A functional variant in the transcriptional regulatory region of gene LOC344967 cosegregates with disease phenotype in familial nasopharyngeal carcinoma.

Nasopharyngeal carcinoma is a common malignancy in Southeast Asian countries, and genetic background is a well-known component of the complexity underlying its tumorigenic process. We have mapped a nasopharyngeal carcinoma susceptibility locus to chromosome 4p15.1-q12 in a previous linkage study on nasopharyngeal carcinoma pedigrees. In this study provided in this communication, we screened all the genes in this region, with a focus on exons, promoters, and the exon-intron boundary to identify nasopharyngeal carcinoma-associated mutations or functional variants. Importantly, we found a novel gene (LOC344967) with a single nucleotide polymorphism -32G/A in the promoter region. This gene is a member of the acyl CoA thioesterase family that plays an important role in fatty acid metabolism and is involved in the progression of various types of tumors. The -32A variant was found cosegregated with the disease phenotype in the nasopharyngeal carcinoma pedigrees that we previously used for the linkage study. Moreover, this -32A variant creates an activator protein (AP-1)-binding site in the transcriptional regulatory region of LOC344967, which significantly enhanced the binding of AP-1 to the promoter region and the transcription activity of the promoter in vivo. Furthermore, the expression of LOC344967 was significantly up-regulated at both mRNA and protein levels in nasopharyngeal carcinoma cells sharing the -32G/A genotype compared with nasopharyngeal carcinoma cells with the -32G/G genotype. Collectively, these results provide evidence that the -32A variant is a functional sequence change and may be related to nasopharyngeal carcinoma susceptibility in the families studied.

Adenoviral expression of a truncated S1 subunit of SARS-CoV spike protein results in specific humoral immune responses against SARS-CoV in rats.

The causative agent of severe acute respiratory syndrome (SARS) has been identified as SARS-associated coronavirus (SARS-CoV), but the prophylactic treatment of SARS-CoV is still under investigation. We constructed a recombinant adenovirus containing a truncated N-terminal fragment of the SARS-CoV Spike (S) gene (from--45 to 1469, designated Ad-S(N)), which encoded a truncated S protein (490 amino-acid residues, a part of 672 amino-acid S1 subunit), and investigated whether this construct could induce effective immunity against SARS-CoV in Wistar rats. Rats were immunized either subcutaneously or intranasally with Ad-S(N) once a week for three consecutive weeks. Our results showed that all of the immunized animals generated humoral immunity against the SARS-CoV spike protein, and the sera of immunized rats showed strong capable of protecting from SARS-CoV infection in vitro. Histopathological examination did not find evident side effects in the immunized animals. These results indicate that an adenoviral-based vaccine carrying an N-terminal fragment of the Spike gene is able to elicit strong SARS-CoV-specific humoral immune responses in rats, and may be useful for the development of a protective vaccine against SARS-CoV infection.

Transcriptional gene expression profile of human nasopharynx.

Inflammation in the nasopharynx is very common worldwide and nasopharyngeal carcinoma (NPC) results in significant morbidity and mortality in southeast Asia and north Africa. To facilitate the understanding of pathogenesis of these diseases as well as normal nasopharynx biology, transcriptional gene expression profile of normal human nasopharynx, from undissected biopsies, was established in this study by generating a large amount of ESTs, followed by bioinformatics analysis. A total of 27,209 ESTs generated from human nasopharynx cDNA library were integrated into 8,420 non-redundant clusters, of which, 6,070 (72.09%) corresponded to known genes, 156 (1.85%) to known ESTs, and 2,194 (26.06%) to novel sequences, respectively. Functional classification revealed that transcripts constituting enzymes (2,284, 28.30%) and those participating in cell growth/maintenance (3,306, 46.33%) were the largest population expressed in the nasopharynx, followed by genes coding for binding proteins (2,135, 26.45%) and involved in cell communication process (1,832, 25.68%). In addition, through comparison of the nasopharynx gene expression profile with those of 7 other human tissues, 59 transcripts preferentially expressed and 22 transcripts unique in the nasopharynx were identified. Our study acquired an initial assessment of qualitative diversity of gene expression in the nasopharynx, providing the first step toward comprehensive characterization of the molecular features of the nasopharyngeal disorders.

Genetic polymorphisms of CYP2A13 and its relationship to nasopharyngeal carcinoma in the Cantonese population.

