{"id":1742,"date":"2019-05-09T13:11:06","date_gmt":"2019-05-09T11:11:06","guid":{"rendered":"http:\/\/www.newslab.sk\/2019\/05\/09\/vyuzitie-ngs-next-generation-sequencing-v-rutinnej-diagnostike\/"},"modified":"2019-05-09T13:17:57","modified_gmt":"2019-05-09T11:17:57","slug":"application-of-ngs-next-generation-sequencing-in-routine-diagnostics","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/application-of-ngs-next-generation-sequencing-in-routine-diagnostics\/","title":{"rendered":"Application of NGS (next generation sequencing) in routine diagnostics"},"content":{"rendered":"

*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper.<\/strong><\/span><\/p>\n

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\u00davod<\/strong><\/p>\n

Term\u00edn myeloproliferat\u00edvne neopl\u00e1zie (MPN) ozna\u010duje skupinu ochoren\u00ed, ktor\u00fdch patogen\u00e9za sa za\u010d\u00edna transform\u00e1ciou pluripotentnej kme\u0148ovej bunky. V\u00a0transformovanej bunke doch\u00e1dza k somatick\u00fdm mut\u00e1ci\u00e1m, ktor\u00e9 poskytuj\u00fa selekt\u00edvnu v\u00fdhodu pre patologick\u00fd klon, ktor\u00fd potl\u00e1\u010da fyziologick\u00fa<\/p>\n

hemopo\u00e9zu. D\u00f4sledkom tohto procesu doch\u00e1dza aj k v\u00fdvoju krvn\u00fdch elementov z jedn\u00e9ho hematologick\u00e9ho radu, ktor\u00fd v\u00fdrazne prevy\u0161uje ostatn\u00e9(1). Z\u00e1kladn\u00e9 rozdelenie MPN je zalo\u017een\u00e9 na pr\u00edtomnosti, resp. nepr\u00edtomnosti Philadelphia (Ph) chromoz\u00f3mu. Medzi Ph pozit\u00edvne neopl\u00e1zie patr\u00ed chronick\u00e1 myelocytov\u00e1 leuk\u00e9mia (CML) s\u00a0pr\u00edtomnos\u0165ou BCR<\/em>–ABL1 <\/em>f\u00fazie. Ph negat\u00edvne MPN zvy\u010dajne nes\u00fa somatick\u00fa driver <\/em>mut\u00e1ciu v g\u00e9noch JAK2<\/em>, CALR <\/em>alebo MPL<\/em>, ktor\u00e9 s\u00fa hlavn\u00fdmi diagnostick\u00fdmi markermi spolu s\u00a0hematologick\u00fdmi a morfologick\u00fdmi abnormalitami. Transform\u00e1cia ochorenia do myelodysplastick\u00e9ho syndr\u00f3mu (MDS) alebo sekund\u00e1rnej ak\u00fatnej myeloidnej leuk\u00e9mie (AML) je mo\u017en\u00e1 pri Ph+ aj Ph\u2013 MPN(2).<\/p>\n

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Ph negat\u00edvne MPN<\/strong><\/p>\n

