The contribution of a novel PHEX gene mutation to X-linked hypophosphatemic rickets: a case report and an analysis of the gene mutation dosage effect in a rat model.

A Chinese family was identified to have two patients with rickets, an adult female and a male child (proband), both exhibiting signs related to X-linked hypophosphatemic rickets (XLH). Gene sequencing analysis revealed a deletion of adenine at position 1985 (c.1985delA) in the PHEX-encoding gene. To investigate the relationship between this mutation and the pathogenicity of XLH, as well as analyze the effects of different dosages of PHEX gene mutations on clinical phenotypes, we developed a rat model carrying the PHEX deletion mutation. The CRISPR/Cas9 gene editing technology was employed to construct the rat model with the PHEX gene mutation (c.1985delA). Through reproductive procedures, five genotypes of rats were obtained: female wild type (X/X), female heterozygous (-/X), female homozygous wild type (-/-), male wild type (X/Y), and male hemizygous (-/Y). The rats with different genotypes underwent analysis of growth, serum biochemical parameters, and bone microstructure. The results demonstrated the successful generation of a stable rat model inheriting the PHEX gene mutation. Compared to the wild-type rats, the mutant rats displayed delayed growth, shorter femurs, and significantly reduced bone mass. Among the female rats, the homozygous individuals exhibited the smallest body size, decreased bone mass, shortest femur length, and severe deformities. Moreover, the mutant rats showed significantly lower blood phosphorus concentration, elevated levels of FGF23 and alkaline phosphatase, and increased expression of phosphorus regulators. In conclusion, the XLH rat model with the PHEX gene mutation dosage demonstrated its impact on growth and development, serum biochemical parameters, and femoral morphology.

A Unique Mechanism of a Novel Synonymous PHEX Variant Causing X-Linked Hypophosphatemia.

CONTEXT: Synonymous mutations are usually nonpathogenic. OBJECTIVE: We report here a family with X-linked hypophosphatemia (XLH) due to a novel synonymous PHEX variant with a unique mechanism. METHODS: We studied a 4-member family (a mother, a son, and 2 daughters), all affected with XLH. Genomic DNA was extracted from peripheral leucocytes. Whole exome sequencing (WES) was used to identify the underlying genetic variant in the proband (the son). Sanger sequencing was used to confirm this variant in the proband and his family members. RT-PCR and sequencing of the cDNA revealed the effect of this variant on the PHEX structure and function. RESULTS: A synonymous variant in the PHEX gene (c.1701A>C) was identified in all affected members. This variant changes the first nucleotide of exon 17 from adenine to cytosine. Using RT-PCR, this variant was shown to interfere with splicing of exons 16 with 17 resulting in a single shorter PHEX transcript in the proband compared to normal control. Sanger sequencing of the cDNA revealed a complete skipping of exon 17 and direct splicing of exons 16 and 18. This led to a frameshift and an introduction of a new stop codon in the next codon (codon 568), which ultimately led to truncation and loss of the final 183 amino acids of PHEX. CONCLUSION: This novel variant shows how a synonymous exonic mutation may induce a complex series of changes in the transcription and translation of the gene and causes a disease, a mechanism that is not commonly recognized.

X-linked hypophosphatemic rickets: Description of seven new variants in patients followed up in reference hospitals in Rio de Janeiro.

