Contribution of Genetic Changes in Hereditary & Sporadic Forms of Head & Neck Cancer

Abstract

CYP1A1, GSTM1, GSTT1 and GSTP1 are major detoxifying enzymes related to head and neck cancer (HNC). Polymorphisms in these genes have frequently been reported in literature and are known to follow diverse pattern in relation to different populations. The current study was designed to screen these genes in HNC patients and controls at DNA, mRNA and protein levels. A total of 437 pathologically confirmed HNC patients and 507 normal healthy controls were recruited. The results revealed that the mean age of cancer patients included in the study was 48 +16.59 years. PCR-SSCP was used for genetic screening followed by sequencing. Novel substitution (tyrosine to serine) and frameshift mutation (insertion of T) were found in HNC patients in CYP1A1 gene. GSTM1 and GSTT1 null genotypes were found significantly higher in HNC patients compared to controls (OR 2.3, 95% CI 1.5-5.5 and OR 2.04, 95% CI 1.3-3.1 respectively). GSTM1 deletion mapping revealed an amplified region 98 bp upstream and 293 bp downstream the gene, deleting 6 Kbp segment containing the entire gene. Similarly in GSTT1 gene a region spanning 537 bp upstream and 333 bp downstream was deleted and the total size of this deletion was approximately 9 Kbp. A new technique for mapping of deletion has been introduced that opens new ideas for researchers. Novel mutations in GSTP1 gene in exonic region (substitution A2848T and G2849A) were found in 9.5% HNC patients whereas the controls did not show these mutations. In addition, two intronic deletions of C at nucleotide 1074 and 1466 were also found in patients. Therefore it was found that exonic as well as intronic variations may be involved in HNC risk. Reverse transcriptase PCR was used for mRNA expression variation screening in 4 selected genes. Expression analysis showed that CYP1A1 mRNA expression was markedly reduced in tissues of head and neck carcinoma compared to adjacent normal tissue (OR 4.5, CI 1.5-13.4). Partial loss of expression of GSTM1 and GSTT1 mRNA was also observed at a higher rate in HNC tissues compared to controls (OR 4.5, CI 1.5- 13.4 and OR 3.2, CI 1.1- 9.6 respectively). GSTM1 and GSTT1 expression was also down regulated that was directly correlated with stages of cancer. GSTP1 mRNA expression was significantly higher in cancerous tissue compared to control tissue (OR 4.2, CI 1.2- 15.3). GSTP1 over expression was also observed to be directly correlated with stages of cancer. It was found that 5 patients had variation in GSTP1 mRNA with a large product size than expected. Sequencing revealed insertion of intronic segment between 6th and 7th exon of GSTP1 gene. Germline screening was performed showing mobility shifts which suggested mutation at DNA level resulting in intronic portion retention. ELISA was performed to check the serum GSTs level and significant decrease was observed in head and neck cancer patients compared to controls (P<0.001). Immunohistochemistry was performed to check the protein expression of these genes. CYP1A1 was expressed in cancer tissues as well as controls; however mild expression was observed in patients compared to controls. Regarding the level of GSTT1 mRNA loss and mild expression was common among HNC tissues compared to controls. GSTP1 was overexpressed in most of the tissues compared with controls. From the current study, GSTs and CYP1A1 were found to be one of the factors responsible for systematic progression of HNC. Expression regulation of CYP1A1, GSTM1, GSTT1 and GSTP1 genes is an area which can be further explored. Establishing a marker of prognostic significance as well as its potential role in HNC can help in designing most promising gene therapy for patients