Comparative Studies on Tumor Protein 53 Gene Polymorphism, Demographic Attributes and Associated Risk Factors Among Hepatocellular Carcinoma and Liver Cirrhosis Patients in Calabar, Nigeria

doi:10.21203/rs.3.rs-4487937/v1

Mutations in the TP53 gene had been attributed to the development of liver cancer. Hepatocellular carcinoma (HCC) and liver cirrhosis are liver diseases having high mortality rates in several populations. There is no information on the TP53 gene polymorphism among liver diseases patients in Calabar, Nigeria. This study investigated the genetic polymorphism of TP53 among HCC and liver cirrhosis in Calabar. This research was carried out in the University of Calabar Teaching Hospital, Calabar. Blood was collected from 35 clinically diagnosed hepatocellular carcinoma and 10 cirrhosis patients and 10 healthy controls. DNA was extracted from all blood samples. Polymerase Chain Reaction (PCR) was performed. The amplicon was digested using Hae III restriction enzyme and the genotypic and allelic frequencies were determined. In liver diseases patients, male was 68.9% (31), female (31.1%; 14), sex ratio (2.2: 0.5), mean age was 41.51 ± 2.13 years and odd ratio of 1.25. Marital status of patients were 33(73.3%), 10(22.2%), and 2(4.4%) for married, single, and widowed respectively. Ethnicity of patients were Yakurr, Efik, Boki, Ogoja, Annang, Ibibio and Igbo having 22.2%(10), 20%(9), 13.3%(6), 13.3%(6), 8.8% (4), 2.2%(1) and 2.2%(1) respectively. Approximately, 64.7% (30) of the chronic liver diseases were from the Central and Northern part of Cross River State. The risk factors were HCV infection, HBsAg⁺, alcoholism, smoking, consumption of groundnuts that may have been contaminated with aflatoxin and family history of the disease. PCR product yielded 254bp and digested PCR product showed homozygous TT mutation (27), heterozygous GT mutation (17) and homozygous GG wild type (1) in cases. The overall TP53 gene mutation frequency was 46.32% (44). The frequency of G allele, T allele, GG, GT and TT genotypes were 0.21, 0.79, 0.04, 0.33 and 0.62 respectively among cases, while GG (wild type) was only detected among controls in the study population. The genotypic and allelic frequencies conform to Hardy-Weinberg equilibrium meaning that the forces of evolution were not acting on the locus. There were significant differences in the genotypic proportions of the TP53 gene polymorphism among patients and controls. This study on the TP53 gene polymorphism will serve as baseline information on the molecular etiology of hepatocellular carcinoma and liver cirrhosis in Cross River State.

Mutations

Tumor protein gene

Hepatocellular carcinoma

Liver Cirrhosis

patients

Hepatocellular carcinoma (HCC) is a global health problem, with an increasing incidence worldwide [1]. This disease can be associated with risk factors identified by epidemiological studies which include chronic infection with hepatitis B and C viruses (HBV, HCV), non-alcoholic fatty liver disease (NAFLD), and ethanol consumption [2]. Studies by some oncologists and molecular biologists have reported on the contributions of TP53 gene mutation in the molecular etiology of Hepatocellular Carcinoma [3, 4 &5]. In HCC, TP53 mutational frequency has been reported to range between 22 and 33% [6, 7, 8, 9 &10]. The frequency of TP53 mutations varies within geographic regions, etiological factors and carcinogen exposure, with higher frequencies in regions where hepatitis B virus (HBV) infection is endemic [6, 11]. Liver cancer shows no symptoms in the early stage, but symptoms are evident in the advanced stage, which leads to third most lethal cancer type. Early diagnosis of HCC and treatment are likely to prolong the lifespan of liver cancer patients. There is need for the identification of high sensitivity and specificity markers for HCC [12].

