Hepatitis B is a viral disease characterized by potentially fatal inflammation of the liver. The hepatitis B virus (HBV) is an enveloped, DNA virus belonging to the Hepadnaviridae family [1]. The virus is responsible for its most often acute or even fulminant inflammations, which can either resolve or become chronic. It is a highly endemic disease that poses a major public health problem worldwide: a leading cause of mortality and morbidity [2][3]. According to the WHO 2020, approximately 296 million people suffer from chronic hepatitis B infection [4]; approximately 820,000 people die each year, primarily from liver cirrhosis or hepatocellular carcinoma (i.e., primary liver cancer) [5].

Chronic hepatitis B infection increases the risk of developing a liver tumor tenfold, thus constituting one of the main risk factors for the occurrence of HCC [6], [7]. In Africa, particularly in sub-Saharan Africa, the prevalence rate of HBV is between 8% and 18%, and it constitutes an area of high endemicity [8]. The prevalence of HBV in the general population in Togo varies between 13% and 16% according to the Global Alliance for Vaccines and Immunization [9]. Bitty-Anderson et al. found a prevalence of 9.9% in a study conducted in Lomé [10]. This prevalence was further estimated at 8.76% by Dossim et al. in Kara, a city located in the northern region of the country [11].

These findings underscore the high endemicity of the virus in the country [12] despite the implementation, since 2008, of a universal HBV vaccination as part of the Expanded Program on Immunization (EPI). These data highlight the urgent need to further investigate the mechanisms explaining the diversity of clinical outcomes of HBV in this region. In this context, the analysis of genetic factors appears particularly relevant for identifying prognostic markers and for better understanding the progression of the infection [13]. Studies have revealed that numerous human genes can confer protection or promote the progression of the infection to severe forms of the disease, specifically the p53, RB1 [14][15],GST[7], [16], ECM1 [17], and interleukins (IL6 and IL10) [18].

Indeed, during HBV infection, an infiltration of immune cells occurs, leading to hepatocyte damage [19][20] and cause extracellular matrix (ECM) deposition, which increases liver tissue damage [21]. The ECM1 protein (85 kDa glycoprotein) exists in two forms (interstitial and basal) and regulates extracellular matrix bonds. Its decrease exacerbates liver fibrosis [22][23]. Massive remodeling of the interstitial extracellular membrane generally promotes tumor progression, notably by inducing biochemical and biophysical changes affecting cell signaling, cell migration, tumor progression, and extracellular matrix stiffness [24]. The ECM1 gene controls tissue remodeling and liver homeostasis, inhibiting fibrogenesis by regulating stellate hepatocytes. These polymorphisms (e.g., rs3754217 and rs3834087) could alter their function, modulating the risk of liver complications in patients with hepatitis B [25]. The rs3834087 gene involves a three-base insertion/deletion (GAG) in ECM1. The rs3754217 gene results from the substitution of guanine (G) for thymine (T) on chromosome 1 [22]. In Togo, the correlation between the ECM1 gene polymorphisms (rs3834087 and rs3754217) and the progression of HBV infection remains unexplored. The present research activities aim to investigate the correlation between the ECM1 gene polymorphisms rs3834087 and rs3754217 and HBV progression.

This study, conducted in Togo, included participants comprising both healthy HBV carriers (controls) and patients with various clinical forms of the infection, including chronic hepatitis B (HBC), cirrhosis, and hepatocellular carcinoma (HCC). It allowed us not only to estimate the allelic and genotypic frequencies of the ECM1 gene polymorphisms rs3834087 and rs3754217 within this population, but also to assess their impact on the progression of HBV infection in Togo.

In the cohort, the mean age increased with the severity of clinical forms: 33.48 years for Chronic Hepatitis B (HBC), 45.54 years for cirrhotic patients, and 53.37 years for hepatocellular carcinoma (HCC). Individually, for example, the mean age of patients with cirrhosis was 45.54 ± 12.75 years. The present results are similar to previous studies conducted in Burkina Faso, which found mean ages of cirrhosis patients of 46.5 and 46.9 years in 2002 and 2020, respectively [27][28]. However, our results differ from those of a study conducted in America, where the mean age was approximately 60 years [29]. These figures show that our patients were younger, compared to those in developed countries. The age difference observed between our West African population (predominance of viral liver infections) and the American population (predominance of the risk factor alcohol) could be explained by the etiology of cirrhosis (the same applies to the average age of patients with HCC in our study population, compared to that of developed countries).

