{"id":1117,"date":"2016-12-04T22:05:47","date_gmt":"2016-12-04T21:05:47","guid":{"rendered":"http:\/\/www.newslab.sk\/2016\/12\/04\/sekvenovanie-novej-generacie-a-jeho-vyuzitie-v-klinickej-genetike\/"},"modified":"2017-10-03T14:49:16","modified_gmt":"2017-10-03T12:49:16","slug":"next-generation-sequencing-and-its-application-in-clinical-genetics","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/next-generation-sequencing-and-its-application-in-clinical-genetics\/","title":{"rendered":"Next generation sequencing and its application in clinical genetics"},"content":{"rendered":"<pre><span style=\"color: #ff0000;\"><strong>*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf <\/strong><\/span>\r\n <span style=\"color: #ff0000;\"><strong>attachment at the end of the paper. <\/strong><\/span><\/pre>\n<p>&nbsp;<\/p>\n<p><strong>Sekvena\u010dn\u00e9 technol\u00f3gie prvej a\u00a0druhej gener\u00e1cie<\/strong><\/p>\n<p>Met\u00f3da DNA sekvenovania publikovan\u00e1 F. Sangerom koncom 70. rokov minul\u00e9ho storo\u010dia (1) ozna\u010dovan\u00e1 aj ako dideoxynukleotidov\u00e9 sekvenovanie, priniesla nov\u00e9 a\u00a0v \u010dase svojho vzniku e\u0161te netu\u0161en\u00e9 mo\u017enosti anal\u00fdzy jednotliv\u00fdch g\u00e9nov a\u00a0nesk\u00f4r cel\u00fdch gen\u00f3mov. Prvogenera\u010dn\u00e1 Sangerova met\u00f3da sa postupne vyvinula na v\u00fdkonn\u00fa automatizovan\u00fa technol\u00f3giu vyu\u017e\u00edvaj\u00facu polymer\u00e1zov\u00fa re\u0165azov\u00fa reakciu (PCR), fluorescen\u010dn\u00e9 zna\u010denie dideoxynukleotidov, kapil\u00e1rnu elektrofor\u00e9zu a\u00a0komplexn\u00e9 po\u010d\u00edta\u010dov\u00e9 spracovanie d\u00e1t a\u00a0metodicky st\u00e1le predstavuje tzv. zlat\u00fd \u0161tandard. Napriek ur\u010dit\u00fdm obmedzeniam automatiz\u00e1cia Sangerovho postupu umo\u017enila \u00faspe\u0161n\u00fa realiz\u00e1ciu projektu sekvenovania \u013eudsk\u00e9ho gen\u00f3mu a etablovanie sekvena\u010dnej anal\u00fdzy v rutinnej molekulovo-genetickej diagnostike. Program sekvenovania cel\u00e9ho \u013eudsk\u00e9ho gen\u00f3mu v\u0161ak dramaticky zv\u00fd\u0161il n\u00e1roky na kapacitu sekvena\u010dn\u00fdch technol\u00f3gi\u00ed. Projekt sa realizoval v \u201etov\u00e1rensky\u201c organizovan\u00fdch sekvena\u010dn\u00fdch centr\u00e1ch so \u0161pecifickou infra\u0161trukt\u00farou, ktor\u00e1 zah\u0155\u0148ala stovky ve\u013ekokapacitn\u00fdch sekven\u00e1torov, \u010fal\u0161\u00edch automatizovan\u00fdch pr\u00edstrojov a po\u010d\u00edta\u010dov, ako aj\u00a0ve\u013ek\u00e9 mno\u017estvo person\u00e1lu. Tak\u00fdto typ zv\u00fd\u0161enia v\u00fdkonnosti sekvenovania ale nebol perspekt\u00edvne posta\u010duj\u00faci pre nov\u00e9 v\u00fdskumn\u00e9 a\u00a0diagnostick\u00e9 v\u00fdzvy v\u00a0hum\u00e1nnej genetike a genomike a\u00a0postupne doch\u00e1dzalo k\u00a0v\u00fdvoju nov\u00fdch, \u010doraz komplexnej\u0161\u00edch sekvena\u010dn\u00fdch postupov.