GENOMICS
genetics dabeecadda
Isku xigxiga-dheer-akhriska ayaa tilmaamaya GGC ku celcelinta balaadhinta ee NOTCH2NLC ee la xidhiidha cudurka ka mid noqoshada neuronal intranuclear
ONT isku xigxiga |Illumina |Isku xigxiga exome oo dhan |CRISPR-Cas9 ONT taxanaha la beegsaday |RNA-seq |ONT 5mC methylation call
Tilmaamaha
1. Marka la eego falanqaynta Linkage ee qoys weyn oo NIID ah, laba gobol ayaa la aqoonsaday.
2.ONT-ku-salaysan taxanaha dheer-akhrinta iyo Cas-9 dhexdhexaadinta kobcinta ONT isku xigxiga ayaa daahfuray sabab iman kara hidde ee NIID, GGC ku celi balaadhinta 5′ UTR ee NOTCH2NLC.Daraasadani waxa ay sheegtay in ay ku soo noqnoqotay balaadhinta hiddo-wadaha gaarka ah ee bini’aadamka markii ugu horreysay kuwaas oo ka soo baxay qaybo kala duwan.
3.RNA isku xigxiga ayaa daaha ka qaaday qoraalada lidka ku ah aan caadiga ahayn bilawgii ama gudaha GGC ku celceli gobolada fidinta ee NOTCH2NLC.
Asalkii hore
NCudurka ku darida intranuclear-ka ee euronal (NIID) waa cudur horusocod ah oo dilaa ah neurodegenerative, kaas oo lagu garto joogitaanka eosinophilic hyaline intranuclear inclusions ee habka dhexe iyo habdhiska dareenka.Tilmaamaheeda caafimaad ee aadka u kala duwan ayaa kor u qaadaya dhibaatooyin weyn oo lagu ogaanayo cudurka ilaa laga soo saaro biopsiga maqaarka.Si kastaba ha ahaatee, hababka ku salaysan histopathology-ga ayaa wali la ildaran ogaanshaha khaldan, kaas oo ku baaqaya fahamka hidda-socodka NIID.
Guulihii
Falanqaynta isku xirka
Staxanaha hort-akhri ku salaysan oo dhan genome sequencing (WGS) iyo gabi ahaan exome sequencing (WES) ayaa lagu sameeyay qoys weyn oo NIID ah (13 ay saamaysay iyo 7 xubnood oo aan la taaban).Falanqaynta isku xirka SNP-yada laga soo saaray xogtan ayaa muujisay laba gobol oo keliya oo isku xidhan: gobolka 3.5 Mb ee 1p36.31-p36.22 (ugu badnaan LOD=2.32) iyo gobolka 58.1 Mb ee 1p22.1-q21.3 (ugu badnaan LOD: 4.21) ).Si kastaba ha ahaatee, ma jiraan SNP-yada cudur-sidaha ama CNV-yada lagu aqoonsaday gobolladan ku xiran.
GGC ku celi balaadhinta NOTCH2NLC
Nisku xigxiga anopore-ku-saleysan ayaa lagu farsameeyay 13 ay saameysay iyo 4 xubnood oo aan saameynin oo ka kala socday 8 qoys (xubin kale oo ay saameysay waxaa iskuxigay Pacbio madal taxanaha akhriska dheer.).Xogta dheer ee la akhriyay ayaa shaaca ka qaaday cudurada la xidhiidha GGC ku celcelinta balaadhinta 5′ UTR ee khariidaynta hiddasidaha NOTCH2NLC ilaa 58.1 Mb ee gobolka isku xidhan (Jaantuska 1).Balaadhintan soo noqnoqota ayaa sidoo kale lagu aqoonsaday dhammaan 40 kiis oo goos goos ah oo NIID ah oo ay tijaabisay RP-PCR.
Cas-9 dhexdhexaadinta bartilmaameedka isku xigxiga ee madal nanopore ayaa la shaqaaleysiiyay si loo gaaro caynsanaanta akhriska sare ee NOTCH2NLC ku celiska (100 X-1,795 X).Taxanahan la isku afgartay ayaa si wanaagsan ugu raacay natiijooyinkii hore ee ku celcelinta GGC.Waxaa intaa dheer, {(GGA)n (GGC)n} soo noqnoqoshada ayaa loo aqoonsaday inay yihiin calaamad muujinaysa daciifnimada-xukunka phenotype (Jaantuska 2).
Jaantuska 1. Cudurada la xidhiidha balaadhinta soo noqnoqda ee lagu aqoonsaday exon 1 ee NOTCH2NLC isoforms.
