LITŠOANTŠISO TSA BATHO
liphatsa tsa lefutso tsa tlhaho
Tatelano ea nako e telele e bonts'a keketseho ea GGC ho NOTCH2NLC e amanang le lefu la neuronal intranuclear inclusion.
ONT requencing |Ilumina |Exome sequencing |CRISPR-Cas9 ONT e lebisitsoeng ho latellana |RNA-seq |Ho letsetsa ONT 5mC methylation
Lintlha-khōlō
1.Ka tlhahlobo ea Khokahano lelapeng le leholo la NIID, ho ile ha khetholloa libaka tse peli tse hokahaneng.
Tatelano ea nako e telele ea ho bala 2.ONT le Cas-9 mediated e ruisang ONT sequencing e fumane sesosa se ka bang teng sa lefutso sa NIID, GGC e pheta katoloso ho 5′ UTR ea NOTCH2NLC.Boithuto bona bo tlalehile katoloso ea liphatsa tsa lefutso tse ikhethileng khetlo la pele tse bileng teng ka ho ikatisa ha likaroloana.
Tatelano ea 3.RNA e senotse lingoliloeng tse sa tloaelehang tsa antisense qalong kapa ka hare ho GGC e pheta libaka tsa katoloso ho NOTCH2NLC.
Ka morao
Neuronal intranuclear inclusion disease (NIID) ke lefu le tsoelang pele le le bolaeang la neurodegenerative, le khetholloang ka ho ba teng ha eosinophilic hyaline intranuclear inclusions tsamaisong ea methapo ea bohareng le ea peripheral.Lipontšo tsa eona tse fapaneng haholo tsa kliniki li baka mathata a maholo ho hlahlojoa ho fihlela ho hlahisoa ha biopsy ea letlalo.Leha ho le joalo, mekhoa e thehiloeng ho histopathology e ntse e tšoeroe ke tlhahlobo e fosahetseng, e hlokang kutloisiso ea liphatsa tsa lefutso tsa NIID.
Katleho
Tlhahlobo ea Khokahano
SHort-read sequencing based whole genome sequencing (WGS) le whole exome sequencing (WES) e ile ea etsoa ho lelapa le leholo la NIID (13 e amehileng le litho tse 7 tse sa amehang).Tlhahlobo ea khokahano ho li-SNP tse ntšitsoeng ho data ena e senotse libaka tse peli feela tse hokahaneng: sebaka sa 3.5 Mb ho 1p36.31-p36.22 (boholo ba LOD = 2.32) le sebaka sa 58.1 Mb ho 1p22.1-q21.3 (boholo ba LOD: 4.21 ).Leha ho le joalo, ha ho li-SNP kapa li-CNV tsa pathogenic tse ileng tsa khetholloa libakeng tsena tse hokahaneng.
Katoloso e phetoang ea GGC ho NOTCH2NLC
Ntatellano e thehiloeng ho anopore e ile ea sebetsoa ho 13 e amehileng le litho tsa 4 tse sa ameheng tse tsoang malapeng a 8 (setho se seng se amehileng se ne se hlophisitsoe ke Pacbio nako e telele ea ho bala tatellano.).Lintlha tse baliloeng ka nako e telele li senoletse lefu le amanang le GGC le pheta-pheta katoloso ho 5' UTR ea NOTCH2NLC 'mapa oa liphatsa tsa lefutso ho ea sebakeng se hokahaneng sa 58.1 Mb (Setšoantšo sa 1).Likatoloso tsena tse pheta-phetoang li boetse li khetholloa maemong ohle a 40 a nakoana a NIID a lekiloeng ke RP-PCR.
Cas-9 mediated target tatellano sethaleng sa nanopore se ile sa sebelisoa ho fihlela litaba tse baloang ka holimo ho NOTCH2NLC pheta (100 X-1,795 X).Litumellano tsena tsa litumellano li lumellane hantle le liphuputso tsa nakong e fetileng mabapi le katoloso ea phetisetso ea GGC.Ho feta moo, {(GGA)n (GGC)n} ho pheta-pheta ho ile ha khetholloa e le letšoao le ka bang teng la liphatsa tsa lefutso bakeng sa phenotype e matla haholo (Setšoantšo sa 2).
Setšoantšo sa 1. Katoloso e amanang le ho pheta-pheta e tšoauoang ho exon 1 ea NOTCH2NLC isoforms.