Medzi Ph\u2013 MPN s\u00fa zaraden\u00e9 neopl\u00e1zie bez BCR<\/em>–ABL1 <\/em>f\u00fazie. T\u00e1to skupina zah\u0155\u0148a prav\u00fa polycyt\u00e9miu (PV), esenci\u00e1lnu trombocyt\u00e9miu (ET) a prim\u00e1rnu myelofibr\u00f3zu (PMF). WHO klasifik\u00e1cia MPN je zalo\u017een\u00e1 najm\u00e4 na morfologick\u00fdch zmen\u00e1ch v kostnej dreni a po\u010dte krvn\u00fdch elementov v\u00a0perif\u00e9rnej krvi. Trombocyt\u00f3za je hlavn\u00fdm znakom pre ET, ale b\u00fdva pr\u00edtomn\u00e1 aj pri in\u00fdch MPN. Erytrocyt\u00f3za je diagnostick\u00fdm znakom pre PV a leukocyt\u00f3za sa vyskytuje u\u00a0pacientov s pokro\u010dil\u00fdm \u0161t\u00e1diom ochorenia. Pacienti s PMF maj\u00fa \u010dasto kombin\u00e1ciu prejavov, av\u0161ak v signifikantnej \u010dasti sa vyskytuje najm\u00e4 an\u00e9mia. Na diferenci\u00e1lnu diagnostiku uveden\u00fdch ochoren\u00ed sa v rutinnej praxi vyu\u017e\u00edva detekcia somatick\u00fdch mut\u00e1ci\u00ed na molekulovej \u00farovni. Vo viac ako 95 % pr\u00edpadov s\u00fa za vznik MPN zodpovedn\u00e9 driver <\/em>mut\u00e1cie v\u00a0g\u00e9noch JAK2<\/em>, CALR <\/em>a MPL<\/em>(3). \u0160tandardn\u00fdm postupom pri ur\u010den\u00ed diagn\u00f3zy je detekcia mut\u00e1cie JAK2 <\/em>(V617F) a v pr\u00edpade negativity sa pokra\u010duje detekciou mut\u00e1ci\u00ed JAK2 <\/em>v ex\u00f3ne 12 \u2013 15. Pozit\u00edvny n\u00e1lez je v takomto pr\u00edpade indik\u00e1torom PV. V\u00a0pr\u00edpade negat\u00edvneho v\u00fdsledku pokra\u010duje vy\u0161etrenie anal\u00fdzou g\u00e9nu CALR<\/em>, v ktorom sa vyskytuj\u00fa najm\u00e4 frame-shift <\/em>(FS) mut\u00e1cie posledn\u00e9ho ex\u00f3nu, \u010do ma za n\u00e1sledok vznik alternat\u00edvneho C-konca v peptide. Ak nie s\u00fa pr\u00edtomn\u00e9 ani mut\u00e1cie v\u00a0g\u00e9ne CALR<\/em>, vy\u0161etrenie je v poslednom kroku zameran\u00e9 na detekciu MPL <\/em>(W515L\/K). Pr\u00edtomnos\u0165 somatick\u00fdch mut\u00e1ci\u00ed v\u00a0g\u00e9noch CALR <\/em>a MPL <\/em>je v priamej s\u00favislosti s diagn\u00f3zami ET a PMF, ktor\u00fdch patogen\u00e9za postihuje megakaryocytov\u00fa l\u00edniu buniek. ET s\u00favis\u00ed najm\u00e4 s megakaryocytovou prolifer\u00e1ciou a PMF s diferenci\u00e1ciou \u2013 myelodyspl\u00e1ziou(4). Na z\u00e1klade poslednej WHO reklasifik\u00e1cie myeloproliferat\u00edvnych neopl\u00e1zi\u00ed z roku 2016 bolo nevyhnutn\u00e9 zahrn\u00fa\u0165 do rutinnej molekulovogeneticke diagnostiky \u010fal\u0161ie g\u00e9ny pre spr\u00e1vne zaradeni pacientov do jednotliv\u00fdch skup\u00edn, ur\u010denie lie\u010debnej strat\u00e9gie a progn\u00f3zy, monitorovanie MRD (minim\u00e1lna rezidu\u00e1lna choroba) a na pos\u00fadenie zaradenia do transplanta\u010dn\u00e9ho programu. Po\u017eadovan\u00e9 g\u00e9ny zah\u0155\u0148aj\u00fa ASXL1<\/em>, IDH1\/IDH2<\/em>, SF3B1<\/em>, SRSF2<\/em>, DNMT3A<\/em>, TET2 <\/em>a EZH2<\/em>(5). Vhodnou alternat\u00edvou simult\u00e1nneho vy\u0161etrenia viacer\u00fdch g\u00e9nov, resp. vybran\u00fdch ex\u00f3nov a hotspotov\u00fdch oblast\u00ed bolo zavedenie panelov\u00e9ho NGS do rutinnej diagnostiky myeloproliferat\u00edvnych neopl\u00e1zi\u00ed.<\/p>\n

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Next generation sequencing <\/em><\/strong>(NGS)<\/strong><\/p>\n