Jaantuska 2. Taxanayaasha la isku raacsan yahay ee NPTCH2NLC waxay ku celceliyaan bukaanada NIID ee leh(*) ama aan lahayn daciifnimo-xun phenotype
NHidde-sidayaasha OTCH2NL waa hiddo-sidaha gaarka ah ee bini'aadamka, kuwaas oo la rumeysan yahay inay door muhiim ah ka ciyaaraan korriinka maskaxda aadanaha iyo cudurrada neerfaha.Si kastaba ha ahaatee, saddex hidde-sideyaal NOTCH2 la xidhiidha (NOTCH2NLA, NOTCH2NLB iyo NOTCH2NLC) oo leh> 99.1% aqoonsiga isku xigxiga lama xalin ilaa shirkii ugu dambeeyay ee genome-ga aadanaha.Isku xigxiga bilaashka ah iyo akhrinta dheer ee isku xigxiga ee nanopore waxay muujisay faa'iidooyin la taaban karo oo lagu xallinayo gobollada isku midka ah iyo (GGC) n ku celceliya 100% GC-hodanka ah.
GGC ku celi balaadhinta NOTCH2NLC
Ttaxanaha ranscriptome ayaa lagu farsameeyay 2 ay saamaysay iyo 2 xubnood oo aan la taaban.Qoto dheer ee akhriska caadiga ah waxaa lagu xisaabiyay dareenka iyo xargaha dareenka lidka ku ah ee kor u kaca ee ugu horreeya ee NOTCH2NL paralogs.Qoraallada aan caadiga ahayn ee ka-hortagga dareenka ayaa laga helay kaliya kiisaska la saameeyay, kuwaas oo ku fadhiya bilowga ama gudaha gobolka ballaarinta soo noqnoqda (Purple peaks ee F1-14 iyo F1-16 ee Jaantuska 3.).Intaa waxaa dheer, 54 DEG ayaa la aqoonsaday oo dhamaantood lagu hodmay ereyada GO iyo MPO ee la xiriira hawlaha neerfaha.
Jaantus 3. Qoto dheer oo akhris ah oo caadi ah xagga sare ee exonka koowaad ee NOTCH2NLC ee aan saamaynin (korka) iyo kuwa saameeya (hoos)
Farsamada
Oxford Nanopore Teghnologies (ONT)
Nisku xigxiga anopore waxa uu ka soocaa qaybaha kale ee isku xigxiga, taas oo ah in nucleotide-yada si toos ah loo akhriyo iyada oo aan la helin habka DNA-da.Sida DNA-da hal xarig ah u dhex marto dalool borotiin ah oo nano ah (nanopore), nucleotide kala duwan ayaa abuura qulqulo ionic oo kala duwan, kuwaas oo la qabsan karo loona wareejin karo isku xigxiga saldhigyada.Madal isku xigxiga ONT lafteedu ma muujinayso xaddid farsamo oo muuqata dhererka akhriska DNA.Sidaa darteed, akhrinta-dheer-dheer (ULRs) ayaa diyaar u ah isu-ururinta genome ee tayada sare leh.Intaa waxaa dheer, akhristayaashan aadka u dheer, kuwaas oo ku filan in ay ka gudbaan sifooyinka isku xigxiga ee kakan ama kala duwanaanshiyaha qaabdhismeedka, ayaa gacan ka geysta in ay ka gudbaan xaddidaadaha akhrinta gaaban ee halkan.
Isku xigxiga Nanopore
Kala duwanaanshaha qaab dhismeedka (SV) aqoonsiga
Sisku xigxiga-free ynthesis-free si weyn loo ilaaliyo macluumaadka methylation DNA on template.Methylated A, T, C iyo G waxay abuuraan qulqulo ionic oo kala duwan kuwa aan-methylated ahayn, kuwaas oo si toos ah loogu akhriyi karo goobta.Isku xigxiga Nanopore wuxuu awood siinayaa sifada-genome-ga dhan ee 5mC iyo 6mA ee xallinta hal-nucleotide.
Tixraac
Jun Sone, iyo.al.Isku xigxiga-dheer-akhriska ayaa tilmaamaya GGC ku celcelinta balaadhinta ee NOTCH2NLC ee la xidhiidha cudurka ka mid noqoshada neuronal intranuclear.Hiddaha Dabeecada (2019)
Farsamada iyo Waxyaabaha ugu muhiimsan ujeedadeedu tahay in la wadaago adeeggii ugu dambeeyay ee lagu guulaystay ee tignoolajiyada kala duwan ee isku xigxiga-sare ee saaxadda cilmi-baarista ee kala duwan iyo sidoo kale fikrado cajiib ah oo ku saabsan naqshadaynta tijaabada iyo macdanta xogta.
Waqtiga boostada: Jan-06-2022