<\/p>\n<p>Technol\u00f3gie sekvenovania novej gener\u00e1cie (NGS) sa od automatizovanej Sangerovej met\u00f3dy odli\u0161uj\u00fa najm\u00e4 svojou vysokou v\u00fdkonnos\u0165ou. Podstata zv\u00fd\u0161enej efektivity spo\u010d\u00edva v\u00a0mas\u00edvnej paraleliz\u00e1cii biochemick\u00fdch a\u00a0merac\u00edch krokov, ako aj v\u00fdraznom zv\u00fd\u0161en\u00ed r\u00fdchlosti sekvenovania. Nov\u00e9 met\u00f3dy umo\u017e\u0148uj\u00fa simult\u00e1nnu anal\u00fdzu mili\u00f3nov sekvena\u010dn\u00fdch \u201e\u010d\u00edtan\u00ed\u201c v jednej vzorke. \u010eal\u0161ie rozdiely zah\u0155\u0148aj\u00fa odli\u0161n\u00fa formu v\u00fdchodiskov\u00e9ho templ\u00e1tu (tzv. fragmentov\u00e9 kni\u017enice, vi\u010f ni\u017e\u0161ie) a\u00a0o\u00a0nie\u010do krat\u0161iu d\u013a\u017eku sekvenovania analyzovan\u00fdch fragmentov v\u00a0porovnan\u00ed so Sangerovou met\u00f3dou. Obmedzenia sp\u00f4soben\u00e9 krat\u0161\u00edmi \u010d\u00edtaniami ale kompenzuje mas\u00edvna h\u013abka pokrytia a paraleliz\u00e1cia, t.j. mnohon\u00e1sobn\u00e9 opakovanie anal\u00fdzy tej istej cie\u013eovej oblasti gen\u00f3mu v\u00a0 zmesi obrovsk\u00e9ho mno\u017estva s\u00fabe\u017ene zoraden\u00fdch DNA fragmentov r\u00f4zneho typu (Obr.1).<\/p>\n<p>V\u00a0s\u00fa\u010dasnosti pou\u017e\u00edvan\u00e9 NGS technol\u00f3gie druhej gener\u00e1cie (popis met\u00f3d tretej gener\u00e1cie, ktor\u00e9 sa st\u00e1le e\u0161te len profiluj\u00fa, je nad r\u00e1mec tohto preh\u013eadov\u00e9ho \u010dl\u00e1nku) z\u00a0chemick\u00e9ho h\u013eadiska vyu\u017e\u00edvaj\u00fa DNA synt\u00e9zu alebo lig\u00e1ciu. Detekcia sign\u00e1lu z\u00a0mili\u00f3nov chemick\u00fdch reakci\u00ed, ktor\u00fd je n\u00e1sledne transformovan\u00fd do sekven\u010dn\u00fdch d\u00e1t, je zabezpe\u010den\u00e1 sn\u00edman\u00edm emitovanej fluorescencie (z fluorescen\u010dne zna\u010den\u00fdch nukleotidov), enzymaticky vyvolanej chemiluminiscencie (pri degrad\u00e1cii pyrofosf\u00e1tu) a\u00a0pH zmien (pri uvo\u013e\u0148ovan\u00ed prot\u00f3nov po\u010das inkorpor\u00e1cie nukleotidov do DNA re\u0165azca). Bez oh\u013eadu na chemick\u00fa podstatu sekvenovania, v\u0161etky NGS met\u00f3dy vy\u017eaduj\u00fa komplexn\u00fa predsekvena\u010dn\u00fa pr\u00edpravu DNA templ\u00e1tu \u00a0a\u00a0n\u00e1sledn\u00fa bioinformatick\u00fa anal\u00fdzu sekvena\u010dn\u00fdch d\u00e1t (Obr.2). Predsekvena\u010dn\u00e9 