BACKGROUND: X-linked hypophosphatemic rickets (XLHR) is a rare genetic disease, often delayed in diagnosis due to the low degree of suspicion and limited access to sophisticated diagnostic tools that confirm the diagnosis, such as genetic testing. METHODS: Through a cross-sectional and observational study, 26 patients with a previously presumptive diagnosis of X-linked hypophosphatemic rickets (based on clinical history, laboratory findings, and physical examination), were followed for approximately 12 months. During 12 months of follow-up, only 16 patients underwent genetic testing and enrolled in the study. Previous data were analyzed, such as clinical history (e.g., gender, current age, age of clinical diagnosis, age of admission to hospital, family history, and previous orthopedic surgery), physical exam, imaging tests (e.g., radiological changes) and laboratory tests (e.g., tubular maximum reabsorption rate of phosphate to glomerular filtration rate, alkaline phosphatase, and phosphate levels) at the time of the patient's admission to IEDE and UFRJ, to corroborate and substantiate our research. These data were extracted from the medical records of the patients. RESULTS: Among the 16 patients analyzed by molecular biology techniques, the new generation sequencing (NGS), using DNA samples from oral swabs, we obtained seven variants never previously described, which were verified by Sanger sequencing. Among the seven variants never previously described, the most common coding impact was the nonsense mutation. We found two frameshift, one intronic splicing variant, three nonsense, and one deletion splice junction loss. Among patients with new mutations who presented data in the medical record, 100% showed a reduction in TmP/GFR (average of 1.98 mg/dl), the most sensitive laboratory parameter at the time of diagnosis, as well as serum phosphorus (100% had hypophosphatemia on arrival at the referral hospitals--average of 2.4 mg/dl and median 2.3 mg/dl). We also performed NGS on three mothers of the patients with identified mutations. Among these mothers, only one tested negative for the mutation and no family history was reported as well. This mother had serum phosphate of 3.5 mg/dl (normal range: 2.5-4.5 mg/dl) at the time of genetic test collection. The others had a positive test, low serum phosphorus at the time of the molecular test, in addition to a positive family history. CONCLUSION: This study describes seven new variants in the PHEX gene and aims to increase the knowledge of the scientific community about the types of mutations involving this gene, increasing information on the genetic basis of this condition, enabling future considerations about genotype-phenotype correlation, in addition to diagnosis accurate and early.

X-linked hypophosphataemic rickets: Report of a novel PHEX mutation and cinacalcet as adjuvant therapy in the mineral metabolism control.

X-linked hypophosphataemic rickets (XLH) is a rare disease caused by a mutation in the phosphate-regulating neutral endopeptidase (PHEX) gene, located on the X chromosome. This gene encodes the phosphate-regulating endopeptidase, and its inactivation leads to increased levels of circulating phosphatonins responsible for renal phosphate loss. The treatment for XLH is still carried out with long-term administration of phosphate and calcitriol, which can be complicated by hyperparathyroidism, nephrocalcinosis, renal failure, and hypertension. We describe the case of a four-decade follow-up patient with XLH. When she was diagnosed, at 19 years, due to bone pain and deformities, she was put on therapy with phosphorus and cholecalciferol. Despite the clinical improvement, serum phosphorus remained difficult to control. At the age of 44 years, she developed tertiary hyperparathyroidism and was submitted to parathyroidectomy. Five years later, parathyroid hyperfunction recurred. This time, cinacalcet was started, 30 mg alternating with 60 mg/day. Currently, she is 59-years old and remains with controlled mineral metabolism. The genetic study of this patient revealed a nonsense heterozygous mutation (c.501G>A) in PHEX gene that was not previously described. In this case, the off-label use of cinacalcet resulted in the normalisation of serum parathormone and phosphorus levels, eliminated recurrent secondary hyperparathyroidism, which aggravates the bone fragility inherent to XLH, and prevented a new parathyroidectomy. This report also adds important information to the genetic basis of XLH with the identification of a new nonsense mutation of the PHEX gene.

Adult rheumatologic features, treatment and complications of X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is a rare genetic phosphate disorder caused mainly by PHEX mutations. Unlike for children, knowledge of the disease's manifestations in adults is limited. Musculoskeletal symptoms are the main feature of the disease in young adults associated with a heavy burden on patients' life. They include fractures and pseudofractures, pain, joint stiffness, osteoarthritis, enthesopathies, and muscle weakness, eventually leading to impaired quality of life. Conventional treatment with phosphate supplements and vitamin D analogs is indicated in symptomatic patients. Appropriate rehabilitation is also a key to the management of the disease to improve physical function and decrease pain, stiffness, and fatigue. Regarding the incidence and consequences of musculoskeletal features in XLH, all patients should be assessed by a bone disease specialist and, if necessary, managed by a multidisciplinary team.

