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Platelet activation and platelet-derived cytokines contribute to the vascular inflammation and increased thrombotic activity known to occur in patients with sickle cell anemia (SCA). CD40 ligand (CD40L), a platelet-associated pro-inflammatory molecule that promotes endothelial cell activation, is elevated in the circulation of SCA patients. We sought to evaluate the association of CD40L and inflammation with sickle-related clinical complications and laboratory variables in SCA patients. Soluble CD40L, thrombospondin (TSP)-1 and tumor necrosis factor (TNF)-α were determined in the platelet-poor plasma of healthy individuals and steady-state SCA patients by ELISA. Lifetime clinical complications were verified by detailed review of patients' medical records. We found that plasma CD40L was associated with acute chest syndrome (ACS), and that SCA patients with a lifetime history of ACS (ACS+) presented significantly higher plasma CD40L and TSP-1 than patients who had never experienced ACS (ACS-). In the ACS+ group, both platelet-derived proteins (CD40L and TSP-1) correlated with mean corpuscular volume, mean corpuscular hemoglobin and reticulocyte hemoglobin, while in the ACS- group, CD40L correlated with low red blood cell counts, hemoglobin, hematocrit and lactate dehydrogenase, and TSP-1 correlated with reticulocyte percentage and white blood cell count. As expected, CD40L and TSP-1 correlated with platelet counts in both groups. These data highlight the possible role of platelet activation in ACS and suggest that plasma sCD40L, together with TSP-1, may represent a potential marker of susceptibility to ACS in SCA.

Cancer risk can be influenced by the exposure to endogenous or environmental toxins. Polymorphic enzymes involved in the metabolic activation/detoxification of carcinogens may account for individual variations of risk. We studied the polymorphisms of five enzymes of the P450 superfamily, CYP1A1, CYP1A2, CYP2D6, CYP2E1 and CY3A4, as risk factors for liver disease progression and cancer in hepatitis C virus-infected patients. CYP genotyping was performed by polymerase chain reaction (PCR) restriction fragment length polymorphism or allele-specific PCR. Different stages of disease were considered, as follows: 90 asymptomatic carriers and 87 chronic hepatitis, 92 cirrhosis and 91 hepatocellular carcinoma (HCC) cases. Reference allele frequencies were obtained from 99 blood donors. Allele distributions among categories were compared using the chi(2) test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to express relative risks. Independent associations were modeled by correspondence analysis and logistic regression. Frequencies of the CYP1A1 highly inducible alleles, MspI m2 and Val, were increased in liver disease patients compared with carriers; no specific association with HCC was found. The high-activity CYP2E1 c2 allele was underrepresented among HCC patients with respect to other HCV categories, including cirrhosis. CYP2D6 poor metabolizer (PM) genotypes were significantly more frequent in healthy subjects (7.1%) and carriers (11.1%) than in hepatitis/cirrhosis (4.6%) and HCC (1.2%) patients. This was confirmed by multivariable analysis. PM genotypes protected against progressive disease as ORs reduced proportionally to stage. The age at diagnosis for HCC was anticipated in non-PM individuals. No differences were seen for CYP1A2 and CYP3A4 genes. Polymorphic variants of CYP genes may contribute to the progression of liver disease and HCC risk in HCV-infected subjects.