kroky zah\u0155\u0148aj\u00fa pr\u00edpravu tzv. sekvena\u010dnej (fragmentovej) kni\u017enice, pod\u013ea potreby aj vr\u00e1tane tzv. cielen\u00e9ho obohatenia (target enrichment) a\u00a0n\u00e1sledne klon\u00e1lnu amplifik\u00e1ciu pripravenej sekvena\u010dnej kni\u017enice. Pr\u00edprava kni\u017enice sa zvy\u010dajne sklad\u00e1 z\u00a0dvoch krokov, fragment\u00e1cie v\u00fdchodiskovej DNA na ve\u013ekos\u0165 150-500 bp fyzik\u00e1lnym (napr. sonik\u00e1ciou) alebo enzymatick\u00fdm sp\u00f4sobom (nukle\u00e1zou, transpoz\u00f3nami) a lig\u00e1cie adaptorov\u00fdch primerov na fragmenty. Cielen\u00e9 obohatenie, t.j. \u0161pecifick\u00e1 selekcia \u017eelan\u00fdch DNA fragmentov sa vykon\u00e1va v\u00a0pr\u00edpade potreby sekvenova\u0165 len ur\u010dit\u00e9 oblasti gen\u00f3mu v\u00a0sum\u00e1rne men\u0161om rozsahu (napr. len niektor\u00e9 ex\u00f3ny alebo sady g\u00e9nov ap.) a naj\u010dastej\u0161ie sa realizuje pomocou PCR alebo hybridiz\u00e1cie so \u0161pecifick\u00fdmi oligonukleotidov\u00fdmi pr\u00f3bami. Klon\u00e1lna amplifik\u00e1cia kni\u017enice prebieha pomocou \u0161pecifick\u00fdch typov PCR \u2013 naj\u010dastej\u0161ie tzv. emulznej alebo most\u00edkovej (bridge) PCR. Posekvena\u010dn\u00e1 bioinformatick\u00e1 anal\u00fdza zah\u0155\u0148a spracovanie nasn\u00edman\u00e9ho sign\u00e1lu, jeho transform\u00e1ciu do \u010diastkov\u00fdch nukleotidov\u00fdch sekvenci\u00ed a\u00a0 porovnanie v\u00fdslednej \u201eposkladanej\u201c DNA sekvencie s\u00a0referen\u010dnou za \u00fa\u010delom z\u00e1vere\u010dnej identifik\u00e1cie a\u00a0anot\u00e1cie variantn\u00fdch nukleotidov (2).<\/p>\n<p>Po\u010das sekvena\u010dnej reakcie sa vygeneruj\u00fa mili\u00f3ny a\u017e miliardy tzv. \u010d\u00edtan\u00ed. \u010c\u00edtanie (read) je\u00a0nukleotidov\u00e1 sekvencia ur\u010ditej d\u013a\u017eky (read length) z\u00edskan\u00e1 z DNA fragmentu nach\u00e1dzaj\u00faceho sa v\u00a0sekvena\u010dnej kni\u017enici. Pre presnos\u0165 NGS anal\u00fdzy a\u00a0spr\u00e1vnu interpret\u00e1ciu d\u00e1t je nevyhnutn\u00e9 dosiahnu\u0165 dostato\u010dn\u00fa h\u013abku pokrytia (depth of coverage). H\u013abka pokrytia je po\u010det \u010d\u00edtan\u00ed, ktor\u00fd pokr\u00fdva konkr\u00e9tny cie\u013eov\u00fd nukleotid a\u00a0be\u017ene sa vyjadruje vo forme Nx (napr. 40x, 100x, t.j. \u0161tyridsa\u0165, sto \u010d\u00edtan\u00ed cie\u013eov\u00e9ho nukleotidu). \u010eal\u0161\u00edm d\u00f4le\u017eit\u00fdm parametrom je h\u013abka sekvenovania (sequencing depth), t.j. celkov\u00e9 mno\u017estvo sekvena\u010dn\u00fdch d\u00e1t (pre\u010d\u00edtan\u00fdch nukleotidov), ktor\u00e9 vy\u017eaduje dan\u00e1 vzorka, aby sa dosiahla po\u017eadovan\u00e1 priemern\u00e1 h\u013abka pokrytia. Naj\u010dastej\u0161ie sa ud\u00e1va ako po\u010det potrebn\u00fdch \u010d\u00edtan\u00ed na vzorku (napr. 40 mili\u00f3nov, 1 miliarda) alebo po\u010det nukleotidov (b\u00e1z), ktor\u00e9 je nutn\u00e9 sekvenova\u0165 (napr. 100 Mb, 4 Gb).