Regulatory T cells (Tregs) suppress immune responses in vivo in an antigen-specific manner. Of clinical relevance, Tregs can be isolated and expanded in vitro while maintaining immunoregulatory function. Tregs are classified as CD4+CD25highCD127low FOXP3+ cells. Demethylation of the Treg-specific demethylation region (TSDR) of FOXP3 is found in natural Tregs (nTregs). We report a method for the characterization of the differential methylation pattern of the FOXP3 TSDR in patient-derived and expanded nTregs. Human TSDR sequences from nTregs (unmethylated sequence) and pancreatic (methylated sequence) cells were amplified and cloned into plasmids. A droplet digital TaqMan probe-based qPCR (ddPCR) assay using methylation-specific primers and probes was employed to quantify unmethylated and methylated sequences. The methylation-specific droplet digital PCR (ddMSP) assay was specific and selective for unmethylated DNA in mixtures with methylated DNA in the range of 5000 copies/μL to less than 1 copy/μL (R 2 = 0.99) even in the presence of non-selective gDNAs. CD4+CD25highCD127lowFOXP3+ human nTregs, in the presence of Dynabeads or activators, were expanded for 21 days. There was a decrease in the unmethylated ratio of Tregs after expansion with essentially the same ratio at days 10, 14, and 17. However, the activator expanded group showed a significant decrease in unmethylated targets at day 21. The suppression activity of activator-expanded nTregs at day 21 was decreased compared to cells expanded with Dynabeads. These data suggest that the ddMSP can quantitatively monitor nTreg expansion in vitro. These data also indicate that the assay is sensitive and specific at differentiating nTregs from other cells and may be useful for rapid screening of nTregs in clinical protocols.

Abstract Background Human pancreata contain many types of cells, such as endocrine islets, acinar, ductal, fat, and mesenchymal stromal cells (MSCs). MSCs are important and shown to have a promising therapeutic potential to treat various disease conditions. Methods We investigated intra-pancreatic tissue-derived (IPTD) MSCs isolated from tissue fractions that are routinely discarded during pancreatic islet isolation of human cadaveric donors. Furthermore, whether pro-angiogenic and anti-inflammatory properties of these cells could be enhanced was investigated. Results IPTD-MSCs were expanded in GMP-compatible CMRL-1066 medium supplemented with 5% human platelet lysate (hPL). IPTD-MSCs were found to be highly pure, with > 95% positive for CD90, CD105, and CD73, and negative for CD45, CD34, CD14, and HLA-DR. Immunofluorescence staining of pancreas tissue demonstrated the presence of CD105+ cells in the vicinity of islets. IPTD-MSCs were capable of differentiation into adipocytes, chondrocytes, and osteoblasts in vitro, underscoring their multipotent features. When these cells were cultured in the presence of a low dose of TNF-α, gene expression of tumor necrosis factor alpha-stimulated gene-6 (TSG-6) was significantly increased, compared to control. In contrast, treating cells with dimethyloxallyl glycine (DMOG) (a prolyl 4-hydroxylase inhibitor) enhanced mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and vascular endothelial growth factor (VEGF). Interestingly, a combination of TNF-α and DMOG stimulated the optimal expression of all three genes in IPTD-MSCs. Conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α contained higher levels of pro-angiogenic (VEGF, IL-6, and IL-8) compared to controls, promoting angiogenesis of human endothelial cells in vitro. In contrast, levels of MCP-1, a pro-inflammatory cytokine, were reduced in the conditioned medium of IPTD-MSCs treated with a combination of DMOG and TNF-α. Conclusions The results demonstrate that IPTD-MSCs reside within the pancreas and can be separated as part of a standard islet-isolation protocol. These IPTD-MSCs can be expanded and potentiated ex vivo to enhance their anti-inflammatory and pro-angiogenic profiles. The fact that IPTD-MSCs are generated in a GMP-compatible procedure implicates a direct clinical application.

