Mistargeting of Elastase in Bone Marrow Failure

描述（由申请人提供）： 嗜中性粒细胞是吞噬性白色血细胞，其数量的缺乏（“嗜中性粒细胞减少症”）易于发生细菌和真菌感染。人类血小板减少性骨髓衰竭的两种主要遗传形式是周期性中性粒细胞减少症（CN）和白血病易感的重度先天性中性粒细胞减少症（SCN）。“良性种族中性粒细胞减少症”在非洲人后裔中很常见，也可能对健康产生影响。编码嗜中性粒细胞弹性蛋白酶（NE）的基因ELA 2的突变导致CN，并且是SCN的最常见原因。最近的研究发现，犬CN是由AP 3转运蛋白突变引起的，一些SCN可能是由ELA 2启动子变体或转录抑制因子Gfi 1突变引起的，这两者都导致NE的过度表达，这表明在这些和其他骨髓衰竭综合征中中性粒细胞减少症的假设：NE是AP 3的跨膜“货物”蛋白。NE跨膜结构域的突变导致过多的NE在颗粒中积累，导致CN。NE的AP 3识别信号或AP 3本身的突变使NE错误定位于质膜。类似地，NE的过度表达导致ELA 2启动子变体或Gfi 1突变破坏了正常的AP 3介导的运输途径，并将NE转移到质膜。三个具体目的是针对测试这一假设，确定在非裔美国人群体中常见的ELA 2启动子变体是否与良性种族中性粒细胞减少症相关，并鉴定另外的中性粒细胞减少症基因：1表征NE的膜关系：1.1开发针对NE的预测细胞质和管腔表面的抗体; 1.2测试膜结合的NE对模型和生物底物的改变的催化; 1.3研究其他物种和其他蛋白酶中AP 3相互作用的进化保守性。2评价NE的误分泌作为中性粒细胞减少症的一般机制：2.1识别误定位NE的潜在底物和辅因子; 2.2检查其他血小板减少症疾病中的NE运输。3发现可能通过NE过表达引起中性粒细胞减少症的其他突变：3.1确定ELA 2启动子变异是否有助于中性粒细胞减少症; 3.2测量患有良性种族中性粒细胞减少症的非洲裔个体中ELA 2 C-199 A等位基因的频率; 3.3鉴定新的中性粒细胞减少症基因。

DESCRIPTION (provided by applicant): Neutrophils are phagocytic white blood cells, and a deficiency of their numbers ("neutropenia") predisposes to bacterial and fungal infection. The two principal inherited forms of human neutropenic bone marrow failure are cyclic neutropenia (CN) and leukemia-predisposing, severe congenital neutropenia (SCN). "Benign ethnic neutropenia" is common to individuals of African descent and may also have health consequences. Mutations of the gene ELA2, encoding neutrophil elastase (NE), cause CN and are the most frequent cause of SCN. Recent findings that canine CN is caused by mutations of the AP3 transporter and that some SCN may result from ELA2 promoter variants or mutations of the transcriptional repressor Gfi1, both of which lead to over-expression of NE, suggest a hypothesis for neutropenia in these and other bone marrow failure syndromes: NE is a transmembrane "cargo" protein for AP3. Mutation of NE's transmembrane domains causes too much NE to accumulate in granules, resulting in CN. Mutation of NE's AP3-recognition signal, or of AP3 itself, mislocalizes NE to the plasma membrane. Similarly, over-expression of NE consequent to ELA2 promoter variants or Gfi1 mutations overwhelms normal AP3-mediated trafficking pathways and diverts NE to the plasma membrane. Three Specific Aims are directed toward testing this hypothesis, determining if a common ELA2 promoter variant in the African-American population associates with benign ethnic neutropenia, and identifying additional neutropenia genes: 1 Characterize NE's membrane relationship: 1.1 Develop antibodies to predicted cytoplasmic and luminal surfaces of NE; 1.2 Test membrane-bound NE for altered catalysis on model and biological substrates; 1.3 Examine the evolutionary conservation of AP3 interactions in other species and for other proteases. 2 Evaluate mistrafficking of NE as a general mechanism for neutropenia: 2.1 Identify potential substrates and cofactors of mislocalized NE; 2.2 Inspect NE trafficking in other neutropenic disorders. 3 Discover other mutations that may cause neutropenia through over-expression of NE: 3.1 Determine if ELA2 promoter variation contributes to neutropenia; 3.2 Measure the frequency of the ELA2 C-199A allele in individuals of African descent with benign ethnic neutropenia; 3.3 Identify new neutropenia genes.