Comparison of haploidentical-allogeneic hematopoietic stem cell transplantation and intensive immunosuppressive therapy for patients with severe aplastic anemia with an absolute neutrophil count of zero: a retrospective study.

A retrospective analysis was conducted based on the clinical data from 60 patients older than 16 years from January 2016 to January 2021. All the patients were newly diagnosed with severe aplastic anemia (SAA) with an absolute neutrophil count (ANC) of zero. We compared the hematological response and survival of haploidentical-allogeneic hematopoietic stem cell transplantation (HID-HSCT) (n = 25) and intensive immunosuppressive therapy (IST) (n = 35) treatments. At six months, the overall response rate and complete response were significantly higher in the HID-HSCT group than those in the IST group (84.0% vs. 40.0%, P = 0.001; 80.0% vs. 17.1%, P = 0.001). With a median follow-up of 18.5 months (4.3~30.8 months), patients in the HID-HSCT group had longer overall survival and event-free survival (80.0% vs. 47.9%, P = 0.0419; 79.2% vs. 33.5%, P = 0.0048). These data suggested that HID-HSCT might be an effective alternative treatment option for adult patients with SAA with an ANC of zero, which requires further validation in an additional prospective study.

Frontline Therapy Options for Adults With Newly Diagnosed Severe Aplastic Anemia: Intensive immunosuppressive Therapy Plus Eltrombopag or Matched Sibling Donor Hematopoietic Stem Cell Transplantation?

Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disease. Allogeneic hematopoietic stem cell transplantation from a matched sibling donor (MSD-HSCT) and intensive immunosuppressive therapy (IST) are 2 major comparable treatments for SAA. As the addition of eltrombopag (EPAG) to standard IST therapy has greatly improved the survival prognosis of SAA, whether MSD-HSCT or IST/EPAG is the better choice has become a matter of debate. A study was performed involving 99 patients with newly diagnosed acquired SAA from 5 medical centers, including 48 MSD-HSCT cases and 51 IST/EPAG cases, which consisted of rabbit antithymocyte globulin or porcine-antilymphocyte globulin, cyclosporine plus eltrombopag. The results suggested that patients treated with MSD-HSCT or IST/EPAG had similar overall survival (OS) rates exceeding 95% (P = .97). However, the event-free survival rate (EFS) of IST/EPAG (71.0%) was significantly lower than that of MSD-HSCT (89.6%), P = .04. Subgroup analysis indicated that the OS of the MSD-HSCT group was superior to that of the IST/EPAG group (100% versus 85.7%, P = .04) among those with very severe aplastic anemia (VSAA). Both the complete response rate (CR) and overall response rate (OR) with MSD-HSCT were significantly higher than those with IST/EPAG (CR: 79.2% versus 15.7%, P < .001; OR: 97.9% versus 72.6%, P = .001). In conclusion, IST/EPAG or MSD-HSCT treatment achieves an equally high OS in SAA, but MSD-HSCT leads to a better OS in patients with VSAA and shows advantages in improving EFS and accelerating hematopoietic reconstruction in patients with SAA.

Efficacy and safety of sirolimus early conversion protocol in liver transplant patients with hepatocellular carcinoma: A single-arm, multicenter, prospective study.

Mammalian target of rapamycin (mTOR) inhibitor as an attractive drug target with promising antitumor effects has been widely investigated. High quality clinical trial has been conducted in liver transplant (LT) recipients in Western countries. However, the pertinent studies in Eastern world are paucity. Therefore, we designed a clinical trial to test whether sirolimus can improve recurrence-free survival (RFS) in hepatocellular carcinoma (HCC) patients beyond the Milan criteria after LT. This is an open-labeled, single-arm, prospective, multicenter, and real-world study aiming to evaluate the clinical outcomes of early switch to sirolimus-based regimens in HCC patients after LT. Patients with a histologically proven HCC and beyond the Milan criteria will be enrolled. The initial immunosuppressant regimens are center-specific for the first 4-6 weeks. The following regimens integrated sirolimus into the regimens as a combination therapy with reduced calcineurin inhibitors based on the condition of patients and centers. The study is planned for 4 years in total with a 2-year enrollment period and a 2-year follow-up. We predict that sirolimus conversion regimen will provide survival benefits for patients particular in the key indicator RFS as well as better quality of life. If the trial is conducted successfully, we will have a continued monitoring over a longer follow-up time to estimate indicator of overall survival. We hope that the outcome will provide better evidence for clinical decision-making and revising treatment guidelines based on Chinese population data. Trial register: Trial registered at http://www.chictr.org.cn: ChiCTR2100042869.