We previously reported the development of an oral vaccine for diabetes based on live attenuated Salmonella-expressing preproinsulin (PPI) as the autoantigen. When combined with host cell-expressed TGFβ, the vaccine prevented the onset of diabetes in non-obese diabetic (NOD) mice. Herein, we investigated factors that could affect vaccine efficacy including vaccination number, optimization of the autoantigen codon sequence, Salmonella SPI2-TTSS promoter/effector combinations, concurrent short-course low-dose anti-CD3. We also evaluated autoantigen GAD65 and cytokine IL10 treatment upon vaccine efficacy. T-cells we employed to elucidate the mechanism of the vaccine action. Our results showed that GAD65+TGFβ or PPI+TGFβ+IL10 prevented the onset of diabetes in the NOD mice and maintained glucose tolerance. However, increasing the number of vaccine doses, codon-optimization of the autoantigen(s) or use of other Salmonella promoter/effector combinations had no in vivo effect. Interestingly, two doses of vaccine (PPI+TGFβ+IL10) combined with a sub-therapeutic dose of anti-CD3 prevented diabetes and decreased hyperglycemia in mice. The combined therapy also increased splenic Tregs and local Tregs in pancreatic lymph nodes (PLN) and increased regulatory (IL10 and IL2) but reduced inflammatory (IFNγ and TNFα) cytokines. Together, these results indicate that the combination of low vaccine dose number, less vaccine autoantigen expression and short-course low-dose anti-CD3 can increase regulatory mechanisms and suppress autoimmunity.

Abstract It is well documented that, both Foxp3+ regulatory T cells (Treg) and antigen-expanded Foxp3lo/- Treg play critical roles in regulating immunity and autoimmunity, as well as in preventing autoimmune diseases. However, the molecular mechanisms and genes responsible for modulating function of these Treg still remain incompletely understood. WRNIP1 (Werner helicase-interacting protein 1) was identified as a protein that interacts with WRN, the Werner syndrome responsible gene product, and may participate in DNA synthesis, damage and repair. Here, we report an unexpected finding that WRNIP1 plays an important role in regulating the function of autoantigen-expanded Foxp3lo/- Treg. Down regulation of WRNIP1 in these Treg significantly increased their proliferation and reduced activation-induced cell death. More importantly, WRNIP1-downregulated Treg showed markedly enhanced suppressive function in vitro, partly due to an increased production of IL-10 and TGF-β. These Treg also significantly improved their ability in preventing diabetes development in NOD mice. To understand how WRNIP1 regulates Treg function, we found that elevated Foxo1 expression may contribute to the enhanced Treg function through promoting IL-10 and TGF-β production. These studies demonstrate that WRNIP1 negatively regulates Treg function and suggest previously undisclosed molecular pathways and targets for improving Treg-mediated immune tolerance to treat autoimmune diseases like type 1 diabetes.

The British Journal of Haematology publishes original research papers in clinical, laboratory and experimental haematology. The Journal also features annotations, reviews, short reports, images in haematology and Letters to the Editor.

MicroRNAs (miRNAs) play a vital role in the regulation of immunological functions and prevention of autoimmune disease. The abnormal expressions of several miRNAs in patients with the acquired autoimmune disease, immune thrombocytopenia (ITP), have been reported. However, the exact mechanism of miRNAs in the pathogenesis of ITP is currently not well understood. This study examined the miRNA expression profile of peripheral blood mononuclear cells (PBMCs) in ITP patients by miRNA array and TaqMan real-time polymerase chain reaction. MIR130A expression was found to be significantly decreased in PBMCs from patients with active chronic ITP compared with that of normal controls. Subsequently, dualluciferase reporter gene analysis was used to validate that MIR130A targeted the transforming growth factor-beta1 (TGFB1) and interleukin 18 (IL18) genes. In addition, we also monitored the dynamic expression of MIR130A and its targeted genes pre- and post-treatment of ITP patients and determined that the expression of MIR130A and TGFB1 was up-regulated, whereas IL18 expression was down-regulated after effective treatment. In conclusion, this study suggests that reduced MIR130A is involved in ITP via targeting of TGFB1 and IL18 expression.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by autoantibody-mediated platelet destruction. Multiple factors have been implicated in ITP pathogenesis, including T-lymphocyte dysfunctions. The protein tyrosine phosphatase, nonreceptor type 22 (PTPN22) gene encodes lymphoid-specific phosphatase (LYP), a critical negative regulator of T cell activation. Single nucleotide polymorphisms (SNPs) of PTPN22 have been broadly associated with susceptibilities to various autoimmune disorders. Here we conducted a case-control study investigating whether the PTPN22 -1123G>C SNP contributes to the risk of ITP in Chinese population. The study included 191 ITP cases and 216 ethnically matched normal controls. Genotyping of -1123G>C SNP was performed using a single-base extension (SBE) and mass spectrometry method. Allelic and genotypic frequencies were compared between the case-control groups by the chi-square test. We observed significant overrepresentation of -1123G allele (p = 0.034, odds ratio (OR) = 1.374, 95% confidence interval (CI) [1.024-1.843]) and GG genotype (P = 0.038, OR = 1.951, 95% CI [1.031-3.694]) in the patients compared with the controls. Stratified analysis by gender and age of disease onset revealed comparable observations in both male and adult ITP cohorts. These data suggest a moderate association of PTPN22 -1123G>C SNP with susceptibility to ITP. Together with previous reports, our finding provides further evidence for PTPN22 being a general autoimmunity gene.

