Discussion

We have identified SNPs in the D-AKAP2 gene that are asso- ciated with morbidity by using a genome-wide association study from an age-stratified healthy population and 6,500 gene-based SNPs. The combined genetic and biochemical evidence points to the Ile(cid:1) Val variant as the functional polymorphism. The

PKA phosphorylates numerous substrates in response to - adrenergic stimulation that influence the contractility of cardiac myocytes (37). Some of these substrates include the L-type Ca2(cid:10)

channel, the ryanodine receptor, and phospholamban of the sar- coplasmic reticulum. AKAP-mediated targeting of PKA in cardiac myocytes has also been directly implicated in regulating cell con- tractility (38). Three AKAPs have been shown to interact with PKA in cardiac myocytes; muscle-selective AKAP (mAKAP), AKAP18 and Yotiao. mAKAP targets PKA to the perinuclear region of differentiated myocytes, coordinating both PKA and phosphodies- terase activity in a single complex (39). AKAP18 couples PKA to L-type Ca2(cid:10) channels, which enhances Ca2(cid:10) influx through the  channel after -adrenergic stimulation (40). Yotiao, an AKAP associated with N-methyl-D-aspartate (NMDA) receptors (41), has JES | been shown to interact with the KCNQ1-KCNE1 K channel subunits in human hearts (42). Mutations in this channel associated with hereditary long QT syndrome abolish Yotiao interactions with the channel, and attenuate the response mediated by cAMP (42).

channel or ion exchanger. The D-AKAP2 variants could impact the phosphorylation state of the ion channel or ion exchanger by etlaa recruiting different amounts of PKA-RI and might thereby mod- ulate heart contraction. This hypothesis fits the observed associa- tion with an EKG phenotype. The shorter depolarization intervals for Val(cid:1) Val individuals might be due to increased activation of ion channels in cardiac myocytes. However, the identification of this phenotypic association does not exclude the possibility that other phenotypic traits might be associated with this variant. Because it was identified by a general morbidity approach, it is possible that this functional variant has an impact on several biological pathways and can potentially lead to multiple clinical phenotypes.

In summary, we have demonstrated that screening for SNPs by using age-stratified healthy individuals is a useful and efficient way to identify functional variants in genes associated with morbidity. Because it is based on samples from the general population, this strategy may be able to identify major genetic components with significant health impacts and high penetrance. Through subse- quently analyzing significant morbidity markers in another well- phenotyped healthy twin population, we were able to associate a cardiac phenotype with the D-AKAP2 genotypes. This genetics approach may be able to establish early entry points into under- standing critical pathways that affect human health.

Direct involvement of PKA in developing heart disease comes from a recent study in which transgenic mice overexpressing the catalytic subunit of PKA developed dilated cardiomyopathy with reduced cardiac contractility and increased risk of arrhythmias (43). These cardiac abnormalities correlated with PKA-mediated hyper- phosphorylation of the ryanodine receptor and Ca2(cid:10) release from wepTa i the sarcoplasmic reticulum (43). RI is likely to play a role in cardiac physiology because it is the predominant isoform associated withthesarcolemmaincardiacmyocytes(44,45).Infact,mutations oakTa g in the RI gene are associated with both familial cardiac myxomas and Carney complex, implicating this isoform in cardiac growth and differentiation (32, 33).

We thank T. D. Spector from St. Thomas Hospital, J. L. Fridey and D. B. Perez from the Blood Bank of San Bernardino, CA, and D. Witthaus and N.BrownfromtheBloodBankCenterinRanchoMirage,CA,fortheirkind collaboration.The genetic analysis was partially supported by National Institutes of Health Grant CA83429 (to A.B.). The functional work was supported by National Institutes of Health Grant DK-54441 (to S.S.T.). L.L.B.-H. was supported by National Institutes of Health Training Grant 5T32 DK-07233. Sequenom, Inc., has an ownership interest in patent applications that have been filed and are directed to the subject matter of this paper.

The PKA signaling pathways associated with D-AKAP2 are not known. However, D-AKAP2 contains a PDZ binding motif (TKL) at the C terminus (Fig. 2a), which could serve as a targeting domain to membrane-bound receptors, ion channels or ion exchangers (26), ws? and two RGS domains, which could coordinate upstream G signaling with downstream PKA signaling. D-AKAP2 might there- fore be part of a signaling complex associated with a cardiac ion

D

ries,L.,Zheng,B.,Fischer,T.,Elenko,E.&Farquhar,M.G.(

)Annu.Rev. Pharmacol. Toxicol.

,

–

Harris, B. Z. & Lim, W. A. (

) J. Cell Sci.

,

–

Huang,L.J.,Durick,K.,Weiner,J.A.,Chun,J.&Taylor,S.S.(

)J.Biol.Chem.

,

–

Chen, Q., Lin, R. Y. & Rubin, C. S. (

) J. Biol. Chem.

,

–

Burns-Hamuro, L. L., Ma, Y., Kammerer, S., Reineke, U., Self, C., Cook, C., Olson, G.L.,Cantor,C.R.,Braun,A.&Taylor,S.S.(

)Proc.Natl.Acad.Sci.USA

,

–

Skalhegg, B. S. & Tasken, K. (

) Front. Biosci.

, D

–D

Cho Chung, Y. S., Pepe, S., Clair, T., Budillon, A. & Nesterova, M. (

) Crit. Rev. Oncol. Hematol.

,

–

Casey, M., Vaughan, C. J., He, J., Hatcher, C. J., Winter, J. M., Weremowicz, S., Montgomery, K., Kucherlapati, R., Morton, C. C. & Basson, C. T. (

) J. Clin. Invest.

, R

–R

Kirschner, L. S., Carney, J. A., Pack, S. D., Taymans, S. E., Giatzakis, C., Cho, Y. S., Cho-Chung, Y. S. & Stratakis, C. A. (

) Nat. Genet.

,

–

Torgersen, K. M., Vang, T., Abrahamsen, H., Yaqub, S. & Tasken, K. (

) Cell Signaling

,

–

Amieux, P. S., Howe, D. G., Knickerbocker, H., Lee, D. C., Su, T., Laslo, G. S., Idzerda, R. L. & M

night, G. S. (

) J. Biol. Chem.

,

–