Mitochondrial haplogroups and lifespan in a population isolate

Journal Pre-proofs Mitochondrial Haplogroups and Lifespan in a Population Isolate Joseph Bonner, Rachel Fisher, Ellen Wilch, Debra Schutte, Brian Schutte PII: DOI: Reference:

S1567-7249(19)30104-7 https://doi.org/10.1016/j.mito.2019.12.004 MITOCH 1433

To appear in:

Mitochondrion

Received Date: Revised Date: Accepted Date:

27 June 2019 29 November 2019 16 December 2019

Please cite this article as: Bonner, J., Fisher, R., Wilch, E., Schutte, D., Schutte, B., Mitochondrial Haplogroups and Lifespan in a Population Isolate, Mitochondrion (2019), doi: https://doi.org/10.1016/j.mito.2019.12.004

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier B.V.

Mitochondrial Haplogroups and Lifespan in a Population Isolate

Authors: Dr. Joseph Bonner (corresponding author) City of Hope National Medical Center Center for Precision Medicine 1500 E. Duarte Road Duarte, CA 91010-3000 Phone: 517-256-6748 [email protected] Dr. Rachel Fisher Michigan State University, College of Human Medicine (Retired) Dr. Ellen Wilch (Unaffiliated) Dr. Debra Schutte Wayne State University, College of Nursing Dr. Brian Schutte Michigan State University, College of Natural Science Abstract Physiochemical differences between mitochondrial DNA (mtDNA) haplogroups that favor oxidative phosphorylation efficiency during periods of caloric limitation can lead to lifespan lengthening when food calories are less abundant. For example, prior work demonstrated that older female haplogroup H carriers had modestly lengthened lifespans beyond 60 years during the Great Depression, a time of caloric limitation in North America. The objective of the current study is to replicate the prior findings in an independent cohort that includes both sexes and younger ages. By determining and cross-referencing the mtDNA genotypes of a culturally homogeneous population isolate to the lifespans of their ancestors, we found that between 1930 and 1939, haplogroup H compared to haplogroup U carriers had a modestly lengthened lifespan (3 years) past 60 years (hazard ratio 2.35; CI95 1.41-3.90; p-value: 0.0029). The lifespan-lengthening association was apparent in both sexes but only after the age of 60. Our results provide further support for the role of mitochondrial genetics in lengthening human lifespan. 1. Introduction Mitochondria power cellular life by converting chemical energy from carbon:carbon bonds into adenosine triphosphate (ATP) or heat through oxidative phosphorylation (OXPHOS). OXPHOS can generate reactive oxygen species (ROS) as a byproduct. ROS act as cellular signals to maintain homeostasis; however, excess production of ROS can damage nearby biomolecules including nuclear and mitochondrial DNA (mtDNA). mtDNA encodes for 37 genes, 13 of which are essential for OXPHOS. mtDNA is maternally inherited [1, 2] and phylogenic trees of mtDNA haplotypes show global/regional groupings called haplogroups that connect the carrier with their distant maternal lineages with predictable fidelity [3].

In a previous study, Beckstead et. al. presented bioinformatic evidence for OXPHOS physiochemical variation at mtDNA T14766C. This variation distinguishes haplogroup H from haplogroup U and changes amino acid 7 of cytochrome B from threonine in haplogroup H to isoleucine in haplogroup U. The different physiochemical properties of threonine and isoleucine favor greater OXPHOS efficiency during calorie-limited periods for haplogroup H [4]. Beckstead et. al. posited that during the Great Depression in North America calories were limited, and their bioinformatic evidence predicted a lifespan lengthened phenotype in haplogroup H carriers during the periods of caloric restriction. They tested their prediction in a study of lifespan using death dates from genealogic records and haplogroups obtained from contemporary individuals. To minimize potential competing effects, Beckstead et. al. studied only females who lived beyond the age of 60 and died in North America. They found that among females who died over the age of 60 between 1920 and 1940 haplogroup H carriers lived 2.6 years longer than haplogroup U carriers [4]. This work extends the Beckstead et. al. findings in three ways: including both sexes, including younger ages, and analyzing other haplogroups in a different population. In this work, we studied lifespan variation during a specific epoch among relatives of known haplogroup carriers. The relatives within this work come from genealogic records of a population isolate from a rural area in mid-Michigan. Most members of this population descend from a defined group of founders who settled the region starting in 1835. For over 150 years, descendants have farmed the same land and shared a similar diet, environment, and climate [5]. Using genealogic records, we assessed the haplogroup and lifespan association across “time” in a defined “place” focusing on the decade of the Great Depression 19301939. 2. Materials and Methods 2.1 Genealogic data The population isolate used in this study lived in a 90 square mile area circumscribing three rural municipalities in two contiguous counties in mid-Michigan [5]. Members of the community are partners in the community-based Cooperative for Studies Across Generations (CoSAGE) [6, 7]. CoSAGE maintains a pedigree database of descendants of the founders and a registry to which community members they may volunteer and donate a DNA sample. The pedigree database contains 37,315 people; 14,204 were born in the study century between 1820 and 1919 and are now all deceased. A small set of 344 distant ancestors covers 67% of the extant genome[5]. 2.2 Study cohorts Individuals were recruited who previously consented to the CoSAGE registry [6, 7]. Potential recruits received a study packet containing an introductory letter and a consent document for permission to use their banked DNA and corresponding genealogic data. Consenting CoSAGE registry participants comprised the study Direct Cohort. Deceased relatives of the Direct Cohort who had connected matrilineages in the pedigree database formed a larger, Indirect Cohort. Michigan State University Institutional Review Board approved the Informed Consent documents. 2.3 Lifespan phenotype The lifespan phenotype of the Indirect Cohort was quantified by the age of death. To maximize the sample size and avoid censoring data, individuals missing a death date were assigned the median age of death in the pedigree database for their birth year [8]. 2.4 DNA isolation and banking

