Fig. 4 Impact of polymorphisms in LASS1 on gene expression. A luciferase reporter assay was used for determining the promoter activity in transfected HeLa cells. Error bars denote SEM for six independent experiments with at least six replicates for each of the four promoter constructs shown. The LASS1 promoter variants CCT and CGT associated with exceptional longevity were compared to an AGC variant. The control is a reporter lacking inserted promoter sequences.

Discussion

We have found a combination of variants in three genes that is associated not only with exceptional longevity but also with healthy aging. This is a novel finding. An association of HRAS1 with longevity was found before, and an interaction with the mitochondrial genome was demonstrated (Bonafe et al., 2002). A compensatory effect in late life of variants in one gene for the deleterious effects of variants in a second gene has also been described (Bergman et al., 2007). The combined effect described here involves three genes and extends the association to old age. Genetic interactions play an important role in complex traits, but they cansubjects. The Georgia sample (N = 650) was recruited from 44 counties in Northern Georgia through voters registration files, surveys of nursing homes and personal care homes in the catchment areas, and random digit dialing for younger subjects. The Louisiana sample (N = 869) was recruited from eight parishes within a 40-mile radius of Baton Rouge through the Center for Medicare and Medicaid Services enrollment database for subjects 65 and older and voters registration files for younger individuals. Age of subjects was verified using demographic questionnaires and documentary evidence, and mortality data were collected using Social Security Death Index search. Subjects provided informed consent according to protocols approved by the respective institutional review boards. The demographic characteristics of the samples are tabulated (Table S7).

Small subsets of samples from Georgia and Louisiana were first independently genotyped to identify all detectable variation in the selected genomic regions. Only polymorphisms present at a frequency of over 0.09 were chosen for subsequent analysis in all samples. The haplotypes in the three genes are all present at high frequency in the European subpopulation in both Georgia and Louisiana.

The Alu sequences were amplified by PCR using locus-specific primers, and the PCR fragments were separated by agarose gel electrophoresis. The primers and methods used have been described previously (Bamshad et al., 2003; Watkins et al., 2003; et al., 2005). Subjects were classified as one or the other of possible homozygotes based on the presence of a particular sequence at a given locus or as heterozygotes. The Alu types thus obtained were analyzed to assign ethnicity using Structure (Pritchard et al., 2000). For this purpose, the same Alu genotypes from 715 independent DNA samples of subjects from around the world were used (Bamshad et al., 2003; Watkins et al., 2003; Ray et al., 2005). These spanned the geographic regions of Europe, Africa, Asia, India, and included sampling of individuals from many nations in each region (31 total). Subjects were then assigned populations of origin corresponding to these geographic regions using an assignment probability of 0.9. This provided sufficient power (> 0.95) to detect associations with exceptional longevity (Dai et al., 2007).

Genomic regions of interest were then amplified by the polymerase chain reaction (PCR) using specific primers for each gene. Amplicons were verified by agarose gel electrophoresis, and cleaned DNA was subjected to cycle-sequencing using the Big-Dye Terminator Reagent version 3.1 (Applied Biosystems, Foster City, CA, USA) and the same primers, and the 3130xl DNA sequencing system (Applied Biosystems). Identification of alleles was aided by the SNP detection and base-calling software SEQSCAPE v2.5 (Applied Biosystems). To minimize errors, both strands of DNA were sequenced twice independently. The genotyped loci included SNP: rs449647, rs769446, rs405509, rs429358, rs7412, rs8176330, rs8176331, rs8176332, rs8176333, rs8176334, rs8176335, rs12628, rs60774903, and rs3746263.

Haplotype blocks for the three genes were examined using Haploview. The standard color scheme is used for DLOD values: bright red (D = 1, LOD ≥ 2); blue (D = 1, LOD < 2); shades of pink/red (D < 1, LOD ≥ 2); white (D < 1, LOD < 2). The numbers shown inside the boxes are D values ≥ 100 (empty boxes represent the D value of 1). Haplotype blocks are defined based on confidence bounds on D. If the one-sided upper 95% confidence bound on D lies between 0.7 and 0.98, the SNP pairs involved are considered to be in strong LD (Gabriel et al., 2002). Haploview was also used to examine the Hardy-Weinberg equilibrium. None of the 15 SNPs examined in this study showed significance for departure from Hardy-Weinberg proportions in the Georgia and Louisiana samples stratified by Structure at a 0.9 assignment probability. Haploview is available at: http://www.broad.mit.edu/mpg/haploview.