Hepatocellular carcinoma is the deadly complication of liver diseases and usually develops from liver cirrhosis in most cases. In Asia and Africa, hepatitis B viral infection (HBV), with or without exposure to aflatoxin B1, is the most frequent etiology, whereas hepatitis C viral infection (HCV), severe alcohol abuse is most frequently related to HCC in Western countries [13, 14 & 15]. HCV infection is a global health problem, as well as been the major cause of end-stage liver disease such as liver cirrhosis (LC) and HCC. Most people that are infected with HCV become chronic hepatitis sufferers, while some of them may develop liver cirrhosis after years of follow up, which will be followed by HCC [16]. Aside from the known risk factors of HCC, which include dietary Aflatoxin B1 consumption, alcohol consumption; several genetic disorders such as hemochromatosis (an iron overload disease) that are associated with an increased risk of HCC [17]. Regardless of its etiology, cirrhosis alone is also an independent risk factor for HCC. Polymorphisms of TP53 gene have been associated with HCC, but their impacts on the progression of liver disease are receiving constant attention by scientists [18, 19]. Tumour suppressor gene (TP53) codes for a protein that regulates the cell cycle and hence functions as tumour suppressant [19]. It is located on chromosome 17p and encodes a 53-KDa nuclear phosphoprotein, which plays a major role in safeguarding the human genome [20]. When tumor suppressor genes do not function properly, cells can grow out of control, which will lead to cancer. According to [21] it is important for cells in multicellular organisms to suppress cancer. TP53 has been described as the guardian of the genome, which refers to its role in conserving stability by preventing genome mutation. The development of HCC is a multistage process and related to viral infection and chemical carcinogens. [22] reported that in the molecular aspects, receptors, deregulation of growth factors and their downstream signaling pathway components represents a central pro-tumorigenic principle in human hepatocarcinogenesis.

A specific TP53 gene mutation at codon 249 in exon 7 was associated with Aflatoxin B1 (AFB₁) inducing HCC in certain population with high AFB1 contamination. The wild-type TP53 gene exhibits a polymorphism at codon 72 in exon 4, with a single nucleotide change that causes a substitution of proline for arginine (Arg72Pro). The two polymorphism variants of TP53 are functionally distinct, and these differences may influence cancer risk. The polymorphism consists of a single base pair change of either arginine or proline which creates 3 distinct genotypes: Homozygous for Arginine (Arg/Arg), Homozygous for proline (Pro/Pro) and Heterozygous (Pro/Arg). TP53 codon 72 have been reported to be associated with cancer of the lung, esophagus, stomach, colorectal, breast, bladder and cervix [22]. TP53 is the most frequently mutated gene in human cancer. According to [23, 24], mutations in the TP53 gene may lead to three types of cellular outcome like formation of many mutant TP53 isoforms (can exert dominant-negative effects over wild type TP53 expressed from the remaining wildtype allele), Some mutant TP53 isoforms carry new pro-oncogenic activities referred to as ‘gain of function’ and lastly TP53 gene mutation can result in loss of wildtype activities (activation of TP53 target genes) and loss of tumor suppressor function as published [24]. [25], reported that mutations of TP53 gene or positive immunostaining for mutated TP53 protein can be used as a significant indicator of poor prognosis in patients with HCC.

Researches in different populations have revealed the association of candidate genes to the molecular etiology of hepatocellular carcinoma [26, 27, 28, 29]. Results in some studied populations highlighted the association of TP53 gene polymorphism in the pathogenesis of the disease, but few others observed no association in the populations investigated [30, 31, 32, 33]. There is paucity of documented molecular research on the genetic polymorphism of TP53 gene and the association with hepatocellular carcinoma in Nigeria. Thus, this research seeks to investigate the genetic polymorphism of TP53 gene and association with liver cirrhosis and hepatocellular carcinoma in Calabar, Nigeria.

2.1 The study location

The study was carried out at the Department of Internal Medicine, Gastroenterology and Hepatology Unit, University of Calabar Teaching Hospital, Calabar, Nigeria from September 2017 to September 2020.

2.2 Ethical approval

Ethical approval was obtained from the University of Calabar Teaching Hospital Ethical Review Committee before the commencement of this study.

