Mutation analysis of WASP gene and prenatal diagnosis of Wiskott-Aldrich syndrome / 中华儿科杂志

<p><b>OBJECTIVE</b>Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and an increased incidence of autoimmunity and malignancies. The patients always have a severe clinical phenotype that can result in death if not diagnosed and treated early in life. The treatment of choice with the best outcome is hematopoietic stem cell transplantation, preferably from a matched related donor. But uncertain treatment effect and high treatment cost limit its clinical application. It is the best strategy that avoiding birth of a fetus with defect through prenatal diagnosis at present. This study aimed to analyze the mutation of WASP gene in 4 Chinese families with WAS and to provide prenatal diagnosis for the high-risk fetus.</p><p><b>METHOD</b>The probands of the four WAS families were all males, one of whom was deceased but had a family history and clinical datas integrated. All the patients were detected with blood routine tests, immunological tests and bone marrow examination. PCR and bilateral direct sequencing of PCR product was carried out in the regions of exon and exon-intron boundaries of WASP gene for 3 probands, 4 mothers and 100 unrelated healthy individuals as control. Prenatal diagnosis was provided for the two fetuses at the first trimester by mutation analysis.</p><p><b>RESULT</b>Four WASP gene mutations were detected: c.91A > G (p.E31K), c.665C > T (p.R211X), c.397G > A (p.E133K), c.952-953delCC (p. P317fsX18), among which c.952-953delCC (p. P317fsX18) was first reported. Mothers in Family 2, 3 and 4 were carriers of WASP gene mutation, but family 1 was considered as a de-novo mutation. None of the 100 unaffected subjects had the above mutants. Prenatal diagnosis indicated that the fetus in family 2 was male and carried the same mutation as the proband, so the fetus was presumably to be a patient. The parents decided to receive an induced abortion. Following the termination of the pregnancy, the result of gene analysis of the aborted tissues was consistent with prenatal diagnosis. The fetus in family 3 was normal male confirmed by normal test results six months after birth.</p><p><b>CONCLUSION</b>The 4 mutations of the WASP gene probably were causative to the families of WAS, among which c.952-953delCC was reported for the first time. Prenatal diagnosis by DNA sequencing is the effective method to avoid birth of WAS patient.</p>

Effects of aspirin on production of nitric oxide and inducible nitric oxide synthase mRNA expression under inflammatory conditions in human vascular endothelial cells / 中国病理生理杂志

AIM: To explore the effect of aspirin on inducible nitric oxide synthesis and gene expression under inflammation in endothelial cells. METHODS: Using NADPH, Griess methods and RT-PCR, the activity of isozymes of NO synthase (NOS), nitric oxide (NO) level, and iNOS mRNA expression were examined respectively. Also, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content and cell viability were measured. RESULTS: Aspirin (3 mmol/L) reduced inducible NO production and NOS activity(P

The cytotoxicity of nitric oxide induced by inflammatory cytokine in combination with LPS in endothelial cells / 中国病理生理杂志

AIM: To explore the mechanism underlying inducible nitric oxide (NO) caused injury of endothelial cells during inflammation. METHODS: The activity of iso-enzymes of NO synthase (NOS), NO level and iNOS expression were examined using NADPH method, Griess reaction and RT-PCR, respectively. Furthermore, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content were also measured. RESULTS:Co-administration of cytokines (TNF-? 5?10 5 U/L, IL-1? 2?10 5 U/L, INF-? 2?10 5 U/L) and LPS (10 mg/L) caused an obvious increase in NOS activity, NO levels (about two-fold) and a significant injury of the cells. At the same time, a significant increase in iNOS mRNA was also detected. Wheareas, treatment of the cells separately with cytokines or LPS for 24 h had no significant effect on NOS activity and NO level in cell lysates, however, it caused a significant increase in LDH release and MDA content. Also, the effect of cytokines and LPS on cell viability was concentration-and time-dependent. L-NMMA, a inhibitor of NOS, can suppress inducible NO production and protect cells against NO induced injury. CONCLUSION:Co-administration of cytokines (TNF-?, IL-1? and INF-?) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.

Regulatory mechanism of cellular iron metabolism on aspirin resistance to oxidative damage / 中国病理生理杂志

AIM: To explore the regulatory effects of ferritin expression and intracellular iron change on aspirin resistance to oxidative damage in endothelial cells. METHODS: Using ELISA to measure the levels of ferritin expression under different aspirin concentrations, in the presence of iron cheltor desferioxamine and add to FeCl 3. Then using RNA-protein bandshift assay and RT-PCR to examine the activation of IRP and the expression of IRP 2 mRNA onaspirin induced ferritin formation. RESULTS: Aspirin at low concentration (0.1mmol/L) induced significant increase in ferritin expression in a concentration-dependent fashion up to 25% over basal levels. Aspirin induced cytoprotection from H 2O 2 damage increased significantly following ferritin formation in endothelial cells.However, in the presence of iron chelator desferrioxamine, aspirin enhanced ferritin synthesis was abrogated with a 3 fold increase in the activity of IRP and significant increase in IRP 2 mRNA level. In contrast, FeCl 3 and aspirin both increased the level of induced ferritin synthesis with significant decrease in IRP activity and IRP 2 mRNA level. CONCLUSION: The effect of aspirin induced ferritin synthesis on resistance to oxidative damage in endothelium was operated through down-regulating IRP activation and IRP 2 mRNA level.