Naj\u010dastej\u0161ie vyu\u017e\u00edvan\u00fdm postupom pri NGS je tzv. sequencing by synthesis <\/em>(SBS) od firmy Illumina. Proces spo\u010d\u00edva v tom, \u017ee po\u010das cyklu DNA synt\u00e9zy s\u00fa fluorescen\u010dne zna\u010den\u00e9 dNTP inkorporovan\u00e9 do templ\u00e1tov\u00e9ho vl\u00e1kna pomocou DNA polymer\u00e1zy. V ka\u017edom cykle je dan\u00fd nukleotid identifikovan\u00fd prostredn\u00edctvom excit\u00e1cie fluorof\u00f3ru. Pred samotn\u00fdm sekvenovan\u00edm je potrebn\u00e1 pr\u00edprava tzv. kni\u017enice. Z\u00e1kladn\u00fdm princ\u00edpom pr\u00edpravy kni\u017en\u00edc je fragment\u00e1cia DNA, pr\u00edpadne cDNA s n\u00e1slednou modifik\u00e1ciou 3\u2019 a 5\u2019 koncov vl\u00e1kien, na ktor\u00fdch doch\u00e1dza k lig\u00e1cii adapt\u00e9rov. K\u00a0jednotliv\u00fdm vzork\u00e1m sa po\u010das pr\u00edpravy prid\u00e1va unik\u00e1tna kombin\u00e1cia adapt\u00e9rov, \u010do umo\u017e\u0148uje multiplexov\u00fa anal\u00fdzu (do 96 vzoriek). Fragmenty DNA s viazan\u00fdmi adapt\u00e9rmi s\u00fa n\u00e1sledne amplifikovan\u00e9 pomocou PCR reakcie a pre\u010disten\u00e9 pod\u013ea pr\u00edslu\u0161n\u00e9ho protokolu(6). Na tvorbu klastrov je potrebn\u00e1 hybridiz\u00e1cia amplifikovan\u00fdch kni\u017en\u00edc na flow-cell. Na jej povrchu sa nach\u00e1dzaj\u00fa oligonukleotidy komplement\u00e1rne k\u00a0sekvenci\u00e1m adapt\u00e9rov. Po hybridiz\u00e1cii nast\u00e1va tvorba klastrov prostredn\u00edctvom most\u00edkovej amplifik\u00e1cie, po\u010das ktorej DNA polymer\u00e1za vytvor\u00ed komplement\u00e1rne vl\u00e1kno k templ\u00e1tu. P\u00f4vodn\u00e9 vl\u00e1kno je odmyt\u00e9 a ponechan\u00e9 je nov\u00e9, reverzn\u00e9. Na konci reverzn\u00e9ho vl\u00e1kna sa nach\u00e1dza adapt\u00e9rov\u00e1 sekvencia, pomocou ktorej sa nov\u00e9 vl\u00e1kno prichyt\u00ed na komplement\u00e1rny oligonukleotid na povrchu flow-cell. DNA polymer\u00e1za tak vytvor\u00ed nov\u00e9 komplement\u00e1rne vl\u00e1kno identick\u00e9 s p\u00f4vodn\u00fdm templ\u00e1tom. Vzniknut\u00e1 dsDNA je denaturovan\u00e1, a tak sa m\u00f4\u017ee samostatne ka\u017ed\u00e9 vl\u00e1kno op\u00e4\u0165 prichyti\u0165 adapt\u00e9rom na pr\u00edslu\u0161n\u00fd oligonukleotid (obr\u00e1zok 1)<\/em><\/strong>. Uveden\u00fdm sp\u00f4sobom vznikaj\u00fa naraz vo vzorke tis\u00edcky klastrov, ktor\u00e9 s\u00fa sekvenovan\u00e9 v\u00a0priebehu 2-3 dn\u00ed. Pr\u00edtomnos\u0165 konkr\u00e9tnej b\u00e1zy vo vl\u00e1kne DNA je detegovan\u00e1 pomocou \u0161tyroch reverzibiln\u00fdch termin\u00e1torov, ktor\u00e9 s\u00fa zna\u010den\u00e9 fluroescen\u010dn\u00fdmi farbivami s\u00a0rozdielnou emisiou. Sekven\u00e1tor (MiSeq System, Illumina) po ka\u017edom cykle odfot\u00ed fluorescen\u010dn\u00e9 sign\u00e1ly na flow-cell a po ukon\u010den\u00ed anal\u00fdzy posklad\u00e1 v\u00fdsledn\u00fa sekvenciu(8).<\/p>\n

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S\u00fabor pacientov <\/strong><\/p>\n

