Molecular Mechanism Research Of Chinese Patient With Myeloperoxidase Complete Deficiency In Neutrophils

[Objective]Through systematic molecular genetics research, we try to clarify the moleculargenetics characteristic and the molecular mechanism of this completemyeloperoxidase deficient family. In addition, we also expounded how the moleculargenetics change of myeloperoxidase will influences its physiological and pathologicalfunction. All of these researches offer a powerfully experimental material for furtherresearch of myeloperoxidase in physiology, pathology and synthetic mechanisms.[Methods]1. Blood samples of the complete deficient family were collected, then neutrophiliswere isolated and genomes were extracted from the blood. Primers to amplify themyeloperoxidase gene transcription regulatory region and the twelve exons ofmyeloperoxidase were designed. Differences between the sequences of experimentgroups were compared with that of control group. The family tree of the family wasdrawn.2. Myeloperoxidase’s oxidation activity, expression and location in neutrophiliswere analysed through myeloperoxidase oxidation activity test, cytochemical stainand immunofluorescence stain. The proteins of neutrophils homogenates wereseparated through SDS-PAGE, then the existence of myeloperoxidase in neutrophiliswere analysed through Western-Blot by using myeloperoxidase antibody.3. Total RNA was extracted from neutrophils treated by CHX or not. Then themRNA levels of myeloperoxidase were relatively quantified by real-time RT PCR, toclarify the molecular mechanism of this nosense mutation. [Results]1. Genome sequences of the deficient family were analysed. A guanine （G） toadenine （A） substitution in the exon11region of the patient myeloperoxidase genewas found, which located at position1805from adenine of first ATG and resulted inan amino acid conversion from tryptophan （TGG） to termination codon （TAG）,W602X. The W602X was homozygous in the patient, but heterozygous in the motherand son of the patient. And the family tree displayed that the deficiency was inheritedin an autosomal recessive manner.2. Myeloperoxidase oxidation activity in neutrophilis of the patient was less than5%of the healthy control. Cytochemical staining, fluorescence microscope andWestern-Blot show the myeloperoxidase result of the patient were all completelydefect, but the result of patient’s mother and son were all nearly half of the healthycontrol.3. Real-time RT PCR indicates that the quantity of myeloperoxidase mRNA in thepatient was less than1%of the control group, but this result rise obviously afterneutrophilis with treatment of the CHX. These indicate myeloperoxidase mRNAdecay was existed in the neutrophilis, and this decay was closely related with NMD.[Conclusions]In the present study, we found a complete myeloperoxidase deficient patient.And this defect was resulted in a G to A substitution at position1805in exon11of thepatient myeloperoxidase gene （NG₀09629.1:c.1805G>A）. The W602X washomozygous and generates PTC ahead of time, the PTC-containing myeloperoxidasemRNA triggers NMD and then decays this mRNA in return. As a result of it, thetranscription of myeloperoxidase was abnormal and led to complete defection.