Methods for predicting the health status of human subjects

Abstract

A method for predicting the health status of a human subject includes: (a) obtaining genomic DNA from a blood or saliva sample of the human subject; (b) observing unmethylated CG loci, which includes performing a bisulfite conversion treatment on the genomic DNA of the human subject; (c) comparing the unmethylated CG loci observed in (b) with unmethylated CG loci observed in genomic DNA taken from a reference group of human individuals; and (d) predicting the health status of the human subject by correlating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in a reference group of human individuals.

Description

Technical Field

[0001] Related Applications

[0001] This patent application claims the priority of U.S. Provisional Patent Application No. 63 / 472,428, filed on June 12, 2023, the disclosure of which is incorporated herein by reference.

[0002]

[0002] This specification generally relates to the construction and validation of a DNA reference matrix focused on specific unmethylated cytosine-phosphate-guanine sites / CG loci, and aims to (a) predict the levels of multiple proteins and metabolites in a human subject under test, and (b) correlate the predicted levels of multiple proteins and metabolites with the disease risk of the human subject. This enables better correlation of patterns with diseases using more information through methylation and creation of more complex phenotype (disease) predictions.

Background Art

[0003]

[0003] When the body ages faster than the passage of calendar time, it is in a state of accelerated biological aging, which is the greatest predictor of chronic diseases. Fortunately, there are lifestyle habits and medical interventions that have been proven to help slow down, stop, and even reverse the body's biological aging process. This specification relates to a method for measuring the baseline and changes in the aging rate of an individual, and gives the individual the power to guide their anti-aging or health promotion journey in the right direction.

Summary of the Invention

[0004]

