Mitochondriogenesis Genes and Extreme Longevity

Introduction

M itochondria are one of the most important organelles for understanding the aging process. ¹ 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 accompany the age-related loss of mitochondrial function. ² Because mtDNA haplotypes or haplogroups (i.e., non-pathogenic mtDNA populations that form a series of population-specific lineages) might influence individual susceptibility to mtDNA damage, they could theoretically influence human aging. Yet their association with extreme longevity, i.e., reaching ≥100 years of age, is controversial. ^{3 –8} Because extreme longevity is partly influenced by genetic factors, ⁹ other genetic variants related to mitochondrial function might be involved in the heritability of this rare phenotype.

A constant renewal of mitochondria is crucial for maintaining their normal function as well as for ensuring the functional and structural integrity of post-mitotic tissues. ¹⁰ Yet the capacity for mitochondrial biogenesis (or mitochondriogenesis) diminishes with age, ^11,12 and is responsible for many of the deleterious effects of aging, including loss of muscle mass and function, i.e., sarcopenia. ^13,14 Mitochondrial synthesis is stimulated by the proliferator-activated receptor delta (PPARD)-peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A, also termed PGC1-α)-nuclear respiratory factor (NRF)-mitochondrial transcription Factor A (TFAM) pathway. Briefly, PPARD induces promotion of PPARGC1A, ¹⁵ which is the first stimulator of mitochondriogenesis. NRF1 and 2 (the latter being also known as GA-binding protein alpha chain [GABPA]) are intermediate transcription factors that stimulate the synthesis of TFAM, which is the final effector activating the duplication of mitochondrial DNA molecules. ^{16 –18}

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 PPARGC1A gene has been repeatedly associated with type 2 diabetes ^{19 –22} and with related traits, including insulin resistance, ²³ obesity, ^24,25 hypertension, ^{26 –28} dyslipidemia, ²⁹ or aerobic fitness. ³⁰ The aforementioned polymorphism and the A/G (rs2267668) variation in PPARD are independently implicated in the modulation of insulin sensitivity or aerobic fitness, ³¹ and rs2267668 has also been associated with muscle mass. ³² Common variants in NRF genes (NRF1 A/G [rs6949152] and NRF2 A/C [rs12594956]) influence aerobic fitness phenotypes, ^{33 –35} whereas the TFAM Ser12Thr (rs1937) variation has been associated with late-onset ^{36 –38} or sporadic Alzheimer disease. ³⁹ 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, rs2267668, rs6949152, and rs12594956) 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 the 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. ⁴⁰ These recommendations include among others, the following items: Indicating time period and location of subject recruitment, success rate for DNA acquisition, and sample tracking methods.

Results

Genotype success was 100% for rs2267668, rs6949152, and rs1937, 99.7% for rs8192678, and 99.2% for rs12594956. Parallel genotyping results showed 100% concordance between the two laboratories. All genotype distributions met Hardy–Weinberg equilibrium in both the control and the centenarians' group (p>0.05), except for TFAM Ser12Thr (rs1937) in the centenarians' group (p<0.001). Figures 1 and 2 show the allele and genotype frequencies, respectively, in the two study groups. When studying men and women together in the two cohorts, we found no significant between-group differences (all p>0.1), except for the genotype frequency of rs1937 (χ²=11.440, p=0.003), with no representation of the CC genotype in the control group (0%) versus 2.8% in centenarians (2 men, 1 woman). This marginal association remained when studying only men in both cohorts, i.e., χ²=15.696, p<0.001 for the between-group comparison of the frequency of CC genotype, but not when studying only women (χ²=2.940, p=0.230). We found no significant odds ratio for any of the genotypes or alleles we studied.

OPEN IN VIEWER

FIG. 1.

Allele distribution of the study polymorphisms. No between-group differences were found. PPARD, Proliferator-activated receptor delta; PPARGC1A, peroxisome proliferator-activated receptor γ coactivator 1α; NRF, nuclear respiratory factor; TFAM, mitochondrial transcription Factor A.

OPEN IN VIEWER

FIG. 2.

Genotype distribution of the study polymorphisms. (*) p=0.003 for the between-group difference. PPARD, Proliferator-activated receptor delta; PPARGC1A, peroxisome proliferator-activated receptor γ coactivator 1α; NRF, nuclear respiratory factor; TFAM, mitochondrial transcription Factor A.

Discussion

The polymorphisms we studied have been associated with several health/disease phenotypes, many of which are related to mitochondrial function, notably the variant Ser allele of the PPARGC1A Gly482Ser (rs8192678) variation is associated with risk of type 2 diabetes,^19°22 insulin resistance, ²³ obesity, ^24,25 , dyslipidemia, ²⁹ hypertension,^26°28 and low aerobic fitness. ³⁰ Yet, we found no association of genetic variants in the PPARD-PPARGC1A-NRF-TFAM pathway and extreme longevity. We only found a marginal association for the TFAM Ser(G)12Thr(C) (rs1937) polymorphism when studying the two sexes together or only men separately, with no representation of the CC genotype in the control cohort versus a total frequency of ∼3% in centenarians, included among them the oldest person in Europe until September, 2012 (disease-free man, aged 111 years). Interestingly, the frequency of the CC genotype in our centenarians' cohort was above that previously reported for control European cohorts, with a virtual absence of the CC genotype, e.g., 0% in adolescents of both genders from Argentina (European descent), ⁴⁶ 0.7% in Russian men and women, ⁴⁷ 1.0% in European (German, Swiss, Italian) men and women (0% in men), ³⁹ and 1.8% in Spanish men and women. ³⁶

