Study subjects

The Georgia sample (N = 650) was recruited in the Georgia Centenarian Study from 44 counties in Northern Georgia. The sampling frame consisted of voter’s registration files and lists prepared from a survey of nursing homes and personal care homes in the catchment area for centenarians and random digit dialing for younger subjects. The Louisiana sample (N = 869) was recruited in the Louisiana Healthy Aging Study from eight parishes within a 40-mile radius of Baton Rouge. The sampling frame consisted of the Center for Medicare and Medicaid Ser- vices enrollment database for subjects 65 and older and voter’s registration files for younger individuals. Age of subjects was verified using both demographic questionnaires and documen- tary evidence. Mortality data were collected using Social Security Death Index search. Subjects provided informed consent accord- ing to protocols approved by the respective institutional review boards. The demographic characteristics of the samples are tabulated (Table S7).

Genotyping

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 ‡ 0.09 frequency were chosen for subsequent analysis in all

The haplotypes in the three genes are all present at high fre- quency in the European subpopulation in both Georgia and Lou-

the genome. The Alu sequences were amplified by PCR locus-specific primers. The PCR fragments were separated agarose gel electrophoresis. The primers and methods used been described (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 ethnic using Structure (Pritchard et al., 2000). For this purpose, same Alu genotypes from 715 independent DNA samples subjects from around the world were used (Bamshad et 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 tions in each region (31 total). Subjects were then assigned populations of origin corresponding to these geographic using an assignment probability of 0.9.

samples. This provided sufficient power (> 0.95) to detect asso- ciations with exceptional longevity (Dai et al., 2007). Genomic regions of interest were then amplified by the polymerase chain reaction (PCR). The primers, forward and reverse respectively, used to amplify the DNA regions of interest by the PCR were as follows: (i) the promoter region of the APOE gene, 5¢- GCGTCTGAGCGTTCACTGT-3¢ and 5¢-GTCCCAGTCTCGCAT- TCCT-3¢; (ii) exon 4 of APOE, 5¢-CTTGGGTCTCTCTGGCTCATC- 3¢ and 5¢-GCAGCCTGCACCTTCTCC-3¢; (iii) promoter of LASS1, 5¢-CAGCAAGTGACCCTCAGAGA-3¢ and 5¢-GACCTGGACCC- CGAGAGA-3¢; (iv) exon1 of LASS1, 5¢-GCCTGGGTTTCTCTG- CTG-3¢ and 5¢-GCCCGAGAGACCTTATCCTG-3¢; (v) promoter of HRAS1, 5¢-ATCCCAGCCTTTCCCCAG-3¢ and 5¢-TTCGCCCC- GCGCATGGGCT-3¢; (vi) exon 1 of HRAS1, 5¢-CAGGAGACCC- TGTAGGAGGA-3¢ and 5¢-CCTATCCTGGCTGTGTCCTG-3¢.

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, rs3746263, and an SNP in exon 1 (+234) in LASS1 that is not

Plot of pairwise linkage disequilibrium (LD) comparison

Haplotype blocks for the three genes were examined using Haploview. The standard color scheme is used for D¢⁄LOD 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 SNP examined in this study showed significance for departure from Hardy–Weinberg proportions in the Georgia and Louisi- ana samples stratified by Structure at 0.9 assignment proba- bility. Haploview is available at: http://www.broad.mit.edu/ mpg/haploview.

Some of the Louisiana samples were genotyped using the SNPlex technology (Applied Biosystems), which is based on the oligonucleotide ligation assay combined with multiplex PCR amplification. Primers were designed according to the SNPlex Design Pipeline for the following SNP: rs449647, rs769446, rs405509, rs8176330, and rs3746263. Size fractionation of genotype-specific products was performed by capillary electro- phoresis using the 3130xl DNA Analyzer, and genotype calls were made using the GENEMAPPER v4.0 software (Applied Bio- systems). The SNPlex genotyping was repeated twice indepen- dently.

Promoter analysis of LASS1 haplotypes