V roku 2018 bolo do rutinnej diagnostiky zaveden\u00e9 panelov\u00e9 sekvenovanie TruSight Myeloid Sequencing Panel, Illumina. Do s\u00faboru bolo zatia\u013e zaraden\u00fdch 56 pacientov. Pou\u017eit\u00fdm materi\u00e1lom bola gDNA z\u00edskan\u00e1 izol\u00e1ciou z plnej kostnej drene a perif\u00e9rnej krvi. Do vy\u0161etrenia boli zahrnut\u00ed pacienti s diagn\u00f3zou zo skupiny myeloproliferat\u00edvnych neopl\u00e1zi\u00ed. Naj\u010dastej\u0161ie i\u0161lo o pacientov nereaguj\u00facich na lie\u010dbu s podozren\u00edm na pr\u00edtomnos\u0165 mut\u00e1cie v in\u00fdch g\u00e9noch, ako JAK2<\/em>, CALR <\/em>a MPL<\/em>, u pacientov s \u0165a\u017eko definovate\u013enou diagn\u00f3zou z\u00a0d\u00f4vodu polyfenotypov\u00e9ho prejavu ochorenia alebo v\u00a0pr\u00edpade predikcie \u00faspe\u0161nosti transplanta\u010dn\u00e9ho procesu.<\/p>\n

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Materi\u00e1l a met\u00f3dy<\/strong><\/p>\n

Na oddelen\u00ed lek\u00e1rskej genetiky, Medirex, a. s., bolo medzi rutinn\u00e9 vy\u0161etrenia pacientov s MPN zaveden\u00e9 panelov\u00e9 sekvenovanie TruSight Myeloid Sequencing Panel, Illumina. Uveden\u00e1 met\u00f3da sl\u00fa\u017ei na detekciu somatick\u00fdch variantov, ktor\u00fdch v\u00fdskyt je typick\u00fd najm\u00e4 pre ak\u00fatnu myeloidn\u00fa leuk\u00e9miu (AML), myelodysplastick\u00fd syndr\u00f3m (MDS), myeloproliferat\u00edvne neopl\u00e1zie (MPN), chronick\u00fa myeloidn\u00fa leuk\u00e9miu (CML), chronick\u00fa myelomonocytov\u00fa leuk\u00e9miu (CMML) a\u00a0juveniln\u00fa myelomonocytov\u00fa leuk\u00e9miu (JMML). Uveden\u00fd panel pokr\u00fdva 15 g\u00e9nov (ex\u00f3ny) a v 39 g\u00e9noch zah\u0155\u0148a hotspotov\u00e9 oblasti (tabu\u013eka 1)<\/em><\/strong>. Okrem g\u00e9nov JAK2<\/em>, CALR <\/em>a MPL <\/em>zah\u0155\u0148a v\u0161etky ostatn\u00e9 g\u00e9ny potrebn\u00e9 na diagnostiku MPN pod\u013ea najnov\u0161ej WHO klasifik\u00e1cie z roku 2016. Vstupn\u00fdm materi\u00e1lom je gDNA s koncentr\u00e1ciou 50 ng na reakciu. Pou\u017eit\u00e1 bola ch\u00e9mia MiSeq Reagent Kit v3 v kombin\u00e1cii s nano-flow-cell, \u010do umo\u017e\u0148uje simult\u00e1nnu anal\u00fdzu 8 vzoriek. Ve\u013ekos\u0165 v\u00fdsledn\u00fdch amplik\u00f3nov je ~250 bp. Z\u00edskan\u00e9 v\u00fdsledky boli hodnoten\u00e9 v softv\u00e9roch Finalist Dx, Ingenuity a IGV. Do spr\u00e1vy pre klinikov boli uveden\u00e9 iba sekven\u010dn\u00e9 varianty s\u00a0patog\u00e9nnym a pravdepodobne patog\u00e9nnym v\u00fdznamom, s po\u010dtom \u010d\u00edtan\u00ed aspo\u0148 500x a alelovou frekvenciou nad 5 %.<\/p>\n

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V\u00fdsledky<\/strong><\/p>\n