Foxp3+ regulatory T cells (Treg) play a crucial role in regulating immune tolerance. The use of Treg to restore immune tolerance is considered an attractive novel approach to inhibit autoimmune disease, including type 1 diabetes (T1D), and to prevent rejection of organ transplants. In view of the goal of developing autologous Treg-based cell therapy for patients with long-term (>15 years) T1D, it will be necessary to expand a sufficient amount of functional Treg in vitro in order to study and compare Treg from T1D patients and healthy subjects. Our results have demonstrated that there is a comparable frequency of Treg in the peripheral blood lymphocytes (PBLs) of patients with long-term T1D relative to those in healthy subjects; however, Th1 cells, but not Th17 cells, were increased in the T1D patients. Further, more Treg in PBLs from T1D patients than from healthy subjects expressed the CD45RO+ memory cell phenotype, suggesting they were antigen-experienced cells. After isolation, Treg from both T1D patients and healthy subjects were successfully expanded with high purity. Although there was no difference in Helios expression on Treg in PBLs, in vitro expansion led to fewer Helios-expressing Treg from T1D patients than healthy subjects. While more Th1-like Treg expressing IFN-γ or TNF-α were found in the PBLs of T1D patients than healthy controls, there was no such difference in the expanded Treg. Importantly, expanded Treg from both subject groups were able to suppress autologous or allogeneic CD8+ effector T cells equally well. Our findings demonstrate that a large number of ex vivo expanded functional Treg can be obtained from long-term T1D patients, although fewer expanded Treg expressed a high level of Helios. Thus, based on the positive outcomes, these potent expanded Treg from diabetic human patients may be useful in treating T1D or preventing islet graft rejection.

Background/Aims: Human umbilical cord mesenchymal stem cells (hUC-MSCs) possess immunosuppressive activities but the mechanisms of such activities are not fully understood. Here, we investigated the role of IL-6, one of the characteristic factors of MSCs, in the immunoregulating effect of hUC-MSCs on CD4+ T lymphocytes. Methods: The condition media from human peripheral blood mononuclear cells (hPBMCs) or CD14+/- cell were tested if stimulating IL-6 production by hUC-MSCs. The related signaling pathway of IL-6, and the immunosuppressive activity of IL-6 on CD4+ T lymphocytes were studied. Result: IL-6 production was dramatically increased by hUC-MSCs when co-culturing with resting or activated hPBMCs. CD14+ monocytes-paracrined IL-1β promoted the secretion of IL-6 by hUC-MSCs via JNK and NF-ĸB signaling pathway. Blocking of PGE2 synthesis did not affect the secretion of IL-6, anti-IL-6 antibody was not able to reverse hUC-MSCs-mediated inhibition on CD4+ T lymphocytes. IL-6 did not mediate the suppressive activity of IL-1β-hUC-MSCs- PGE2 on CD4+ T cell. Conclusion: CD14+ monocytes-paracrined IL-1β promotes IL-6 secretion by hUC-MSCs through activating JNK and NF-ĸB signaling pathway. However, increased IL-6 production does not contribute to immunosuppressive activity of IL-1β-hUC-MSCs- PGE2 on CD4+ T cells.

