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Twenty-five years ago, a pathogenic variant of the phosphate-regulating endopeptidase homolog X-linked (PHEX) gene was identified as the cause of X-linked hypophosphatemic rickets (XLH). Subsequently, the overproduction of fibroblast growth factor 23 (FGF23) due to PHEX defects has been found to be associated with XLH pathophysiology. However, the mechanism by which PHEX deficiency contributes to the upregulation of FGF23 and the function of PHEX itself remain unclear. To date, over 700 pathogenic variants have been identified in patients with XLH, and functional assays and genotype–phenotype correlation analyses based on pathogenic variant data derived from XLH patients have been reported. Genetic testing for XLH is useful for the diagnosis. Not only have single-nucleotide variants causing missense, nonsense, and splicing variants and small deletion/insertion variants causing frameshift/non-frameshift alterations been observed, but also gross deletion/duplication variants causing copy number variants have been reported as pathogenic variants in PHEX. With the development of new technologies including next generation sequencing, it is expected that an increasing number of pathogenic variants will be identified. This chapter aimed to summarize the genotype of PHEX and related analyses and discusses the pathophysiology of PHEX defects to seek clues on unsolved questions.

Pachydermoperiostosis (PDP) is a rare hereditary disease characterized by digital clubbing, pachydermia, and periostosis. We describe a Japanese male patient with PDP who was differentially diagnosed with acromegaly by identification of compound heterozygous variants in SLCO2A1. Recent studies have reported various clinical manifestations, as well as skeletal and dermal features, in patients with PDP. Genetic testing provided not only PDP diagnosis and differentiation from acromegaly, but also information about possible complications and comorbidities throughout life.

Osteogenesis imperfecta (OI) is a hereditary skeletal disease characterized by bone fragility. Areal bone mineral density (BMD), evaluated by dual-energy X-ray absorptiometry (DXA), is used to assess bone brittleness. The height-adjusted BMD Z-score (BMDHAZ) is calculated in children and adolescents with OI to reduce the confounding factor of short stature. However, even with the BMDHAZ, severity evaluation in children and adolescents with OI is challenging because certain abnormalities in bone quality cannot be accurately assessed by BMD analysis. The trabecular bone scores (TBS) and bone mineral apparent density (BMAD), which represent the structural integrity of bone and bone-size-associated BMD, respectively, are associated with fracture risk. Recently, age- and sex-specific reference ranges have been reported, enabling the calculation of Z-scores for children. To evaluate which density measurements show the highest correlation with fracture risk, we analyzed the associations between the Z-scores of TBS, BMAD, and BMDHAZ, fracture rate, and genetic variants. We retrospectively reviewed 42 participants with OI aged 5 to 20 years who underwent DXA. COL1A1/2 pathogenic variants were detected in 41 of the 42 participants. In participants with nonsense and frameshift variants (n = 17) resulting in haploinsufficiency and mild phenotype, the TBS Z-score was negatively correlated with fracture rate (FR) (r = -0.50, p = 0.042). In participants with glycine substitution (n = 9) causing the severe phenotype, the BMAD Z-scores were negatively correlated with FR (r = -0.74, p = 0.022). No correlation between the BMDHAZ and FR was observed in both groups. These findings suggest that the TBS and BMAD are useful in assessing children and adolescents with OI with specific genetic variants.