2.3 Subject enrolment

A total of 55 participants from different ethnic groups were included in this study. The participants were grouped based on their ethnicity, age, marital status, sex. 35 were patients with hepatocellular carcinoma & 10 were patients with liver cirrhosis.

2.4 Inclusion criteria

All patients were individuals suffering from liver disease.

2.5 Exclusion criteria

The individuals with a history of any liver disease (familial or personal) were excluded from the study.

2.6 Instrument for data and sample collection

Informed consent was obtained from the patients, guardian or parent before data collection and sample collection was initiated. Detailed medical history, age, race or ethnicity, social history/risk factors like alcohol intake, multiple sex partners/unprotected sex, dietary habits and family history of liver cancer case(s) was collected from the subjects recruited for this study. A 2-3ml of whole blood was collected from both patients and controls, put into EDTA bottle and labeled properly. Blood samples was stored in deep freezer (-20⁰c) before onward transportation to the virology and molecular diagnostic units, International Institute for Tropical Agriculture (IITA), Ibadan, Oyo State, Nigeria for molecular analysis.

2.7 Genomic DNA extraction

DNA was extracted from all the blood samples. The DNA extraction, DNA quantification, Polymerase Chain Reaction (PCR), restriction fragment length polymorphism (RFLP) of the targeted genes under consideration (TP53, genes) were carried out at virology and molecular diagnostic units, International Institute for Tropical Agriculture (IITA), Ibadan, Oyo State, Nigeria. DNA extraction was carried out according to Dellaporta et al., (1983) protocol with some modifications as reported by [34]. About 150μl of whole blood was transfered into 1.5ml eppendorf tube placed on a rack and 350μl of extraction buffer was added to the tube. 40µl of 20% Sodium dodecyl sulphate (SDS) was added and the tubes were inverted 3-4 times before incubation using water bath at 65 ⁰C for 10 minutes. The tubes were brought out of the incubator and allow to cool at room temperature. Potassium acetate (160μl of the 5M solution) was added to the tubes. The tubes were inverted 2-3 times and centrifuged at 10,000 rcf for 10 minutes. Four hundred micro liters of supernatant was carefully transferred into new eppendorf tubes and 200μl of cold isopropanol was added. The tubes were inverted gently 5-6 times to precipitate DNA. The tubes were stored in the fridge at 4°C for 15-20 minutes.

The tubes were then centrifuged at 10,000 rcf for 10 minutes to sediment the DNA. The supernatant was decanted gently in order to ensure the pellets are not disturbed, 500μl of cold ethanol was added to the pellet to wash the DNA. It was centrifuged again at 10,000 rcf for 5-10 minutes. The ethanol was removed and the DNA was allowed to air-dry at room temperature until no trace of alcohol was observed in the tubes.

The DNA was re-suspended in 50μl of T.E buffer and stored in the freezer as stock solution. An aliquot of the DNA stock solution was run on agarose gel electrophoresis to check the quality of DNA. Each sample of genomic DNA (5μl) was mix with 2ul of loading dye and transfer into the wells of the gel. A voltage of 110V was applied for about 45 minutes. The samples were score as faint, +, 2+, 3+ depending on the intensity of the bands to indicate the amount of DNA extracted after mounting and viewing in the automated gel documentation system.

2.8 Primer for the TP53 gene

The forward primer used to amplify the TP53 gene on exon 7 is: 5¹-CTTGCCACAGGTCTCCCCAA-3¹ and reverse primer 5¹–AGGGGTCACCGGCAAGCAGA-3¹[36,37].

2.9 Constitution of PCR cocktail

The PCR cocktail was a total volume of 25µl reaction, which comprises of 18.3µl of distilled water (H20), 2.5µl of 10× buffer, 1µl of dNTPs, 0.5µl of forward primers, 0.5µl of reverse primers, 0.2µl of Taq polymerase and 2µl of genomic DNA. The cycling conditions for PCR reaction was denaturation for 5mins at 94⁰c, annealing was carried out at 55⁰c for 60seconds and final extension at 72⁰c for 10 mins The amplicons of 254 base-pair were visualized by staining the gel with ethidium bromide, after electrophoresis on 2% agarose gel [37].

