Elucidation of molecular basis of May-Hegglin anomaly and its related disordes

对10例Myh9疾病患者中性粒细胞NMMHCA的定位进行了研究。5例患者外周血涂片May-Grunwald-Giemsa染色可见白细胞包涵体。在其余5例中，白细胞包涵体的存在是不明确的。中性粒细胞NMMHCA染色均异常。随后的Myh9基因突变分析显示，所有病例都有Myh9杂合突变。免疫荧光分析中性粒细胞NMMHCA定位是检测微小白细胞内含物和诊断由Myh9突变引起的常染色体显性遗传性大血小板减少症的一种明确和明确的替代方法。Myh9基因敲打小鼠的建立从129SvJ来源的基因组文库中分离小鼠基因组DNA克隆，并利用跨越外显子16区域的克隆构建靶向载体。通过定点突变引入R702C点突变。在3‘端插入loxP-neo-loxP和DTA盒，分别用于电穿孔ES细胞的阳性和阴性选择。将靶向载体电穿孔至ES细胞，通过聚合酶链式反应和Southern blotting筛选同源重组子。通过杂交嵌合体创建者，敲入小鼠正在构建中。在建立Myh9 R702C敲门模型后，将对小鼠的血细胞、肾脏和内耳进行详细的病理组织学和生理检查。

Analysis of MYH9 disordersWe studied the neutrophil NMMHCA localization in 10 patients with MYH9 disorders. In five cases, leukocyte inclusion bodies were observed on May-Grunwald-Giemsa stained peripheral blood smears. In the rest five cases, the presence of leukocyte inclusion bodies were ambiguous. Abnormal staining of neutrophil NMMHCA was detected in all cases. Subsequent mutational analysis of the MYH9 gene showed that all cases had a heterozygous MYH9 mutation. Immunofluorescence analysis of neutrophil NMMHCA localization represents a clear and unambiguous alternative to conventional staining for the detection of minute leukocyte inclusions and the diagnosis of the autosomal dominant macrothrombocytopenias caused by MYH9 mutations.Establishment of MYH9 Knock in miceMouse genomic DNA clones were isolated from the 129SvJ-derived genomic library, and the clones spanning the exon 16 region were used to construct the targeting vector. A R702C point mutation was introduced by site-directed mutagenesis. At the 3' end, a LoxP-Neo-LoxP and DTA cassettes were inserted for positive and negative selection of electroporated ES cells, respectively. The targeting vector was electroporated to ES cells, and the homologous recombinants were selected by PCR and Southern blotting. The knock-in mice are under construction by crossing chimeric founders. After establishment of MYH9 R702C knock in mice, detailed pathohistological and physiological examinations of blood cells, kidney and inner ear will be investigated.