Identification of functional variants in FOXO3A, a validated candidate gene influencing human longevity.
Final Report Abstract
The forkhead box O3A (FOXO3A) transcription factor is a key regulator of the insulin receptor/insulin-like growth factor-I signaling (IIS) pathway. FOXO3A is convincingly annotated as a human longevity gene, but the functional variants influencing the phenotype still remain to be identified. The main objective of this study was to detect and characterize such variants using Next Generation Sequencing (SOLiD, Illumina) and Sanger technologies as well as association tests and functional studies. Sequencing of exons was greatly hampered by the fact that they show 99% sequence homology with the FOXO3B pseudogene. To this end, we developed an experimental set-up using long-range PCR products for reliable and reproducible data generation. Exons and the promoter were sequenced by Sanger in 300 individuals. Sequencing of the whole FOXO3A gene region was performed by SOLiD and Illumina technologies in 150 samples. Despite the presence of the FOXO3B pseudogene, we successfully sequenced the whole FOXO3A gene region including exons, introns, the promoter and ~15 kb in 5’ and 3’ direction (in total ~170 kb). Altogether, 1,108 known and novel single nucleotide variants (SNVs) were detected. To select SNVs for subsequent experiments, we first evaluated all identified variants by in silico bioinformatic analyses for functional effects. For the genotyping, we considered all SNVs located in regulatory regions (e.g. transcription factor binding sites, microRNA regulatory sites) and in the exons. In total, 209 variants of interest were successfully genotyped in 1,109 German long-lived individuals (LLI) (centenarians and nonagenarians, age range: 95-110 years) and 918 younger controls (age range: 60-75 years) using iPLEX® and TaqMan® technologies. We identified 54 statistically significant SNVs. The strongest association signal was caused by an intronic SNV near the 3’UTR. The alleles of this SNV were predicted by bioinformatic tools to influence the binding affinity for two transcription factors, PDX1 (pancreatic and duodenal homeobox 1, for which the wild-type allele has a high affinity) and SRF (serum response factor, for which the derived minor allele has a high affinity). The minor SNV allele is more common in LLI than in younger controls and is, therefore, supposed to have a beneficial (i.e. longevity-enabling) effect. All three genes FOXO3A, PDX1 and SRF are expressed in pancreas and play important roles in the glucose-dependent IIS pathway. A dual luciferase assay revealed that the minor SNV allele increases the promoter activity in the pancreas cell line panc1 compared to the wild-type allele. We hypothesize that the binding of SRF to the minor allele may lead to higher FOXO3A expression, which was previously shown to be associated with life-span extension in model organisms. Further functional studies are needed to gain more insights into the FOXO3A SNV-allele mediated mechanisms and their influence on human longevity.
Publications
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(2013) Genetic investigation of FOXO3A requires special attention due to sequence homology with FOXO3B. Eur J Hum Genet 21:240-2
Flachsbart F., Möller M., Däumer C., Gentschew L., Kleindorp R., Krawczak M., Caliebe A., Schreiber S., Nebel A.
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(2014) Adjustment for smoking does not alter the FOXO3A association with longevity. Age (Dordr) 36:911-21
Däumer C., Flachsbart F., Caliebe A., Schreiber S., Nebel A., Krawczak M.
