Molecular and genetic characterization of the Sorting Nexin 10 R51Q mutation and other mutations, causing osteopetrosis in infancy in Palestinian clans

常染色体隐性遗传性骨质疏松症(ARO)是一种严重的遗传性疾病，由骨吸收缺陷引起的骨生长异常引起。ARO是一种罕见的疾病，但在有血缘关系的人群中发病率很高。2012年，在巴勒斯坦流行人群中发现了分类连接蛋白10(SNX10)基因点突变，导致精氨酸/谷氨酰胺交换(SNX10 R51Q)。从那时起，世界各地都发现了导致骨化症的SNX10基因的不同突变。ARO的金标准治疗方法是移植健康的造血干细胞，以野生型细胞修复骨吸收破骨细胞的缺陷。然而，疾病的延迟诊断和随后的延迟移植、供体细胞杂合性的未知影响、移植时供体嵌合率较低以及该突变可能导致移植后患者的病理变化的未知影响，阻碍了成功的治疗。因此，我们产生了一种高度翻译的方法来解开SNX10 R51Q引起的ARO的病因学，通过调查群体遗传学，开发SNX10 R51Q病的转基因小鼠模型，并通过分析破骨细胞衰竭的细胞生物学机制。在第一个资助期内，我们在前16个月里，我们来自巴勒斯坦、德国和以色列的三方跨学科研究联盟取得了实质性进展。我们提高了人们和当地政府对这种疾病的遗传咨询的认识。我们成功地将SNPs和STR定位到该基因座上，发现了新的受累家系，并在ARO中发现了迄今未知的突变。随着SNX10 R51Q敲入小鼠的产生，我们建立了第一个与患者表型非常相似的ARO突变的敲入小鼠模型。我们发现了一种新的破骨细胞表型，它表现出无限融合，从而导致非功能性吸收。这一发现指向了破骨细胞融合的基本问题，这些问题仍然不清楚，但除了骨质疏松和癌症等常见疾病的骨化症之外，这些问题也很重要。我们现在的目标是从长凳到床边的转换和反向方法来表征SNX10 R51Q-和巴勒斯坦人口中的一个新突变的遗传频率。我们将使用骨髓嵌合小鼠模型来解剖SNX10 R51Q敲入和SNX10完全敲除小鼠的破骨细胞自主和非自主功能。这些实验将深入了解即使在移植后患者中也发生的SNX10相关病理。最后，我们将仔细分析SNX10特别是SNX10 R51Q在破骨细胞融合过程中的分子机制，以及野生型细胞和突变细胞的混合融合如何影响融合过程，这一情况可能发生在移植患者身上。我们的见解将对患者有直接的好处，并将对一般的骨生物学产生影响。

Autosomal recessive osteopetrosis (ARO) is a severe inherited disease in infants due to aberrant bone growth caused by defects in bone resorption. ARO is a rare disease, but with high prevalence in consanguineous populations. 2012 a point mutation in the Sorting Nexin 10 (SNX10) gene was discovered in Palestinian endemic populations leading to an arginin/glutamin exchange (SNX10 R51Q). Since then different mutations in the SNX10 gene causing osteopetrosis were identified all over the world. Gold-standard treatment of ARO is the transplantation of healthy hematopoietic stem cells in order to rescue the defect of the bone resorbing osteoclasts with wild type cells. However, a successful therapy is hampered by late diagnosis of the disease and subsequent late transplantation, the unknown effect of heterozygous donor cells, a low donor chimerism upon transplantation, and unknown effects of the mutation possibly contributing to pathologies in patients after transplantation. We therefore generated a highly translational approach to unravel the etiology of ARO caused by SNX10 R51Q, by investigating population genetics, developing transgenic mouse models for the SNX10 R51Q disease and by analysing the cell biological mechanisms of osteoclast failure. In the first funding period we achieved in the first 16 months substantial progress in our trilateral interdisciplinary research consortium from Palestine, Germany and Israel. We increased awareness of the population and local government towards genetic counseling for the disease. We successfully mapped SNPs and STRs to the locus, detected new affected families and discovered so far unknown mutations in ARO. With the generation of SNX10 R51Q Knock-In mice we established the first Knock-In mouse model for an ARO mutation closely resembling the patient phenotype. We discovered a novel osteoclast phenotype that displays unlimited fusion and thus leads to non-functional resorption. This discovery points towards fundamental questions of osteoclast fusion that are still unclear but are important beyond osteopetrosis for common diseases such as osteoporosis and cancer. We now aim in a bench to bed side translation and back approach to characterize genetic frequencies of the SNX10 R51Q- and one novel mutation in the Palestine population. We will dissect the osteoclast autonomous and non-autonomous function in SNX10 R51Q Knock-In and SNX10 complete Knock-Out mice using bone marrow chimeric mouse models. These experiments will give insight in SNX10-related pathologies occuring in patients even after transplantation. And finally we will carefully analyze the molecular mechanisms of SNX10 in general and SNX10 R51Q in particular in the osteoclast fusion process and how mixed fusions of wild type cells and mutant cells affect the fusion process, a situation that might occur in patients upon transplantations. Our insights will be of direct benefit for the patients and will give implications for bone biology in general.