Martins C, Kedda MA, Kew MC, MRC/CANSA/University Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand Medical School, 7 York Road, Parkto wn 2193, Johannesburg, South Africa.
Supported in part by grants/bursaries from the University of the Witwatersrand, and the Foundation for Research Development, Pretoria, South Africa
Correspondence to: Professor M.C. Kew, Department of Medicine, Medical School, 7 York Road, Parktown, 2193, Johannesburg, South Africa.
Telephone: +27(0)11 488 3626, Fax. +27(0)11 643 4318
Email:014anna＠chiron.wits.ac.za
Received: 1999-08-10

Subject headings: carcinoma, hepatocellular; southern African b lacks; cumulative LOH; tumor suppressor genes; microsatellite genomic instabilit y; liver neoplasms

Martins C, Kedda MA, Kew MC. Characterization of six tumor suppressor genes and microsatellite instability in hepatocellular carcinoma in southern African blacks. World J Gastroentero, 1999;5(6):470-476

Abstract
AIM: To analyse cumulative loss of heterozygosity (LOH) of ch romosomal regions and tumor suppressor genes in hepatocellular carcinomas (HCC s) from 20 southern African blacks.

METHODS: p53, RB1, BRCA1, BRCA2, WT1 and E-cadherin- ge nes were analysed for LOH, and p53 gene was also analysed for the codon 249 mutation, in tumor and adjacent non-tumorous liver tissues using molecular tech niques and 10 polymorphic microsatellite markers.

RESULTS: p53 codon 249 mutation was found in 25% of the subj ects, as was expected, because many patients were from Mozambique, a country wit h high aflatoxin B₁ exposure. LOH was found at the RB1, BRCA2 and WT1 loci in 20%(4/20) of the HCCs, supporting a possible role of these genes in HC C. No LOH was evident in any of the remaining genes. Reports of mutations of p 53 and RB1 genes in combination, described in other populations, were not confirmed in this study. Change in microsatellite repeat number was noted at 9 /10 microsatellite loci in different HCCs, and changes at two or more loci w ere detected in 15%(3/20) of subjects.

CONCLUSION: We propose that microsatellite/genomic instability may play a role in the pathogenesis of a subset of HCCs in black Africans.