Nasopharyngeal carcinoma (NPC) is characterized by a high prevalence in Southern China, especially among Cantonese individuals of the Guangdong Province. Epidemiological studies have suggested that frequent exposure to high levels of nitrosamine from preserved foods such as salted fish could be a risk factor for NPC. Cytochrome P450 encompasses a family of enzymes that metabolize carcinogens and CYP2A13, a member of this family, is expressed predominantly in the respiratory tract with the highest levels in the nasal mucosa. In an effort to test whether a correlation exists between CYP2A13 genetic polymorphism and the risk of developing NPC, we sequenced all nine exons and the exon-intron junctions of the CYP2A13 gene in 45 NPC patients. We identified a total of 21 single nucleotide polymorphism (SNPs), including 7 novel SNPs. The most frequent functional variant allele was 74A-1757G-3375T-7233G with a haplotype frequency of 7.8% in the 45 NPC cases. In addition, a stop codon mutation was detected in one case. We then selected the 3 most frequent SNPs and one stop codon mutation to expand our study to a case-control analysis within the Cantonese population. A novel haplotype consisting 8 SNPs in introns, and four additional novel SNPs were identified; but no correlation between CYP2A13 genetic polymorphism and individual susceptibility to NPC was observed.

[Mutation and amplification of RIT1 gene in hepatocellular carcinoma].

OBJECTIVE: To explore the mutation and amplification of RIT1 gene and their correlation with carcinogenesis of hepatocellular carcinoma (HCC). METHODS: The polymerase chain reactioindirect sequencing method was used for detecting the mutations in the sequence of all 6 exons in the RIT1 gene of 50 HCC tissues and paratumor tissues. And the amplification of RIT1 gene was examined by fluorescence quantitative polymerase chain reaction method. RESULTS: A nucleotide 241 G --> C substitution in exon 5 of RIT1 gene was detected in one patient's HCC tissue, but not in paratumor tissue; this 241 G --> C substitution leads to Glu81Gln amino acid alteration in the conservative domain binding GTP. A nucleotide G --> C substitution in 5'-UTR (-21 bp from initial codon) was detected in all of the 50 HCC tissues and paratumor tissues, and 2- to 297-fold amplification of RIT1 gene was detected in 11 of 43 qualified cases, the amplification frequency being 25.6%. CONCLUSION: Gene amplification is one of the main activating ways of RIT1 gene in HCC, and its amplification might be correlated with HCC carcinogenesis, while point mutation might be not.

[Amplification of RIT1 in hepatocellular carcinoma and its clinical significance].

BACKGROUND & OBJECTIVE: Previous study has demonstrated that high frequent gain of 1q was detected in hepatocellular carcinoma (HCC), 1q21-22 was identified as the minimum overlapping amplified region and might contain the candidate oncogenes involved in HCC. RIT1 gene is located in 1q21.3 region and is a member of Ras subfamily. RIT1 protein is similar to Ras protein in molecular structure and functions. It was speculated that RIT1 gene might be a candidate oncogene in HCC. So, the amplification of RIT1 gene was examined in HCC and was linked with the clinical indicators in this study to explore the possible functions of RIT1 gene in HCC development and progression. METHODS: The fluorescence quantitative polymerase chain reaction(FQ-PCR) method was established successfully. The number of RIT1 gene DNA copies was examined in the tumor tissues and its paratumor tissues from 43 patients with HCC by PE ABI 7000 Sequence Detector. The ratio of the number of RIT1 gene DNA copies between the tumor tissue and its paratumor tissue represented the extent of amplification of RIT1 gene DNA. RESULTS: RIT1 gene DNA was amplified in 11 cases (25.6%)among 43 patients. The mean survival time (15 months) of the RIT1 gene-amplification group is significantly shorter than that (34 months) of the non-amplification group (P = 0.0009); furthermore, the pathological grade and the extent of liver cirrhosis were significantly different between the RIT1 gene-amplification group and the non-amplification group (P< 0.01). CONCLUSION: The amplification of RIT1 gene might be one of the activation ways in HCC and might play an important role in HCC development and progression.

Alterations of BLU, a candidate tumor suppressor gene on chromosome 3p21.3, in human nasopharyngeal carcinoma.