From buccal samples, DNA was isolated using Oragene™ DNA Self-Collection Kit (DNA Genotek Inc., Ottawa ON, Canada). From venous blood samples, DNA was isolated using the Quick-Gene -810 following manufacturer recommendations (AutoGen, Holliston, MA, USA). Extracted DNA was banked in a freezer at -80o C until consent for retrieval. 2.5 Genotyping mtDNA from the non-coding/hypervariable region was amplified using a forward primer (mt15596 5’AAG TCT TTA ACT CCA TTA GCA-3’) and a reverse primer (mt326 5’- GGC TGG TGT TAG GGT TCT TTG- 3’). The PCR amplification condition was an initial temperature elevation to 94⁰C (3 minutes), followed by 35 cycles of 94⁰C (30 seconds), 60⁰C (3 minutes), and 72⁰C (1 minute). After 25 cycles, reactions were held at 72⁰C for 1 minute followed by 15⁰C until retrieval. Sequencing aliquots of each PCR were prepared with the primers listed above and an internal primer (mt16523 5’-ACT TCA GGG CCA TAA AGC CT-3’). PCR products were sequenced using an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). 2.6 Bioinformatics Methods DNA sequencing data files were imported into Sequencher™ (Gene Codes, Ann Arbor, MI, USA). DNA sequences were trimmed, and ambiguous calls were reviewed by two investigators independently (JB and EW). Trimmed sequences were aligned with the revised Ptolemaic Cambridge Reference Sequence (rCRS) [9, 10]. mtDNA sequence variants from rCRS were uploaded into HaploGrep [11] for haplogroup scoring. 2.7 Data Management and Statistical Analysis The lifespan phenotype was analyzed using proportional hazards models and survival curves [8]. Data management, manipulations, and some statistical analyses were executed in SAS™ [12]. Statistical analyses and graphical output were composed in R™ [13]. 3. Results 3.1 Pedigree Data The pedigree database contains 37,315 individuals born between 1511 to 2013; and 14,204 individuals were born in the study century that spans 1820 and 1919 and died in the past 150 years, 48% were female. The proportion of female births varied throughout the decades (p for trend <0.0001). Death dates were available for 72% of the pedigree database. Most individuals lived into adulthood with only 7% dying before 20. Of those born in the study century, 3,506 (27%) individuals lived past 79 years old, 1,134 (7.9%) lived past 89, and 115 (0.8%) lived past 99. Table 1 presents the characteristics of the pedigree data for the study century by birth decade. 3.2 The Direct Cohort The CoSAGE registry contained 256 potential participants. We received consent and recruited 118 individuals into the Direct Cohort. Of those, 98 had a banked DNA sample. Fifty-nine participants were from matrilineages contributing births after the study century. Thirty-nine donor samples were from 29 distinct matrilineages contributing births during the study century. Nine samples are replicates of the 29 matrilineages. 3.2.1 Genotyping and Haplogrouping of the Direct Cohort

We extracted, amplified, sequenced, aligned, and assigned mtDNA haplogroups for 38/39 samples from the Direct Cohort. All nine replicate samples matched the haplogroups of other samples within their matrilineage. We used the major clade name to ascribe haplogroups to matrilineages. Table 2 presents the frequency of haplogroup clades of both cohorts. 3.3 The Indirect Cohort The 29 matrilineages of the Direct Cohort pedigrees map to an Indirect Cohort of 2,902/14,204 (20%) of individuals born during the study century. The proportion of derived death dates, shortened lifespans, and lengthened lifespans differs by birth decade intervals. The proportion of females born by birth decade is 50% in the Indirect Cohort. Table 1 presents characteristics of the Indirect Cohort by birth decade. There are fewer derived death dates in the Indirect Cohort than in the pedigree database in general (13% vs 30%). Table 2 presents the characteristics of the Indirect Cohort stratified by haplogroup. The Indirect Cohort contains 20 centenarians, eight from haplogroup H, seven from haplogroup T, three from haplogroup J, and one each from haplogroups K and U. 3.3.1 Lifespan by death decade in the Indirect Cohort The median lifespan of the Indirect Cohort increased between 1880 and 2009 (Figure 1). There is a modest difference in lifespan when contrasting all six haplogroups and across all death ages (0-120) (pvalue: 0.017) (Figure 2). We stratified and tested lifespan within each study decade individually. We found that the decade of 1930-1939, corresponding to the Great Depression, is one of two decades presenting differences in lifespan (beyond 60 years of age) between haplogroups H and U. (Table 3) (hazard ratio: 2.35; CI95: 1.413.90; p-value: 0.0029). In the target period of 1930-1939, the median lifespan of haplogroup H carriers was lengthened by 3 years compared to haplogroup U carriers. The lifespan lengthening difference was apparent in both sexes (Figure 3). There was also a lengthening of 1 year in the 2000-2009 decade. 4. Discussion In this study, we replicated and extended the findings of Beckstead et. al. Consistent with their report, we found that during the decade of the Great Depression haplogroup H carriers lived modestly longer (3 years) than did haplogroup U carriers beyond the age of 60. We extended the findings by observing lifespan lengthening in carriers of haplogroup H in both sexes. We also show a modest effect when contrasting all six haplogroups across all death ages; however, the effect fails to sustain significance when corrected for multiple comparisons. This study has limitations. First, the closed study cohort negates the need to censor because all members were deceased. However, the closed cohort also does not contribute younger deaths because by 2000 the youngest cohort members were in their ninth decade of life. An open cohort with members born after 1920 and living into 2009 could potentially attenuate the differences in lifespan presented by haplogroups H and U. Second, to keep the full Indirect Cohort intact and avoid the need to censor data in the survival analysis, we chose to impute missing death dates rather than exclude those with missing data. Our chosen method was naïve to haplogroup carrier status. The naivety should not have introduced any bias. However, any bias introduced would have been non-differential and favored a null hypothesis of no effect between haplogroups. Previous work has well-established the increased likelihood of living to centenarian in many populations including Italians [14, 15], Irish [16], Finns [17, 18], Danes [19], Japanese [18, 20], Tunisians [21], Ashkenazi [22] ,Costa Ricans [23, 24], Uyghurs [25] , Spaniards [26, 27], Poles [28], Chinese [29, 30],

Amish [31], and Turks [32]. A recent pooled analysis of case-control studies showed centenarians are more likely to carry haplogroup J than are contemporaneous younger controls (Odds Ratio 1.7) [33]. However, our indirect cohort identified only one matrilineage carrying haplogroup J and only three centenarians disallowing any meaningful association testing. In addition, numerous studies show associations with mtDNA, longevity, and other phenotypes in crosssectional study settings. Beckstead et. al. was the first to show a lifespan lengthening using proportional hazard models during a specific period. Our replication of the Beckstead et. al. findings present strength for the time-period specificity of the mtDNA haplogroup association in a cohort study setting. The difference in lifespan between 2000 and 2009 is outside the original objectives of this work. Our findings can be interpreted within the context of the Thrifty Gene Hypothesis and the Wallace Hypothesis. The Thrifty Genotype Hypothesis postulates that genotypes efficient in extracting energy from alimentation during times of famine might contribute to chronic diseases (specifically diabetes) during times of plenty [34]. At first, the postulate mentioned nothing of mitochondrial contributions; however, later additions to the hypothesis included mtDNA variation and metabolic influences [35]. Our work contributes historic evidence in support of a thrifty mitochondrial genotype. Additionally, during periods of global human migration and settlement, populations adapted to new climates and diets. The Wallace Hypothesis states that accumulation of mutations in mtDNA that are advantageous to new environments drives evolution of new mtDNA haplogroups; variation in mtDNA-encoded OXPHOS genes has been correlated with increased thermogenesis at the expense of efficiency of ATP generation [3643]. Our work lends evidence, albeit observational and ecological, that differences in energy efficiency corresponding to haplogroup supports both the Thrifty Genotype Hypothesis and the Wallace Hypothesis. Our genealogic data lacks cause of death and chronic disease diagnoses. Thus, it cannot offer direct evidence about variations in diabetes risks correlated with the mtDNA haplogroup association. To build on our replicated findings, future studies of genealogic records or population isolates might correlate causes of death with year of death and year of birth by mtDNA haplogroup. By correlating mtDNA haplogroups, phenotypes, and historical records future investigations can seek evidence for mtDNA contributions to adult onset chronic diseases. Additionally, future work might link multiple generations across time and mtDNA haplogroup and study and potentially trans-generational effects of parental environmental exposures like caloric limitation [44]. 5. Acknowledgements We acknowledge the Michigan State University through the Families and Communities Together (FACT) Coalition [PI: Schutte, B.C.; Schutte, D.L.] and a Strategic Partnership funding mechanisms [PI: Schutte, B.C.; Schutte, D.L.] for graciously funding this work. We also acknowledge Chris Ghandi of City of Hope National Medical Center scientific writing staff for valuable manuscript editing. And the reviewers for the helpful insights and persistence toward this final document. 6. Tables and Figures Table 1: Characteristics of Pedigree Database and Indirect Cohort by Birth Decade for the Study Century. Pedigree Database

