Method for predicting health of a human subject

Abstract

A method for predicting the health of a human subject comprising the steps of: (a) obtaining genomic DNA from a blood or saliva sample of a human subject, (b) observing unmethylated CG loci, wherein the observing comprises performing a bisulfite conversion process on the genomic DNA of the human subject, (c) comparing the unmethylated CG loci observed in (b) to unmethylated CG loci observed in genomic DNA collected from a reference group of human individuals, and (d) correlating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in the reference group of human individuals to predict the health of the human subject.

Description

Related applications

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

[0002] This paper generally involves constructing and validating a DNA reference matrix, focusing on certain unmethylated cytosine-phosphate-guanine sites / CG loci, in order to (a) predict the levels of multiple proteins and metabolites in human subjects under test, and (b) associate the predicted levels of multiple proteins and metabolites with the disease risk of that human subject. In this way, it is possible to better associate patterns with diseases and create more complex phenotypic (disease) predictions with more information through methylation. Background Technology

[0003] When the body ages faster than the calendar advances, individuals experience accelerated biological aging, a leading predictor of chronic diseases. Fortunately, lifestyle and medical interventions have been shown to help slow, halt, and even reverse the body's biological aging process. This article discusses a method for measuring an individual's rate of aging and its changes, enabling that individual to take control of their anti-aging or health journey in the right direction. Summary of the Invention

[0004] A novel and improved method is provided for predicting the health of human subjects. The method includes, comprises, or substantially comprises the following steps: (a) Obtaining genomic DNA from a blood or saliva sample of a human subject in a manner known in the art; (b) Observe the unmethylated CG locus in the genomic DNA of the human subject, wherein the observation includes performing a bisulfite conversion process on the genomic DNA of the human subject, such that cytosine residues in the genomic DNA of the human subject are converted to uracil, while 5-methylcytosine residues in the genomic DNA of the human subject are not converted to uracil; (c) Compare the unmethylated CG loci observed in (b) with unmethylated CG loci observed in genomic DNA collected from human individual reference sets; and (d) Associating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in the human individual reference group to predict the health of the human subjects, wherein the CG loci are multiple CG loci selected from the group consisting of: cg00011943、cg00014020、cg00056433、cg00067133、cg00071360、cg00074145、cg00080105、cg00120123、cg00122614、cg00153759、cg00177243、cg00178119、cg00182994、cg00209129、cg00215550、cg00217225、cg00236988、cg00237714、cg00243480、cg00251125、cg00262132、cg00275686、cg00278547、cg00283022、cg00303773、cg00322993、cg00342468、cg00358895、cg00458337、cg00465312、cg00481382、cg00530438、cg00538458、cg00548856、cg00593298、cg00594167、cg00602326、cg00615271、cg00631329、cg00639633、cg00646555、cg00687674、cg00687962、cg00698355、cg00712841、cg00737155、cg00748449、cg00756943、cg00767058、cg00800095、cg00815399、cg00910115、cg00935026、cg00935421、cg00964035、cg00973876、cg00993310、cg01020263、cg01025883、cg01067216、cg01089474、cg01094440、cg01098936、cg01113710、cg01134012、cg01166985、cg01169463、cg01178063、cg01224520、cg01243823、cg01253289、cg01266159、cg01271129、cg01274656、cg01282852、cg01313513、cg01320510、cg01346718、cg01377932、cg01380346、cg01402735、cg01410359、cg01446515、cg01457077、cg01541424、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、cg08573404、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、cg19297245、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、cg23549902、cg23561053、cg23570968、cg23574427、cg23586821、cg23615892、cg23643435、cg23793194、cg23838503、cg23879118、cg23883522、cg23894058、cg23910413、cg23923562、cg23956385、cg23964820、cg24003560、cg24027965、cg24055030、cg24055461、cg24112628、cg24141036、cg24185013、cg24220948、cg24236938、cg24241488、cg24262996、cg24266506、cg24293344、cg24317857、cg24337318、cg24354818、cg24362016、cg24365657、cg24372256、cg24392971、cg24397183、cg24432675、cg24463073、cg24468603、cg24492140、cg24507955、cg24520070、cg24523415、cg24530234、cg24543255、cg24577941、cg24589102、cg24601404、cg24674445、cg24678611、cg24697099、cg24721873、cg24769628、cg24796651、cg24799795、cg24835295、cg24920358、cg24925701、cg24967099、cg24976744、cg24982343、cg24997750、cg25047618、cg25063538、cg25114 611、cg25146575、cg25148589、cg25156198、cg25161899、cg25181997、cg25 189904, cg25202593, cg25259149, cg25259707, cg25330725, cg25331593, cg25361850, cg25375711, cg25397922, cg25418001, cg25418670, cg254204 82、cg25420747、cg25436217、cg25499552、cg25504214、cg25560772、cg25 563198、cg25569590、cg25623035、cg25638549、cg25639566、cg25670171、c g25671708, cg25703588, cg25717260, cg25738786, cg25745729, cg25784308, cg25786980, cg25843115, cg25843873, cg25903319, cg25916759, cg259 27838, cg25928819, cg25943804, cg25945293, cg25952581, cg25952596, cg26029902, cg26044490, cg26071551, cg26093148, cg26116542, cg2611655 6、cg26156626、cg26229024、cg26239485、cg26267310、cg26276120、cg263 01353、cg26314853、cg26315261、cg26441486、cg26442630、cg26443646、cg 26479667、cg26499822、cg26500093、cg26501477、cg26528000、cg2654485 7、cg26577320、cg26599630、cg26606789、cg26676765、cg26691434、cg2670 2770, cg26760942, cg26764761, cg26767387, cg26805839, cg26818257, cg26839512, cg26883048, cg26962439, cg26992381, cg27021634, cg27066201cg27079776, cg27127090, cg27128734, cg27130665, cg27131142, cg27152890, cg27154679, cg271 54725, cg27208536, cg27229100, cg27245649, cg27256066, cg27296119, cg27315314, cg27336261, cg27348423, cg27354537, cg27357902, cg27368039, cg27374247, cg27395675, cg27449200, cg274 54650, cg27461310, cg27488658, cg27498980, cg27500647, cg27553457, cg27589058, cg27632248. ,

