Background

Human hepatocellular carcinoma (HCC) is one of the leading cancer-related causes of death; it is a common malignancy in develo** countries with rising incidence as it has a high prevalence in Southeast Asia, China, and sub-Saharan Africa with low incidence in the USA and Europe [1]. The process of HCC carcinogenesis is complex and multistep. Several risk factors are believed to have hepatocarcinogenesis contribution, such as chronic hepatitis C and B virus (HCV, HBV) infection, cirrhosis, carcinogen exposure, and many single nucleotide polymorphisms (SNPs) [2,3,4,5]. The HCC progression and metastasis mechanisms are still not fully clarified. Moreover, the prognosis of HCC is still poor due to tumor cell frequent intrahepatic spread, invasiveness, and metastasis [6].

Heparanase, a mammalian endo-β-D-glycosidase, particularly cleaves the heparin glycosaminoglycans sulfate side chains, the most abundant basement membrane and extracellular matrix macromolecules [7]. Heparanase activity can influence several biological and pathological processes, including tissue repair, inflammation, tumor angiogenesis, invasion, and metastasis [8, 9]. Different studies have studied the clinical significance of heparanase in patients with HCC using immunohistochemistry, RT-PCR and qPCR, in situ hybridization, tissue microarrays (TMAs), and western blotting with upregulation in HCC [10,Data analysis

IBM SPSS computer software package version 20.0. (Armonk, NY: IBM Corp) was used to analyze the obtained data. Qualitative data analysis was calculated using percent and number. The distribution normality was verified by the Kolmogorov-Smirnov test. Quantitative data analysis was done by using mean, standard deviation, and range (minimum and maximum). The results significance was judged at the level of 0.5%. The P values, odds ratios (ORs), and 95% confidence intervals (95% CIs) were then determined. Further, the used tests were Fisher’s exact or Monte Carlo correction chi-square test, Mann-Whitney test, Student’s t test, and odds ratio (OR). P value < 0.5 is considered statistically significant.

Results

Characteristics of the study population

The selected attributes of the case-control subjects are mentioned in Table 1. No significant differences were found between patients with HCC and control in terms of sex and age. The values of AFP, TBIL, AST, and ALT increased significantly in HCC patients than in healthy individuals (P < 0.001). The hematological parameters show that the values of the hemoglobin (Hb), RBCs, WBCs, platelets, and prothrombin concentration were significantly decreased in HCC than in control groups (P < 0.001), while the creatinine and total bilirubin levels were not different significantly among the two groups (P > 0.05).

Table 1 Selected clinical and demographic characteristics of patients and controls
Full size table

Genotypic and allelic frequencies of HPSE SNPs in the study groups

The main goal was to gain the distribution of the HPSE polymorphisms within the Egyptian HCC patients. To obtain the knowledge concerning the HPSE SNP distribution among the Egyptian HCC patients, initially, the genomic extracted DNA from 30 control subjects were analyzed. The frequencies of rs12331678 genotypes within this control group were 56.7% CC, 43.3% CA, and 0.00% AA, while for the rs12503843, they were 73.3% CC, 26.7% CT, and 0.0% TT (Table 2 and Fig. 1). SNP rs12331678 and rs12503843 in the control group showed to be in Hardy-Weinberg equilibrium (χ2 tests, P ≥ 0.05), which gives the allowance to proceed with the HCC patient genotype distribution. Next, the distribution of HPSE polymorphisms within an Egyptian cohort of 70 HCC patients was done. SNP genoty** in the HCC subjects showed that the distributions for the rs12331678 genotypes were 67.1% CC, 31.4% CA, and 1.4% AA, while distributions for the rs12503843 CC, GT, and TT genotypes were 52.9%, 41.4%, and 5.7%, respectively (Fig. 1 and Table 2). The genotype frequencies corresponding to rs12503843 (CC, TT, CT) among the control and the HCC cohort (Fig. 1b) demonstrated to be non-statistical difference (P > 0.05), but regarding allelic distribution among the two groups, analysis showed to be a statistically significant difference with a higher prevalence of the unfavorable (T) allele within the HCC group (Table 2). The same analysis was performed for HPSE rs12331678; no statistically significant differences were observed between both cohorts either in genotypic or allelic distribution (Fig. 1a and Table 2). Furthermore, the SNPs stratification based on HCV carrier in HCC groups were analyzed, and the results revealed that there were no statistically significant differences between the HCV carrier and non-carrier individuals in the HCC group regarding the HPSE rs12331678 and rs12503843 (Table 3).