Factors influencing the progression of liver disease and the development of hepatocellular carcinoma (HCC) in chronic hepatitis C virus (HCV) infection are poorly understood. Inherited variations of drug-metabolizing enzyme (DME) activities may affect liver damage and cancer risk by modifying individual susceptibility to endogenous or exogenous toxic compounds. We investigated the association of liver disease severity with common alleles of microsomal epoxide hydrolase (mEH), an enzyme involved in the metabolism of highly reactive epoxide intermediates. Three polymorphisms (Tyr113His, His139Arg, and −613C/T) were analyzed by polymerase chain reaction (PCR) restriction fragment length polymorphisms (RFLPs) in 394 patients at different stages of disease, including 92 asymptomatic carriers, 109 patients with chronic hepatitis, 100 patients with cirrhosis, and 93 patients with HCC. Reference allele frequencies were obtained from 99 healthy blood donors. Allele distributions between categories were compared using the [Chi ]2 test; odds ratios (ORs) and 95% CI were calculated to express relative risks. Allele frequencies among 99 healthy controls were as follows: 15.1% for 113His/His, 4.0% for 139Arg/Arg, and 46.5% for −613C/T. mEH 113His/His homozygotes were overrepresented in advanced stages of disease, in particular among HCC patients (27.9%; P = .03; OR, 2.2; 95% CI, 1.0-4.6). Differences were more pronounced among men and between extreme patient categories. When mEH genotypes were combined to express a metabolic phenotype, very slow metabolizers were highly prevalent among cirrhotic and HCC patients (18% vs. 3.3% in carriers; P < .001). In conclusion, mEH gene polymorphisms were significantly associated with HCV-related liver disease severity and HCC risk. Men were at higher risk than women; this might be explained by hormonal regulation of gene expression or by differential exposure to environmental toxins. (HEPATOLOGY 2002;36:195-201.)

BACKGROUND: We have previously described a unique English family with beta-thalassemia trait which was not linked to the β-globin gene locus (Thein, Wood, Wickramasinghe, & Galvin, 1993). This suggested involvement of a trans-acting factor required for full activation of the β-globin gene locus. Such a factor is likely to be a modulator of disease severity in sickle cell disease and beta-thalassemia which could provide insights for novel therapeutic targets in the beta-globinopathies. RESULTS: We applied whole genome scan (WGS) to 2 affected and 2 unaffected subjects of the English family. The familial segregation suggested a dominant transmission mode; WGS identified 15 genes as potentially causative to the phenotype, with four genes located on chromosome 1, four on chromosome 3, three on chromosome 20, and one on chromosome 6, chromosome 8, chromosome 10 and chromosome 19. Sanger sequence analysis on 23 family members spanning three generations, including the 4 individuals that were subjected to WGS, revealed that the 15 variants were not artefacts of the WGS and that all variants were present in the 2 affected but not in the 2 unaffected individuals. Furthermore we found that 4 of the 15 variants were consistently and uniquely present in all 9 affected but absent in the unaffected family members. We performed association linkage analysis using the 15 markers in the whole family, and confirmed that the phenotype was closely linked to the 4 genes that were inherited as a block spanning the centromere on chromosome 1. We concluded that the region containing these 4 genes most likely harbours the mutation causing the phenotype. Among the 4 candidate genes, 2 were not expressed in erythroid cells, but the other 2 - one encoding an integral membrane protein (LRIG2) and the other one encoding a methyl transferase (ASH1L)- were expressed in erythroid cells. Functional studies for these two genes were performed on primary human erythroid progenitor cells (hEPCs) in culture. In following the kinetics of the 2 candidates during differentiation of hEPCs, we observed that the expression of ASH1L increased at later stages of differentiation, where LRIG2 displayed a less dramatic change of expression. Moreover, ASH1L has previously been found to occupy transcribed chromatin domains and methylate histone tails in vitro (Gregory et al., 2007; Miyazaki et al., 2013; Tanaka et al., 2011). In undifferentiated mouse embryonic stem cells there is no ASH1L recruitment to the β-globin gene locus but upon erythroid differentiation the protein is recruited to the transcribed portion of the gene (Gregory et al., 2007). This suggests an involvement of ASH1L in beta-globin activation in erythroid lineages. We used shRNA lentiviruses to generate knock-down (KD) of ASH1L and obtained over 65-75% KD of the gene. In hEPCs treated with the shRNA lentivirus, we observed a slight decrease in beta-globin expression compared to the control hEPCs. The α/β-globin and α/(β+γ) globin ratios were also affected by the gene knock-down. ChIP-qPCR was performed to assess the enrichment of the ASH1L protein at β-globin promoter region. The results show that enrichment of ASH1L at the β-globin promoter correlates with the β-globin expression in cells. CONCLUSIONS: These results suggest that ASH1L is responsible for the phenotype observed in the English family and act in differentiating hEPCs as a trans-acting factor for full beta-globin gene activation. Further ChIP analysis to assess the binding of the protein to the beta-globin locus during hEPCs differentiation and under KD condition will provide us with a better understanding of the influence of the methyl transferase on β-globin activation. The replication of the patient mutation in vitro using CRISPR technology will provide the model to study fully the impact of the mutation on the phenotype described in the original paper. These findings could provide new insights for therapeutic targets for beta-globinopathies. Disclosures No relevant conflicts of interest to declare.