<\/p>\n<p>Proces anal\u00fdzy NGS d\u00e1t m\u00f4\u017eeme v\u0161eobecne rozdeli\u0165 na tri stupne (Obr.3). Prim\u00e1rna anal\u00fdza, tzv. stanovenie b\u00e1z (base-calling) zah\u0155\u0148a konvertovanie biochemick\u00fdch sign\u00e1lov, \u010di\u017ee hrub\u00fdch d\u00e1t, ktor\u00e9 boli z\u00edskan\u00e9 sn\u00edman\u00edm zmien svetelnej intenzity (fluoresencia, chemiluminiscencia) alebo zmien pH, do kr\u00e1tkych nukleotidov\u00fdch sekvenci\u00ed \u2013 \u010d\u00edtan\u00ed. Sekund\u00e1rna anal\u00fdza zabezpe\u010duje porovnanie (alignment) sekvenci\u00ed s\u00a0referenciou a\u00a0stanovenie variantn\u00fdch nukleotidov a\u00a0ich anot\u00e1ciu. Terci\u00e1rna anal\u00fdza znamen\u00e1 interpret\u00e1ciu, t.j. anal\u00fdzu zisten\u00fdch variantov z\u00a0h\u013eadiska p\u00f4vodu, jedine\u010dnosti a\u00a0funk\u010dn\u00e9ho dopadu. Ka\u017ed\u00fd z\u00a0t\u00fdchto krokov vy\u017eaduje \u0161peci\u00e1lne vytvoren\u00e9 datab\u00e1zy, algoritmy, softv\u00e9r a\u00a0sk\u00fasenosti. Procesy prim\u00e1rnej anal\u00fdzy s\u00fa u\u017e v\u00a0s\u00fa\u010dasnosti \u00faspe\u0161ne zvl\u00e1dnut\u00e9 a\u00a0postupne sa automatizuj\u00fa. Aj v\u00a0r\u00e1mci sekund\u00e1rnej anal\u00fdzy sa \u0161tandardizuj\u00fa met\u00f3dy mapovania \u010d\u00edtan\u00ed ku najnov\u0161ej referen\u010dnej sekvencii \u013eudsk\u00e9ho gen\u00f3mu. Limit\u00e1ciu \u0161ir\u0161ej klinickej aplik\u00e1cie NGS predstavuje pr\u00e1ve biomedic\u00ednska interpret\u00e1cia identifikovan\u00fdch variant, ktor\u00e1 sa zatia\u013e e\u0161te len vyv\u00edja \u010do sa t\u00fdka algoritmov, analytick\u00fdch postupov a\u00a0 identifik\u00e1cie \u010di predikcie kauz\u00e1lnych genotypovo-fenotypov\u00fdch korel\u00e1ci\u00ed (3).<\/p>\n<p>V\u00a0s\u00fa\u010dasnosti dostupn\u00e9 NGS technol\u00f3gie vyu\u017e\u00edvan\u00e9 v\u00a0klinick\u00fdch aplik\u00e1ci\u00e1ch s\u00fa Roche GS, Solid, Illumina a\u00a0IonTorrent, pri\u010dom posledn\u00e9 dve zmienen\u00e9 s\u00fa najroz\u0161\u00edrenej\u0161ie. Z\u00e1kladn\u00e1 charakteristika konkr\u00e9tnych NGS platforiem druhej gener\u00e1cie je uveden\u00e1 v\u00a0Tab.1.