[Allogeneic Hematopoietic Stem Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria].

OBJECTIVE: To analyze the clinical efficacy and safety of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for paroxysmal nocturnal hemoglobinuria (PNH), and preliminarily explore the role of an improved post-transplantation cyclophosphamide (PTCy) based conditioning regimen in PNH patients receiving transplantation. METHODS: Clinical related data of PNH sufferers receiving allo-HSCT in Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology were collected, and hematopoietic reconstitution, chimerism, PNH cloning, graft-versus-host disease (GVHD), infection, and survival were analyzed. RESULTS: Totally five PNH patients receiving allo-HSCT were enrolled, including 1 case with classic PNH, 3 cases with aplastic anemia-PNH syndrome, 1 case with myelodysplastic syndrome, three of them (case 1-3) received the improved PTCy based conditioning regimen before HSCT. All sufferers engrafted successfully within 28 days, the median time of neutrophil and platelet engraftment was 11 days and 12 days, respectively, no patient occurred acute or chronic GVHD, after a median follow-up of 16 months, all recipients survived and completely eliminated PNH cloning. CONCLUSION: Allo-HSCT can completely clear PNH cloning and restore hematopoietic function with controllable complications, and the improved PTCy based conditioning regimen is proved to be effective in PNH transplantation.

Current Treatment Patterns of Aplastic Anemia in China: A Prospective Cohort Registry Study.

Aplastic anemia (AA) is a hematologic disease characterized by pancytopenia and hypocellular bone marrow, potentially leading to chronic anemia, hemorrhage, and infection. The China Aplastic Anemia Committee and British Committee for Standards in Haematology guidelines recommend hematopoietic stem-cell transplantation (HSCT) or immunosuppressive therapy (IST) comprising antithymocyte globulin (ATG) with cyclosporine (CsA) as initial treatment for AA patients. With limited epidemiological data on the clinical management of AA in Asia, a prospective cohort registry study involving 22 AA treatment centers in China was conducted to describe the disease characteristics of newly diagnosed AA patients and investigate real-world treatment patterns and patient outcomes. Of 340 AA patients, 72.9, 12.6, and 3.5% were receiving IST, traditional Chinese medicine, and HSCT, respectively, at baseline; only 22.2% of IST-treated patients received guideline-recommended ATG with CsA initially. Almost all patients received supportive care (95.6%) as blood transfusion (97.8%), antibiotics (63.7%), and/or hematopoietic growth factors (58.2%). Overall, 64.8% achieved a partial or complete response, and 0.9% experienced relapse. No new safety concerns were identified; serious adverse events were largely unrelated to the treatment regimen. These results demonstrate the need to identify and minimize treatment barriers to standardize and align AA management in China with treatment guideline recommendations and further improve patient outcomes.

Genome-wide characterization of the seasonal H3N2 virus in Shanghai reveals natural temperature-sensitive strains conferred by the I668V mutation in the PA subunit.

Seasonal H3N2 influenza viruses are recognized as major epidemic viruses, exhibiting complex seasonal patterns in regions with temperate climates. To investigate the influence of viral evolution and mutations on the seasonality of influenza, we performed a genome-wide analysis of samples collected from 62 influenza A/H3N2-infected patients in Shanghai during 2016-2017. Phylogenetic analysis of all eight segments of the influenza A virus revealed that there were two epidemic influenza virus strains circulating in the 2016-2017 winter season (2016-2017win) and 2017 summer season (2017sum). Replication of the two epidemic viral strains at different temperatures (33, 35, 37, and 39 °C) was measured, and the correlation of the mutations in the two epidemic viral strains with temperature sensitivity and viral replication was analyzed. Analysis of the replication kinetics showed that replication of the 2016-2017win strains was significantly restricted at 39 °C compared with that of the 2017sum strains. A polymerase activity assay and mutational analysis demonstrated that the PA I668V mutation of the 2016-2017win viruses suppressed polymerase activity in vitro at high temperatures. Taken together, these data suggest that the I668V mutation in the PA subunit of the 2016-2017win strains may confer temperature sensitivity and attenuate viral replication and polymerase activity; meanwhile, the 2017sum strains maintained virulence at high temperatures. These findings highlight the importance of certain mutations in viral adaptation and persistence in subsequent seasons.