Autoreactive pathogenic T cells (Tpaths) and regulatory T cells (Tregs) express a distinct gene profiles; however, the genes and associated genetic/signaling pathways responsible for the functional determination of Tpaths vs. Tregs remain unknown. Here we show that Skp2, an E3 ubiquitin ligase that affects cell cycle control and death, plays a critical role in the function of diabetogenic Tpaths and Tregs. Down-regulation of Skp2 in diabetogenic Tpaths converts them into Foxp3-expressing Tregs. The suppressive function of the Tpath-converted Tregs is dependent on increased production of TGF-β/IL-10, and these Tregs are able to inhibit spontaneous diabetes in NOD mice. Like naturally arising Foxp3 + nTregs, the converted Tregs are anergic cells with decreased proliferation and activation-induced cell death. Skp2 down-regulation leads to Tpath–Treg conversion due at least in part to up-regulation of several genes involved in cell cycle control and genes in the Foxo family. Down-regulation of the cyclin-dependent kinase inhibitor p27 alone significantly attenuates the effect of Skp2 on Tpaths and reduces the suppressive function of converted Tregs; its effect is further improved with concomitant down-regulation of p21, Foxo1, and Foxo3. In comparison, Skp2 overexpression does not change Tpath function, but significantly decreases Foxp3 expression and abrogates the suppressive function of nTregs. These findings support the critical role of Skp2 in functional specification of Tpaths and Tregs, and demonstrate an important molecular mechanism mediating Skp2 function in balancing immune tolerance during autoimmune disease development.

Both Foxp3+regulatory T cells (Tregs) and antigen-expanded Foxp3−Tregs play an important role in regulating immune responses as well as in preventing autoimmune diseases and graft rejection. Molecular mechanisms modulating Treg function remain largely unclear, however. We report here on the expression and function of an inhibitory killer cell Ig-like receptor, KIR3DL1, in a nonobese diabetic (NOD) mouse-derived autoantigen-specific Treg (2D2), which protects from type 1 diabetes (T1D) in adoptive transfer experiments. This gene is not expressed in T1D pathogenic T cells (Tpaths) or non-Tpath T cells. KIR genes are known to play an important role in regulating natural killer (NK) cell function, but their role in Tregs and T1D is unknown. To examine whether KIR3DL1 expression may modulate Treg function, we used shRNA to down-regulate KIR3DL1 expression (2D2-shKIR). We find that KIR3DL1 down-regulation enhances in vitro function, as measured by improved suppression of target cell proliferation. Antibody blockade of IL-10 but not IL-4 partially abrogated suppressive function. In vivo function is also improved. Adoptive transfer of 2D2-shKIR into 10-wk-old NOD mice prevented spontaneous insulitis and T1D, and the inhibitory effect was further improved if the cells were transferred earlier into 6-wk-old NOD mice. These studies indicate that KIR3DL1 expression may negatively regulate Treg function and suggest a previously undescribed target for improving immune tolerance for potential treatment of autoimmune diseases like T1D.

Mesenchymal stem cells (MSCs), which are poorly immunogenic and have potent immunosuppressive activities, have emerged as a promising candidate for cellular therapeutics for the treatment of disorders caused by abnormal immune responses. In this study we investigated whether human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) could ameliorate colitis in a trinitrobenzene sulfonic acid (TNBS)-induced colitis model. TNBS-treated colitic mice were infused with hUC-MSCs or vehicle control. The mice were sacrificed on day 1, 3, and 5 after infusion, and their clinical and pathological conditions were evaluated by body weight, colon length, and histological analysis. The expression levels of proinflammatory cytokine proteins in colon were examined by ELISA. The homing of hUC-MSCs was studied by live in vivo imaging and immunofluorescent microscopy. hUC-MSCs were found to migrate to the inflamed colon and effectively treated the colitic mice with improved clinical and pathological signs. The levels of IL-17 and IL-23 as well as IFN-γ and IL-6 were significantly lower in the colon tissues of the hUC-MSC-treated mice in comparison with the vehicle-treated mice. Coculture experiments showed that hUC-MSCs not only could inhibit IFN-γ expression but also significantly inhibit IL-17 production by lamina propria mononuclear cells (LPMCs) or splenocytes of the colitic mice or by those isolated from normal animals and stimulated with IL-23. Systemically infused hUC-MSCs could home to the inflamed colon and effectively ameliorate colitis. In addition to the known suppressive effects on Th1-type immune responses, hUC-MSC-mediated modulation of IL-23/IL-17 regulated inflammatory reactions also plays an important role in the amelioration of colitis.