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Aplik\u00e1cie NGS<\/strong><\/p>\n<p>Sekvenovanie tzv. g\u00e9nov\u00fdch panelov (panelov\u00e9 sekvenovanie, PS) je zameran\u00e9 na konkr\u00e9tne ochorenia, nako\u013eko sa analyzuje stanoven\u00e1 sada zn\u00e1mych, klinicky v\u00fdznamn\u00fdch g\u00e9nov. Dosahuje sa t\u00fdm v\u00e4\u010d\u0161ia h\u013abka pokrytia (t.j. viac \u010d\u00edtan\u00ed v\u00a0\u017eelan\u00fdch oblastiach) a\u00a0n\u00e1sledne aj vy\u0161\u0161ia analytick\u00e1 senzitivita a\u00a0\u0161pecificita. Ke\u010f\u017ee PS analyzuje len g\u00e9ny so zn\u00e1mou asoci\u00e1ciou s konkr\u00e9tnymi ochoreniami, umo\u017e\u0148uje lep\u0161iu interpret\u00e1ciu n\u00e1lezov v\u00a0klinickom kontexte. V\u00a0porovnan\u00ed s\u00a0ex\u00f3mov\u00fdm a\u00a0gen\u00f3mov\u00fdm sekvenovan\u00edm (vi\u010f ni\u017e\u0161ie) je PS kompatibiln\u00e9 s\u00a0ekonomicky a\u00a0\u010dasovo v\u00fdhodnej\u0161\u00edm vyu\u017eit\u00edm men\u0161\u00edch stolov\u00fdch sekven\u00e1torov (ni\u017e\u0161ia cena pr\u00edstroja, mo\u017enos\u0165 anal\u00fdzy viacer\u00fdch vzoriek v\u00a0jednom behu) a\u00a0jednoduch\u0161\u00edm mana\u017eovan\u00edm objemu d\u00e1t. V\u00a0Tab.2 s\u00fa uveden\u00e9 pr\u00edklady g\u00e9nov\u00fdch panelov, ktor\u00e9 v\u00a0s\u00fa\u010dasnosti komer\u010dne pon\u00fakaj\u00fa v\u00fdrobcovia dvoch NGS platforiem (Illumina, Life Technologies) na priame pou\u017eitie v diagnostike.<\/p>\n<p>Ex\u00f3mov\u00e9 sekvenovanie (ES) znamen\u00e1 stanovenie DNA sekvencie ex\u00f3mu, teda v\u00e4\u010d\u0161iny z\u00a0pribli\u017ene 160.000 prote\u00edn-k\u00f3duj\u00facich ex\u00f3nov, ktor\u00e9 s\u00edce predstavuj\u00fa len 1-2% \u013eudsk\u00e9ho gen\u00f3mu (~30-60 Mb), ale zah\u0155\u0148aj\u00fa ~85% zn\u00e1mych kauz\u00e1lnych mut\u00e1ci\u00ed (8). ES sa pou\u017e\u00edva na detekciu variantov (mut\u00e1ci\u00ed) v\u00a0u\u017e zn\u00e1mych, s\u00a0ochoreniami asociovan\u00fdch g\u00e9noch, ako aj na zis\u0165ovanie nov\u00fdch etiopatogenetick\u00fdch asoci\u00e1ci\u00ed. Objavovanie a\u00a0charakteriz\u00e1cia \u201enov\u00fdch\u201c g\u00e9nov boli e\u0161te doned\u00e1vna dom\u00e9nou v\u00fdskumn\u00fdch laborat\u00f3ri\u00ed, ale postupne doch\u00e1dza k\u00a0presunu identifik\u00e1cie kandid\u00e1tnych kauz\u00e1lnych g\u00e9nov aj do klinicko-diagnostick\u00fdch laborat\u00f3ri\u00ed, hoci na jednozna\u010dn\u00e9 potvrdenie asoci\u00e1cie s\u00fa zvy\u010dajne potrebn\u00e9 \u010fal\u0161ie \u0161t\u00fadie, \u010dasto v\u00a0spolupr\u00e1ci s\u00a0v\u00fdskumn\u00fdmi pracoviskami.