Non-HFE mutations in haemochromatosis in China: combination of heterozygous mutations involving HJV signal peptide variants.

INTRODUCTION: Hereditary haemochromatosis (HH) caused by a homozygous p.C282Y mutation in haemochromatosis (HFE) gene has been well documented. However, less is known about the causative non-HFE mutation. We aimed to assess mutation patterns of haemochromatosis-related genes in Chinese patients with primary iron overload. METHODS: Patients were preanalysed for mutations in the classic HH-related genes: HFE, HJV, HAMP, TFR2 and SLC40A1. Whole exome sequencing was conducted for cases with variants in HJV signal peptide region. Representative variants were analysed for biological function. RESULTS: None of the cases analysed harboured the HFE p.C282Y; however, 21 of 22 primary iron-overload cases harboured at least one non-synonymous variant in the non-HFE genes. Specifically, p.E3D or p.Q6H variants in the HJV signal peptide region were identified in nine cases (40.9%). In two of three probands with the HJV p.E3D, exome sequencing identified accompanying variants in BMP/SMAD pathway genes, including TMPRSS6 p.T331M and BMP4 p.R269Q, and interestingly, SUGP2 p.R639Q was identified in all the three cases. Pedigree analysis showed a similar pattern of combination of heterozygous mutations in cases with HJV p.E3D or p.Q6H, with SUGP2 p.R639Q or HJV p.C321X being common mutation. In vitro siRNA interference of SUGP2 showed a novel role of downregulating the BMP/SMAD pathway. Site-directed mutagenesis of HJV p.Q6H/p.C321X in cell lines resulted in loss of membrane localisation of mutant HJV, and downregulation of p-SMAD1/5 and HAMP. CONCLUSION: Compound heterozygous mutations of HJV or combined heterozygous mutations of BMP/SMAD pathway genes, marked by HJV variants in the signal peptide region, may represent a novel aetiological factor for HH.

Stool vs. Serum Hepatitis B Virus DNA in Patients with Chronic Hepatitis B.

BACKGROUND Serum hepatitis B virus (HBV) DNA and hepatitis B e antigen (HBeAg) liver function in patients with chronic hepatitis B (CHB) are significantly associated. A comparison of clinical significance of fecal HBV DNA and serum HBV DNA has not yet been reported. MATERIAL AND METHODS Stool and serum samples were collected from 66 patients with CHB. Fecal HBV DNA, serum HBV DNA, and intestinal microbiota DNA were detected by real-time quantitative fluorescence polymerase chain reaction (PCR). Liver function and HBeAg were analyzed. RESULTS The stool and serum HBV DNA were positively correlated (r=0.57, P=0.001). Fecal HBV DNA was higher in the HBeAg-positive group than in the HBeAg-negative group (P=0.02). Fecal HBV DNA was negatively correlated with alkaline phosphatase (ALP) (r=-0.41, P=0.001) and TBIL (r=-0.29, P=0.02), and was positively correlated with Enterococcus (r=0.38, P=0.002). Serum HBV DNA was negatively correlated with alanine aminotransferase (ALT) (r=-0.30,P=0.02), aminotransferase (AST) (r=-0.26, P=0.049), and Lactobacillus (r=-0.31, P=0.01). CONCLUSIONS These observations suggest that fecal HBV DNA and serum HBV DNA in patients with CHB have different effects. Fecal HBV DNA might be associated with changes in Enterococcus concentrations, but serum HBV DNA is not.

Myeloid-derived suppressor cells suppress CD4⁺ and CD8⁺ T cell responses in autoimmune hepatitis.

Myeloid-derived suppressor cells (MDSCs) have been demonstrated with possess the ability to suppress T­cell responses. Therefore, MDSCs are an attractive candidate for immune intervention aimed at reconstituting self­tolerance in autoimmune conditions. The present study investigated the frequency and function of MDSCs in the peripheral blood of patients with autoimmune hepatitis (AIH), and examined its correlation with disease progression. Peripheral blood samples were obtained from 48 patients diagnosed with AIH and 24 healthy controls. The frequency of MDSCs was analyzed using flow cytometry, and its correlation with liver biochemical indicators was assessed. The sorted peripheral blood mononuclear cells and MDSCs, cocultivated with CD3 and CD28 monoclonal antibodies, were labeled with carboxylfluorescein succinimidyl ester and detected using flow cytometry for the proliferation of T cells. T cell apoptosis was detected using annexin V and 7­aminoactinomycin D. Interferon γ and nitric oxide were detected using ELISA, and inducible nitric oxide synthase (iNOS) was detected using immunohistochemical staining. The frequency of MDSCs in the patients with non­cirrhotic AIH was significantly higher, compared with the healthy controls and patients with cirrhotic AIH (P<0.05). However, no significantly differences were observed between the patients with cirrhotic AIH and the healthy controls (P>0.05). In addition, the frequency of MDSCs in the peripheral blood was positively correlated with alanine transaminase and aspartate transaminase in patients with AIH. The T cells of the incubation system were suppressed by the MDSCs, which was associated with the iNOS expressed on MDSCs. In patients with non­cirrhotic AIH, the peripheral frequency of MDSCs was increased through a feedback loop and autoimmune responses were inhibited. However, a variety of causes led to a decrease in the number of MDSCs in patients with cirrhotic AIH, therefore, accelerating the progression of liver injury and liver cirrhosis.