Indirect Cohort

Birth Decade

Births

Death Date Derived n (%)

Death Before 20 n (%)

Females n (%)

Alive Past 60 n (%)

Births n (%)

Death Date Derived n (%)

Death Before 20 n (%)

Females n (%)

Alive Past 60 n (%)

1820-1829

444

140 (32)

24 (5)

201 (45)

350 (79)

53 (12)

9 (17)

8 (15)

28 (53)

34 (64)

1830-1839

532

165 (31)

40 (8)

255 (48)

397 (75)

73 (14)

11 (15)

5 (7)

38 (52)

52 (71)

1840-1849

716

214 (30)

49 (7)

350 (49)

526 (73)

121 (17)

20 (17)

18 (15)

61 (50)

78 (64)

1850-1859

996

314 (32)

64 (6)

469 (47)

773 (78)

146 (15)

30 (21)

15 (10)

75 (51)

108 (74)

1860-1869

1,216

343 (28)

79 (6)

576 (47)

892 (73)

221 (18)

34 (15)

17 (8)

115 (52)

164 (74)

1870-1879

1,597

447 (28)

165 (10)

787 (49)

1139 (71)

320 (20)

69 (22)

44 (14)

172 (54)

212 (66)

1880-1889

1,731

394 (23)

109 (6)

836 (48)

1353 (78)

356 (21)

44 (12)

27 (8)

167 (47)

272 (76)

1890-1899

2,046

368 (18)

134 (7)

990 (48)

1657 (81)

461 (23)

32 (7)

51 (11)

222 (48)

348 (75)

1900-1909

2,362

446 (19)

133 (6)

1,167 (49)

1970 (83)

551 (23)

53 (10)

38 (7)

294 (53)

453 (82)

1910-1919

2,564

574 (22)

131 (5)

1,237 (48)

2141 (84)

600 (23)

72 (12)

53 (9)

291 (49)

462 (77)

6,868 (48)

11,198 (78)

2,902 (20)

374 (13)

Totals (%)

14,204

3,405 (24)

928 (7)

276 (9.5)

1,470 (50)

2,183 (75)

Table 2: Haplogroup assignments of Direct Cohort and Indirect Cohort Haplogroup

Direct Cohort

Indirect Cohort

Haplogroup Sub-Clade (n)

Individual Samples n(%)

Matrilineages n (%)

H

H1+152 (1), H1a1 (2), H1b (1), H1e1a (1), H1e1a1 (1), H1e1a4 (1), H2a2a (10), H2a2a1g (1), H4a1a (1), H6 (5)

24 (63)

16 (55)

1,442 (50)

179 (12)

126 (9)

1082 (75)

749 (51)

I

I (1)

1 (3)

1(3)

216 (7)

12 (6)

25 (12)

162 (75)

108 (50)

J

J1c2 (1)

1 (3)

1 (3)

235 (8)

22 (9)

24 (10)

171 (73)

116 (49)

K

K (1), K2a (1), K2b1b (1)

3 (8)

3(10)

159 (5)

29 (18)

8 (5)

130 (82)

87 (55)

T

T2 (4), T2a1b (1), T2b (1)

6 (16)

5(18)

383 (13)

57 (15)

45 (12)

276 (72)

189 (49)

U

U4a1 (1), U5a1 (1), U7 (1)

3(8)

3(10)

467 (16)

75 (16)

48 (10)

362 (72)

221 (47)

38

29

374 (13)

276 (22)

2,283 (75)

1,470 (50)

Totals (row %)

Births N (%)

2,902

Death Date Derived n (%)

Death Before 20 n (%)

Alive Past 60 n (%)

Females n (%)

Figure 1: Lifespan (age at death by decade of the indirect cohort)

Figure 2: Lifespan by Haplogroup

Table 3: Hazard Ratios contrasting lifespan of haplogroups H and U within decade of death. Lifespan after 60 for Haplogroup H vs U Death Decade 1880-1889

Hazard Ratio

CI95

P-value 0.9985

1890-1899

1.85

(0.07-4.37)

0.6597

1900-1909

1.79

(0.76-3.82)

0.2385

1910-1919

1.95

(0.97-3.92)

0.0732

1920-1920

1.37

(0.83-2.26)

0.2443

1930-1939

2.35

(1.41-3.90)

0.0029 *

1940-1949

1.10

(0.72-1.70)

0.682

1950-1959

1.13

(0.74-1.70)

0.6179

1960-1969

1.13

(0.81-1.57)

0.482

1970-1979

1.31

(0.95-1.81)

0.1167

1980-1989

0.95

(0.70-1.27)

0.7262

1990-1999

1.16

(0.85-1.59)

0.3839

2000-2009 1.93 (1.12-3.32) 0.0304 * * p-value < 0.05 within the decade. Crude/unadjusted hazard ratios are presented.

Figure 3: Survival Curves of lifespan past 60 between 1930 and 1939.

