2.1. Subject selection

The Leiden 85-plus Study is a population-based study in which all inhabitants of Leiden, The Netherlands, aged 85 years and over were invited to take part. Between 1986 and 1988 a total number of 977 participants were enrolled in cohort’87 (Slagboom et al., 2000). Blood samples were taken at baseline. Survival data for all participants were available until May 2001 at which date only 15 had survived. Between 1997 and 1999 another 599 subjects were enrolled in cohort’97 on their 85th birthday (Bootsma-van der Wiel et al., 2002). Survival data were available until

We investigated the association between SHC1 gene variants and longevity in the Leiden 85-plus Study, which comprises of two independent cohorts of community dwell- ing citizens aged 85 years and over who were prospectively followed for survival. First, as sequence variation that would result in a survival benefit would be most easily detected in a sample of the oldest subjects, while sequence variation that would result in a survival disadvantage would be most easily detected in the sample of young subjects, we amplified and sequenced the p66SHC1 specific region of the coding sequences and the promoter of the SHC1 gene in a sample of extreme survivors of the Leiden 85-plus Study as well as in a young control group. In both cases, we set the limit for detecting sequence variations at a population frequency of at least 5%, as lower frequencies wouldstrongly limit the power of further analyses on the association of the sequence variation with longevity. Second, we tested for association between the Met410Val variant and mortality among these oldest old using a prospective follow-up design.

Based on the set limit for detecting sequence variations at a population frequency of at least 5%, thirty of the longest- lived subjects (median age 98 years) and 30 younger volunteers (median age 31 years) were selected for screen- ing for new polymorphisms in the p66 specific region of the SHC1 gene and the p66SHC1 promoter. The Met410Val polymorphism was determined individually in all Leiden

The medical ethical committee of the Leiden University Medical Center approved this study.

2.2. DNA isolation

For cohort’87 DNA was extracted from frozen mono- cytes shortly after inclusion by protein precipitation using potassium acetate and chloroform extraction. To increase sample size and DNA quantity, we performed new DNA extractions from stored frozen monocytes for this study,

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probe assay (Applied Biosystems) was used according to the manufacturer’s specifications with the following modifications. A qPCR core kit was used (Eurogentec) and half the amount of probes in a volume of 18 ml. As forward primer Met410Val-for1 and as reverse primer Met410Val-rev1 were used, resulting in a 122 bp DNA fragment (Table 1). Real time PCR was performed on an ABI 7700 (Applied Biosystems) using the standard protocol except 55 cycles in stage 3.

Table 1

Sequence of oligonucleotides used

Name Sequence Type Met410Val-for1 50-GGGCTTCTGGTCTCACCTTA-30 Primer Met410Val -rev1 50-CTCGGCTCCCTTCATACCT-30 Primer p66-for1 50-TGTGTCCGGTTTTGAGTGAC-30 Primer p66-rev1 50-CTGCATCCCAACGACAAAG-30 Primer Met410Val-probe1 50-TGGCAtCTGTTTGC-30 FAM probe Met410Val-probe2 50-AGGTGGCAcCTGTT-30 VIC probe

Bases in lower case demarcate the position of the SNP. All primers were designed using Primer3 software (Roze and Skaletsky, 2001). All probes were designed using PrimerExpress (Applied Biosystems).

2.6. Statistical analysis

The frequencies of variations in the p66SHC1 sequences were calculated and are presented as percentage with their 95% confidence interval. In all cases of a frequency of zero the ‘rule of zero equals three’ was applied. This rule states that the confidence interval of an observed frequency of zero reaches from zero to three (Hanley and Lippman-Hand,

using the QIAamp blood minikit (Qiagen). This yielded a total sample size of 736.

For cohort’97 DNA was extracted from buffy coats the QIAamp blood midikit (Qiagen). This yielded a size of 563.

2.3. PCR and purification

As virtually all subjects from cohort’87 had died, they were stratified for age at death in three groups: 85–90 years, 90–95 years and 95 and over. Frequencies of the different Met410Val genotypes were calculated for the separate groups and are presented as percentages. Significance was tested using a Chi-square and linear by linear correlation.

A standard PCR was performed with 10 ng of genomic DNA, using Amplitaq gold (Applied Biosystems). We used primers p66-for1 and p66-rev1 (Table 1), resulting in a 981 base pairs (bp) fragment. The PCR product was purified using the QIAquick PCR purification kit (Qiagen).

Survival curves from age 85 to 90 years were plotted for valine allele carriers and non-carriers in combined cohorts. The Cox proportional hazard model was used to calculate sex-adjusted mortality risks and their 95% confidence interval for both cohorts combined and separately. For cohort’87 a left truncated model was used.

2.4. Sequencing and assembly of

et al., 1977), with the following modifications. A 12 ml solution of 10 ng of PCR product and 6 pmol of primer in 10% DMSO was used. Primers p66-for1 or p66-rev1 were used. Sequencing was performed on an ABI PRISMw 3700 DNA Analyzer (Applied Biosystems). Sample files were processed using the STADEN Package (Staden, 1996). Sequences were assembled using a normal shotgun assembly and compared to the previously published sequence (GenBank accession number: AF455141). All trace files were reviewed by hand for mutations.

All calculations were performed with the SAS system, RELEASE 8.0.

3. Results