GENETIC BASES FOR DISEASES OF THE RANK SIGNALING PATHWAY

描述(申请人提供)：青少年Paget‘s病(JPD)，一种常染色体隐性遗传性骨病，以编织骨快速重塑、骨质减少、骨折和进行性骨骼畸形为特征，可在年轻时致命。编码护骨素(OPG)的TNFRSF11B基因纯合缺失是两个纳瓦霍家族JPD的原因。以前，RANK(核因子受体激活剂？-B)基因的缺陷被确定为一种相关疾病--家族性扩张性骨溶解(FEO)的原因。因此，涉及OPG/RANKL/RANK/NF-1B信号通路的基因突变与JPD及相关的骨转换障碍有关。这项研究的长期目标是确定OPG/RANKL/RANK/NF-？B通路成员中导致JPD和相关疾病的其他突变，并使用体外细胞建模系统来评估这些突变的下游影响，以阐明这些疾病的病理生物学。该项目的具体目标是：具体目标1：确定和评估世界各地更多患有青少年Paget病(JPD)、家族性扩张性骨溶解(FEO)和相关疾病的患者。具体目标2：在患有JPD、FEO和相关疾病的新患者中，确定导致这些骨骼疾病的基因缺陷。具体目标3：研究突变型OPG、RANK和OPG/RANKL/RANK/NF-1B信号通路的其他成员在破骨细胞形成和作用中的下游作用。更多的患者将被确定，并通过放射学、生化和分子学进行评估。DNA和RNA样本将使用聚合酶链式反应和毛细管DNA测序以及RT-PCR筛查编码OPG的TNFRSF11B、编码RANKL的TNFRSF11A和编码RANKL的TNFSF11的突变。我们还将使用基于微阵列的拷贝数分析来识别这些患者中潜在的基因组缺陷，并识别新的候选基因。对于没有OPG、RANK或RANKL基因突变的患者，大规模Solexa测序将用于在OPG/RANKL/RANK/NF-？B信号通路中寻找其他候选基因的致病突变，包括SQSTM1、VCP、TRAF6、aPKC、Nik等。还将研究RANK信号的调节器，包括TRAIL、IL-1、MCSF、c-FMS和其他细胞因子及其同源受体。体外破骨细胞培养试验将用于确定突变对破骨细胞形成和功能的下游影响。这些研究将进一步阐明JPD、FEO和相关的骨转换障碍的遗传学基础，这些疾病是由OPG/RANKL/RANK/NF-？B信号通路缺陷引起的，而OPG/RANKL/RANK/NF-？B信号通路对破骨细胞的形成和作用至关重要。

DESCRIPTION (provided by applicant): Juvenile Paget's disease (JPD), an autosomal recessive osteopathy, features rapidly remodeling woven bone, osteopenia, fractures, and progressive skeletal deformity that can be fatal by young adult life. Homozygous deletion of the TNFRSF11B gene, encoding osteoprotegerin (OPG), was identified as the cause of JPD in two Navajo families. Previously, a defect in the RANK (receptor activator of nuclear factor ?-B) gene was determined to be the cause of a related disorder, familial expansile osteolysis (FEO). Hence, mutations in genes involved in the OPG/RANKL/RANK/NF-?B signaling pathway are responsible for JPD and related disorders of bone turnover. The long-range goal of this study is to identify other mutations in members of the OPG/RANKL/RANK/NF-?B pathway that cause JPD and related diseases in newly-ascertained patients, and to use in vitro cell modeling systems to assess the downstream effects of these mutations, to elucidate the pathobiology of these disorders. The Specific Aims of this project are: Specific Aim 1: Ascertain and evaluate additional patients worldwide with juvenile Paget's disease (JPD), familial expansile osteolysis (FEO), and related disorders. Specific Aim 2: In new patients with JPD, FEO, and related disorders, identify the genetic defects that cause these skeletal diseases. Specific Aim 3: Characterize the downstream effects of mutant OPG, RANK, and other members of the OPG/RANKL/RANK/NF-?B signaling pathway on osteoclast formation and action. Additional patients will be ascertained, and evaluated radiographically, biochemically, and molecularly. DNA and RNA samples will be screened for mutations in TNFRSF11B, encoding OPG, TNFRSF11A, encoding RANK, and TNFSF11, encoding RANKL, using PCR and capillary DNA sequencing, and RT-PCR. We will also use a microarray-based copy number analysis to identify potential genomic defects in these patients, and identify new candidate genes. For patients without mutations in the OPG, RANK, or RANKL genes, large-scale Solexa sequencing will be used to search other candidate genes in the OPG/RANKL/RANK/NF-?B signaling pathway for disease-causing mutations, including SQSTM1, VCP, TRAF6, aPKC, NIK, and others. Modulators of RANK signaling will also be examined, including TRAIL, IL-1, MCSF, c-FMS and additional cytokines and their cognate receptors. In vitro osteoclast culture assays will be used to determine downstream effects of mutations on osteoclast formation and function. These studies will further elucidate the genetic bases for JPD, FEO, and related disorders of bone turnover, caused by defects in the OPG/RANKL/RANK/NF-?B signaling pathway, which is critical for osteoclast formation and action.