Since we could not predict what minimum age requirements would provide optimal power, ten sets of gender-specific minimum expected age at death requirements were applied to all subjects, with the hypothesis that as the cutoffs increased, the loss of power due to the decreasing number of families might be partially offset by an increase in genetic homogeneity and/or magnitude of effects in older subjects. As designated hereafter, Categories 1 to 10 cover the range from the upper 5% to the upper 0.2% tail of the birth cohort, corresponding to a minimum expected age at death of 90 to 100 for males and 95 to 104 for females (Table 1). Subjects that did not meet the age criteria for a category were removed from the analysis; for sibships with more than two siblings, individual siblings were excluded while retaining the sibship, whereas once either member of a sibling pair was eliminated, the entire sibship was removed.

Table 1. Subject characteristics by age category for Total group.

Age, gender, and sibship composition statistics for the Total group are given across ten categories defined by gender-specific minimum requirements for expected age at death. The designations 2-ship, 3-ship, 4-ship, and 5-ship refer to sibships with two, three, four, or five siblings, respectively. doi:10.1371/journal.pone.0012432.t001

LOD scores were noted only in genomic regions for which the nonparametric LOD scores were considered interesting (LOD$2). The hLOD scores were used to verify that the nonparametric results were robust to different assumptions and provide information about the possible mode of inheritance of the putative longevity allele. Empirical P values were determined solely on the basis of nonparametric LOD scores.

hypothesis of no linkage, applying the same marker map, allele frequencies, LD-corrected cluster definitions and haplotype frequen- cies (generated from the original scan with the --cfreq option), and missing data pattern as the actual data. The replicate pedigree files were successively trimmed nine times each to produce the corresponding pedigree files for Categories 2 to 10. These 1000 sets of ten replicates were analyzed identically to the actual data, and the number of distinct replicates or genomic locations in like replicates across all age categories that met or exceeded the observed LOD score for a given peak was recorded. If a replicate exceeded the observed LOD score in multiple categories at the same location, it was counted only once, just as in the original analysis.

We used the --rsq option in MERLIN [35] to accommodate linkage disequilibrium (LD) between markers. SNPs for which the pairwise coefficients of determination (r2) exceeded 0.16, above which LOD score inflation due to inter-marker LD becomes appreciable when parental genotypes are unobserved [36], were clustered with intervening SNPs and treated as a single multi- allelic marker. The error checking and Pedwipe functions of MERLIN were used to remove unlikely genotypes as implied by close double crossovers. Mendelian inheritance errors could not be identified due to the lack of parental genotypes.

We used the formula P=(r+1)/(n+1) to calculate empirical P values, where r is the number of replicates reaching the observed LOD score, and n is the number of replicates analyzed [38]. This formula produces a biased estimate of the true underlying P value, but provides a more accurate estimate of the type I error rate than the conventional, unbiased (yet anticonservative) formula P=r/n [38–41]. Upper 95% confidence limits for empirical P values were calculated using the conservative Clopper-Pearson exact method for a binomial distribution [42], as implemented in an online calculator [43].

Empirical P Values

To account for the multiple partially-dependent hypotheses represented by the ten age categories, we calculated an Overall empirical P value for linkage peaks from any category in the Total group that met the significance threshold of LOD =3.6 for a single genomewide scan with a fully informative marker panel [37]. Based on the error-wiped pedigrees from Category 1, MERLIN was used to generate 1000 replicates of simulated genotype data under the null

Results

In the Total group we identified a region on chromosome 3p24- 22 with an Overall maximum LOD score of 4.02 in Category 4,

, Age category 1 , Age category 6   lzopieg x                                                                          categoplata ls tals le] sie] o| mw] as} 2/19) 4} 15/16 |e |10|19|20b04 xcM, Age category 34 || 4a 32 2LoD LoD1 1oT 0a 1ola ta ls tals els |e] o| w] ar) | 19/14] 15/16] 07/18 |19|20b0d x ola tats tals tol sels! sla} {19} 10] 5]16|17 {10 10]a0dd xom cM, Age category 4 , Age category 94 4a 32 2LoD LoD1 10 °4 1plata lelals|els {elo | la] 2/19} 15/s5/s5|e7|10lrlaooeed x plata lelalste| sels] sla] ls} 1) s|s6| |e lsolaobder xom om, Age category 5 , Age category 104 4a 32 2LoD LoD1 1oF oa alt ha tats tals tel sels | sols) {10} 1) 15|16 |17|10 ol20pded x omWFR oD) RC ANIC | ocananss nlneann nee 7 Reece FAIN | Valen Cl lees @ | 219499

Figure 1. Genomewide nonparametric LOD scores by age category for Total group. Chromosome number is indicated at the bottom each plot. Red line denotes empirical genomewide significance threshold across all age categories of LOD=3.9. doi:10.1371/journal.pone.0012432.g001