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Effect of X-linked hypophosphatemia (the Hyp mutation) on gene expression in the mid-shaft of the mouse femur

ABSTRACT: The pathophysiology of the osteomalacia in X-linked hypophosphatemia is uncertain. In this project, genomic DNA microarrays were used to identify novel genes with abnormal mRNA expression levels in mice with the dominant Hyp mutation of the Phex gene. Femoral shafts from five-week-old C57BL/6J mice, male and female, normal and Hyp (hemizygous male and heterozygous female), were flushed with saline to remove the marrow. RNA was extracted from each bone, pooled between two mice for each array, processed to cRNA, and hybridized to Affymetrix Mouse 430 2.0 GeneChip microarrays with probe sets for 45,101 genes. Twenty microarrays (40 mice) were done with 5 arrays for each treatment group (normal male, Hyp male, normal female and Hyp female). For each gene, factorial analysis of variance was performed for the main effects of genotype (normal vs. Hyp), sex (male vs. female), and genotype-by-sex interaction. The mRNA levels for 54 % of the genes on each array were scored as present. At P < 0.01, 2,635 genes were significant for genotype, 1,488 for sex, and 509 for genotype-by-sex interaction. There were two probes sets for the Phex gene. Probe 1450445, at the 3’ end of the coding sequence, was low in normal samples (246 ± 37 (10), mean ± SEM (n)) and absent in Hyp samples. Probe 1421979, at the far 3’ end of the untranslated region of the cDNA, 3,000 base pairs from the coding sequence, was high in normal mice (3,915 ± 315 (10); 8x brighter than the average gene), undetectable in Hyp males, and 725 ± 93 (5) in Hyp females. Both probe sets were scored as absent in kidney tissue. In Hyp bone, male and female, there was significant down-regulation of markers of osteoblasts and bone matrix synthesis with significant up-regulation of markers of blood vessel formation and cytoskeleton. No prominent skeletal gene was up-regulated in Hyp to attempt to compensate for the low skeletal mineralization. The genes with significant genotype-by-sex interaction did not show a marked fold difference between male and female Hyp mice. In conclusion, male and female Hyp mice showed similar depression of mRNA levels of genes related to bone synthesis in the femoral shaft. There was a high signal level from probes for a sequence in the 3’ untranslated region of the Phex gene of normal, but not Hyp, mice, suggesting the need for further study of the molecular organization of this gene. Keywords: 2x2 factorial design with complete blocks

ORGANISM(S): Mus musculus

PROVIDER: GSE5657 | GEO | 2007/02/06

SECONDARY ACCESSION(S): PRJNA96865

REPOSITORIES: GEO

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Project description:The pathophysiology of the osteomalacia in X-linked hypophosphatemia is uncertain. In this project, genomic DNA microarrays were used to identify novel genes with abnormal mRNA expression levels in mice with the dominant Hyp mutation of the Phex gene. Femoral shafts from five-week-old C57BL/6J mice, male and female, normal and Hyp (hemizygous male and heterozygous female), were flushed with saline to remove the marrow. RNA was extracted from each bone, pooled between two mice for each array, processed to cRNA, and hybridized to Affymetrix Mouse 430 2.0 GeneChip microarrays with probe sets for 45,101 genes. Twenty microarrays (40 mice) were done with 5 arrays for each treatment group (normal male, Hyp male, normal female and Hyp female). For each gene, factorial analysis of variance was performed for the main effects of genotype (normal vs. Hyp), sex (male vs. female), and genotype-by-sex interaction. The mRNA levels for 54 % of the genes on each array were scored as present. At P < 0.01, 2,635 genes were significant for genotype, 1,488 for sex, and 509 for genotype-by-sex interaction. There were two probes sets for the Phex gene. Probe 1450445, at the 3â end of the coding sequence, was low in normal samples (246 Â± 37 (10), mean Â± SEM (n)) and absent in Hyp samples. Probe 1421979, at the far 3â end of the untranslated region of the cDNA, 3,000 base pairs from the coding sequence, was high in normal mice (3,915 Â± 315 (10); 8x brighter than the average gene), undetectable in Hyp males, and 725 Â± 93 (5) in Hyp females. Both probe sets were scored as absent in kidney tissue. In Hyp bone, male and female, there was significant down-regulation of markers of osteoblasts and bone matrix synthesis with significant up-regulation of markers of blood vessel formation and cytoskeleton. No prominent skeletal gene was up-regulated in Hyp to attempt to compensate for the low skeletal mineralization. The genes with significant genotype-by-sex interaction did not show a marked fold difference between male and female Hyp mice. In conclusion, male and female Hyp mice showed similar depression of mRNA levels of genes related to bone synthesis in the femoral shaft. There was a high signal level from probes for a sequence in the 3â untranslated region of the Phex gene of normal, but not Hyp, mice, suggesting the need for further study of the molecular organization of this gene. Experiment Overall Design: Equal amounts of RNA from two mice, matched for genotype and sex were pooled to create each sample for microarray analysis. Four treatment groups were done: (1) Normal male mice, (2) Hyp male mice, (3) normal female mice, and (4) Hyp female mice. Five replicates were done with each replicate containing one sample from each of the four treatment groups for a total of 20 independent samples (40 mice total). Each replicate was matched for littermates and parallel processing.

Project description:We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts regulating Fgf23 production and mineralization in Hyp mice. We discovered that activation of Fgf receptor- and Wnt-pathways contribute to increased Ffg23 gene transcription in Hyp bone. We found evidence in Hyp bone for increased expression of Fgf1, Fgf7, and Egr2 in the Fgf-signaling pathway and decrements in Sost and Cpz and increments in Sfrp1 and 4 in the Wnt-signaling pathway. Moreover, activation of Fgf and Wnt-signaling stimulated, whereas Tgf β inhibited Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the Fgf23 regulatory extracellular matrix protein Dmp1. These findings suggest that elevation of Fgf23 expression in osteocytes is regulated by interactions between cell surface expression of Phex, extracellular matrix proteins and paracrine effects of Fgf and Wnt. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced post-translational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regards to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone. We isolated total RNAs from long bones of both WT and Hyp mice at 12 days of age. Since the RNA yields from the long bones are very low, we combined 2 bone samples with same genotype (WT or Hyp) for one RNA extraction. We will compare the difference of the gene expressions between Hyp and WT. We will use 4 samples in each animal condition.

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Effect of X-linked hypophosphatemia (the Hyp mutation) on gene expression in the mid-shaft of the mouse femur

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