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

MYH9疾病分析我们研究了10例MYH9疾病患者的中性粒细胞NMMHCA定位。5例患者外周血涂片可见白细胞包涵体。在其余5例中，白细胞包涵体的存在是模糊的。所有病例均检测到中性粒细胞NMMHCA染色异常。随后对MYH9基因的突变分析表明，所有病例都有MYH9杂合突变。中性粒细胞NMMHCA定位的免疫荧光分析是一种清晰而明确的替代传统染色的方法，用于检测微小的白细胞包涵体和诊断由MYH9突变引起的常染色体显性巨血小板减少症。从129svj衍生的基因组文库中分离小鼠基因组DNA克隆，利用跨外显子16区的克隆构建靶向载体。通过定点诱变，引入R702C点突变。在3'端插入LoxP-Neo-LoxP和DTA盒，分别对电穿孔ES细胞进行阳性和阴性选择。将目标载体电穿孔至胚胎干细胞，通过PCR和Southern blotting筛选同源重组子。基因敲入小鼠正在通过杂交嵌合创始人进行构建。在小鼠MYH9 R702C敲型建立后，对血细胞、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.