Pseudohypoparathyroidism 1A (PHP1A) and pseudopseudohypoparathyroidism (PPHP) are caused by loss-of-function variants of GNAS, which encodes Gsα. We present two unrelated Japanese families with PHP1A and PPHP harboring unreported pathogenic variants of GNAS (c.1141delG, p.Asp381Thrfs*23 and c.1117delC, p.Arg373Alafs*31). These variants introduce abnormal amino acids in the β6 strand/α5 helix of Gsα, which interact with G protein coupling receptor (GPCR). We conclude that these variants alter the association of Gsα with GPCR and cause PHP1A or PPHP.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare, inherited autosomal recessive disorder caused by fibroblast growth factor-23 (FGF23), N-acetylgalactosaminyltransferase 3 (GALNT3), or Klotho (KL) gene variants. Here, we report the case of a Japanese boy who presented with a mass in his left elbow at the age of three. Laboratory test results of the patient revealed normocalcemia (10.3 mg/dL) and hyperphosphatemia (8.7 mg/dL); however, despite hyperphosphatemia, serum intact FGF23 level was low, renal tubular reabsorption of phosphate (TRP) level was inappropriately increased, and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) level was inappropriately normal. Genetic analysis revealed maternal uniparental disomy (UPD) of chromosome 2, which included a novel GALNT3 variant (c.1780-1G>C). Reverse transcription-polymerase chain reaction (RT-PCR) analysis of GALNT3 mRNA confirmed that this variant resulted in the destruction of exon 11. We resected the mass when the patient was five years old, owing to its gradual enlargement. No relapse or new pathological lesions were observed four years after tumor resection. This is the first case report of a Japanese patient with HFTC associated with a novel GALNT3 variant, as well as the first case of HFTC caused by maternal UPD of chromosome 2 that includes the GALNT3 variant. [ABSTRACT FROM AUTHOR]

Brachydactyly mental retardation syndrome (BDMR) or chromosome 2q37 deletion syndrome is a genetic disorder caused by 2q37 deletion or haploinsufficiency of histone deacetylase 4 (HDAC4). The HDAC4 gene is responsible for major BDMR phenotypes. The symptoms of BDMR include mild-to-moderate intellectual disability, seizures, autism spectrum disorder, short stature, obesity, and facial dysmorphism. Here, we report a family (n = 5) with BDMR who had a missense variant of HDAC4. Four affected individuals [5-yr-old girl (index case); 15- and 3-yr-old siblings; and father] had mild intellectual disability, three of the four affected individuals had short stature and mild cardiac anomalies, and two of the four affected individuals had hypothyroidism. Wholeexome sequencing and analyses of the index case and her family revealed an allelic variant in the HDAC4 gene (NM_001378414.1:c.2204G>A:p. Arg735Gln). A healthy family member (mother) did not have the missense variant. To our knowledge, this is the first report of a missense variation in HDAC4 that is associated with BDMR. [ABSTRACT FROM AUTHOR]

Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH), Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT) control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi) co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10-8 M 1,25(OH)2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10-8 M 1,25(OH)2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.

We describe a three-generation family with tall stature, scoliosis and macrodactyly of the great toes and a heterozygous p.Val883Met mutation in Npr2, the gene that encodes the CNP receptor NPR2 (natriuretic peptide receptor 2). When expressed in HEK293A cells, the mutant Npr2 cDNA generated intracellular cGMP (cyclic guanosine monophosphate) in the absence of CNP ligand. In the presence of CNP, cGMP production was greater in cells that had been transfected with the mutant Npr2 cDNA compared to wild-type cDNA. Transgenic mice in which the mutant Npr2 was expressed in chondrocytes driven by the promoter and intronic enhancer of the Col11a2 gene exhibited an enhanced production of cGMP in cartilage, leading to a similar phenotype to that observed in the patients. In addition, blood cGMP concentrations were elevated in the patients. These results indicate that p.Val883Met is a constitutive active gain-of-function mutation and elevated levels of cGMP in growth plates lead to the elongation of long bones. Our findings reveal a critical role for NPR2 in skeletal growth in both humans and mice, and may provide a potential target for prevention and treatment of diseases caused by impaired production of cGMP.

Hypophosphatasia (HPP) is a rare bone disease caused by inactivating mutations in the ALPL gene, which encodes tissue-nonspecific alkaline phosphatase (TNSALP). Patients with HPP have varied clinical manifestations and are classified based on the age of onset and severity. Recently, enzyme replacement therapy using bone-targeted recombinant alkaline phosphatase (ALP) has been developed, leading to improvement in the prognosis of patients with life-threatening HPP. Considering these recent advances, clinical practice guidelines have been generated to provide physicians with guides for standard medical care for HPP and to support their clinical decisions. A task force was convened for this purpose, and twenty-one clinical questions (CQs) were formulated, addressing the issues of clinical manifestations and diagnosis (7 CQs) and those of management and treatment (14 CQs). A systematic literature search was conducted using PubMed/MEDLINE, and evidence-based recommendations were developed. The guidelines have been modified according to the evaluations and suggestions from the Clinical Guideline Committee of The Japanese Society for Pediatric Endocrinology (JSPE) and public comments obtained from the members of the JSPE and a Japanese HPP patient group, and then approved by the Board of Councils of the JSPE. We anticipate that the guidelines will be revised regularly and updated. [ABSTRACT FROM AUTHOR]

