Genes and Extreme Longevity

Catalina Santiago,1,* Nuria Garatachea,2,* Thomas Yvert,1 Gabriel Rodriguez-Romo,3 Alejandro Santos-Lozano,2 Carmen Fiuza-Luces,1 and Alejandro Lucia1.

Abstract:
Genes of the proliferator-activated receptor delta (PPARD)-peroxisome proliferator-activated receptor c coactivator 1a (PPARGC1A, also termed PGC1-a)-nuclear respiratory factor (NRF)-mitochondrial transcription Factor A (TFAM) mitochondriogenesis pathway can influence health/disease phenotypes, yet their association with extreme longevity is not known. We studied the association of five common polymorphisms in genes of this pathway (rs2267668, rs8192678, rs6949152, rs12594956, rs1937) and extreme longevity using a case-control design. We found no between-group differences in allele/genotype frequencies, except for the CC genotype in rs1937 (p=0.003), with no representation in controls (0%), versus 2.8% in centenarians (2 men and 1 woman). In summary, the studied genetic variants of the PPARD-PPARGC1A-NRF-TFAM pathway were not associated with extreme longevity, yet a marginal association could exist for rs1937.

Introduction:
Mitochondria are essential organelles for understanding the aging process. They are both the main source and target of reactive oxygen species, and accumulation of somatic mutations in mitochondrial DNA (mtDNA) is one of the main features that accompanies age-related loss of mitochondrial function. Because mtDNA haplotypes or haplogroups might influence individual susceptibility to mtDNA damage, they could theoretically influence human aging. However, their association with extreme longevity, i.e., reaching 100 years of age, is controversial. Because extreme longevity is partly influenced by genetic factors, other genetic variants related to mitochondrial function might be involved in the heritability of this rare phenotype.

Several genetic variants could affect the PPARD-PPARGC1A-NRF-TFAM pathway and thus influence several important health/disease phenotypes related to mitochondrial function. For instance, a common missense variation (Gly482Ser, rs8192678) at exon 8 of the PPARGC1A gene has been repeatedly associated with type 2 diabetes and with related traits, including insulin resistance, obesity, hypertension, dyslipidemia, or aerobic fitness. The aforementioned polymorphism and the A/G (rs2267668) variation in PPARD are independently implicated in the modulation of insulin sensitivity or aerobic fitness. Common variants in NRF genes (NRF1 A/G [rs6949152] and NRF2 A/C [rs12594956]) influence aerobic fitness phenotypes, whereas the TFAM Ser12Thr (rs1937) variation has been associated with late-onset or sporadic Alzheimer's disease. Yet no candidate gene association study has analyzed whether genetic variants of the PPARD-PPARGC1A-NRF-TFAM mitochondriogenesis pathway can influence extreme longevity. Here, we have studied the association of five common polymorphisms in the aforementioned pathway (rs8192678, rs2267668, rs6949152, rs12594956, and rs1937) and extreme longevity in a Spanish (Caucasian) cohort using a case-control design.

Methods:
The study was designed and carried out in accordance with the recommendations for human genotype-phenotype association studies recently published by the National Cancer Institute-National Human Genome Research Institute (NCI-NHGRI) Working Group on Replication in Association Studies. Genotyping was performed specifically for research purposes. The researchers in charge of genotyping were totally blinded to the participants' identities, and saliva samples were tracked solely with barcoding. We used the classical phenol-chloroform DNA extraction protocol with alcoholic precipitation. Genomic DNA was resuspended in 50 μL of milli-Q H2O and stored at -20°C.

Genotyping was performed in the genomics laboratory of Universidad Europea de Madrid during the summer of 2012. Allelic discrimination analysis was performed with predesigned Applied Biosystems TaqMan SNP Genotyping Assays on demand for the five polymorphisms: PPARD A/G (rs2267668) (ID: C__15872729_10), PPARGC1A Gly482Ser (rs8192678) (ID: C___1643192_20), NRF1 A/G (rs6949152) (ID: C__29144830_10), NRF2 A/C (rs12594956) (ID: C__32072163_20), and TFAM Ser12Thr (rs1937) (ID: C___8975662_10). PCR amplification was performedGenotype results for rs8192678 were confirmed in a second laboratory, Progenika Biopharma in Derio-Vizcaya, Spain, using a new low-density DNA microarray based on allele-specific probes. The arrays were designed, fabricated, validated, and analyzed following a procedure detailed elsewhere with minor modifications. The PCR products were fluorescently labeled and hybridized to the DNA microarray using an automated platform (Tecan HS4800, Mannedorf, Switzerland). The microarrays were scanned (Innopsys S.A., Carbonne, France), and variants were determined using software that converts the intensity of the spots into the genotype of each variant. Cognitive ability was evaluated in the centenarians using the Mini-Mental State Examination (MMSE) and their independence/functional capacity with the Barthel index. The Barthel index is an instrument widely used to measure a person's capacity for the execution of 10 basic activities in daily life, obtaining a quantitative estimation of their level of independence. The sum-score ranges from 0 (totally dependent) to 100 (totally independent). Approximately 12% of the centenarians were virtually independent during daily living without assistance from other persons, i.e., their Barthel and MMSE score was 80 and >23, respectively.