7.0 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Giles, R.E., et al., Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci U S A, 1980. 77(11): p. 6715-9. Rius, R., et al., Biparental inheritance of mitochondrial DNA in humans is not a common phenomenon. Genet Med, 2019. Case, J.T. and D.C. Wallace, Maternal inheritance of mitochondrial DNA polymorphisms in cultured human fibroblasts. Somatic Cell Genet, 1981. 7(1): p. 103-8. Beckstead, W.A., et al., Evolutionary pressure on mitochondrial cytochrome b is consistent with a role of CytbI7T affecting longevity during caloric restriction. PLoS One, 2009. 4(6): p. e5836. Bonner, J.D., et al., Pedigree structure and kinship measurements of a mid-Michigan community: A new North American population isolate identified. Human biology, 2014. 86(1): p. 59-68. Schutte, D.L., et al., Community Health Needs Assessment in a Rural Setting: Foundation for a Community–Academic Partnership. Journal of Higher Education Outreach and Engagement, 2016. 20(2): p. 85-108. Goris, E.D., et al., Community leader perceptions of the health needs of older adults. West J Nurs Res, 2015. 37(5): p. 599-618. Lilienfeld, D.E., P.D. Stolley, and A.M. Lilienfeld, Foundations of epidemiology. 3rd ed. 1994, New York: Oxford University Press. ix, 371 p. Andrews, R.M., et al., Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet, 1999. 23(2): p. 147. Behar, D.M., et al., A "Copernican" reassessment of the human mitochondrial DNA tree from its root. Am J Hum Genet, 2012. 90(4): p. 675-84. Weissensteiner, H., et al., HaploGrep 2: mitochondrial haplogroup classification in the era of high-throughput sequencing. Nucleic Acids Res, 2016. 44(W1): p. W58-63. Institute, T.S., SAS 9.1.3 Help and Documentation. 2004: SAS Institute Inc. R: A language and environment for statistical computing. R Foundation for Statistical

Computing. 2014 October 13, 2019]; Available from: http://www.R-project.org/. 14. De Benedictis, G., et al., Mitochondrial DNA inherited variants are associated with successful aging and longevity in humans. FASEB J, 1999. 13(12): p. 1532-6. 15. Dato, S., et al., Association of the mitochondrial DNA haplogroup J with longevity is population specific. Eur J Hum Genet, 2004. 12(12): p. 1080-2. 16. Ross, O.A., et al., Mitochondrial DNA polymorphism: its role in longevity of the Irish population. Exp Gerontol, 2001. 36(7): p. 1161-78. 17. Niemi, A.K., et al., Mitochondrial DNA polymorphisms associated with longevity in a Finnish population. Hum Genet, 2003. 112(1): p. 29-33. 18. Niemi, A.K., et al., A combination of three common inherited mitochondrial DNA polymorphisms promotes longevity in Finnish and Japanese subjects. Eur J Hum Genet, 2005. 13(2): p. 166-70. 19. Benn, M., et al., Mitochondrial haplogroups: ischemic cardiovascular disease, other diseases, mortality, and longevity in the general population. Circulation, 2008. 117(19): p. 2492-501. 20. Bilal, E., et al., Mitochondrial DNA haplogroup D4a is a marker for extreme longevity in Japan. PLoS One, 2008. 3(6): p. e2421. 21. Costa, M.D., et al., Data from complete mtDNA sequencing of Tunisian centenarians: testing haplogroup association and the "golden mean" to longevity. Mech Ageing Dev, 2009. 130(4): p. 222-6. 22. Shlush, L.I., et al., Ashkenazi Jewish centenarians do not demonstrate enrichment in mitochondrial haplogroup J. PLoS One, 2008. 3(10): p. e3425.

23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

Castri, L., et al., A mitochondrial haplogroup is associated with decreased longevity in a historic new world population. Hum Biol, 2014. 86(4): p. 251-9. Castri, L., et al., Mitochondrial polymorphisms are associated both with increased and decreased longevity. Hum Hered, 2009. 67(3): p. 147-53. Ren, W.H., et al., Mitochondrial DNA haplogroups in a Chinese Uygur population and their potential association with longevity. Clin Exp Pharmacol Physiol, 2008. 35(12): p. 1477-81. Dominguez-Garrido, E., et al., Association of mitochondrial haplogroup J and mtDNA oxidative damage in two different North Spain elderly populations. Biogerontology, 2009. 10(4): p. 435-42. Pinos, T., et al., Are mitochondrial haplogroups associated with extreme longevity? A study on a Spanish cohort. Age (Dordr), 2011. Lorenc, A., et al., Mitochondrial DNA in Polish Centenarians. Toxicology Mechanisms and Methods, 2004. 14(1-2): p. 91-95. Feng, J., et al., Association of mtDNA haplogroup F with healthy longevity in the female Chuang population, China. Exp Gerontol, 2011. Yang, X., et al., Mitochondrial DNA polymorphisms are associated with the longevity in the Guangxi Bama population of China. Mol Biol Rep, 2012. 39(9): p. 9123-31. Courtenay, M.D., et al., Mitochondrial haplogroup X is associated with successful aging in the Amish. Hum Genet, 2012. 131(2): p. 201-8. Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population. Mitochondrion, 2014. 17: p. 7-13. Marom, S., M. Friger, and D. Mishmar, MtDNA meta-analysis reveals both phenotype specificity and allele heterogeneity: a model for differential association. Sci Rep, 2017. 7: p. 43449. Neel, J.V., Diabetes mellitus: a "thrifty" genotype rendered detrimental by "progress"? Am J Hum Genet, 1962. 14: p. 353-62. Enns, G.M., The contribution of mitochondria to common disorders. Mol Genet Metab, 2003. 80(1-2): p. 11-26. Wallace, D.C. and W. Fan, Energetics, epigenetics, mitochondrial genetics. Mitochondrion, 2010. 10(1): p. 12-31. Wallace, D.C., A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet, 2005. 39: p. 359-407. Wallace, D.C., Mitochondria as chi. Genetics, 2008. 179(2): p. 727-35. Wallace, D.C., The human mitochondrion and pathophysiology of aging and age-related diseases, in Molecular Biology of Aging, L.P. Guarente, Editor. 2008, Cold Spring Harbor Laboratory Press: COld Springs Harbor, NY. p. 1-38. Wallace, D.C., The epigenome and the mitochondrion: bioenergetics and the environment [corrected]. Genes Dev, 2010. 24(15): p. 1571-3. Wallace, D.C., Mitochondrial DNA mutations in disease and aging. Environ Mol Mutagen, 2010. 51(5): p. 440-50. Wallace, D.C., Mitochondrial genetic medicine. Nature Genetics, 2018. 50(12): p. 1642-1649. Wallace, D.C., Mitochondrial DNA variation in human radiation and disease. Cell, 2015. 163(1): p. 33-8. Barker, D.J., Fetal programming of coronary heart disease. Trends Endocrinol Metab, 2002. 13(9): p. 364-8.

