The functional significance of these length/sequence changes in the DRD4 protein, in a region that couples to G-proteins, has

DOI:10.1523/JNEUROSCI.3515-12.2013

38©°o      2o                                 2” 17.0%=260 2085z 48bp VNTR ie)Z Vv“50 a i Al rTiw6 40 Exon 1 Exon2 Exon3 Exon4ii}*30 i aos O7648 bp VNTR Roa 0.002"20 (Numerous Re «0.0019 49cSNPs) Room <0,001QR ooo <0,00110R sos <0,001AR ooo <0.001°o°0 10 20 30 40 50 60 70 80 90 100AGE1995 Cohort (Projected)110

Figure1. Survivalcurvesoftheoldest-oldcohort.Theknownmale(blue)andfemale(pink)survivalcurvesofEuropeanancestryindividualsbornin1910(theaveragebirthyearofparticipants) areshown(source:BerkeleyMortalityDatabase,http://www.demog.berkeley.edu/(cid:6)bmd/;UKandUSAdata).Individualsolderthan90yearsofagerepresent1.4%(male)to2.9%(female)oftheir birthcohort.Theprojectedcurveofthe1995birthcohort(orange)suggeststhat17%ofthiscohort(malesandfemales)willreach90(cid:2)yearsofage(SocialSecurityAdministration,AlternativeII Forecast, 1998, http://www.demog.berkeley.edu/(cid:6)bmd/). Inset, Diagrammatic representation of the human DRD4 gene region. Exon positions are indicated by blocks (yellow   noncoding; orange(cid:1)coding),andthepositionsofalurepetitivesequencesarerepresentedbypointedblueblocks.Thepositionofacoding48bpVNTRinexon3isindicatedbyagreentriangle.The2Rto11R variantsofthisrepeatareindicatedbelowexon3,alongwiththeirfrequenciesinEuropeanancestrypopulations(Dingetal.,2002;Gradyetal.,2003;Wangetal.,2004;Gradyetal.,2005;thisstudy).

been studied extensively. The 7R variant shows a blunted re- sponse to dopamine when compared with the 4R variant (As- ghari et al., 1995; Jovanovic et al., 1999; Wang et al., 2004) and does not form heteromers with the short isoform of the presyn- aptic dopamine D2 receptor (Borroto-Escuela et al., 2011; Gonza´lez et al., 2012). The DRD4 protein is expressed in several brain regions, with high levels in prefrontal cortex (Oak et al., 2000). DRD4 knock-out mice display better performance on complex motor tasks and reduced exploration of novel stimuli (Rubinstein et al., 1997; Dulawa et al., 1999). ADHD individuals with a 7R allele have faster reaction times than non-7R individu- als (Swanson et al., 2000; Langley et al., 2004).

is significantly associated with lower mortality risks (15–35%), and is not confounded by smoking, alcohol intake, caffeine consumption, body mass index (BMI), or a history of hypertension, angina, heart attack, stroke, dia- betes, rheumatoid arthritis, or cancer (Paganini-Hill et al., 2011).

Cell lines/DNA and genotyping/resequencing. Genotypes were obtained from DNA isolated from blood cell pellets from a subset of 310 subjects wee”» ~6 (91malesand219females, 60%ofindividualsstillalive)representative ake0+ ¢ oftheentire90 cohortwithrespecttogender,education,BMI,exercise, drinking, or medical histories. Two hundred seventy participants had complete information available from the Leisure World Cohort Study (Paganini-Hill et al., 2011). Lymphoblastoid cell lines were established for all 310 participants, using a modified protocol involving up to 5 months of cell culture (Grady et al., 2003). Detailed ancestry information was col- 0+ lectedfromthe90 Studyparticipantstoconfirmself-declaredancestryfor matchingtoEuropeanancestrypopulation-basedcontrolsampleDRD4ge-  notypes (N 2902) from ongoing studies (Ding et al., 2002; Grady et al., 2003, 2005; Wang et al., 2004). Controls ranged in age from 7 to 45 years of age (mostly adolescents and college students). Previously established meth- odstogenotypeandresequencetheDRD4VNTRvariantwereused(Dinget al., 2002; Grady et al., 2003, 2005; Wang et al., 2004). Single-nucleotide polymorphisms in other genes used as controls were genotyped by DNA sequencing. The Institutional Review Board of the University of California, Irvine, approved all procedures.

Given that the DRD4 7R allele is associated with increased activity/novelty-seeking in children and young adults (Faraone et al., 2001; Kluger et al., 2002; Grady et al., 2003, 2005; Li et al., 2006), we hypothesized that this allele would be associated with increased physical activity in the elderly. In addition, since phys- ical activity is associated with increased lifespan and health (Adlard et al., 2005; Larson et al., 2006; Willcox et al., 2006; Paganini-Hill et al., 2011), we hypothesized that the DRD4 7R allele would be over-represented in the oldest old.

Genotypes obtained for the 90 sample were as follows: 6 2R/2R, 27 2R/4R, 4 2R/7R, 4 3R/3R, 25 3R/4R, 7 3R/7R, 135 4R/4R, 1 4R/5R, 3 4R/6R, 82 4R/7R, 1 4R/11R, 1 5R/6R, 1 5R/7R, 1 6R/6R, 11 7R/7R, and 1 7R/8R. The genotypes of the control individuals were as follows: 60 2R/ 2R, 16 2R/3R, 345 2R/4R, 1 2R/5R, 58 2R/7R, 1 2R/8R, 4 3R/3R, 127 3R/4R, 17 3R/7R, 1715 4R/4R, 5 4R/5R, 1 4R/6R, 493 4R/7R, 1 4R/8R, 1 4R/9R, 2 5R/5R, 2 5R/7R, 2 6R/7R, and 51 7R/7R. There were no signif- icant deviations from the expected Hardy–Weinberg equilibrium.  Observed allele frequencies for the oldest-old sample were 2R 0.07,  aks       3R 0.06, 4R 0.66, 5R 0.005, 6R 0.009, 7R 0.189, and 8R 0.0016. Observed allele frequencies for the population and ancestry-       oan  a aeR< matched controls were 2R 0.09, 3R 0.03, 4R 0.76, 5R 0.002, 6R    atk R< ( 0.001, 7R 0.12, 8R 0.001, and 9R 0.001. The DRD4 7R/x category contains individuals with at least one 7R (or derived 7R, i.e., 5R, 6R, 8R, 9R, 10R, and 11R) allele, and the non-7R/x category the remaining individuals (Dingetal.,2002;Wangetal.,2004).Arepresentativesampleof140oldest- old individuals was resequenced at the DRD4 exon 3 polymorphism to de-

Materials and Methods