[0005] The number of CG loci observed from a specified group of CG loci can vary depending on the implementation of the method. Therefore, the number of CG loci can be any number between 2 CG loci in the specified group and 1352 (all) CG loci.

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

[0007] The observation of the unmethylated CG locus in the genomic DNA of the human subject may include hybridizing the genomic DNA with a bead chip to create a genomic DNA hybrid bead chip, and then imaging the genomic DNA hybrid bead chip.

[0008] The method may include using a regression algorithm to associate the unmethylated CG locus observed in (b) with the unmethylated GC locus observed in the human individual reference group.

[0009] The method may include executing a penalized regression model via a computing device to create a CpG regression algorithm with relative weights, wherein the methylation risk score (MRS) is defined by a linear combination of the β values ​​c of m CpG sites and the weights w, where: MRS = ∑ i =1 killer . Detailed Implementation

[0010] A method for predicting the health of human subjects includes the following steps: (a) Obtaining genomic DNA from blood or saliva samples of human subjects; (b) Observe unmethylated CG loci in the genomic DNA of human subjects, wherein the observation includes performing a bisulfite conversion process on the genomic DNA of human subjects, such that cytosine residues in the genomic DNA of human subjects are converted to uracil, while 5-methylcytosine residues in the genomic DNA of human subjects are not converted to uracil; (c) Compare the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in genomic DNA collected from human individual reference sets; and (d) Associate the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in a human individual reference group to predict the health of human subjects, wherein the CG loci are multiple CG loci selected from the following groups: cg00011943、cg00014020、cg00056433、cg00067133、cg00071360、cg00074145、cg00080105、cg00120123、cg00122614、cg00153759、cg00177243、cg00178119、cg00182994、cg00209129、cg00215550、cg00217225、cg00236988、cg00237714、cg00243480、cg00251125、cg00262132、cg00275686、cg00278547、cg00283022、cg00303773、cg00322993、cg00342468、cg00358895、cg00458337、cg00465312、cg00481382、cg00530438、cg00538458、cg00548856、cg00593298、cg00594167、cg00602326、cg00615271、cg00631329、cg00639633、cg00646555、cg00687674、cg00687962、cg00698355、cg00712841、cg00737155、cg00748449、cg00756943、cg00767058、cg00800095、cg00815399、cg00910115、cg00935026、cg00935421、cg00964035、cg00973876、cg00993310、cg01020263、cg01025883、cg01067216、cg01089474、cg01094440、cg01098936、cg01113710、cg01134012、cg01166985、cg01169463、cg01178063、cg01224520、cg01243823、cg01253289、cg01266159、cg01271129、cg01274656、cg01282852、cg01313513、cg01320510、cg01346718、cg01377932、cg01380346、cg01402735、cg01410359、cg01446515、cg01457077、cg01541424、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、cg08573404、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、cg19297245、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