Ruqia Mehmood Baig

Genetic and expressional variations of APEX1 are associated with increased risk of head and neck cancer

The aetiology of head and neck cancer (HNC) has been shown to be associated with genetic and certain environmental factors that produce DNA damage. Base excision repair (BER) genes are responsible for repair of DNA damage caused by reactive oxygen species and other electrophiles and therefore are good candidate susceptibility genes for HNC. Apurinic/apyrimidinic endonuclease-1 (APEX1) proteins have important functions in the BER pathway. In this case-control study, all exons of the APEX1 gene and its exon/intron boundaries were amplified in 300 HNC cases and 300 matched healthy controls and then analysed by single-stranded conformational polymorphism. Amplified products showing altered mobility patterns were sequenced and analysed. To confirm our observations, we examined APEX1 expression at mRNA level on 50 head and neck squamous cell carcinoma (HNSCC) and 50 normal control samples by quantitative real-time polymerase chain reaction. At germ line level, three novel mutations (13T > G, Ser129Arg and Val131Gly) of APEX1 were observed. The homozygous and heterozygous genotypes of APEX1 13T > G, Ser129Arg and Val131Gly appear to be significantly involved in the development of HNC. In the case of expressional level, APEX1 mRNA expression was positively correlated with tumour size, clinical stage and positive lymph node metastasis. Statistical analysis showed a significantly higher APEX1 mRNA level in HNC tumour tissue than in control samples. Our study demonstrated that APEX1 mutations and deregulation of APEX1 are associated with increased risk of HNC in the Pakistani population.

Mutational spectrum of Gelsolin and its down regulation is associated with breast cancer.

Cytoskeletal rearrangement occurs in variety of cellular processes and involves a wide spectrum of proteins. Gelsolin super family proteins control actin organization by severing and capping filament ends and nucleating actin assembly. Gelsolin is the founding member of this family and plays important role in pathogenesis of human neoplasia. This study aimed to investigate the germline mutations and expressional profile of Gelsolin in human breast cancer tissues. For germ line screening PCR-SSCP technique was used while expression was analyzed through quantitative real time PCR. Different types of mutations were observed in Gelsolin coding regions on exons 4, 10, 11, 14 and 15. These mutations include 3 missense nonsynonymous substitution mutations, 2 deletions, 1 insertion and 1 synonymous substitution mutation. Gelsolin transcript level was found significantly lower in breast tumor tissues compared to control samples (p=0.03). Low level of Gelsolin was found in metastatic patients (p=0.002) and patients who died from breast cancer (P=0.03) compared to disease free patients at final follow up. This study shows that level of Gelsolin is down regulated in breast cancer tissues and is linked with metastasis development and death in patients. It is concluded that genetic changes in coding regions of Gelsolin can potentially contribute to genetic instability. These genetic variations and expressional correlation with patient survival may prove to be of significant importance.

Correlation between selected XRCC2, XRCC3 and RAD51 gene polymorphisms and primary breast cancer in women in Pakistan.

Genetic polymorphisms in homologous recombination repair genes cause an abnormal development of cancerous cells. In the present study we evaluated the possibility of breast cancer association with single nucleotide polymorphisms of RAD51, XRCC2 and XRCC3 genes. Polymorphisms selected in this study were RAD51 135G/C, XRCC2 Arg188His; and XRCC3 Thr241Met. Each polymorphism was genotyped using Polymerase chain reaction-restriction fragment length polymorphism in study cohort of 306 females (156 breast cancer patients and 150 controls). We observed that heterozygous variant genotype (GC) of RAD51 135 G/C polymorphism was associated with a significantly (OR=2.70; 95%CI (0.63-1.79); p<0.03) increased risk of breast cancer. In case of the XRCC3 gene we observed that frequency of heterozygous (OR=2.88; 95%CI (1.02-8.14); p<0.02) and homozygous (OR=1.46; 95%CI (0.89-2.40); p<0.04) genotype of Thr241Met polymorphism were significantly higher in breast cancer patients. For the Arg188His polymorphism of XRCC2, ~2fold increase in breast cancer risk (OR=1.6, 95%CI = 0.73-3.50) was associated with GA genotype with a p value for trend of 0.03. Our results suggest that the 135G/C polymorphism of the RAD51, Thr241Met polymorphism of XRCC3 and Arg188His polymorphism of XRCC2 can be independent markers of breast cancer risk in Pakistan.

