Altered Splicing in Stable Cell Strains Expressing Mini-hF9 Gene with Nonsense Mutation / 中国实验血液学杂志

OBJECTIVE@#To investigate the molecular mechanism in stable cell strains expressing Mini-hF9 gene with nonsense mutation.@*METHODS@#Mini-hF9 gene and its nonsense mutants were transfected into HeLa cells independently, and stable cell strains were obtained after G418 resistance screening and monoclonal transformation. The altered splicing and protein expression of mRNA in Mini-hF9 gene in stable cell strains were detected by using RT-PCR and Western blot.@*RESULTS@#The wild type and nonsense mutated human coagulation factor IX stable cell strains were constructed successfully, which were named HeLa-F9-WT, HeLa-F9-M1 and HeLa-F9-M2. Only normal splicing Norm was detected in the wild-type cell strain HeLa-F9-WT; Norm and Alt-S1 splicing were detected in HeLa-F9-M1; while Norm, Alt-S1 and Alt-S2 splicing were detected in HeLa-F9-M2.@*CONCLUSION@#The nonsense associated altered splicing (NAS) pathway, which generated alternately spliced transcripts, might be triggered in coagulation factor IX gene with nonsense mutation.

Detection of intron 22 inversion of factor VIII gene in severe hemophilia A patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the incidence of intron 22 inversion (INV22) of factor VIII (FVIII) gene in severe hemophilia A (HA) patients, clarify its pathological mechanism, and identify INV22 carrier in the female family members.</p><p><b>METHODS</b>One-stage method was used to assay the FVIII activity (FVIII:C)in 126 severe HA patients with a median age of 14 years old (range: 4 months-63 years). INV22 was analyzed by long-distance polymerase chain reaction (LD-PCR) and pulsed field gel electrophoresis (PFGE), and pedigree were conducted in 3 involved HA families.</p><p><b>RESULTS</b>Of all the 126 severe HA, 52 (41.3%) cases had the INV22. Four females including 3 mothers and 1 sister of probands were diagnosed as INV22 carriers among 11 suspected carrier mosaicisms from 3 INV22 positive HA families. In 8 females from one family without HA history, the patient's mother was a INV22 carrier, but her maternal grandmother, 2 maternal aunts, 2 female siblings and 1 elder female cousin were negative.</p><p><b>CONCLUSION</b>LD-PCR and PFGE could be used to diagnose severe HA patients with INV22 and identify the carriers.</p>

Expressions of Th17 cells and interleukin 17 in patients with primary immune thrombocytopenia and their clinical significance / 中国实验血液学杂志

The aim of this study was to detect the rate of T-helper (Th)17 cells and interleukin (IL)-17 level in peripheral blood of patients with primary immune thrombocytopenia (ITP) and to explore their clinical significance. The proportion of Th17 cells from 48 patients with ITP and 28 healthy controls was detected by flow cytometry, and the IL-17 level was evaluated by enzyme-linked immunosorbent assay (ELISA). The results showed that the percentage of Th17 cells in ITP group was (1.40 ± 1.35)%, which was significantly higher than that in healthy control group (P < 0.05), but in the glucocorticoid hormone-treated group it was significantly lower than that in treated group without glucocorticoid hormone(P < 0.05). The level of IL-17 expressed by Th17 cells in ITP patients was (19.624 ± 5.187) pg/ml, which was higher than that in the healthy control group (P < 0.05), it was lower in the glucocorticoid hormone treated group than that in treated group without glucocorticoid hormone, but there was no statistically significant difference between the glucocorticoid treated and treated group without glucocorticoid hormone (P > 0.05). It is concluded that the Th17 cells may involve in the pathogenesis of ITP, and the glucocorticoid hormone probably plays a therapeutic role through inhibiting Th17 cells.

Screening and analysis of coagulation factor VIII inhibitor in patients with hemophilia A / 中国实验血液学杂志

In order to detect coagulation factor VIII (FVIII) inhibitor in patients with severe hemophilia A (HA) and preliminarily study the genetic mutation in patients with inhibitor positive. Totally 58 patients with HA (FVIII: C < 1%) were enrolled. FVIII: C activity was measured by one-stage coagulation assay. FVIII inhibitor was screened by using APTT method and FVIII inhibitor in screened positive patients with HA was quantitatively analyzed by using Bethesda method. Using genomic DNA as template, 12, 14, 16 exons of FVIII in screened positive patients were amplified, and the mutations of amplified products were detected by direct sequencing. The results indicated that the FVIII inhibitor could be detected in 4 patients (6.9%) from 58 HA patients, no gene mutations in 12, 14, 16 exons of FVIII were found. It is concluded that the positive rate of FVIII inhibitor in HA patients is lower than that reported in literature. The causes of inhibitor production needs to further investigate.