Achondroplasia (ACH) is a skeletal dysplasia that presents with limb shortening, short stature, and characteristic facial configuration. ACH is caused by mutations of the FGFR3 gene, leading to constantly activated FGFR3 and activation of its downstream intracellular signaling pathway. This results in the suppression of chondrocyte differentiation and proliferation, which in turn impairs endochondral ossification and causes short-limb short stature. ACH also causes characteristic clinical symptoms, including foramen magnum narrowing, ventricular enlargement, sleep apnea, upper airway stenosis, otitis media, a narrow thorax, spinal canal stenosis, spinal kyphosis, and deformities of the lower extremities. Although outside Japan, papers on health supervision are available, they are based on reports and questionnaire survey results. Considering the scarcity of high levels of evidence and clinical guidelines for patients with ACH, clinical practical guidelines have been developed to assist both healthcare professionals and patients in making appropriate decisions in specific clinical situations. Eleven clinical questions were established and a systematic literature search was conducted using PubMed/MEDLINE. Evidence-based recommendations were developed, and the guidelines describe the recommendations related to the clinical management of ACH. We anticipate that these clinical practice guidelines for ACH will be useful for healthcare professionals and patients alike. [ABSTRACT FROM AUTHOR]

Congenital generalized lipodystrophy type 4 (CGL4) is a rare disease caused by mutations in the gene polymerase I and transcript release factor (PTRF), the main symptoms of which are systemic reductions in adipose tissue and muscular dystrophy. The strategy of treating CGL4 is to improve the insulin resistance and hypertriglyceridemia that result from systemic reductions in adipose tissue. Metreleptin, a synthetic analog of human leptin, is effective against generalized lipodystrophies; however, there are no reports of the use of metreleptin in the treatment of CGL4. Herein, we discuss the treatment of a six-year-old boy diagnosed with CGL4 due to a homozygous mutation in PTRF with metreleptin. His serum triglyceride level and homeostasis model assessment of insulin resistance (HOMA-IR) value decreased after two months of metreleptin treatment. However, the efficacy of metreleptin gradually decreased, and the treatment was suspended because anaphylaxis occurred after the dosage administered was increased. Subsequently, his serum triglyceride level and HOMA-IR value significantly increased. Anti-metreleptin-neutralizing antibodies were detected in his serum, which suggested th a t these antibodies reduced the efficacy of metreleptin and caused increased hypersensitivity. Thus, metreleptin appeared to be efficacious in the treatment of CGL4 in the short term, although an adverse immune response resulted in treatment suspension. Further studies are needed to evaluate metreleptin treatments for CGL4. [ABSTRACT FROM AUTHOR]

Acrodysostosis is characterized by distinctive facial features and severe brachydactyly. Mutations in PRKAR1A or PDE4D are known to be responsible for this disease. Cases of hormonal resistance have been reported, particularly in patients with PRKAR1A mutations. The physical characteristics and endocrine function of pseudohypoparathyroidism type Ia is known to resemble acrodysostosis. We report the case of a 4-yr-old patient with a PRKAR1A mutation. He had characteristic facies with an upturned nose and cone-shaped epiphyses of most phalanges. These findings have not been reported as extensive for cases of pseudohypoparathyroidism type Ia. He also had TSH resistance from birth. We performed endocrinological stimulation tests to further evaluate his endocrine status. These examinations revealed resistance to TSH and PTH, but there was normal secretion of ACTH, GH and cortisol. An Ellsworth-Howard test resulted in normal urinary cAMP excretion. This response differs from that of pseudohypoparathyroidism type Ia. In summary, the constellation of an upturned nose, cone-shaped epiphyses of most if not all phalanges, and PTH resistance with a normal urinary cAMP response may satisfactorily enable clinical diagnosis of acrodysostosis. [ABSTRACT FROM AUTHOR]