Genome-wide hypomethylation has been confirmed in patients with primary immune thrombocytopenia (ITP). Proteins containing methylcytosine-binding domain (MBD) are involved in promoter methylation as transcriptional repressors and promote the gene-silencing effect of DNA methylation. The purpose of this study was to investigate the methylation pattern of T cells and the relationship between genomic methylation and the expression of MBD2 and MBD4 in ITP patients. DNA deoxymethylcytosine content of CD4(+) cells from peripheral blood mononuclear cells was measured by enzyme-linked immunoassay. Real-time polymerase chain reaction was performed to quantify the transcription levels of MBD2 and MBD4 in peripheral blood mononuclear cells and CD4(+) cells. DNA dmC content in CD4(+) cells of ITP patients was significantly lower than in the controls (p = 0.001). The mRNA level of MBD2 and MBD4 in CD4(+) cells of ITP patients was statistically lower than those of the controls (p < 0.001). Positive correlations between methylation indexes and expression of each enzyme were observed in the control group (r(2) = 0.718, p = 0.004 for MBD2; r(2) = 0.608, p = 0.015 for MBD4). However, inverse correlations were found in ITP patients (r(2) = 0.604, p = 0.008 for MBD2; r(2) = 0.498, p = 0.027 for MBD4). Our results indicate that decreased expression of MBD2 and MBD4 might involve in the pathogenesis of ITP.

In order to evaluate whether mesenchymal stem cells (MSCs) from non-hematopoietic tissues are able to regulate megakaryocytopoiesis, we identified human MSCs from adult bone marrow (ABM), fetal pancreas (FPan) and umbilical cord (UC), and their abilities to support megakaryocyte (MK) differentiation from CD34+ hematopoietic progenitor cells (HPCs) were comparatively studied. First, MSCs were isolated from ABM, FPan and UC then their growth kinetics, molecular characterization and mesodermal differentiation capacity were determined. ABM-MSCs, FPan-MSCs and UC-MSCs were irradiated and cocultured with human umbilical cord blood (UCB) CD34+ cells, and the expansion efficiency of MK progenitor cells and MK formation were analysed and compared. Finally, SCF, IL-6 and GM-CSF expression by the three types of MSCs were also examined. Our results showed that FPan-MSCs and UC-MSCs shared most of the characteristic of ABM-MSCs, including morphology, immunophenotype, adipogenic and osteogenic differentiation potentials. Compared with ABM-MSCs, fetal MSCs had higher proliferative capacity. After 7 days' coculture, the maximal production of CD34+/CD41a+ cells was obtained in a group of CD34+ HPCs + ABM-MSCs. Furthermore, this group produced more MK colonies than other groups (p < 0.05). Surface antigen and ploidy analysis morphological observation demonstrated that a proportion of expanded cells in each group differentiated into mature MKs. ABM-MSCs, FPan-MSCs and UC-MSCs were revealed to express SCF, IL-6 and GM-CSF at mRNA level. We conclude that FPan-MSCs and UC-MSCs have the ability to promote megakaryocytopoiesis, while ABM-MSCs expand more MK progenitor cells from CD34+ HPCs than MSCs from non-hematopoietic tissues and CD34+ cells alone.