2.10 Purification of the amplicons

Amplicons were purified at the Virology and Molecular Diagnostics Unit, International Institute for Tropical Agriculture (IITA), Ibadan, Oyo State, Nigeria using the alcohol-based method [38]. 21μl of amplicons were put into newly neatly PCR tubes and 52.5μl of 95% ethanol (21μl x 2.5μl) was added to precipitate the amplicons. It was then centrifuged at 12,000 rcf for 10 minutes and gently decanted; remaining the pellets attached to the wall of the tube. Approximately, 500μl of 70% alcohol were added and then centrifuged at 12,000 rcf for five minutes. It was then carefully decanted and the amplicon pellets were air dry at room temperature (25⁰C- 28⁰C) [38,39]. Finally, 22μl of distilled water was added to each PCR tube pellets of purified amplicons and stored in deep freezer for further usage. The suspended purified amplicons tubes were wrapped in foil and packed in ice containers for shipment to Inqaba Biotec Laboratory, South Africa.

2.11 Restriction fragment length polymorphism for TP53 gene

The TP53 amplicons were digested with restriction endonuclease named Hae III (Haemophilus aegyptius). The cocktail constituted 0.5µl of Hae III enzyme, 5µl of PCR product, 1µl 10× buffer, 3.5µl of sterile distilled water. Then the components were mixed by gentle pipetting and tapping. The tubes were incubated at 37⁰c for 18 hours (overnight) using a thermal cycler. The incubated samples were run on 1.5% agarose gel. Wild type (GG) was detected as having 92bp, 66bp, while heterozygous mutation was detected as having un-cleaved 158bp, 92bp, 66bp (GT) and homozygous mutation as un-cleaved 158bp (TT) [37,40].

2.12 Statistical analysis

The socio-demographic variables like age, ethnicity, marital status, sex etc, were computed and analyzed using Statistical Package for Social Sciences (SPSS) version 20.0. Demographic and clinical variables were compared using chi-square (X²) test. Simple percentage was used to determine frequency. Allele and genotype frequencies were calculated. Significance levels of comparison were determined at 0.05 level of significance.

3.1 Demographic characteristics of cases and controls

The polymorphism of TP53 gene associated with hepatocellular carcinoma and liver cirrhosis in University of Calabar Teaching Hospital (UCTH) was carried out using 35 HCC cases, 10 liver cirrhosis patients with or without hepatitis viral infections, and 50 controls. Hepatocellular carcinoma (HCC) and liver cirrhosis patients were 31 (68.9%) and 14 (31.1%) for males and females respectively as represented on Table 1. The mean age of cases was 41.51±2.13 years, the minimum and maximum age of patients was 18years and 75 years respectively. The male to female ratio was 2.2:0.5 and the odd ratio was 1.25. In the HCC cases, male and female patients were 25(55.5%) and 10(22.2%), 7(15.6%) and 3(6.7%) respectively. The healthy controls consisted of 32(6.4%) males and 18(36%) females with the mean age of 40.28±2.11 (Table 1). The marital status of patients were 33(73.3%), 10(22.2%) and 2(4.4%) for married subjects single and widowed respectively (Table 1). The ethnicity distribution of patients was Yakurr (22.2%; 10 patients), Boki (13.3%; 6 patients), Ogoja (13.3%; 6 patients), Efik (20%; 9 patients), Annang (8.8%; 4 patients), Ibibio (2.2%; 1 patient) and Igbo (2.2%; 1 patient). The majority of the cases were from Northern and Central Cross River with a total proportion of 64.7% (30 cases) as shown on Table 1.