The rs1937 variation in TFAM could affect disease phenotypes and thus, at least partly, longevity owing the fact that: (1) This polymorphism could be of relevant functional significance, because it predicts an amino acid change (Ser12Thr) in the sequence of the mitochondrial signal peptide TFAM ⁴⁸ ; and (2) the putative role of this gene. The TFAM gene, located at 10q21, encodes the key protein responsible for replication and transcription of mitochondrial DNA and protects cells against oxidative stress, the latter being involved in the development of disease phenotypes, notably neurodegenerative disorders. ^49,50 TFAM knockout mice exhibit depletion of mtDNA and abolished oxidative phosphorylation, ⁵¹ whereas heart-specific TFAM gene knockout mice display a progressive heart phenotype with depletion of mtDNA and an accompanying severe decline of respiratory chain enzyme activities and decreased mitochondrial adenosine triphosphate (ATP) production. ⁵² Wredenberg et al. ⁵³ reported accumulation of abnormally appearing mitochondria, progressively deteriorating respiratory chain function, and reduced muscle-force production in muscle fibers of mice with skeletal muscle-specific disruption of the TFAM gene. In contrast, over-expression of this gene in cardiac myocytes is associated with a two-fold increase in the number of mtDNA copies and elevated ATP production. ⁵⁴

Human research has shown an association between rs1937 and neurodegenerative diseases characterized by disturbance of mtDNA integrity and mitochondrial dysfunction, i.e., late-onset ^{36 –38} or sporadic Alzheimer disease, ³⁹ with the rare CC genotype conferring a protective effect ³⁸ and the GG being a risk genotype. ^36,39 Furthermore, the rare C allele has been recently associated with an ‘aerobic’ or ‘endurance-oriented phenotype’, with elite endurance athletes showing higher frequency of the C allele than non-athletic controls. ⁴⁷ Regarding this, a shift toward a more ‘aerobic phenotype’ could have a beneficial influence on health-related phenotypes and longevity, with classic studies in rats by Koch's and Britton's groups showing improved mitochondrial fitness may be a common factor linking physical fitness and decreased disease risk. ^55,56

The renewal of mitochondria through the process of biogenesis is vital for maintaining mitochondrial integrity, and a diminished capacity for organelle biogenesis has been implicated in the pathogenesis of several diseases as well as in the aging process. ^57,58 A role for mitochondrial dysfunction in aging is evidenced by mtDNA mutator mice that express a proofreading-deficient mitochondrial DNA polymerase gamma. ⁵⁹ These mutant mice have increased mtDNA mutations and display age-related phenotypes, including severe sarcopenia that is likely due to defects in the assembly of functional electron chain transport complexes. ⁶⁰ Similar findings of increased mtDNA mutagenesis and reduced mitochondrial enzyme activities have been reported in human skeletal muscle with age. ⁶¹ However, our findings and those recently reported by our group with part of the present centenarians' cohort do not support a major role of genetic variants in mtDNA ⁶² or in the PPARD-PPARGC1A-NRF-TFAM mitochondriogenesis pathway on extreme longevity. Other genetic variants not studied here or other non-genetic factors that are known to influence mitochondriogenesis, such as exercise or calorie restriction, ^1,63 might be more influential to extreme longevity.

We believe the results of our study are valid overall, because all of the following criteria were met ⁶⁴ : The phenotype ‘exceptional longevity’ was properly defined with all cases being centenarians (living 100+ years is still a rare phenotype, i.e., ≤1 every 10,000 people ⁹ ), both groups of cases and controls were ethnically matched, genetic assessment was unbiased, all genotype distributions were in Hardy–Weinberg equilibrium in the control group, and we adjusted our statistical analyses for multiple comparisons. The relatively low sample size of the centenarians' cohort is a limitation. Future research is needed with larger sample sizes, especially with more centenarian men, to corroborate the existence of an association between rs1937 and extreme longevity or whether this association is sex-dependent. The lack of data in a ‘replication’ cohort of a different ethnic background also limits the ‘external validity’ (and therefore generalizability) of our results. On the other hand, while keeping in mind the aforementioned limitations, we believe the major strength of our study stems in its novelty and in the rationale for studying genetic variants in the mitochondriogenesis pathway.

In summary, common genetic variants in the PPARD-PPARGC1A-NRF-TFAM pathway (rs2267668, rs8192678, rs6949152, rs12594956, rs1937) are not associated with extreme longevity, at least in the Spanish population, yet a marginal association could exist for the TFAM Se12Thr (rs1937) polymorphism, with a certain survival benefit existing for the rare, low-risk CC genotype. More research is needed on other genetic variants potentially associated with maintenance of mitochondrial function.