Immune thrombocytopenia (ITP) is an acquired organ-specific autoimmune disease with a polarization of T(h)1. Both the T(h)1 chemokine CXCL10 and T(h)2 chemokine CCL2 have been studied in several autoimmune diseases, but the status of these chemokines in ITP is still unknown. The aims of this study were to determine the expression of CXCL10 and CCL2 and their receptors, CXCR3 and CCR2, in ITP patients, and to conduct a preliminary study of the pathogenic roles of these factors in ITP. Plasma samples from 49 patients with ITP and 24 normal healthy subjects were assayed for CXCL10 and CCL2 plasma concentration by enzyme-linked immunosorbent assay. Real-time quantitative polymerase chain reaction was performed to determine the mRNA expression of these chemokines and their receptors in the PBMNC of 24 normal controls and 28 active ITP patients as well as splenocytes of nine ITP patients. The CXCL10 levels in the plasma samples from patients with active ITP were significantly higher than those from healthy controls (p = 0.007) and decreased to normal levels in patients with remission ITP. In contrast, CCL2 levels were similar in patients with active disease, patients in remission, and control subjects. PBMNC of patients with active disease expressed more CXCL10 mRNA (p = 0.031) but less CCR2 mRNA (p = 0.005). Lower peripheral platelet count correlated with higher CXCL10 levels and CXCL10/CCL2 ratios. Our study demonstrated that plasma levels of CXCL10 and CXC10/CCL2 ratio were higher in patients with active ITP than in healthy donors, and had an association with platelet counts of the patients. CXCL10 might be a pathogenic factor of this disorder.

Epigenetics might contribute to autoimmune diseases including immune thrombocytopenic purpura (ITP). Methyl-CpG binding domain protein 4 (MBD4) plays an important role in DNA methylation and transcriptional regulation of gene expression. The polymorphism of the MBD4 gene may influence MBD4 activity on gene expression profiles, thereby influencing individual susceptibility to ITP. To verify this hypothesis, we investigated the association between the MBD4 polymorphism and the risk for ITP in the Chinese population. The polymorphism of MBD4 rs140693 was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In this study, there was no significant difference in genotype and alleles distribution between the ITP patients and the controls. Similar results were observed between the two groups when stratified by age and disease course including acute childhood, chronic childhood, acute adult and chronic adult. In the conclusion, MBD4 polymorphism may not be a stratification marker to predict the susceptibility to ITP, at least in Chinese population.

OBJECTIVE To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase. METHODS The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR. RESULTS The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes. CONCLUSION Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.

Idiopathic thrombocytopenic purpura (ITP) is an acquired organ-specific autoimmune hemorrhagic disease with many immune dysfunctions. Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T-lymphocyte surface molecule that can down modulate and terminate immune responses. Recently, several studies have confirmed that some polymorphisms of this gene can influence its expression level, therefore speculating that they might be associated with autoimmune diseases. In order to investigate the role of the CTLA-4 gene in ITP, we investigated -318 and CT60 polymorphisms of the CTLA-4 gene in 186 ITP patients and 162 healthy controls through polymerase chain reaction (PCR)–restriction fragment length polymorphism. No significant differences were revealed in genotypes and allele distributions between the patients with ITP and the controls in both sites. Similar results were observed between the two groups when stratified by first onset age and disease course including acute childhood, chronic childhood, acute adult, and chronic adult. In the conclusion, these two single-nucleotide polymorphisms in CTLA-4 are not associated with susceptibility to ITP in a Chinese population.

Combined deficiency of factor V and VIII (F5F8D) is a rare, autosomal recessive disorder caused by mutations of either lman1 or mcfd2. To identify mutations of these two genes in a Chinese F5F8D family, the samples of peripheral blood were collected from the proband and her parents. Coagulation tests were carried out, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fg) and coagulate activity of FV, FVIII (FV:C, FVIII:C). The genomic DNA was extracted, then all the exons and intron/exon boundaries of these two genes were amplified by polymerase chain reaction (PCR). The products were finally analyzed by direct sequencing. The results showed that the proband's APTT, PT, TT, Fg, FV:C and FVIII:C were 82.2 sec, 19.6 sec, 18.6 sec, 2.9 g/L, 7.1% and 18.7% respectively, while those parameters of the parents were all within the normal range. Two pathogenic mutations were identified in lman1 gene of the proband: one was the heterozygous c.912_913insA in exon 8 resulting in a frameshift of p.Glu305fsX20; the other was the heterozygous c.1366C > T in exon 11 resulting in p.Arg456X. The proband's father and mother were heterozygous for c.1366C > T and c.912_913insA respectively. It is concluded that F5F8D of the proband is caused by a novel compound heterozygous mutation of the lman1 gene, which has never been reported.