[0004] A novel and improved method for predicting the health status of a human subject is provided. The method comprises (a) obtaining genomic DNA from a blood or saliva sample of a human subject by a method well known in the art, (b) A step of observing unmethylated CG loci in the genomic DNA of a human subject, which includes performing a bisulfite conversion treatment on the genomic DNA of the human subject, thereby converting cytosine residues in the genomic DNA of the human subject to uracil, while 5-methylcytosine residues in the genomic DNA of the human subject are not converted to uracil. (c) The step of comparing the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in genomic DNA taken from a reference group of human individuals, and (d) A step of predicting the health status of a human subject by correlating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in a reference group of human individuals. It includes, consists of, or essentially comes from The CG loci are cg00011943, cg00014020, cg00056433, cg00067133, cg00071360, cg00074145, cg00080105, cg00120123, cg00122614, cg00153759, cg0017 7243, cg00178119, cg00182994, cg00209129, cg00215550, cg00217225, cg 00236988, cg00237714, cg00243480, cg00251125, cg00262132, cg00275686 , cg00278547, cg00283022, cg00303773, cg00322993, cg00342468, cg0035 8895, cg00458337, cg00465312, cg00481382, cg00530438, cg00538458, cg0 0548856, cg00593298, cg00594167, cg00602326, cg00615271, cg00631329 , cg00639633, cg00646555, cg00687674, cg00687962, cg00698355, cg00712 841, cg00737155, cg00748449, cg00756943, cg00767058, cg00800095, cg0 0815399, cg00910115, cg00935026, cg00935421, cg00964035, cg00973876 , cg00993310, cg01020263, cg01025883, cg01067216, cg01089474, cg0109 4440, cg01098936, cg01113710, cg01134012, cg01166985, cg01169463, cg0 1178063, cg01224520, cg01243823, cg01253289, cg01266159, cg01271129 , cg01274656, cg01282852, cg01313513, cg01320510, cg01346718, cg01377 932, cg01380346, cg01402735, cg01410359, cg01446515, cg01457077, cg0 1541424, cg01545493, cg01549940, cg01579765, cg01586635, cg01606057,cg01610915、cg01645869、cg01652244、cg01660630、cg01715525、cg01770232、cg01809675、cg01824466、cg01826979、cg01844642、cg01849284、cg01857379、cg01858517、cg01865937、cg01892689、cg01893681、cg01894498、cg01929138、cg01930417、cg01956293、cg02001279、cg02007867、cg02042600、cg02048674、cg02055264、cg02056921、cg02058108、cg02081263、cg02084118、cg02084211、cg02094681、cg02106682、cg02132714、cg02217269、cg02246605、cg02310386、cg02339455、cg02345572、cg02371935、cg02380750、cg02390624、cg02415029、cg02426324、cg02444928、cg02447542、cg02471378、cg02474731、cg02489377、cg02500990、cg02509300、cg02524236、cg02534780、cg02605776、cg02613370、cg02618485、cg02643782、cg02672830、cg02675560、cg02720346、cg02721394、cg02731293、cg02740457、cg02762363、cg02768694、cg02798801、cg02867402、cg02909068、cg02942382、cg02981003、cg02987388、cg03007462、cg03031520、cg03044367、cg03082247、cg03137472、cg03138091、cg03139710、cg03240800、cg03245912、cg03261737、cg03328727、cg03340356、cg03340754、cg03356492、cg03357219、cg03368099、cg03400443、cg03401077、cg03417454、cg03423767、cg03441242、cg03443888、cg03458265、cg03463465、cg03474430、cg03514545、cg03517760、cg03546163、cg03549506、cg03549779、cg03550384、cg03558379、cg03587144、cg03593336、cg03605735、cg03626541、cg03636183、cg03644691、cg03650179、cg03654623、cg03676636、cg03707183、cg03732728、cg03741185、cg03741498、cg03743982、cg03760060、cg03772253、cg03785619、cg03796580、cg03807316、cg03834947、cg03839794、cg03858365、cg03874914、cg03876766、cg03877420、cg03926921、cg03935495、cg03945604、cg03989967、cg04030486、cg04045009、cg04046848、cg04060356、cg04064054、cg04079779、cg04083076、cg04114590、cg04115418、cg04137003、cg04139465、cg04148839、cg04156293、cg04161236、cg04162034、cg04172953、cg04181107、cg04204356、cg04206342、cg04213565、cg04214966、cg04225172、cg04227949、cg04230438、cg04235071、cg04241623、cg04276953、cg04285652、cg04322572、cg04393397、cg04398451、cg04411086、cg04436120、cg04439218、cg04444415、cg04460372、cg04480106、cg04484376、cg04545003、cg04567124、cg04584675、cg04595658、cg04615668、cg04707706、cg04733838、cg04742282、cg04757168、cg04768425、cg04781339、cg04789125、cg04799410、cg04836038、cg04846343、cg04847826、cg04859706、cg04875128、cg04885881、cg04902443、cg04907325、cg04980928、cg05021355、cg05024939、cg05067995、cg05169665、cg05185749、cg05187965、cg05196720、cg05201093、cg05230392、cg05236757、cg05239544、cg05258736、cg05270922、cg05280814、cg05298224、cg05331789、cg05338009、cg05352464、cg05380920、cg05399221、cg05418947、cg05422344、cg05423392、cg05426134、cg05442477、cg05481257、cg05485520、cg05498943、cg05506600、cg05517880、cg05551469、cg05575921、cg05616010、cg05635388、cg05666515、cg05669298、cg05671603、cg05676441、cg05679108、cg05715050、cg05720678、cg05721485、cg05726118、cg05729185、cg05777089、cg05786053、cg05802075、cg05820243、cg05828191、cg05830220、cg05845765、cg05901141、cg05910038、cg05913921、cg05941525、cg05951558、cg05987787、cg05989533、cg06001484、cg06030695、cg06047828、cg06062604、cg06068545、cg06072257、cg06083423、cg06159131、cg06169746、cg06213468、cg06232874、cg06251791、cg06279632、cg06295952、cg06319999、cg06335143、cg06374715、cg06401979、cg06429010、cg06454296、cg06478823、cg06519334、cg06535156、cg06572713、cg06572849、cg06580943、cg06602086、cg06604227、cg06610988、cg06621358、cg06630425、cg06639320、cg06642354、cg06645033、cg06660395、cg06665890、cg06667732、cg06672424、cg06675893、cg06679261、cg06683719、cg06692384、cg06696596、cg06753303、cg06784991、cg06789517、cg06812822、cg06870213、cg06874063、cg06878994、cg06888566、cg06926961、cg06958563、cg06975196、cg06984463、cg06997226、cg07015424、cg07023764、cg07057549、cg07076816、cg07097098、cg07151966、cg07193470、cg07196593、cg07202981、cg07304986、cg07323488、cg07351787、cg07363137、cg07365979、cg07393369、cg07404706、cg07426101、cg07434188、cg07462861、cg07529122、cg07541744、cg07558704、cg07569063、cg07601816、cg07613945、cg07620230、cg07624842、cg07629158、cg07640648、cg07643355、cg07668501、cg07672814、cg07674095、cg07675998、cg07692435、cg07702236、cg07714289、cg07768696、cg07778205、cg07828121、cg07855914、cg07871442、cg07871717、cg07899016、cg07951728、cg08075719、cg08094551、cg08103988、cg08113373、cg08125436、cg08126148、cg08132054、cg08173263、cg08182270、cg08259413、cg08281455、cg08284801、cg08312389、cg08335107、cg08342619、cg08350092、cg08369164、cg08375139、cg08421807、cg08424491、cg08439752、cg08443038、cg08506743、cg08551532、cg0857340、 4、cg08609249、cg08640901、cg08684580、cg08709765、cg08743947、cg08750459、cg08762603、cg08772194、cg08812936、cg08830214、cg08856764、cg08885224、cg08893036、cg08968830、cg08992229、cg09082985、cg09087222、cg09087669、cg09094866、cg09103812、cg09129997、cg09146197、cg09167379、cg09169335、cg09191731、cg09196193、cg09211372、cg09228052、cg09267188、cg09313734、cg09317728、cg09365138、cg09391799、cg09422291、cg09428537、cg09434029、cg09435875、cg09450907、cg09463001、cg09507782、cg09536390、cg09554406、cg09576223、cg09601629、cg09621460、cg09681594、cg09702859、cg09703048、cg09716613、cg09717927、cg09717936、cg09788524、cg09823288、cg09823713、cg09831942、cg09842685、cg09862733、cg09868598、cg09874992、cg09931939、cg09963257、cg09969145、cg09977126、cg09993780、cg10043414、cg10060499、cg10116893、cg10123377、cg10127085、cg10133386、cg10157184、cg10168894、cg10201382、cg10208550、cg10235817、cg10251070、cg10290474、cg10325507、cg10326287、cg10407585、cg10459672、cg10472537、cg10501210、cg10514580、cg10518439、cg10573018、cg10583266、cg10601230、cg10621420、cg10624914、cg10662975、cg10703784、cg10716444、cg10759025、cg10766585、cg10859114、cg10866445、cg10874644、cg10878114、cg10882522、cg10884112、cg10949164、cg10961323、cg10977392、cg10996589、cg11020001、cg11031278、cg11052535、cg11104177、cg11113469、cg11120021、cg11130780、cg11149163、cg11240908、cg11241278、cg11270070、cg11290628、cg11338041、cg11341981、cg11362848、cg11400286、cg11408477、cg11409205、cg11415498、cg11426563、cg11479653、cg11532431、cg11540692、cg11620909、cg11624211、cg11640253、cg11649376、cg11671400、cg11691908、cg11725835、cg11735780、cg11737758、cg11749817、cg11752250、cg11765003、cg11777043、cg11793132、cg11801086、cg11804239、cg11819402、cg11824202、cg11833003、cg11844835、cg11921111、cg11935615、cg11951427、cg11971423、cg11976052、cg12016314、cg12016809、cg12018436、cg12030941、cg12038299、cg12050358、cg12100785、cg12112853、cg12135976、cg12145721、cg12146151、cg12151545、cg12158389、cg12159504、cg12168623、cg12170419、cg12195980、cg12242038、cg12272488、cg12282371、cg12307092、cg12347617、cg12364808、cg12378187、cg12394821、cg12416359、cg12434362、cg12463857、cg12518665、cg12538421、cg12560256、cg12569593、cg12586992、cg12606517、cg12615235、cg12637448、cg12655260、cg12662084、cg12743638、cg12744031、cg12797254、cg12853426、cg12935715、cg12941754、cg12978573、cg12986327、cg13017801、cg13055484、cg13073141、cg13083251、cg13087259、cg13099106、cg13154925、cg13167828、cg13169967、cg13176778、cg13188098、cg13206721、cg13224583、cg13229291、cg13234653、cg13257436、cg13295463、cg13298859、cg13312094、cg13340220、cg13343932、cg13367272、cg13369416、cg13372562、cg13393601、cg13399544、cg13431929、cg13473880、cg13507563、cg13543436、cg13545297、cg13563074、cg13609948、cg13671730、cg13672444、cg13676215、cg13690812、cg13697829、cg13702196、cg13714975、cg13719443、cg13742305、cg13779009、cg13814747、cg13897675、cg13914486、cg13941682、cg13959647、cg13979305、cg13987110、cg14030785、cg14047961、cg14117377、cg14118991、cg14157578、cg14167430、cg14172633、cg14175748、cg14209041、cg14224028、cg14234520、cg14251509、cg14252140、cg14257435、cg14262357、cg14264125、cg14280181、cg14339287、cg14354398、cg14384748、cg14391737、cg14413904、cg14441271、cg14442890、cg14484844、cg14534474、cg14615491、cg14639293、cg14640588、cg14675598、cg14688662、cg14708893、cg14710896、cg14738170、cg14751023、cg14781394、cg14794281、cg14801398、cg14837598、cg14850945、cg14873958、cg14920334、cg14942542、cg14962363、cg14975009、cg14994914、cg15033140、cg15047582、cg15059204、cg15109292、cg15128785、cg15141757、cg15143202、cg15175143、cg15179011、cg15194100、cg15208687、cg15223345、cg15234627、cg15246131、cg15288815、cg15318748、cg15374686、cg15391651、cg15405515、cg15418593、cg15436354、cg15467112、cg15471501、cg15480367、cg15498435、cg15506060、cg15575700、cg15592143、cg15603964、cg15625940、cg15690995、cg15709989、cg15710198、cg15720821、cg15733713、cg15821589、cg15827092、cg15837235、cg15885703、cg15909933、cg15961042、cg15968648、cg15994304、cg16014202、cg16014412、cg16026114、cg16070035、cg16084969、cg16097191、cg16141165、cg16195970、cg16198075、cg16211224、cg16240781、cg16306900、cg16314089、cg16369168、cg16408865、cg16430267、cg16488565、cg16489878、cg16517790、cg16529483、cg16551483、cg16569522、cg16570202、cg16649588、cg16661213、cg16679001、cg16733203、cg16736896、cg16769369、cg16795184、cg16842060、cg16861968、cg16882373、cg16891431、cg16908208、cg16969368、cg16979095、cg16987876、cg16994082、cg17017746、cg17030092、cg17050472、cg17077421、cg17107501、cg17117243、cg17120358、cg17129809、cg17133012、cg17148219、cg17210497、cg17219432、cg17225923、cg17232357、cg17251114、cg17275047、cg17408993、cg17435901、cg17464298、cg17464436、cg17465569、cg17476910、cg17485717、cg17514701、cg17569235、cg17576228、cg17591595、cg17652174、cg17660663、cg17665268、cg17691612、cg17695543、cg17699330、cg17761940、cg17786697、cg17825846、cg17872672、cg17885794、cg17944885、cg17945419、cg17967443、cg17979595、cg17981101、cg17988830、cg18014413、cg18023598、cg18031708、cg18031880、cg18071894、cg18133309、cg18181703、cg18190506、cg18251449、cg18262201、cg18319024、cg18380497、cg18398019、cg18425341、cg18516816、cg18587063、cg18597252、cg18598029、cg18604560、cg18669861、cg18699242、cg18740583、cg18771034、cg18784500、cg18795169、cg18817318、cg18822243、cg18856359、cg18859711、cg18882407、cg18885210、cg18976649、cg19006431、cg19009870、cg19058685、cg19089073、cg19091784、cg19172278、cg19182279、cg19224645、cg19225755、cg19266329、cg19284211、cg19296509、cg19、 297245、cg19302996、cg19304797、cg19308663、cg19320294、cg19380156、cg19474865、cg19536401、cg19548511、cg19558933、cg19604384、cg19611570、cg19635897、cg19636673、cg19652271、cg19665696、cg19693031、cg19726712、cg19730248、cg19774733、cg19800670、cg19822434、cg19859910、cg19909400、cg19935065、cg19941446、cg19977924、cg19992304、cg20004147、cg20025086、cg20037601、cg20059597、cg20119148、cg20128319、cg20143092、cg20155714、cg20162239、cg20185773、cg20192259、cg20207443、cg20217702、cg20244340、cg20249402、cg20270599、cg20272789、cg20282550、cg20299697、cg20348196、cg20351734、cg20353489、cg20370296、cg20376953、cg20432211、cg20442128、cg20446824、cg20515136、cg20534585、cg20587151、cg20594802、cg20596493、cg20597382、cg20597477、cg20601028、cg20623853、cg20628696、cg20656525、cg20663241、cg20668177、cg20675702、cg20720208、cg20737582、cg20773320、cg20802942、cg20865054、cg20874261、cg20891771、cg20907051、cg20914958、cg20918218、cg20921972、cg20972814、cg20973476、cg21000454、cg21077074、cg21082913、cg21112943、cg21123355、cg21124655、cg21132686、cg21136789、cg21185661、cg21204522、cg21232671、cg21253043、cg21259215、cg21305555、cg21306240、cg21312434、cg21314942、cg21321148、cg21346818、cg21356998、cg21410629、cg21447975、cg21455901、cg21489638、cg21533743、cg21566642、cg21622536、cg21628390、cg21640508、cg21658235、cg21693883、cg21717369、cg21723004、cg21725954、cg21730080、cg21733098、cg21739934、cg21761881、cg21783047、cg21791657、cg21824343、cg21834645、cg21870393、cg21907521、cg21934504、cg21947254、cg21962907、cg21985831、cg21986679、cg21998324、cg22002034、cg22019884、cg22022821、cg22023206、cg22041417、cg22061523、cg22061769、cg22069247、cg22171714、cg22178367、cg22200330、cg22220710、cg22232492、cg22243260、cg22282672、cg22322653、cg22339775、cg22344631、cg22356226、cg22363327、cg22416596、cg22434421、cg22442430、cg22454769、cg22556008、cg22563987、cg22581353、cg22594985、cg22691256、cg22711187、cg22719651、cg22729726、cg22736354、cg22792461、cg22822140、cg22822803、cg22945467、cg22956922、cg22993154、cg23059366、cg23063236、cg23080422、cg23192918、cg23196035、cg23234847、cg23235141、cg23248055、cg23265105、cg23276290、cg23302570、cg23313424, cg23366493, cg23371851, cg23378546, cg23410824, cg23424407, cg23447131, cg23465508, cg23486701, cg23500537, cg23547619, cg23 549902, cg23561053, cg23570968, cg23574427, cg23586821, cg23615892, cg23643435, cg23793194, cg23838503, cg23879118, cg23883522, cg238940 58、cg23910413、cg23923562、cg23956385、cg23964820、cg24003560、cg24 027965、cg24055030、cg24055461、cg24112628、cg24141036、cg24185013、c g24220948、cg24236938、cg24241488、cg24262996、cg24266506、cg242933 44、cg24317857、cg24337318、cg24354818、cg24362016、cg24365657、cg243 72256、cg24392971、cg24397183、cg24432675、cg24463073、cg24468603、c g24492140、cg24507955、cg24520070、cg24523415、cg24530234、cg2454325 5、cg24577941、cg24589102、cg24601404、cg24674445、cg24678611、cg246 97099、cg24721873、cg24769628、cg24796651、cg24799795、cg24835295、cg 24920358, cg24925701, cg24967099, cg24976744, cg24982343, cg24997750, cg25047618, cg25063538, cg25114611, cg25146575, cg25148589, cg2515 6198, cg25161899, cg25181997, cg25189904, cg25202593, cg25259149, cg25259707, cg25330725, cg25331593, cg25361850, cg25375711, cg25397922cg25418001、cg25418670、cg25420482、cg25420747、cg25436217、cg25499 552、cg25504214、cg25560772、cg25563198、cg25569590、cg25623035、cg25 638549, cg25639566, cg25670171, cg25671708, cg25703588, cg25717260, cg25738786, cg25745729, cg25784308, cg25786980, cg25843115, cg258438 73, cg25903319, cg25916759, cg25927838, cg25928819, cg25943804, cg25945293, cg25952581, cg25952596, cg26029902, cg26044490, cg26071551, c g26093148、cg26116542、cg26116556、cg26156626、cg26229024、cg262394 85、cg26267310、cg26276120、cg26301353、cg26314853、cg26315261、cg264 41486、cg26442630、cg26443646、cg26479667、cg26499822、cg26500093、c g26501477、cg26528000、cg26544857、cg26577320、cg26599630、cg2660678 9、cg26676765、cg26691434、cg26702770、cg26760942、cg26764761、cg267 67387、cg26805839、cg26818257、cg26839512、cg26883048、cg26962439、cg 26992381, cg27021634, cg27066201, cg27079776, cg27127090, cg27128734, cg27130665, cg27131142, cg27152890, cg27154679, cg27154725, cg2720 8536, cg27229100, cg27245649, cg27256066, cg27296119, cg27315314, cg27336261, cg27348423, cg27354537, cg27357902, cg27368039, cg27374247These are multiple CG gene loci selected from the group consisting of cg27395675, cg27449200, cg27454650, cg27461310, cg27488658, cg27498980, cg27500647, cg27553457, cg27589058, and cg27632248.

