Phex (gene responsible for X-linked hypophosphatemic rickets (XLH)/osteomalacia) expression. Further, sKL-FGF23-dependent hypomineralization in vitro was rescued by soluble PHEX. These data suggest that exogenously added sKL directly participates in FGF23 signaling in bone and that PHEX is a downstream effector of the sKL-FGF23-FGFR axis in bone.

Multiple unexplained fractures in infants and child physical abuse.

When an infant presents with X-rays showing multiple unexplained fractures in various stages of healing (MUFVSH), the child is usually diagnosed with child abuse based on criteria of the Academy of Pediatrics' Committee on Child Abuse and Neglect (AAPCCAAN). Almost always, the infant is subsequently removed from the home and civil or criminal proceeding commence. It may be that healing infantile rickets or other poorly understood metabolic bone disorders of infancy are responsible for these x-rays. Activated vitamin D is a seco-steroid hormone, whose mechanism of action is genetic regulation. Lack of it can result in musculoskeletal defects known as rickets. Low calcium can also cause rickets. However, it is clear that experts for the state believe that the x-rays in these cases are so definitive as to be pathognomonic for child abuse. Therefore, if the caregivers deny abusing their infants, experts following American Academy of Pediatric's Committee on Child Abuse and Neglect. guidelines are essentially claiming that x-rays showing multiple unexplained fractures in various stages of healing are lie detector tests. However, it is not widely appreciated that the gold standard for the diagnosis of rickets is a bone biopsy, not x-rays, as radiologists miss biopsy proven rickets 80% of the time; that is, 4 out of 5 infants with rickets will have normal x-rays. In this article we provide reports of two cases and their outcomes. We discuss information about healing infantile rickets and an example of common sense medical conclusions in these cases. This information could lead to a significant reduction in the number of innocent parents having their infant removed or sent to prison.

Rickets in a 6-year-old girl resulting in extreme deformities.

Rickets remains endemic in low- and middle-income countries (LMIC) and has re-emerged in high-income countries. Poverty, ignorance and poor healthcare in LMIC still contribute to development of the disease and, if untreated, its progression to severe forms. A 6-year-old girl from a poor background presented with malnutrition, anaemia, rachitic rosary and severe deformities of the upper and lower limbs owing to vitamin D deficiency. Although she responded well to treatment initially, some of the deformities required surgical intervention. Unfortunately, she was lost to follow-up.

Transgenic Expression of the Vitamin D Receptor Restricted to the Ileum, Cecum, and Colon of Vitamin D Receptor Knockout Mice Rescues Vitamin D Receptor-Dependent Rickets.

Although the intestine plays the major role in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action on calcium homeostasis, the mechanisms involved remain incompletely understood. The established model of 1,25(OH)2D3-regulated intestinal calcium absorption postulates a critical role for the duodenum. However, the distal intestine is where 70% to 80% of ingested calcium is absorbed. To test directly the role of 1,25(OH)2D3 and the vitamin D receptor (VDR) in the distal intestine, three independent knockout (KO)/transgenic (TG) lines expressing VDR exclusively in the ileum, cecum, and colon were generated by breeding VDR KO mice with TG mice expressing human VDR (hVDR) under the control of the 9.5-kb caudal type homeobox 2 promoter. Mice from one TG line (KO/TG3) showed low VDR expression in the distal intestine (<50% of the levels observed in KO/TG1, KO/TG2, and wild-type mice). In the KO/TG mice, hVDR was not expressed in the duodenum, jejunum, kidney, or other tissues. Growth arrest, elevated parathyroid hormone level, and hypocalcemia of the VDR KO mice were prevented in mice from KO/TG lines 1 and 2. Microcomputed tomography analysis revealed that the expression of hVDR in the distal intestine of KO/TG1 and KO/TG2 mice rescued the bone defects associated with systemic VDR deficiency, including growth plate abnormalities and altered trabecular and cortical parameters. KO/TG3 mice showed rickets, but less severely than VDR KO mice. These findings show that expression of VDR exclusively in the distal intestine can prevent abnormalities in calcium homeostasis and bone mineralization associated with systemic VDR deficiency.

Unexpected widespread hypophosphatemia and bone disease associated with elemental formula use in infants and children.