[Genetic and clinical analysis of X-linked hypophosphatemic rickets].

Objective: To investigate the clinical and genetic features, and treatment of X-linked hypophosphatemic rickets (XLH). Methods: In this retrospective study, we reviewed the medical records of 25 pediatric patients with XLH who were admitted to Department of Endocrinology Genetics and Metabolism,Beijing Children's Hospital from January 2010 to January 2020. The clinical characteristics, PHEX gene variants, as well as clinical outcome of the patients were summarized. To analyze the correlation between genotype and phenotype, the patients were divided into different subgroups according to the location of the variants, including N-terminal-located vs. C-terminal-located variant, and Zn-binding domain exon 17 or 19 variant vs. non-exon 17 or 19 variant. The age at onset, height standard deviation score (HtSDS), intercondylar or intermalleolar distance, fasting serum phosphorus, and HtSDS and intercondylar or intermalleolar distance at the final follow-up were compared by rank sum test or t text. Results: Among the 25 children with XLH, 8 were boys and 17 were girls. The median age of onset was 1.2 (1.0, 1.8) years, and the median age of diagnosis was 2.5 (1.5, 4.3) years. The main clinical manifestations were abnormal gait and lower limb deformity. The HtSDS was -2.0(-3.2, -0.8), and the intercondylar or intermalleolar distance was 4.5 (3.0, 6.0) cm. The fasting serum phosphorus level was 0.8 (0.7, 0.9) mmol/L, while the serum alkaline phosphatase level was (721±41) U/L and the serum calcium level was (2.5±0.1) mmol/L. Three patients (12%) had parathyroid hormone levels above the upper limit of the normal range. Twenty-five patients (100%) showed radiographic changes of active rickets. Nephrocalcinosis was found in 2 cases (9%). Twenty-four different PHEX variations were detected in 25 patients, among whom 11 (44%) had not been reported previously. No hot spot variation was found. No statistical differences (all P>0.05) were identified in clinical features and outcomes either in comparing patients with N-terminal (21 cases) and C-terminal (4 cases) variants, or in comparing patients with variant located in exon 17 or 19 (4 cases) or not (21 cases). Twenty-four cases (96%) were treated regularly with phosphate supplements and active vitamin D. After 2.7 (1.6, 5.0) years of follow-up, clinical symptoms were relieved in 96% (24/25) of the patients. The HtSDS after treatment had no significant difference compared to that before treatment (-2.0(-3.2, -0.8) vs.-2.0(-2.8, -1.1),Z =-0.156, P>0.05), while the intercondylar or intermalleolar distance after treatment was significantly reduced compared to that before treatment (4.5(3.0, 6.0) vs. 1.5(0, 3.3) cm, Z =-3.043, P<0.05). Bone X-rays were reexamined in 17 cases after treatment, and radiographic signs of rickets were improved. Eighteen cases had secondary hyperparathyroidism and 7 cases had nephrocalcinosis. Conclusions: The main clinical manifestations of XLH are abnormal gait, lower limb deformity and short stature. A high proportion of novel variations of PHEX gene but no hot spot variation neither genotype-phenotype correlation are found. Regular treatment with phosphate supplements and active vitamin D can significantly improve the symptoms except for the height. However, the rate of adverse events including secondary hyperparathyroidism and nephrocalcinosis seems to be high.

A mosaic mutation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) in X-linked hypophosphatemic rickets with mild bone phenotypes.

