Telomere length can also be maintained through alternative lengthening mechanisms in the absence of telomerase activity, as described in cancer cells (Bryan et al. 1997; Reddel et al. 2001; Reddel and Bryan 2003). These alternative mechanisms include homologous recombination (HR) or non-homologous end joining (NHEJ). In NHEJ, which involves the Ku heterodimer and DNA ligase complex, DNA ends are directly fused without the need for substantial homol- ogy (Moore and Haber 1996). Ku consists of Ku70 and Ku80 encoded by XRCC6 and XRCC5, respectively (Critchlow and Jackson 1998; Riha et al. 2006). In normal cells with intact telomeres, Ku promotes genomic stability by repressing intra- or inter-chro- matid exchange via the telomeric repeats (Celli et al. 2006). In human somatic cells, induced loss of either Ku subunit results in cell death with substantial telomere loss, indicating that Ku is essential in

Circulating blood leukocytes have been a resource in telomere biology because of their abundance and the relative ease with which they are obtained. Leukocyte telomere length can be used as a reasonable estimator for the relative telomere length in other tissues, but this may not be applicable to all tissues (Friedrich et al. 2000; Thomas et al. 2008; Young 2010). It reflects the length of telomeres in cells

sample are shown in Online Resource 1. For the longevity association study, we examined 517 Cauca- sians consisting of 293 young controls, which were 21–59 years old, and 224 nonagenarian cases, which were 90-103 years old. All of these relatively-healthy, community-dwelling individuals are part of the Lou- isiana Healthy Aging Study (LHAS) (Jazwinski et al. 2010). Only the Caucasian subjects in LHAS were included to avoid confounding by population admix- ture and because of sample size considerations. To replicate the results of the longevity association study, 390 Caucasian (European-origin) subjects from the Georgia Centenarian Study (GCS) were examined, including 170 subjects C98 years of age and 220 young controls 20–59 years old (Jazwinski et al. 2010). The samples for the longevity association and telomere studies are summarized in Table 1. Subjects provided informed consent according to protocols approved by the institutional review boards.

maintaining normal telomere length (d’Adda di Fagagna et al. 2001; Wang et al. 2009).

The Ku complex interacts with a number of proteins. For example, Ku interacts with hTERT, the catalytic subunit of telomerase reverse transcriptase (Chai et al. 2002). Ku also interacts with SIRT1, as shown by co-immunoprecipitation (Jeong et al. 2007). SIRT1 is recruited to DSBs and is required for efficient DSB repair via HR (Oberdoerffer et al. 2008). Increased expression of SIRT1 in mice results in longer telomeres, an effect dependent on telomerase, and increased HR throughout the genome, including telomeres (Palacios et al. 2010). A catalytically inactive form of SIRT1 undermines deacetylation of Ku70 and its role in DNA repair (Jeong et al. 2007). These findings indicate that SIRT1 and Ku interact protecting from DNA damage and maintaining telo- mere integrity.

In this study, we have examined the association of two key genes in telomere maintenance pathways with age-related decline in telomere length. This telomere endophenotype was used to identify candidate genes for association with exceptional survival. Unlike XRCC6, SIRT1 was significantly associated with longevity.

Population stratification

Ethnic affiliation for the LHAS (N = 869) and GCS (N = 650) subjects was inferred genetically. One hundred informative, identical-by-descent Alu inser- tion polymorphisms were used with 715 reference subjects of known geographic ancestry (Europe, Asia, Africa, India) (Ray et al. 2005) employing the Struc- ture program (Pritchard et al. 2000), as described in detail previously (Jazwinski et al. 2010). Stratification of subjects was according to origin-population assign- ment probability greater than 0.80. Additionally, the mean frequencies of each of the individual Alu’s were compared in the Caucasian (European-origin) subjects

Materials and methods

For the telomere analyses, we examined 357 Cauca- sian (European-origin) subjects who were C60 years old. The phenotypic summary characteristics of this

Table 1 Demographic characteristics of population samples in longevity and telomere analysesa

Sample Caseb Controlc Additionald Total Male Female Male Female Male Female LHASe 84 140 100 193 0 0 517 (80) (130) (0) (0) (68) (79) 357 GCSf 25 145 84 136 0 0 390

a Number of subjects in telomere analyses are in parentheses b Nonagenarian in LHAS, centenarian in GCS c

–

years old d

–

years old e Louisiana Healthy Aging Study f Georgia Centenarian Study

(TRF) lengths determined by Southern blot analysis (Cawthon 2002, 2009; Pavesi et al. 2009). However, there seems to be a large between-study variation in the correlation of the two measures: One unit of T/S corresponds to approximately 6–10 kb in TRF length, depending on the reference sample used and other experimental conditions.

Telomere length determination

Average telomere length in white blood cells was estimated using a quantitative polymerase chain reaction method (q-PCR), as described (Cawthon 2002; Terry et al. 2008; Cawthon 2009) with modi- fications. Briefly, genomic DNA was extracted from whole blood and quantified using the Picogreen assay (Ahn et al. 1996). To estimate telomere length, two q-PCRs were applied to each sample, a single-copy- gene-specific q-PCR for b-globin and a telomere- specific q-PCR. A telomere/single-copy gene (T/S) ratio was calculated for each sample using the q-PCR results and normalized to the T/S ratio of a reference sample. The normalized T/S ratio was used as an estimate of the relative telomere length.

Other phenotypic variables