OBJECTIVE: Hypophosphatemia occurs with inadequate dietary intake, malabsorption, increased renal excretion, or shifts between intracellular and extracellular compartments. We noticed the common finding of amino-acid based elemental formula [EF] use in an unexpected number of cases of idiopathic hypophosphatemia occurring in infants and children evaluated for skeletal disease. We aimed to fully characterize the clinical profiles in these cases. METHODS: A retrospective chart review of children with unexplained hypophosphatemia was performed as cases accumulated from various centres in North America and Ireland. Data were analyzed to explore any relationships between feeding and biochemical or clinical features, effects of treatment, and to identify a potential mechanism. RESULTS: Fifty-one children were identified at 17 institutions with EF-associated hypophosphatemia. Most children had complex illnesses and had been solely fed Neocate® formula products for variable periods of time prior to presentation. Feeding methods varied. Hypophosphatemia was detected during evaluation of fractures or rickets. Increased alkaline phosphatase activity and appropriate renal conservation of phosphate were documented in nearly all cases. Skeletal radiographs demonstrated fractures, undermineralization, or rickets in 94% of the cases. Although the skeletal disease had often been attributed to underlying disease, most all improved with addition of supplemental phosphate or change to a different formula product. CONCLUSION: The observed biochemical profiles indicated a deficient dietary supply or severe malabsorption of phosphate, despite adequate formula composition. When transition to an alternate formula was possible, biochemical status improved shortly after introduction to the alternate formula, with eventual improvement of skeletal abnormalities. These observations strongly implicate that bioavailability of formula phosphorus may be impaired in certain clinical settings. The widespread nature of the findings lead us to strongly recommend careful monitoring of mineral metabolism in children fed EF. Transition to alternative formula use or implementation of phosphate supplementation should be performed cautiously with as severe hypocalcemia may develop.

Nutritional rickets around the world: an update.

Worldwide, nutritional rickets continues to be an evolving problem with several causes. This paper provides an updated literature review characterising the prevalence, aetiology, pathophysiology and treatment of nutritional rickets worldwide. A systematic review of articles on nutritional rickets from various geographical regions was undertaken. For each region, key information was extracted, including prevalence, cause of rickets specific to the region, methods of confirming the diagnosis and current treatment and preventive measures. Calcium deficiency continues to be a major cause of rickets in Africa and Asia. Vitamin D deficiency rickets is perhaps increasing in the Americas, Europe and parts of the Middle East. There continues to be a distinct presentation of calcium-predominant versus vitamin D predominant rickets, although there are overlapping features. More careful diagnosis of rickets and reporting of 25-OHD concentrations has improved accurate knowledge of rickets prevalence and better delineated the cause. Nutritional rickets continues to be an evolving and multi-factorial problem worldwide. It is on a spectrum, ranging from isolated vitamin D deficiency to isolated calcium deficiency. Specific areas which require emphasis include a consistent community approach to screening and diagnosis, vitamin D supplementation of infants and at-risk children, prevention of maternal vitamin D deficiency and the provision of calcium in areas with low calcium diets.

Sex-related differences in the skeletal phenotype of aged vitamin D receptor global knockout mice.

The role of the vitamin D receptor (VDR) in maintaining skeletal health appears to be complex and dependent on the physiological context. Global Vdr deletion in a mouse model (Vdr-/-) results in hypocalcemia, secondary hyperparathyroidism and bone features typical of vitamin D-dependent rickets type II. When weanling Vdr-/- mice are fed a diet containing high levels of calcium, phosphorus and lactose, termed the rescue diet, normalisation of serum calcium, phosphate and parathyroid hormone levels results in prevention of rickets at 10 weeks of age. However, 17 week old male Vdr-/- mice, fed the rescue diet, have been reported as osteopenic due to a decrease in bone formation when compared to wild type mice. We now report confirmation of this finding with further data on the effect of the rescue diet on appendicular and axial skeletal structures in male and female Vdr-/- mice at 26 weeks of age compared to Vdr+/- controls. All Vdr-/- mice were normocalcemic with no evidence of any mineralization defect. However, male Vdr-/- mice exhibited significantly reduced mineral in femoral and vertebral bones when compared to control littermate Vdr+/- mice, consistent with the previously reported data. In contrast, 26-week-old female Vdr-/- mice demonstrated significantly increased femoral trabecular bone volume although there was decreased vertebral trabecular bone volume, similar to males, and femoral cortical bone volume was unchanged. Thus, the Vdr-/- mouse model displays sex- and site-specific differences in skeletal structures with long-term feeding of a rescue diet. Although the global Vdr-/- ablation does not permit the determination of skeletal mechanisms producing these differences, these data confirm skeletal changes even when fed the rescue diet.