X-linked hypophosphatemic rickets (XLH) is primarily characterized by renal phosphate wasting with hypophosphatemia, short stature, and bone deformity of the leg. Here we present a male case of XLH with relatively mild bone deformity caused by a mosaic mutation of the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX). Polymerase chain reaction (PCR) direct sequencing revealed a novel in-frame deletion, NM-000444.6:c.671-685del p.Gln224-Ser228del, at exon 6 in PHEX as a mosaic pattern. This mutation was not found in any database and may result in a significant change in higher-order protein structure and function. TA cloning of the PCR product and clone sequencing estimated the mutation allele frequency at 21%. Literature review of the previously reported three cases with novel mosaic mutations in PHEX, together with the present case, suggests that the rates of the mutation allele correlate with phenotype severity to some extent. We initially treated him with nutritional vitamin D supplements and phosphate salts. However, to avoid the development of secondary/tertiary hyperparathyroidism, we had switched nutritional to active vitamin D supplementation with reduced phosphorus salts. The present report contributes to understanding the relationship between the mosaic rate, in addition to the mutation locus, of the PHEX gene, and clinical features of XLH.

Consensus Recommendations for the Diagnosis and Management of X-Linked Hypophosphatemia in Belgium.

X-linked hypophosphatemia (XLH) is the most common genetic form of hypophosphatemic rickets and osteomalacia. In this disease, mutations in the PHEX gene lead to elevated levels of the hormone fibroblast growth factor 23 (FGF23), resulting in renal phosphate wasting and impaired skeletal and dental mineralization. Recently, international guidelines for the diagnosis and treatment of this condition have been published. However, more specific recommendations are needed to provide guidance at the national level, considering resource availability and health economic aspects. A national multidisciplinary group of Belgian experts convened to discuss translation of international best available evidence into locally feasible consensus recommendations. Patients with XLH may present to a wide array of primary, secondary and tertiary care physicians, among whom awareness of the disease should be raised. XLH has a very broad differential-diagnosis for which clinical features, biochemical and genetic testing in centers of expertise are recommended. Optimal care requires a multidisciplinary approach, guided by an expert in metabolic bone diseases and involving (according to the individual patient's needs) pediatric and adult medical specialties and paramedical caregivers, including but not limited to general practitioners, dentists, radiologists and orthopedic surgeons. In children with severe or refractory symptoms, FGF23 inhibition using burosumab may provide superior outcomes compared to conventional medical therapy with phosphate supplements and active vitamin D analogues. Burosumab has also demonstrated promising results in adults on certain clinical outcomes such as pseudofractures. In summary, this work outlines recommendations for clinicians and policymakers, with a vision for improving the diagnostic and therapeutic landscape for XLH patients in Belgium.

Phenotypic characterization of X-linked hypophosphatemia in pediatric Spanish population.

BACKGROUND: X-linked hypophosphatemia (XLH) is a hereditary rare disease caused by loss-of-function mutations in PHEX gene leading tohypophosphatemia and high renal loss of phosphate. Rickets and growth retardation are the major manifestations of XLH in children, but there is a broad phenotypic variability. Few publications have reported large series of patients. Current data on the clinical spectrum of the disease, the correlation with the underlying gene mutations, and the long-term outcome of patients on conventional treatment are needed, particularly because of the recent availability of new specific medications to treat XLH. RESULTS: The RenalTube database was used to retrospectively analyze 48 Spanish patients (15 men) from 39 different families, ranging from 3 months to 8 years and 2 months of age at the time of diagnosis (median age of 2.0 years), and with XLH confirmed by genetic analysis. Bone deformities, radiological signs of active rickets and growth retardation were the most common findings at diagnosis. Mean (± SEM) height was - 1.89 ± 0.19 SDS and 55% (22/40) of patients had height SDS below-2. All cases had hypophosphatemia, serum phosphate being - 2.81 ± 0.11 SDS. Clinical manifestations and severity of the disease were similar in both genders. No genotype-phenotype correlation was found. Conventional treatment did not attenuate growth retardation after a median follow up of 7.42 years (IQR = 11.26; n = 26 patients) and failed to normalize serum concentrations of phosphate. Eleven patients had mild hyperparathyroidism and 8 patients nephrocalcinosis. CONCLUSIONS: This study shows that growth retardation and rickets were the most prevalent clinical manifestations at diagnosis in a large series of Spanish pediatric patients with XLH confirmed by mutations in the PHEX gene. Traditional treatment with phosphate and vitamin D supplements did not improve height or corrected hypophosphatemia and was associated with a risk of hyperparathyroidism and nephrocalcinosis. The severity of the disease was similar in males and females.

