DMP1 C-Terminal Mutant Mice Recapture the Human ARHR Tooth Phenotype

DMP1 mutations in autosomal recessive hypophosphatemic rickets (ARHR) patients and mice lacking Dmp1 display an overlapping pathophysiology, such as hypophosphatemia. However, subtle differences exist between the mouse model and human ARHR patients. These differences could be due to a species specificity of human versus mouse, or it may be that the mutant DMP1 in humans maintains partial function of DMP1. In this study we report a deformed tooth phenotype in a human DMP1 deletion mutation case. Unexpectedly, the deletion of nucleotides 1484 to 1490 (c.1484_1490delCTATCAC, delMut, resulting in replacement of the last 18 residues with 33 random amino acids) showed a severe dentin and enamel defect similar to a dentinogenesis imperfecta (DI) III–like phenotype. To address the molecular mechanism behind this phenotype, we generated delMut transgenic mice with the endogenous Dmp1 gene removed. These mutant mice did not recapture the abnormal phenotype observed in the human patient but displayed a mild rachitic tooth phenotype in comparison with that in the Dmp1-null mice, suggesting that the DI III–like phenotype may be due to an as-yet-undetermined acquired gene modifier. The mechanism studies showed that the mutant fragment maintains partial function of DMP1 such as stimulating MAP kinase signaling in vitro. Last, the in vitro and in vivo data support a role of odontoblasts in the control of fibroblast growth factor 23 (FGF-23) regulation during early postnatal development, although this regulation on Pi homeostasis is likely limited. © 2010 American Society for Bone and Mineral Research

Long-Term Clinical Outcome and Carrier Phenotype in Autosomal Recessive Hypophosphatemia Caused by a Novel DMP1 Mutation

Homozygous inactivating mutations in DMP1 (dentin matrix protein 1), the gene encoding a noncollagenous bone matrix protein expressed in osteoblasts and osteocytes, cause autosomal recessive hypophosphatemia (ARHP). Herein we describe a family with ARHP owing to a novel homozygous DMP1 mutation and provide a detailed description of the associated skeletal dysplasia and carrier phenotype. The two adult patients with ARHP, a 78-year-old man and his 66-year-old sister, have suffered from bone pain and lower extremity varus deformities since early childhood. With increasing age, both patients developed severe joint pain, contractures, and complete immobilization of the spine. Radiographs showed short and deformed long bones, significant cranial hyperostosis, enthesopathies, and calcifications of the paraspinal ligaments. Biochemistries were consistent with hypophosphatemia owing to renal phosphate wasting; markers of bone turnover and serum fibroblast growth factor 23 (FGF-23) levels were increased significantly. Nucleotide sequence analysis of DMP1 revealed a novel homozygous mutation at the splice acceptor junction of exon 6 (IVS5-1G > A). Two heterozygous carriers of the mutation also showed mild hypophosphatemia, and bone biopsy in one of these individuals showed focal areas of osteomalacia. In bone, DMP1 expression was absent in the homozygote but normal in the heterozygote, whereas FGF-23 expression was increased in both subjects but higher in the ARHP patient. The clinical and laboratory observations in this family confirm that DMP1 has an important role in normal skeletal development and mineral homeostasis. The skeletal phenotype in ARHP may be significantly more severe than in other forms of hypophosphatemic rickets. © 2010 American Society for Bone and Mineral Research

Three Novel Mutations in the PHEX Gene in Chinese Subjects with Hypophosphatemic Rickets Extends Genotypic Variability

Mutations in the phosphate-regulating endopeptidase homolog, X-linked, gene (PHEX), which encodes a zinc-dependent endopeptidase that is involved in bone mineralization and renal phosphate reabsorption, cause the most common form of hypophosphatemic rickets, X-linked hypophosphatemic rickets (XLH). The distribution of PHEX mutations is extensive, but few mutations have been identified in Chinese with XLH. We extracted genomic DNA and total RNA from leukocytes obtained from nine unrelated Chinese subjects (three males and six females, age range 11–36 years) who were living in Taiwan. The PHEX gene was amplified from DNA by PCR, and the amplicons were directly sequenced. Expression studies were performed by reverse-transcription PCR of leukocyte RNA. Serum levels of FGF23 were significantly greater in the patients than in normal subjects (mean 69.4 ± 18.8 vs. 27.2 ± 8.4 pg/mL, P < 0.005), and eight of the nine patients had elevated levels of FGF23. Germline mutations in the PHEX gene were identified in five of 9 patients, including novel c.1843 delA, donor splice site mutations c.663+2delT and c.1899+2T>A, and two previously reported missense mutations, p.C733Y and p.G579R. These data extend the spectrum of mutations in the PHEX gene in Han Chinese and confirm variability for XLH in Taiwan

A Novel Nonsense Mutation in the DMP1 Gene Identified by a Genome-Wide Association Study Is Responsible for Inherited Rickets in Corriedale Sheep