Because hepatitis C virus (HCV) genotypes have raised considerable interest as variables that influence chronic hepatitis C progression, a case-control study was conducted to estimate their effects on patients with cirrhosis. Case patients (n = 46) had tested positive for anti-HCV antibody and HCV RNA and were residents of the study area who had cirrhosis recently diagnosed. Controls (n = 138) were drawn randomly from a residents' cohort from the same area. Demographic and other information were recorded. Presence of HCV infection, presence of HCV RNA, and HCV genotypes were assessed. Crude, stratified, and logistic regression analyses were performed. HCV genotype 2a/c occurred in 84 controls (60.9%) and 9 case patients (19.6%); HCV genotype 1b was found in 45 controls (32.6%) and 34 case patients (73.9%). HCV 1b genotype showed an independent effect on the risk of cirrhosis (odds ratio, 7.49; 95% confidence interval, 3.15--17.81). No significant effects related to other variables were observed. These results indicate that the genetic diversity of HCV phylogenetic variants may explain differences in biological behaviors.

In a cohort of subjects from Italy, anti-hepatitis C virus (HCV) and HCV RNA [HCV(+) subgroup] prevalences were 24.6 and 79.6%, respectively. HCV types 1b and 2a/c accounted for 95% of infections. Adjusted alanine aminotransferase levels were higher in males than in females and in RNA-positive subjects than in RNA-negative subjects regardless of HCV type. Genotype distribution was unrelated to demographic variables.

Abstract 5287 INTRODUCTION: Ferroportin (FPN) is the sole mammalian iron exporter protein known and it plays a critical role in iron metabolism. It is expressed in various types of cells including duodenal enterocytes, hepatocytes, erythroblasts cells, syncytiotrophoblasts and reticuloendothelial macrophages. Ferroportin is expressed in multiple alternative transcripts: with (FPN1A) or without (FPN1B) an iron-responsive element (IRE). The expression of one form rather than the other depends on cell type and iron availability. The expression of ferroportin in thalassemia intermedia (TI), characterized by iron overload, is not yet fully elucidated. AIM: To investigate the different expression profile of ferroportin isoforms during erythroid differentiation in control and TI cell cultures. METHODS: After informed consent, the CD34+ cells were obtained from peripheral blood of healthy volunteers and from patients with thalassemia intermedia by positive selection using anti-CD34-tagged magnetic beads and cultured for 14 days with a medium containing stem cell factor (SCF), interleukin 3 (IL-3) and erythropoietin to induce erythroid differentiation. The expression profiling of FPN1A and FPN1B was evaluated at baseline, day 7 and day 14 of culture by real-time PCR (2̂-dCt). RESULTS: In control cultures, FPN1A isoform was highly expressed at erythroid progenitors stage (day 0 of culture), decreased at early erythroblasts stage (day 7) and increased again at late erythroblasts stage (day 14). In TI cultures, the FPN1A isoform expression remained high even in early erythroblasts (Table 1). In control cultures the FPN1B isoform expression was very low at any stage of erythroid differentiation, whereas in TI cultures it was highly expressed at baseline and, althoug decreased during differentiation, remained always higher than control (Table 2). CONCLUSIONS: In thalassemic conditions the FPN1B is the major expressed ferroportin isoform, possibly contributing to iron overload. In control cultures, FPN1A was mainly expressed in undifferentiated erythroid progenitors and in mature erythroblasts, suggesting a functional role at these stages of erythroid differentiation. In TI cultures, the persistent expression of FPN1A at early erythroblasts stage was probably due to thalassemic erythropoiesis. These data suggest that in TI condition other signals, such as the erythropoiesis status, can override iron overload in regulating ferroportin expression. Disclosures: No relevant conflicts of interest to declare.