Physiochemical differences between mitochondrial DNA (mtDNA) haplogroups that favor oxidative phosphorylation efficiency during periods of caloric limitation can lead to lifespan lengthening when food calories are less abundant. For example, prior work demonstrated that older female haplogroup H carriers had modestly lengthened lifespans beyond 60 years during the Great Depression, a time of caloric limitation in North America. The objective of the current study is to replicate the prior findings in an independent cohort that includes both sexes and younger ages. By determining and cross-referencing the mtDNA genotypes of a culturally homogeneous population isolate to the lifespans of their ancestors, we found that between 1930 and 1939, haplogroup H compared to haplogroup U carriers had a modestly lengthened lifespan (3 years) past 60 years (hazard ratio 2.35; CI95 1.41-3.90; p-value: 0.0029). The lifespan-lengthening association was apparent in both sexes but only after the age of 60. Our results provide further support for the role of mitochondrial genetics in lengthening human lifespan.

Highlighted changes between revision 2 and revision 3 50 differences: 232 lines, 378 inline differences in 208 changed lines Added in Revision 3(12,118) Deleted from Revision 2(12,77) Changed Lines in Revision 3(208) Changes within changed paragraphs(183)

Revision 2

Revision 3

Abstract

Abstract

Previous work presented physiochemical difference s between mitochondrial DNA (mtDNA) haplogroups that might favor oxidative phosphorylation efficiency during periods of caloric limitation. The efficiency differences might offer carriers a lifespan lengthening when food calories are less abundant. The prior work demonstrate d that female haplogroup H carriers had modestly lengthened lifespans beyond 60 years, during the Great Depression. The current study r eplicates the findings of the prior work. We find that between 1930 and 1939, haplogroup H compared to haplogroup U carriers had a modestly lengthened lifespan past 60 years (hazard ratio 2.35; CI95 1.41-3.90; p-value: 0.0029 ). The lifespan-lengthening association was apparent in both sexes and principally in the decade of the Great Depression.

Physiochemical differences between mitochondrial DNA (mtDNA) haplogroups that favor oxidative phosphorylation efficiency during periods of caloric limitation can lead to lifespan lengthening when food calories are less abundant. For example, prior work demonstrate d that older female haplogroup H carriers had modestly lengthened lifespans beyond 60 years during the Great Depression, a time of caloric limitation in North America . The objective of the current study is to replicate the prior findings in an independent cohort that includes both sexes and younger ages. By determining and cross-referencing the mtDNA genotypes of a culturally homogen eous population isolate to the lifespans of their ancestors, we found that between 1930 and 1939, haplogroup H compared to haplogroup U carriers had a modestly lengthened lifespan (3 years) past 60 years (hazard ratio 2.35; CI95 1.41-3.90; p-value: 0.0029 ). The lifespan-lengthening association was apparent in both sexes but only after the age of 60 . Our results provide further support for the role of mitochondrial

1.0 Introduction Mitochondria power cellular life. They convert chemical energy from carbon:carbon bonds into adenosine tri-phosphate or heat through oxidative phosphorylation (OXPHOS). OXPHOS sometimes generates reactive oxygen species (ROS). In homeostasis, ROS act as cellular signals. In excess, they damage nearby biomolecules including DNA. Mitochondria possess their own DNA outside the nucleus. Mitochondrial DNA (mtDNA) is a circular molecule of 16,569 bases. mtDNA encodes for 37 genes, 13 of which are essential for OXPHOS. mtDNA shows consistent maternal inheritance.[1, 2] Phylogenic trees of mtDNA haplotypes show global/regional groupings called haplogroups. Haplogroups connect the carrier with their distant maternal lineage s with predictable fidelity. Beckstead et. al. presented bioinformatic evidence for OXPHOS physiochemical variation at mtDNA T14766C. T14766C distinguishes haplogroup H from haplogroup U. This polymorphism codes the amino acid 7 of cytochrome B. Haplogroup H encodes a threonine in the seventh amino acid position and haplogroup U encodes for an isoleucine. The physiochemical properties between the threonine and isoleucine might favor greater OXPHOS efficiency during calorie-limited periods. [3] Beckstead et. al. posit that during the Great Depression in North America calories were limited. Their bioinformatic evidence predicted a lifespan lengthened phenotype in haplogroup H carriers when during the period of limited calories. They tested their prediction in a study of lifespan using death dates from genealogic records and haplogroups obtained from contemporary individuals. In order to minimize the potential competing effects, Beckstead et. al. studied only females , who lived beyond the age of 60 and died in North America. They found that between 1920 and 1940 females carrying haplogroup H lived 2.6 years longer on average than haplogroup U carriers.[3]

genetics in lengthening human lifespan. 1. Introduction Mitochondria power cellular life by converting chemical energy from carbon:carbon bonds into adenosine triphosphate (ATP) or heat through oxidative phosphorylation (OXPHOS). OXPHOS can generate reactive oxygen species (ROS) as a byproduct. ROS act as cellular signals to maintain homeostasis; however, excess production of ROS can damage nearby biomolecules including nuclear and mitochondrial DNA (mtDNA) . mtDNA encodes for 37 genes, 13 of which are essential for OXPHOS. mtDNA is maternally inherited [1, 2] and phylogenic trees of mtDNA haplotypes show global/regional groupings called haplogroups that connect the carrier with their distant maternal lineage s with predictable fidelity [3].

In a previous study, Beckstead et. al. presented bioinformatic evidence for OXPHOS physiochemical variation at mtDNA T14766C. This variation distinguishes haplogroup H from haplogroup U and changes amino acid 7 of cytochrome B from threonine in haplogroup H to isoleucine in haplogroup U. The different physiochemical properties of threonine and isoleucine favor greater OXPHOS efficiency during calorie-limited periods for haplogroup H [4]. Beckstead et. al. posited that during the Great Depression in North America calories were limited, and their bioinformatic evidence predicted a lifespan lengthened phenotype in haplogroup H carriers during the periods of caloric restriction. They tested their prediction in a study of lifespan using death dates from genealogic records and haplogroups obtained from contemporary individuals. To minimize potential competing effects, Beckstead et. al. studied only females who lived beyond the age of 60 and died in North America. They found that among females who died over the age of 60 between 1920 and 1940 haplogroup H carriers lived 2.6 years longer than haplogroup U carriers [4].

