A novel SATB2 mutation illuminates bone anabolism

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性状（如骨量）的重要见解。评价了一名50岁白色女性，因为其骨量极高（T评分+6 - +10），放射学检查显示骨正常，但皮质非常厚，骨形成和骨吸收标志物长期显著升高。高转换率通常与低骨量有关，特别是在成年女性中，因此该患者非常罕见。(It值得注意的是，LRP 5功能获得性突变的患者P1 NP或CTX水平不升高。全外显子组测序鉴定了该个体中富含特异性AT的结合蛋白2（SATB 2）基因的一种新变体。在位置chr 2：200193607（hg 19）处的单个碱基对变化（G至C）导致氨基酸变化（赖氨酸400至天冬酰胺（K400 N））。这种等位基因变异在父母双方、2500名正常个体或公开数据库中都没有发现。SATB 2是与DNA中的基质附着区（MAR）结合的蛋白质家族的成员，并且先前已报道SATB 2在骨形成中具有关键作用。赖氨酸400位于结合DNA的SATB 2的高度保守的CUT 1结构域中。赖氨酸400在进化上是严格保守的，这表明这种氨基酸在功能上具有重要作用。原位杂交研究证明SABT 2在成骨细胞和骨细胞中有高水平的特异性表达，但在软骨或骨髓中没有表达。MC 3 T3-E1细胞中K400 N变体的过表达导致体外自发矿化。由于目前骨合成代谢治疗的靶点很少，因此探索SATB 2 K400 N突变导致高骨量的机制非常重要。在实验模型中忠实地概括这个个体的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SATB 2等位基因，即，通过创造一个敲入的老鼠。为了开始鉴定SATB 2中该突变所携带的代谢途径，我们将追求以下具体目标：1.使用已通过重组引入突变等位基因的工程化ES细胞克隆产生携带SATB 2-K400 N突变的敲入小鼠。2.通过使用密度测定、组织形态测定和生物力学方法以及通过研究从这些动物中分离的MSC、成骨细胞和破骨细胞来表征SATB 2-K400 N敲入小鼠的表型。3.使用从野生型和基因敲入小鼠的原代颅骨成骨细胞培养物中分离的RNA和Affytron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转换率通常仅见于儿童和青少年时期，这是骨骼快速增长的时期。发生这种情况的分子和代谢途径尚不清楚。SATB 2 K400 N突变是否携带了类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新的分子途径调节骨锚定和新的药物发现的目标。

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.