Analysis of the data for the two ECM1 gene polymorphisms in the general population of our study reveals highly relevant results. For rs3834087, we found that the GAG/GAG, GAG/-, and -/- genotypes had respective frequencies of 5.67%, 7.80%, and 86.52%. Furthermore, the frequency of the wild-type GAG allele was 9.57%, while that of the mutated allele was 90.42%. Our results highlight the predominance of the homozygous -/- and the mutated allele. These results differ from those found by Traore et al., who found the predominance of the heterozygous GAG/- and the wild-type GAG allele in Burkina Faso in 2024 [22]. These two studies also differ from that conducted by He et al. in China, where the wild-type GAG/GAG genotype and the GAG allele were predominant [30]. By examining the polymorphisms of the rs3754217 gene, we found that the GG, GT, and TT genotypes had frequencies of 31.91%, 58.86%, and 9.21%, respectively. Furthermore, the frequency of the wild-type G allele was 61.34%, while that of the mutated T allele was 38.65%. Our results, and those of Traore et al., highlight the predominance of the heterozygous GT genotype over the other genotypes, where they had found a GT predominance of 70.1% [22]. Our results corroborate those of He et al., who showed that the wild-type GG and heterozygous GT genotypes were more prevalent, with frequencies of approximately 45% - 50%, and that the mutated genotype represented about 6% [30]. Furthermore, the wild-type G allele was found to be predominant compared to the mutated allele. Such disparities highlight the influence of geographic and racial factors, climatic exposures, and occupational factors on genetic variability.

Examining our polymorphisms according to sex revealed that the T mutation of the rs3754217 gene appears to confer a non-significant risk of progression of viral hepatitis B to severe forms in men (OR = 1.02, CI = 0.55 to 1.90; p = 0.93) but seems to show protection in women (OR = 0.82, CI = 0.33 to 2.03; p = 0.68). This coincides with the analysis performed by Traore et al. The mutated GAG allele of the rs3834087 polymorphism appears to increase the risk of severe viral hepatitis B in both sexes, but more pronounced in men (OR = 1.67, CI = 0.65 to 4.27; p = 0.27). These two analyses suggest that men have a genetic predisposition to developing severe forms of hepatitis B infection. This could be explained by the role of sex hormones in chronic HBsAg carriage and the severity of the infection. Indeed, according to some studies, the virus’s genome contains a specific DNA sequence that interacts with the androgen receptor. This could explain why men infected with the virus are more likely to develop severe forms of the infection [22][25][31].

Analysis of our data according to clinical subgroups revealed some very intriguing trends. It emerged that a potentially significant protective effect against the progression of severe infection was associated with the GT genotype of rs3754217 in the subgroup of chronic HBV to severe forms, particularly liver cirrhosis, with an OR of 0.33 and a CI of 0.10 - 1.03, p = 0.04. Our results corroborate the findings of He et al. in China, who found that the GT genotype of rs3754217 was significantly associated with a reduced risk of progression in all patients with chronic HBV infection (OR = 0.75, 95% CI: 0.56 - 1.00) [30]. Our results differ from those found by Traore et al. in 2024 in Burkina Faso [22]. This protection against severe forms of the infection could be explained by the fact that a mutation in the ECM1 gene leads to an increase in the ECM1 protein. Indeed, ECM1 has been shown to be systematically downregulated during liver damage, and strategies for ECM1 re-expression in hepatocytes could be used to treat liver fibrosis [22][30]. Similarly, research conducted by Fan et al. on liver fibrosis in mouse models confirmed this hypothesis by showing that liver damage reduced ECM1 production levels during fibrogenic activity, and ECM1 re-expression prevented the progression of liver fibrosis [17].

