Khadija Rebbani

Genetic Variation in the Interleukin-28B Gene Is Associated with Spontaneous Clearance and Progression of Hepatitis C Virus in Moroccan Patients

Background Genetic variation in the IL28B gene has been strongly associated with treatment outcomes, spontaneous clearance and progression of the hepatitis C virus infection (HCV). The aim of the present study was to investigate the role of polymorphisms at this locus with progression and outcome of HCV infection in a Moroccan population. Methods We analyzed a cohort of 438 individuals among them 232 patients with persistent HCV infection, of whom 115 patients had mild chronic hepatitis and 117 had advanced liver disease (cirrhosis and hepatocellular carcinoma), 68 individuals who had naturally cleared HCV and 138 healthy subjects. The IL28B SNPs rs12979860 and rs8099917 were genotyped using a TaqMan 5′ allelic discrimination assay. Results The protective rs12979860-C and rs8099917-T alleles were more common in subjects with spontaneous clearance (77.9% vs 55.2%; p = 0.00001 and 95.6% vs 83.2%; p = 0.0025, respectively). Individuals with clearance were 4.69 (95% CI, 1.99–11.07) times more likely to have the C/C genotype for rs12979860 polymorphism (p = 0.0017) and 3.55 (95% CI, 0.19–66.89) times more likely to have the T/T genotype at rs8099917. Patients with advanced liver disease carried the rs12979860-T/T genotype more frequently than patients with mild chronic hepatitis C (OR = 1.89; 95% CI, 0.99–3.61; p = 0.0532) and this risk was even more pronounced when we compared them with healthy controls (OR = 4.27; 95% CI, 2.08–8.76; p = 0.0005). The rs8099917-G allele was also associated with advanced liver disease (OR = 2.34; 95% CI, 1.40–3.93; p = 0.0100). Conclusions In the Moroccan population, polymorphisms near the IL28B gene play a role both in spontaneous clearance and progression of HCV infection.

Polymorphic APOBEC3 modulates chronic hepatitis B in Moroccan population

The cytidine deaminase apolipoprotein B mRNA editing catalytic subunit‐3 (APOBEC3) induces G‐to‐A hypermutation in hepatitis B virus (HBV) genomes and operates as part of the innate antiviral immune system. We investigated the associations between the presence of APOBEC3 variants and HBV carriage in a case–control study in the Moroccan population. A polymorphic deletion affecting the APOBEC3B gene and the H186R variant of APOBEC3G were genotyped in 179 HBV chronic carriers and 216 healthy control subjects. In addition, to assess the overall impact of APOBEC3 deaminases on circulating HBV, we looked for hyperedited forms of the viral genome using the 3DPCR technique and analysed editing context. Data analysis showed that there was no significant difference in the frequencies of deleted APOBEC3B alleles (P = 0.261) or genotypes (P = 0.333) between patients with chronic hepatitis B and control subjects. By contrast, subjects bearing deleted genotype had a faster progression of liver disease than those with the insertion genotype (adjusted OR, 3.72; 95% CI, 0.38–36.12). The analysis of the APOBEC3G H186R polymorphism revealed that R/R genotype frequencies were not significantly different in HBV infected patients and in healthy subjects. 3DPCR was positive in 26 samples (14%) among 179. Amplified viral segments displayed monomorphic G>A transitions highly reminiscent of APOBEC3G activity. Most intriguingly, hemi/homozygous carriers of the APOBEC3B deletion had significantly lower virus loads than patients with the wild type (median 539 vs. 2213 IU/mL, P = 0.0023). This result suggests that genetic variations in APOBEC3 cytidine deaminases do not predispose to chronicity but may modulate the course of persistent HBV infection.