Pacient 1.<\/em><\/strong><\/p>\n

46-ro\u010dn\u00e1 pacientka lie\u010den\u00e1 od roku 2009 s\u00a0diagn\u00f3zou PV vo FNsP F. D. Roosevelta v Banskej Bystrici. V roku 2017 progresia ochorenia do sekund\u00e1rnej myelofibr\u00f3zy. V\u00a0marci 2018 na oddelen\u00ed lek\u00e1rskej genetiky, Medirex, a. s., bola panelov\u00fdm NGS detegovan\u00e1 pr\u00edtomnos\u0165 sekven\u010dn\u00e9ho variantu JAK2 <\/em>(V617F) s patog\u00e9nnym v\u00fdznamom a alelovou frekvenciou 50 %. V tom istom mesiaci bola pr\u00edtomnos\u0165 uvedenej mut\u00e1cie potvrden\u00e1 met\u00f3dou real-time PCR s 53 % zast\u00fapen\u00edm. V apr\u00edli 2018 bola pacientka zaraden\u00e1 do transplanta\u010dn\u00e9ho programu v Nemocnici sv. Cyrila a Metoda v Bratislave. V tom \u010dase bola kvantita mutovan\u00e9ho JAK2 <\/em>u\u017e 73 %. V\u00a0j\u00fani 2018 bol prv\u00fdkr\u00e1t vy\u0161etren\u00fd chim\u00e9rizmus po transplant\u00e1cii. Hodnota p\u00f4vodnej autol\u00f3gnej krvotvorby klesla pod 2 % a JAK2 <\/em>(V617F) na 0,64 %.<\/p>\n

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Pacient 2.<\/em><\/strong><\/p>\n

49-ro\u010dn\u00fd pacient bol v apr\u00edli 2018 odoslan\u00fd na genetick\u00e9 vy\u0161etrenie do centr\u00e1lneho laborat\u00f3ria Medirex, a. s., s\u00a0podozren\u00edm na CML. V\u00fdsledok anal\u00fdzy BCR<\/em>–ABL1 <\/em>zlomov bol bez n\u00e1lezu. V tom istom mesiaci boli pomocou FISH <\/em>vy\u0161etren\u00e9 g\u00e9ny MLL<\/em>, CRLF2, ABL2 <\/em>a molekulovou MLPA anal\u00fdzou g\u00e9n IKZF1<\/em>. Uveden\u00e9 vy\u0161etrenia mali negat\u00edvny v\u00fdsledok. U pacienta bol z\u00e1rove\u0148 vy\u0161etren\u00fd vstupn\u00fd panel pre AML, z ktor\u00e9ho pozit\u00edvny v\u00fdsledok mala iba hodnota expresie WT1 <\/em>(NCN = 0,05). V m\u00e1ji 2018 bol u pacienta vy\u0161etren\u00fd myeloidn\u00fd NGS panel, pomocou ktor\u00e9ho bola detegovan\u00e1 pr\u00edtomnos\u0165 sekven\u010dn\u00e9ho variantu v g\u00e9ne CSF3R <\/em>(T618I) s\u00a0patog\u00e9nnym v\u00fdznamom. Alelov\u00e1 frekvencia bola 39 %. V\u00a0septembri 2018 bolo realizovan\u00e9 kontroln\u00e9 NGS vy\u0161etrenie, ktor\u00fdm bola dok\u00e1zan\u00e1 st\u00fapaj\u00faca hodnota mutovan\u00e9ho g\u00e9nu a\u017e na 48 %. Hladina expresie WT1 <\/em>g\u00e9nu mala tie\u017e st\u00fapaj\u00faci charakter. U pacienta je podozrenie na relaps.<\/p>\n

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Pacient 3.<\/em><\/strong><\/p>\n

47-ro\u010dn\u00fd pacient lie\u010den\u00fd v Nemocnici sv. Cyrila a\u00a0Metoda s diagn\u00f3zou PV. V roku 2018 prechod do sekund\u00e1rnej myelofibr\u00f3zy. V janu\u00e1ri 2018 bol vy\u0161etren\u00fd vstupn\u00fd panel pre MPN. Pomocou real-time PCR bola detegovan\u00e1 pr\u00edtomnos\u0165 mut\u00e1cie JAK2 <\/em>(V617F), kvantita 86 %. U pacienta bol n\u00e1sledne vy\u0161etren\u00fd myeloidn\u00fd NGS panel, ktor\u00fd potvrdil pr\u00edtomnos\u0165 sekven\u010dn\u00fdch variantov v g\u00e9noch DNMT3A <\/em>(R659C) s\u00a0alelovou frekvenciou 40,2 % a JAK2 <\/em>(V617F) s alelovou frekvenciou 60,7 %. U pacienta je zva\u017eovan\u00e1 alog\u00e9nna transplant\u00e1cia.<\/p>\n