<\/p>\n<p>Celogen\u00f3mov\u00e9 sekvenovanie (whole genome sequencing, WGS) stanovuje DNA sekvenciu prakticky cel\u00e9ho gen\u00f3mu a\u00a0pokr\u00fdva teda k\u00f3duj\u00face aj nek\u00f3duj\u00face oblasti. V\u00fdhodou tohto pr\u00edstupu je relat\u00edvne nekomplikovan\u00e1 predsekvena\u010dn\u00e1 pr\u00edprava vzoriek, ktor\u00e1 nevy\u017eaduje PCR amplifik\u00e1ciu alebo obohacovanie cie\u013eov\u00fdch oblast\u00ed. Kv\u00f4li obmedzeniam pri interpret\u00e1cii variantov v\u00a0nek\u00f3duj\u00facich oblastiach sa \u010dasto vol\u00ed strat\u00e9gia, pri ktorej sa najprv analyzuj\u00fa k\u00f3duj\u00face regi\u00f3ny (t.j. ex\u00f3m). V\u00a0pr\u00edpade, \u017ee sa nen\u00e1jdu kauz\u00e1lne mut\u00e1cie, d\u00e1ta sa reanalyzuj\u00fa a\u00a0h\u013eadaj\u00fa sa varianty v\u00a0regula\u010dn\u00fdch nek\u00f3duj\u00facich oblastiach, ktor\u00e9 by mohli ovplyv\u0148ova\u0165 expresiu klinicky v\u00fdznamn\u00fdch g\u00e9nov. WGS pri porovnan\u00ed s\u00a0cielen\u00fdm PS vy\u017eaduje r\u00e1dovo desa\u0165n\u00e1sobky h\u013abky sekvenovania (sequencing depth), t.j. mno\u017estva sekvenovania, ktor\u00e9 vy\u017eaduje dan\u00e1 vzorka, a\u00a0to pri podstatne ni\u017e\u0161ej h\u013abke pokrytia (depth of coverage), \u010do prin\u00e1\u0161a ve\u013ek\u00e9 n\u00e1roky na kvalitu a kapacitu technol\u00f3gie (konkr\u00e9tneho pr\u00edstroja) aj\u00a0d\u00e1tovej anal\u00fdzy. Ke\u010f\u017ee sekvena\u010dn\u00e1 h\u013abka analyzovanej vzorky je n\u00e1sobkom objemu sekvenovanej DNA a \u017eelanej h\u013abky pokrytia, tak pri ve\u013ekosti g\u00e9nov\u00e9ho panela napr. 3 Mb (3 x 10<sup>6 <\/sup>bp) a\u00a0h\u013abke pokrytia 100x je sekvena\u010dn\u00e1 h\u013abka 0,3 Gb (3 Mb x 100), zatia\u013e\u010do pri sekvenovan\u00ed cel\u00e9ho gen\u00f3mu s\u00a0ve\u013ekos\u0165ou 3 Gb (3 x 10<sup>9 <\/sup>bp) a h\u013abke pokrytia 30x je po\u017eadovan\u00e1 sekvena\u010dn\u00e1 h\u013abka a\u017e 90 Gb (3 Gb x 30). Celogen\u00f3mov\u00e9 sekvenovanie tak v\u00a0s\u00fa\u010dasnosti predstavuje finan\u010dne najviac n\u00e1ro\u010dn\u00fa NGS aplik\u00e1ciu s\u00a0najni\u017e\u0161ou priemernou h\u013abkou pokrytia, hoci v bud\u00facnosti sa tieto nev\u00fdhody bud\u00fa postupne redukova\u0165.<\/p>\n<p><strong>\u00a0<\/strong><\/p>\n<p><strong>Klinick\u00e9 aspekty implement\u00e1cie NGS<\/strong><\/p>\n<p>Vzh\u013eadom na pomerne ned\u00e1vne etablovanie NGS technol\u00f3gi\u00ed a r\u00f4znorodos\u0165 klinick\u00fdch aplik\u00e1ci\u00ed sa NGS testovanie (najm\u00e4 panelov\u00e9) vykon\u00e1va preva\u017ene pomocou vlastn\u00fdch testov vyvinut\u00fdch v\u00a0 jednotliv\u00fdch laborat\u00f3ri\u00e1ch. Vyu\u017e\u00edvaj\u00fa sa najrozli\u010dnej\u0161ie kombin\u00e1cie pr\u00edstrojov, chemik\u00e1li\u00ed a\u00a0 postupov d\u00e1tovej anal\u00fdzy. Niekedy s\u00fa jednotliv\u00e9 \u010dasti anal\u00fdzy (napr. zostava predsekvena\u010dn\u00e9ho postupu od jedn\u00e9ho v\u00fdrobcu alebo softv\u00e9ry pre terci\u00e1rnu anal\u00fdzu d\u00e1t) komer\u010dne, resp. verejne dostupn\u00e9, ale n\u00e1sledne musia by\u0165 validovan\u00e9 pre diagnostick\u00e9 pou\u017eitie (9).