This work attempts to replicate the Beckstead et. al. findings in a different population and extend to both sexes, younger ages and other haplogroup co mparisons. In this work, we study lifespan variation during a specific epoch among relatives of known haplogroup carriers . The relatives within this work come from genealogic records of a population isolate. The population isolate is a community and extended family from a rural area in midMichigan . Most community and family members descend from a defined group of founders who settled the region in the mid-1830s. For two centuries, the descendants have farmed the same land , shar ed a similar diet, environment and climate. [4] Using the genealogic records of the population isolate, we assessed the haplogroup and lifespan association acros s “time” in a defined “place” focusing on the decade of the Great Depression 1930-1939. 2. Materials and Methods. 2.1 Genealogic data We previously presented the population isolate as a large extended family and community from a 90 square mile area circumscribing three rural municipalities in two contiguous counties in mid-Michigan [4]. The families and community are partners in the community-based Cooperative for Studies Across Generations (CoSAGE; www.cosage.org). The cooperative maintains a registry to which individuals may volunteer and donate a DNA sample and a pedigree database of descendants of the original immigrants. The pedigree database contains 37,315 people; 14,204 were born in the century between 1820 and 1919 and are now all deceased. In the study century, individuals were likely to have shared the same agricultural, cultural, economic conditions because they shared the same “place” during “time”. We define matrilineage as all the maternal descendants of a distant female ancestor. A matrilineage includes everyone who shares a female descendant of the distant maternal ancestor. A matrilineage includes males; but excludes their descendants. A small set of distant

This work extends the Beckstead et. al. findings in three ways: including both sexes, including younger ages, and analyzing other haplogroups in a different population . In this work, we studied lifespan variation during a specific epoch among relatives of known haplogroup carriers . The relatives within this work come from genealogic records of a population isolate from a rural area in midMichigan. Most members of this population descend from a defined group of founders who settled the region starting in 1835. For over 150 years, descendants have farmed the same land and shar ed a similar diet, environment , and climate [5]. Using genealogic records, we assessed the haplogroup and lifespan association across “time” in a defined “place” focusing on the decade of the Great Depression 19301939.

2. Materials and Methods 2.1 Genealogic data The population isolate used in this study lived in a 90 square mile area circumscribing three rural municipalities in two contiguous counties in mid-Michigan [5]. Members of the community are partners in the community-based Cooperative for Studies Across Generations (CoSAGE) [6, 7]. CoSAGE maintains a pedigree database of descendants of the founders and a registry to which community members they may volunteer and donate a DNA sample. The pedigree database contains 37,315 people; 14,204 were born in the study century between 1820 and 1919 and are now all deceased. A small set of 344 distant ancestors covers 67% of the extant genome[5].

maternal ancestors covers a large proportion of members of the pedigree. [4] 2.2 Two study cohorts The CoSAGE registry project recruited individuals through referrals, informational sessions, community newspaper advertisements, community bulletin boards, church bulletins, health fairs, community events, an internet web page, Facebook.com ®, and routine “walk-in” information sessions at the project office. At the events interested individuals could complete a contact request card. A CoSAGE team member would schedule a meeting to inform the potential participant about the project and discuss the informed consent. After consenting, participants would complete a questionnaire. A CoSAGE team member collected anthropometric assessments and a sample of venous whole blood or buccal cavity swab to bank their DNA.

2.2 Study cohorts Individuals were recruited who previously consented to the CoSAGE registry [6, 7]. Potential recruits received a study packet containing an introductory letter and a consent document for permission to use their banked DNA and corresponding genealogic data. Consenting CoSAGE registry participants comprised the study Direct Cohort. Deceased relatives of the Direct Cohort who had connected matrilineages in the pedigree database formed a larger, Indirect Cohort. Michigan State University Institutional Review Board approved the Informed Consent documents .

This study obtained an informed consent document distinct from main the CoSAGE registry. The Informed Consent documents were approved by the Michigan State University Institutional Review Board. For this project, we recruited individuals who previously consented to the CoSAGE registry. Potential recruits received a study packet containing an introductory letter and a consent document through the USPS . The consent document sought permission to use banked DNA and correlate genealogic data. The CoSAGE registry participants who returned a signed consented form comprise the Direct Cohort for this analysis. Deceased relatives of the Direct Cohort with connected matrilineages in the pedigree database form a larger, Indirect Cohort. 2.3 Lifespan phenotype We quantified the lifespan phenotype of the Indirect Cohort by the age of death. To maximize the sample size and avoid censoring data , we included those missing a death date by ascribing to each presumed dead individual the median death age of everybody else in the pedigree database who was born in their birth year.

2.3 Lifespan phenotype The lifespan phenotype of the Indirect Cohort was quantified by the age of death. To maximize the sample size and avoid censoring data , individuals missing a death date were assigned the median age of death in the pedigree database for their birth year [8].

2.4 DNA isolation and banking From buccal samples, DNA was isolated using Oragene™ DNA Self-Collection Kit following manufacturer recommendations (DNA Genotek Inc., Ottawa ON, Canada). From venous blood samples, we isolated DNA using the Quick-Gene -810 as recommended ( AutoGen, Holliston, MA, USA). Extracted DNA was banked in a freezer at -80o C until further consent allows retrieval. 2.5 Genotyping To amplify mtDNA from the noncoding/hypervariable region, we designed a forward primer (mt15596 5’-AAG TCT TTA ACT CCA CCA TTA GCA3’), and a reverse primer (mt326 5’GGC TGG TGT TAG GGT TCT TTG- 3’). The PCR amplification condition was an initial temperature elevation to 94⁰C held for 3 minutes, followed by 25 cycles of 94⁰C for 30 seconds, 60⁰C for 3 minutes and 72⁰C for 1 minute, and a final cycle at 72⁰C for 1 minute and held at 15⁰C. Sequencing aliquots of each PCR were prepared with the primers listed above and an internal primer (mt16523 5’-ACT TCA GGG CCA TAA AGC CT-3’). PCR products were sequenced using an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). 2.6 Bioinformatics Methods The files of sequence data were imported into Sequencher V2.1.6 (Gene Codes, Ann Arbor, MI, USA). DNA sequences were trimmed and ambiguous calls were reviewed by two investigators independently (JB and EW). Trimmed sequences were aligned with the revised Ptolemaic Cambridge Reference Sequence ( rCRS). [5, 6] mtDNA sequence variants from rCRS into HaploGrep[7] for Haplogroup scoring. 2.7 Data Management and Statistical Analysis We analyzed the lifespan phenotype using proportional hazards models and survival curves. Data management, manipulations and some statistical analyses were executed in SAS™ [8].

2.4 DNA isolation and banking From buccal samples, DNA was isolated using Oragene™ DNA Self-Collection Kit (DNA Genotek Inc., Ottawa ON, Canada). From venous blood samples, DNA was isolated using the Quick-Gene -810 following manufacturer recommendations (AutoGen, Holliston, MA, USA). Extracted DNA was banked in a freezer at -80o C until consent for retrieval. 2.5 Genotyping mtDNA from the noncoding/hypervariable region was amplified using a forward primer (mt15596 5’-AAG TCT TTA ACT CCA TTA GCA-3’) and a reverse primer (mt326 5’- GGC TGG TGT TAG GGT TCT TTG- 3’). The PCR amplification condition was an initial temperature elevation to 94⁰C (3 minutes), followed by 35 cycles of 94⁰C (30 seconds), 60⁰C (3 minutes), and 72⁰C (1 minute). After 25 cycles, reactions were held at 72⁰C for 1 minute followed by 15⁰C until retrieval . Sequencing aliquots of each PCR were prepared with the primers listed above and an internal primer (mt16523 5’-ACT TCA GGG CCA TAA AGC CT-3’). PCR products were sequenced using an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). 2.6 Bioinformatics Methods DNA sequencing data files were imported into Sequencher™ (Gene Codes, Ann Arbor, MI, USA). DNA sequences were trimmed , and ambiguous calls were reviewed by two investigators independently (JB and EW). Trimmed sequences were aligned with the revised Ptolemaic Cambridge Reference Sequence ( rCRS) [9, 10]. mtDNA sequence variants from rCRS were uploaded into HaploGrep [11] for haplogroup scoring. 2.7 Data Management and Statistical Analysis The life span phenotype was analyzed using proportional hazards models and survival curves [8]. Data management, manipulations, and some statistical analyses were executed in SAS ™ [12].