[0005]

[0005] The number of CG loci observed from the designated group of CG loci may vary depending on the embodiment of this method. Therefore, the number of CG loci in the designated group can be any number between 2 and 1,352 (total).

[0006]

[0006] The method may include amplifying the genomic DNA of a blood or saliva sample by polymerase chain reaction.

[0007] Observation of unmethylated CG loci in human genomic DNA may involve hybridizing genomic DNA to a bead chip to create a genomic DNA hybridized bead chip, and then imaging the genomic DNA hybridized bead chip.

[0007]

[0008] The method may involve using a regression algorithm to correlate the unmethylated CG loci observed in (b) with the unmethylated GC loci observed in a reference group of human individuals.

[0008]

[0009] The method may involve running a penaltyed regression model on a computing device to create a regression algorithm for relative weighted CpGs, where the methylation risk score (MRS) is defined by a linear combination of the beta values ​​c and weights w of m CpG sites, and MRS = Σi = 1mwici. [Modes for carrying out the invention]

[0009]

[0010] Methods for predicting the health status of human subjects are: (a) A step of obtaining genomic DNA from a blood or saliva sample of a human subject, (b) A step of observing unmethylated CG loci in the genomic DNA of a human subject, which includes performing a bisulfite conversion treatment on the genomic DNA of the human subject, thereby converting cytosine residues in the genomic DNA of the human subject to uracil, while 5-methylcytosine residues in the genomic DNA of the human subject are not converted to uracil. (c) The step of comparing the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in genomic DNA taken from a reference group of human individuals, and (d) A step of predicting the health status of a human subject by correlating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in a reference group of human individuals. Includes, The CG loci are cg00011943, cg00014020, cg00056433, cg00067133, cg00071360, cg00074145, cg00080105, cg00120123, cg00122614, cg00153759, cg0017 7243, cg00178119, cg00182994, cg00209129, cg00215550, cg00217225, cg 00236988, cg00237714, cg00243480, cg00251125, cg00262132, cg00275686 , cg00278547, cg00283022, cg00303773, cg00322993, cg00342468, cg0035 8895, cg00458337, cg00465312, cg00481382, cg00530438, cg00538458, cg0 0548856, cg00593298, cg00594167, cg00602326, cg00615271, cg00631329 , cg00639633, cg00646555, cg00687674, cg00687962, cg00698355, cg00712 841, cg00737155, cg00748449, cg00756943, cg00767058, cg00800095, cg0 0815399, cg00910115, cg00935026, cg00935421, cg00964035, cg00973876 , cg00993310, cg01020263, cg01025883, cg01067216, cg01089474, cg0109 4440, cg01098936, cg01113710, cg01134012, cg01166985, cg01169463, cg0 1178063, cg01224520, cg01243823, cg01253289, cg01266159, cg01271129 , cg01274656, cg01282852, cg01313513, cg01320510, cg01346718, cg01377 932, cg01380346, cg01402735, cg01410359, cg01446515, cg01457077, cg0 1541424, cg01545493, cg01549940, cg01579765, cg01586635, cg01606057,cg01610915、cg01645869、cg01652244、cg01660630、cg01715525、cg01770232、cg01809675、cg01824466、cg01826979、cg01844642、cg01849284、cg01857379、cg01858517、cg01865937、cg01892689、cg01893681、cg01894498、cg01929138、cg01930417、cg01956293、cg02001279、cg02007867、cg02042600、cg02048674、cg02055264、cg02056921、cg02058108、cg02081263、cg02084118、cg02084211、cg02094681、cg02106682、cg02132714、cg02217269、cg02246605、cg02310386、cg02339455、cg02345572、cg02371935、cg02380750、cg02390624、cg02415029、cg02426324、cg02444928、cg02447542、cg02471378、cg02474731、cg02489377、cg02500990、cg02509300、cg02524236、cg02534780、cg02605776、cg02613370、cg02618485、cg02643782、cg02672830、cg02675560、cg02720346、cg02721394、cg02731293、cg02740457、cg02762363、cg02768694、cg02798801、cg02867402、cg02909068、cg02942382、cg02981003、cg02987388、cg03007462、cg03031520、cg03044367、cg03082247、cg03137472、cg03138091、cg03139710、cg03240800、cg03245912、cg03261737、cg03328727、cg03340356、cg03340754、cg03356492、cg03357219、cg03368099、cg03400443、cg03401077、cg03417454、cg03423767、cg03441242、cg03443888、cg03458265、cg03463465、cg03474430、cg03514545、cg03517760、cg03546163、cg03549506、cg03549779、cg03550384、cg03558379、cg03587144、cg03593336、cg03605735、cg03626541、cg03636183、cg03644691、cg03650179、cg03654623、cg03676636、cg03707183、cg03732728、cg03741185、cg03741498、cg03743982、cg03760060、cg03772253、cg03785619、cg03796580、cg03807316、cg03834947、cg03839794、cg03858365、cg03874914、cg03876766、cg03877420、cg03926921、cg03935495、cg03945604、cg03989967、cg04030486、cg04045009、cg04046848、cg04060356、cg04064054、cg04079779、cg04083076、cg04114590、cg04115418、cg04137003、cg04139465、cg04148839、cg04156293、cg04161236、cg04162034、cg04172953、cg04181107、cg04204356、cg04206342、cg04213565、cg04214966、cg04225172、cg04227949、cg04230438、cg04235071、cg04241623、cg04276953、cg04285652、cg04322572、cg04393397、cg04398451、cg04411086、cg04436120、cg04439218、cg04444415、cg04460372、cg04480106、cg04484376、cg04545003、cg04567124、cg04584675、cg04595658、cg04615668、cg04707706、cg04733838、cg04742282、cg04757168、cg04768425、cg04781339、cg04789125、cg04799410、cg04836038、cg04846343、cg04847826、cg04859706、cg04875128、cg04885881、cg04902443、cg04907325、cg04980928、cg05021355、cg05024939、cg05067995、cg05169665、cg05185749、cg05187965、cg05196720、cg05201093、cg05230392、cg05236757、cg05239544、cg05258736、cg05270922、cg05280814、cg05298224、cg05331789、cg05338009、cg05352464、cg05380920、cg05399221、cg05418947、cg05422344、cg05423392、cg05426134、cg05442477、cg05481257、cg05485520、cg05498943、cg05506600、cg05517880、cg05551469、cg05575921、cg05616010、cg05635388、cg05666515、cg05669298、cg05671603、cg05676441、cg05679108、cg05715050、cg05720678、cg05721485、cg05726118、cg05729185、cg05777089、cg05786053、cg05802075、cg05820243、cg05828191、cg05830220、cg05845765、cg05901141、cg05910038、cg05913921、cg05941525、cg05951558、cg05987787、cg05989533、cg06001484、cg06030695、cg06047828、cg06062604、cg06068545、cg06072257、cg06083423、cg06159131、cg06169746、cg06213468、cg06232874、cg06251791、cg06279632、cg06295952、cg06319999、cg06335143、cg06374715、cg06401979、cg06429010、cg06454296、cg06478823、cg06519334、cg06535156、cg06572713、cg06572849、cg06580943、cg06602086、cg06604227、cg06610988、cg06621358、cg06630425、cg06639320、cg06642354、cg06645033、cg06660395、cg06665890、cg06667732、cg06672424、cg06675893、cg06679261、cg06683719、cg06692384、cg06696596、cg06753303、cg06784991、cg06789517、cg06812822、cg06870213、cg06874063、cg06878994、cg06888566、cg06926961、cg06958563、cg06975196、cg06984463、cg06997226、cg07015424、cg07023764、cg07057549、cg07076816、cg07097098、cg07151966、cg07193470、cg07196593、cg07202981、cg07304986、cg07323488、cg07351787、cg07363137、cg07365979、cg07393369、cg07404706、cg07426101、cg07434188、cg07462861、cg07529122、cg07541744、cg07558704、cg07569063、cg07601816、cg07613945、cg07620230、cg07624842、cg07629158、cg07640648、cg07643355、cg07668501、cg07672814、cg07674095、cg07675998、cg07692435、cg07702236、cg07714289、cg07768696、cg07778205、cg07828121、cg07855914、cg07871442、cg07871717、cg07899016、cg07951728、cg08075719、cg08094551、cg08103988、cg08113373、cg08125436、cg08126148、cg08132054、cg08173263、cg08182270、cg08259413、cg08281455、cg08284801、cg08312389、cg08335107、cg08342619、cg08350092、cg08369164、cg08375139、cg08421807、cg08424491、cg08439752、cg08443038、cg08506743、cg08551532、cg0857340、 