A novel SATB2 mutation illuminates bone anabolism

一种新的 SATB2 突变阐明了骨合成代谢

基本信息

批准号：
8758799
负责人：
KARL Leonard INSOGNA
金额：
$ 21.98万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8758799
关键词：
Adolescence Adult Affect Alleles Amino Acids Anabolism Animals Asparagine Back Base Pairing Binding Proteins Biomechanics Bone Density Bone Resorption Bone remodeling Calvaria Cartilage Cells Childhood Clinical Trials Cyclophosphamide DNA DNA Binding Data Data Set Databases Development Dideoxy Chain Termination DNA Sequencing Dual-Energy X-Ray Absorptiometry Engineering Experimental Models Family member Female Genes Genetic Recombination Genetic Transcription Genomic DNA Genotype In Situ Hybridization In Vitro Individual Knock-in Mouse Lead Lysine Marrow Matrix Attachment Regions Measures Mesenchymal Stem Cells Metabolic Pathway Metabolism Methodology Molecular Mus Mutation Osteoblasts Osteoclasts Osteocytes Osteogenesis Osteoporosis Parents Pathway interactions Patients Phase Phenotype Play Positioning Attribute Protein Binding RNA Rare Diseases Recording of previous events Regulator Genes Reporting Role Signal Transduction Spectroscopy, Fourier Transform Infrared Testing Thick Variant Woman Work bone bone mass bone metabolism bone strength bone turnover clinical phenotype drug discovery embryonic stem cell exome exome sequencing fly gain of function mutation genetic variant insight kindred mineralization mouse genome mutant neutralizing antibody novel novel therapeutics older women overexpression public health relevance research study skeletal trait

项目摘要

DESCRIPTION (provided by applicant): The study of uncommon clinical phenotypes can sometimes provide important insights into common genetically influenced traits, such as bone mass. A 50 yo white female was evaluated because of extremely high bone mass (T-scores +6 - +10), radiographically normal appearing bones except for very thick cortices, and chronically and markedly elevated markers of both bone formation and bone resorption. High turnover is usually associated with low bone mass, particularly in adult women, so this patient is very unusual. (It is worth noting that patients with gain-of-function mutations in LRP5 do not have elevated levels of P1NP or CTX.) Whole exome sequencing identified a novel variant in the gene Special AT rich Binding Protein 2 (SATB2) in this individual. A single base pair change, G to C, at position chr2:200193607 (hg19) resulted in an amino acid change, lysine 400 to asparagine (K400N). This allelic variant was not seen in either parent, in 2500 normal individuals or in publicly available databases. SATB2 is a member of the family of proteins that bind to matrix attachment regions (MAR) in DNA and SATB2 has been previously reported to have a critical role in bone formation. Lysine 400 is in the highly conserved CUT1 domain of SATB2 that binds DNA. Lysine 400 is strictly evolutionarily conserved back to flies, indicating a functionally important role for this amino acid. In situ hybridization studies documented high-level specific expression of SABT2 in osteoblasts and osteocytes, but not in cartilage or marrow. Overexpression of the K400N variant in MC3T3-E1 cells led to spontaneous in vitro mineralization. Since at present there are very few targets for bone anabolic therapies, exploring the mechanisms by which the SATB2 K400N mutation results in high bone mass is important. The only way to faithfully recapitulate the genotype of this individual in an experimental model is to replace one wild type SATB2 allele in a mouse with the mutant allele, i.e., by creating a knock-in mouse. To begin to identify the metabolic pathways entrained by this mutation in SATB2, we will pursue the following Specific Aims: 1. Generate knock-in mice bearing the SATB2-K400N mutation using an engineered ES cell clone in which the mutant allele has already been introduced by recombination. 2. Characterize the phenotype of the SATB2- K400N knock-in mice by using densitometric, histomorphometric and biomechanical methodologies and by studying MSCs, osteoblasts and osteoclasts isolated from these animals. 3. Undertake gene profiling studies using RNA isolated from primary calvarial osteoblast cultures from wild type and knock-in mice and Affymetrix whole mouse genome 430 2.0 arrays. High rates of skeletal turnover associated with increasing bone mass are normally only seen during childhood and adolescence, a period of rapid skeletal accrual. The molecular and metabolic pathways by which this occurs are unclear. Whether pathways similar to those are entrained by the SATB2 K400N mutation remains to be determined, but is an intriguing possibility. The proposed studies will help identify new molecular pathways regulating bone anabolism and new targets for drug discovery.

描述（由申请人提供）：对不常见临床表型的研究有时可以提供对常见遗传影响特征（例如骨量）的重要见解。对一名 50 岁的白人女性进行了评估，因为其骨量极高（T 值 +6 - +10），除了非常厚的皮质外，放射学上骨骼外观正常，并且骨形成和骨吸收的标记物长期显着升高。高周转率通常与低骨量有关，特别是对于成年女性，因此该患者非常不寻常。（值得注意的是，LRP5 功能获得性突变的患者的 P1NP 或 CTX 水平并未升高。）全外显子组测序在该个体的富含特殊 AT 结合蛋白 2 (SATB2) 的基因中发现了一个新的变异。 Chr2:200193607 (hg19) 位置处的单个碱基对变化（G 变为 C）导致氨基酸变化，即第 400 位赖氨酸变为天冬酰胺 (K400N)。在父母、2500 名正常人或公开数据库中都没有发现这种等位基因变异。 SATB2 是与 DNA 中的基质附着区 (MAR) 结合的蛋白质家族的一员，此前曾报道 SATB2 在骨形成中具有关键作用。赖氨酸 400 位于 SATB2 的高度保守的 CUT1 结构域中，可结合 DNA。赖氨酸 400 在进化上是严格保守的，这表明该氨基酸在功能上发挥着重要作用。原位杂交研究记录了 SABT2 在成骨细胞和骨细胞中的高水平特异性表达，但在软骨或骨髓中没有。 MC3T3-E1 细胞中 K400N 变体的过度表达导致自发的体外矿化。由于目前骨合成代谢治疗的靶点很少，因此探索SATB2 K400N突变导致高骨量的机制具有重要意义。在实验模型中忠实重现该个体基因型的唯一方法是用突变等位基因替换小鼠中的一个野生型 SATB2 等位基因，即通过创建敲入小鼠。为了开始确定 SATB2 中这种突变所带来的代谢途径，我们将追求以下具体目标： 1. 使用已引入突变等位基因的工程 ES 细胞克隆生成携带 SATB2-K400N 突变的敲入小鼠通过重组。 2. 通过使用光密度、组织形态测量和生物力学方法以及研究从这些动物中分离的 MSC、成骨细胞和破骨细胞来表征 SATB2-K400N 敲入小鼠的表型。 3. 使用从野生型和敲入小鼠的原代颅骨成骨细胞培养物中分离的 RNA 以及 Affymetrix 全小鼠基因组 430 2.0 阵列进行基因分析研究。与骨量增加相关的高骨骼更新率通常只出现在儿童期和青春期，这是骨骼快速增长的时期。这种情况发生的分子和代谢途径尚不清楚。 SATB2 K400N 突变是否会产生与这些相似的途径仍有待确定，但这是一个有趣的可能性。拟议的研究将有助于确定调节骨合成代谢的新分子途径和药物发现的新靶点。