Statistical analyses and graphical output were composed in R™.[9]

Statistical analyses and graphical output were composed in R™ [13].

3.0 Results

3. Results

3.1 Pedigree Data

3.1 Pedigree Data

The pedigree database contains 37,315 individuals born between 1511 to 2013. There are 14,204 individuals born in the century that spans 1820 and 1919 and are likely to have died in the century that past between 1919 and 2019. Of the births that span the study century, 48% were female. The proportion of female births varied throughout the decades (p for trend <0.0001). Death dates were available for 72% of the pedigree database. Most individuals lived into adulthood with only 30% dying before 20. Of those born in the study century, 3,506/14,204 (27%) individuals lived past 79 years old, 1,134/14,204 (7.9%) lived past 89, and 115/14,204 (0.8%) lived past 99. Table 1 presents the characteristics of the pedigree data by birth decade.

The pedigree database contains 37,315 individuals born between 1511 to 2013; and 14,204 individuals were born in the study century that spans 1820 and 1919 and died in the past 150 years, 48% were female. The proportion of female births varied throughout the decades (p for trend <0.0001). Death dates were available for 72% of the pedigree database. Most individuals lived into adulthood with only 7% dying before 20. Of those born in the study century, 3,506 (27%) individuals lived past 79 years old, 1,134 (7.9%) lived past 89, and 115 (0.8%) lived past 99. Table 1 presents the characteristics of the pedigree data for the study century by birth decade.

3.2 The Direct Cohort We received consent and recruited 118 individuals of the 250 registry participants into the Direct Cohort. Of those eligible, 98 had a banked DNA sample. Fifty-nine of the donors were from matrilineages contributing births outside the study century. Thirty-nine donor samples were from 29 distinct matrilineages contributing births in the study century. N ine samples are replicates of the 29 matrilineages. 3.3 Genotyping and Haplogrouping We successfully extracted, amplified, sequenced, aligned and assigned mtDNA haplogroups for 38/39 samples from the Direct Cohort. All nine replicate samples matched the haplogroups of other samples within their matrilineage. We used the major clade name to ascribe haplogroups to matrilineages. Table 2 presents the frequency of haplogroup clades of both cohorts. 3.4 The Indirect Cohort The 29 matrilineages of the Direct Cohort pedigrees map to an Indirect Cohort of 2,902/14,204 (20%) of the

3.2 The Direct Cohort The CoSAGE registry contained 256 potential participants. We received consent and recruited 118 individuals into the Direct Cohort. Of those, 98 had a banked DNA sample. Fifty-nine participants were from matrilineages contributing births after the study century. Thirty-nine donor samples were from 29 distinct matrilineages contributing births during the study century. Nine samples are replicates of the 29 matrilineages. 3.2.1 Genotyping and Haplogrouping of the Direct Cohort We extracted, amplified, sequenced, aligned, and assigned mtDNA haplogroups for 38/39 samples from the Direct Cohort. All nine replicate samples matched the haplogroups of other samples within their matrilineage. We used the major clade name to ascribe haplogroups to matrilineages. Table 2 presents the frequency of haplogroup clades of both cohorts. 3.3 The Indirect Cohort The 29 matrilineages of the Direct Cohort pedigrees map to an Indirect

presumably dead individuals born during the study century. The proportion of derived death dates, shortened lifespans, and lengthened lifespans differs by birth decade intervals. The proportion of females born by birth decade is 50% in the Indirect Cohort. Table 1 presents characteristics of the Indirect Cohort by birth decade. There are fewer derived death dates in the Indirect Cohort than in the pedigree database in general (13% vs 30%). Table 2 presents the characteristics of the Indirect Cohort stratified by haplogroup. The Indirect Cohort contains 20 centenarians, eight from haplogroup H, seven from haplogroup T, three from haplogroup J and one each from haplogroup K and U. 3.5 Lifespan by death decade in the indirect cohort. The median lifespan of the indirect cohort increased between 1880 and 2009. (Figure 1). There is a modest difference in lifespan when contrasting all six haplogroups and death ages (0-120) (p-value: 0.017) (Figure 2) . We stratified and tested lifespan within each study decade individually. We find that the decade of 1930-1939 is one of two presenting differences in lifespan between haplogroups H and U. (Table 3) ( hazard ratio: 2.35; CI95: 1.41-3.90; p-value: 0.0029). In the target period of the Great Depression, the median lifespan of haplogroup H carriers was lengthened by 3 years. The lifespan lengthening difference was apparent in both sexes (Figure 3). There was also a modest lengthening of 1 year in the 20002009 decade.

4.0 Discussion In this study, we replicate and extend the findings of Beckstead et. al. We replicate the finding that during the Great Depression female haplogroup H carriers lived modestly longer than did haplogroup U carriers beyond the age of 60. We extend the findings by observing the lifespan lengthening in both sexes.

Cohort of 2,902/14,204 (20%) of individuals born during the study century. The proportion of derived death dates, shortened lifespans, and lengthened lifespans differs by birth decade intervals. The proportion of females born by birth decade is 50% in the Indirect Cohort. Table 1 presents characteristics of the Indirect Cohort by birth decade. There are fewer derived death dates in the Indirect Cohort than in the pedigree database in general (13% vs 30%). Table 2 presents the characteristics of the Indirect Cohort stratified by haplogroup. The Indirect Cohort contains 20 centenarians, eight from haplogroup H, seven from haplogroup T, three from haplogroup J , and one each from haplogroups K and U. 3.3.1 Lifespan by death decade in the Indirect Cohort The median lifespan of the Indirect Cohort increased between 1880 and 2009 (Figure 1) . There is a modest difference in lifespan when contrasting all six haplogroups and across all death ages (0-120) (pvalue: 0.017) (Figure 2) . We stratified and tested lifespan within each study decade individually. We found that the decade of 1930-1939, corresponding to the Great Depression, is one of two decades presenting differences in lifespan (beyond 60 years of age) between haplogroups H and U. (Table 3) (hazard ratio: 2.35; CI95: 1.413.90; p-value: 0.0029). In the target period of 1930-1939, the median lifespan of haplogroup H carriers was lengthened by 3 years compared to haplogroup U carriers . The lifespan lengthening difference was apparent in both sexes (Figure 3). There was also a lengthening of 1 year in the 2000-2009 decade.