、cg23549902、cg23561053、cg23570968、cg23574427、cg23586821、cg23615892、cg23643435、cg23793194、cg23838503、cg23879118、cg23883522、cg23894058、cg23910413、cg23923562、cg23956385、cg23964820、cg24003560、cg24027965、cg24055030、cg24055461、cg24112628、cg24141036、cg24185013、cg24220948、cg24236938、cg24241488、cg24262996、cg24266506、cg24293344、cg24317857、cg24337318、cg24354818、cg24362016、cg24365657、cg24372256、cg24392971、cg24397183、cg24432675、cg24463073、cg24468603、cg24492140、cg24507955、cg24520070、cg24523415、cg24530234、cg24543255、cg24577941、cg24589102、cg24601404、cg24674445、cg24678611、cg24697099、cg24721873、cg24769628、cg24796651、cg24799795、cg24835295、cg24920358、cg24925701、cg24967099、cg24976744、cg24982343、cg24997750、cg25047618、cg25063538、cg25114 611、cg25146575、cg25148589、cg25156198、cg25161899、cg25181997、cg25 189904, cg25202593, cg25259149, cg25259707, cg25330725, cg25331593, cg25361850, cg25375711, cg25397922, cg25418001, cg25418670, cg254204 82、cg25420747、cg25436217、cg25499552、cg25504214、cg25560772、cg25 563198、cg25569590、cg25623035、cg25638549、cg25639566、cg25670171、c g25671708, cg25703588, cg25717260, cg25738786, cg25745729, cg25784308, cg25786980, cg25843115, cg25843873, cg25903319, cg25916759, cg259 27838, cg25928819, cg25943804, cg25945293, cg25952581, cg25952596, cg26029902, cg26044490, cg26071551, cg26093148, cg26116542, cg2611655 6、cg26156626、cg26229024、cg26239485、cg26267310、cg26276120、cg263 01353、cg26314853、cg26315261、cg26441486、cg26442630、cg26443646、cg 26479667、cg26499822、cg26500093、cg26501477、cg26528000、cg2654485 7、cg26577320、cg26599630、cg26606789、cg26676765、cg26691434、cg2670 2770, cg26760942, cg26764761, cg26767387, cg26805839, cg26818257, cg26839512, cg26883048, cg26962439, cg26992381, cg27021634, cg27066201cg27079776, cg27127090, cg27128734, cg27130665, cg27131142, cg27152890, cg27154679, cg271 54725, cg27208536, cg27229100, cg27245649, cg27256066, cg27296119, cg27315314, cg27336261, cg27348423, cg27354537, cg27357902, cg27368039, cg27374247, cg27395675, cg27449200, cg274 54650, cg27461310, cg27488658, cg27498980, cg27500647, cg27553457, cg27589058, cg27632248. ,

[0011] Deletion of methylation at any of the identified CG loci is associated with and predicts one or more of the following diseases, conditions, or illnesses: alcoholic liver disease, allergic rhinitis, aortic aneurysm dissection, arterial embolism, asthma, bladder cancer, breast cancer, bronchiectasis, cancer, cardiomyopathy, chronic bronchitis, chronic kidney disease, chronic liver disease, chronic pulmonary heart disease, chronic viral hepatitis, cirrhosis, coin-shaped lesion lung disease, colorectal cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), coronary artery disease, cardiovascular disease (CVD) (excluding stroke), depression, emphysema, hypertensive heart disease, interstitial lung disease, leukemia, liver cancer, lung cancer, melanoma, myocardial infarction, non-alcoholic fatty liver disease (NAFLD), and non-Hodgkin lymphoma. 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, as verified by genomic DNA collected from individual reference groups.

