Weeks and Lange also describe a more powerful multipoint variation of the APM statistic [32]. The multipoint statistic is simply the sum of S for a set of adjacent markers, but the ij appropriate null distribution must then account for non-indepen- dence among markers. To accommodate this requirement, the gene-dropping algorithm (above) was modified so that ‘‘chromo- somes’’ – sets of linked markers– passed from parents to offspring with between-marker recombination probabilities assigned by Haldane’s mapping function [33]. We simulated 500 null values per chromosome. Z scores and p-values were computed as described above. Multipoint APM (MAPM) scores were also computed for all pairs of markers adjacent to a locus on either side, at 1 centimorgan (cM) intervals; the technique weights the contribution of each marker inversely by the relative probability of recombination with the adjoining locus.

To assess the degree of sensitivity of the linkage signal on chromosome 3 to the criteria we used to assign ELP ‘‘affection’’ status, we repeated the APM analysis and reassigned affected individuals 3 ways, depending on their attained age: 100, 98, or 95 years (regardless of sex or family history). Results are shown in Figure 2 where the highest peak corresponds to D3S3547 (56.45 cM), the same marker with the highest signal by the original APM test and definition of ELP affected. Shifting the criterion up for attained age and reassigning affection status accordingly, yielded peaks for the same markers, although the magnitude of each peak was considerably diminished. A similar reduction in evidence for linkage on 3p with increasing affection threshold was observed by Boyden and Kunkel [1]. The true centenarians (attained age of 100 yrs) exhibited a second peak at marker D3S3521 (64.35 cM), sufficiently close to the linked region reported here to warrant further investigation. It should be noted that increasing the attained age threshold for ELP substantially reduced the number of individuals counted as affected, especially among true centenarians (195 individuals, 67 related pairs aged $95; 88 individuals, 13 related pairs aged $98; 49 individuals, 9 related pairs aged $100). Therefore, the added linkage peak effect

To adjust for multiple comparisons, we refer to the distribution of simulated Z-scores across all markers and multipoint intervals, and compute the experiment-wise p-values as described by Churchill and Doerge [34].

For selected regions that showed evidence of linkage in both the HMS and FLAG data (3p and 18q), we generated meta-nominal p-values following the method of Dempfle and Loesgen [35]. These p-values were adjusted for multiple comparisons by reference to the quantiles of the simulated Z-score distribution described above.

Results

Figure 1 shows p-values for linkage across the genome from the APM scan. Table 2 lists 19 markers with unadjusted asymptotic p- values,0.01. Table 3 lists 17 multipoint regions with multiple comparison-adjusted p-values,1.0. The most noteworthy linkage scores indicate regions of interest on chromosomes 3p24 and 18q22. In each of these two regions, markers adjacent to the one giving the highest linkage signal, also show evidence of linkage. The strongest linkage signal was observed at D3S3547, on

 19 22T17       

Figure 1. FLAG study linkage results for all autosomes. A) nominal singlepoint p-values; B) nominal multipoint p-values. doi:10.1371/journal.pone.0034746.g001

Table 2. Markers linked to exceptional longevity with nominal p-value,0.01.

Asymptotic Chromosome Marker Map Position (cM) Z p-val Adjusted p-value 1 D1S2667 20.36 2.84 0.00223 0.966 1 D1S2628 168.68 2.76 0.00288 0.976 3 D3S1297 5.05 2.35 0.00928 1.000 3 D3S1266 52.22 2.60 0.00469 0.998 3 D3S3547 56.45 4.19 0.00001 0.090 4 D4S1615 129.75 2.49 0.00634 1.000 5 D5S424 93.23 3.14 0.00085 0.752 7 D7S502 80.86 2.55 0.00545 1.000 8 D8S585 47.96 2.62 0.00438 0.996 8 D8S531 63.93 2.45 0.00705 1.000 8 D8S281 119.66 2.80 0.00254 0.970 10 D10S196 71.95 3.15 0.00082 0.740 15 D15S1507 68.15 2.67 0.00375 0.992 15 D15S216 75.09 2.41 0.00796 1.000 17 D17S1795 77.3 3.27 0.00054 0.626 18 D18S469 106.38 3.00 0.00137 0.890 18 D18S1161 111.05 3.30 0.00048 0.592 20 D20S432 49.66 2.57 0.00504 1.000 21 D21S1898 38.62 2.71 0.00338 0.988

Singlepoint scores per marker. doi:10.1371/journal.pone.0034746.t002

after adjustment may be due (all or in part) to an increase in noise as a result of a decrease in sample size.

Discussion

Table 3. Multipoint linked regions with adjusted p- value,1.0.

One immediate result of the FLAG study scan is an independent replication of Boyden and Kunkel’s finding of a genetic linkage between excess longevity and a locus in the 3p22- 24 region in the HMS data. Figure 3 compares the HMS and FLAG linked regions, based on Boyden and Kunkel’s Age Category 4 (upper 2.5% of the 1900 birth cohort). Superficially, the HMS linked region appears broader than the linked region we have observed. This may be a consequence of design differences between the FLAG and HMS studies: a sib pair analysis led to the HMS result, while an analysis of a wide range of kin relationships led to the FLAG result, for approximately the same total number of pairs in each study. Our sample from UPDB’s large multiplex families ensures greater variation in shared chromosomal segment length, and shorter average segments, among relative pairs contributing to linkage peaks. On the other hand, the microsat- ellite markers used in this study are less precise markers of location because the distance between them is much greater than for the SNP panel used by Boyden and Kunkel [1]. To remedy this, we are conducting fine mapping studies to more precisely define the shape and location of the linkage signal within the 3p22-24 region.