2. Materials and methods

2.1. Older subjects

Stessman and his colleagues have surveyed the social and medical conditions of this heterogeneous elderly population

medical conditions of this heterogeneous elderly population (Bursztyn et al., 1996, 1999; Cohen et al., 1995; Dresner- Pollak et al., 1996; Eliakim et al., 1996; Ginsberg et al., 1999; Maaravi et al., 1996; Stessman et al., 1995, 1996, 2000). From a representative systematic sample (from a geographically sorted electoral register) information was gathered on migration history, dwelling conditions, health status, health service utilization, employment status, activities of daily living, social support, use of drugs and war experience. Additional information was obtained from in-depth anamnesis and physical examination, as well as cognitive and psychological tests. In addition, a battery of biochemical and hematological blood tests were performed as well as urine analysis and culture, ECG and pulmonary function tests. DNA samples are available on approximately 400 of these subjects. The age of the population at the time of DNA sampling was 75 years. There were 150 males and 74 females in the older Ashkenazi group.

SNPs were assayed using an ABI SNaPshot kit and the products analyzed in an ABI 310 DNA analyzer. The primers for the first and second PCR reactions are shown below. A Reddy Mix master mix was used (Abgene, Surrey, UK) at a magnesium concentration of 1.5–2.5 mM MgCl . 2 The first PCR reaction was carried out as follows. The sample was initially heated at 95 8C for 5 min followed by 35 cycles of 95 8C (30 s), 55 8C (30 s), 72 8C (90 s) and a final extension step of 72 8C for 10 min.

MHTFR Forward GCTTTGAGGCTGACCTGAAG Reverse GGAGCTTATGGGCTCTCCTG Probe (23T)-CTGCGTGATGATGAAATCG CAD Forward CTGCACAAGTTCACGTCCAT Reverse TCTTGAGCACCTCGGGAAT Probe (34T)-CTCGGTCACGGCTGGCACC SOD2 Forward GGCTGTGCTTTCTCGTCTTC Reverse GTGCTTGCTGTGGTGCAG Probe TCTGCCTGGAGCCCAGATACCCCAAA Leiden factor 5 Forward GCCCCATTATTTAGCCAGGA Reverse GCCCAGTGCTTAACAAGACC Probe TTCAAGGACAAAATACCTGTATTCCT

2.2. Younger subjects

The younger (21.89 years, range 13–33 years) non- clinical population was recruited in ongoing studies of normal personality (Ebstein et al., 2000; Zohar et al., 2003) and eating behavior (Bachner-Melman et al., 2004). There were 121 males and 320 females in the young Ashkenazi group.

percentage of the apoE 4 allele (logistic regression: likelihood ratio = 9.236 P = 0.01, parameter estimate Wald statistic allele 2 = 5.438 P = 0.02, allele 3 = 8.628 P = 0.003; chi-square: male chi-square = 7.409, P = 0.025, 2d.f.; female chi-square = 5.453, P = 0.065, 2d.f.).

3.2. Methylenetetrahydrofolate reductase (MTHFR)

There is an increase in C allele frequency (67.4% versus 55.3%) in the older Ashkenazi females (logistic regression: likelihood ratio = 6.001 P = 0.014, parameter estimate Wald statistic allele C = 5.95 P = 0.015; chi-square: male chi- square = 0.227, P = NS, 2d.f.; female chi-square = 4.76, P = 0.029, 1d.f.). There is an increase in the percentage of CC homozygotes (logistic regression: likelihood ratio = 7.33, P = 0.026; chi-square = 6.199, P = 0.045) in the older female Ashkenazi group (50% versus 30.6%).

Superoxide dismutase 2 (SOD2)

The only significant result was an increase in the percentage of T allele (51% versus 33.3%) in older Ashkenazi male subjects (chi-square = 6.749, P = 0.009, 1d.f.) and a corresponding increase in the percentage (45.5% versus 25%) of TT homozygotes (chi-square = 6.545, P = 0.038, 2d.f.).

2.4. Statistical analysis

Comparison of allele and genotype frequencies was carried out using chi-square analysis and logistic regression as provided for in the SPSS Windows program. All polymorphisms, except for Factor V (young group), were in Hardy Weinberg equilibrium in both the young and old cohorts, examined separately. HWE was calculated using a convenient website: (http://www.kursus.kvl.dk/shares/vet- gen/Popgen/genetik/applets/kitest.htm). Considering that 11 alleles were studied and that we compared controls versus an older cohort, a conservative stand would be to conclude that a difference is significant for P-values < 0.05/11 = 0.0045. Nevertheless, the P-values reported in the results were uncorrected for multiple testing.

The only significant result was decrease in the percentage (30.6% versus 19.2%) of the A allele in older Ashkenazi male subjects (chi-square = 4.027, P = 0.045, 1d.f.) and a corresponding marked decrease (9.7–2.1%) in the percen- tage of AA homozygotes (chi-square = 5.790, P = 0.055,

3.5. Insulin-like growth factor 2 (IGF2) ApaI

An increase in the A allele was observed in older Ashkenazi females (chi-square = 3.455, P = 0.063) and a highly significant increase (3.8–15.7%) was observed in AA genotype in these subjects (chi-square = 10.972, P = 0.004, 2d.f.).

3. Results

years and used chi-square and logistic regression analysis to compare allele and genotype frequency for each of these candidate genes. In the chi-square analysis, results were stratified by sex whereas in the logistic regression sex was entered as a covariate. Sex was used to stratify the sample since a number of associations between common poly- morphisms and longevity appear to be gender specific (Bonafe et al., 2001; Ivanova et al., 1998; Lio et al., 2002a, 2002b, 2002c)

There was no significant association for any of the other polymorphisms examined.

Only significant findings are presented below.

As summarized in Table 1, we have examined 11 polymorphisms for association with a single outcome, survival to 75 years, by comparing a young Ashkenazi group to an older cohort. Although nominally significant associa- tions were observed it should be noted that only one is robust

Ashkenazi older subjects were characterized by an percentage of the apoE 2 allele and a decreased

Table 1

Summary of findings in the Ashkenazi ethnic group