The serum levels of calcium (Ca) and phosphate are maintained higher in the fetus than in the pregnant mother, especially in late gestation, to meet the demands of fetal bone development. In order to maintain this fetal stage-specific mineral homeostasis, the placenta plays a critical role through active transcellular mineral transport. Although the molecular mechanism of transplacental Ca transport has been well studied, little is known about the transport mechanism of phosphate and magnesium. Maternal mineral homeostasis is also altered during pregnancy to supply minerals to the fetus. In the lactating mother, osteocytic osteolysis is suggested to be involved in the supply of minerals to the baby. The levels of some calcitropic and phosphotropic (Ca- and phosphate-regulating, respectively) hormones in the fetus are also different from those in the adult. The PTH level in the fetus is lower than that in the mother and nonpregnant adult. It is suggested, however, that low fetal PTH plays an important role in fetal mineral metabolism. The concentration of PTHrP in the fetus is much higher than that of PTH and plays a critical role in perinatal Ca homeostasis. Uncovering the molecular mechanisms for fetal stage-specific mineral metabolism will lead to better management of perinatal patients with mineral abnormalities. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) is a heritable brittle bone disease mainly caused by mutations in the two type I collagen genes. Collagen synthesis is a complex process including trimer formation, glycosylation, secretion, extracellular matrix (ECM) formation, and mineralization. Using OI patient-derived fibroblasts and induced pluripotent stem cells (iPSCs), we investigated the effect of 4-phenylbutyric acid (4-PBA) on collagen synthesis to test its potential as a new treatment for OI. Endoplasmic reticulum (ER) retention of type I collagen was observed by immunofluorescence staining in OI patientderived fibroblasts with glycine substitution and exon skipping mutations. Liquid chromatography-mass spectrometry analysis revealed excessive glycosylation of secreted type I collagen at the specific sites in OI cells. The misfolding of the type I collagen triple helix in the ECM was demonstrated by the incorporation of heat-dissociated collagen hybridizing peptide in OI cells. Type I collagen was produced excessively by OI fibroblasts with a glycine mutation, but this excessive production was normalized when OI fibroblasts were cultured on control fibroblast-derived ECM. We also found that mineralization was impaired in osteoblasts differentiated from OI iPSCs. In summary, treatment with 4-PBA normalizes the excessive production of type I collagen, reduces ER retention, partially improves misfolding of the type I collagen helix in ECM, and improves osteoblast mineralization. Thus, 4-PBA may improve not only ER retention, but also type I collagen synthesis and mineralization in human cells from OI patients. [ABSTRACT FROM AUTHOR]

Paget's disease (PD) is characterized by focal and dramatic bone resorption and formation. Treatments that target osteoclasts (OCLs) block both pagetic bone resorption and formation; therefore, PD offers key insights into mechanisms that couple bone resorption and formation. Here, we evaluated OCLs from 3 patients with PD and determined that measles virus nucleocapsid protein (MVNP) was expressed in 70% of these OCLs. Moreover, transgenic mice with OCL-specific expression of MVNP (MVNP mice) developed PD-like bone lesions that required MVNP-dependent induction of high IL-6 expression levels in OCLs. In contrast, mice harboring a knockin of p62P394L (p62-KI mice), which is the most frequent PD-associated mutation, exhibited increased bone resorption, but not formation. Evaluation of OCLs from MVNP, p62-KI, and WT mice revealed increased IGF1 expression in MVNP-expressing OCLs that resulted from the high IL-6 expression levels in these cells. IL-6, in turn, increased the expression of coupling factors, specifically ephrinB2 on OCLs and EphB4 on osteoblasts (OBs). IGF1 enhanced ephrinB2 expression on OCLs and OB differentiation. Importantly, ephrinB2 and IGF1 levels were increased in MVNP-expressing OCLs from patients with PD and MVNP-transduced human OCLs compared with levels detected in controls. Further, anti-IGF1 or anti-IGF1R blocked Runx2 and osteocalcin upregulation in OBs cocultured with MVNP-expressing OCLs. These results suggest that in PD, MVNP upregulates IL-6 and IGF1 in OCLs to increase ephrinB2-EphB4 coupling and bone formation. [ABSTRACT FROM AUTHOR]