MAIMULT A MINIATUROMUTUUS010011863B2 TI WON HUN (12 ) United States Patent (10 ) Patent No. : US 10 ,011 , 863 B2 Lipsky et al. ( 45 ) Date of Patent : Jul. 3 , 2018 (54 ) METHODS FOR DETECTING ENHANCED Grafman , Vietnam Head Injury Study, Aug. 2007 ( Year: 2007 ). * NMDA RECEPTOR FUNCTION AND USES Lipsky et al . (2011 - 2012 ARDRAF Final ProjectReport, abstract ) . ( Year : 2011 ). * THEREOF Ioannidis (Nature Genetics , vol . 29 , pp . 306 - 309 , Nov. 2001) ( Year: 2001 ). * (71 ) Applicant: INOVA HEALTH SYSTEM , Falls Hirschhorn et al. (Genetics in Medicine . vol . 4 , No. 2 , pp . 45 -61 , Church , VA (US ) Mar. 2002) ( Year: 2002) . * dbSNP, SS4950068 , https: / /www . ncbi. nlm .nih .gov / projects / SNP / (72 ) Inventors : Robert H . Lipsky , Kensington , MD snp _ ss .cgi ? subsnp _ id = 4950068) ( Year : 2001 ) . * Lipsky, Robert H . , ‘ Functional Characterization of Promoter Po (US ) ; Mingkuan Lin , Fairfax , VA Lymor - Ph I Sms of the Human Grin2b Glutamate Receptor Gene (US ) ; Yang Jiang , Lexington , KY (US ) Associated With Altered Memory Functioning in Older Adults ' In : 2011 -2012 Ardraf Final Project Report, 2012 , 2 Page See p . 2 , 2nd ( 73 ) Assignee : INOVA HEALTH SYSTEM , Falls Paragraph . ??????? Church , VA (US ) Lipsky , Robert H ., “Understanding the Development of Alzheimer ' s Disease From the Perspective of the Aging Brain ' In : George Mason ( * ) Notice: Subject to any disclaimer, the term of this University College of Science Biology Department Seminar Fall patent is extended or adjusted under 35 2015 , Oct. 20 , 2015 , Johnson Center Room , 1st Sheet See 1st Sheet . U . S . C . 154 (b ) by 0 days . Yoo , Hee Jeong et al. , ' Family Based Association of Grin2a and Grin2b With Korean Autism Spectrum Disorders ' Neuroscience (21 ) Appl. No. : 15 /589 , 257 Letters . 2012 , vol . 512 , pp . 89 -93 Scc Abstract; Supplement Table Andreoli, Virginia et al ., ' Potential Involvement of Grin2b Encod (22 ) Filed : May 8 , 2017 ing the Nmda Receptor Subunit Nr2b in the Spectrum of Alzheim er ' s Disease ', Journal of Neural Transmission , 2014 , vol . 121, pp . (65 ) Prior Publication Data 533 - 542 See the Whole Document . US 2017 /0349943 A1 Dec . 7 , 2017 Jiang , Yang et al. , “ Functional Human Grin2b Promoter Polymor phism and Variation of Mental Processing Speed in Older Adults ' , Aging (Albany Ny) , Epub . Apr. 24 , 2017 , vol. 9 , No . 4 , pp . Related U . S . Application Data 1293 - 1305 See Abstract; p . 1294 , Right Column , 2nd -4th Para ( 60 ) Provisional application No . 62/ 341 , 883 , filed on May graphs ; p . 1296 , Right Column; p . 1299 , Left Column, 3rd Para graph ; p . 1300 , Right Column , 3rd . 26 , 2016 . International Search Report of PCT/ US2017 /031540 dated Aug . 8 , 2017 . (51 ) Int. CI. Lipsky H . Robert ; “ Understanding the Development of Alzheimer ' s CO7H 21/ 04 ( 2006 . 01 ) Disease from the Perspective of the Aging Brain " , George Mason C120 1 /68 ( 2018 . 01 ) University , College of Science , Biology Department Seminar, Fall C120 1 /6837 ( 2018 .01 ) (52 ) U . S . CI. 2015 . CPC . C12Q 1 /6837 (2013 . 01 ); C12Q 2600 /156 * cited by examiner ( 2013 .01 ) ( 58 ) Field of Classification Search Primary Examiner — Jeanine A Goldberg None (74 ) Attorney , Agent, or Firm — Polsinelli PC See application file for complete search history . (57 ) ABSTRACT (56 ) References Cited A method of determining the risk of cognitive decline in an aging subject is provided . Themethod includes analyzing an U . S . PATENT DOCUMENTS MRNA transcript including a GRIN2B nucleic acid 2007 / 0048767 Al 3 /2007 Martucci et al . sequence for the presence of the A allele in a biological 2009 /0253585 Al 10 / 2009 Diatchenko et al . sample obtained from the subject . The method also includes identifying the subject as having a decreased risk of cogni OTHER PUBLICATIONS tive decline when the A allele is present. Syvanen (Nature , vol. 2 , pp . 930 - 942 , Dec . 2001) . ( Year: 2001) . * 8 Claims, 7 Drawing Sheets carmenatent somJul. 3 , 2018 Sheetom 1 of 7 US 10 ,011 , 863 B2 5'noncoding DOXA FIG.1 ETS dqOIE. * GGAA ggGGGAcac 717**** * * atent Jul. 3 , 2018 Sheet 2 of 7 US 10 ,011 , 863 B2 GGAA/T rs3764030 A>G Coresequence Gallele GGGA Aallele GGAA ETScoresequence: )-(GTGT )-(GTGT )-(GGGA )+(GGAA : Stainedfor protein )+(GGAT FIG.2 )-(GGGA )+(GGAA Stainedfor dsDNA )+(GGAT CoresequenceETSsite(+or -) bufferpH6.3, ETS HEPES polydldcno Freeprobe atent Jul. 3 , 2018 Sheet 3 of 7 US 10 ,011 , 863 B2 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * hmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmin .*. ?????????????????? * 3 .* ** in * * * te - V444444444444 s' werden . * prostitutionen . * *-1* . 10 . w.wewe . NMDA(UM) V . *..