Inherited rickets of Corriedale sheep is characterized by decreased growth rate, thoracic lordosis and angular limb deformities. Previous outcross and backcross studies implicate inheritance as a simple autosomal recessive disorder. A genome wide association study was conducted using the Illumina OvineSNP50 BeadChip on 20 related sheep comprising 17 affected and 3 carriers. A homozygous region of 125 consecutive single-nucleotide polymorphism (SNP) loci was identified in all affected sheep, covering a region of 6 Mb on ovine chromosome 6. Among 35 candidate genes in this region, the dentin matrix protein 1 gene (DMP1) was sequenced to reveal a nonsense mutation 250C/T on exon 6. This mutation introduced a stop codon (R145X) and could truncate C-terminal amino acids. Genotyping by PCR-RFLP for this mutation showed all 17 affected sheep were “T T” genotypes; the 3 carriers were “C T”; 24 phenotypically normal related sheep were either “C T” or “C C”; and 46 unrelated normal control sheep from other breeds were all “C C”. The other SNPs in DMP1 were not concordant with the disease and can all be ruled out as candidates. Previous research has shown that mutations in the DMP1 gene are responsible for autosomal recessive hypophosphatemic rickets in humans. Dmp1_knockout mice exhibit rickets phenotypes. We believe the R145X mutation to be responsible for the inherited rickets found in Corriedale sheep. A simple diagnostic test can be designed to identify carriers with the defective “T” allele. Affected sheep could be used as animal models for this form of human rickets, and for further investigation of the role of DMP1 in phosphate homeostasis

The Biological Function of DMP-1 in Osteocyte Maturation Is Mediated by Its 57-kDa C-terminal Fragment

Dentin matrix protein 1 (DMP-1) is a key molecule in controlling osteocyte formation and phosphate homeostasis. Based on observations that full-length DMP-1 is not found in bone, but only cleaved fragments of 37 and 57 kDa are present, and in view of the finding that mutations in the 57-kDa fragment result in disease, we hypothesized that the 57-kDa C-terminal fragment is the functional domain of DMP-1. To test this hypothesis, a 3.6-kb type I collagen promoter was used to express this 57-kDa C-terminal fragment for comparison with full-length DMP-1 in Dmp1 null osteoblasts/osteocytes. Not only did expression of the full-length DMP-1 in bone cells fully rescue the skeletal abnormalities of Dmp1 null mice, but the 57-kDa fragment also had similar results. This included rescue of growth plate defects, osteomalacia, abnormal osteocyte maturation, and the abnormal osteocyte lacunocanalicular system. In addition, the abnormal fibroblast growth factor 23 (FGF-23) expression in osteocytes, elevated circulating FGF-23 levels, and hypophosphatemia were rescued. These results show that the 57-kDa C-terminal fragment is the functional domain of DMP-1 that controls osteocyte maturation and phosphate metabolism. © 2011 American Society for Bone and Mineral Research

FGF-23: More Than a Regulator of Renal Phosphate Handling?

Fibroblast growth factor 23 (FGF-23) is likely to be the most important regulator of phosphate homeostasis, which mediates its functions through FGF receptors and the coreceptor Klotho. Besides reducing expression of the sodium-phosphate cotransporters NPT2a and NPT2c in the proximal tubules, FGF-23 inhibits the renal 1α-hydroxylase and stimulates the 24-hydroxylase, and it appears to reduce parathyroid hormone (PTH) secretion in short-term studies. FGF-23 synthesis and secretion by osteocytes and osteoblasts is upregulated through 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and through an increased dietary phosphate intake. FGF-23 levels are elevated or inappropriately normal in patients with tumor-induced osteomalacia and several inherited hypophosphatemic disorders, but the most significant increases are found in patients with chronic kidney disease (CKD). During the early stages of CKD, increased FGF-23 production enhances urinary phosphate excretion and thus prevents the development of hyperphosphatemia, reduces the circulating levels of 1,25(OH)2D3, and therefore contributes to the development of secondary hyperparathyroidism. In patients with end-stage renal disease (ESRD), FGF-23 levels can be extremely high and were shown to be predictors of bone mineralization, left ventricular hypertrophy, vascular calcification, and mortality. It remains to be determined, however, whether FGF-23 represents simply a sensitive biomarker of an abnormal phosphate homeostasis or has, independent of serum phosphate levels, potentially negative “off-target” effects. Nonetheless, reducing the production and/or the biologic activity of FGF-23 may be an important therapeutic goal for this patient population. © 2010 American Society for Bone and Mineral Research

Identification of FOXP1 Deletions in Three Unrelated Patients with Mental Retardation and Significant Speech and Language Deficits

Mental retardation affects 2-3% of the population and shows a high heritability. Neurodevelopmental disorders that include pronounced impairment in language and speech skills occur less frequently. For most cases, the molecular basis of mental retardation with or without speech and language disorder is unknown due to the heterogeneity of underlying genetic factors. We have used molecular karyotyping on 1523 patients with mental retardation to detect copy number variations (CNVs) including deletions or duplications. These studies revealed three heterozygous overlapping deletions solely affecting the forkhead box P1 (FOXP1) gene. All three patients had moderate mental retardation and significant language and speech deficits. Since our results are consistent with a de novo occurrence of these deletions, we considered them as causal although we detected a single large deletion including FOXP1 and additional genes in 4104 ancestrally matched controls. These findings are of interest with regard to the structural and functional relationship between FOXP1 and FOXP2. Mutations in FOXP2 have been previously related to monogenic cases of developmental verbal dyspraxia. Both FOXP1 and FOXP2 are expressed in songbird and human brain regions that are important for the developmental processes that culminate in speech and language. ©2010 Wiley-Liss, Inc

A protective role for FGF-23 in local defence against disrupted arterial wall integrity?