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Diskusia<\/strong><\/p>\n

WHO klasifik\u00e1cia MPN ochoren\u00ed z roku 2016 zah\u0155\u0148a nov\u00e9 diagnostick\u00e9 entity pod\u013ea pr\u00edtomnosti genetick\u00fdch variantov (mut\u00e1ci\u00ed) vybran\u00fdch g\u00e9nov. Diagnostick\u00e9 krit\u00e9ri\u00e1 boli roz\u0161\u00edren\u00e9 v\u010faka zav\u00e1dzaniu nov\u00fdch citlivej\u0161\u00edch met\u00f3d molekulovej diagnostiky a cielen\u00e9mu sekvenovaniu klinicky relevantn\u00fdch g\u00e9nov. \u010eal\u0161ie mut\u00e1cie v g\u00e9noch, ktor\u00e9 sa podie\u013eaj\u00fa na epigenetickej regul\u00e1cii a signaliz\u00e1cii, zohr\u00e1vaj\u00fa k\u013e\u00fa\u010dov\u00fa \u00falohu v patogen\u00e9ze MPN. Mut\u00e1cie v epigenetick\u00fdch regul\u00e1toroch s\u00fa zapojen\u00e9 do inici\u00e1cie a progresie ochorenia. Cie\u013eom pr\u00e1ce bolo zavedenie panelov\u00e9ho NGS do rutinnej molekulovogenetickej diagnostiky t\u00fdchto ochoren\u00ed. Na anal\u00fdzu pacientov bol pou\u017eit\u00fd TruSight Myeloid Sequencing Panel od firmy Illumina, ktor\u00fdm mo\u017eno analyzova\u0165 54 g\u00e9nov v jednej reakcii. Pomocou uveden\u00e9ho vy\u0161etrenia s\u00fa zachyten\u00e9 aj mut\u00e1cie s predikt\u00edvnym v\u00fdznamom pre progn\u00f3zu a pre\u017e\u00edvanie pacientov, ale aj na pos\u00fadenie \u00faspe\u0161nosti transplanta\u010dn\u00e9ho procesu. Zavedenie panelov\u00e9ho NGS m\u00e1 v rutinnej diagnostike v\u00fdznamn\u00e9 postavenie, preto\u017ee umo\u017e\u0148uje aj detekciu tak\u00fdch mut\u00e1ci\u00ed, ktor\u00e9 by pri be\u017enom vy\u0161etrovacom algoritme v\u00f4bec neboli zachyten\u00e9. Pri PMF sa v \u010dase diagn\u00f3zy vyskytuj\u00fa in\u00e9 mut\u00e1cie ako v JAK2<\/em>, CALR <\/em>a MPL <\/em>vo viac ako 50 %. Pri PV a ET s\u00fa \u010fal\u0161ie mut\u00e1cie detegovan\u00e9 pribli\u017ene v 10 % pr\u00edpadov.<\/p>\n