Association of RAD 51 135 G/C, 172 G/T and XRCC3 Thr241Met Gene Polymorphisms with Increased Risk of Head and Neck Cancer

Homologous recombination repair (HRR) plays an important role in protection against carcinogenic factors. Genes regulating the HRR mechanisms may impair their functions and consequently result in increased cancer susceptibility. RAD 51 and XRCC3 are key regulators of the HRR pathway and genetic variability in these may contribute to the appearance and progression of various cancers including head and neck cancer (HNC). The aim of the present study was to compare the distribution of genotypes of RAD51 (135G/C, 172 G/T) and XRCC3 (Thr241Met) polymorphisms between HNC patients and controls. Each polymorphism was genotyped using the polymerase chain reaction-restriction fragment length polymerase (PCR-RFLP) technique in 200 pathologically confirmed HNC patients along with 150 blood samples from normal, disease free healthy individuals. We observed that homozygous variant CC genotype of RAD51 135G/C was associated with a 2.5 fold increased HNC risk (OR=2.5; 95%CI=0.69-9.53; p<0.02), while second polymorphism of RAD 51 172 G/T, heterozygous variant GT genotype was associated with a 1.68 fold (OR=1.68; 95%CI=1.08-2.61; p<0.02) elevation when compared with controls. In the case of the Thr241Met polymorphism of XRCC3, we observed a 16 fold (OR=16; 95% CI= 3.78-69.67; p<0.0002) increased HNC risk in patients compared to controls. These results further suggested that RAD51 (135G/C, 172 G/T) and XRCC3 (Thr241Met) polymorphisms may be effective biomarkers for genetic susceptibility to HNC. Larger studies are needed to confirm our findings and identify the underlying mechanisms.

Expression of CYP1A1 and GSTP1 in Human Brain Tumor Tissues in Pakistan

Most of the exogenous and endogenous chemical compounds are metabolized by enzymes of xenobiotic processing pathways, including the phase I cytochrome p450 species. Carcinogens and their metabolites are generally detoxified by phase II enzymes like glutathione-S-transferases (GST). The balance of enzymes determines whether metabolic activation of pro-carcinogens or inactivation of carcinogens occurs. Under certain conditions, deregulated expression of xenobiotic enzymes may also convert endogenous substrates to metabolites that can facilitate DNA adduct formation and ultimately lead to cancer development. In this study, we aimed to test the association between deregulation of metabolizing genes and brain tumorigenesis. The expression profile of metabolizing genes CYP1A1 and GSTP1 was therefore studied in a cohort of 36 brain tumor patients and controls using Western blotting. In a second part of the study we analyzed protein expression of GSTs in the same study cohort by ELISA. CYP1A1 expression was found to be significantly high (p<0.001) in brain tumor as compared to the normal tissues, with ~4 fold (OR=4, 95%CI=0.43-37) increase in some cases. In contrast, the expression of GSTP1 was found to be significantly low in brain tumor tissues as compared to the controls (p<0.02). This down regulation was significantly higher (OR=0.05, 95%CI=0.006-0.51; p<0.007) in certain grades of lesions. Furthermore, GSTs levels were significantly down-regulated (p<0.014) in brain tumor patients compared to controls. Statistically significant decrease in GST levels was observed in the more advanced lesions (III-IV, p<0.005) as compared to the early tissue grades (I-II). Thus, altered expression of these xenobiotic metabolizing genes may be involved in brain tumor development in Pakistani population. Investigation of expression of these genes may provide information not only for the prediction of individual cancer risk but also for the prevention of cancer.