Nonrandom allelic loss on chromosome 3p is a common event in nasopharyngeal carcinoma (NPC) with the implication that certain tumor suppressor gene(s) in this region are involved in the pathogenesis of these tumors. The BLU gene, located at 3p21.3, has recently been identified as a candidate tumor suppressor gene due to the occurrence of missense mutations and loss of its expression in lung cancer. To investigate the involvement of BLU gene in NPC, we examined both genetic and epigenetic changes of BLU in NPC primary tumors and cell lines. No pathogenic mutations were detected in the entire coding region of this gene in 45 primary NPC tumors and 5 NPC cell lines. While BLU was expressed in 100% (15 of 15) of noncancerous nasopharyngeal epithelia, its transcripts were missing in all 5 NPC cell lines, and absent or reduced mRNA levels were observed in 78% (28 of 36) of the primary tumors. In the NPC cell lines, loss of BLU expression correlated with hypermethylation of the CpG island promoter sequence, and expression was restored after treatment with 5'-aza-2'-deoxycytidine. Methylation specific PCR analysis revealed that the BLU promoter was highly methylated in 74% (17 of 23) of primary tumors in which BLU was downregulated, whereas only 2 of 9 non-neoplastic nasopharyngeal epithelia exhibited hypermethylation in the BLU promoter region. The high incidence of BLU alterations suggests that it may be one of the critical tumor suppressor genes on chromosome 3p21.3 involved in the development of NPC.

[Mutation and expression of SEMA3B and SEMA3F gene in nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: Though the molecular etiology of nasopharyngeal carcinoma(NPC) is currently unknown, evidence from both loss of heterozygosity analysis and functional studies suggested that there are NPC-associated tumor suppressor genes(TSGs) residing in chromosome 3p21.3. Recently, two members of semaphorin family, SEMA3B and SEMA3F gene, located at 3p21.3, were characterized as TSGs. Studies showed that SEMA3B and SEMA3F are capable of suppressing the growth of tumor cells and inducing apoptosis. Loss of SEMA3B mRNA expression or aberrant SEMA3F cellular localization were found in lung cancers. In order to investigate the involvement of SEMA3B and SEMA3F in NPC, the authors examined both mutation and expression of these two genes in NPC. METHODS: The entire coding regions, the splice donor/acceptor sites, and partial regulatory regions of SEMA3B and SEMA3F gene were screened for mutations by PCR-sequencing in 21 primary NPC tumors and 2 NPC cell lines(CNE2 and SUNE1). The mRNA expression levels were determined by semi-quantitative RT-PCR analysis. RESULTS: No somatic mutation was found in either SEMA3B or SEMA3F gene. However, two missense polymorphisms including Thr415Ile and lle242Met were found in SEMA3B in NPC. For the Thr415Ile polymorphism, the Ile allele type which leads to SEMA3B function defects was predominant in NPC with the allele frequency of 64% (27/42). SEMA3B mRNA was expressed in all 6 non-neoplastic nasopharyngeal epithelia, but was absent or down-regulated in 76% (16/21) of primary NPC tumors. No significant difference of SEMA3B expression was observed between NPC and noncancerous controls. CONCLUSION: High frequency of SEMA3B expression alterations suggests that the inactivation of this gene was strongly associated with NPC. SEMA3B may be a tumor suppressor on 3p21.3 involved in NPC.

[Mutation analysis of KLF6 gene in human nasopharyngeal carcinomas].

BACKGROUND & OBJECTIVE: Nasopharyngeal carcinoma (NPC) is one of the most common cancers in South China and Southeast Asia. The etiological factor is believed to be the interaction between genetic susceptibility, EBV infection and environmental factors, involved in the multi-step process of carcinogenesis and development of NPC. However, the molecular pathology of NPC is unclear yet. Kruppel-like factor 6(KLF6) is a ubiquitously expressed nuclear transcription factor, which is deleted and/or mutated with high frequency in a subset of prostate cancer. This study was designed to investigate KLF6 mutation in NPC tumors and NPC cell lines. METHODS: Genomic DNAs from 19 NPC tumor biospies and 3 NPC cell lines were used in mutation detection of KLF6 coding region and splice sites by PCR-sequencing. 100 chromosomes from 50 random healthy individuals were used as control. RESULTS: In 3 of 19 NPC tissues, 3 different mutations (Glu75Val, Ser136Arg, Arg243 Lys) in KLF6 gene were found by PCR-sequencing. None of the 3 mutations were detected in the 50 random healthy individuals. CONCLUSION: KLF6 gene may be involved in carcinogenesis of sporadic NPC.