4、cg08609249、cg08640901、cg08684580、cg08709765、cg08743947、cg08750459、cg08762603、cg08772194、cg08812936、cg08830214、cg08856764、cg08885224、cg08893036、cg08968830、cg08992229、cg09082985、cg09087222、cg09087669、cg09094866、cg09103812、cg09129997、cg09146197、cg09167379、cg09169335、cg09191731、cg09196193、cg09211372、cg09228052、cg09267188、cg09313734、cg09317728、cg09365138、cg09391799、cg09422291、cg09428537、cg09434029、cg09435875、cg09450907、cg09463001、cg09507782、cg09536390、cg09554406、cg09576223、cg09601629、cg09621460、cg09681594、cg09702859、cg09703048、cg09716613、cg09717927、cg09717936、cg09788524、cg09823288、cg09823713、cg09831942、cg09842685、cg09862733、cg09868598、cg09874992、cg09931939、cg09963257、cg09969145、cg09977126、cg09993780、cg10043414、cg10060499、cg10116893、cg10123377、cg10127085、cg10133386、cg10157184、cg10168894、cg10201382、cg10208550、cg10235817、cg10251070、cg10290474、cg10325507、cg10326287、cg10407585、cg10459672、cg10472537、cg10501210、cg10514580、cg10518439、cg10573018、cg10583266、cg10601230、cg10621420、cg10624914、cg10662975、cg10703784、cg10716444、cg10759025、cg10766585、cg10859114、cg10866445、cg10874644、cg10878114、cg10882522、cg10884112、cg10949164、cg10961323、cg10977392、cg10996589、cg11020001、cg11031278、cg11052535、cg11104177、cg11113469、cg11120021、cg11130780、cg11149163、cg11240908、cg11241278、cg11270070、cg11290628、cg11338041、cg11341981、cg11362848、cg11400286、cg11408477、cg11409205、cg11415498、cg11426563、cg11479653、cg11532431、cg11540692、cg11620909、cg11624211、cg11640253、cg11649376、cg11671400、cg11691908、cg11725835、cg11735780、cg11737758、cg11749817、cg11752250、cg11765003、cg11777043、cg11793132、cg11801086、cg11804239、cg11819402、cg11824202、cg11833003、cg11844835、cg11921111、cg11935615、cg11951427、cg11971423、cg11976052、cg12016314、cg12016809、cg12018436、cg12030941、cg12038299、cg12050358、cg12100785、cg12112853、cg12135976、cg12145721、cg12146151、cg12151545、cg12158389、cg12159504、cg12168623、cg12170419、cg12195980、cg12242038、cg12272488、cg12282371、cg12307092、cg12347617、cg12364808、cg12378187、cg12394821、cg12416359、cg12434362、cg12463857、cg12518665、cg12538421、cg12560256、cg12569593、cg12586992、cg12606517、cg12615235、cg12637448、cg12655260、cg12662084、cg12743638、cg12744031、cg12797254、cg12853426、cg12935715、cg12941754、cg12978573、cg12986327、cg13017801、cg13055484、cg13073141、cg13083251、cg13087259、cg13099106、cg13154925、cg13167828、cg13169967、cg13176778、cg13188098、cg13206721、cg13224583、cg13229291、cg13234653、cg13257436、cg13295463、cg13298859、cg13312094、cg13340220、cg13343932、cg13367272、cg13369416、cg13372562、cg13393601、cg13399544、cg13431929、cg13473880、cg13507563、cg13543436、cg13545297、cg13563074、cg13609948、cg13671730、cg13672444、cg13676215、cg13690812、cg13697829、cg13702196、cg13714975、cg13719443、cg13742305、cg13779009、cg13814747、cg13897675、cg13914486、cg13941682、cg13959647、cg13979305、cg13987110、cg14030785、cg14047961、cg14117377、cg14118991、cg14157578、cg14167430、cg14172633、cg14175748、cg14209041、cg14224028、cg14234520、cg14251509、cg14252140、cg14257435、cg14262357、cg14264125、cg14280181、cg14339287、cg14354398、cg14384748、cg14391737、cg14413904、cg14441271、cg14442890、cg14484844、cg14534474、cg14615491、cg14639293、cg14640588、cg14675598、cg14688662、cg14708893、cg14710896、cg14738170、cg14751023、cg14781394、cg14794281、cg14801398、cg14837598、cg14850945、cg14873958、cg14920334、cg14942542、cg14962363、cg14975009、cg14994914、cg15033140、cg15047582、cg15059204、cg15109292、cg15128785、cg15141757、cg15143202、cg15175143、cg15179011、cg15194100、cg15208687、cg15223345、cg15234627、cg15246131、cg15288815、cg15318748、cg15374686、cg15391651、cg15405515、cg15418593、cg15436354、cg15467112、cg15471501、cg15480367、cg15498435、cg15506060、cg15575700、cg15592143、cg15603964、cg15625940、cg15690995、cg15709989、cg15710198、cg15720821、cg15733713、cg15821589、cg15827092、cg15837235、cg15885703、cg15909933、cg15961042、cg15968648、cg15994304、cg16014202、cg16014412、cg16026114、cg16070035、cg16084969、cg16097191、cg16141165、cg16195970、cg16198075、cg16211224、cg16240781、cg16306900、cg16314089、cg16369168、cg16408865、cg16430267、cg16488565、cg16489878、cg16517790、cg16529483、cg16551483、cg16569522、cg16570202、cg16649588、cg16661213、cg16679001、cg16733203、cg16736896、cg16769369、cg16795184、cg16842060、cg16861968、cg16882373、cg16891431、cg16908208、cg16969368、cg16979095、cg16987876、cg16994082、cg17017746、cg17030092、cg17050472、cg17077421、cg17107501、cg17117243、cg17120358、cg17129809、cg17133012、cg17148219、cg17210497、cg17219432、cg17225923、cg17232357、cg17251114、cg17275047、cg17408993、cg17435901、cg17464298、cg17464436、cg17465569、cg17476910、cg17485717、cg17514701、cg17569235、cg17576228、cg17591595、cg17652174、cg17660663、cg17665268、cg17691612、cg17695543、cg17699330、cg17761940、cg17786697、cg17825846、cg17872672、cg17885794、cg17944885、cg17945419、cg17967443、cg17979595、cg17981101、cg17988830、cg18014413、cg18023598、cg18031708、cg18031880、cg18071894、cg18133309、cg18181703、cg18190506、cg18251449、cg18262201、cg18319024、cg18380497、cg18398019、cg18425341、cg18516816、cg18587063、cg18597252、cg18598029、cg18604560、cg18669861、cg18699242、cg18740583、cg18771034、cg18784500、cg18795169、cg18817318、cg18822243、cg18856359、cg18859711、cg18882407、cg18885210、cg18976649、cg19006431、cg19009870、cg19058685、cg19089073、cg19091784、cg19172278、cg19182279、cg19224645、cg19225755、cg19266329、cg19284211、cg19296509、cg19、 297245、cg19302996、cg19304797、cg19308663、cg19320294、cg19380156、cg19474865、cg19536401、cg19548511、cg19558933、cg19604384、cg19611570、cg19635897、cg19636673、cg19652271、cg19665696、cg19693031、cg19726712、cg19730248、cg19774733、cg19800670、cg19822434、cg19859910、cg19909400、cg19935065、cg19941446、cg19977924、cg19992304、cg20004147、cg20025086、cg20037601、cg20059597、cg20119148、cg20128319、cg20143092、cg20155714、cg20162239、cg20185773、cg20192259、cg20207443、cg20217702、cg20244340、cg20249402、cg20270599、cg20272789、cg20282550、cg20299697、cg20348196、cg20351734、cg20353489、cg20370296、cg20376953、cg20432211、cg20442128、cg20446824、cg20515136、cg20534585、cg20587151、cg20594802、cg20596493、cg20597382、cg20597477、cg20601028、cg20623853、cg20628696、cg20656525、cg20663241、cg20668177、cg20675702、cg20720208、cg20737582、cg20773320、cg20802942、cg20865054、cg20874261、cg20891771、cg20907051、cg20914958、cg20918218、cg20921972、cg20972814、cg20973476、cg21000454、cg21077074、cg21082913、cg21112943、cg21123355、cg21124655、cg21132686、cg21136789、cg21185661、cg21204522、cg21232671、cg21253043、cg21259215、cg21305555、cg21306240、cg21312434、cg21314942、cg21321148、cg21346818、cg21356998、cg21410629、cg21447975、cg21455901、cg21489638、cg21533743、cg21566642、cg21622536、cg21628390、cg21640508、cg21658235、cg21693883、cg21717369、cg21723004、cg21725954、cg21730080、cg21733098、cg21739934、cg21761881、cg21783047、cg21791657、cg21824343、cg21834645、cg21870393、cg21907521、cg21934504、cg21947254、cg21962907、cg21985831、cg21986679、cg21998324、cg22002034、cg22019884、cg22022821、cg22023206、cg22041417、cg22061523、cg22061769、cg22069247、cg22171714、cg22178367、cg22200330、cg22220710、cg22232492、cg22243260、cg22282672、cg22322653、cg22339775、cg22344631、cg22356226、cg22363327、cg22416596、cg22434421、cg22442430、cg22454769、cg22556008、cg22563987、cg22581353、cg22594985、cg22691256、cg22711187、cg22719651、cg22729726、cg22736354、cg22792461、cg22822140、cg22822803、cg22945467、cg22956922、cg22993154、cg23059366、cg23063236、cg23080422、cg23192918、cg23196035、cg23234847、cg23235141、cg23248055、cg23265105、cg23276290、cg23302570、cg23313424, cg23366493, cg23371851, cg23378546, cg23410824, cg23424407, cg23447131, cg23465508, cg23486701, cg23500537, cg23547619, cg23 549902, cg23561053, cg23570968, cg23574427, cg23586821, cg23615892, cg23643435, cg23793194, cg23838503, cg23879118, cg23883522, cg238940 58、cg23910413、cg23923562、cg23956385、cg23964820、cg24003560、cg24 027965、cg24055030、cg24055461、cg24112628、cg24141036、cg24185013、c g24220948、cg24236938、cg24241488、cg24262996、cg24266506、cg242933 44、cg24317857、cg24337318、cg24354818、cg24362016、cg24365657、cg243 72256、cg24392971、cg24397183、cg24432675、cg24463073、cg24468603、c g24492140、cg24507955、cg24520070、cg24523415、cg24530234、cg2454325 5、cg24577941、cg24589102、cg24601404、cg24674445、cg24678611、cg246 97099、cg24721873、cg24769628、cg24796651、cg24799795、cg24835295、cg 24920358, cg24925701, cg24967099, cg24976744, cg24982343, cg24997750, cg25047618, cg25063538, cg25114611, cg25146575, cg25148589, cg2515 6198, cg25161899, cg25181997, cg25189904, cg25202593, cg25259149, cg25259707, cg25330725, cg25331593, cg25361850, cg25375711, cg25397922cg25418001、cg25418670、cg25420482、cg25420747、cg25436217、cg25499 552、cg25504214、cg25560772、cg25563198、cg25569590、cg25623035、cg25 638549, cg25639566, cg25670171, cg25671708, cg25703588, cg25717260, cg25738786, cg25745729, cg25784308, cg25786980, cg25843115, cg258438 73, cg25903319, cg25916759, cg25927838, cg25928819, cg25943804, cg25945293, cg25952581, cg25952596, cg26029902, cg26044490, cg26071551, c g26093148、cg26116542、cg26116556、cg26156626、cg26229024、cg262394 85、cg26267310、cg26276120、cg26301353、cg26314853、cg26315261、cg264 41486、cg26442630、cg26443646、cg26479667、cg26499822、cg26500093、c g26501477、cg26528000、cg26544857、cg26577320、cg26599630、cg2660678 9、cg26676765、cg26691434、cg26702770、cg26760942、cg26764761、cg267 67387、cg26805839、cg26818257、cg26839512、cg26883048、cg26962439、cg 26992381, cg27021634, cg27066201, cg27079776, cg27127090, cg27128734, cg27130665, cg27131142, cg27152890, cg27154679, cg27154725, cg2720 8536, cg27229100, cg27245649, cg27256066, cg27296119, cg27315314, cg27336261, cg27348423, cg27354537, cg27357902, cg27368039, cg27374247These are multiple CG gene loci selected from the group consisting of cg27395675, cg27449200, cg27454650, cg27461310, cg27488658, cg27498980, cg27500647, cg27553457, cg27589058, and cg27632248.