Mut\u00e1cia v g\u00e9ne TET2 <\/em>sa pri PMF vyskytuje v 19 % a ASXL1 <\/em>a\u017e v 40 % pr\u00edpadov. Obe s\u00fa pri MPN asociovan\u00e9 so zlou progn\u00f3zou( 10). V\u00fdskyt patog\u00e9nnych variantov v g\u00e9noch ASXL1<\/em>, EZH2<\/em>, SRSF2 <\/em>a IDH1\/2 <\/em>indikuje u pacientov v\u00fdrazne skr\u00e1ten\u00fd \u010das pre\u017e\u00edvania. V pr\u00edpadoch s dvomi a viac mut\u00e1ciami je medi\u00e1n do\u017eitia 2,6 roka, pri jednej mut\u00e1cii 7 rokov a v\u00a0pr\u00edpadoch bez mut\u00e1ci\u00ed v uveden\u00fdch g\u00e9noch je medi\u00e1n pre\u017e\u00edvania 12,3 roka(11). Hlavnou v\u00fdhodou NGS anal\u00fdzy je simult\u00e1nna anal\u00fdza ve\u013ek\u00e9ho mno\u017estva DNA s vysokou citlivos\u0165ou v\u00a0porovnan\u00ed s klasick\u00fdm Sangerov\u00fdm sekvenovan\u00edm. Sekvenovan\u00edm mnoh\u00fdch g\u00e9nov naraz mo\u017eno vy\u0161etri\u0165 aj viacer\u00e9 diagn\u00f3zy, \u010do zna\u010dne skracuje \u010das nasadenia vhodnej terapie. NGS anal\u00fdzou je stanoven\u00e1 aj alelov\u00e1 frekvencia danej mut\u00e1cie, \u010do sl\u00fa\u017ei najm\u00e4 na sledovanie \u00fa\u010dinku lie\u010dby a MRD (minim\u00e1lna rezidu\u00e1lna choroba). Zatia\u013e najv\u00e4\u010d\u0161ou nev\u00fdhodou v\u0161etk\u00fdch NGS anal\u00fdz je ve\u013ek\u00fd objem z\u00edskan\u00fdch d\u00e1t, ktor\u00e9 musia by\u0165 okrem in\u00e9ho vyhodnoten\u00e9 so zrete\u013eom na dan\u00fd druh ochorenia a\u00a0p\u00f4vod zachytenej mut\u00e1cie (somatick\u00e1 vs germin\u00e1lna). V\u00a0neposlednom rade je pri hodnoten\u00ed d\u00e1t potrebn\u00e9 bra\u0165 oh\u013ead aj na etick\u00fa str\u00e1nku a citlivos\u0165 z\u00edskan\u00fdch inform\u00e1ci\u00ed o danom pacientovi v s\u00favislosti s diagn\u00f3zou.<\/p>\n

 <\/p>\n

Z\u00e1ver<\/strong><\/p>\n

Sekvenovanie novej gener\u00e1cie sa v rutinnej diagnostike pou\u017e\u00edva v \u010doraz vy\u0161\u0161ej miere a predstavuje v\u00fdznamn\u00fd n\u00e1stroj na potvrdenie, resp. vyl\u00fa\u010denie viacer\u00fdch diagn\u00f3z v\u00a0jednej anal\u00fdze. Detekcia patog\u00e9nnych variantov v dan\u00fdch g\u00e9noch m\u00e1 diagnostick\u00fd a prognostick\u00fd v\u00fdznam u\u00a0pacientov s myeloproliferat\u00edvnymi neopl\u00e1ziami. NGS vy\u0161etrenie je tie\u017e d\u00f4le\u017eit\u00e9 na stanovenie klon\u00e1lneho charakteru ochorenia. Najv\u00e4\u010d\u0161ou v\u00fdzvou do bud\u00facnosti je zefekt\u00edvnenie vyhodnotenia ve\u013ek\u00e9ho objemu z\u00edskan\u00fdch d\u00e1t a zavedenie \u0161tandardn\u00e9ho postupu na hodnotenie ich kvality.<\/p>\n

\u00a0<\/strong><\/p>\n

LITERAT\u00daRA<\/strong><\/p>\n

    \n
  1. Mead AJ, Mullally A. Myeloproliferative neoplasm stem cells. Blood 2017, 129(12): 1607-161.<\/li>\n
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  4. Schalling M, Gleiss A, Gisslinger B, et al. Essential thrombocythemia vs. pre-fibrotic\/early primary myelofibrosis: discrimination by laboratory and clinical data. Blood Cancer J 2017; 7(12): 643-647.<\/li>\n
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  6. Liu L, Li Y, Li S, et al. Comparison of Next-Generation Sequencing Systems. J Biomed Biotechnol 2012; 2012: 251364.<\/li>\n
  7. Chandran A. Overview of Next-Generation Sequencing Technologies and Its Application in Chemical Biology. In: Advancing Development of Synthetic Gene Regulators. Springer These 2018.<\/li>\n
  8. Heather JM, Chain B. The sequence of sequencers: The history of sequencing DNA. Genomics 2016; 107(1): 1-8.<\/li>\n
  9. https:\/\/www.illumina.com\/products\/by-type\/clinical-research-products\/<\/a> trusight-myeloid.html#gene-list<\/li>\n
  10. Brecqueville M, Rey J, Bertucci F, et al. Mutation analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 in myeloproliferative neoplasms. Genes Chromosomes Cancer 2012; 51: 743-755.<\/li>\n
  11. Guglielmelli P, Lasho TL, Rotunno G, et al. The number of prognostically detrimental mutations and prognosis in primary myelofibrosis: an international study of 797 patients. Leukemia 2014; 28(9): 1804-1810.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