Alterations of bone material properties in adult patients with X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) caused by PHEX mutations results in elevated serum FGF23 levels, renal phosphate wasting and low 1,25-dihydroxyvitamin D. The glycophosphoprotein osteopontin, a potent inhibitor of mineralization normally degraded by PHEX, accumulates within the bone matrix. Conventional therapy consisting of supplementation with phosphate and vitamin D analogs is burdensome and the effects on bone material poorly characterized. We analyzed transiliac bone biopsies from four adult patients, two of them severely affected due to no diagnosis and no treatment until adulthood. We used light microscopy, qBEI and FTIRI to study histology, histomorphometry, bone mineralization density distribution, properties of the organic matrix and size of hypomineralized periosteocytic lesions. Non-treatment resulted in severe osteomalacia, twice the amount of mineralized trabecular volume, multiple osteon-like perforations, continuity of lamellae from mineralized to unmineralized areas and distinctive patches of woven bone. Periosteocytic lesions were larger than in treated patients. The latter had nearly normal osteoid thicknesses, although surface was still elevated. The median calcium content of the matrix was always within normal range, although the percentage of lowly mineralized bone areas was highly increased in non-treated patients, resulting in a marked heterogeneity in mineralization. Divalent collagen cross-links were evident independently of the mineral content of the matrix. Broad osteoid seams lacked measurable pyridinoline, a mature trivalent cross-link and exhibited considerable acidic lipid content, typically found in matrix vesicles. Based on our results, we propose a model that possibly integrates the relationship between the observed mineralization disturbances, FGF23 secretion and the known osteopontin accumulation in XLH.

Novel variants and uncommon cases among southern Chinese children with X-linked hypophosphatemia.

PURPOSE: X-linked hypophosphatemia (XLH) is the most common inherited renal phosphate wasting disorder and is often misdiagnosed as vitamin D deficiency. This study aims to provide clinical and mutational characteristics of 65 XLH pediatric patients in southern China. METHODS: In this work, a combination of DNA sequencing and qPCR analysis was used to study the PHEX gene in 80 pediatric patients diagnosed with hypophosphatemia. The clinical and laboratory data of confirmed 65 XLH patients were assessed and analyzed retrospectively. RESULTS: In 65 XLH patients from 61 families, 51 different variants in the PHEX gene were identified, including 23 previously reported variants and 28 novel variants. In this cohort of XLH patients, the c.1601C>T(p.Pro534Leu) variant appears more frequently. Fourteen uncommon XLH cases were described, including four boys with de novo mosaic variants, eight patients with large deletions and a pair of monozygotic twins. The clinical manifestations in this cohort are very similar to those previously reported. CONCLUSION: This study extends the mutational spectrum of the PHEX gene, which will contribute to accurate diagnosis. This study also suggests a supplementary qPCR or MLPA assay may be performed along with classical sequencing to confirm the gross insertion/deletion.

Mineralized tissues in hypophosphatemic rickets.