[0010]

[0011] A lack of methylation at any of the CG loci identified above is associated with and predicts one or more of the following diseases, disorders, or conditions: alcoholic liver disease, allergic rhinitis, aortic aneurysm dissection, arterial embolism and thrombosis, asthma, bladder cancer, breast cancer, bronchiectasis, cancer, cardiomyopathy, chronic bronchitis, chronic kidney disease, chronic liver disease, chronic cor pulmonale, chronic viral hepatitis, cirrhosis, coin-shaped shadow lung disease, and colon disease. Rectal cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), coronary artery disease, cardiovascular disease excluding stroke (CVD), depression, emphysema, hypertensive heart disease, interstitial lung disease, leukemia, liver cancer, lung cancer, melanoma, myocardial infarction, non-alcoholic fatty liver disease (NAFLD), non-Hodgkin lymphoma, other chronic hepatitis, ovarian cancer, pancreatic cancer, peripheral vascular disease, prostate cancer, gastric cancer, stroke, type 1 diabetes, type 2 diabetes, and endometrial cancer. These were validated using genomic DNA collected from individuals in the reference group.

[0011]

[0012] The number of CG loci observed from a specified group of CG loci may vary depending on the embodiment of this method. Therefore, the number of CG loci in the specified group can be any number between 2 and 1,352 (total). In one possible embodiment, at least 5 CG loci are selected from the group. In another embodiment, at least 10 CG loci are selected from the group. In yet another embodiment, at least 15 CG loci are selected from the group.

[0012]

[0013] In yet another embodiment, at least 25 multiple CG loci are selected from the group. In another possible embodiment, at least 50 multiple CG loci are selected from the group. In another possible embodiment, at least 75 multiple CG loci are selected from the group. In another possible embodiment, at least 100 multiple CG loci are selected from the group. In another possible embodiment, at least 150 multiple CG loci are selected from the group.

[0013]

[0014] In another possible embodiment, at least 200 multiple CG loci are selected from the group. In yet another possible embodiment, at least 250 multiple CG loci are selected from the group. In yet another possible embodiment, at least 500 multiple CG loci are selected from the group. In yet another possible embodiment, at least 1,000 multiple CG loci are selected from the group. In yet another possible embodiment, all 1,352 CG loci in the group are selected.

[0014]

[0015] The bisulfite conversion process mentioned in step (b) is well known in the art. Kits useful for bisulfite conversion are commercially available from several manufacturers, including Human Genetic Signatures' Methyleasy and Chemicon's CpGenome Modification Kit. See also WO04096825A1, which describes the bisulfite modification method, and Olek et al., Nuc. Acids Res. 24:5064~6 (1994), which discloses a method for performing bisulfite treatment and subsequent amplification.

