Method: Next Generation Sequencing (NGS) of one African American and one Caucasian family was performed to search for gene mutations with major effects on disease risk. DNAs were enriched for protein coding regions of the human genome using Agilent’s SureSelect exon capture method. Sequencing of fragment libraries was performed on Applied Biosystems’ SOLiD platform. Sequence reads were mapped to the human reference genome and Single Nucleotide Polymorphisms (SNPs) identified using LifeScope (Applied Biosystems) software. We also assayed the Illumina Omni2.5 arrays (2.4 million SNPs). Non-synonymous (changing amino acid), stop codon (premature termination of the protein) and mRNA splice site gain/loss SNPs were mapped using Golden Helix’s SNP Variation Suite software. Effects of variants on protein function were predicted using SIFT and PolyPhen algorithms. Variants were further prioritized by known gene functions and observation of transmission in the families.
Result: After quality control procedures, our NGS and SNP array genotype call agreement was >99.4%. One mutation in axonemal heavy chain dynein gene DNAH5 and two mutations in DNAH9 were identified as strong candidates for AgP susceptibility in African Americans. Population surveys show these variants are very rare in Caucasians (0 or 1 heterozygotes out of 2,700 subjects) versus frequencies of 0.6%, 4.0% and 16.6% in African Americans. DNAH9 is also implicated by our analysis of supplementary data from a previously-reported GWAS study of AgP. In the mouse, dynein-mediated transport of lysosomes has been shown to be critical for phagosomal maturation in neutrophils and impairment of this innate immune defense leads to periodontitis.
Conclusion: Exome sequencing has implicated a novel mechanism of AgP pathogenesis based on dynein’s role in host defense.