Esapa Christopher T, Piret Sian E, Nesbit M Andrew, Loh Nellie Y, Thomas Gethin, Croucher Peter I, Brown Matthew A, Brown Steve D M, Cox Roger D, Thakker Rajesh V
Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.
MRC Mammalian Genetics Unit and Mary Lyon Centre, MRC Harwell, Harwell Science and Innovation Campus, Harwell, United Kingdom.
PLoS One. 2016 Dec 13;11(12):e0167916. doi: 10.1371/journal.pone.0167916. eCollection 2016.
Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis.
非综合征性脊柱后凸是一种常见疾病，与显著的发病率相关且有很强的遗传因素参与；然而，致病基因仍有待确定，因为此类研究受到遗传异质性、小家庭规模和多种遗传模式的阻碍。为克服这些局限性，我们使用包括畸形学、放射学和双能X线吸收法在内的表型评估方法，对用化学诱变剂N-乙基-N-亚硝基脲（ENU）处理的小鼠的12周龄后代进行了研究。这鉴定出一只患有常染色体隐性脊柱后凸（KYLB）的小鼠。与未受影响的同窝小鼠相比，KYLB小鼠有：胸腰段脊柱后凸、椎骨更大、体长增加以及骨面积增加。此外，雌性KYLB小鼠的骨矿物质含量和血浆碱性磷酸酶活性增加。重组定位将Kylb基因座定位于15A1染色体上一个5.5Mb的区域，该区域包含51个基因，包括利钠肽受体3（Npr3）基因。对Npr3的DNA序列分析鉴定出一个错义突变Tyr209Asn，该突变引入了一个N-连接糖基化共有序列。野生型NPR3和与KYLB相关的Tyr209Asn NPR3突变体在COS-7细胞中的表达表明，该突变体与异常的N-连接糖基化以及在内质网中的滞留相关，导致其不在质膜上表达。NPR3是C型利钠肽（CNP）的诱饵受体，CNP也与NPR2结合并刺激丝裂原活化蛋白激酶（MAPK）信号传导，从而增加肥大软骨细胞的数量和大小。对KYLB椎骨和胫骨的组织形态计量学分析显示，软骨内骨化延迟且生长板肥大区扩大，免疫组织化学显示KYLB椎骨整个生长板中p38 MAPK磷酸化增加。因此，我们建立了一种由于新型NPR3突变导致的脊柱后凸模型，其中质膜NPR3表达缺失导致MAPK途径激活增加，引起椎骨伸长并导致脊柱后凸。