INTRODUCTION
The evolution of cancer is thought to occur from the stepwise accumulation of ge netic aberrations in the same cell. These include loss of function of tumor supp ressor genes, activation of proto-oncogenes, faulty DNA mismatch repair, and th e integration of viral DNA^［1，2］. Hepatocellular carcinoma (HCC) is a le ading cause of death in both Africa and the Far East, resulting in at least 310000 deaths worldwide each year^［3］. HCC is multifactorial in aet iology and its pathogenesis is complex. The major risk factors involved in the development of the tumor are chronic HBV and HCV infections, cirrhosis and aflatoxin B₁ (AFB) exposure^［4,5］.
      Heavy dietary AFB intake is thought to cause a guanine (G) to thymine (T) transversion at the third base of codon 249 of the p53 gene, and for this reason clustering of this point mutation occurs in HCCs from Africa and China^［6-8］. In addition, other mutations in, or deletions of, the p53 gene (on chromosome 17p13.1) are found with relatively high frequency in human HCCs in other countries. The functional loss of this tumor suppressor gene, as well as its abnormal expression, have been proposed to play a significant role in HCC development^［9］ or at least in the development of a subset of HCCs. The majority of p53 alterations reported to date have loss of one allele accompanied by mutations of the second allele^［10］. Abnormalities of the p53 gene, such as gene mutation, deletion, or the nuclear accumulation of mutant p53 protein have also been found to correlate with increased allelic loss at the Breast Cancer Susceptibility Gene 1 (BRCA1) locus (17q21). This gene is thought to encode a transcription factor which acts as a tumor suppressor^［11］. LOH of the BRCA1 gene in HCC was reported in a Korean study^［12］. The Breast Cancer Susceptibility Gene 2 (BRCA2) (13q12-13) product is thought to be a tumor suppressor^［13］ involved in cellular proliferation and differentiation^［14］, and may be involved in the development of HCC^［15］. LOH at the BRCA2 locus has been reported in HCC^［15,16］ and it has been suggested that mutations of the BRCA2 gene may be involved in hepatocarcinogenesis^［15］. The retinoblastoma (RB1) gene (13q14.2) product (pRB) functions as a cell cycle regulator^［17］, and its absence leads to unrestricted cell growth. Although there is no definite evidence that mutations of the RB gene are involved in HCC, LOH of the RB1 gene has been documented in human HCCs^［18,12］. LOH of WT1 and 11p13 have been reported in human HCCs^［12,16］. WT1 appears to be involved in proliferation, differentiation and apoptosis^［19,20］. The product of the E-cadherin (Uvomorulin) gene (16q221) is the primary adhesion molecule in epithelium^［21］. Loss of function of E-cadherin may lead to decreased cell-cell adhesion^［22］, cellular phenotypic changes, and the development of invasive properties^［23］. In HCC, multicentric development and the formation of intrahepatic metastases is common^［24］. LOH on chromosome 16q has been previously reported to be important in the initiation or progression of HCC^［25,26］.
      Polymerase chain reaction (PCR) amplification of microsatellites (sequences uniformly distributed throughout the human genome) provides a simple and effective method of rapidly detecting loss of heterozygosity/microsatellite instability (LOH/MI)^［27］. Microsatellite instability is defined as the loss or gain o f microsatellite repeats at 2 or more loci and is detected by the presence of extra bands or band shifts between tumor and non-tumorous tissue DNA.
      In this study, we examined the G-T transversion at codon 249 of the p53 gene, LOH of the p53, RB1, BRCA1, BRCA2, WT1 and E-cadherin genes, and micr osatellite instability at 10 loci flanking these genes, in HCC and adjacent non -tumorous liver, from 20 southern African blacks.

MATERIALS AND METHODS
Subjects
The subjects included 20 southern African black men, aged between 20 and 40 years. HCC tissue and matched non-tumorous liver were obtained at necropsy or durin g surgical resection. DNA was extracted from the tissues using a modified “salt ing-out” procedure^［28］.

HBV markers
The HBV status of the subjects was determined previously using commercially available kits to detect HBV markers in serum (Abbott Labs, Chicago, IL, USA).

LOH and microsatellite instability
Microsatellite instability (MI) and loss of heterozygosity (LOH) studies were carried out by PCR and gel electrophoresis using polymorphic repeat markers (Table 1).
      PCR products of the polymorphic loci p53, D17S846 and RB1.20 were resolved on 4% composite agarose gels, while radioactively labeled PCR products of the remaining loci were resolved on polyacrylamide gels, and viewed by autoradiography. Band mobility shifts between tumor and matched non-tumorous liver DNA wer e scored as a change in allele repeat number. LOH was characterized by the disappearance of one band or a considerable (＞80%) decrease in band intensity in heterozygotes, whilst microsatellite instability was determined by expansion and/or contraction of microsatellite sequences.

PCR for LOH and MI
A standard PCR protocol (primers, Table 1) was followed for the p53, WT1, D1 3S137, RB(1.20), D13S120, D13S127, D17S855, and D17S846 loci. Each PCR reaction consisted, at final volume, of 100ng DNA, 1U Taq DNA polymerase (Prom ega, Madison, USA), 1×buffer, 1mM each dATP, dTTP, dGTP, 0.1mM d CTP, 0.025μCi α³² P dCTP, and 50pmol of each pr imer; in a total volume of 50μL, amplification for 30 cycles of denaturation at 94℃ for 30s, annealing 55℃ for 30s, extension at 72℃ for 1min, and a final cycle of 72℃ for 10 minutes.
      The PCR reaction for the D16S301 and D16S260 loci (primers, Table 1) consisted, at final volume, of 100ng DNA, 1U Taq- DNA Polymerase, 1×buffer, 0.1 % gelatin, 1mM each dGTP, dATP, dTTP, 0.1mM dCTP, and 0.025μCi α ³²P dCTP, 50pmol of each primer; in a total volume of 25μL, amplification for 25 cycles of denaturation at 94℃ for 1min, annealing at 55℃ for 2min, extension at 72℃ for 2.5min, and a final cycle of 72℃ for 10 minutes.