4. Discussion In this study, we replicated and extend ed the findings of Beckstead et. al. Consistent with their report, we found that during the decade of the Great Depression haplogroup H carriers lived modestly longer (3 years) than did haplogroup U carriers beyond the age of 60. We extended

We also show a modest difference when contrasting all six haplogroups across all death ages. However, the effect fails to sustain significance when corrected for multiple comparisons. The difference in lifespan between 2000 and 2009 is outside the original objectives of this work to deductively replicate the Beckstead et. al. findings. The closed study cohort negates the need to censor because all members were dead. However, the closed cohort also does not contribute younger deaths since by 2000 the youngest cohort members were in their ninth decade of life. An open cohort with members born after 1920 and living into 2009 could potentially dilute the differences in lifespan presented by haplogroups H and U. Numerous studies show associations with mtDNA, longevity , and other phenotypes in cross-sectional study settings. Beckstead et. al. was the first to show a period specificity in lifespan and mtDNA patterns. Our replication of the Beckstead et. al. findings present strength for the time-period specificity of the mtDNA haplogroup association in a cohort study setting. To keep the full Indirect Cohort assembled and avoid the need to censor data in the survival analysis, we chose to impute missing death dates rather than exclude those with missing data . Our chosen method was naïve to haplogroup carrier status. The naivety should not have introduced any bias. However, any bias introduce d would have been nondifferential and favored a null hypothesis of no effect between haplogroups . Our rural and agricultural population isolate might not have had the same calorie-limiting pressures during the Great Depression. Nonetheless, our findings replicate a similar time-period specific effect. The original concept of this study was to test the well-established increased likelihood of living to centenarian as widely tested in many populations including Italians [10, 11], Irish[12], Finns[13, 14],

the findings by observing lifespan lengthening in carriers of haplogroup H in both sexes. We also show a modest effect when contrasting all six haplogroups across all death ages; however, the effect fails to sustain significance when corrected for multiple comparisons. This study has limitations. First, the closed study cohort negates the need to censor because all members were deceased. However, the closed cohort also does not contribute younger deaths because by 2000 the youngest cohort members were in their ninth decade of life. An open cohort with members born after 1920 and living into 2009 could potentially attenuate the differences in lifespan presented by haplogroups H and U. Second, to keep the full Indirect Cohort intact and avoid the need to censor data in the survival analysis, we chose to impute missing death dates rather than exclude those with missing data . Our chosen method was naïve to haplogroup carrier status. The naivety should not have introduced any bias. However, any bias introduced would have been nondifferential and favored a null hypothesis of no effect between haplogroups . Previous work has well-established the increased likelihood of living to centenarian in many populations including Italians [14, 15], Irish [16], Finns [17, 18], Danes [19], Japanese [18, 20], Tunisians [21], Ashkenazi [22] ,Costa Ricans [23, 24], Uyghurs [25] , Spaniards [26, 27], Poles [28], Chinese [29, 30], Amish [31], and Turks [32]. A recent pooled analysis of case-control studies showed centenarians are more likely to carry haplogroup J than are contemporaneous younger controls (Odds Ratio 1.7) [33]. However, our indirect cohort identified only one matrilineage carrying haplogroup J and only three centenarians disallowing any meaningful association testing. In addition, numerous studies show associations with mtDNA, longevity, and other phenotypes in crosssectional study settings. Beckstead et. al. was the first to show a lifespan lengthening using proportional hazard models during a specific period. Our replication of

Danes[15], Japanese [14, 16], Tunisians[17], Ashkenazi[18] ,Costa Ricans [19, 20], Uyghurs[21] , Spaniards [22, 23], Poles[24], Chinese[25, 26], Amish[27], and Turks[28]. A recent pooled analysis of case-control studies showed centenarians are more likely to carry haplogroup J than are contemporaneous younger controls (Odds Ratio 1.7).[29]. However, our indirect cohort harvested only one matrilineage carrying haplogroup J and three centenarians disallowing any meaningful association testing.

the Beckstead et. al. findings present strength for the time-period specificity of the mtDNA haplogroup association in a cohort study setting. The difference in lifespan between 2000 and 2009 is outside the original objectives of this work. Our findings can be interpreted within the context of the Thrifty Gene Hypothesis and the Wallace Hypothesis. The Thrifty Genotype Hypothesis postulates that genotypes efficient in extracting energy from alimentation during times of famine might contribute to chronic diseases (specifically diabetes) during times of plenty [34]. At first, the postulate mentioned nothing of mitochondrial contributions; however, l ater additions to the hypothesis included m tDNA variation and metabolic influences [35]. Our work contributes historic evidence in support of a thrifty mitochondrial genotype. Additionally, during periods of global human migration and settlement, populations adapted to new climate s and diets. The Wallace Hypothesis states that accumulation of mutations in mtDNA that are advantageous to new environments drives evolution of new mtDNA haplogroups; variation in mtDNAencoded OXPHOS genes has been correlated with increased thermogenesis at the expense of efficiency of ATP generation [3643]. Our work lends evidence, albeit observational and ecological , that differences in energy efficiency corresponding to haplogroup supports both the Thrifty Genotype Hypothesis and the Wallace Hypothesis. Our genealogic data lacks cause of death and chronic disease diagnoses . Thus, it cannot offer direct evidence about variations in diabetes risks correlated with the mtDNA haplogroup association. To build on our replicated findings, future studies of genealogic records or population isolates might correlate causes of death with year of death and year of birth by mtDNA haplogroup. By correlating mtDNA haplogroups, phenotypes, and historical records future investigations can seek evidence for mtDNA contributions to adult onset chronic diseases. Additionally, future work might link multiple generations across time and mtDNA haplogroup and study and

potentially trans-generational effects of parental environmental exposures like caloric limitation [44]. 5.0 Acknowledgements We acknowledge the Michigan State University through the Families and Communities Together (FACT) Coalition and a Strategic Partnership funding mechanisms for graciously funding this work .

5. Acknowledgements We acknowledge the Michigan State University through the Families and Communities Together (FACT) Coalition [PI: Schutte, B.C.; Schutte, D.L.] and a Strategic Partnership funding mechanisms [PI: Schutte, B.C.; Schutte, D.L.] for graciously funding this work. We also acknowledge Chris Ghandi of City of Hope National Medical Center scientific writing staff for valuable manuscript editing. And the reviewers for the helpful insights and persistence toward this final document. 6. Tables and Figures Table 1: Characteristics of Pedigree Database and Indirect Cohort by Birth Decade for the Study Century .