Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects

Background α-Klotho (αKl) regulates mineral metabolism such as calcium ion (Ca2+) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type αKl has been demonstrated, less is known regarding the physiological importance of soluble-type αKl (sαKl) in circulation. Objectives The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum sαKl, and (2) to determine reference values for sαKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects. Results We successively developed an ELISA to measure serum sαKl in healthy volunteers (n = 142, males 66) of ages (61.1 ± 18.5 year). The levels (mean ± SD) in these healthy control adults were as follows: total calcium (Ca; 9.46 ± 0.41 mg/dL), Pi (3.63 ± 0.51 mg/dL), blood urea nitrogen (BUN; 15.7 ± 4.3 mg/dL), creatinine (Cre; 0.69 ± 0.14 mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)2D; 54.8 ± 17.7 pg/mL), intact parathyroid hormone (iPTH; 49.2 ± 20.6 pg/mL), calcitonin (26.0 ± 12.3 pg/mL) and intact fibroblast growth factor (FGF23; 43.8 ± 17.6 pg/mL). Serum levels of sαKl ranged from 239 to 1266 pg/mL (mean ± SD; 562 ± 146 pg/mL) in normal adults. Although sαKl levels were not modified by gender or indices of mineral metabolism, sαKl levels were inversely related to Cre and age. However, sαKl levels in normal children (n = 39, males 23, mean ± SD; 7.1 ± 4.8 years) were significantly higher (mean ± SD; 952 ± 282 pg/mL) than those in adults (mean ± SD; 562 ± 146, P < 0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that sαKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum sαKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of α-klotho gene. In this patient, sαKl level was notably lower than those of age-matched controls. Conclusion We established a detection system to measure human serum sαKl for the first time. Age, Ca and Pi seem to influence serum sαKl levels in a normal population. This detection system should be an excellent tool for investigating sαKl functions in mineral metabolism

Japanese nationwide survey of hypophosphatasia reveals prominent differences in genetic and dental findings between odonto and non-odonto types.

Hypophosphatasia (HPP) is a rare and intractable metabolic bone disease caused by mutations in the ALPL gene. Here, we undertook a nationwide survey of HPP in Japan, specifically regarding the prominent genetic and dental manifestations of odonto (n = 16 cases) and other (termed "non-odonto") (n = 36 cases) types. Mean serum alkaline phosphatase (ALP) values in odonto-type patients were significantly greater than those of non-odonto-type patients (P<0.05). Autosomal dominant and autosomal recessive inheritance patterns were detected, respectively, in 89% of odonto-type and 96% of non-odonto-type patients. The ALPL "c.1559delT" mutation, associated with extremely low ALP activity, was found in approximately 70% of cases. Regarding dental manifestations, all patients classified as odonto-type showed early exfoliation of the primary teeth significantly more frequently than patients classified as non-odonto-type (100% vs. 56%; P<0.05). Tooth hypomineralisation was detected in 42% of non-odonto-type patients, but not in any odonto-type patients (0%; P<0.05). Collectively, these results suggest that genetic and dental manifestations of patients with odonto-type and non-odonto-type HPP are significantly different, and these differences should be considered during clinical treatment of patients with HPP

Association of trabecular bone score and bone mineral apparent density with the severity of bone fragility in children and adolescents with osteogenesis imperfecta: A cross-sectional study.

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

Functional analysis of monocarboxylate transporter 8 mutations in Japanese Allan-Herndon-Dudley syndrome patients.

Monocarboxylate transporter 8 (MCT8) facilitates T3 uptake into cells. Mutations in MCT8 lead to Allan-Herndon-Dudley syndrome (AHDS), which is characterized by severe psychomotor retardation and abnormal thyroid hormone profile. Nine uncharacterized MCT8 mutations in Japanese patients with severe neurocognitive impairment and elevated serum T3 levels were studied regarding the transport of T3. Human MCT8 (hMCT8) function was studied in wild-type (WT) or mutant hMCT8-transfected human placental choriocarcinoma cells (JEG3) by visualizing the locations of the proteins in the cells, detecting specific proteins, and measuring T3 uptake. We identified 6 missense (p.Arg445Ser, p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, and p.Gly312Arg), 2 frameshift (p.Arg355Profs*64 and p.Tyr550Serfs*17), and 1 deletion (p.Pro561del) mutation(s) in the hMCT8 gene. All patients exhibited clinical characteristics of AHDS with high free T3, low-normal free T4, and normal-elevated TSH levels. All tested mutants were expressed at the protein level, except p.Arg355Profs*64 and p.Tyr550Serfs*17, which were truncated, and were inactive in T3 uptake, excluding p.Arg445Ser and p.Pro561del mutants, compared with WT-hMCT8. Immunocytochemistry revealed plasma membrane localization of p.Arg445Ser and p.Pro561del mutants similar with WT-hMCT8. The other mutants failed to localize in significant amount(s) in the plasma membrane and instead localized in the cytoplasm. These data indicate that p.Arg445Ser and p.Pro561del mutants preserve residual function, whereas p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, p.Gly312Arg, p.Arg355Profs*64, and p.Tyr550Serfs*17 mutants lack function. These findings suggest that the mutations in MCT8 cause loss of function by reducing protein expression, impairing trafficking of protein to plasma membrane, and disrupting substrate channel

An overgrowth disorder associated with excessive production of cGMP due to a gain-of-function mutation of the natriuretic peptide receptor 2 gene.

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