Clinical Features Analysis, Mutation Screening And Pathogenic Mechanism Study Of A Family With MYH9-Related Disease

PART I Family collection, diagnosis and clinical features analysis of non-myosin heavy chain 9 related diseaseObjective To collect the detailed genetic resource in this family with non-muscle myosin heavy chain 9 related disease(MYH9-RD) and make a diagnosis for this family by analyzing the clinical manifestations,inheritance, laboratory features to explore the pathgenic mechanism of MYH9-RD in this family.Methods All of the family members were enrolled in the current study and a detailed screening including clinical and laboratory features were made for this family members. Family members were screened for the presence or absence of clinical manifestations including nephritis, sensorineural hearing loss, cataract, abnormal bleeding or renal disease etc, and other complications and laboratory evaluation. Platelet counts were examined by automatic blood cell counter and morphology, respectively. Meanwhile characteristic and percentage of giant platelet and neutrophil inclusion bodies were analyzed by morphology.Technique of chromosome karyotyping was applied to carry diagnostic cytogenetics, technique of light microscope and electron microscope were applied to analyze the cell morphology and ultrastructure of platelet and inclusion bodies in granulocytes.Results Based on the laboratory evulation of peripheral blood and clinical manifestations,15 patients with a autosomal dominant disorder markedly showed thrombocytopenia, giant platelets and inclusion bodies in granulocytes among 32 menbers of four generations investigated in this family. All the patients not only have thrombocytopenia, giant platelets and neutrophils inclusion, but also have easy bruising and mild to moderate bleeding tendency which is not associated with platelet count or diameter. Moreover, most patients presented complicated clinical manifestations, and some even suffered from serious phenotypes, such as leucocythemia, glaucoma, cataract, proteinuria, abnormal hepatic function, hyperlipemia and disordered action of heart etc, which is distinguished from those families reported previously. Chromosome karyotyping of patients are normal. Platelet count from cell counter was lower than that from microscope (P<0.01). Ultrastructurally, giant platelets and neutrophil inclusions were clearly observed and neutrophil inclusions were oval or spindle-like, localized in the periphery of the neutrophils. Meanwhile, the unaffected members from the family were chosen as control among which normal platelet count, normal platelet volume and without neutrophils inclusions.Conclusion The family patients were diagnosed as MYH9-RD based on clinical and laboratory features. Also not only this family is large, but also the phenotypes of the patients from the family had complicated clinical manifestations which were more serious than those families ever reporeted. Therefore, this large family is established and will lay the foundation for the further studying of MYH9-RD. Moreover,microscopic cell counter rather than automatic blood cell counter should be used to examine the correct platelet counts for the giant platelets patients. PARTⅡMutation screening and analysis of non-myosin heavy chain 9 gene of the MYH9-RD familyObjective Mutation screening and analysis of non-myosin heavy chain 9 (MYH9) gene in this family patients with MYH9-RD were given to explore the pathogenic mechanism of MYH9-RD in this family.Methods After informed consent, we obtained blood samples from fifteen patients and their normal family members in the family with MYH9-RD. Under standard PCR conditions, genomic DNA was extract-ed from peripheral blood leukocytes. All the 40 exons and franking regions of MYH9 gene from 15 patiens and the normal family member were amplified by polymerase chain reaction (PCR), and PCR products were analyzed by using direct sequcing method.The normal family members were chosen as control subjects, and results of sequence was analyzed comparing with the normal MYH9 gene in the Genebank by DNAMAN software.Results Of all the patients, there is no pathogenic mutation in the exons and franking regions of MYH9 gene except a synonymous mutation in the 25 and 18 exon in patients. Meanwhile, there is no mutation in the 40 exons and franking regions of MYH9 gene in normal family members.Conclusion There