Alam Imranul, McQueen Amie K, Acton Dena, Reilly Austin M, Gerard-O'Riley Rita L, Oakes Dana K, Kasipathi Charishma, Huffer Abigail, Wright Weston B, Econs Michael J
Medicine, Indiana University School of Medicine, IN, USA.
Medicine, Indiana University School of Medicine, IN, USA.
Bone. 2017 Jan;94:34-41. doi: 10.1016/j.bone.2016.10.016. Epub 2016 Oct 14.
Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic bone disorder due to dysfunctional osteoclast activity. ADO2 is caused by missense mutations in the chloride channel 7 (CLCN7) gene characterized by osteosclerosis with multiple fractures. ADO2 can result in osteomyelitis, visual loss and bone marrow failure. Currently, there is no cure for ADO2, and until recently no appropriate animal model of ADO2 existed to understand better the pathogenesis of this disease and to test new therapies. Therefore, we created ADO2 knock-in mouse model with a G213R (human homolog of G215R) missense mutation in the Clcn7 gene on 129S1 background, and demonstrated that this mouse model phenocopies human ADO2. As ADO2 gives rise to incomplete penetrance (66%) in human and marked phenotypic variability is observed among patients with the same mutation, we hypothesized that the severity and penetrance of ADO2 will also vary in mouse models on different genetic backgrounds. To test this, we created ADO2 mouse models in DBA/D2, C57BL/6J/B6 and Balb/c strains, and compared bone phenotypes and performed serum biochemical analysis between strain- and age-matched wild-type (WT) and ADO2 mice. At 3months of age, whole body aBMD was higher (4-7% in male; 1-5% in female) in the ADO2 mice compared to their wild-type littermates. In addition, ADO2 male mice on 129 background displayed highest percent increase of BV/TV (106%), followed by D2 (92%), B6 (46%), and Balb/c (33%) compared to strain-matched wild-type mice. We observed similar differences for BV/TV between ADO2 and wild-type mice on different genetic backgrounds in female: 129 (96%)>D2 (73%)>Balb/c (39%) and B6 (36%). Serum calcium, phosphorus, alkaline phosphatase and P1NP levels were similar in the WT and ADO2 mice on all genetic backgrounds but TRAP was higher (76% to 220% in male; 33-95% in female) and CTX/TRAP ratio was lower (39-65% in male and 3-41% in female) in the ADO2 mice compared to their strain-matched wild-type littermates. We also found that young (3months) ADO2 mice on 129S1 background exhibited 200% higher trabecular BV/TV whereas old (18months) ADO2 mice displayed 400-700% higher BV/TV compared to their age-matched wild-type controls. In summary, phenotypic severity in ADO2 mice varied markedly on different genetic backgrounds (129>D2>Balb/c>B6) and became more pronounced with age, which resembles the wide variations in phenotype observed in ADO2 patients. These mouse models will help us to identify genes/factors that influence severity and penetrance of ADO2, and test innovative therapies to treat this disease.
常染色体显性II型骨硬化症（ADO2）是一种由于破骨细胞活性功能失调引起的遗传性骨硬化性骨病。ADO2由氯离子通道7（CLCN7）基因中的错义突变导致，其特征为骨硬化并伴有多处骨折。ADO2可导致骨髓炎、视力丧失和骨髓衰竭。目前，尚无治愈ADO2的方法，并且直到最近还没有合适的ADO2动物模型来更好地理解该疾病的发病机制并测试新的治疗方法。因此，我们在129S1背景下创建了在Clcn7基因中带有G213R（G215R的人类同源突变）错义突变的ADO2基因敲入小鼠模型，并证明该小鼠模型模拟了人类ADO2。由于ADO2在人类中表现出不完全外显率（66%），并且在具有相同突变的患者中观察到明显的表型变异性，我们推测ADO2的严重程度和外显率在不同遗传背景的小鼠模型中也会有所不同。为了验证这一点，我们在DBA/D2、C57BL/6J/B6和Balb/c品系中创建了ADO2小鼠模型，并比较了不同品系和年龄匹配的野生型（WT）及ADO2小鼠之间的骨表型并进行了血清生化分析。在3个月大时，与野生型同窝小鼠相比，ADO2小鼠的全身骨密度（aBMD）更高（雄性高4 - 7%；雌性高1 - 5%）。此外，与品系匹配的野生型小鼠相比，129背景的ADO2雄性小鼠的骨体积分数（BV/TV）增加百分比最高（106%），其次是D2（92%）、B6（46%）和Balb/c（33%）。在雌性中，我们在不同遗传背景的ADO2和野生型小鼠之间观察到类似的BV/TV差异：129（96%）>D2（73%）>Balb/c（39%）和B6（36%）。在所有遗传背景下，野生型和ADO2小鼠的血清钙、磷、碱性磷酸酶和I型前胶原氨基端前肽（P1NP）水平相似，但与品系匹配的野生型同窝小鼠相比，ADO2小鼠的抗酒石酸酸性磷酸酶（TRAP）更高（雄性高76%至220%；雌性高33 - 95%），而I型胶原交联C末端肽（CTX）/TRAP比值更低（雄性低39 - 65%，雌性低3 - 41%）。我们还发现，与年龄匹配的野生型对照相比，129S1背景的年轻（3个月）ADO2小鼠的小梁骨BV/TV高200%，而老年（18个月）ADO2小鼠的BV/TV高400 - 700%。总之，ADO2小鼠的表型严重程度在不同遗传背景（129>D2>Balb/c>B6）下有显著差异，并且随着年龄增长变得更加明显，这类似于在ADO2患者中观察到的广泛表型变异。这些小鼠模型将有助于我们识别影响ADO2严重程度和外显率的基因/因素，并测试治疗该疾病的创新疗法。