<\/p>\n<p>Pri tradi\u010dnom diagnostickom testovan\u00ed je \u00falohou klinick\u00e9ho genetika stanovi\u0165 diagn\u00f3zu zalo\u017een\u00fa na klinickom obraze, objekt\u00edvnom vy\u0161etren\u00ed a rodinnej anamn\u00e9ze. Genetick\u00fd test b\u00fdva indikovan\u00fd na potvrdenie, resp. vyl\u00fa\u010denie diagn\u00f3zy. Vy\u0161etruj\u00face laborat\u00f3rium vo v\u00fdsledkovej spr\u00e1ve uvedie v\u0161etky potenci\u00e1lne patologick\u00e9 varianty (mut\u00e1cie) v\u00a0analyzovanom g\u00e9ne, ako aj z\u00e1kladn\u00fd popis pou\u017eit\u00fdch met\u00f3d vr\u00e1tane analytick\u00fdch parametrov (senzitivita, \u0161pecificita, detek\u010dn\u00fd limit ap.). Panelov\u00e9 sekvenovanie predstavuje z\u00a0poh\u013eadu klinick\u00e9ho genetika logick\u00e9 roz\u0161\u00edrenie s\u00fa\u010dasn\u00fdch sekvena\u010dn\u00fdch testov v\u00a0pr\u00edpade tzv. geneticky heterog\u00e9nnych ochoren\u00ed. Indik\u00e1ciou PS je teda klinick\u00e1 diagn\u00f3za ochorenia, ktor\u00e9ho genetick\u00e1 etiol\u00f3gia je ve\u013emi heterog\u00e9nna, konkr\u00e9tny kauz\u00e1lny g\u00e9n sa ned\u00e1 nijak\u00fdm sp\u00f4sobom vyselektova\u0165 a\u00a0s\u00fabe\u017en\u00e1 anal\u00fdza ve\u013ek\u00e9ho po\u010dtu asociovan\u00fdch g\u00e9nov preto predstavuje najefekt\u00edvnej\u0161\u00ed diagnostick\u00fd postup. Korektn\u00e1 indik\u00e1cia PS tak m\u00f4\u017ee prispie\u0165 k\u00a0podstatn\u00e9mu zr\u00fdchleniu identifik\u00e1cie kauz\u00e1lnej mut\u00e1cie u\u00a0novodiagnostikovan\u00fdch pacientov, resp. \u00faspe\u0161n\u00e9mu zav\u0155\u0161eniu diagnostick\u00e9ho procesu u\u00a0dlhodobo nedorie\u0161en\u00fdch pr\u00edpadov. Testovanie pomocou PS tie\u017e najlep\u0161ie vyhovuje s\u00fa\u010dasn\u00fdm modelom finan\u010dnej \u00fahrady (zdravotn\u00fdmi pois\u0165ov\u0148ami i samoplatcovsky) za molekul\u00e1rne diagnostick\u00e9 testy. Z\u00a0t\u00fdchto d\u00f4vodov je pre laborat\u00f3ri\u00e1 aj indikuj\u00facich klinick\u00fdch genetikov najv\u00fdhodnej\u0161ie za\u010da\u0165 zo z\u00edskavan\u00edm sk\u00fasenost\u00ed najprv prostredn\u00edctvom PS pred postupn\u00fdm prechodom k ES a WGS aplik\u00e1ci\u00e1m.