Serum M2BPGi level is a novel predictive biomarker for the responses to pegylated interferon-α treatment in HBeAg-positive chronic hepatitis B patients.

Serum Mac-2-binding protein glycosylation isomer (M2BPGi) level was found to be a useful prognostic marker for hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients treated with nucleoside/nucleotide analogs (NUCs) therapy, and the aim of our study is to evaluate the clinical implementation of M2BPGi level in the prediction of antiviral responses to pegylated-interferon-α (PEG-IFN-α) treatment in HBeAg-positive CHB patients. Ninety-six CHB patients who received PEG-IFN-α treatment for at least 48 weeks were recruited. The serum M2BPGi, alanine aminotransferase (ALT), hepatitis B surface antigen (HBsAg), HBeAg, and HBV DNA levels at baseline, weeks 4, 12, and 24 after PEG-IFN-α treatment were determined and their associations with antiviral responses were evaluated and the virological response (VR) rate and serological response (SR) rate after 48 weeks of treatment were 65.6% and 35.4%, respectively. Baseline serum M2BPGi level was significantly different between VR and non-VR (P = 0.002) or SR and non-SR groups (P = 0.012). Multivariate analyses suggested that baseline serum M2BPGi level was independently associated with VR and SR of PEG-IFN-α treatment at week 48. The area under the ROC curve (AUC) of baseline M2BPGi was 0.682 in predicting VR, which was superior to HBsAg (AUC = 0.566) or HBV DNA (AUC = 0.567). The AUC of baseline M2BPGi in predicting SR was 0.655, which was also higher than that of HBsAg (AUC = 0.548) or HBV DNA (AUC = 0.583). These results suggested that baseline serum M2BPGi level was a novel predictor of VR and SR for PEG-IFN-α treatment in HBeAg-positive CHB patients.

Clinical features and viral quasispecies characteristics associated with infection by the hepatitis B virus G145R immune escape mutant.

Coexistence of the hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs) is an uncommon phenomenon, and the underlying mechanisms remain largely unknown. Amino-acid (aa) substitution from glycine to arginine at aa 145 (G145R), in the major hydrophilic region, has been reported in patients with HBsAg and anti-HBs coexistence. However, there is limited knowledge about the clinical features and viral quasispecies characteristics associated with G145R mutant hepatitis B virus (HBV) infection. We herein describe the dynamic changes in the serological and virological markers in a case of hepatitis B with coexisting HBsAg and anti-HBs, caused by a G145R immune escape mutant (genotype C). Entecavir was administered during the 4th week after admission. Alanine aminotransferase peaked in the 16th week, while both the HBsAg and HBeAg declined rapidly. HBsAg clearance and hepatitis B e antigen (HBeAg)/hepatitis B e antibody (anti-HBe) seroconversion were achieved in the 36th week, and then entecavir was withdrawn. A follow-up of 96 weeks showed that HBV DNA remained undetectable and that anti-HBs was maintained above 100 mIU/mL. The quasispecies characteristics of the G145R mutant HBV were investigated via ultra-deep sequencing. The complexity and genetic distance of the S and RT regions were much higher in the 8th week than at baseline or in the 4th week. Moreover, the frequencies of mutations (L173P, Q181R and A184V) in cytotoxic T lymphocyte epitopes increased before entecavir treatment. These findings extend understanding of the evolution of HBV under host immune pressure and of the clinical outcomes of affected patients.

