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

描述（由申请人提供）：佩吉特骨病是一种快速骨形成的疾病，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 （SQSTM1）基因座，在40%的家族病例中发现了种系突变。在一些明显散发的佩吉特病病例中也发现了种系SQSTM1突变，这表明这些病例可能是新的家族性病例。除一例外，所有的构象突变都是杂合的。这表明突变可能以显性方式起作用。然而，也有可能这种突变是显性负突变或单倍不全突变，或者是隐性突变，在受影响的骨骼中失去了杂合性。我们对受影响的骨头中突变的性质很好奇。我们选择检测散发性患者患骨中的SQSTM1位点，以观察：1)这些骨中是否发生SQSTM1的体细胞突变，2)该突变在患骨中是杂合的还是纯合的。当我们对受影响的骨骼和匹配的外周血中的SQSTM1基因进行测序时，我们发现5个受影响的骨骼样本中有4个存在P392L突变，而匹配的血液样本中没有P392L突变。更令人惊讶的是，在受影响的骨骼中，样本中P392L突变的相对频率在10%到28%之间，这表明骨骼是为突变而镶嵌的。在重复重采样和重测序后，每个样本中突变的相对频率是一致的。其次，由于骨肉瘤是Paget病的并发症，我们很想知道SQSTM1突变是否存在于Paget骨肉瘤中。当我们在这些肿瘤中筛选SQSTM1基因时，我们发现5个骨肉瘤中有4个具有纯合子P392L突变。同样，在匹配的正常骨骼中没有检测到突变。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SQSTM1突变是家族性佩吉特病的易感因素，但它们并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SQSTM1突变的成骨细胞，并且成骨细胞在Paget病的病因学中起作用。我们的假设是，SQSTM1突变发生在成骨细胞样细胞亚群中，这些体细胞突变在成骨肉瘤发生过程中变为纯合子。为了验证这一假设，我们提出了以下具体目的：1)验证体细胞SQSTM1突变在异位骨中是杂合的还是克隆的。2)检测SQSTM1位点杂合性缺失是否发生在异位骨或异位肿瘤发生过程中。

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.