p53 codon 249 mutation
The p53 codon 249 mutation was detected by PCR-RFLP using primer sequences F3 and R3 (Table 1), and confirmed by sequence analysis. The PCR reaction consis ted of, at final volume^［6］, 100ng DNA, 2.5U of Taq-DNA polymerase (Promega), 1×buffer, 1mM MgCl₂, 0.8mM each of dCTP, dATP, dGTP, dTTP, and 50pmol of each primer; in a total volume of 50μL, amplification for 30 cycles of denaturation at 94℃ for 15s, annealing at 56℃ for 15s, and extension at 72℃ for 30s. The 110bp PCR product was sized on ethidium bromide stained agarose gels against a 100bp DNA ladder (Promega). A G to T transversion at the third base of codon 249 was detected by the presence or absence of a HaeⅢ restriction site^［6］.All samples shown by digestion to have the codon 249 mutation were sequenced in both directions both upstream and downstream in separate reactions, to confirm the presence of the mutation.

Table 1 PCR primers

Note: WT1: Wilm′s tumor gene; RB1: Retinoblastoma gene; BRCA1: Brea st cancer susceptibility gene 1; BRCA2: Breast cancer susceptibility gene 2; bp: base pairs.

Sequencing
All sequencing was carried out using the Sequenase PCR Product Sequencing Kit (United States Biochemical Corp., Cleveland, Ohio), according to the manufacture r′s instructions.

RESULTS
HBV status
Seven patients were currently infected with HBV (5 of these were HBsAg-positive ; HBeAg-negative; the HBeAg status of the remaining 2 was unknown), and 6 were previously infected (anti-HBc and anti-HBs-positive). The HBV status of the remaining patients was not known (Table 2).

LOH/MI analyses
LOH was noted for the WT1 (1/13 subjects), RB (1.20) (1/10 subjects), D13S120 (1/20 subjects) and D13S127 (2/14 subjects) loci (Table 2).
      The D13S137 and D13S127 loci flank the RB1 gene, while the RB (1.20) repeat sequence is within intron 20 of the same gene. LOH at the D13S127 locus suggests loss of at least a portion of the RB1 gene as shown in 2/14 informative subjects. LOH at RB (1.20) indicated loss of the RB1 gene in a further 1/ 10 informative subjects. The RB1 gene was thus lost in 3/18 informativ e subjects (Table 2). LOH at the D13S120 and D13S127 loci flanking the BRC A2- gene was shown in 2/20 informative subjects (Table 2). No LOH was found for any of the remaining loci (Table 2).
      Microsatellite/genomic instability (or a gain/loss of microsatellite repeats ) was found in 15% (3/20) of subjects.

p53 gene codon 249 analysis
The p53 codon 249 mutation was detected in 25% of the subjects using PCR-RFLP analysis, and confirmed by sequencing. The p53 codon 249 mutation was det ected in the tumor tissue of 3 subjects, in the non-tumorous liver of 1 subject , and in both the tumor and non-tumorous liver tissue of 1 subject (Table 2) .
      Sequencing gel electrophoresis of the p53 gene product revealed a gel artifact, in all subjects with wild-type chromosomes, previously described by Kapelne r et al(1994).
      All tumors were at an advanced stage. No attempt was made to correlate the presence of LOH or microsatellite instability with clinical or other features.

Table 2 LOH, SSCP and sequence analysis