GoKawiilThe UM-HET3 sibship
UM-HET3 mice are progeny of a cross between two types of F 1 hybrids—female F 1 mice from matings of BALB/cByJ dams to C57BL/6J sires and male F 1 mice from matings of C3H/HeJ dams to DBA/2J sires. These four inbred progenitors are abbreviated CBy, B6, C3H and D2 when referring to mice and strains, and abbreviated C, B, H and D when referring to genotypes and haplotypes. These four fully inbred strains were selected to maximize phenotypic diversity. Young virgin F 1 males and females bred at the Jackson Laboratory (JL) JAX facility were transferred to ITP ageing colonies at JL in Bar Harbor Maine, the University of Michigan in Ann Arbor Michigan (UM) and the University of Texas Health Science Center in San Antonio (UT). Breeding cages were set up in spring. First litters were not used. All subsequent litters were used at UT, litters of 6 or more pups were used at JL, and those with 7 or more pups were used at UM. Weanlings were entered into the study over the next 7–8 months.
This study is based on tail samples acquired initially at the three ITP sites in accordance with standards of the Association for the Assessment and Accreditation of Laboratory Animal Care and recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, including annual reviews and approvals of all protocols. Links to data for ITP papers and data are available at the Mouse Phenome Database75.
All mice in the first four cohort years were born between April 2004 and January 2008. In these cohort years we have numerically well-balanced DNA samples from all sites. Almost no mice were generated in 2008 owing to a funding gap. All mice in the final cohort years used in this study were born between July 2009 and March 2013. The 2009 cohort includes mice from all sites, but 2010 and 2011 include mice only from UM and UT, and cohort years 2012 and 2013 include mice only from UM. The last UM-HET3 mouse in this study died in 21 December 2015. In all years, only a small percentage of mice (<10%) were born between January and April. Mice were weighed at 42 ± 2 days, and at 183 days (6 months), 365 days (12 months), 548 days (18 months) and at 730 days (24 months) with a timing error of ~7 days.
The UM-HET3 sibship segregates for ~10.6 million sequence variants (Supplementary Table 1). All UM-HET3 mice inherit C and B haplotypes from their F 1 mothers and H and D haplotypes from their fathers. As a result, the entire sibship segregates for four genotypes, the four two-way combinations of maternal and paternal haplotypes, on all autosomes—CH, CD, BH and BD. Females inherit one entire non-recombinant H-type chromosome X from their paternal grandmothers and a potentially recombined chromosome X from their mothers (recombinations between C and B haplotypes only; Fig. 3g). As a result, females have either CH or BH chromosome X genotypes. Hemizygous males inherit a potentially recombined C or B chromosome X from their mothers. All mitochondria and their genomes in all animals are derived from maternal grandmothers (C) and male chromosome Y is derived from paternal grandfathers (D).
We genotyped 6,872 UM-HET3 mice for which we had full lifespan estimates (n = 3,252 females, n = 3,620 males). Of these genetic siblings 6,438 passed all genotype quality control steps at 891 markers (n = 3401 females, n = 3037 males; see Fig. 1c and ‘Genotype quality control’). To ensure balanced numbers by sex later in life, every ITP cohort initially consists of 51 male and 44 female weanlings per year, per site and treatment category, but with twofold more common untreated controls at each site. This was done to enable overly aggressive males and any wounded mice to be removed while balancing male and female numbers later in life—almost always before 550 days. The earliest minimum inclusion age in this study is T 42 , the pubescent age at which tails were docked for tissue acquisition, an age equivalent to about 12 years in humans. For numerical convenience we set the first T-age at T 35 . To compensate for twofold higher male mortality in the first 2 years of life (33% versus 16%), we included 12% more males than females in the T 42 survivorship. The earliest death was at 46 days. This left us with a small surplus of 227 males at T 365 , but by T 560 , numerical balance was restored and there were 2,930 females and 2,929 males. This age corresponds to roughly 56 years of age in humans. All individual-level data, metadata, survivorships and mortalities per 15-day interval are provided in Supplementary Table 2.
We genotyped two major categories of mice—those not treated with any dietary intervention and mice treated with a dietary supplement that did not modify lifespan significantly on a per-drug basis using standard statistical criteria76,77,78. These latter mice have been referred to as ‘no drug effect’ (NDE) cases. However, when data are combined across the entire class of these individually ‘ineffective’ agents from 2004 to 2013, there is a highly significant combined effect that is positive on lifespan (Fig. 2i,j).
Husbandry
Mice were weaned into same-sex cages—three males or four females per cage—at 20 ± 1 days (ref. 22). They lived together from weaning to death without any replacements within cages. As a result, most mice lived alone after ~1,000 days, a factor to consider with respect to mortality risks, but not yet integrated into our analysis. From 2004 until 2013, all sites used NIH-31 standard diets. For breeding cages, UM used Purina 5008, UT used Teklad 7912, and JL used Purina 5K52. For mice up to ~122 days, UM used Purina 5001, UT used Teklad 7912, and JL used Purina 5LG6. After 2004, a single control diet—LabDiet 5LG6—was used by all sites. Mice were monitored daily for signs of ill health and aggression and euthanized if moribund. Tails were obtained at 42 ± 2 days for DNA extraction. Body mass at this age was acquired for 2,459 mice, and at half-year intervals for 4,688 mice at 183 days (6 months), and down to 2,208 mice at 730 days (24 months). Our analysis includes four experimental variables—sex, site, dietary drug treatment and cohort year (Supplementary Table 3). The class of nominally ineffective treatments included in this study are listed on Mouse Phenome Database ITP Portal under the acronyms 4OHPBN, CAPEhi, CAPElo, Cur, Enal, FOhi, FOlo, GTE, HBX, I767d, MB, MCTO, MET, NFP, OAA, Res07, Reshi3, Reslo3, Simhi, Simlo and UA.
DNA extraction, sequencing and sequence alignment
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