TABLE 1 Socio-demographic characteristics of subjects recruited for the study

Variables	Distribution in cases (%) (n=45)	Controls (%) (n=10)	Odd ratio	x²cal	x²tab

Sex Male Female	31(68.9) 14(31.1)	6(60) 4(40)	1.25 (0.30- 2.20)	3.79	0.37

Age (years) Mean age	41.51±2.13	39.16±2.11

Type of liver disease HCC LC	35(77.8) 10(22.2)	0 0		4.72	1.91

Ethnicity Yakurr Boki Yala Ogoja Obubra Obudu Biase Akamkpa Efik Anang Ibibio Igbo	10(22.2) 6(13.3) 1(2.2) 6(13.3) 2(4.4) 2(4.4) 1(2.2) 2(4.4) 9(20.0) 4(8.8) 1(2.2) 1(2.2)	2(20) 1(10) 1(10) 1(10) 1(10) 0(0) 1(10) 0(0) 1(10) 0(0) 1(10) 1(10)		2.01	1.31

Educational status Tertiary Secondary Primary	30(66.7) 12(26.7) 3(6.7)	3(30) 5(50) 2 (20)		1.12	0.52

Marital Status Married Single Widowed	33(73.3) 10(22.2) 2(4.4)	3(30) 6(60) 1(10)		2.21	0.90

Associated risk factors identified for hepatocellular carcinoma and liver cirrhosis

The risk factors associated with hepatocellular carcinoma and liver cirrhosis patients are represented on Table 2. Hepatitis C virus infection was detected in 11 patients (24.4%), HBsAg⁺(hepatitis B virus surface antigen positive) was 27 (60%), co-infection was 11 cases (24.4%), alcoholism and chain smoking (42.2%), occasional alcohol consumption (73.3%), consumption of contaminated groundnut (95.6%) and family history of the disease was 22.2% (10 patients).

TABLE 2 The risks factors for HCC and liver cirrhosis in UCTH, Calabar

Variables	Total (%) n=45

HCV	11 (24.4)

HBsAg⁺	27 (60)

HCV/HBsAg⁺	11 (24.4)

HBeAg⁺	7 (15.6)

Alcoholism and smoking	19 (42.2)

Alcoholic consumption	41 (91)

Smoking	16 (35.6)

Cconsumptions of contaminated groundnuts	43 (95.6)

Family history	10 (22.2)

Use of sharp objects	32 (71.1)

Amplification of TP53 gene

The electropherogram of genomic extracted DNA from hepatocellular carcinoma patients, liver cirrhosis patients and healthy controls are displayed on Plate 1 shows DNA integrity test from lane 1-lane 16. Plate 2 displays the TP53 gene amplicons from patients and controls numbered from 1-16, while lane D is the DNA ladder of 100base pairs as amplified by the primers. Plate 3, displays the Hae III digest of TP53 gene amplicons. Plate 3 showed that Lane 1 is a 50 base pair DNA ladder, lane 2-11 are liver cancer patients having heterozygous GT mutation (un-cleaved 158 base pair, 92 base pair and 66 base pair fragments).

The TP53 gene polymorphism among hepatocellular carcinoma and liver cirrhosis patients.

The overall TP53 gene mutation frequency among liver disease patients (hepatocellular carcinoma and liver cirrhosis) based on the Hae III digest was 46.32% (44/95). The genotype and allelic frequency are represented on Table 3. The frequency of the G allele and T allele in patients were 0.21 and 0.79 respectively. The genotype frequency calculated for GG, GT and TT were 0.04, 0.33 and 0.62 respectively, while their observed percentage were 2.22%, 37.78% and 60% respectively in the patients. The genotypic frequencies observed in the patients and control population conformed to Hardy-Weinberg equilibrium. The genotypic frequency was significant at 5% (P<0.05). The genotypic and allelic frequency of the TP53 gene polymorphism in male liver cancer patients are shown on Table 4. The male G allele and T allele frequencies was 0.26 and 0.74 respectively, while male genotypic frequency was 0.07, 0.38 and 0.55 respectively.

