Genotyping

DNA was isolated from whole blood or blood spot cards using the QIA- amp DNA Mini and Micro Kits (Qiagen, Hilden, Germany). Genotyping of the four candidate TERT SNPs (rs2736098, rs2853669, rs2853691, and rs33954691) and two candidate TERC SNPs (rs12696304 and rs3772190) was carried out by allelic discrimination using predesigned TaqMan(cid:2) SNP genotyping assays (Applied Biosystems, Carlsbad, CA, USA). DNA was amplified in a total volume of 5 lL containing 2.5 lL TaqMan Universal Master Mix (Applied Biosystems), 900 nM of each primer, 200 nM of each probe, and approximately 10 ng template DNA. PCR was performed

rs3772190 experienced reduced survival during old age, in line with the association of this SNP with shortened LTL, although the finding did not remainsignificantafterBonferronistep-downcorrection(P-corrected = 0.108 using a dominant model and P = 0.153 for comparison of GG vs. AG). If instead applying the less conservative Benjamini and Hochberg correction method, the P-values were 0.108 for the dominant model and 0.081 for GG vs. AG, respectively. In any case, this finding seems relevant, since

using the StepOne(cid:2) Real Time PCR instrument (Applied Biosystems) using standard conditions. Because of technical difficulties, genotyping of rs2853691 failed for 13.7% of the samples, probably because of poor sample quality. Although these failures were all because of lack of signal, and not because of insufficient discrimination between genotype clusters, this may potentially introduce bias. However, the 86.3% of the samples genotyped was in Hardy–Weinberg equilibrium, indicating that a differ- ential lack of one of the genotype groups is not the case. Moreover, the mean (age-adjusted and sex-stratified) LTL among the 13.7% nontyped samples was not statistical different from the mean of the 86.3% suc-

Acknowledgments

This study was supported by the Max-Planck Institute for Demo- graphic Research, (Rostock, Germany), the INTERREG 4 A pro- gramme Syddanmark-Schleswig-K.E.R.N. (by EU funds from the European Regional Development Fund), the National Institute on Aging (P01 AG08761 and R01AG030678), the Novo Nordisk Foun- dation, the Aase and Ejnar Danielsen Foundation, the Augustinus Foundation, the Brødrene Hartmann Foundation, the King Christian the 10th Foundation, and the Einar Willumsens Mindelegat Founda- tion. The Danish Aging Research Center is supported by a grant from the VELUX Foundation. Marlene Graff Sørensen, Susanne Knudsen, Steen Gregersen, Ulla Munk, and Shuxia Li are thanked for excellent technical work.

The 11 TERT tagging SNPs and the genotyping of these are described in the Data S1.

Measurement of LTL

Leukocyte telomere length measurements were performed by Southern blot analysis of the terminal restriction fragments, which were generated by HinfI and RsaI restriction enzymes, and the mean terminal restriction fragment length, which represents LTL, was calculated as previously described (Kimura et al., 2010).

Author contributions

Mette Soerensen contributed by generating the study concept and the study design and by acquisition of data, conduction of data clean up and data analysis, interpretation of data, and drafting the manu- script. Mikael Thinggaard contributed by providing data and data cleaning and assisting in establishing and conducting the statistical analysis. Marianne Nygaard contributed by acquisition of data. Qihua Tan contributed by assisting in establishing the statistical methods. Jacob Hjelmborg contributed by assisting in establishing the statisti- cal methods. Karen Andersen-Ranberg contributed by acquisition of data. Serena Dato contributed by generating the study concept and the study design and by acquisition of data. Tinna Stevnsner and Vil- helm A. Bohr contributed by generating the study concept and study design. Abraham Aviv contributed by acquisition of data and inter- pretation of data. Masayuki Kimura contributed by acquisition of data. Kaare Christensen contributed by generating the study concept and the study design and by interpretation of data. Lene Christiansen contributed by generating the conception of the study and the study design and by acquisition of data and interpretation of data. Authors have revised the manuscript and given their final approval.

Statistical analysis

Chi-square test statistics were applied for all cross-sectional associa- tion studies of genotype, allele, and haplotype frequencies using the Plink statistical program (http://pngu.mgh.harvard.edu/purcell/plink; Purcell et al., 2007). Among the subjects genotyped for the TERT and TERC candidate SNPs were 381 intact twin pairs, which possibly can lead to an increased risk of false positive findings because of the non- independency of twin samples. However, repeating all cross-sectional analyses while excluding one arbitrarily chosen twin from each pair completely mirrored the presented findings, thus leading to the same conclusions.

The mortality risk of genotypes in longitudinal data was estimated with STATA 11.1 (Stata Corporation, College Station, TX, USA) using a sex- adjusted, left-truncated Cox proportional hazards model to adjust for late entry into the data set according to age. Because intact twin pairs were included in the study, statistical analyses were performed using the robust estimator of variance assuming independence between the twin pairs. The proportional hazard assumption was evaluated using Schoenfeld residuals and performing an Aalen linear hazard model, and both sug- gested a change in effect with age for rs2853668, rs33954691, rs3772190, and rs3891054. Hence, an extended Cox model was performed splitting the effect up in age spans which the Aalen model supported.

References

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