Department of Orthopaedic Surgery, Boston Children's Hospital, Boston, MA, USA; Department of Genetics, Harvard Medical School, Boston, MA, USA.
J Bone Miner Res. 2013 Oct;28(10):2081-93. doi: 10.1002/jbmr.1946.
Loss-of-function and certain missense mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) significantly decrease or increase bone mass, respectively. These human skeletal phenotypes have been recapitulated in mice harboring Lrp5 knockout and knock-in mutations. We hypothesized that measuring mRNA expression in diaphyseal bone from mice with Lrp5 wild-type (Lrp5(+/+) ), knockout (Lrp5(-/-) ), and high bone mass (HBM)-causing (Lrp5(p.A214V/+) ) knock-in alleles could identify genes and pathways that regulate or are regulated by LRP5 activity. We performed RNA-seq on pairs of tibial diaphyseal bones from four 16-week-old mice with each of the aforementioned genotypes. We then evaluated different methods for controlling for contaminating nonskeletal tissue (ie, blood, bone marrow, and skeletal muscle) in our data. These methods included predigestion of diaphyseal bone with collagenase and separate transcriptional profiling of blood, skeletal muscle, and bone marrow. We found that collagenase digestion reduced contamination, but also altered gene expression in the remaining cells. In contrast, in silico filtering of the diaphyseal bone RNA-seq data for highly expressed blood, skeletal muscle, and bone marrow transcripts significantly increased the correlation between RNA-seq data from an animal's right and left tibias and from animals with the same Lrp5 genotype. We conclude that reliable and reproducible RNA-seq data can be obtained from mouse diaphyseal bone and that lack of LRP5 has a more pronounced effect on gene expression than the HBM-causing LRP5 missense mutation. We identified 84 differentially expressed protein-coding transcripts between LRP5 "sufficient" (ie, Lrp5(+/+) and Lrp5(p.A214V/+) ) and "insufficient" (Lrp5(-/-) ) diaphyseal bone, and far fewer differentially expressed genes between Lrp5(p.A214V/+) and Lrp5(+/+) diaphyseal bone.
Wnt 核心受体低密度脂蛋白受体相关蛋白 5（LRP5）的功能丧失和某些错义突变分别显著降低和增加骨量。这些人类骨骼表型已在携带 Lrp5 敲除和敲入突变的小鼠中重现。我们假设，测量具有 Lrp5 野生型（Lrp5(+/+)）、敲除（Lrp5(-/-)）和高骨量（HBM）引起（Lrp5(p.A214V/+)）的小鼠骨干质骨的 mRNA 表达可以鉴定出调节或受 LRP5 活性调节的基因和途径。我们对来自四只 16 周龄的上述基因型的小鼠的对侧胫骨骨干进行了 RNA-seq。然后，我们评估了不同的方法来控制数据中混杂的非骨骼组织（即血液、骨髓和骨骼肌）。这些方法包括用胶原酶预消化骨干和分别对血液、骨骼肌和骨髓进行转录谱分析。我们发现胶原酶消化减少了污染，但也改变了剩余细胞中的基因表达。相比之下，通过计算从动物左右胫骨和具有相同 Lrp5 基因型的动物的 RNA-seq 数据中过滤高度表达的血液、骨骼肌和骨髓转录本，可以显著增加 RNA-seq 数据之间的相关性。我们得出结论，从小鼠骨干中可以获得可靠和可重复的 RNA-seq 数据，并且缺乏 LRP5 对基因表达的影响比 HBM 引起的 LRP5 错义突变更为明显。我们在 LRP5“充足”（即 Lrp5(+/+)和 Lrp5(p.A214V/+)）和“不足”（Lrp5(-/-)）骨干之间鉴定出 84 个差异表达的蛋白编码转录本，而在 Lrp5(p.A214V/+)和 Lrp5(+/+)骨干之间差异表达的基因则要少得多。
