database. The dual-specific A kinase-anchoring protein 2 (D-AKAP2) gene has a single-nucleotide polymorphism (SNP) resulting in an amino acid change from Ile to Val in the A-kinase-binding domain. This SNP showed the strongest correlation with age in a screen of healthy European-American individuals. An independent sample analysis indicated that the Val variant was associated with a statistically significant decrease in the length of the electrocardiogram PR interval. An in vitro binding assay revealed that the Ile variant bound 3-fold weaker to the protein kinase A (PKA)-RI isoform than the Val variant. This decreased affinity resulted in alterations in the subcellular distribution of recombinantly expressed PKA-RI isoform. Our study suggests that alterations in PKA-RI subcellular localization caused by variation in D-AKAP2 may have a negative health prognosis 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.

The focus of human genetics has shifted towards identifying genes involved in common diseases such as cancer, diabetes, cardiovascular diseases, and Alzheimer's disease. To test the assumption that frequencies of disease susceptibility alleles are expected to decrease in healthy elderly individuals due to their contribution to disease morbidity and mortality, we compared allele frequencies of 6,500 SNPs located in 5,000 genes between DNA pools of age-stratified healthy European-American individuals. We define variants with decreased frequency due to morbidity and mortality as disease susceptibility alleles. Our approach is distinct from previous approaches because our samples are primarily selected as being healthy and not by age.

Efforts to identify susceptibility genes for common diseases have critical limitations such as small numbers of genes and high expenses. Linkage analyses have been proven expensive and ineffective at identifying disease susceptibility genes. However, shared public and private efforts to identify and validate SNPs genome-wide and improvements in SNP measurement technologies have enhanced the feasibility of genome-wide association studies.

DNA was extracted from blood samples of healthy individuals ascertained through blood donation centers. Age-, gender-, and ethnicity-specific pools were generated by pooling equimolar amounts of each DNA sample. All PCR and MassEXTEND reactions were conducted using standard conditions, and both alleles were analyzed to calculate allele frequencies.

Orthologous sequences were identified with PSI-BLAST against the NCBI database. The study suggests that age-stratified samples are useful for screening SNPs to identify functional gene variants that have an impact on health.and old groups. 

We used a non-redundant database and a BLOSUM62 weight matrix to identify orthologues of mouse D-AKAP2 (SwissProt O88845) from various species, including Homo sapiens (SwissProt O43572), Sus scrofa (SwissProt P57770), Drosophila melanogaster (SW:Q9V3I3), and Caenorhabditis elegans (SwissProt Q10955). Partial sequences from Anopheles gambiae (GenBank accession no. AJ281189) and Gallus gallus (GenBank accession no. AJ398916) were obtained from GenBank EST sequences. 

Allele frequencies were estimated based on independent mass spectrometry measurements of four analyte aliquots derived from a single PCR reaction, with females and males analyzed separately. SNPs that had P values of 0.05 among all measured SNPs were identified and validated through individual genotyping. 

Recombinant D-AKAP2 variant construction was carried out by fusing the 40 C-terminal amino acids of D-AKAP2 to the C terminus of GST and subcloning between the NdeI and BamHI sites of pRSET. The constructs were transfected into BL21 cells and expressed for 6 h at 20C. Fluorescence anisotropy was monitored using a Fluoromax-2 equipped with polarizers, with data averaged and fit to a 1:1 binding model using nonlinear regression application in GRAPHPAD PRISM version 3.00. 

D-AKAP2 codes for dual-specific A-kinase anchor protein 2, which is part of a family of scaffold proteins known as A-kinase anchoring proteins (AKAPs). AKAPs bind the regulatory subunit of cAMP-dependent PKA, and target the kinase to various intracellular locations. PKA is a broad specificity kinase and phosphorylates numerous proteins that function in many essential cellular processes such as metabolism, gene transcription, cell division, and neuronal transmission. 

We identified 50 markers of the 6,500 tested markers that show a reproducibly significant allele frequency change between the two age groups in at least one gender. The SNP that demonstrated the strongest association with age in both genders is located within the D-AKAP2 gene. We pursued a strategy that utilizes estimates of allele frequencies in DNA pools to screen large numbers of SNPs in a quick and affordable manner. 

An A 3 G polymorphism in the 3 UTR of D-AKAP2 showed a significant decrease of 8% (P 0.01) of the G allele in the older sample of both genders. The marker was individually genotyped and the frequency differences between young and old individuals calculated from the genotypes were very similar to the pool results. There was a slight skewing of frequencies in the pools likely resulting from uneven PCR amplification of the two alleles. This led to an underestimation of the G allele frequency in all pools but did not impact the significance of the differences between young and old groups.text, there are several errors that need to be corrected. Here is the processed text: