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Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets.

ABSTRACT: We previously demonstrated that the mutations Met1Val (M1V) and the deletion of nucleotides 1484-1490 (1484-1490del) in Dentin matrix protein-1 (DMP1) cause the novel disorder autosomal recessive hypophosphatemic rickets (ARHR), which is associated with elevated fibroblast growth factor-23 (FGF23). To further understand the role of DMP1 in ARHR, we undertook molecular genetic and in vitro expression studies. First, we examined a kindred with a severe hypophosphatemic rickets phenotype and recessive inheritance. Analyses of this family demonstrated that the affected members had elevated serum FGF23 and carried a large, biallelic deletion that removed the majority of DMP1. At a minimum, this deletion encompassed 49 kb between DMP1 exon 3 and an intergenic region 5' to the next telomeric gene, integrin-binding sialoprotein (IBSP). We next performed immunofluorescent studies in cells to understand the effects of the known ARHR mutations on DMP1 cellular processing. These analyses showed that the M1V DMP1 mutant was not sorted to the trans-Golgi network (TGN) and secretory pathway, but filled the entire cytoplasm. In contrast, the 1484-1490del mutant localized to the TGN and was secreted, similar to wild type DMP1. The 1484-1490del mutation replaces the DMP1 18 C-terminal amino acids with 33 non-native residues. Truncation of wild type DMP1 by these native 18 residues followed by Western blot and confocal microscopic analyses demonstrated a wild type expression pattern when compared with the 1484-1490del mutant, indicating that the last 18 residues are not critical for cellular trafficking, but that the 33 additional residues arising from the 1484-1490del mutation likely compromise DMP1 processing. The relationship between DMP1 and FGF23 is unclear. To test endogenous DMP1 response to serum metabolites that also regulate FGF23, UMR-106 cells were treated with 1,25(OH)(2) vitamin D (1x10(-7) M) and showed a 12-fold increase in DMP1 mRNA and protein at 24 h. In summary, we have identified a novel DMP1 deletion as the cause of ARHR, as well as demonstrated that the ARHR mutations alter DMP1 cellular processing, and that DMP1 can be regulated by vitamin D. Taken together, this work expands our understanding of the genetic and molecular mechanisms associated with DMP1 alterations causing ARHR.

SUBMITTER: Farrow EG

PROVIDER: S-EPMC2669955 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets.

Farrow Emily G EG Davis Siobhan I SI Ward Leanne M LM Summers Lelia J LJ Bubbear Judith S JS Keen Richard R Stamp Trevor C B TC Baker Laurence R I LR Bonewald Lynda F LF White Kenneth E KE

Bone 20081025 2

We previously demonstrated that the mutations Met1Val (M1V) and the deletion of nucleotides 1484-1490 (1484-1490del) in Dentin matrix protein-1 (DMP1) cause the novel disorder autosomal recessive hypophosphatemic rickets (ARHR), which is associated with elevated fibroblast growth factor-23 (FGF23). To further understand the role of DMP1 in ARHR, we undertook molecular genetic and in vitro expression studies. First, we examined a kindred with a severe hypophosphatemic rickets phenotype and recess ...[more]

PMID: 19007919

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Project description:Autosomal recessive hypophosphatemic rickets (ARHR) is a heritable disorder characterized by hypophosphatemia, osteomalacia, and poor bone development. ARHR results from inactivating mutations in the DMP1 gene with the human phenotype being recapitulated in the Dmp1 null mouse model which displays elevated plasma fibroblast growth factor 23. While the bone phenotype has been well-characterized, it is not known what effects ARHR may also have on skeletal, cardiac, or vascular smooth muscle function, which is critical to understand in order to treat patients suffering from this condition. In this study, the extensor digitorum longus (EDL-fast-twitch muscle), soleus (SOL-slow-twitch muscle), heart, and aorta were removed from Dmp1 null mice and ex-vivo functional tests were simultaneously performed in collaboration by three different laboratories. Dmp1 null EDL and SOL muscles produced less force than wildtype muscles after normalization for physiological cross sectional area of the muscles. Both EDL and SOL muscles from Dmp1 null mice also produced less force after the addition of caffeine (which releases calcium from the sarcoplasmic reticulum) which may indicate problems in excitation contraction coupling in these mice. While the body weights of the Dmp1 null were smaller than wildtype, the heart weight to body weight ratio was higher. However, there were no differences in pathological hypertrophic gene expression compared to wildtype and maximal force of contraction was not different indicating that there may not be cardiac pathology under the tested conditions. We did observe a decrease in the rate of force development generated by cardiac muscle in the Dmp1 null which may be related to some of the deficits observed in skeletal muscle. There were no differences observed in aortic contractions induced by PGF2? or 5-HT or in endothelium-mediated acetylcholine-induced relaxations or endothelium-independent sodium nitroprusside-induced relaxations. In summary, these results indicate that there are deficiencies in both fast twitch and slow twitch muscle fiber type contractions in this model of ARHR, while there was less of a phenotype observed in cardiac muscle, and no differences observed in aortic function. These results may help explain skeletal muscle weakness reported by some patients with osteomalacia and need to be further investigated.

Project description:Autosomal dominant hypophosphatemic rickets (ADHR) is caused by mutations impairing cleavage of fibroblast growth factor 23 (FGF23). FGF23 gene expression increases during iron deficiency. In humans and mice with the ADHR mutation, iron deficiency results in increased intact FGF23 concentrations and hypophosphatemia. We conducted a prospective open label pilot clinical trial of oral iron replacement over 12?months in ADHR patients to test the hypothesis that oral iron administration would normalize FGF23 concentrations. Eligibility criteria included: FGF23 mutation; and either serum iron <50 ?g/dL; or serum iron 50 to 100 ?g/dL combined with hypophosphatemia and intact FGF23?>30?pg/mL at screening. Key exclusion criteria were kidney disease and pregnancy. Oral iron supplementation started at 65?mg daily and was titrated based on fasting serum iron concentration. The primary outcome was decrease in fasting intact FGF23 by ?20% from baseline. Six adults (three male, three female) having the FGF23-R176Q mutation were enrolled; five completed the 12-month protocol. At baseline three of five subjects had severely symptomatic hypophosphatemia (phosphorus <2.5?mg/dL) and received calcitriol with or without phosphate concurrent with oral iron during the trial. The primary outcome was met by 4 of 5 (80%) subjects all by month 4, and 5 of 5 had normal intact FGF23 at month 12. Median (minimum, maximum) intact FGF23 concentration decreased from 172 (20, 192) pg/mL at baseline to 47 (17, 78) pg/mL at month 4 and 42 (19, 63) pg/mL at month 12. Median ferritin increased from 18.6 (7.7, 82.5) ng/mL at baseline to 78.0 (49.6, 261.0) ng/mL at month 12. During iron treatment, all three subjects with baseline hypophosphatemia normalized serum phosphorus, had markedly improved symptoms, and were able to discontinue calcitriol and phosphate. Oral iron repletion normalized FGF23 and phosphorus in symptomatic, iron-deficient ADHR subjects. Thus, the standard approach to ADHR should include recognition, treatment, and prevention of iron deficiency. © 2019 American Society for Bone and Mineral Research.

Dataset Information

Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets.

Publications

Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets.

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