Table 2 Genotypic and allelic frequencies of HPSE SNP in HCC and control group
Full size table
Fig. 1
figure 1

The HPSE SNP genoty** from Egyptian patients with HCC and control individuals by agarose gel electrophoresis. a genoty** of HPSE rs12331678 by ARMS-PCR as the first 2 lanes are CC genotype and the lanes 3 and 4 are CA genotype. b genoty** of HPSE rs12503843 by RFLP-PCR; CC genotype product 237 bp, while CT genotype 237 and 261 bp and TT genotype 261 bp

Full size image
Table 3 Stratifications of rs12331678 and rs12503843 in correlation to HCV carrier status in HCC group (N = 70)
Full size table

To elucidate the role of HPSE rs12331678 and rs12503843 gene polymorphisms in the HCC patients’ clinicopathologic status, the association of clinical features and distribution of HPSE SNP polymorphisms in HCC subjects were evaluated, including Child-Pugh score, focal lesion size and number, HCV infection (anti-HCV), and the common HCC clinical-pathological features including AFP, ALT, and AST. No significant relation was observed between the HPSE rs12331678 and rs12503843 gene polymorphisms and all clinicopathologic status and markers (Tables 4 and 5).

Table 4 Association between rs12331678 and clinicopathological features
Full size table
Table 5 Association between rs12503843 and clinicopathological markers
Full size table

Discussion

The most common human genome sequence variation is the single nucleotide base substitution, commonly named as an SNP. SNP is a stable nucleotide variation in sequence with an occurrence of more than 1% in at least one population [29]. A single variant effect is probably insignificant, but combinations of different SNPs, either in the same gene or among distant genes, could corporately contribute to disorder occurrence. Studies on genetic associations have begun for studying the SNP effect on the disease outcome, as it changes the phenotypic expression of a recognized gene making the individual more susceptible to a specific disease [30].

Hepatocellular carcinoma (HCC) is among the most frequently occurring malignancies and a prominent cancer cause associated mortalities worldwide. The HCC etiology remains mostly elusive. Presently, the well-known HCC risk factors are chronic viral hepatitis, liver cirrhosis, aflatoxin exposure, alcohol consumption, and smoking [31, 32]. However, a few fractions of individuals with recognized risk factors finally develop HCC, implying that other environmental and genetic mediators may be participating in the development of HCC. In this manner and considering that there was not enough information on genotypes distribution of HPSE SNPs for Egyptian HCC patients, the evaluation of the frequency of HPSE rs12331678 and rs12503843 in a number of Egyptian HCC patients and control individuals was at high interest.

In the present study, 70 HCC patients and 30 control subjects were genotyped for two SNPs of HPSE gene (rs12331678 and rs12503843). Analysis of allele genotype frequencies of rs12331678 revealed that no significant difference was revealed among the HCC patients and control group regarding the frequencies of different genotypes. On the other hand, the unfavorable (T) allele of rs12503843 was found at a high frequency in the HCC group, given a higher prevalence of favorable rs12503843 (C) allele in healthy individuals when compared to HCC patients. This result suggests that rs12503843 may be significantly correlated with HCC susceptibility in Egyptian individuals.

The possible mechanisms which explained the association between the HCC risk and HPSE rs12503843 may include the functional role of this SNP to serve as a marker in tight linkage disequilibrium (LD) with other functional SNPs in the 3′UTR region of HPSE [33]. Ostrovsky et al. have stated that the rs4693602 SNP, which is present in the 3′UTR distal part of the HPSE gene, was correlated with multiple myeloma (MM) disease and may alter the expression of the HPSE gene. The intronic rs12503843polymorphism in tight LD together with rs4693602 might act as genetic markers, possibly because they are located in downstream of the HPSE 3′UTR region [23].

The associations between the HCC risk and these SNPs were assessed with HCV carrier status stratification. There was a non-significant interaction between rs12331678 and rs12503843 and HCV carrier status, indicating that this status did not modify the HCC susceptibility. Finally, the association between the SNPs and clinicopathological features was examined; however, we could not show statistically significant relevance, and this may be attributed to the small population size of our pilot study.

On the contrary to the findings by Ostrovsky et al. [23], no relationship was detected between the SNP rs12331678 and the occurrence of HCC (P = 0.553), but our finding comes in accordance with the results by Winter et al. [28]. A possible explanation is that different genetic mechanisms of the susceptibility of different diseases might be involved in population-specific variations. However, our negative findings could be attributed to genetic variation influence among ethnic groups, e.g., differences in the pattern of LD or allele frequencies of HPSE between populations. Our results come in agreement with recently published data by Yu et al. who stated that the HPSE rs12503843 (T) allele was more susceptible to HCC development in the Chinese population [33].

Conclusion

The current pilot study provides, for the first time, HPSE gene SNP characterization among Egyptian patients diagnosed with HCC and suggests the associations between the HPSE rs12503843 only with the HCC development. Therefore, results of our pilot study offer the rationale for further larger trials to elucidate clinical significance and importance of the HPSE gene polymorphisms in HCC pathogenesis. Furthermore, a direct connection between heparanase expression and HCC-associated variants or function should be investigated more intensely.