Somatic changes in primary liver cancer in Russia: A pilot study

Primary liver cancer (PLC) is a major public health concern worldwide, ranking third among the causes of death from cancer. Molecular pathogenesis of PLC is known to be especially sensitive to ethno-environmental variations that modulate mutation spectra in tumours. Despite a high prevalence of chronic liver diseases, the molecular epidemiology of PLC is still poorly known in Russia. To characterize the major genetic features of liver tumours in Russian populations, we conducted a pilot study on 34 PLC cases (28 hepatocellular, two cholangiocellular, and four mixed cases) among patients attending the Radiology and Roentgenology Hospital in Saint Petersburg. Point mutations were searched in 9 genes that are commonly altered in PLC, viz. TP53, CTNNB1, AXIN1, H/K/N-RAS, BRAF, PTEN, and NFE2L2. The genes TP53 and AXIN1 were mutated in 16% and 10% of the cases, respectively, whereas mutations of β-catenin were present in only 7% of samples, an unusual situation for Europe but common in East Asia. No R249S mutation indicative of exposure to aflatoxin B1 was detected in TP53. A single case harboured an NFE2L2 mutation. The loss of chromosome 18q was associated with early onset of tumours (mean 50 vs 62yrs, p=0.0252) and with the patients place of birth in Caucasus or Siberia. A lack of any risk factor was noted in 47% of the patients, whereas only 23% of the patients were infected either by hepatitis virus B or C. An extension of the present cohort as well as further molecular studies are now warranted in order to understand the processes governing liver carcinogenesis affecting Russian populations.

Common polymorphic effectors of immunity against hepatitis B and C modulate susceptibility to infection and spontaneous clearance in a Moroccan population.

Chronic diseases caused by hepatitis B and C viruses may evolve towards major complications as liver cirrhosis and cancer. Fortunately, only subsets among acutely infected individuals develop a persistent disease suggesting that genetic susceptibility may influence the establishment of chronicity. In the present study we aim to explore variants distribution in genes encoding for important immune response effectors in chronic hepatitis B and C. We intend to identify common features and to establish connections between single nucleotide polymorphisms (SNPs) predisposing to both chronic hepatitis and spontaneous clearance in a Moroccan population. Ten SNPs mapping on seven candidate genes (CD209, TGFβ-1, CCR5, CCL2, CXCL12, SUMO1 and UBC9) were studied in 544 Moroccan subjects grouped in chronically infected patients, spontaneously resolved individuals, liver disease progressors and healthy controls. Among significant associations found between virus infections and genetic variants, we report for the first time an association of rs4804803 (CD209) A and G variants with susceptibility to HBV infection and spontaneous clearance (p<0.001, OR=3.53, 95% CI 2.155; 5.908, and p<0.001, OR=7.75, 95% CI 4.646-13.114, respectively). Other important, albeit previously unknown, association was found between SUMO1 rs10185956T variant and spontaneous clearance of HCV infection (p=0.002, OR=2.71, 95% CI 1.332-5.869). Our observation, that deserves further confirmation with other SNPs and populations, underlines the involvement of selected immune polymorphisms, among which those in CD209, in the natural history of both chronic hepatitis B and C.

The allele 4 of neck region liver-lymph node-specific ICAM-3-grabbing integrin variant is associated with spontaneous clearance of hepatitis C virus and decrease of viral loads.

Abstract L-SIGN is a C-type lectin expressed on liver sinusoidal endothelial cells involved in the capture of hepatitis C virus and trans-infection of adjacent hepatocyte cells. The neck region of L-SIGN is highly polymorphic, with three to nine tandem repeats of 23 residues. This polymorphism is associated with a number of infectious diseases, but has not been explored in HCV. We therefore investigated the impact of L-SIGN neck region length variation on the outcome of HCV infection. We studied 322 subjects, 150 patients with persistent HCV infection, 63 individuals with spontaneous clearance and 109 healthy controls. In healthy subjects, we found a total of nine genotypes, with the 7/7 genotype being the most frequent (33%) followed by the 7/6 (22.9%) and the 7/5 (18.3%). The frequencies of the alleles were as follows: 7-LSIGN (56.4%), 6-LSIGN (20.2%), 5-L-SIGN (18.3%) and 4-L-SIGN (5%). The frequency of the 7/4 genotype was higher in spontaneous resolvers (14.3%) as compared with the persistent group (4%) (OR = 0.25, 95% CI = 0.07–0.82, p 0.022). In addition, we found that 4-L-SIGN was associated with spontaneous resolution of HCV infection (OR = 0.30, 95%CI, 0.12–0.74, p 0.005). Interestingly, patients with 4-L-SIGN had lower viral loads when compared with carriers of the 5 (p 0.001), 6 (p 0.021) and 7-alleles (p 0.048). The results indicate that neck region polymorphism of L-SIGN can influence the outcome of HCV infection and the four-tandem repeat is associated with clearance of HCV infection.