Serum WFA+ -M2BP levels for evaluation of early stages of liver fibrosis in patients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: Accurate evaluation of liver fibrosis is crucial for predicting progression of chronic hepatitis B virus (HBV) infection. We assessed the utility of a novel fibrosis glycobiomarker Wisteria floribunda agglutinin-positive Mac-2-binding protein (WFA+ -M2BP) for evaluating liver fibrosis and disease progression in patients with chronic HBV infection. METHODS: We enrolled 774 patients with chronic HBV infection, with or without fibrosis, diagnosed by liver biopsy/FibroScan. Patients who underwent liver biopsy (n = 297) were divided into training (n = 221) and validation (n = 76) groups. Serum WFA+ -M2BP values were measured and compared with FIB-4 index, aspartate aminotransferase (AST)-to-platelet ratio (APRI) and AST-to-alanine aminotransferase ratio (AAR) using receiver-operating characteristic (ROC) analysis. RESULTS: Serum WFA+ -M2BP levels increased significantly with fibrosis progression (P < 0.0001). Area under the ROC curve of WFA+ -M2BP for diagnosing significant fibrosis was higher than that of FIB-4 (P = 0.198), APRI (P = 0.017) and AAR (P < 0.001), with sensitivity and specificity in the training set of 60.5% and 79.8% and validation set of 59.5% and 82.1%, respectively. Serum WFA+ -M2BP levels were significantly correlated with FibroScan values (P < 0.0001) and improved the accuracy of FibroScan in assessing significant fibrosis. Changes in WFA+ -M2BP levels were parallel with those in FibroScan values during nucleot(s)ide analogues therapy in patients with chronic HBV infection. CONCLUSIONS: WFA+ -M2BP is an accurate serum indicator for assessing early stages of liver fibrosis and may monitor regression of fibrosis during the treatment of chronic HBV infection. WFA+ -M2BP provides a simple and reliable alternative or complementary method to liver biopsy and FibroScan.

Characteristics of CpG Islands and their quasispecies of full-length hepatitis B virus genomes from patients at different phases of infection.

BACKGROUND: CpG islands in hepatitis B virus (HBV) genome are potential targets for methylation mediated gene silencing, and may be involved in the pathogenesis of HBV infection. To date, their characteristics in HBV quasispecies (QS) remain largely unknown. The purpose of this study was to investigate the characteristics of CpG islands in HBV QS. METHODS: Forty patients diagnosed as acute hepatitis B (AHB, n = 10), immune-tolerant HBV carriers (IT, n = 9), chronic hepatitis B (CHB, n = 11), or acute on chronic liver failure (ACLF, n = 10), were enrolled in this case-control study. A total of 599 clones were isolated, and full-length HBV genomes were sequenced. RESULTS: CpG island II (CGII) in AHB group was shorter in length and its QS heterogeneity was lower than that in the chronic infection group. Among the chronic infection subgroups, CGII and CpG island III (CGIII) in IT group were longer and their heterogeneity was lower compared to CHB and ACLF groups. Length of CGII correlated with HBV DNA levels positively while the complexity and diversity of CGII correlated with HBV DNA levels negatively. Moreover, CGII and CGIII were shorter in genotype B than those in genotype C, while QS complexity and diversity of either CGII or CGIII had no significant difference between genotype B and C. CONCLUSIONS: Overall, our results suggest that the distribution, length and QS heterogeneity of CpG islands in full-length HBV genome differ across clinical phases of infection, of which the mechanism warrants further study.

Quantitative hepatitis B surface antigen combined with hepatitis B e antigen as sustained virological response predictors during extended therapy with Peginterferon alfa-2a for hepatitis B e antigen-positive chronic hepatitis B.

BACKGROUND: The best strategy for chronic hepatitis B patients with poor response to 48 weeks of Peginterferon-based therapy has been controversial and the predictive value of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B virus (HBV) DNA levels for determining the sustained virological response (SVR) of these patients is uncertain. OBJECTIVES: To optimize management of these patients and evaluate the use of these serobiomarkers to predict SVR. STUDY DESIGN: Eighty-one patients with an unsatisfactory response after 48 weeks of Peginterferon-based therapy were treated with extended Peginterferon therapy with or without nucleo(s) tide analogues (NAs), for a total of 96 weeks of Peginterferon treatment. HBsAg, HBeAg and HBV DNA levels were measured serially during the treatment and follow-up. RESULTS AND CONCLUSIONS: Twenty-six of 81 patients (32.1%) attained SVR during the 72-week follow-up. The SVR rate was not statistically different between groups receiving 1-year prolongation of Peginterferon with or without NAs. The serum HBsAg cut-off of 1800IU/mL at week 48 had area under curve (AUC) of 0.727, and the serum HBsAg cut-off of 1500IU/mL, combined with HBeAg loss at week 72, had AUC of 0.753 to predict SVR during the follow-up. In conclusion, extended treatment with Peginterferon with or without NAs for patients with unsatisfactory response after 48 weeks of Peginterferon-based therapy is a promising strategy to achieve SVR, and quantitative serum HBsAg at week 48 and HBsAg level combined with HBeAg loss at week 72 of therapy can predict SVR to prolongation therapy with Peginterferon.

