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.