Influence of mutation of the HFE gene on the progression of chronic viral hepatitis B and C in Moroccan patients.

The implication of hemochromatosis (HFE) gene mutations in chronic viral hepatitis remains controversial. The aim of the present study was to measure the frequencies of the common HFE gene mutations in Moroccan subjects with chronic viral hepatitis B and C and to assess their influence on the progression of liver disease. H63D and C282Y mutations were screened by the polymerase chain reaction followed by restriction fragment length polymorphism analysis in 170 chronic hepatitis B patients, 168 chronic hepatitis C patients, and 200 healthy controls. A very small proportion of patients infected with hepatitis B virus (HBV) or hepatitis C virus (HCV; 1.8% and none, respectively) were heterozygous for the C282Y mutation, that is, rates not statistically different from those observed in healthy control (2%, P > 0.05). Similarly, the frequency of the H63D allele was not significantly different between HBV (13.8%) or HCV (14.3%) patients and controls (13.5%, P > 0.05). Multivariate analysis showed that carriers of the H63D mutation infected with HBV are at higher risk to progress towards an advanced liver disease when compared with patients infected with HBV with wild‐type (OR = 2.45, 95% CI = 1.07–5.58). In contrast, no association between HFE mutated HCV‐infected patients and an increased risk of disease progression was found (OR = 1.24, 95% CI = 0.61–2.50, P = 0.547). In conclusion, in Morocco the frequency of the HFE C282Y allele is very low and H63D mutation carriage occurs in almost 14% of the patients, a rate similar in chronic hepatitis patients and healthy controls. In the case of chronic hepatitis B, the carriage of the H63D variant represents a risk factor of evolution towards a more active disease. J. Med. Virol. 83:2096–2102, 2011.

TP53 R72P polymorphism modulates DNA methylation in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is characterized by widespread epidemiological and molecular heterogeneity. Previous work showed that in the western part of North Africa, a region of low incidence of HCC, mutations are scarce for this tumor type. As epigenetic changes are considered possible surrogates to mutations in human cancers, we decided, thus, to characterize DNA methylation in HCC from North-African patients.MethodsA set of 11 loci was investigated in a series of 45 tumor specimens using methylation-specific and combined-bisulfite restriction assay PCR. Results obtained on clinical samples were subsequently validated in liver cancer cell lines.ResultsDNA methylation at tumor suppressor loci is significantly higher in samples displaying chromosome instability. More importantly, DNA methylation was significantly higher in Arg/Arg when compared to Pro/Pro genotype carriers at codon 72 rs1042522 of TP53 (65% vs 20% methylated loci, p = 0.0006), a polymorphism already known to affect somatic mutation rate in human carcinomas. In vitro experiments in cell lines indicated that enzymes controlling DNA methylation were differentially regulated by codon 72 Arg or Pro isoforms of p53. Furthermore, the Arg72-carrying version of p53 was shown to re-methylate DNA more rapidly than the pro-harboring isoform. Finally, Pro-carrying cell lines were shown to be significantly more resistant to decitabine treatment (two-fold, p = 0.005).ConclusionsOur data suggest that Arg72Pro polymorphism in a WT p53 context may act as a primary driver of epigenetic changes in HCC. It suggests, in addition, that rs1042522 genotype may predict sensitivity to epigenetic-targeted therapy. This model of liver tumorigenesis that associates low penetrance genetic predisposition to epigenetic changes emerges from a region of low HCC incidence and it may, therefore, apply essentially to population living in similar areas. Surveys on populations submitted to highly mutagenic conditions as perinatally-acquired chronic hepatitis B or aflatoxin B1 exposure remained to be conducted to validate our observations as a general model.