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection.

Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.

Inherent lipid metabolic dysfunction in glycogen storage disease IIIa.

We studied two patients from a nonconsanguineous family with life-long abnormal liver function, hepatomegaly and abnormal fatty acid profiles. Abnormal liver function, hypoglycemia and muscle weakness are observed in various genetic diseases, including medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and glycogen storage diseases. The proband showed increased free fatty acids, mainly C8 and C10, resembling fatty acid oxidation disorder. However, no mutation was found in ACADM and ACADL gene. Sequencing of theamylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase (AGL) gene showed that both patients were compound heterozygotes for c.118C > T (p.Gln40X) and c.753_756 del CAGA (p.Asp251Glufsx29), whereas their parents were each heterozygous for one of these mutations. The AGL protein was undetectable in EBV-B cells from the two patients. Transcriptome analysis demonstrated a significant different pattern of gene expression in both of patients' cells, including genes involving in the PPAR signaling pathway, fatty acid biosynthesis, lipid synthesis and visceral fat deposition and metabolic syndrome. This unique gene expression pattern is probably due to the absence of AGL, which potentially accounts for the observed clinical phenotypes of hyperlipidemia and hepatocyte steatosis in glycogen storage disease type IIIa.

Effects of metformin on FOXM1 expression and on the biological behavior of acute leukemia cell lines.

Forkhead box M1 (FOXM1) is a typical proliferation­associated transcription factor, which is overexpressed in many types of human cancer. We investigated the expression level of FOXM1 in patients with untreated acute leukemia (AL) and explored the correlation between expression levels and AL type. The relationship between the expression of the genes FOXM1 and mammalian target of rapamycin (mTOR) was determined after treatment of ML-2 cells with thiostrepton. The apoptosis, proliferation and cell-cycle progression of ML-2 lines were examined after treatment with metformin. We found that FOXM1 is expressed in the majority of AL patients and that its expression level was associated with the AL type. Thiostrepton is a specific inhibitor of FOXM1, and by inhibiting the FOXM1 expression via thiostrepton, we observed downregulatiion of mTOR; a significant correlation between FOXM1 and mTOR levels was observed. Thus, metformin may be involved in the downregulation of FOXM1. In addition, our study demonstrated that metformin promotes the apoptosis of ML-2 cells, induces cell-cycle arrest at the G0/G1 and G2/M phases, and inhibits proliferation. The potential role of FOXM1 in tumorigenesis renders it an attractive target for anticancer therapy, and metformin may represent a new agent for the treatment of leukemia.

Survey and analysis of the current state of residency training in medical-school-affiliated hospitals in China.

BACKGROUND: Since the global standards for postgraduate medical education (PGME) were published in January 2003, they have gained worldwide attention. The current state of residency training programs in medical-school-affiliated hospitals throughout China was assessed in this study. METHODS: Based on the internationally recognized global standards for PGME, residents undergoing residency training at that time and the relevant residency training instructors and management personnel from 15 medical-school-affiliated hospitals throughout China were recruited and surveyed regarding the current state of residency training programs. A total of 938 questionnaire surveys were distributed between June 30, 2006 and July 30, 2006; of 892 surveys collected, 841 were valid. RESULTS: For six items, the total proportions of "basically meets standards" and "completely meets standards" were <70% for the basic standards. These items were identified in the fields of "training settings and educational resources", "evaluation of training process", and "trainees". In all fields other than "continuous updates", the average scores of the western regions were significantly lower than those of the eastern regions for both the basic and target standards. Specifically, the average scores for the basic standards on as many as 25 of the 38 items in the nine fields were significantly lower in the western regions. There were significant differences in the basic standards scores on 13 of the 38 items among trainees, instructors, and managers. CONCLUSIONS: The residency training programs have achieved satisfactory outcomes in the hospitals affiliated with various medical schools in China. However, overall, the programs remain inadequate in certain areas. For the governments, organizations, and institutions responsible for PGME, such global standards for PGME are a very useful self-assessment tool and can help identify problems, promote reform, and ultimately standardize PGME.