Hypophosphatemic rickets is caused by renal phosphate wasting that is most commonly due to X-linked dominant mutations in PHEX. PHEX mutations cause hypophosphatemia indirectly, through the increased expression of fibroblast growth factor 23 (FGF23) by osteocytes. FGF23 decreases renal phosphate reabsorption and thereby increases phosphate excretion. The lack of phosphate leads to a mineralization defect at the level of growth plates (rickets), bone tissue (osteomalacia), and teeth, where the defect facilitates the formation of abscesses. The bone tissue immediately adjacent to osteocytes often remains unmineralized ("periosteocytic lesions"), highlighting the osteocyte defect in this disorder. Common clinical features of XLH include deformities of the lower extremities, short stature, enthesopathies, dental abscesses, as well as skull abnormalities such as craniosynostosis and Chiari I malformation. For the past four decades, XLH has been treated by oral phosphate supplementation and calcitriol, which improves rickets and osteomalacia and the dental manifestations, but often does not resolve all aspects of the mineralization defects. A newer treatment approach using inactivating FGF23 antibodies leads to more stable control of serum inorganic phosphorus levels and seems to heal rickets more reliably. However, the long-term benefits of FGF23 antibody treatment remain to be elucidated.

Management of X-linked hypophosphatemia in adults.

X-linked hypophosphatemia (XLH) is caused by mutations in the PHEX gene which result in Fibroblast Growth Factor-23 (FG-F23) excess and phosphate wasting. Clinically, XLH children present with rickets, bone deformities and short stature. In adulthood, patients may still be symptomatic with bone and joint pain, osteomalacia-related fractures or pseudofractures, precocious osteoarthrosis, enthesopathy, muscle weakness and severe dental anomalies. Besides these musculoskeletal and dental manifestations, adult XLH patients are also prone to secondary and tertiary hyperparathyroidism, cardiovascular and metabolic disorders. Pathophysiology of hyperparathyroidism is only partially understood but FGF23 excess and deficient production of calcitriol likely contributes to its development. Similarly, the pathophysiological mechanisms of potential cardiovascular and metabolic involvements are not clear, but FGF-23 excess may play an essential role. Treatment should be considered in symptomatic patients, patients undergoing orthopedic or dental surgery and women during pregnancy and lactation. Treatment with oral phosphate salts and active vitamin D analogs has incomplete efficacy and potential risks. Burosumab, a recombinant human monoclonal antibody against FGF-23, has proven its efficacy in phase 2 and phase 3 clinical trials in adult patients with XLH, but currently its position as first line or second line treatment differ among the countries.

Diagnosis, treatment-monitoring and follow-up of children and adolescents with X-linked hypophosphatemia (XLH).

Early diagnosis, optimal therapeutic management and regular follow up of children with X-linked hypophosphatemia (XLH) determine their long term outcomes and future quality of life. Biochemical screening of potentially affected newborns in familial cases and improving physician's knowledge on clinical signs, symptoms and biochemical characteristics of XLH for de novo cases should lead to earlier diagnosis and treatment initiation. The follow-up of children with XLH includes clinical, biochemical and radiological monitoring of treatment (efficacy and complications) and screening for XLH-related dental, neurosurgical, rheumatological, cardiovascular, renal and ENT complications. In 2018, the European Union approved the use of burosumab, a humanized monoclonal anti-FGF23 antibody, as an alternative therapy to conventional therapy (active vitamin D analogues and phosphate supplements) in growing children with XLH and insufficiently controlled disease. Diagnostic criteria of XLH and the principles of disease management with conventional treatment or with burosumab are reviewed in this paper.

X-linked hypophosphatemia: Management and treatment prospects.

X-linked hypophosphatemia (XLH), due to a PHEX gene mutation, is the most common genetic form of rickets and osteomalacia. Manifestations in children consist of rickets, lower-limb bone deformities, bone pain, failure to thrive, dental abscesses, and/or craniostenosis. Adults may present with persistent bone pain, early osteoarthritis, hairline fractures and Looser zones, enthesopathy, and/or periodontitis. Regardless of whether the patient is an infant, child, adolescent or adult, an early diagnosis followed by optimal treatment is crucial to control the clinical manifestations, prevent complications, and improve quality of life. Treatment options include active vitamin D analogs and phosphate supplementation to correct the 1.25(OH)2 vitamin D deficiency and to compensate for the renal phosphate wasting, respectively. The recently introduced FGF23 antagonist burosumab is designed to restore renal phosphate reabsorption by the proximal tubule and to stimulate endogenous calcitriol production. In Europe, burosumab is licensed for use in pediatric patients older than 1 year who have XLH. This review discusses the diagnosis and treatment of XLH and describes the indications of the various available treatments.

