Analysis of loss of heterozygosity on chromosome 4q in hepatocellular carcinoma using high-throughput SNP array

Abstract

Abstract. To identify tumour suppressor genes (TSGs) associated with hepatocellular carcinoma (HCC) on chromosome 4q using a high-throughput single nucleotide polymorphism (SNP) array, we first scanned for loss of heterozygosity (LOH) of 40 SNPs on chromosome 4q and discovered 2 hot regions: 4q24-26 and 4q34.3-35. We then further scanned for LOH of 338 SNPs in genes around 4q34.3-35 and discovered 3 genes with the most frequent LOH: nei endonuclease VIIIlike 3 (NEIL3), interferon regulatory factor 2 (IRF2) and inhibitor of growth family member 2 (ING2). A review of the literature indicates only ING2 might be a TSG associated with HCC. IntroductionPrimary liver cancer, which consists predominantly of hepatocellular carcinoma (HCC), has risen to become the fifth most common malignancy worldwide and the third leading cause of cancer mortality (1). Previous studies have demonstrated that accumulation of the activation of oncogenes and/or inactivation of tumour suppressor genes (TSGs) is involved in the carcinogenesis of human cancer (2-4). However, the inactivation of TSGs was especially reported to play a more critical role in HCC pathogenesis (5). Recently, loss of heterozygosity (LOH) as an indirect procedure to identify TSGs has been widely used. Frequent allele deletions are observed on chromosomes 1p, 4q, 5q, 8p, 8q, 9p, 10q, 11p, 13q, 14q, 16q, and 17p in HCC (6-13).Whole-genome scans and chromosome analyses have been performed, and the chromosome 4q carries deletions in HCC more frequently than in other tumours (14)(15)(16)(17)(18)(19)(20)(21)(22). In early studies, we investigated LOH on 22 autosomes with 382 sets of microsatellite markers (MS) in 65 cases of HCC. The results showed that the frequency of LOH on 4q was 48.1% and that TSGs associated with HCC might be on 4q.In the present study, we first scanned chromosome 4q with a high-throughput SNP array to analyse the LOH and found the region with the most frequent LOH, then further scanned the SNPs around the region to search for the TSGs associated with HCC. Materials and methodsPatients and tissue specimens. All matched primary hepatocellular carcinoma tissue and adjacent cancer-free tissue specimens (n=69) were obtained from those patients who underwent surgical resection of their diseases in the Sun Yat-sen University Cancer Center between 2005 and 2007. The patients who received any preoperative treatment such as chemotherapy and radiotherapy were excluded. The 69 patients included 60 males and 9 females with a median age of 52 years (range, 21-75 years). The fresh tissues were immediately immersed in RNAlater (Ambion, Inc., USA) after surgical resection, stored at 4˚C overnight to allow thorough penetration of the tissues, then frozen at -80˚C until RNA and DNA extraction. Both cancer and corresponding adjacent cancer-free tissues not less than 2 cm away from the liver were sampled from cancer patients and verified by pathological examination.DNA extraction. Both cancer and corresponding cancer-free liver tissues were digested with proteinase K (1...