Part â… Identification And Characterization Of Clinical Features And Gene Mutation In Patients With Iron-refractory Iron Deficiency Anemia(IRIDA)Part â…¡Interaction Between IASPP And SON And Its Effects On Leukemia Cells

Purpose:To increase the awareness of iron-refractory iron deficiency anemia （IRIDA） by summarizing and analyzing the IRIDA patient’s hematological characteristics and their mutations of TMPRSS6.Methods:Hepcidin levels were measured for the suspected IRIDA patients by enzyme-linked immunosorbent assay （ELISA）. The clinical features and hematological characteristics of the confirmed cases were analyzed. TMPRSS6gene mutation was detected and the mutation consequence was forecasted through Swiss-Model prediction and Alamut software. TMPRSS6mRNA expression was detected by real-time quantitative reverse-transcription polymerase chain reaction （RT-PCR）.Result:Five IRIDA patients from4different families were included in this research. All of them exhibit hypochromic, microcytic anemia （low MCV and MCHC） with low serum iron and transferrin saturation but refractory to oral iron treatment. They also showed an inappropriate elevation of plasma hepcidin level compared to the acquired IDA patients who could recover completely through oral iron therapy. DNA sequencing results showed that one patient harbored a homozygous missense mutation of K253E （c.757A>G） in exon7of TMPRSS6gene （NM₁53609.2） that could change its conformation and function by Swiss-Model prediction. The second patient and his mother both carried a homozygous, the other one had a heterozygous synonymous mutation of T371T （c.1113G>A） in the splice site of exon9and heterozygous rs855791（V736A）. In silico analysis predicted that both mutations in splice site could interfere with the process of mRNA splicing. Real time PCR analysis revealed that TMPRSS6mRNA level in peripheral blood mononuclear cells was lower in the person who harbored the splice junction mutation even with the heterozygous form. The last patient exhibited a typical IRIDA characteristics but without any special mutation of TMPRSS6gene.Three IRIDA patients were receiving intravenous iron treatment and one receiving erythropoietin treatment. As results, the hemoglobin concentration increased to normal range in two patients. One patients received iron treatment had a delayed response, the last one had a remarkable rise of his hemoglobin concentration after receiving erythropoietin treatment. But the erythrocyte parameters of all patients failed recover to normal range after treatment.Conclusion:The clinical features and gene mutation in the5patients with IRIDA from4families in China were characterized and analyzed for the first time. As a result,3mutations of TMPRSS6gene including two novel splice site mutations were found, and one patient without TMPRSS6mutation also showed typical characteristics of IRIDA, indicating that other reasons that inducing hepcidin expression also could cause IRIDA. Intravenous iron treatment could improve the iron deficiency condition of IRIDA patients. However, erythropoietin could help iron mobilizing from the liver to erythroblasts to increase erythropoiesis. Both intravenous iron and erythropoietin treatment could be good to alleviate anemia for IRIDA patients. Purpose:Inhibitory member of the ASPP family （iASPP） is an evolutionarily conserved inhibitor of p53. It could specifically inhibit the apoptotic function of P53. The expression of iASPP is up-regulated in human solid tumors and acute leukemia. iASPPsv was identified by our lab as the short isoform of iASPP, which encoded a407aa protein and highly matched the carboxyl terminus of iASPP. It localized in nuclear and could interact with P53to inhibit the P53-induced apoptosis. SON is a DNA-and RNA-binding protein localized in nuclear speckles. That is highly expressed in undifferentiated hematopoietic stem/progenitor cells and leukemic blasts. SON has a role in maintaining proper nuclear organization of pre-mRNA processing factors in nuclear speckles. It is essential for pre-mRNA splicing process of many genes that involved in fundamental cellular pathways such as integrin-mediated cell adhesion, cell cycle regulation, apoptosis and epigenetic regulation of gene expression. The yeast two-hybrid system has identified that iASPP could interact with SON. In order to confirm this interaction and its effects on leukemia cells, we designed this study.Methods:The interaction and the interactive domain between iASPP and SON were identified by immunopreciptation and confocal flurescent. Nalm6cells with stable expression of SON-C45aa were established and sorted by fluorescent cell sorting. The changes of prolification, apoptosis and cell cycles were detected by MTT, AnnexinV/PI and Flow cytometry, respectively. The changes of mRNA and protein expression level were detected by RQ-PCR and Western blot.Result and conclusion:SON could interact with iASPPsv throught its C-terminus. After interferring the interaction between iASPPsv and SON by SON-C45aa in Nalm6cells, G0/G1phase proportion and the apoptosis rate induced by VP-16were increased compared to the control. It indicated that the interaction betweent iASPP and SON could inhibit apotosis of leukemia cells,...