(Reprinted) JAMA, October 15, 2003—Vol 290, No. 15 2031

©2003 American Medical Association. All rights reserved.

subclasses and 5 HDL subclasses of varying size. From the LDL and HDL subclass levels are calculated weighted- mean LDL and HDL particle sizes (nm diameter) and LDL particle concentra- tions (nmol/L). Lipoprotein sub- classes were grouped as large LDL (21.3-23.0 nm), intermediate LDL (19.8-21.2 nm), small LDL (18.3-19.7 nm), large HDL (8.8-13.0 nm), inter- mediate HDL (8.2-8.8 nm), and small HDL (7.3-8.2 nm). Although the LDL sizes ranged from 18.3 to 23.0 nm and HDL sizes from 7.3 to 13.0 nm, small changes within this range are associ- ated with marked clinical differences in CVD risks.

estimated from the slope of the linear regression of the traits of each parent on the mean value of offspring.17 For a comparison of the difference in CETP- I405V genotype frequency between the groups, Hardy-Weinberg equilibrium aN! was tested and the 2 test was per- Mw AP<. formed. P .05 was considered the threshold for statistical significance. Data are expressed as mean (SD or SE).

chain reaction product pool were then detectedcolorimetricallywithsequence- specific oligonucleotide probes immo- bilized in a linear array on nylon mem- branes stripes. Probe specificities had previously been confirmed by sequenc- ing and through use of DNA genotyped independently through other methods such as restriction length polymor- phism analysis.16 The CETP concentra- tions in human serum were measured by ELISA (Wako Chemicals USA Inc, Richmond, Va).

Lipoprotein Properties in Families With Exceptional Longevity

Statistical Analyses

Because the extreme old age of the pro- bands preempts a proper comparison (control) group, we also recruited the offspring of probands, some of whom presumably inherited longevity genes and thus should manifest the longev- ity phenotype. Candidate longevity phenotypes could then be compared between the offspring and age- matched Ashkenazi controls or non- Ashkenazi participants of the Framing- ham Offspring Study, neither of which are known to have longevity genes. There were no significant differences between the groups for routine blood chemistries,includingelectrolytes,liver function, and kidney function tests. Body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) was similar between offspring and the Ashkenazi control groups (P=.75 and P=.21 in women and men, respectively), both of which were significantly higher than in probands (TABLE 1). Typical mea- sures of lipoproteins, including total cholesterol, HDL, LDL, and triglycer- ideswerequitesimilarinprobandscom- pared with control group as well as between offspring and control groups. Only total cholesterol and LDL levels were lower in female probands vs con- trol groups, and HDL levels were sig- nificantly higher in female offspring compared with Ashkenazi control groups. In contrast, there were marked differences between groups in lipopro- tein particle sizes as determined by NMR. The HDL and LDL particle sizes inprobandsweremarkedlyhighercom- paredwithbothcontrolgroups(P=.001

Pairwise crude comparisons of lipid lev- els and lipid particle sizes among the study groups were performed by us- ing the Mann-Whitney U test because the distributions were skewed. Be- cause of strong correlations among the lipid variables, a multivariate analysis was also needed to disentangle the re- lationships between the different lipid variables and study group member- ship. We conceptualized the study groups as forming an ordered set, with probands containing the highest preva- lence, offspring an intermediate preva- lence, and controls the lowest preva- lence of longevity-promoting genes and phenotypes. Therefore, we used or- dered logistic regression to determine which lipid variables exhibited the strongest ordinal association with study group membership. Study group mem- bership (1 = control, 2 = offspring, 3=proband) was the dependent vari- able, and LDL, HDL, LDL-particle size, and HDL-particle size were the inde- pendent variables. In interpreting the results of this analysis, direct compari- son of the coefficients of the indepen- dent variables is not meaningful be- cause they are measured in different units. The corresponding z statistics are all dimensionless and directly com- mensurable, and were used as mea- sures of the ordinal association be- tween the lipid variables and study group membership. Calculations were performed by using SAS version 6.12 (SAS Institute, Cary, NC) and Stata ver- sion 8.0 (Stata Corp, College Station, Tex). Narrow sense heritability (h2) was

The LDL and HDL subclass distribu- tions and particle sizes determined by NMR are highly correlated with those measured by gradient gel electropho- resis and density gradient ultracentri- fugation.14,15 The analytical reproduc- ibility (given by the coefficient of variability) of LDL and HDL size is less than 0.5%,12 and the stability on re- peated drawing for LDL size was 0.9% and for HDL size was 1.1%.

CETP Genotyping and

Lipoprotein sizes are largely deter- mined by hepatic lipase and CETP, in addition to other possible pathways, therefore providing the rationale to examine variation in these genes. We sequenced the promoter region of hepatic lipase gene in 100 centenarians comparedwithcontrolsanddidnotfind different frequencies in known and unknown polymorphic markers in this region. We then genotyped several known CETP polymorphic markers: –631C/A(NCBIdbSNPrs1800776)and –629C/A (rs1800775) in the promoter; codon 405 isoleucine to valine (I405V) (rs5882) and D442G (rs2303790) in exons 14 and15, respectively; and G/A Amultilocuspolymerasechainreaction– based assay in the first nucleotide in intron 14 was used to genotype these polymorphisms.16 Briefly, DNA was amplifiedusingamultiplexreactioncon- tainingbiotinylatedprimerpairs.Ampli- fied fragments within each polymerase