Over-expression of FoxM1 is associated with adverse prognosis and FLT3-ITD in acute myeloid leukemia.

Forkhead box M1 (FoxM1) drives cell cycle progression and the prevention of growth arrest and is over-expressed in many human malignancies. However, the characteristics of FoxM1 in acute myeloid leukemia (AML) are not clearly understood. We investigated the expression level of FoxM1 and analyzed the correlation of FoxM1 expression with AML patient characteristics and prognoses. Changes in FoxM1 expression were detected after MV4-11 cells, which have an internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 gene (FLT3-ITD), and control THP1 cells (encoding wild-type FLT3) were treated with the FLT3 receptor tyrosine kinase inhibitor AC220 (quizartinib) or FLT3 ligand (FL). Finally, we determined the apoptosis rates after the addition of the FoxM1 inhibitor thiostrepton (TST) to AML cells with or without FLT3-ITD. The expression of FoxM1 in AML patients was correlated with the presence of FLT3-ITD, genetic groups, and possibly overall survival. Inhibition of FLT3-ITD by AC220 down-regulated FoxM1 expression in MV4-11 cells, and stimulation of FLT3 by FL up-regulated FoxM1 expression in MV4-11 and THP1 cells. TST induced the apoptosis of MV4-11 and THP1 cells in a dose-dependent manner. Thus, FoxM1 is a potential prognostic marker and a promising therapeutic target in AML.

Clinical implications of evolutionary patterns of homologous, full-length hepatitis B virus quasispecies in different hosts after perinatal infection.

Chronic hepatitis B virus (HBV) infection via perinatal transmission is common in the Asia-Pacific region, but related quasispecies (QS) characteristics are not yet defined. To investigate the homologous, full-length HBV QS after perinatal infection and their clinical implications, five pairs of mother-daughter patients with chronic HBV infection (one patient with liver cirrhosis, one with immune tolerance, and eight with chronic hepatitis) were included. Full-length HBV were amplified by PCR from serum samples before antiviral treatment and cloned; an average of 17 clones per sample were sequenced, and the QS complexities, diversities, and evolution patterns were analyzed. Full-length HBV sequence similarities within mother-daughter pairs were 91.3 to 98.3%. The QS complexities of full-length HBV were similar between mothers and daughters (median of 0.9734 compared to 0.9688, P>0.05), as were the diversities (median of 3.396×10(-3) compared to 4.617×10(-3) substitutions/site, P>0.05). However, the distribution patterns of QS complexities and diversities within specific genes were different, and QS genetic distances of the mothers were higher than those of daughters, both more evident in pairs with different antiviral responses and different immune phases or stages. The nucleotide substitution rate of full-length HBV was 14.388×10(-5) substitutions/site/year, whereas the preC/C gene rate was the highest. Mutations and indels were more common in clones from the mothers, which decreased the affinity of epitopes by 6- to 89-fold. The various genes from homologous HBV genomes evolved in different patterns despite numerically comparable full-length QS complexities and diversities. The different patterns may correlate with the immune stages of chronic HBV infection, which warrants further study.

A novel homozygous no-stop mutation in G6PC gene from a Chinese patient with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive genetic disorder resulting in hypoglycemia, hepatomegaly and growth retardation. It is caused by mutations in the G6PC gene encoding Glucose-6-phosphatase. To date, over 80 mutations have been identified in the G6PC gene. Here we reported a novel mutation found in a Chinese patient with abnormal transaminases, hypoglycemia, hepatomegaly and short stature. Direct sequencing of the coding region and splicing-sites in the G6PC gene revealed a novel no-stop mutation, p.*358Yext*43, leading to a 43 amino-acid extension of G6Pase. The expression level of mutant G6Pase transcripts was only 7.8% relative to wild-type transcripts. This mutation was not found in 120 chromosomes from 60 unrelated healthy control subjects using direct sequencing, and was further confirmed by digestion with Rsa I restriction endonuclease. In conclusion, we revealed a novel no-stop mutation in this study which expands the spectrum of mutations in the G6PC gene. The molecular genetic analysis was indispensable to the diagnosis of GSD-Ia for the patient.

