Retinoblastoma (RB1) pocket domain mutations and promoter hyper-methylation in head and neck cancer
Cellular Oncology, 2014
The RB1 gene plays a pivotal role in cell cycle regulation. In this case-control study we searche... more The RB1 gene plays a pivotal role in cell cycle regulation. In this case-control study we searched for alterations in the RB1 pocket domain and its promoter region in head and neck cancer (HNC) patients in the Pakistani population. For germline mutation analyses, 380 blood samples from HNC patients and 350 blood samples from control individuals were included. Polymerase chain reaction (PCR) and single strand conformational polymorphism (SSCP) assays, followed by sequence analyses, were used for the RB1 pocket domain mutation screens. For the RB1 promoter methylation screens, 72 HNC tumor samples along with adjacent uninvolved tissues were tested using a methylation-specific polymerase chain reaction (MSP) assay. RB1 (pocket domain and spacer region) sequence analysis revealed one frameshift and seven non-synonymous missense mutations. The frequency of missense mutations in exon 14, i.e., g76474C > T, g76475G > C and g76476A > G, resulting in Arg455Ser, was found to be highest (0.10). Missense mutations…
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Papers by Muhammad Saeed
Aberrant expression of tumor suppressor genes may correspond to the abnormal cell devel- opment and tumorigenesis. Rbl2/p130, a member of retinoblastoma family of proteins, has growth suppressive properties. Numerous studies reported de-regulation of Rbl2/p130 in various types of cancer as a consequence of a number of genetic alterations. However, role of epigenetic mechanisms like DNA methylation in Rbl2/p130 expression remains elusive.
Methods
In the current study, 76 breast cancer tumors along with normal tissues (n = 76), blood (n = 76) of respective individuals and control blood (n = 50) were analyzed. Rbl2/p130 expres- sion was analyzed by quantitative real time PCR (syber green method). Promoter methyla- tion status was studied through methylation specific PCR of bisulfite converted genomic DNA. Data was analyzed using various statistical tests.
Results
We report significantly reduced Rbl2/p130 expression (P = 0.001) in tumors tissues as com- pared to control samples. Similarly, Rbl2/p130 expression varies with age and disease stages (P = 0.022), which suggest its involvement in tumor progression. Aberrant promoter methyla- tion (Δmeth) was found in almost all the diseased samples and that was significantly different (P<0.001) with control samples. Similarly, methylation status varies significantly with tumor progression stages (P = 0.022). Hyper-methylation was observed at -1, +3, +15 and +75 of Rbl2/p130 promoter flanking around the TSS. Statistical analysis revealed that Rbl2/p130 expression negatively correlates to its promoter methylation (r = -0.412) in tumor tissues. Our results reflect an epigenetic regulation of Rbl2/p130 expression in breast cancer. This high- lights the importance of Rbl2/p130 promoter methylation in breast cancer pathogenesis.
Methods For germline mutation analyses, 380 blood samples from HNC patients and 350 blood samples from control individuals were included. Polymerase chain reaction (PCR) and single strand conformational polymorphism (SSCP) as- says, followed by sequence analyses, were used for the RB1 pocket domain mutation screens. For the RB1 promoter meth- ylation screens, 72 HNC tumor samples along with adjacent uninvolved tissues were tested using a methylation-specific polymerase chain reaction (MSP) assay.
Results RB1 (pocket domain and spacer region) sequence analysis revealed one frameshift and seven non-synonymous missense mutations. The frequency of missense mutations in exon 14, i.e., g76474C > T, g76475G > C and g76476A > G, resulting in Arg455Ser, was found to be highest (0.10). Mis- sense mutations g76467G > C (exon14), g76468T > C (exon14), g77041A > T and g77043A > T (exon 16), when analyzed via Alamut biosoftware version 2.0, were found to be present in highly conserved amino acids with Align GVGD scores C15 (GV: 0.00 - GD: 21.82), C65 (GV: 0.00 - GD: 83.33) and C65 (GV: 0.00 - GD: 98.69), respectively. These missense mutations were found to be deleterious by SIFT score: 0.00 (median 3.64). RB1 promoter methylation analysis revealed that 16 % of its cytosines (3 % in CpG) were methylated in the HNC tumor samples.
Conclusion Our findings indicate that both genetic and epi- genetic RB1 changes may contribute to the pathogenesis of HNC in the Pakistani population.