may be a novel mutation or another novel pathogenic gene or other reasons with MYH9-RD which is related to its complex clinicl phenotype of this family. It is of significance to reveal the molecular pathogenesis mechanism of MYH9-RD by further studying this large family. PartⅢLocalization and expression of non-myosin heavy chain-ⅡA in granulocytes of the MYH9-RD familyObjective To infer the formation characterics of neutrophils inclusions and explore the. pathologic mechanism of this MYH9-RD family by detecting and analyzing the localization or distribution and the expression of NMMHCⅡA in neutrophils of MYH9-RD patients.Methods Neutrophils were isolated from periperal blood of MYH9-RD patients and normal family members. Normal family members were chosen as control subjects. Immunofluorescence technique was used to observe localization or distribution of NMMHCⅡA in MYH9-RD patients and normal family members. NMMHCⅡA levels in neutrophils from patients and normal family members were analyzed by westernblot.Results Oval or spindle-like neutrophils inclusions with green fluorescence in MYH9-RD patients were markly showed by immunofluorescence technique, which matched very well in localization, size and shape, compared with the inclusions, displayed by light microscope (Wright-Giemsa’s stain). Meanwhile, no inclusions in the normal family members were observed except a diffusive distribution of fluorescent spot in neutrophils cytoplasm. The result of western blot revealed that the expression of NMMHC-ⅡA in neutrophils of MYH9-RD patients was down regulated than normal family members.Conclusion The distribution and expression of NMMHC-ⅡA in neutrophils between patients and the normal family members are all differently revealed by immunofluorescence technology and westernblot technology. It is concluded that the molecular pathogenesis mechanism of this MYH9-RD family may be also resulted from abnormal MYH9 gene because the major component of neutrophils inclusions from this MYH9-RD family patients are NMMHC-ⅡA, and dominant negative effect of NMMHC-ⅡA is involved in the formation of inclusion bodies even though there is no pathogenic mutation in the 40 exons and franking regions of MYH9 gene in this family.It is of significance to reveal the molecular pathogenic mechanism of this MYH9-RD family by the study of NMMHC-ⅡA. Meanwhile, immunofluorescence technology is more sensitive and specific than Wright-Giemsa’s staining in detecting inclusions of MYH9-RD patients. PartⅣDetection and analy sis of NMMHC-ⅡA and membrane glycoprotein in platelets of the MYH9-RD familyObjective To further explore the pathogenic mechanism by detect ing and analyzing the distribution of NMMHC-ⅡA and expression of membrane glycoprotein in the platelets of MYH9-RD patients.Methods we obtained periperal blood from MYH9-RD patients and normal family members. Meanwhile, platelets were isolated. The platelet membrane glycoprotein including GPⅡb/Ⅲa(CD41/61),GP I a(CD49b), GP I b/IX/V (CD42a) GP I b(CD42b) and GPIV(CD36) was examined by flowcytometry and the distribution of NMMHCⅡA in the platelet was observed by immunofluorescence technique. Normal family members were chosen as control subjects.Results Platelet membrane glycoprotein GPⅡb/Ⅲa(CD41/61), GPIa(CD49b), GP I b/Ⅸ/Ⅴ(CD42a) GP I b(CD42b)和GP IV (CD36)were in normal range in the family patients. There is no difference in the expression of the platelet membrane glycoprotein between patients and normal family members by flowcytometry(P> 0.05),while the activated level of platelet membrane protein among the family patients was different. Immunofluorescence technique showed well-distributed staining of NMMHC-ⅡA in the central zone and intensive stainning in the periphery in platelets of the patients, while only showed well-distributed staining in the normal family members.Conclusion The distribution of NMMHC-ⅡA in platelets between patients and normal family members was differently displayed by immunofluorescence technology. It revealed that the formation of giant platelets may be related to the abnormal distribution of NMMHC-ⅡA. The difference for the level of platelet membrane glycoprotein between family patients and normal family members is normal while the difference among family patients are quite differently.It revealed that the difference for the activated level of platelet may be involved in the clinical manifestations of family patients.