[0015]

[0016] The bissulfite conversion process may include steps of amplifying the genomic DNA of a blood sample by a polymerase chain reaction process, hybridizing the genomic DNA to a bead chip to create a genomic DNA hybridized bead chip, staining the genomic DNA hybridized bead chip, and then imaging the genomic DNA hybridized bead chip.

[0016]

[0017] Imaging of genomic DNA hybridized bead chips may be performed using the Illumina EPIC850k array, which provides clear identification of specific CG loci for the purpose of observing methylation of specific CG loci in the human DNA genome. Alternatively, other epigenetic β-value acquisition methods may be used as desired.

[0017]

[0018] The method also includes the step of correlating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in a reference group of human individuals using a regression algorithm. The method involves running a penaltyed regression model on a computer to create a regression algorithm for relative-weighted CpG, where the methylation risk score (MRS) is defined by a linear combination of the beta values ​​c and weights w of m CpG sites, with MRS = Σi = 1mwici. It should be noted that different relative weights may be assigned to GC loci.

[0018]

[0019] To confirm that the methylation risk score has additional predictive value compared to commonly obtained features (e.g., age, sex), a baseline predictive model was created including patient age, sex, reference-based methylated cell type composition estimates, self-reported race-ethnicity, self-reported smoking status, and the first 10 genetic principal components. Depending on whether the results were continuous or binary variables, the baseline model was fitted using a linear or logistic regression model. The baseline model was compared to a model that included methylation data or genotype data in addition to the baseline features. For MRS, baseline features were treated as fixed covariates, and regression analysis was performed on genomic features using LASSO, elastic net, and ridge regularization. The inventors fitted all models using 10-fold double cross-validation. Additional cross-validation was performed within each training set to select hyperparameters, and then the models cross-validated on this training set were used to predict the reserved test set. The inventors verified the significance of the cross-validated prediction results (i.e., the combined prediction values ​​across all divisions) and the actual results using a correlation test (by linear regression).

[0019]

[0020] Furthermore, this method includes predicting proteomics and metabolomics data as surrogate biomarkers. For example, the inventors have used DNA methylation to predict classical clinical markers such as C-reactive proteins. For this purpose, the method has been used to predict alcoholic liver disease, allergic rhinitis, aortic aneurysm dissection, arterial embolism and thrombosis, asthma, bladder cancer, breast cancer, bronchiectasis, cancer, cardiomyopathy, chronic bronchitis, chronic kidney disease, chronic liver disease, chronic cor pulmonale, chronic viral hepatitis, cirrhosis, coin-shaped opacity lung disease, colorectal cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), coronary artery disease, cardiovascular disease excluding stroke (CVD), depression, emphysema, hypertensive heart disease, interstitial lung disease, and leukemia. Several known proteins and metabolites of medical interest for diagnosing diseases such as liver cancer, lung cancer, melanoma, myocardial infarction, non-alcoholic fatty liver disease (NAFLD), non-Hodgkin lymphoma, other chronic hepatitis, ovarian cancer, pancreatic cancer, peripheral vascular disease, prostate cancer, gastric cancer, stroke, type 1 diabetes, type 2 diabetes, and endometrial cancer are first used to construct a baseline CG locus reference matrix from a reference group of individuals with a known health history. Then, using this method, the levels of those several proteins and metabolites in human subjects are predicted by correlating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in the human individual reference group, thereby predicting the disease risk or health status of human subjects.

[0020]

[0021] As used herein, the following terms, when written in the singular form, “a,” “an,” and “the,” mean “at least one” or “one or more.” The use of the phrase “one or more” herein does not alter this intended meaning of “a,” “an,” and “the.” Thus, as used herein, the terms “a,” “an,” and “the” may refer to and encompass the plural form of the entity or subject described, unless otherwise specifically defined or stated herein, or unless the context clearly indicates otherwise. For example, as used herein, the phrase “a protein” may refer to and encompass multiple proteins.

[0021]

[0022] The following terms: "includes", "including", "has", "having", "comprises", "comprising", and their linguistic / grammatical variations, derivatives, or / and conjugations, when used in this specification, mean "including but not limited to", and should be construed as identifying the recited components, features, characteristics, parameters, integers, or steps, and do not exclude the addition of one or more additional components, features, characteristics, parameters, integers, steps, or groups thereof.

[0022]

[0023] As used in this specification, the phrase "consisting of" means a closed range and excludes elements, steps, or components not specifically recited. As used in this specification, the phrase "consisting essentially of" is a term indicating a semi-closed range and indicates that it is limited to those that do not materially affect the basic and novel characteristics of the specified components and the specified ones.

[0023]

[0024] Approximating expressions such as "about", "substantially", "approximately", etc. used in this specification refer to ±10% of the recited numerical value.

[0025] The methods described in this disclosure are illustrated and presented by specific examples and their instances, but it will be understood by those skilled in the art that many alternatives, modifications, and / or variations will be apparent. Accordingly, all these alternatives, modifications, and / or variations are intended to be included within the spirit of the appended claims and are intended to be encompassed within their broad scope.