TABLE 3 Genotype and allele frequency of the TP53 gene polymorphism in general population (patients and control)

Groups	Allele frequency			Genotype frequency
				GG		GT		TT		X² cal	X² tab
	N	G	T	Obs	Exp	Obs	Exp	Obs	Exp	1972.68	3.84
patients	45	0.21	0.79	1	0.04	17	0.33	27	0.62
%				2.22		37.78		60
controls	10	1	0	10	1	0	0	0	0
%				100		0		0

TABLE 4 Genotype and allele frequency of the TP53 gene polymorphism in male patients

Groups	Allele frequency			Genotype frequency
				GG		GT		TT		X² cal	X² tab
	N	G	T	Obs	Exp	Obs	Exp	Obs	Exp
patients	31	0.26	0.74	1	0.07	14	0.38	16	0.55	930.64	3.84
%				3.23		45.16		51.61
controls	6	1	0	6	1	0	0	0	0
%				100		0		0

TABLE 5 Genotype and allele frequency of the TP53 gene polymorphism in female patients

Groups	Allele frequency			Genotype frequency
				GG		GT		TT		X² cal	X² tab
	N	G	T	Obs	Exp	Obs	Exp	Obs	Exp
patients	14	0.11	0.89	0	0.01	3	0.19	11	0.79	171.72	3.84
%				0		21.43		78.57
controls	4	1	0	4	1	0	0	0	0
%				100		0		0

The genotypic frequencies observed in male patients and control population conformed to Hardy-Weinberg equilibrium. the female genotypic and allelic frequency of the TP53 gene polymorphism in patients are displayed on Table 5. The allelic frequency was 0.11 for G allele and 0.89 for T allele. The genotypic frequency was 0.01, 0.19 and 0.79 respectively and also conformed to Hardy-Weinberg equilibrium. The chi-square was statistically significant with respect to genotypic frequencies (P<0.05).

The prevalence of 46.32% (44) for TP53 gene polymorphism and sex ratio of 2.2:0.5 was observed in this study among HCC and liver cirrhosis patients in University of Calabar Teaching Hospital, Calabar, Cross River State showing that more males have these liver disease presentations. This finding of more males having chronic liver disease is similar to other results in Port Harcourt [39, 41], Ibadan [35, 42], Jos [41, 43], Egypt [21, 42], Kenya [43, 44], Iran [45, 46] and other populations [47, 48, 49]. In contrast, more females having this disease were documented in Port-Harcourt [48, 49] which are not in agreement with this current finding in Calabar. High prevalence of 48.8% was reported among the black race in United States of America (Sun et al., 2021), 94.3% in Jos [41, 50] and 92% in Ibadan [51, 52], which are not similar to 46.32% [44, 53] documented in this current study in Calabar.

Lower prevalence of 0.44% was documented in United States of America among ethnic group comprising the whites, black and other races having hepatocellular carcinoma [54, 55]. Also 6.1% and 7.1% in Abuja and Ibadan respectively [49, 56], 11.8% in Nigerians population [57], 3.6% in Port-Harcourt [39, 58] 0.2% for liver cancer in Akwa-Ibom State [59, 60], while 34.3% for gastrointestinal malignancy in Uyo [61, 62] and 2.5% in Calabar for hepatobillary disease [63]. The 46.32% prevalence documented in this study is similar to 44.3% documented in Enugu among HCC patients [64, 65], which is tandem with prevalence rate of this study. The odd ratio of 1.25 was recorded in this study in Calabar and similar odd ratio of 1.361 was documented in Egyptian population [66], but higher odd ratio of 2.0 reported in Ibadan [67] and 2.461 documented in Mansoura city in another Egyptian population [42, 68]. The reason for differences in the prevalence of the disease may be due to the sample size, differences in the patient’s exposure to risk factors and age/or gender characteristics of participants enrolled in the study. These reasons are similar to those reported in previous studies [68, 69, 70]. In this study in Calabar, married people made up the large proportion of subjects (73.3%) recruited for the study, this is similar to the findings from previously documented study in Calabar [70]. The high proportion of liver cirrhosis and hepatocellular carcinoma among married women may have resulted in high incidence of the disease presentation in men due to transfer of hepatitis virus infection to their spouses who have not received the viral vaccination. The diverse distribution of hepatocellular carcinoma and liver cirrhosis in different ethnic groups have been documented in several population [72.73]. These documented findings are similar to our present results in Calabar where the disease was distributed among the ethnic groups in Northern/central Cross River State, Efiks and Ibibios.