Setšoantšo sa 2. Tatelano ea tumellano ea NPTCH2NLC e pheta-pheta ho bakuli ba NIID ba nang le (*) kapa ntle le bofokoli bo matla ba phenotype
NLiphatsa tsa lefutso tsa OTCH2NL ke liphatsa tsa lefutso tse ipapisitseng le motho, tseo ho lumeloang hore li na le karolo ea bohlokoa kholong ea boko ba motho le mafu a methapo ea kutlo.Leha ho le joalo, liphatsa tsa lefutso tse tharo tse amanang le NOTCH2 (NOTCH2NLA, NOTCH2NLB le NOTCH2NLC) tse nang le >99.1% ea boitsebiso ba tatellano ha lia ka tsa rarolloa ho fihlela ka mor'a kopano ea morao-rao ea genome ea motho.Tatelano e sa sebetseng le ea nako e telele e baloang sethaleng sa nanopore e bonts'itse melemo e hlokomelehang ho rarolleng libaka tse tšoanang haholo le (GGC)n pheta ka 100% GC-rich.
Katoloso e phetoang ea GGC ho NOTCH2NLC
Ttatellano ea ranscriptome e ile ea sebetsoa ho litho tsa 2 tse amehileng le tse 2 tse sa ameheng.Botebo bo tloahelehileng ba ho bala bo ile ba baloa ho latela moelelo le likhoele tsa antisense ho ea holimo ho li-exons tsa pele tsa li-paralog tsa NOTCH2NL.Lingoliloeng tse sa tloaelehang tse khahlanong le maikutlo li ile tsa fumanoa feela maemong a amehileng, a lutseng qalong kapa ka hare ho sebaka sa ho atolosa ho pheta-pheta (litlhōrō tsa Purple ho F1-14 le F1-16 ho Figure 3.).Ho feta moo, li-DEGs tse 54 li ile tsa khetholloa 'me kaofela li ile tsa ntlafatsoa ka mantsoe a GO le a MPO a amanang le mesebetsi ea methapo.
Setšoantšo sa 3. Botebo bo tloaelehileng ba ho bala ka holimo ho exon ea pele ea NOTCH2NLC maemong a sa amehang (ka holimo) le a amehang (ka tlase).
Thekenoloji
Oxford Nanopore Teghnologies (ONT)
Ntatellano ea anopore e ikhetholla ho li-platform tse ling tsa tatellano, ka hore li-nucleotide li baloe ka kotloloho ntle le ts'ebetso ea DNA synthesis.Ha DNA ea khoele e le 'ngoe e feta ka har'a pore ea protheine e boholo ba nano (nanopore), li-nucleotide tse fapaneng li hlahisa maqhubu a ionic a fapaneng, a ka tšoaroang le ho fetisetsoa ka tatellano ea metheo.Sethala sa tatellano sa ONT ka bosona ha se bontše moeli o hlakileng oa tekheniki bolelele ba ho bala DNA.Ka hona, li-Ultra-long reads (ULRs) lia fumaneha bakeng sa kopano ea genome ea boleng bo holimo.Ho feta moo, lipalo tsena tse telele haholo, tse telele ho lekana ho tšela likarolo tse rarahaneng tsa tatellano kapa phapang ea sebopeho, li thusa ho hlola mefokolo ea tatellano e khuts'oane mona.
Nanopore sequencing
Khethollo ea sebopeho (SV).
Statellano ea mahala ea synthesis e bolokile haholo tlhahisoleseling ea DNA methylation ho template.Methylated A, T, C le G li hlahisa maqhubu a ionic a fapaneng ho tloha ho a se nang methylated, a ka baloang ka ho toba ke sethala.Nanopore sequencing e matlafatsa profiling ea genome ea 5mC le 6mA ka qeto ea single-nucleotide.
Referense
Jun Sone, le.al.Tatelano e baloang ka nako e telele e supa ho atolosoa ha GGC ho NOTCH2NLC e amanang le lefu la neuronal intranuclear inclusion.Nature Genetics (2019)
Theknoloji le Lintlha-khōlō e ikemiselitse ho arolelana ts'ebeliso ea morao-rao e atlehileng ea mekhoa e fapaneng ea tatellano ea maemo a holimo mabaleng a fapaneng a liphuputso hammoho le mehopolo e bohlale moralong oa liteko le ho rafa data.
Nako ea poso: Jan-06-2022