Identification of single nucleotide pleomorphisms associated with periodontal disease in head and neck cancer irradiation patients by exome sequencing

Abstract

Objective: Periodontal disease (PD) is a common oral complication in patients with head and neck cancer (HNC) undergoing radiation therapy (RT). Our objective was to identify candidate single nucleotide polymorphisms (SNPs) associated with PD in radiation-treated patients with HNC.

Study design: DNA was extracted from the saliva of patients with HNC (n = 69) before RT. Clinical attachment loss (CAL) increment greater than 0.2 mm over 24 months after RT was used to define PD progression. After exome sequencing, SNPs associated with post-RT PD progression were identified by using logistic regression and homozygosity analyses. The web tools STRING, the Database for Annotation, Visualization and Integrated Discovery (DAVID), GeneCodis, and Ensembl Variant Effect Predictor were used for functional analysis.

Results: Of the 48 patients with HNC with post-RT PD progression, 24 had no tooth with 5 mm or greater pocket depth before RT, whereas of the 21 patients with HNC without progression, 11 had PD initially. A total of 330 SNPs (249 genes) with over-represented homozygous genotype (98.5% variant allele) were found to be associated with post-RT PD. Sixty of these corresponded to PD-related pathways, including previously identified genes. In patients with HNC with post-RT PD progression, SNPs were found in genes (n = 10) in contrast to those without progression (n = 7).

Conclusions: The SNPs of collagen genes were identified, potentially defining susceptibility to PD in patients with HNC, and this could be further investigated to characterize PD drug targets.

Conflict of interest statement

Disclosures: Authors have no conflict of interest.

Copyright © 2020 Elsevier Inc. All rights reserved.

Figures

Figure 1.. Analytical strategy of SNP analysis of Radiation treated HNC patients at risk for developing periodontal disease

a) Filtering and statistical analyses: The combined VCF file, obtained from n=69 patients, was filtered for minor allele frequency >5%, genotype rate >80% and biallelic sites. Logit regression (adjusted p<0.05) and z-test (p<0.05) identified 247 genes (244 alternate variant allele and 3 reference allele) with candidate SNPs potentially associated with periodontal disease. b) Gene network and gene ontology analyses: STRING tool determined 17 gene networks (64 genes) based on the 247 genes input. The 64 genes were subjected to gene ontology analysis using GeneCodis (https://david.ncifcrf.gov/) and DAVID (Database for Annotation, Visualization and Integrated Discovery, https://david.ncifcrf.gov/). As a result, six highly related biological processes with periodontal disease including 26 unique genes were identified. Gene ontology analysis was repeated including genes identified in previous studies as being associated with periodontal disease. This study confirmed HLA-B, MMP2, COL14A1, and HLA-DQB1 genes, previously shown to contain SNPs associated with periodontal disease. CAL= Clinical Attachment Loss; RT= Radiation Therapy

Figure 2.. Molecular network representing six major biological processes with relevance to periodontal disease identified in this study

A molecular interaction network of 64 among 247 candidate genes associated with periodontal disease was identified using STRING tool. The network includes 17 subnetworks. Of these 64 genes, 26 are part of six major biological processes identified using DAVID and GeneCodis tools. Blue line (solid) circled group of 9 genes represents “cell adhesion” biological process and red line (short dash) represents “extracellular matrix organization” (10 genes). Both biological processes include four collagen genes. The pink line (alternate dot and dash) represents the biological process “angiogenesis” (2 genes). Orange line (alternate double-dot and dash) identifies the “DNA repair” process (5 genes). Dark green line (long dash) represents “immune response” (7 genes) and purple line (dotted) “innate immune response” (5 genes).