Transcription profiling of mouse femur in X-linked hypophosphatemia Hyp mutated animals

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. 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.X连锁低磷血症（X-linked hypophosphatemia）相关骨软化症的病理生理学机制尚未明确。本研究利用基因组DNA微阵列，旨在鉴定Phex基因（Phex gene）显性Hyp突变小鼠中mRNA表达异常的新基因。实验取材于5周龄C57BL/6J小鼠的股骨干，涵盖正常雌雄小鼠与Hyp突变小鼠（半合子雄性及杂合子雌性），先用生理盐水冲洗以去除骨髓。提取每根骨骼的RNA，每两只同基因型、同性别的小鼠RNA混合后制备为互补RNA（cRNA），随后与搭载45101个基因探针集的爱费默尼克斯（Affymetrix）Mouse 430 2.0基因芯片微阵列进行杂交。本实验共完成20张芯片杂交，对应40只小鼠，每个处理组（正常雄性、Hyp突变雄性、正常雌性、Hyp突变雌性）各5张芯片。针对每个基因，采用析因方差分析检验基因型（正常vs Hyp突变）、性别（雄性vs雌性）及其交互作用的主效应。每张芯片上54%的基因mRNA水平被判定为存在表达信号。当P<0.01时，共有2635个基因在基因型效应上显著，1488个基因在性别效应上显著，509个基因存在基因型-性别交互效应。Phex基因对应2个探针集：位于编码序列3'端的探针1450445，在正常样本中信号值较低（246±37(10)，均值±标准误（SEM，样本量n）），在Hyp突变样本中无信号；位于cDNA非编码区3'远端、距编码序列3000碱基对的探针1421979，在正常小鼠中信号值较高（3915±315(10)，信号强度为平均基因的8倍），在Hyp突变雄性小鼠中无法检测到，在Hyp突变雌性小鼠中信号值为725±93(5)。两个探针集在肾脏组织中均被判定为无表达信号。在Hyp突变小鼠的骨骼中，无论雌雄，成骨细胞与骨基质合成相关标志物均显著下调，而血管生成与细胞骨架相关标志物显著上调。未发现有显著上调的骨骼基因可代偿Hyp突变导致的骨骼矿化不足。存在基因型-性别交互效应的基因，在雌雄Hyp突变小鼠间未表现出显著的表达倍数差异。综上，无论雌雄，Hyp突变小鼠股骨干中与骨合成相关的基因mRNA水平均呈现相似程度的下调。正常小鼠Phex基因3'非编码区探针信号值较高，而Hyp突变小鼠无此信号，提示需进一步研究该基因的分子组织结构。实验总体设计：将同基因型、同性别的两只小鼠的RNA等量混合，制备为每个微阵列分析的样本。共设置4个处理组：(1)正常雄性小鼠、(2)Hyp突变雄性小鼠、(3)正常雌性小鼠、(4)Hyp突变雌性小鼠。每个处理组设置5次生物学重复，每次重复包含4个处理组的各1个样本，共计20个独立样本（对应40只小鼠）。每次重复均采用同窝小鼠样本并进行平行处理。