Novel germline CDK4 mutations in patients with head and neck cancer

BackgroundCyclin-dependent kinase 4 (CDK4) together with its regulatory subunit cyclin D1, governs cell cycle progression through G1 phase. Cyclin-dependent kinase inhibitors, including p16INK4A in turn regulate CDK4. In particular, deregulation of the p16/CDK4/cyclin D1 complex has been established in a variety of human tumors including gliomas, sarcomas, melanoma, breast and colorectal cancer. However, changes in CDK4 have rarely been observed.MethodIn this study we used a combination of PCR-SSCP and direct sequencing for mutational screening of CDK4. DNA was isolated from peripheral blood leukocyte of patients with squamous cell carcinoma of head and neck, for screening germline mutations in coding regions of CDK4.ResultsVariations observed in exon 2 and 5 were three missense mutations, g5051G > C (Ser52Thr), g5095G > C (Glu67Gln), g5906C > A, g5907C > G (Pro194Ser) and novel frame shift mutations g7321_23delTGA, g7121_7122insG, g7143delG in exon 7 and 3′UTR respectively.ConclusionIn conclusion, two novel mutations were found in N terminal domain which indicates that CDK4 mutation may play a major role in the development and progression of squamous cell carcinoma of head and neck.

Significance of Cyclin D1 Polymorphisms in Patients with Head and Neck Cancer

Cyclin D1 plays a key role in cell cycle control, particularly in the transition from G1 to S phase, regulated by cyclin-dependent kinases. The objective of the present study was to screen the cyclin D1 gene (CCND1) for polymorphisms in patients with head and neck cancer (HNC). Genomic DNA was isolated from blood samples of 380 HNC patients and 350 controls. In a hospital-based case-control study using the PCR-SSCP technique we found 3 novel germline mutations: g3578C>A, g3475G>C and g3383delA. The commonly reported guanine to adenine polymorphisms in exon 4 g7656G>A (rs9344) and g10861C>A (rs7177) in 3′UTR of CCND1 were also observed. The calculated frequencies of the g7656G>A (rs9344) polymorphism in GG, GA and AA genotypes were 27.3%, 38.6%, and 33.9% in HNC cases, and 44.2%, 29.4%, and 26.2% in normal healthy controls, respectively. Adjusted by age (in years), sex and smoking status, multivariate logistic regression analysis showed that the AA and GA genotypes were associated with a significantly increased risk (OR 1.34, 95% CI 1.03-1.64, p=0.028) for HNC. The CCND1 AA genotype variant was associated with an increased risk in individuals who were <40 years old (OR 1.45, 95% CI 1.02-2.08, p=0.04). In conclusion, it is suggested that the CCND1 G/A polymorphism is associated with the early onset of HNC and may contribute to HNC susceptibility in a Pakistani population.

Rb1/105 gene alterations and head and neck carcinogenesis

Retinoblastoma gene (Rb1) is a tumor suppressor gene, which plays a pivotal role in cell cycle regulation, promoting G1/S arrest and growth restriction through inhibition of the E2F transcription factor. Abnormalities in the genes involved in cell cycle, including Rb1, have been reported in head and neck cancer (HNC) patients. Studies regarding Rb1 have been observed in different world populations but data is missing for Pakistani population. This study was aimed to analyze the genetic aberrations of Rb1 and their association with the development of HNC in Pakistani population. Genomic DNA was isolated from blood samples of 300 HNC patients and 270 controls. Salient coding region of gene was amplified by using Polymerase Chain Reaction (PCR). PCR conditions were optimized for each exon separately. Amplified products were analyzed for mutational screening using Single strand confirmation polymorphism (SSCP) technique followed by sequence analysis. Sequence analysis revealed five missense mutations g77082G>C, g77083G>A, g170220A>T, g170221G>C, g170228T>A, two frameshift mutations, two stop codon and two intronic substitutions in this study. The overall frequency of these mutations was 0.71. Frequency of nonsense mutations; Lys462stop (Novel) and Ser834stop (CM952105) were 0.15 and 0.14 respectively. We also report here novel missense mutations, frameshift mutation and a stop codon Lys462stop in HNC patients of Pakistani origin.This study suggests that the Rb1 germline mutations may contribute to genetic susceptibility for HNC. To our knowledge, this is the first report that Rb1 gene may be associated with risk of cancer in Pakistani population.