Sample sizes are the same as those of the corresponding groups in Table frequencies for each SNP are given for the G allele.

tween old males and females. We applied the Bayesian, coalescent theory-based method (20) to construct haplotypes at these three tightly linked sites for each subject. The estimates of the disequi- Aw iliac3 librium (D ) between the 3 UTR and the I646V and In10 SNPs were 0.991 and 0.255 (r2 a2 —  0.55 and 0.03), respectively. The distance (eave3 between the markers in strong disequilibrium, 3 UTR and I646V Ae  which both showed association, is 4 kb, whereas the intron 10 SNP aes  is located 23 kb upstream of I646V (Fig. 4).

As expected, the changes in genotype frequencies between age groups for the three sites showed a similar level of statistical significance as the changes in allele frequencies (data not shown). a At the 3 UTR and the I646V variable sites, GG homozygotes were reducedandAAhomozygotesincreasedintheoldersampleofboth genders. This further supports the hypothesis that the G allele, which determines the Val allele at I646V, is associated with a negative health impact.

Fig. 2. Characterization of the Ile(cid:1)Val variant in the AKB domain of The residues comprising the hydrophobic face of the helix are highlighted (b) Pull-down assays using the C-terminal, AKB-containing portion of GST-AKB proteins were estimated by SDS gel electrophoresis. An excess of AKB was used so that the amount of R subunit bound to the glutathione reflects the binding affinities. Two separate experiments were performed used, a similar amount of the R subunit was pulled down; when half of amount of R protein was used, approximately half as much was bound. RII(cid:7)were used at concentrations of 2 and 20 (cid:2)M, respectively. (c) binding assay using a fluorescently labeled peptide of both the AKB BindingofRI(cid:7)toboththeIle((cid:1) results in an increase in binding to the RI(cid:7)isoform. The curves through the the AKAP binding domain, does not bind to either peptide. (Inset) The tion constants for the regulatory subunit isoforms.

We also genotyped Hispanic-American samples for the I646V variation. Because we had only a small number of Hispanic- American individuals over 60 years of age, we extended the older sample to all individuals older than 50 years of age (Table 2). Although females showed a statistically significant allele frequency   difference between old and young (P 0.008), the males did not, which may be due to the relatively small number of male individuals (Table 3). However, the allele frequency differences in both gen- ders were comparable to those observed in European Americans. The frequency of the G allele decreased by 0.129 in females and 0.061 in males, the GG homozygote by 0.087 in females and 0.072 in males (data not shown). These results further support the d/o association of the Val allele with morbidity and or mortality, and therefore the involvement of this gene in one or several disease processes. We subsequently verified another nonsynonymous D- AKAP2 variation, retrieved from dbSNP. The G–A transversion in exon 4 results in an Arg to His substitution at position 249 (R249H). The Arg was found to be in complete linkage disequilibrium with the Ile at position 646, occurring together in every case, and therefore shows the same age effect.

nominal level of 0.05. The estimate from the quantitative transmis- sion-disequilibrium test model of the average effect of the G allele  (Val) was to decrease the PR interval 6.3 units (P 0.007). The genotype means in the subset of 207 informative twin pairs were   wsaa manenh+s 157 23.4, 152 26.9, and 146 25.4 (mean SD) for genotypes AA, GA, and GG, respectively.

Association of D-AKAP2 Genotypes with a Cardiac Trait. In an effort

Functional Analysis of the D-AKAP2 Amino Acid Variant. The variable position resulting from the SNP (I64V) is located in the AKB domain of D-AKAP2. This domain is the docking site for PKA (13) and is highly conserved among species (Fig. 2a). Similar to other AKAPs the AKB domain is predicted to form an amphipathic helix

Association of D-AKAP2 Genotypes with a Cardiac Trait. In an effort to identify traits correlated with the observed age association of the I646V SNP, we used a cohort of 417 fasting white twin pairs with extensive coverage for a variety of disease-related traits. Of the 97 traits analyzed, only the PR interval was statistically significant at a

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with hydrophobic amino acids on one face of the helix (21, 22). The length of the hydrophobic side chains on the hydrophobic face of the helix can alter PKA binding specificity (23, 24). The AKB domain of D-AKAP2 interacts with RI and RII isoforms of PKA and determines the extent of subcellular localization of PKA. The presence of two potential RGS (regulator of G protein signaling) motifs suggests the involvement of D-AKAP2 in G protein- mediated signal transduction (13, 18, 25). A putative PDZ-binding motif (TKL) at the C terminus is a protein-protein recognition module that plays a central role in organizing diverse cell signaling assemblies (26). The R249H variant is located in a spacer separating two halves of a potential RGS domain. The conservation of this position and surrounding sequence is very low compared with the AKB domain. Because there is no interaction partner identified for this spacer, the potential functional effect of R249H was not analyzed further.