Mode of Action of SQSTM1 Mutations in Paget's Disease Bone

描述（由申请人提供）：骨的佩吉特病是一种骨快速形成的疾病，改变了骨的强度和形状。家族性佩吉特病的易感性与许多基因座有关，包括Sequestosome 1（SQSTM 1）基因座，其中在40%的家族性病例中鉴定出种系突变。在一些明显的散发性佩吉特病病例中也发现了种系SQSTM 1突变，这表明这些可能是新发家族性病例。在所有的情况下，除了一个，宪法突变已被发现是杂合子。这表明突变可能以显性方式起作用。然而，也有可能该突变是作为显性阴性或单倍不足突变，或作为隐性突变，在受影响的骨中经历了杂合性丢失。我们很好奇受影响骨头的变异性质。我们选择检查散发性患者受影响骨骼中的SQSTM 1基因座，以观察：1）SQSTM 1的体细胞突变是否发生在这些骨骼中，以及2）受影响骨骼中的突变是杂合还是纯合。当我们在受影响的骨和匹配的外周血中测序SQSTM 1基因时，我们在5个受影响的骨样本中的4个中发现了P392L突变，而在匹配的血液样本中没有发现。更令人惊讶的是，在受影响的骨骼中，样本中P392L突变的相对频率范围为10%至28%，这表明骨骼是突变的镶嵌体。每个个体样品中突变的相对频率在重复重测序和重测序时是一致的。其次，由于骨肉瘤是佩吉特病的并发症，我们很想知道SQSTM 1突变是否存在于佩吉特性骨肉瘤中。当我们在这些肿瘤中筛选SQSTM 1基因时，我们发现5例骨肉瘤中有4例具有纯合P392L突变。同样，在匹配的正常骨中没有检测到突变。总之，这些证据导致了我们的佩吉特病模型的范式转变。这表明，虽然SQSTM 1突变是家族性佩吉特病的易感因素，但它们不是散发性佩吉特病的起始事件。其次，这表明肿瘤起源于含有SQSTM 1突变的成骨细胞，成骨细胞在佩吉特病的病因学中起作用。我们的假设是SQSTM 1的突变发生在pagetic骨中成骨细胞样细胞的一个亚群中，并且这些体细胞突变在pagetic骨肉瘤肿瘤发生过程中成为纯合的。为了验证这一假设，我们提出了以下具体目标：1）测试体细胞SQSTM 1突变是否是杂合的和克隆的pagetic骨。2)检测SQSTM 1基因座的杂合性丢失是否发生在pagetic骨或pagetic肿瘤发生过程中。

DESCRIPTION (provided by applicant): Paget's Disease of bone is a condition in which rapid bone formation occurs, altering the strength and shape of the bone. Predisposition to familial Paget's Disease has been linked to a number of loci, including the Sequestosome 1 (SQSTM1) locus where germline mutations have been identified in 40% of the familial cases. Germline SQSTM1 mutations have also been found in some apparent sporadic cases of Paget's Disease, suggesting that these may be de novo familial cases. In all but one case, the constitutional mutations have been found to be heterozygous. This suggests that the mutation may be acting in a dominant manner. However, it is also possible that the mutation is acting as a dominant negative or haploinsufficiency mutation, or as a recessive mutation that has undergone loss of heterozygosity in the affected bone. We were curious as to the nature of the mutation in the affected bone. We chose to examine the SQSTM1 locus in the affected bones of sporadic patients to see: 1) whether somatic mutations in SQSTM1 occurred in these bones and 2) whether the mutation was heterozygous or homozygous in the affected bone. When we sequenced the SQSTM1 gene in both the affected bone and matched peripheral blood, we found a P392L mutation in 4 of 5 samples of affected bone and none of the matched blood samples. What was more surprising was that in the affected bone, the relative frequency of the P392L mutation in the samples ranged from 10% to 28%, suggesting that the bone was mosaic for the mutation. The relative frequency of the mutation in each individual sample was consistent upon repeated resampling and resequencing. Next, since osteosarcoma is a complication of Paget's Disease, we were curious to see whether SQSTM1 mutations were present in pagetic osteosarcomas. When we screened the SQSTM1 gene in these tumors, we found that 4 of 5 osteosarcomas had homozygous P392L mutations. Again, no mutations were detected in the matched normal bone. Together, this evidence causes a paradigm shift in our model of Paget's Disease. It suggests that while mutations in SQSTM1 are predisposing in familial Paget's Disease, they are not the initiating event in sporadic Paget's Disease. Secondly, this suggested that the tumors arose from osteoblastic cells that contained the SQSTM1 mutation and that osteoblasts play a role in the etiology of Paget's Disease. Our hypothesis is that mutations in SQSTM1 occur somatically in a subset of osteoblast-like cells in pagetic bone and that these somatic mutations become homozygous during pagetic osteosarcoma tumorigenesis. To test this hypothesis, we propose the following specific aims: 1) To test whether somatic SQSTM1 mutations are heterozygous and clonal in the pagetic bone. 2) To test whether loss of heterozygosity at the SQSTM1 locus occurs in the pagetic bone or during pagetic tumorigenesis.