[0012] The number of CG loci observed from a designated group of CG loci can vary depending on the implementation of the method. Therefore, the plurality of CG loci can be any number between 2 and 1352 (all) CG loci in the designated group. In one possible implementation, at least five CG loci are selected from the group. In another implementation, at least ten CG loci are selected from the group. In yet another implementation, at least 15 CG loci are selected from the group.

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

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

[0015] The bisulfite conversion process mentioned in step (b) is known in the art. Kits 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 a 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.

[0016] Performing a bisulfite conversion process may include the following steps: amplifying genomic DNA from a blood sample via a polymerase chain reaction process, hybridizing the genomic DNA with a bead chip to create a genomic DNA hybridization bead chip, staining the genomic DNA hybridization bead chip, and then imaging the genomic DNA hybridization bead chip.

[0017] Imaging of genomic DNA hybridization bead arrays can be performed using an Illumina EPIC850k array, which provides unambiguous CG locus identification for the purpose of observing methylation at specific CG loci in the human DNA genome. Alternatively, epigenetic β values ​​can be collected using other methods if desired.

[0018] The method also includes the step of associating the unmethylated CG loci observed in (b) with the unmethylated GC loci observed in the human individual reference group using a regression algorithm. This includes performing a penalized regression model via a computing device to create a CpG regression algorithm with relative weights, where the methylation risk score (MRS) is defined by a linear combination of the β values ​​c of m CpG sites and the weights w, where: MRS = ∑ i =1 killer It should be understood that GC loci can be assigned different relative weights.

[0019] To ensure that methylation risk scores enhance predictive value beyond common capture features (e.g., age and sex), we created baseline prediction models incorporating patient age, sex, reference-based methylated cell type composition estimates, self-reported race-ethnicity, self-reported smoking status, and the top ten genetic principal components. We fitted the baseline models using either linear or logistic regression models, depending on whether the outcome was continuous or binary. We compared the baseline models with models incorporating baseline features along with methylation or genotype data. For MRS, we used LASSO, elastic networks, and ridge regularization to regress genomic features while keeping baseline features as fixed covariates. We fitted all models using 10-fold double cross-validation, where additional cross-validation was performed on each training set to select hyperparameters, and the cross-validated model from the training set was then used to predict the reserved test set. We used association tests (via linear regression) to test the significance between the cross-validated predictions (i.e., the tandem predictors across all folds) and the true outcomes.

[0020] Furthermore, this method incorporates proteomics and metabolomics data as alternative biomarkers. For example, we are using DNA methylation to predict classic clinical markers such as C-reactive protein. For this purpose, a baseline CG locus reference matrix was first constructed from a reference set of individuals with a known health history using a variety of known proteins and metabolites of medical interest for the diagnosis of alcoholic liver disease, allergic rhinitis, aortic aneurysm dissection, arterial embolism, asthma, bladder cancer, breast cancer, bronchiectasis, cancer, cardiomyopathy, chronic bronchitis, chronic kidney disease, chronic liver disease, chronic pulmonary heart disease, chronic viral hepatitis, cirrhosis, coin-shaped lesion lung disease, colorectal cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), coronary artery disease, cardiovascular disease (CVD) (excluding stroke), 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. Then, by associating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in the human individual reference group, the method was used to predict the levels of these multiple proteins and metabolites in human subjects to predict disease risk or health in human subjects.