Claims

1. (a) A step of obtaining genomic DNA from a blood or saliva sample of a human subject, (b) A step of observing unmethylated CG loci in the genomic DNA of a human subject, which includes performing a bisulfite conversion treatment on the genomic DNA of the human subject, thereby converting cytosine residues in the genomic DNA of the human subject to uracil, while 5-methylcytosine residues in the genomic DNA of the human subject are not converted to uracil. (c) The step of comparing the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in genomic DNA taken from a reference group of human individuals, and (d) A step of predicting the health status of a human subject by correlating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in a reference group of human individuals. A method including, The CG loci are cg00011943, cg00014020, cg00056433, cg00067133, cg00071360, cg00074145, cg00080105, cg00120123, cg00122614, cg00153759, cg0017 7243, cg00178119, cg00182994, cg00209129, cg00215550, cg00217225, cg 00236988, cg00237714, cg00243480, cg00251125, cg00262132, cg00275686 , cg00278547, cg00283022, cg00303773, cg00322993, cg00342468, cg0035 8895, cg00458337, cg00465312, cg00481382, cg00530438, cg00538458, cg0 0548856, cg00593298, cg00594167, cg00602326, cg00615271, cg00631329 , cg00639633, cg00646555, cg00687674, cg00687962, cg00698355, cg00712 841, cg00737155, cg00748449, cg00756943, cg00767058, cg00800095, cg0 0815399, cg00910115, cg00935026, cg00935421, cg00964035, cg00973876 , cg00993310, cg01020263, cg01025883, cg01067216, cg01089474, cg0109 4440, cg01098936, cg01113710, cg01134012, cg01166985, cg01169463, cg0 1178063, cg01224520, cg01243823, cg01253289, cg01266159, cg01271129 , cg01274656, cg01282852, cg01313513, cg01320510, cg01346718, cg01377 932, cg01380346, cg01402735, cg01410359, cg01446515, cg01457077, cg0 1541424, cg01545493, cg01549940, cg01579765, cg01586635, cg01606057,cg01610915、cg01645869、cg01652244、cg01660630、cg01715525、cg01770232、cg01809675、cg01824466、cg01826979、cg01844642、cg01849284、cg01857379、cg01858517、cg01865937、cg01892689、cg01893681、cg01894498、cg01929138、cg01930417、cg01956293、cg02001279、cg02007867、cg02042600、cg02048674、cg02055264、cg02056921、cg02058108、cg02081263、cg02084118、cg02084211、cg02094681、cg02106682、cg02132714、cg02217269、cg02246605、cg02310386、cg02339455、cg02345572、cg02371935、cg02380750、cg02390624、cg02415029、cg02426324、cg02444928、cg02447542、cg02471378、cg02474731、cg02489377、cg02500990、cg02509300、cg02524236、cg02534780、cg02605776、cg02613370、cg02618485、cg02643782、cg02672830、cg02675560、cg02720346、cg02721394、cg02731293、cg02740457、cg02762363、cg02768694、cg02798801、cg02867402、cg02909068、cg02942382、cg02981003、cg02987388、cg03007462、cg03031520、cg03044367、cg03082247、cg03137472、cg03138091、cg03139710、cg03240800、cg03245912、cg03261737、cg03328727、cg03340356、cg03340754、cg03356492、cg03357219、cg03368099、cg03400443、cg03401077、cg03417454、cg03423767、cg03441242、cg03443888、cg03458265、cg03463465、cg03474430、cg03514545、cg03517760、cg03546163、cg03549506、cg03549779、cg03550384、cg03558379、cg03587144、cg03593336、cg03605735、cg03626541、cg03636183、cg03644691、cg03650179、cg03654623、cg03676636、cg03707183、cg03732728、cg03741185、cg03741498、cg03743982、cg03760060、cg03772253、cg03785619、cg03796580、cg03807316、cg03834947、cg03839794、cg03858365、cg03874914、cg03876766、cg03877420、cg03926921、cg03935495、cg03945604、cg03989967、cg04030486、cg04045009、cg04046848、cg04060356、cg04064054、cg04079779、cg04083076、cg04114590、cg04115418、cg04137003、cg04139465、cg04148839、cg04156293、cg04161236、cg04162034、cg04172953、cg04181107、cg04204356、cg04206342、cg04213565、cg04214966、cg04225172、cg04227949、cg04230438、cg04235071、cg04241623、cg04276953、cg04285652、cg04322572、cg04393397、cg04398451、cg04411086、cg04436120、cg04439218、cg04444415、cg04460372、cg04480106、cg04484376、cg04545003、cg04567124、cg04584675、cg04595658、cg04615668、cg04707706、cg04733838、cg04742282、cg04757168、cg04768425、cg04781339、cg04789125、cg04799410、cg04836038、cg04846343、cg04847826、cg04859706、cg04875128、cg04885881、cg04902443、cg04907325、cg04980928、cg05021355、cg05024939、cg05067995、cg05169665、cg05185749、cg05187965、cg05196720、cg05201093、cg05230392、cg05236757、cg05239544、cg05258736、cg05270922、cg05280814、cg05298224、cg05331789、cg05338009、cg05352464、cg05380920、cg05399221、cg05418947、cg05422344、cg05423392、cg05426134、cg05442477、cg05481257、cg05485520、cg05498943、cg05506600、cg05517880、cg05551469、cg05575921、cg05616010、cg05635388、cg05666515、cg05669298、cg05671603、cg05676441、cg05679108、cg05715050、cg05720678、cg05721485、cg05726118、cg05729185、cg05777089、cg05786053、cg05802075、cg05820243、cg05828191、cg05830220、cg05845765、cg05901141、cg05910038、cg05913921、cg05941525、cg05951558、cg05987787、cg05989533、cg06001484、cg06030695、cg06047828、cg06062604、cg06068545、cg06072257、cg06083423、cg06159131、cg06169746、cg06213468、cg06232874、cg06251791、cg06279632、cg06295952、cg06319999、cg06335143、cg06374715、cg06401979、cg06429010、cg06454296、cg06478823、cg06519334、cg06535156、cg06572713、cg06572849、cg06580943、cg06602086、cg06604227、cg06610988、cg06621358、cg06630425、cg06639320、cg06642354、cg06645033、cg06660395、cg06665890、cg06667732、cg06672424、cg06675893、cg06679261、cg06683719、cg06692384、cg06696596、cg06753303、cg06784991、cg06789517、cg06812822、cg06870213、cg06874063、cg06878994、cg06888566、cg06926961、cg06958563、cg06975196、cg06984463、cg06997226、cg07015424、cg07023764、cg07057549、cg07076816、cg07097098、cg07151966、cg07193470、cg07196593、cg07202981、cg07304986、cg07323488、cg07351787、cg07363137、cg07365979、cg07393369、cg07404706、cg07426101、cg07434188、cg07462861、cg07529122、cg07541744、cg07558704、cg07569063、cg07601816、cg07613945、cg07620230、cg07624842、cg07629158、cg07640648、cg07643355、cg07668501、cg07672814、cg07674095、cg07675998、cg07692435、cg07702236、cg07714289、cg07768696、cg07778205、cg07828121、cg07855914、cg07871442、cg07871717、cg07899016、cg07951728、cg08075719、cg08094551、cg08103988、cg08113373、cg08125436、cg08126148、cg08132054、cg08173263、cg08182270、cg08259413、cg08281455、cg08284801、cg08312389、cg08335107、cg08342619、cg08350092、cg08369164、cg08375139、cg08421807、cg08424491、cg08439752、cg08443038、cg08506743、cg08551532、cg0857340、 4、cg08609249、cg08640901、cg08684580、cg08709765、cg08743947、cg08750459、cg08762603、cg08772194、cg08812936、cg08830214、cg08856764、cg08885224、cg08893036、cg08968830、cg08992229、cg09082985、cg09087222、cg09087669、cg09094866、cg09103812、cg09129997、cg09146197、cg09167379、cg09169335、cg09191731、cg09196193、cg09211372、cg09228052、cg09267188、cg09313734、cg09317728、cg09365138、cg09391799、cg09422291、cg09428537、cg09434029、cg09435875、cg09450907、cg09463001、cg09507782、cg09536390、cg09554406、cg09576223、cg09601629、cg09621460、cg09681594、cg09702859、cg09703048、cg09716613、cg09717927、cg09717936、cg09788524、cg09823288、cg09823713、cg09831942、cg09842685、cg09862733、cg09868598、cg09874992、cg09931939、cg09963257、cg09969145、cg09977126、cg09993780、cg10043414、cg10060499、cg10116893、cg10123377、cg10127085、cg10133386、cg10157184、cg10168894、cg10201382、cg10208550、cg10235817、cg10251070、cg10290474、cg10325507、cg10326287、cg10407585、cg10459672、cg10472537、cg10501210、cg10514580、cg10518439、cg10573018、cg10583266、cg10601230、cg10621420、cg10624914、cg10662975、cg10703784、cg10716444、cg10759025、cg10766585、cg10859114、cg10866445、cg10874644、cg10878114、cg10882522、cg10884112、cg10949164、cg10961323、cg10977392、cg10996589、cg11020001、cg11031278、cg11052535、cg11104177、cg11113469、cg11120021、cg11130780、cg11149163、cg11240908、cg11241278、cg11270070、cg11290628、cg11338041、cg11341981、cg11362848、cg11400286、cg11408477、cg11409205、cg11415498、cg11426563、cg11479653、cg11532431、cg11540692、cg11620909、cg11624211、cg11640253、cg11649376、cg11671400、cg11691908、cg11725835、cg11735780、cg11737758、cg11749817、cg11752250、cg11765003、cg11777043、cg11793132、cg11801086、cg11804239、cg11819402、cg11824202、cg11833003、cg11844835、cg11921111、cg11935615、cg11951427、cg11971423、cg11976052、cg12016314、cg12016809、cg12018436、cg12030941、cg12038299、cg12050358、cg12100785、cg12112853、cg12135976、cg12145721、cg12146151、cg12151545、cg12158389、cg12159504、cg12168623、cg12170419、cg12195980、cg12242038、cg12272488、cg12282371、cg12307092、cg12347617、cg12364808、cg12378187、cg12394821、cg12416359、cg12434362、cg12463857、cg12518665、cg12538421、cg12560256、cg12569593、cg12586992、cg12606517、cg12615235、cg12637448、cg12655260、cg12662084、cg12743638、cg12744031、cg12797254、cg12853426、cg12935715、cg12941754、cg12978573、cg12986327、cg13017801、cg13055484、cg13073141、cg13083251、cg13087259、cg13099106、cg13154925、cg13167828、cg13169967、cg13176778、cg13188098、cg13206721、cg13224583、cg13229291、cg13234653、cg13257436、cg13295463、cg13298859、cg13312094、cg13340220、cg13343932、cg13367272、cg13369416、cg13372562、cg13393601、cg13399544、cg13431929、cg13473880、cg13507563、cg13543436、cg13545297、cg13563074、cg13609948、cg13671730、cg13672444、cg13676215、cg13690812、cg13697829、cg13702196、cg13714975、cg13719443、cg13742305、cg13779009、cg13814747、cg13897675、cg13914486、cg13941682、cg13959647、cg13979305、cg13987110、cg14030785、cg14047961、cg14117377、cg14118991、cg14157578、cg14167430、cg14172633、cg14175748、cg14209041、cg14224028、cg14234520、cg14251509、cg14252140、cg14257435、cg14262357、cg14264125、cg14280181、cg14339287、cg14354398、cg14384748、cg14391737、cg14413904、cg14441271、cg14442890、cg14484844、cg14534474、cg14615491、cg14639293、cg14640588、cg14675598、cg14688662、cg14708893、cg14710896、cg14738170、cg14751023、cg14781394、cg14794281、cg14801398、cg14837598、cg14850945、cg14873958、cg14920334、cg14942542、cg14962363、cg14975009、cg14994914、cg15033140、cg15047582、cg15059204、cg15109292、cg15128785、cg15141757、cg15143202、cg15175143、cg15179011、cg15194100、cg15208687、cg15223345、cg15234627、cg15246131、cg15288815、cg15318748、cg15374686、cg15391651、cg15405515、cg15418593、cg15436354、cg15467112、cg15471501、cg15480367、cg15498435、cg15506060、cg15575700、cg15592143、cg15603964、cg15625940、cg15690995、cg15709989、cg15710198、cg15720821、cg15733713、cg15821589、cg15827092、cg15837235、cg15885703、cg15909933、cg15961042、cg15968648、cg15994304、cg16014202、cg16014412、cg16026114、cg16070035、cg16084969、cg16097191、cg16141165、cg16195970、cg16198075、cg16211224、cg16240781、cg16306900、cg16314089、cg16369168、cg16408865、cg16430267、cg16488565、cg16489878、cg16517790、cg16529483、cg16551483、cg16569522、cg16570202、cg16649588、cg16661213、cg16679001、cg16733203、cg16736896、cg16769369、cg16795184、cg16842060、cg16861968、cg16882373、cg16891431、cg16908208、cg16969368、cg16979095、cg16987876、cg16994082、cg17017746、cg17030092、cg17050472、cg17077421、cg17107501、cg17117243、cg17120358、cg17129809、cg17133012、cg17148219、cg17210497、cg17219432、cg17225923、cg17232357、cg17251114、cg17275047、cg17408993、cg17435901、cg17464298、cg17464436、cg17465569、cg17476910、cg17485717、cg17514701、cg17569235、cg17576228、cg17591595、cg17652174、cg17660663、cg17665268、cg17691612、cg17695543、cg17699330、cg17761940、cg17786697、cg17825846、cg17872672、cg17885794、cg17944885、cg17945419、cg17967443、cg17979595、cg17981101、cg17988830、cg18014413、cg18023598、cg18031708、cg18031880、cg18071894、cg18133309、cg18181703、cg18190506、cg18251449、cg18262201、cg18319024、cg18380497、cg18398019、cg18425341、cg18516816、cg18587063、cg18597252、cg18598029、cg18604560、cg18669861、cg18699242、cg18740583、cg18771034、cg18784500、cg18795169、cg18817318、cg18822243、cg18856359、cg18859711、cg18882407、cg18885210、cg18976649、cg19006431、cg19009870、cg19058685、cg19089073、cg19091784、cg19172278、cg19182279、cg19224645、cg19225755、cg19266329、cg19284211、cg19296509、cg19、 297245、cg19302996、cg19304797、cg19308663、cg19320294、cg19380156、cg19474865、cg19536401、cg19548511、cg19558933、cg19604384、cg19611570、cg19635897、cg19636673、cg19652271、cg19665696、cg19693031、cg19726712、cg19730248、cg19774733、cg19800670、cg19822434、cg19859910、cg19909400、cg19935065、cg19941446、cg19977924、cg19992304、cg20004147、cg20025086、cg20037601、cg20059597、cg20119148、cg20128319、cg20143092、cg20155714、cg20162239、cg20185773、cg20192259、cg20207443、cg20217702、cg20244340、cg20249402、cg20270599、cg20272789、cg20282550、cg20299697、cg20348196、cg20351734、cg20353489、cg20370296、cg20376953、cg20432211、cg20442128、cg20446824、cg20515136、cg20534585、cg20587151、cg20594802、cg20596493、cg20597382、cg20597477、cg20601028、cg20623853、cg20628696、cg20656525、cg20663241、cg20668177、cg20675702、cg20720208、cg20737582、cg20773320、cg20802942、cg20865054、cg20874261、cg20891771、cg20907051、cg20914958、cg20918218、cg20921972、cg20972814、cg20973476、cg21000454、cg21077074、cg21082913、cg21112943、cg21123355、cg21124655、cg21132686、cg21136789、cg21185661、cg21204522、cg21232671、cg21253043、cg21259215、cg21305555、cg21306240、cg21312434、cg21314942、cg21321148、cg21346818、cg21356998、cg21410629、cg21447975、cg21455901、cg21489638、cg21533743、cg21566642、cg21622536、cg21628390、cg21640508、cg21658235、cg21693883、cg21717369、cg21723004、cg21725954、cg21730080、cg21733098、cg21739934、cg21761881、cg21783047、cg21791657、cg21824343、cg21834645、cg21870393、cg21907521、cg21934504、cg21947254、cg21962907、cg21985831、cg21986679、cg21998324、cg22002034、cg22019884、cg22022821、cg22023206、cg22041417、cg22061523、cg22061769、cg22069247、cg22171714、cg22178367、cg22200330、cg22220710、cg22232492、cg22243260、cg22282672、cg22322653、cg22339775、cg22344631、cg22356226、cg22363327、cg22416596、cg22434421、cg22442430、cg22454769、cg22556008、cg22563987、cg22581353、cg22594985、cg22691256、cg22711187、cg22719651、cg22729726、cg22736354、cg22792461、cg22822140、cg22822803、cg22945467、cg22956922、cg22993154、cg23059366、cg23063236、cg23080422、cg23192918、cg23196035、cg23234847、cg23235141、cg23248055、cg23265105、cg23276290、cg23302570、cg23313424、cg23366493、cg23371851、cg23378546、cg23410824、cg23424 407, cg23447131, cg23465508, cg2346701, cg23500537, cg23547619cg23 549902、cg23561053、cg23570968、cg2354427、cg235892、 cg23643435、cg23793194、cg23838503、cg23879118、cg23883522、cg238940 58、cg23910413、cg23923562、cg2395 6385、cg23964820、cg24003560、cg24 027965、cg24055030、cg24055461、cg 24112628、cg24141036、cg24185013、c g24220948、cg24236938、cg24241488 、cg24262996、cg24266506、cg242933 44, cg24317857, cg24337318, cg24354818, cg24362016, cg24365657, cg243 72256、cg24392971、cg24397183、cg2 4432675、cg24463073、cg24468603、c g24492140、cg24507955、cg24520070 、cg24523415、cg24530234、cg2454325 5、cg24577941、cg24589102、cg24601 404、cg24674445、cg24678611、cg246 97099、cg24721873、cg24769628、cg2 4796651、cg24799795、cg24835295、cg 24920358、cg24925701、cg24967099、cg24976744、cg2499775 0、cg25047618、cg25063538、cg25114 611、cg25146575、cg25148589、cg2515 6198, cg2516189997cg25181997cg25189904cg252025933cg25259149cg2 5259707、cg25330725、cg25331593、cg25361850、cg25375711、cg25397922、cg25418001、cg25418670、cg2542048 2、cg25420747、cg25436217、cg25499 552、cg25504214、cg25560772、cg25563198、cg25569590、cg25623035、cg25 638549、cg25639566、cg25670171、cg25671708、cg25703588、cg25717260、 cg25738786、cg25745729、cg25784308、cg25786980、cg258438 73、cg25903319、cg25916759、cg25927838、cg25928819、cg25943804、cg25 945293, cg25952581、cg25952596、cg26029902、cg26044490、cg26071551、c g26093148、cg26116542、cg26116556 、cg26156626、cg26229024、cg262394 85、cg26267310、cg26276120、cg2630 1353、cg26314853、cg26315261、cg264 41486、cg26442630、cg26443646、cg2 6479667、cg26499822、cg26500093、c g26501477、cg26528000、cg26544857 、cg26577320、cg26599630、cg2660678 9、cg26676765、cg26691434、cg26702 770、cg26760942、cg26764761、cg267 67387、cg26805839、cg26818257、cg2 6839512、cg26883048、cg26962439、cg 26992381、cg27021634、cg27066201、cg27079776、cg2712873 4、cg27130665、cg27131142、cg27152 890、cg27154679、cg27154725、cg2720 8536、cg27229100、cg27245649、cg27 256066、cg27296119、cg27315314、cg2 7336261、cg27348423、cg27354537、cg27357902、cg27368039、cg27374247、The above method, wherein at least five multiple CG gene loci are selected from the group consisting of cg27395675, cg27449200, cg27454650, cg27461310, cg27488658, cg27498980, cg27500647, cg27553457, cg27589058, and cg27632248.

