Pyrosequencing-based DNA, methylation profiling of Fanconi anemia/BRCA pathway genes in laryngeal squamous cell carcinoma

Fanconi anemia (FA) associated genes [FANCA, -B, -C, FANCDI(BRCA2), -D2, -E, -F, -G, -I, -L, -M, FANCN (PALB2), FANCJ(BRIPI) and FA-linked BRCA1] encode proteins of DNA damage response pathways mutated in FA patients. FA is characterized by congenital malformations, chromosomal instability and high cancer susceptibility. FA patients have a 500-700 times higher risk of head and neck squamous cell carcinoma (HNSCC) compared to the non-FA population. As DNA methylation comprises one of the known gene inactivation mechanisms in cancer we have investigated the methylation status of 13 FA and one FA-linked gene in order to assess the role of FA in sporadic laryngeal squamous cell carcinoma (LSCC) tumor samples. Thirteen laryngeal squamous carcinoma cell lines (UT-SCC) and 64 primary laryngeal carcinoma cases were analyzed by bisulfite pyrosequencing. DNA from buccal swabs of 10 healthy volunteers was used as a control group. Promoter regions of FANCA, BRCA1 and BRCA2 displayed recurrent alterations in the methylation levels in cancer samples as compared to buccal swabs controls. For FANCA, hypomethylation was observed in 11/13 cell lines (p < 0.0003) and all 64 primary larynx samples (p < 0.001) compared to buccal swabs. For BRCA1, 4/13 cell lines (p=0.04) and 3/58 primary laryngeal cases (p=0.22) showed hypomethylation. BRCA2, all 13 cell lines (p < 0.0001) 4/63 primary LSCC (p < 0.01) showed hypermethylation as compared to controls. In conclusion, we show recurrent alterations of DNA methylation levels in three Fanconi anemia genes which might contribute to the pathogenesis of LSCC.