Genetic variability of Hepatitis B virus in Morocco

Results The mean age of patients was 44 ± 12.2. Most of them were HBeAg negative (90%). The mean HBV DNA was 475104 ± 160591 UI/mL. Phylogenetic analysis identified 90% isolates in genotype D and 10% in genotype A. Most genotypes D isolates belonged to subgenotype D7 (80%) followed by subgenotype D1 (25%) and one isolate belonged to subgenotype D2. All genotype A strains belonged to subgenotype A2 and specified subtype adw2. In genotypes D strains, subtypes ayw2 (91.7%), ayw3 (3.3%) and ayw4 (3.3%) were identified. A significance prevalence of mutations in the Major Hydrophilic Region (MHR) of HBsAg was found with P120T/S the most frequent. In the core promoter region, the most frequent mutations are G1757A (48.9%), T1773C (42.8%), C1766GÂ /T (40.8%), T1753V (30.7%) and A1762T/G1764A (24.4%). In the precore region, the most common mutations are G1896A (55.1%) and G1899A (34.6%). Double mutation in the core promoter A1762T/G1764A was found more frequently in HCC patients than that in non HCC patients (66.6 % vs 16.2%; p<0.001). In addition, the prevalence of C1653T, T1753V, and G1862T mutations was significantly higher in HCC patients compared with non HCC. However, the prevalence of the G1896A precore mutation was not different between patients with HCC and HBV carriers without HCC (55.5% vs 55%; p>0.05).

Myxovirus resistance 1 gene polymorphisms and outcomes of viral hepatitis B and C infections in Moroccan patients.

Host genetic factors may influence the establishment of chronicity or spontaneous clearance in viral hepatitis B and C infections. More light was shed on the role played by interferon‐stimulated genes in the innate immunity. Myxovirus resistance 1 (MX1) is one of those key genes that have reported to inhibit several viruses. The present study aims to explore the possible association of −88G/T and −123C/A promoter variants of MX1 with susceptibility to chronic hepatitis B and C and/or with spontaneous clearance in a Moroccan population. The −88G/T and −123C/A SNPs were genotyped by PCR‐RFLP in 538 individuals stratified into HBV chronically infected patients (n = 120), HCV‐chronically infected patients (n = 115), HBV spontaneously resolved subjects (n = 114), HCV spontaneously resolved group (n = 52), and healthy controls (n = 137). A significant association of −123C allele with HBV spontaneous clearance has been found (P = 0.002, OR = 2.34; 95%CI [1.36–4]). In addition, a significant correlation between the MX1‐GC haplotype and HBV spontaneous clearance (P < 0.001) was found. No significant association of −88G/T and −123C/A polymorphisms with regard to HCV infection was observed in this study. Here, we show that for North African patients with chronic hepatitis, MX1 gene variation at position −123 may influence the outcome of HBV infection but not HCV infection. J. Med. Virol. 89:647–652, 2017.

P2-75 Haemochromatosis gene mutations in Moroccan patients with chronic viral hepatitis B and C

The implication of haemochromatosis (HFE) gene mutations in chronic viral hepatitis remains controversial. The aim of the present study was to assess the frequencies of the common haemochromatosis gene mutations in Moroccan subjects with chronic viral hepatitis B and C. H63D and C282Y mutations were screened by using polymerase chain reaction followed by restriction fragment length polymorphism analysis in 170 chronic hepatitis B patients, 168 chronic hepatitis C patients and 200 healthy controls. The distribution of allele frequency was then compared between different groups of patients. No subject homozygous for the C282Y mutation was found while 1.76% and 0% were heterozygous for this mutation in HBV and HCV patients, that is, rates not statistically different from those observed in healthy control (2%, 0.129< p<1.000). Similarly, the frequency of the H63D allele was not significantly different between HBV (13.8%) or HCV (14.3%) patients and controls (13.5%, 0.60< p<0.89). Although they do not reach the significance threshold, serum ferritin levels, indicative of body iron content, were higher in HBV or HCV patients than in control individuals with HFE mutations (110.7±43.61 and 149.67±43.52 ng/ml respectively vs 80.84±21.38 ng/ml, 0.229< p<0.607). In conclusion, in Morocco the frequency of the HFE C282Y allele is very low and H63D mutation carriage occurs in <14% of the subjects, a rate similar in chronic hepatitis patients and control individuals. Thus, we assume that the carriage of the common HFE mutations does not represent a risk factor for evolution towards chronic hepatitis B or C in the genetic and environmental context of North Africa.