Amino acid variant in the kinase binding domain of dual-specific A kinase-anchoring protein 2: A disease susceptibility polymorphism

Stefan Kammerer*, Lora L. Burns-Hamuro†, Yuliang Ma†, Sara C. Hamon‡, Jaume M. Ca`naves†, Michael M. Matthew R. Nelson*, Charles F. Sing‡, Charles R. Cantor*, Susan S. Taylor†, and Andreas Braun*§

*Sequenom, Inc., San Diego, CA 92121; †Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093; and ‡Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109

Contributed by Susan S. Taylor, December 31, 2002

The focus of human genetics in recent years has shifted toward identifying genes that are involved in the development of common diseases such as cancer, diabetes, cardiovascular diseases, and Alz- heimer’s disease. Because many complex diseases are late-onset, the frequencies of disease susceptibility alleles are expected to decrease in the healthy elderly individuals of the population at large because of their contribution to disease morbidity and(cid:1)or mortality. To test this assumption, we compared allele frequencies of 6,500 single- nucleotide polymorphisms (SNPs) located in (cid:1)5,000 genes between DNA pools of age-stratified healthy, European-American individuals. A SNP that results in an amino acid change from Ile to Val in the dual-specific A kinase-anchoring protein 2 (D-AKAP2) gene, showed the strongest correlation with age. Subsequent analysis of an inde- pendent sample indicated that the Val variant was associated with a statistically significant decrease in the length of the electrocardio- gram PR interval. The Ile(cid:1)Val SNP is located in the A-kinase-binding domain. An in vitro binding assay revealed that the Ile variant bound (cid:1)3-foldweakertotheproteinkinaseA(PKA)-RI(cid:1)isoformthantheVal variant. This decreased affinity resulted in alterations in the subcel- lular distribution of the recombinantly expressed PKA-RI(cid:1) isoform. OurstudysuggeststhatalterationsinPKA-RI(cid:1)subcellularlocalization causedbyvariationinD-AKAP2mayhaveanegativehealthprognosis in the aging population, which may be related to cardiac dysfunction. Age-stratified samples appear to be useful for screening SNPs to identify functional gene variants that have an impact on health.

likely to be absent from such a sample because they are less likely to be eligible for donating blood. Hence, the frequency of a disease susceptibility allele is expected to be lower in samples of older blood donors. Similarly, a protective allele would be seen at a higher frequency in the older sample. We define variants with a decreased frequency due to morbidity and, to a small extent, mortality as diseasesusceptibilityalleles.Theidentificationoflongevitygenesby comparing genetic markers between young and elderly individuals, usually octogenarians or centenarians, has become more popular in recent years (3). The well known age-related associations of Alzheimer’s disease and risk of cardiovascular disease with the “Mu  4 allele of the apolipoprotein E (ApoE) gene provides the initial proof of concept (4, 5). However, our approach is clearly distinct from the previous approaches because our samples are primarily selected as being healthy and not by age.

In this paper, we report the results of a screen comparing allele ae  frequenciesof6,500SNPslocatedin 5,000genesbetweensamples of young and elderly healthy individuals. This resulted in the identificationofageneencodingafunctionalvariantwithanimpact on morbidity that may be involved in the etiology of cardiac dysfunction.

Materials and Methods

Study Sample. All subjects involved in our studies signed a written informed consent and the institutional ethics committees of par- ticipating institutions approved the experimental protocols. Sub- jects for the disease susceptibility genome screen were part of a sample that was recruited during a routine blood donation from private blood collection centers in San Bernardino and Rancho Mirage, California. The staff of the blood collection agencies invited all healthy blood donors to participate, and helped the subjectsfilloutaconsentformandasimplepersonaldatacollection form before sample collection. The data collection form included information about age, sex, body size, personal and family disease history, and ethnic background of both parents. Information about the identity of the study participants was not recorded. Subjects would be excluded if they failed to meet the blood donation eligibility guidelines established by the American Red Cross. A summary of the samples collected is presented in Table 1. Ethnicity was defined for each subject if they identified both parents as having wenc/g the same ethnic geographic background, otherwise they are indi- cated as ‘‘Other.’’ For the purpose of identifying disease suscepti- bility associated SNPs, we derived from this collection a discovery cohort consisting of male and female European Americans divided (26 into young (18–39 years) and old ( 60 years) groups. These groups and others used in this study are shown in Table 2.

single-nucleotide polymorphism  protein kinase A  health risk factor  cardiac dysfunction   morbidity