Association of UDP-Glucoronyl Transferase 1-A7 Polymorphism at Codon 208 with Hepatocellular Carcinoma and Liver Cirrhosis

: Introduction: Universally, hepatocellular carcinoma (HCC) is one of the foremost broad malignancies. UDP-glucuronosyltransferase (UGT) enzymes are found in a wide range of living things, including humans and microbes. Uridinediphosphoglucuronic acid's transfer (UDPGlcA) glucuronic acid group to a particular substrate's functional group is catalyzed by membrane-bound conjugating enzymes called UGTs. The several UDP-glucuronosyltransferases that are encoded by this complicated locus include this gene. Aim of the study: Determine whether hepatocellular cancer and liver cirrhosis are associated with the UDP-GLUCORONYL TRANSFERASE1-A7 polymorphism at codon 208. Subjects and Methods: Seventy patients were drawn from the Kasr El Ainy tropical unit at Cairo University. A full blood sample was used to obtain genomic DNA. Melting curve analysis in real-time PCR was used to identify the genetic variations in UGT1A7. finding 208 codon mutations was done by screening. Fluorescence resonance energy transfer (FRET) probes and primers flanking the polymorphisms intrigued in UGT1A7's exon 1 were created using the issued nucleotide sequence (GenBank U39570). Results: The findings demonstrated that HCC cases had higher levels of codon 208 (heterozygous (1,3) and homozygous (3,3) genotypes as compared to wild type (1, 1). On the other hand, homozygous and heterozygous liver cirrhosis patients have the same frequency as wild type. When compared to heterozygous genotype, the frequency of wild genotype is higher in the control group. Conclusion: Polymorphism at codon 208 in UGT1A7 has been linked to hepatocellular carcinoma (HCC) and is therefore regarded as a risk factor for both HCC and liver cirrhosis.


Introduction
Over 500,000 persons worldwide receive a diagnosis of hepatocellular carcinoma each year, with about 20,000 of those instances occurring in the United States [1].For men, liver cancer is fifth in incidence, whereas for women, it ranks seventh.The majority of the disease burden (85%) is carried by poor nations, with the greatest incidence rates recorded in areas where hepatitis B virus (HBV) infection is endemic: Sub-Saharan Africa and Southeast Asia [2].Hepatocellular carcinoma usually manifests after the age of forty and peaks around seventy years of age.Men are two to four times as likely as women to develop liver cancer.where the UGT1A7, UGT1A8, and UGT1A10 genes are expressed [4,5].
Numerous substances that are significant to toxicology and medicine are metabolized by UGT1A7.The variant UGT1A73 allele (Lys129Lys131Arg208) is produced by the three missense variants at codons 129/131 and 208.UGT1A7 has been linked to the glucose uptake of carcinogens, including heterocyclic amines produced from food and polycyclic aromatic hydrocarbons [5,6].The UGT1A73 allele encodes a protein with lower enzymatic activity against benzopyrene metabolites than the UGT1A71 allele [7].Research on disease susceptibility has revealed that the UGT1A73 allele is a risk gene for the occurrence of HCC, and oro-laryngeal cancer [8][9][10], colorectal cancer [10,11], and pancreatic cancer [12].
The aim of this study was to find the association between the UDP-GLUCORONYL TRANSFERASE1-A7 polymorphism at codon 208 with hepatocellular carcinoma and liver cirrhosis.

2.1.Subjects
Seventy patients recruited from the tropical unit of Kasr El Ainy, Cairo University were incorporated into this study and classified as follows: 

DNA extraction and Real time PCR
A sample of whole blood was used to extract genomic DNA.Using real-time PCR (polymerase chain reaction), the genetic polymorphisms in UGT1A7 were identified as follows: DNA was isolated from whole blood using the high pure PCR template preparation kit.In codon 208, the wild genotype (WW) was higher in the control group than in liver cirrhosis and HCC groups.The mutant heterozygous genotype (WR) was higher in the HCC group than both liver cirrhosis and control group, the homozygous (RR) was absent in the control group (Figure 1).which is matching our study [17].