    *All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper.     \u00davod Term\u00edn myeloproliferat\u00edvne neopl\u00e1zie (MPN) ozna\u010duje skupinu ochoren\u00ed, ktor\u00fdch patogen\u00e9za sa za\u010d\u00edna transform\u00e1ciou pluripotentnej kme\u0148ovej bunky. V\u00a0transformovanej bunke doch\u00e1dza k somatick\u00fdm mut\u00e1ci\u00e1m, ktor\u00e9 poskytuj\u00fa selekt\u00edvnu v\u00fdhodu pre patologick\u00fd<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_mi_skip_tracking":false,"footnotes":""},"categories":[290],"tags":[1208,1206,1209],"class_list":["post-1742","post","type-post","status-publish","format-standard","hentry","category-genetics","tag-myeloproliferative-neoplasms","tag-ngs-en","tag-routine-diagnostics","typ_clanku-original-work"],"acf":{"abstrakt":"

    Next generation sequencing (NGS) also known as massive parallel sequencing is able to detect a large amount of data in a single assay. More than 90% sequence data is obtained from sequencing by synthesis (SBS) principle from Illumina. Including myeloid NGS panel into routine examination was the most significant diagnostic milestone for patients with myeloproliferative neoplasms on the department of medical genetics, Medirex, a. s. in 2018. Standard diagnostic procedure is cascade examimnation of JAK2, CALR and MPL genes to distinguish polycythemia vera (PV), essential trombocythemia (ET) and primary myelofibrosis (PMF). Due to new WHO reclassification of myeloproliferative diseases in 2016, it was necessary to extend the panel of examined genes for correct assesment of diagnosis, monitoring of treatment response and inclusion into transplantation process.<\/p>\n

    Keywords: NGS, myeloproliferative neoplasms, routine diagnostics<\/p>\n","casopis":[{"ID":1633,"post_author":"7","post_date":"2019-05-09 08:56:48","post_date_gmt":"2019-05-09 06:56:48","post_content":"

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    • Importance and diagnostic of anti-phospholipid antibodies in women with reproductive disorders<\/li>\r\n \t
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    • HCV infection \u2013 more than 20 years of drug development<\/li>\r\n<\/ul>","post_title":"newsLab","post_excerpt":"","post_status":"publish","comment_status":"closed","ping_status":"closed","post_password":"","post_name":"newslab-3","to_ping":"","pinged":"","post_modified":"2019-05-09 08:56:48","post_modified_gmt":"2019-05-09 06:56:48","post_content_filtered":"","post_parent":0,"guid":"http:\/\/www.newslab.sk\/?post_type=casopis&p=1633","menu_order":0,"post_type":"casopis","post_mime_type":"","comment_count":"0","filter":"raw"}],"strana":"9-12","upload_clanok":{"ID":1740,"id":1740,"title":"Vyu\u017eitie NGS (next generation sequencing) v rutinnej diagnostike","filename":"Vyu\u017eitie-NGS-next-generation-sequencing-v-rutinnej-diagnostike.pdf","filesize":208088,"url":"https:\/\/www.newslab.sk\/wp-content\/uploads\/2019\/05\/Vyu\u017eitie-NGS-next-generation-sequencing-v-rutinnej-diagnostike.pdf","link":"https:\/\/www.newslab.sk\/en\/application-of-ngs-next-generation-sequencing-in-routine-diagnostics\/vyuzitie-ngs-next-generation-sequencing-v-rutinnej-diagnostike-2\/","alt":"","author":"7","description":"","caption":"","name":"vyuzitie-ngs-next-generation-sequencing-v-rutinnej-diagnostike-2","status":"inherit","uploaded_to":1742,"date":"2019-05-09 11:07:09","modified":"2019-05-09 11:07:09","menu_order":0,"mime_type":"application\/pdf","type":"application","subtype":"pdf","icon":"https:\/\/www.newslab.sk\/wp-includes\/images\/media\/document.png"}},"_links":{"self":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1742","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/comments?post=1742"}],"version-history":[{"count":0,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1742\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/media?parent=1742"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/categories?post=1742"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/tags?post=1742"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}