Linkage Analysis. DNA was extracted from whole blood with QIAamp DNA Blood Maxi Kit from Qiagen (Chatsworth, CA) and dispensed into 384-well plates with a Hydra 9600 robot (Robbins, Sunnyvale, CA). The ABI Prism Linkage Mapping Set, Version 2 (Applied Biosystems), and True Allele PCR Premix (Applied Biosystems, Foster City, CA) were used for the genome-wide scan reactions on affected individuals as well as on control DNA from Centre d’E´tude du Polymorphisme Humain y (Paris) (no. 1347 02), to standardize the allele sizes during the editing process. The ABI Prism Linkage Mapping Set, Version 2, True Allele PCR Premix (Applied Biosystems, Foster City, CA), and PCR conditions are described in Applied Biosystems user manuals. Reactions were generated in an ABI 9700 PCR machine. The reactions were pooled with a Genesis RSP 150 robot (Tecan, Durham, NC), and products were separated on an ABI Prism 377 sequencer under standard conditions (gels were poured with Long Ranger Single Packs from BMA Biomedi-

Abbreviations: cM, centimorgans; apo, apolipoprotein; lod, logarithm of odds; hlod, heterogeneity, lod; MLS, maximum lod score.

heterogeneity, lod; MLS, maximum lod score. †To whom reprint requests should be addressed at: Howard Hughes Medical Institute, GeneticsDivision,Children’sHospitalandHarvardMedicalSchool,300LongwoodAvenue, Boston, MA 02115. E-mail: apuca@rascal.med.harvard.edu. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

†To whom reprint requests should be addressed at: Howard Hughes Medical Institute, GeneticsDivision,Children’sHospitalandHarvardMedicalSchool,300LongwoodAvenue, Boston, MA 02115. E-mail: apuca@rascal.med.harvard.edu.

GENOME SCAN of 137 SIBSHIPSchr20:     Segcgpes se genes oees geeesEJ vibe            iidil 89MThu Oct 12 10:58:39 EDT 2000

Fig. 1. Multipoint lod scores for the genome-wide scan. Individuals in 137 sibships demonstrating exceptional longevity were genotyped at 400 marker lines on each graph represent MLS 5 2.0, a score slightly higher than the average maximum score expected by chance once in a genome scan.

are available for the study. Because no prior hypothesis of a specific genetic mechanism for extreme longevity existed, and the limited information from most of these families precluded a meaningful estimate of such a model, an affected-only multi- point nonparametric analysis was performed (9). This analysis tested for excess sharing of chromosomal regions that were identical by descent in the sibships. The nonparametric analysis [maximum lod score (MLS)] was performed by using the GENE- HUNTER 2.0 program (10).

cals). GENESCAN ANALYSIS Software 3.1 (Applied Biosystems) was used for size processing together with ROX 400 size standard (Applied Biosystems). The allele-calling software was GENOTYPER 2.5, from Applied Biosystems.

DNA samples of individuals were initially genotyped with 400 simple tandem repeat markers with an average heterozygosity of 0.70 and an average marker density of 10 centimorgans (cM). Subsequently, loci suggestive for linkage were genotyped with additional markers (8). Sibships in which the observed genotype data were 105-fold more likely to have arisen if the individuals were unrelated, or half-siblings were excluded from the genetic analysis. In addition, those families with aberrantly high numbers of Mendelian inheritance errors were excluded (six families and one individual, a half-sib of a larger sibship, were excluded), thus resulting in 137 sibships and 303 individuals in the final analysis. Size standards and alleles were determined by using Applied Biosystems software. Allele frequencies were computed by using PEDMANAGER software (M.J.D., unpublished work), which esti- mates them from alleles present in founder individuals in each pedigree. Calculated allele frequencies did not differ from the Centre d’E´tude du Polymorphisme Humain (Paris) frequencies database. In suggestively linked regions, a higher-density map was analyzed, genotypes that implied double recombinants were identified, and the raw data were reexamined or retyped.

The genome screen of 308 individuals belonging to 137 families identified a region on chromosome 4 between D4S1572 5 and D4S406 as highly suggestive of linkage (MLS 2.67) (see Fig. 1). Fine mapping at an average of 1 marker every 3 cM was performed in a 20-cM region around this peak, resulting in an increased MLS (3.65) at marker D4S1564 (see Table 1).

To estimate the significance of the observation of linkage to chromosome 4, simulations under the hypothesis of no linkage were designed that match the family structure and marker heterozygosity of our study. One thousand genome scans were simulated with a genome-wide map density of 1 marker every 3 cM (matching the fine-mapping density performed in the region of maximum linkage), and in only 44 of those 1,000 genome scans 5 was the observed MLS of 3.65 exceeded (P 0.044).

Results

Previous studies indicate that parametric linkage analyses by using both a dominant and recessive inheritance model may be more powerful in some cases than nonparametric studies for

An obvious consequence of studying the exceptional longevity phenotype is that neither unaffected family members nor parents

Table 1. Nonparametric MLS and parametric hlod scores within the exceptional longevity susceptibility locus relative to markers in the chromosome 4 region

appropriately summarized and corrected by multiplying the more significant associated P value by 2, thus producing a final parametric P value of 0.052. These analyses provide significant evidence for heterogeneity, with odds in favor of heterogeneity of 1,800:1. For this dataset and trait, the parametric and non- parametric analyses corroborate each other and demonstrate that the results are robust to different sets of assumptions.