Farrow Emily G, Davis Siobhan I, Ward Leanne M, Summers Lelia J, Bubbear Judith S, Keen Richard, Stamp Trevor C B, Baker Laurence R I, Bonewald Lynda F, White Kenneth E
Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut St., IB130, Indianapolis, IN 46202, USA.
Bone. 2009 Feb;44(2):287-94. doi: 10.1016/j.bone.2008.10.040. Epub 2008 Oct 25.
We previously demonstrated that the mutations Met1Val (M1V) and the deletion of nucleotides 1484-1490 (1484-1490del) in Dentin matrix protein-1 (DMP1) cause the novel disorder autosomal recessive hypophosphatemic rickets (ARHR), which is associated with elevated fibroblast growth factor-23 (FGF23). To further understand the role of DMP1 in ARHR, we undertook molecular genetic and in vitro expression studies. First, we examined a kindred with a severe hypophosphatemic rickets phenotype and recessive inheritance. Analyses of this family demonstrated that the affected members had elevated serum FGF23 and carried a large, biallelic deletion that removed the majority of DMP1. At a minimum, this deletion encompassed 49 kb between DMP1 exon 3 and an intergenic region 5' to the next telomeric gene, integrin-binding sialoprotein (IBSP). We next performed immunofluorescent studies in cells to understand the effects of the known ARHR mutations on DMP1 cellular processing. These analyses showed that the M1V DMP1 mutant was not sorted to the trans-Golgi network (TGN) and secretory pathway, but filled the entire cytoplasm. In contrast, the 1484-1490del mutant localized to the TGN and was secreted, similar to wild type DMP1. The 1484-1490del mutation replaces the DMP1 18 C-terminal amino acids with 33 non-native residues. Truncation of wild type DMP1 by these native 18 residues followed by Western blot and confocal microscopic analyses demonstrated a wild type expression pattern when compared with the 1484-1490del mutant, indicating that the last 18 residues are not critical for cellular trafficking, but that the 33 additional residues arising from the 1484-1490del mutation likely compromise DMP1 processing. The relationship between DMP1 and FGF23 is unclear. To test endogenous DMP1 response to serum metabolites that also regulate FGF23, UMR-106 cells were treated with 1,25(OH)(2) vitamin D (1x10(-7) M) and showed a 12-fold increase in DMP1 mRNA and protein at 24 h. In summary, we have identified a novel DMP1 deletion as the cause of ARHR, as well as demonstrated that the ARHR mutations alter DMP1 cellular processing, and that DMP1 can be regulated by vitamin D. Taken together, this work expands our understanding of the genetic and molecular mechanisms associated with DMP1 alterations causing ARHR.
我们之前证明，牙本质基质蛋白1（DMP1）中的Met1Val（M1V）突变和核苷酸1484 - 1490缺失（1484 - 1490del）会导致一种新型疾病——常染色体隐性低磷血症佝偻病（ARHR），该病与成纤维细胞生长因子23（FGF23）升高有关。为了进一步了解DMP1在ARHR中的作用，我们进行了分子遗传学和体外表达研究。首先，我们研究了一个患有严重低磷血症佝偻病表型且呈隐性遗传的家族。对这个家族的分析表明，受影响的成员血清FGF23升高，并且携带一个大的双等位基因缺失，该缺失去除了大部分DMP1。至少，这个缺失涵盖了DMP1外显子3和下一个端粒基因整合素结合唾液蛋白（IBSP）5'端的一个基因间区域之间的49 kb。接下来，我们在细胞中进行免疫荧光研究，以了解已知的ARHR突变对DMP1细胞加工的影响。这些分析表明，M1V DMP1突变体没有被分选到反式高尔基体网络（TGN）和分泌途径，而是充满了整个细胞质。相比之下，1484 - 1490del突变体定位于TGN并被分泌，类似于野生型DMP1。1484 - 1490del突变用33个非天然残基取代了DMP1的18个C末端氨基酸。用这18个天然残基截断野生型DMP1，随后进行蛋白质印迹和共聚焦显微镜分析，结果表明与1484 - 1490del突变体相比，其具有野生型表达模式，这表明最后18个残基对于细胞运输并不关键，但1484 - 1490del突变产生的另外33个残基可能会损害DMP1的加工。DMP1与FGF23之间的关系尚不清楚。为了测试内源性DMP1对也调节FGF23的血清代谢物的反应，用1,25(OH)(2)维生素D（1x10(-7) M）处理UMR - 106细胞，结果显示在24小时时DMP1 mRNA和蛋白质增加了12倍。总之，我们已经确定了一种新的DMP1缺失是ARHR的病因，并且证明了ARHR突变会改变DMP1细胞加工，而且DMP1可以被维生素D调节。综上所述，这项工作扩展了我们对与导致ARHR的DMP1改变相关的遗传和分子机制的理解。