Increasing interest is focusing on the role of the FGF-23/Klotho axis in mediating vascular calcification. However, the underpinning mechanisms have yet to be fully elucidated. Murine VSMCs were cultured in calcifying medium for a 21d period. FGF-23 mRNA expression was significantly up-regulated by 7d (1.63 fold; P<0.001), with a concomitant increase in protein expression. mRNA and protein expression of both FGFR1 and Klotho were confirmed. Increased FGF-23 and Klotho protein expression was also observed in the calcified media of Enpp1(−/−) mouse aortic tissue. Reduced calcium deposition was observed in calcifying VSMCs cultured with recombinant FGF-23 (10ng/ml; 28.1% decrease; P<0.01). Calcifying VSMCs treated with PD173074, an inhibitor of FGFR1 and FGFR3, showed significantly increased calcification (50nM; 87.8% increase; P<0.001). FGF-23 exposure induced phosphorylation of ERK1/2. Treatment with FGF-23 in combination with PD98059, an ERK1/2 inhibitor, significantly increased VSMC calcification (10μM; 41.3% increase; P<0.01). Use of FGF-23 may represent a novel therapeutic strategy for inhibiting vascular calcification

Development of a long noncoding RNA-based machine learning model to predict COVID-19 in-hospital mortality

Tools for predicting COVID-19 outcomes enable personalized healthcare, potentially easing the disease burden. This collaborative study by 15 institutions across Europe aimed to develop a machine learning model for predicting the risk of in-hospital mortality post-SARS-CoV-2 infection. Blood samples and clinical data from 1286 COVID-19 patients collected from 2020 to 2023 across four cohorts in Europe and Canada were analyzed, with 2906 long non-coding RNAs profiled using targeted sequencing. From a discovery cohort combining three European cohorts and 804 patients, age and the long non-coding RNA LEF1-AS1 were identified as predictive features, yielding an AUC of 0.83 (95% CI 0.82-0.84) and a balanced accuracy of 0.78 (95% CI 0.77-0.79) with a feedforward neural network classifier. Validation in an independent Canadian cohort of 482 patients showed consistent performance. Cox regression analysis indicated that higher levels of LEF1-AS1 correlated with reduced mortality risk (age-adjusted hazard ratio 0.54, 95% CI 0.40-0.74). Quantitative PCR validated LEF1-AS1's adaptability to be measured in hospital settings. Here, we demonstrate a promising predictive model for enhancing COVID-19 patient management.</p

Genetic Evidence of Serum Phosphate-Independent Functions of FGF-23 on Bone

Maintenance of physiologic phosphate balance is of crucial biological importance, as it is fundamental to cellular function, energy metabolism, and skeletal mineralization. Fibroblast growth factor-23 (FGF-23) is a master regulator of phosphate homeostasis, but the molecular mechanism of such regulation is not yet completely understood. Targeted disruption of the Fgf-23 gene in mice (Fgf-23−/−) elicits hyperphosphatemia, and an increase in renal sodium/phosphate co-transporter 2a (NaPi2a) protein abundance. To elucidate the pathophysiological role of augmented renal proximal tubular expression of NaPi2a in Fgf-23−/− mice and to examine serum phosphate–independent functions of Fgf23 in bone, we generated a new mouse line deficient in both Fgf-23 and NaPi2a genes, and determined the effect of genomic ablation of NaPi2a from Fgf-23−/− mice on phosphate homeostasis and skeletal mineralization. Fgf-23−/−/NaPi2a−/− double mutant mice are viable and exhibit normal physical activities when compared to Fgf-23−/− animals. Biochemical analyses show that ablation of NaPi2a from Fgf-23−/− mice reversed hyperphosphatemia to hypophosphatemia by 6 weeks of age. Surprisingly, despite the complete reversal of serum phosphate levels in Fgf-23−/−/NaPi2a−/−, their skeletal phenotype still resembles the one of Fgf23−/− animals. The results of this study provide the first genetic evidence of an in vivo pathologic role of NaPi2a in regulating abnormal phosphate homeostasis in Fgf-23−/− mice by deletion of both NaPi2a and Fgf-23 genes in the same animal. The persistence of the skeletal anomalies in double mutants suggests that Fgf-23 affects bone mineralization independently of systemic phosphate homeostasis. Finally, our data support (1) that regulation of phosphate homeostasis is a systemic effect of Fgf-23, while (2) skeletal mineralization and chondrocyte differentiation appear to be effects of Fgf-23 that are independent of phosphate homeostasis