Maimoona Sabir

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.

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.

OGG1 Mutations and Risk of Female Breast Cancer: Meta-Analysis and Experimental Data

In first part of this study association between OGG1 polymorphisms and breast cancer susceptibility was explored by meta-analysis. Second part of the study involved 925 subjects, used for mutational analysis of OGG1 gene using PCR-SSCP and sequencing. Fifteen mutations were observed, which included five intronic mutations, four splice site mutations, two 3′UTR mutations, three missense mutations, and a nonsense mutation. Significantly (p < 0.001) increased (~29 fold) breast cancer risk was associated with a splice site variant g.9800972T>G and 3′UTR variant g.9798848G>A. Among intronic mutations, highest (~15 fold) increase in breast cancer risk was associated with g.9793680G>A (p < 0.009). Similarly ~14-fold increased risk was associated with Val159Gly (p < 0.01), ~17-fold with Gly221Arg (p < 0.005), and ~18-fold with Ser326Cys (p < 0.004) in breast cancer patients compared with controls, whereas analysis of nonsense mutation showed that ~13-fold (p < 0.01) increased breast cancer risk was associated with Trp375STOP in patients compared to controls. In conclusion, a significant association was observed between OGG1 germ line mutations and breast cancer risk. These findings provide evidence that OGG1 may prove to be a good candidate of better diagnosis, treatment, and prevention of breast cancer.

Acetylation of retinoblastoma like protein2 (Rb2/p130) in tumor tissues.

The activity of Rb proteins is controlled by post-translational modifications, especially through phosphorylation. Acetylation of Rb2/p130 was reported recently in NIH3T3 cells but its physiological relevance in cell cycle control and tumorigenesis is still unknown. Efforts are underway to investigate possible interplay between Rb2/p130 phosphorylation and acetylation. Here we hypothesized that Rb2/p130 acetylation, like p53 acetylation, may play a role in development of the tumor phenotype. The proposed hypothesis regarding acetylation of Rb2/p130 in tumor VS normal cells was found to be true in our case study of 36 tumor samples. Statistical analysis of results suggest strong correlation among Rb2/p130 acetylation and cancer phenotype.

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.