Following analysis of the other subgroups, no significant association was found between the two polymorphisms regarding the progression or protection of chronic hepatitis B towards cirrhosis and HCC. Our results are similar to those found by He et al., but show some differences compared to the results of Traore et al., who found some associations between chronic carriers and controls with the mutated allele that conferred a low risk of progression to severity. Based on the associations observed within our study groups and the discrepancies with the results of Traore et al. and He et al., we can speculate on the influence of regional epigenetic differences or environmental determinants. Our study was conducted in Togo, and the observed differences in genotypic and allelic frequencies could be due to regional epigenetic differences or differences in environmental exposure. These could include dietary exposures, lifestyles, and environmental factors specific to Togo that may have influenced ECM1 gene expression. Given these findings, further research could be undertaken to explore the potential influence of these factors on ECM1 gene expression. This research could provide valuable insights into the underlying mechanisms contributing to the observed differences in genotypic and allelic frequencies and could help explain discrepancies with the results of other researchers. It could also contribute to the development of personalized interventions and treatments tailored to specific populations based on their unique genetic and environmental profiles.

The main limitation of our study lies in the sampling process. Due to a lack of information or awareness, almost all patients are admitted to the hepato-gastro-enterology department at very advanced stages of the disease (with death occurring within a maximum of 24 hours of admission). Secondly, the frequency of cases was relatively low (barely three patients per week, and even then, inclusion and exclusion criteria had to be applied). Finally, a lack of financial resources prevented us from considering alternative sample collection strategies, such as visiting all hospitals in Togo with hepato-gastro-enterology departments.

Our study was the first to examine the association between the ECM1 gene polymorphisms rs3834087 and rs3754217 and the occurrence of severe forms of HBV infection in the Togolese population. From a genetic standpoint, the frequencies of the GAG/GAG, GAG/-, and -/- genotypes of the ECM1 gene polymorphism rs3834087 were 5.67%, 7.80%, and 86.52%, respectively. Furthermore, the frequency of the wild-type GAG allele was 9.57%, while that of the mutated allele was 90.42%. The rs3754217 gene polymorphism exhibited GG, GT, and TT genotype frequencies of 31.91%, 58.86%, and 9.21%, respectively. Its wild-type allele G was 61.34%, while that of the mutated allele T was 38.65%. This study showed that a potential protective effect against the progression of severe infection might be associated with the GT genotype of rs3754217 in the chronic HBV subgroup towards severe forms, including liver cirrhosis. However, no association was observed between these two polymorphisms and the development of cirrhosis or its progression to HCC.

It is possible that an interaction between several factors could better explain the emergence of severe forms of HBV infection in Togo. Therefore, it is important to conduct further research to better understand and identify other factors that may contribute to the development of these severe forms. This would help identify new therapeutic targets, promote the development of new anti-hepatitis drugs and liver biomarkers, and guide clinicians in the management of severe cases, particularly in high-prevalence regions like Togo.

The data used and/or analyzed during this study are available from the corresponding author upon reasonable request.

This study has received approval from the Togolese Bioethics Committee for Health Research (Reference: Opinion No. 054/2023/CBRS of 22/11/2023).

Our study complies with the Declaration of Helsinki, adopted by the World Medical Association (WMA), on the ethical principles governing medical research involving human subjects. Participants gave their free and informed consent. Every effort was made to preserve not only the privacy, but also the confidentiality, dignity, and honor of the patients.

This study received financial support from the West African Economic and Monetary Union (UEMOA) through the UEMOA Excellence Scholarship.

The authors sincerely thank the Laboratory of Molecular Biology and Genetics (LABIOGENE), the National Blood Transfusion Center of Togo, and the Campus Lomé University Hospital for their invaluable contribution to the success of this study. We are grateful to all the patients who voluntarily agreed to participate in this study.

Concept and design of the study: LT, KSD,FWDandJS.

Sampling and laboratory analyses: LT,SFD,KD,LLM,NLK,FL,DGA,GO,KIMLT,KS,TL,KSD,FWDandJS.

Statistical analysis and interpretation of data: LT, DGA,GO,KIMLTandKS.

Manuscript written by: LT, DGA,GO,KIMLTandKS.

Critical review of the manuscript for intellectual content: FL,OD,NBM, KSD, FWDandJS.

Administrative, technical and material support: LT,KSD, FWDandJS.

The corresponding author states that the manuscript has been read and approved by all the named authors and that the order of authorship in the manuscript has been approved by all of us.