2. The method according to claim 1, wherein at least 10 multiple CG gene loci are selected from the group.

3. The method according to claim 1, wherein at least 15 multiple CG gene loci are selected from the group.

4. The method according to claim 1, wherein at least 25 multiple CG gene loci are selected from the group.

5. The method according to claim 1, wherein at least 50 multiple CG gene loci are selected from the group.

6. The method according to claim 1, wherein at least 75 multiple CG gene loci are selected from the group.

7. The method according to claim 1, wherein at least 100 multiple CG gene loci are selected from the group.

8. The method according to claim 1, wherein at least 150 multiple CG gene loci are selected from the group.

9. The method according to claim 1, wherein at least 200 multiple CG gene loci are selected from the group.

10. The method according to claim 1, wherein at least 250 multiple CG gene loci are selected from the group.

11. The method according to claim 1, wherein at least 500 multiple CG gene loci are selected from the group.

12. The method according to claim 1, wherein at least 1,000 multiple CG gene loci are selected from the group.

13. The method according to claim 1, wherein all 1,352 CG gene loci are selected from the group.

14. The method according to item 1, comprising amplifying genomic DNA from a blood or saliva sample by polymerase chain reaction.

15. Observation of unmethylated CG loci in human genomic DNA is performed according to item 14, comprising hybridizing genomic DNA to a bead chip to create a genomic DNA hybridized bead chip, and then imaging the genomic DNA hybridized bead chip.

17. The method of item 16, comprising using a regression algorithm to correlate the unmethylated CG loci observed in (b) with the unmethylated GC loci observed in a reference group of human individuals.

19. The method according to claim 18, comprising using a computing device to run a penaltyed regression model for creating a regression algorithm for relative weighted CpG, wherein the methylation risk score (MRS) is defined by a linear combination of beta values ​​c and weights w for m CpG sites, and MRS = Σi = 1mwici.

20. Observation of unmethylated CG loci in human genomic DNA is performed according to item 1, comprising hybridizing genomic DNA to a bead chip to create a genomic DNA hybridized bead chip, and then imaging the genomic DNA hybridized bead chip.

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