The risk factors associated with liver cirrhosis and hepatocellular carcinoma in the present study in Calabar were HBsAg, HCV, alcoholism, smoking, diets contaminated with aflatoxins and family history of the disease. Risk factors are documented in USA [70], African Populations [65, 66, 67], also in Zaria, Nigeria [63], Jos [67], Enugu [68, 69], Bayelsa state [70, 71], Rivers State [71], in Calabar and Lafia [61, 71]. Hepatitis B and hepatitis C co-infections was previously reported as a risk factors in Calabar by researchers [64, 70] among chronic liver disease subjects, while others [68] documented HIV patients and pregnant women, which are similar to the results of this present research in Calabar. The main risk factor for the disease presentation in this research were HBV, HCV, diets contaminated with AFB1 (probability), which are in tandem with other studies in Italy [66], Turkey [66], Egyptian populations [68]. The same risk factors mentioned above are also reported in Morocco [70], Kenyan Population [72] and Ibadan [73] all among liver cirrhosis and hepatocellular carcinoma patients. The Hepatitis B and Hepatitis C virus leads to the disease presentation by destroying the healthy liver cells and turning-on-TP53 gene mutations and these are confirmed from documented studies [37, 67, 74].

The genotype and allelic frequency of TP53 gene polymorphism among liver cirrhosis and HCC patients observed in this study were 0.21, 0.79, 0.04, 0.33 and 0.62 for G alleles, T alleles, GG genotype, GT genotype and TT genotype respectively and similar genotypes with varying allelic frequencies were documented in other populations like Iran [71], Italy [72]. Also similar genotypes with varying allelic frequencies were published in Egyptian populations [64, 69, 70]. In Morocco population, the same genotypes were reported [64, 72], Kenyan population [68, 71, 75] and in Ibadan, Nigeria [69, 75] among HCC and liver cirrhosis patients although in different proportions. The detection of Homozygous mutant TT and heterozygous mutant GT in 44 cases of liver cancer patients and not in healthy controls point to the fact that TP53 gene was associated with the molecular etiology of the disease in a sample population of Calabar. The genotype and allelic frequency conformed to Hardy-Weinberg equilibrium principle, meaning that the evolutionary forces like mutation, genetic drift, selection, were not acting on the loci under consideration. This finding is similar to the documented research in Egypt (Zaky et al., (2020) where the genotypic and allelic frequency significantly conformed to Hardy-Weinberg equilibrium principle. The GT (Heterozygous mutations) and TT (Homozygous mutations) are causal alleles in our studied populations and are associated in the molecular etiology of liver cirrhosis in Calabar, Nigeria, similar to other published results in Ibadan, Nigeria [65], Egyptians populations [53.64] and Kenya [59, 68]. The TP53 gene polymorphisms were detected in more males than female cases in our present study in Calabar, agreeing to other documented reports [68, 69]. The mutation of TP53 gene may lead to expression of a mutant TP53 protein that has lost wild type function (controlling of cell cycles) and may deploy a dominant negative regulation over the rest of wild type TP53 that suppress tumour through hetero-oligomerizing with wild type TP53. Therefore acting as a competitive inhibitor of wild type TP53 gene, supported by other published researches [54, 66, 69, 70].

