GENETIC BASES FOR DISEASES OF THE RANK SIGNALING PATHWAY

描述（由申请方提供）：青少年佩吉特病（JPD）是一种常染色体隐性遗传性骨病，特征为编织骨快速重塑、骨质减少、骨折和进行性骨骼畸形，可能在年轻成人时致命。编码骨保护素（OPG）的TNFRSF 11B基因的纯合缺失被确定为两个纳瓦霍家族JPD的原因。以前，RANK（核因子受体激活剂？B）基因被确定为一个相关的疾病，家族性膨胀性骨质溶解（FEO）的原因。因此，OPG/RANKL/RANK/NF-？B信号通路参与了JPD及相关骨转换障碍的发生。本研究的长期目标是确定OPG/RANKL/RANK/NF-？B途径导致JPD和相关疾病的新确定的患者，并使用体外细胞建模系统来评估这些突变的下游效应，以阐明这些疾病的病理生物学。本项目的具体目标是：具体目标1：确定并评价全球范围内患有幼年型佩吉特病（JPD）、家族性膨胀性骨质溶解（FEO）和相关疾病的其他患者。 具体目标2：在JPD、FEO和相关疾病的新患者中，确定导致这些骨骼疾病的遗传缺陷。 具体目标3：表征突变OPG、RANK和OPG/RANKL/RANK/NF-？B信号通路对破骨细胞形成和作用的影响。 将确定其他患者，并进行放射学、生物化学和分子学评价。将使用PCR和毛细管DNA测序以及RT-PCR筛选DNA和RNA样本中的TNFRSF 11B（编码OPG）、TNFRSF 11 A（编码RANK）和TNFSF 11（编码RANKL）突变。我们还将使用基于微阵列的拷贝数分析来确定这些患者中潜在的基因组缺陷，并确定新的候选基因。对于OPG、RANK或RANKL基因无突变的患者，将使用大规模Solexa测序在OPG/RANKL/RANK/NF-？致病突变的B信号通路，包括SQSTM 1、VCP、TRAF 6、aPKC、NIK等。还将检查RANK信号传导的调节剂，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和骨转换相关疾病的遗传基础，这些疾病是由OPG/RANKL/RANK/NF-？B信号通路，其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.