Targeted resequencing of phosphorus metabolism­related genes in 86 patients with hypophosphatemic rickets/osteomalacia.

Hypophosphatemic rickets/osteomalacia is characterized by defective renal phosphate reabsorption and abnormal bone mineralization. Hypophosphatemic rickets/osteomalacia consists of inherited and acquired forms, many of which have unknown aetiology. In the present study, next­generation sequencing­based resequencing was used on samples from Chinese subjects with hypophosphatemic rickets/osteomalacia, aiming to detect the spectrum of pathogenic genes in these patients. A total of 86 hypophosphatemic rickets/osteomalacia patients (ranging from 3 to 70 years old) were recruited. Patients with tumour­induced osteomalacia (TIO), renal tubular acidosis, renal osteodystrophy, and adefovir­induced Fanconi syndrome were excluded. Targeted massively parallel resequencing of 196 candidate genes for hypophosphatemic rickets/osteomalacia was performed in the 86 affected unrelated individuals (cases) and in 100 unrelated healthy controls to identify new genes and mutations in known genes that cause hypophosphatemic rickets/osteomalacia. The results identified seven phosphate­regulating gene with homologies to endopeptidases on the X chromosome (PHEX) mutations (of which two were novel) and one novel dentin matrix protein 1 (DMP1) mutation in eight patients. Following targeted exome sequencing data analysis, 14 candidate disease­related gene loci were selected, two of which were of most concern regarding disease severity. Further validation of the present results is warranted, with additional sequencing projects and functional tests. To our knowledge, the present study is the largest cohort of cases with hypophosphatemic rickets/osteomalacia to undergo targeted resequencing. The diagnosis and understanding of the molecular aetiologies of these disorders will be improved by this fast and efficient approach.

Targeted Pth4-expressing cell ablation impairs skeletal mineralization in zebrafish.

Skeletal development and mineralization are essential processes driven by the coordinated action of neural signals, circulating molecules and local factors. Our previous studies revealed that the novel neuropeptide Pth4, synthesized by hypothalamic cells, was involved in bone metabolism via phosphate regulation in adult zebrafish. Here, we investigate the role of pth4 during skeletal development using single-cell resolution, two-photon laser ablation of Pth4:eGFP-expressing cells and confocal imaging in vivo. Using a stable transgenic Pth4:eGFP zebrafish line, we identify Pth4:eGFP-expressing cells as post-mitotic neurons. After targeted ablation of eGFP-expressing cells in the hypothalamus, the experimental larvae exhibited impaired mineralization of the craniofacial bones whereas cartilage development was normal. In addition to a decrease in pth4 transcript levels, we noted altered expression of phex and entpd5, genes associated with phosphate homeostasis and mineralization, as well as a delay in the expression of osteoblast differentiation markers such as sp7 and sparc. Taken together, these results suggest that Pth4-expressing hypothalamic neurons participate in the regulation of bone metabolism, possibly through regulating phosphate balance during zebrafish development.

FGF23 Is Not Required to Regulate Fetal Phosphorus Metabolism but Exerts Effects Within 12 Hours After Birth.