[0021] As used herein, each of the following terms written in singular grammar—"a," "an," and "the / described"—means "at least one" or "one or more." The use of the phrase "one or more" in this document does not alter the intended meaning of "a," "an," or "the / described." Therefore, unless explicitly defined or stated herein, or unless the context clearly indicates otherwise, the terms "a," "an," and "the / described" may also refer to and encompass multiple stated entities or objects, as used herein. For example, the phrase "protein," as used herein, may also refer to and encompass multiple proteins.

[0022] As used herein, each of the following terms, “including,” “having,” and “comprising,” as well as their language / grammatical variations, derivatives, and / or affixes, means “including but not limited to,” and should be understood to specifically identify the stated component, feature, property, parameter, integer, or step, without excluding the addition of one or more additional components, features, properties, parameters, integers, steps, or groups thereof.

[0023] As used herein, the phrase “consisting of” is a closed term and excludes any elements, steps, or ingredients not explicitly mentioned. As used herein, the phrase “consisting essentially of” is a semi-closed term indicating that something is limited to the specified components and that these components do not materially affect the essential and novel characteristics of the specified content.

[0024] As used herein, approximate terms such as approximately, substantially, about, etc., refer to ±10% of the stated value.

[0025] Although the methods of this disclosure have been illustratively described and demonstrated through specific exemplary embodiments and their examples, it will be apparent to those skilled in the art that many alternatives, modifications, and / or variations will occur to them. Therefore, all such alternatives, modifications, and / or variations are intended to fall within the spirit of the appended claims and be covered within their broad scope.

Claims

1. A method comprising: (a) Obtaining genomic DNA from blood or saliva samples of human subjects; (b) Observe the unmethylated CG locus in the genomic DNA of the human subject, wherein the observation includes performing a bisulfite conversion process on the genomic DNA of the human subject, such that cytosine residues in the genomic DNA of the human subject are converted to uracil, while 5-methylcytosine residues in the genomic DNA of the human subject are not converted to uracil; (c) Compare the unmethylated CG loci observed in (b) with unmethylated CG loci observed in genomic DNA collected from human individual reference sets; as well as (d) Associating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in the human individual reference group to predict the health of the human subjects, wherein the CG loci are at least five CG loci selected from the group consisting of: cg00011943、cg00014020、cg00056433、cg00067133、cg00071360、cg00074145、cg00080105、cg00120123、cg00122614、cg00153759、cg00177243、cg00178119、cg00182994、cg00209129、cg00215550、cg00217225、cg00236988、cg00237714、cg00243480、cg00251125、cg00262132、cg00275686、cg00278547、cg00283022、cg00303773、cg00322993、cg00342468、cg00358895、cg00458337、cg00465312、cg00481382、cg00530438、cg00538458、cg00548856、cg00593298、cg00594167、cg00602326、cg00615271、cg00631329、cg00639633、cg00646555、cg00687674、cg00687962、cg00698355、cg00712841、cg00737155、cg00748449、cg00756943、cg00767058、cg00800095、cg00815399、cg00910115、cg00935026、cg00935421、cg00964035、cg00973876、cg00993310、cg01020263、cg01025883、cg01067216、cg01089474、cg01094440、cg01098936、cg01113710、cg01134012、cg01166985、cg01169463、cg01178063、cg01224520、cg01243823、cg01253289、cg01266159、cg01271129、cg01274656、cg01282852、cg01313513、cg01320510、cg01346718、cg01377932、cg01380346、cg01402735、cg01410359、cg01446515、cg01457077、cg01541424、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、cg08573404、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、cg19297245、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、cg23549902、cg23561053、cg23570968、cg23574427、cg23586821、cg23615892、cg23643435、cg23793194、cg23838503、cg23879118、cg23883522、cg23894058、cg23910413、cg23923562、cg23956385、cg23964820、cg24003560、cg24027965、cg24055030、cg24055461、cg24112628、cg24141036、cg24185013、cg24220948、cg24236938、cg24241488、cg24262996、cg24266506、cg24293344、cg24317857、cg24337318、cg24354818、cg24362016、cg24365657、cg24372256、cg24392971、cg24397183、cg24432675、cg24463073、cg24468603、cg24492140、cg24507955、cg24520070、cg24523415、cg24530234、cg24543255、cg24577941、cg24589102、cg24601404、cg24674445、cg24678611、cg24697099、cg24721873、cg24769628、cg24796651、cg24799795、cg24835295、cg24920358、cg24925701、cg24967099、cg24976744、cg24982343、cg24997750、cg25047618、cg25063538、cg25114 611、cg25146575、cg25148589、cg25156198、cg25161899、cg25181997、cg25 189904, cg25202593, cg25259149, cg25259707, cg25330725, cg25331593, cg25361850, cg25375711, cg25397922, cg25418001, cg25418670, cg254204 82、cg25420747、cg25436217、cg25499552、cg25504214、cg25560772、cg25 563198、cg25569590、cg25623035、cg25638549、cg25639566、cg25670171、c g25671708, cg25703588, cg25717260, cg25738786, cg25745729, cg25784308, cg25786980, cg25843115, cg25843873, cg25903319, cg25916759, cg259 27838, cg25928819, cg25943804, cg25945293, cg25952581, cg25952596, cg26029902, cg26044490, cg26071551, cg26093148, cg26116542, cg2611655 6、cg26156626、cg26229024、cg26239485、cg26267310、cg26276120、cg263 01353、cg26314853、cg26315261、cg26441486、cg26442630、cg26443646、cg 26479667、cg26499822、cg26500093、cg26501477、cg26528000、cg2654485 7、cg26577320、cg26599630、cg26606789、cg26676765、cg26691434、cg2670 2770, cg26760942, cg26764761, cg26767387, cg26805839, cg26818257, cg26839512, cg26883048, cg26962439, cg26992381, cg27021634, cg27066201cg27079776, cg27127090, cg27128734, cg27130665, cg27131142, cg27152890, cg27154679, cg27154725, cg27208536, cg27229100, cg27245649, cg27256066, cg27296119, cg27315314, cg27336261 cg27348423, cg27354537, cg27357902, cg27368039, cg27374247, cg27395675, cg27449200, cg27454650, cg27461310, cg27488658, cg27498980, cg27500647, cg27553457, cg27589058, cg27632248.