-W FIG.3 com La * *W . * * . * . wwwwwwww . Aven . smo s . .4 Ratio Renilla/ Luciferase Firefly U. S . Patentatent JulJul.. 33 , , 2018 Sheet 4 of 7 US 10 ,011 , 863 B2 86 81 76Age ReactiontimevsAgeforrs3764030G/carriers FIG.4A 71 .. .. .. .. .. ) msec( time reaaction Mean atent Jul. 3 , 2018 Sheet 5 of 7 US 10 ,011 , 863 B2 w 85 wwde - - 80 ReactiontimevsAgeforrs3764030A/G+ wwwwalewwwaaa AgeAge carriers 75 FIG.4B verewewewewewewewewewewewewewewe'#** 70 .. .. .. .. .. .. ) msec( time reaction Mean 144444444444444444444444444444444444444444444444444444 atent Jul. 3 , 2018 SheetSheet6 6 ofof 7 7 US 10 ,011 , 863 B2 . Age ReactiontimesvsAgeforrs3764030GG FIG.5A . .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' .' . target to Times eactionReaction Mean U. S . Patent atent TopJul. "3 ,8102 2018 Sheet 7 of 7 US 10 ,011 , 863 B2 * www * * « * aoy VpueOVOCO9/CSIJOJBOVSAownuopoean * asbl * wwwwwwwwwwwwwwwwwww. * MYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYX * target to Times Reaction Mean US 10 ,011 , 863 B2 METHODS FOR DETECTING ENHANCED ing the single nucleotide polymorphism (SNP ) rs3764030 NMDA RECEPTOR FUNCTION AND USES variant A allele from the human GRIN2B gene promoter. THEREOF Double - stranded DNA targets (Methods ) were sequentially incubated with 1 ug recombinant human Elk - 1 protein in CROSS -REFERENCE TO RELATED 5 EMSA buffer, then incubated with the Elk - 1 antibody 20 APPLICATIONS minutes . DNA - protein complexes were Samples were elec trophoresed through 6 % polyacrylamide gels in HEPES This application claims priority to U . S . Provisional Patent buffer pH 6 . 3 , without polydldC . The gel was then stained Application No . 62 / 341, 883 , which was filed in the U . S . with the SYBR Green to visualize DNA and SYPRO Ruby Patent and Trademark Office on May 26 , 2016 , all of which 10 for protein bound to DNA . The stained gel was scanned is incorporated herein by reference in its entirety for all using G :BOX ( Syngene , US ) for imaging . Elk - 1 bound to purposes. DNA is indicated by the arrow . The instant application contains a Sequence Listing which FIG . 3 depicts a concentration dependent response of has been submitted in ASCII format via EFS - Web andLis is human GRIN2B promoter in N2a cells carrying either the A hereby incorporated by reference in its entirety . The 15 or G alleles of SNP rs3764030 to NMDA . Luciferase Sequence Listing is being concurrently submitted via EFS reporter gene plasmids were constructed in PGL 4 . 10 ( to Web as an ASCII text file named 084453 569289 Sequence produce Firefly luciferase, Promega , US) . Luciferase Listing . Txt, created Jun . 8 , 2017 . reporter plasmids and the PGL4 .75 plasmid (to produce Renilla luciferase as a reference ) were co - transfected into GOVERNMENTAL RIGHTS 20 retinoic acid differentiated N2a cells and assayed for (Meth ods ). After 24 hours of transfection , the cells were incubated This invention was made with government support under for six hours with 0 , 30 , 50 , 70 or 90 UM NMDA for grants P30 AG028383 and K01 AG000986 awarded by NIH . transcription factors binding and then replaced with com The government has certain rights in the invention . plete media (with 10 % FBS ) and cultured for an additional 25 40 -44 hrs. Data are presented as means + SE . FIELD OF THE INVENTION FIG . 4A and FIG . 4B depict graphs showing the mean reaction time with increasing age . Data was examined by The disclosure provides methods for predicting memory analysis of covariance ( ANCOVA ) , based on stratification performance and determining the risk for cognitive decline . by A allele carrier genotype group differences . Performance 30 as mean reaction time versus age in GG homozygotes is BACKGROUND OF THE INVENTION shown in FIG . 4A , while mean reaction time versus age in A allele carriers ( AA and AG genotypes combined ) is shown The earliest and the most severe memory loss in old age in FIG . 4B . A significant difference in the slopes of the and dementia occur in recentmemory and working memory . regression lines was observed ( - 3 . 994 + SE 3 . 460 for A allele Compared to young adults , older adults exhibit deficits in 35 carriers vs 5 .848 - SE 2 . 281 for G /G ; P = 0 .026 ) . working memory , an online memory mechanism that allows FIGS. 5A and 5B depict graphs showing the mean reac manipulation of a current target image amongst distracting tion time with increasing age . Data was examined by analy stimuli . Remote memory or long -term is better retained . sis of covariance (ANCOVA ) , based on stratification by A Evidence from neuropsychological and neuroimaging stud - allele carrier genotype group differences. Performance as ies of visual working memory indicates that working 40 mean reaction time versus