Bone Disease in the Common Marmoset: Radiographic and Histological Findings.

The common marmoset (Callithrix jacchus) is a New World primate that is used in biomedical research due to its small size and relative ease of handling compared with larger primates. Although bone disease in common marmosets is well recognized, there are very few detailed descriptions in the literature that cover the range of lesions seen in these animals. For all animals used to model human disease, it is important to be aware of background lesions that may affect the interpretation of study findings. This retrospective study details bone diseases encountered in marmoset breeding colonies at 2 different institutions. Affected marmosets at Johns Hopkins University had lesions compatible with diagnoses of rickets, fibrous osteodystrophy and osteopenia. Affected marmosets at the Wisconsin National Primate Research Center exhibited severe lesions of osteoclastic bone resorption and remodeling that had an unusual distribution and were not easily categorized into a known disease entity. The purpose of this report is to document these naturally occurring skeletal lesions of common marmosets and suggest an approach to evaluating skeletal disease in prospective studies of these animals that will allow the most accurate diagnoses.

Rickets or abuse? A histologic comparison of rickets and child abuse-related fractures.

PURPOSE: The bone changes of vitamin D deficiency rickets have been invoked as an alternate explanation for child-abuse related fractures identified through medical imaging. The lack of modern histopathologic comparisons between these two entities limits the abilities of the forensic pathologist to address this differential diagnosis, both in their autopsy reports and on the witness stand. METHODS: We report a comparison of the histologic appearance of the bones in a two year old child with vitamin D deficiency rickets with fractures occurring in three young children with child abuse. RESULTS: In the case of rickets, there was marked architectural disorganization of endochondral ossification at the costochondral junctions and growth plates of long bones. The child abuse-related fractures showed osteochondral callus at different stages of healing, either centered on a discrete fracture line or at metaphyses (e.g. classical metaphyseal lesions). In many instances, the healing fractures disrupted the line of endochondral ossification. In none of the child abuse-related fractures was there any similarity to the histologic appearance of rickets. CONCLUSION: The maturation disturbance in the growth plate that occurs in rickets is a distinctive entity that cannot be confused histologically with healing fractures, including the classical metaphyseal lesion.

Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl-/- mice by administration of soluble (non-targeted) chimeric alkaline phosphatase.

Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl(-/-)) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl(-/-) mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16 mg/kg, from birth for up to 53 days. Lifespan and body weight of Alpl(-/-) mice were normalized, and vitamin B6-associated seizures were absent with 16 mg/kg/day of ChimAP. Radiographs, µCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl(-/-) mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP.

Toxicity of aflatoxin B1 towards the vitamin D receptor (VDR).

This research describes an unexpected toxicity of the aflatoxin B1 towards the vitamin D receptors. Rickets is a childhood disease, and calcium deficiency is the aetiological cause in Africa, being primarily associated with nutritional problems; in this research the contribution of aflatoxin B1 exposure during the early months of life is an interesting factor to deepen in order to prevent liver damages or the development of rickets. The results show that the expression of vitamin D receptor in osteosarcoma cell line SAOS-2 is significantly down-modulated by exposure to aflatoxin B1. This seems to suggest that Aflatoxin B1, toxic towards the vitamin D receptor, interferes with the actions of the vitamin D on calcium binding gene expression in the kidney and intestine. Experimental data indicate a 58% and 86% decrease if the cells are exposed to 5 ng/mL and 50 ng/mL of aflatoxin B1, respectively. These results seem to indicate that natural occurrence of the aflatoxin B1 and allelic variant of vitamin D receptor on (F allele) increase the risk of developing rickets of African children.