N-glycosylation mutations within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape.

BACKGROUND & AIMS: HBV immune escape represents a challenge to prevention, diagnosis, and treatment of hepatitis B. Here, we analyzed the molecular and clinical characteristics of HBV immune escape mutants in a Chinese cohort of chronically infected patients. METHODS: Two hundred sixteen patients with HBsAg and anti-HBs were studied, with one hundred eighty-two HBV carriers without anti-HBs as a control group. Recombinant HBsAg bearing the most frequent N-glycosylation mutations were expressed in CHO and HuH7 cells. After confirming N-glycosylation at the most frequent sites (129 and 131), together with inserted mutations, functional analysis were performed to study antigenicity and secretion capacity. RESULTS: One hundred twenty-three patients had the wild-type HBs gene sequence, 93 patients (43%) had mutants in the major hydrophilic region (MHR), and 47 of the 93 patients had additional N-glycosylation mutations, which were transmitted horizontally to at least 2 patients, one of whom was efficiently vaccinated. The frequency of N-glycosylation mutation in the case group was much higher than that of the control group (47/216 vs. 1/182). Compared with wild-type HBsAg, HBsAg mutants reacted weakly with anti-HBs using a chemiluminescent microparticle enzyme immunoassay. Native gel analysis of secreted virion in supernatants of transfected HuH7 cells indicated that mutants had better virion enveloping and secretion capacity than wild-type HBV. CONCLUSIONS: Our results suggest that specific HBsAg MHR N-glycosylation mutations are implicated in HBV immune escape in a high endemic area.

[Functional analysis of hepatitis B virus immune escape mutants with insertion mutations in the surface antigen].

OBJECTIVE: To evaluate the influence of insertion mutations occurring in the hydrophobic region, between amino acids 114 and 115, of the hepatitis B surface antigen (HBsAg) on viral antigenicity and replication. METHODS: Hepatitis B virus (HBV) DNA was obtained from patients with HBsAg-positive chronic hepatitis B (CHB) infection and subjected to sequence analysis and comparison to GenBank reference sequences for HBV genotype B (AB073826) and genotype C (AF286594). Insertion mutations detected in the HBsAg region were used to make recombinant expression plasmids via site-directed mutagenesis. After transfecting the recombinant HBsAg into Huh7 cells, the mutants' effects on viral antigenicity and replication were evaluated by chemiluminescence microparticle immunoassay (CMIA) and Southern blot hybridization, respectively. The viral antigenicity of each mutant was predicted by bioinformatic analysis, using the Jameson-Wolf method to predict the antigenic index, the Hopp-Woods method to predict hydrophilicity, the Emini method to predict the probability of a region lying of the protein's surface, and the Karplus-Schulz method to predict the flexibility of the protein backbone. RESULTS: Two CHB patients harbored HBV with insertion mutations in HBsAg: one with two (NT) and one with three (NTT) inserted amino acids between 114 and 115. The NTT recombinant HBsAg mutant showed no impact on viral replication and reacted weakly with anti-HBs in CMIA (P = 0.02). The antigen indices for the insertion of NTT were 1.00, -0.16, and 0.18, and insertion of the three amino acids affected the index values of five proximal amino acid sites (with an average increase of 0.13). The hydrophilic indices for the insertion of NTT were 0.2, -0.4, and -0.4, with no significant effect on the proximal amino acids. The insertion of the three amino acids changed both the surface probability (range: -0.55 to 2.97; affecting eight proximal amino acids) and the flexibility (range: -0.01 to 1.1; affecting five proximal amino acids). CONCLUSION: The NTT three amino acids insertion in the HBsAg open reading frame, between 114 and 115 of the wild-type sequence, detected in a CHB patient may play a role in HBV immune escape without influencing the viral replicative capacity.