Specifically, TP53 gene mutations variants detected in this study among HCC and liver cirrhosis patients were associated with the disease etiology. [70] reported no association of TP53 gene to the molecular etiology of cancers in Iran, disagreeing with this present finding in Calabar among HCC and liver cirrhosis patients. The high prevalence of mutant alleles of the TP53 gene polymorphism among HCC and liver cirrhosis patient in Calabar may be associated with consumption of food heavily contaminated by aflatoxin B1 and HBV/HCV co-infections endemic city in Northern and Central Cross River State. Groundnut has been reported to be the most heavily contaminated food item by aflatoxin [47,79]. Groundnut consumption and groundnut soup are some of the local delicacies of the natives of North/Central Cross River State, and these may be attributed to the high prevalence of HCC among the people of the region. Researchers have associated the amount of groundnut ingested daily with significant levels of serum aflatoxin and the presence of this genetic mutation [34.71], supporting our findings in Calabar for high prevalence of this TP53 gene polymorphism.

The mutant alleles of the TP53 gene polymorphism were also detected in liver cirrhosis patients in our present findings in Calabar. The alteration of the TP53 gene in mutant alleles may have altered critical cell-cycle arrest, apoptosis after DNA damage, thereby promoting the progression from chronic liver diseases to hepatocellular carcinoma. This have been supported by previous studies conducted [45, 72 73]. The mutations of TP53 gene individuals are also fast track by chronic hepatitis infections, Aflatoxin-B1, alcoholism/smoking, family history of cancer and other environmental factors. These factors are previously reported in different studies [71, 74, 75]. The case-control research have associated gene polymorphism to the etiology of liver cirrhosis and hepatocellular carcinoma in Calabar, Nigeria.

Tumor suppressor gene polymorphism was investigated among HCC and liver cirrhosis patients attending the University of Calabar Teaching Hospital. This study revealed the genetic polymorphism of TP53 gene among hepatocellular carcinoma and liver cirrhosis patients in Calabar. The prevalence of 46.32% (44) and odd ratio of 1.25 were observed among chronic liver patients. The Restriction fragment length polymorphism digest showed homozygous TT mutation (27 patients), heterozygous GT mutation (17 patients) and homozygous GG wild type in 0ne patient. The overall TP53 gene polymorphism was 46.32% (44). The frequency of G allele, T allele, GG, GT and TT genotypes were 0.21, 0.79, 0.04, 0.33 and 0.62 respectively among the study population. The genotypic and allelic frequencies conform to Hardy-Weinberg equilibrium. Hepatitis viral infections, aflatoxins, alcoholism, smoking were possible risk factors for the disease etiology in Calabar. This study has provided base line information on the frequency of TP53 gene among hepatocellular carcinoma and liver cirrhosis patients in Calabar.

The screening of patients for TP53 gene polymorphism will help in molecular diagnosis and management of these diseases, therefore reducing prevalence and mortality rate. The study revealed the frequency of TP53 gene mutations among hepatocellular carcinoma and liver cirrhosis patients in Calabar. The mutant forms of TP53 (homozygous TT mutation and heterozygous GT mutation) gene variants were detected among among hepatocellular carcinoma and liver cirrhosis patients, which may be involved in the presentations of these diseases in Calabar.

Data Availability Statement:

All data used in this study can be found in the text or its supplementary files. Additional data sets related to metabolites can be accessed by directly contacting the corresponding author.

Conflicts of Interest:

The authors declare no conflict of interest.

Funding Declarations:

The authors declare that no funding support was provided for this research.

Author Contribution

MEK and BBE conceived the topic idea. BBE and GMU wrote and drafted the manuscript. F.B.U search the literatures. BBE collated the data while MEK, BBE and GMU analyze the data. All authors read and approved the manuscript before submission.

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Plates 1 to 3 are available in the Supplementary Files section

No competing interests reported.

Comparative Studies on Tumor Protein 53 Gene Polymorphism, Demographic Attributes and Associated Risk Factors Among Hepatocellular Carcinoma and Liver Cirrhosis Patients in Calabar, Nigeria

Status:

Version 1

Abstract

Introduction

Materials And Methods

Results

Discussion

Conclusion

Declarations

References

Plates

Additional Declarations

Supplementary Files

Status:

Version 1