Note: MTTP ¼ Microsomal triglyceride transfer protein; SNP ¼ single- nucleotide polymorphism; NS ¼ not significant.

and 75 years and older. Between 1994 and 1996, all sur- viving participants were invited to provide a blood sample. The mean age at blood collection was 77.9 6 0.2 (standard error of the mean [SEM]) years. DNA samples were obtained from 1451 participants, and 1398 were success- fully genotyped for all SNPs. All participants provided written informed consent according to procedures required by the Local Research Ethics Committee, which also ap- proved the study protocol. Analyses of DNA and pheno- typic data were carried out anonymously to the identifiers of individual participants in the study.

R ESULTS

Successful genotyping was ascertained in 98.7 6 0.4% of all 11 SNPs in the MTTP gene with a genotyping error of 0.3% based on the replicate samples. All SNPs were in Hardy–Weinberg equilibrium in both the younger and the older age groups. The allele frequencies of the MTTP Q95H variant and the MTTP I128T variants are shown in Table 1. There were no statistically significant differences in any of these SNPs between the younger and older populations, indicating a lack of survival benefit for any of the variants. The haplotype distribution was based on 11 SNPs in the MTTP gene and yielded eight haplotypes with a frequency greater than 1%. The LD statistics for all 14 SNPs used in the younger control population are graphically plotted in Figure 1. The r2 values show that the SNPs MTTP-5038C/G, MTTP-164C/T, and MTTP-I128T are in complete LD. Of these three SNPs, only MTTP-I128T was therefore typed for the older population. Similarly, SNPs MTTP D98E and MTTP S166N were in complete LD, and MTTP D98E alone was typed in the older population. The D’ values from Figure 1 show that LD extends across the whole 52-kb region of the gene. The previously reported (7) MTTP hap- lotype nr 5 was split into two new haplotypes as the MTTP- 410G/A (MTTP_rs6532821) has not been used before. Table 2 shows the haplotype analysis using all the available information on genetic variability in the MTTP gene and demonstrates no differences in the distribution of these haplotypes between the younger and older populations (p ¼ .45), respectively. Table 2 also shows the minor allele frequency for each SNP. None of these frequencies showed a statistically significant difference between old people and the younger control cohort.

The younger control population was recruited from the Oxford Biobank (10), which is a population-based study of healthy 30- to 50-year-old men and women residing in Oxfordshire. The mean age at blood collection was 40.6 6 0.2 (SEM) years. A total of 777 participants had successful genotyping at all loci tested.

Genotyping

istries (11) at the high-throughput facility (KBioscience, Hoddesdon, U.K.). DNA was in 96-well plates, and every 10th sample was replicated as internal control to control for genotyping error. Haplotype calculations were based on 14 SNPs in the younger cohort and 11 SNPs in the older cohort. It was clear from the younger cohort that 4 SNPs were in complete LD; therefore, only one of these was used in the older cohort. Assessment of the haplotype structure and frequency was made according to Stephens and colleagues (12) using the PhaseV2 program with a 1000 iteration setting; the assessment was repeated five times.

Participants with more than two missing SNP data points were removed before running the PhaseV2 program. This was made to ensure correct input variables as the Phase program fills in missing data. In this process the program tends to bias towards the common variant. The genotype error was well below 1% but, due to the stringent criteria on missing data, the reported numbers represent 89% (haplo- type table) or 91% (Table 1) of all participants originally included. In addition, approximately 10 extremely rare (frequency less than 1%) haplotypes were removed. Al- though some of these haplotypes are likely to represent very uncommon variants, it is also recognized that such haplo- types may have arisen from genotyping errors, thus not representing true genetic variation of the sample.

We also looked at the frequency distribution for each of the 11 SNPs in 5-year age bands within the older people to see if there was an underlying age-dependent trend for any particular SNP frequency. No association of any the 11 SNPs with age was detected within the population.

D ISCUSSION

The MTTP gene associated (5) with longevity was identified after fine-mapping of a locus on chromosome 4 that had been identified by a genome-wide scan in a long- lived North American cohort (4). This finding was recently challenged in a large German case–control study that found

           255119rs881980a °5aa=Q244ES166N1128T098EQ95H-164-388-468-416  -5638g           6TISSzzs4es86TSss.0262!aeNOSTS g§é