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Retinoblastoma (RB1) pocket domain mutations and promoter hyper-methylation in head and neck cancer

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Abstract

Background

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.

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) 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.

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). Missense 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 epigenetic RB1 changes may contribute to the pathogenesis of HNC in the Pakistani population.

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Acknowledgments

The authors acknowledge the financial and infrastructural support from the Higher Education Commission (HEC) and the COMSATS Institute of Information Technology (CIIT), Islamabad. The authors are thankful to the patients and the staff of the Nuclear Oncology and Radiotherapy Institute (NORI), the Pakistan Institute of Medical Sciences (PIMS), the Allied Hospital and the Military Hospital (MH), Islamabad for their contributions to this research.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

It is declared that experiments in this manuscript submitted for publication comply with the current laws of the country in which they were performed. This study was conducted with a prior approval from ethical committees of the COMSATS Institute of Information Technology, Islamabad (CIIT) and the affiliated hospitals.

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Authors and Affiliations

  1. Cancer Genetics Lab, Department of Biosciences, COMSATS Institute of Information Technology, Park Road Chak shahzad, Islamabad, Pakistan

    Maimoona Sabir, Ruqia Mehmood Baig, Kashif Ali, Ishrat Mahjabeen, Muhammad Saeed & Mahmood Akhtar Kayani

  2. School of Medicine, Cardiff University-Peeking University Cancer Institute, Heath Park, Cardiff, UK

    Kashif Ali

  3. Department of Zoology, PMAS Arid Agriculture University, Rawalpindi, Pakistan

    Ruqia Mehmood Baig

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  1. Maimoona Sabir
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Correspondence to Mahmood Akhtar Kayani.

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Sabir, M., Baig, R.M., Ali, K. et al. Retinoblastoma (RB1) pocket domain mutations and promoter hyper-methylation in head and neck cancer. Cell Oncol. 37, 203–213 (2014). https://doi.org/10.1007/s13402-014-0173-9

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