Conclusion
The polymorphism at codon 208 in UGT1A7 is linked to hepatocellular carcinoma (HCC) and is therefore regarded as a risk factor for both liver cirrhosis and HCC.
Funding: This study is not funded.Conflicts of Interest: All authors declare they have no conflicts of interest.
, Anti HCV, AFP level, and Liver functions (AST, ALT, Albumin, Bilirubin total and direct).Subjects had their 2 mm of venous blood taken into sterile EDTA vacutainer tubes in an aseptic manner.Samples were kept at -20°C until extraction.
Windows, SPSS 15.0 (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 15 was utilized for data management and analysis.The mean + SD was the depiction of the statistical data.The means of the three groups were compared using the Post Hoc test and one-way analysis of variance (ANOVA), whereas the means of the two groups were compared using the Student's ttest.To express non-parametric quantitative data, the median (25th-75th quartiles) was utilized, and medians were compared using the Mann-Whitney and Kruskall-Wallis tests.Pearson's correlation coefficient (r) for parametric data and Spearman's correlation coefficient (r) for non-parametric data were utilized to find out the relation between quantitative variables.Frequency and percentage were used to represent qualitative data.Affiliation between qualitative data was done using the Chisquare test.Risk estimate was done by odds ratio.P-value was considered significant at 0.05.

Figure 1 :
Figure 1: Comparison between WW, RR, WR in HCC, liver cirrhosis and control.
Hepatocellular carcinoma (HCC)positions among the foremost prevalent cancers around the world, HCC is the fifth most predominant malignancy in men and the ninth most common in women, with over 500,000 new cases found each year.Trp208Arg are the sites at which the nine alleles (UGT1A7*1-UGT1A7*9) of the UGT1A7 gene differ[5,12].At codons 129/131 and 208, three nonsynonymous SNPs result in the variant allele UGT1A7*3, two of which are closely related (129 and 131).The population of *3/*3 (17%) usually carries this allele.the UGT1A7*4 allele only carries the Trp208Arg mutation.In comparison to the UGT1A7*3 allele in the general population, the additional genetic changes at codons 115 and 139 were shown to be less prevalent.The objective of this study was to find the association between the UDP-Glucoronyl Transferase1-A7 polymorphism at codon 208 with hepatocellular carcinoma and liver cirrhosis.So, this study was conducted on 70 patients; 30 of them were suffering from HCC proved by U/S or C.T and Alpha-fetoprotein (AFP); 20 patients suffering from liver cirrhosis proved by histopathological examination, clinical lab investigations and by imaging using U/S or C.T in addition to 20 individuals selected as a control group that proved by the absence of clinical, laboratory and imaging investigations.The genetic polymorphisms in UGT1A7 were determined by real-time PCR (polymerase chain reaction).Screening for the mutations in codons 208.Fluorescence resonance energy transfer (FRET) probes and primers surrounding the polymorphisms of interest in exon 1 of UGT1A7 were employed.The present study showed a significant association between liver cirrhosis, HCC and UGT1A7 polymorphism (codon 208).Tang et al (2007) found that the probability of developing liver cirrhosis was increased by the interaction between low-activity UGT1A7 genotypes and HBV (or HCV) infection [observed odds ratio (OR =54.59) more than the anticipated (OR =18.05).Advanced liver cirrhosis (Child-Pugh grades C and/or B) was also associated with a low/low genotype of UGT1A7 (OR =7.50, p =0.009) [15].According to Wang et al. (2004), With odds ratios of 2.73 (95% confidence interval, 1.40-5.35)and 1.

Table 2 :
Risk estimation of codon 208 genotypes in HCC and liver cirrhosis groups.

Table 3 :
Risk estimation of codon 208 genotypes in HCC and control groups.

Table 4 :
Haplotype frequency in HCC and liver cirrhosis versus control.