2. The method of claim 1, wherein at least ten CG loci are selected from the group described above.

3. The method of claim 1, wherein at least 15 CG loci are selected from the group described above.

4. The method of claim 1, wherein at least 25 CG loci are selected from the group described above.

5. The method of claim 1, wherein at least 50 CG loci are selected from the group described above.

6. The method of claim 1, wherein at least 75 CG loci are selected from the group described above.

7. The method of claim 1, wherein at least 100 CG loci are selected from the group described above.

8. The method of claim 1, wherein at least 150 CG loci are selected from the group described above.

9. The method of claim 1, wherein at least 200 CG loci are selected from the group described above.

10. The method of claim 1, wherein at least 250 CG loci are selected from the group described above.

11. The method of claim 1, wherein at least 500 CG loci are selected from the group described above.

12. The method of claim 1, wherein at least 1000 CG loci are selected from the group described above.

13. The method of claim 1, wherein all 1,352 CG loci are selected from the group described above.

14. The method of claim 1, further comprising amplifying the genomic DNA of the blood or saliva sample by polymerase chain reaction.

15. The method of claim 14, wherein the observation of the unmethylated CG locus in the genomic DNA of the human subject comprises hybridizing the genomic DNA with a bead chip to create a genomic DNA hybrid bead chip, and then imaging the genomic DNA hybrid bead chip.

16. The method of claim 16, further comprising using a regression algorithm to associate the unmethylated CG locus observed in (b) with the unmethylated GC locus observed in the human individual reference group.

17. The method of claim 18, further comprising executing a penalized regression model via a computing device to create a CpG regression algorithm with relative weights, wherein the methylation risk score (MRS) is defined by a linear combination of β values ​​c of m CpG sites and weights w, wherein: MRS=∑ i =1 mwici 。 18. The method of claim 1, wherein the observation of the unmethylated CG locus in the genomic DNA of the human subject comprises hybridizing the genomic DNA with a bead chip to create a genomic DNA hybrid bead chip, and then imaging the genomic DNA hybrid bead chip.