Loss of fibroblast growth factor-23 (FGF23) causes hyperphosphatemia, extraskeletal calcifications, and early mortality; excess FGF23 causes hypophosphatemia with rickets or osteomalacia. However, FGF23 may not be important during fetal development. FGF23 deficiency (Fgf23 null) and FGF23 excess (Phex null) did not alter fetal phosphorus or skeletal parameters. In this study, we further tested our hypothesis that FGF23 is not essential for fetal phosphorus regulation but becomes important after birth. Although coreceptor Klotho null adults have extremely high FGF23 concentrations, intact FGF23 was normal in Klotho null fetuses, as were fetal phosphorus and skeletal parameters and placental and renal expression of FGF23 target genes. Pth/Fgf23 double mutants had the same elevation in serum phosphorus as Pth null fetuses, as compared with normal serum phosphorus in Fgf23 nulls. We examined the postnatal time courses of Fgf23 null, Klotho null, and Phex null mice. Fgf23 nulls and Klotho nulls were normal at birth, but developed hyperphosphatemia, increased renal expression of NaPi2a and NaPi2c, and reduced renal phosphorus excretion between 5 and 7 days after birth. Parathyroid hormone remained normal. In contrast, excess FGF23 exerted effects in Phex null males within 12 hours after birth, with the development of hypophosphatemia, reduced renal expression of NaPi2a and NaPi2c, and increased renal phosphorus excretion. In conclusion, although FGF23 is present in the fetal circulation at levels that may equal adult values, and there is robust expression of FGF23 target genes in placenta and fetal kidneys, FGF23 itself is not an important regulator of fetal phosphorous metabolism.

Hypertension is a characteristic complication of X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is a group of rare disorders caused by defective proximal tubular reabsorption of phosphate. Mutations in the PHEX gene are responsible for the majority of cases. There are very few reports of long-term complications of XLH other than skeletal and dental diseases. The aim of this study was to identify the phenotypic presentation of XLH during adulthood including complications other than skeletal and dental diseases. The clinical and biochemical phenotype of 22 adult patients with a PHEX gene mutation were examined retrospectively from their medical records. 6 patients had hypertension. The average age of hypertension onset was 29.0 years. Secondary hyperparathyroidism preceded the development of hypertension in 5 patients. 1 patient developed tertiary hyperparathyroidism. 15 patients had nephrocalcinosis. 2 patients had chronic renal dysfunction. Patients with hypertension had a significantly lower eGFR (p=0.010) compared to patients without hypertension. No significant difference was found in any other parameters. To examine the genotype-phenotype correlation, 10 adult males were chosen for analysis. No significant genotype-phenotype correlation analysis was revealed in any of the complications. However, there was a possibility that the age at nephrocalcinosis onset was younger in the non-missense mutation group than in the missense mutation group (p=0.063). This study corroborated the view that early-onset hypertension could be one of the characteristic complications seen in XLH patients. Considering the limited number of our patients, further study is necessary to address a potential cause of hypertension. XLH patients require careful lifelong treatment.

Cholic acid therapy in Zellweger spectrum disorders.

INTRODUCTION: Zellweger spectrum disorders (ZSDs) are characterized by a failure in peroxisome formation, caused by autosomal recessive mutations in different PEX genes. At least some of the progressive and irreversible clinical abnormalities in patients with a ZSD, particularly liver dysfunction, are likely caused by the accumulation of toxic bile acid intermediates. We investigated whether cholic acid supplementation can suppress bile acid synthesis, reduce accumulation of toxic bile acid intermediates and improve liver function in these patients. METHODS: An open label, pretest-posttest design study was conducted including 19 patients with a ZSD. Participants were followed longitudinally during a period of 2.5 years prior to the start of the intervention. Subsequently, all patients received oral cholic acid and were followed during 9 months of treatment. Bile acids, peroxisomal metabolites, liver function and liver stiffness were measured at baseline and 4, 12 and 36 weeks after start of cholic acid treatment. RESULTS: During cholic acid treatment, bile acid synthesis decreased in the majority of patients. Reduced levels of bile acid intermediates were found in plasma and excretion of bile acid intermediates in urine was diminished. In patients with advanced liver disease (n = 4), cholic acid treatment resulted in increased levels of plasma transaminases, bilirubin and cholic acid with only a minor reduction in bile acid intermediates. CONCLUSIONS: Oral cholic acid therapy can be used in the majority of patients with a ZSD, leading to at least partial suppression of bile acid synthesis. However, caution is needed in patients with advanced liver disease due to possible hepatotoxic effects.