<\/p>\n<p>Pou\u017eitie ES alebo WGS umo\u017e\u0148uje vo\u013enej\u0161\u00ed pr\u00edstup k\u00a0testovaniu pacientov, bez nutnosti presnej inici\u00e1lnej diagn\u00f3zy, ale na korektn\u00fa interpret\u00e1ciu d\u00e1t vy\u017eaduj\u00fa obe aplik\u00e1cie \u00fazku spolupr\u00e1cu klinick\u00e9ho genetika s\u00a0laborat\u00f3riom, najm\u00e4 pri vo\u013ebe vhodnej strat\u00e9gie filtrovania variantov a\u00a0n\u00e1slednej interpret\u00e1cii v\u00fdsledkov. Indik\u00e1ciou ES, resp. WGS s\u00fa pr\u00edpady, pri ktor\u00fdch je klinicky ve\u013emi pravdepodobn\u00e9 genetick\u00e9 ochorenie, ale dostupn\u00e9 cielen\u00e9 testy pre konkr\u00e9tny g\u00e9n alebo g\u00e9ny asociovan\u00e9 s\u00a0dan\u00fdm fenotypom (napr. aj pomocou PS) neidentifikovali kauz\u00e1lnu mut\u00e1ciu, alebo ak klinick\u00fd obraz, resp. \u00fadaje z rodinnej anamn\u00e9zy silne suponuj\u00fa genetick\u00fa etiol\u00f3giu, ale fenotyp nekore\u0161ponduje so \u0161pecifick\u00fdm ochoren\u00edm, pre ktor\u00e9 je dostupn\u00fd cielen\u00fd genetick\u00fd test. Pred indik\u00e1ciou ES\/WGS testovania mus\u00ed klinick\u00fd genetik d\u00f4kladne zv\u00e1\u017ei\u0165 aj potenci\u00e1lny dopad tzv. n\u00e1hodn\u00fdch n\u00e1lezov (vi\u010f ni\u017e\u0161ie).<\/p>\n<p>Predtestov\u00e1 konzult\u00e1cia klinick\u00fdm genetikom m\u00e1 zah\u0155\u0148a\u0165 form\u00e1lny informovan\u00fd s\u00fahlas a\u00a0pacienti maj\u00fa by\u0165 podrobne informovan\u00ed o\u00a0o\u010dak\u00e1vanom v\u00fdsledku testovania, pravdepodobnosti a\u00a0type potenci\u00e1lnych n\u00e1hodn\u00fdch n\u00e1lezov a kateg\u00f3ri\u00e1ch v\u00fdsledkov, ktor\u00e9 bud\u00fa, resp. nebud\u00fa uveden\u00e9 vo v\u00fdsledkovej spr\u00e1ve. Takisto je potrebn\u00e9 pacientom jasne vysvetli\u0165 rozdiel medzi v\u00fdlu\u010dne klinicko-diagnostick\u00fdm a\u00a0(potenci\u00e1lne) v\u00fdskumne orientovan\u00fdm testovan\u00edm, hoci oba typy sa \u010dasto prekr\u00fdvaj\u00fa, resp. dop\u013a\u0148aj\u00fa. Nevyhnutn\u00fdm d\u00f4sledkom testovania pomocou ES\/WGS je identifik\u00e1cia sekven\u010dn\u00fdch variantov, ktor\u00e9 sa nevz\u0165ahuj\u00fa priamo k\u00a0p\u00f4vodn\u00e9mu z\u00e1meru testu. Za tzv. prim\u00e1rny n\u00e1lez sa pova\u017euje patog\u00e9nny variant (mut\u00e1cia) v\u00a0g\u00e9ne, ktor\u00fd je relevantn\u00fd k\u00a0diagnostickej indik\u00e1cii NGS testu. Naopak, n\u00e1hodn\u00fd alebo sekund\u00e1rny n\u00e1lez znamen\u00e1 ne\u010dakan\u00e9 zistenie patog\u00e9nnej zmeny v\u00a0g\u00e9ne zjavne nes\u00favisiacom s\u00a0p\u00f4vodnou indik\u00e1ciou. Ur\u010dit\u00e9 typy n\u00e1hodn\u00fdch n\u00e1lezov je mo\u017en\u00e9 pova\u017eova\u0165 za dostato\u010dne z\u00e1va\u017en\u00e9 a ich uvedenie vo v\u00fdsledkovej spr\u00e1ve sa jednozna\u010dne doporu\u010duje (10). Medzi v\u00fdznamn\u00e9 n\u00e1hodn\u00e9 n\u00e1lezy patr\u00ed najm\u00e4 identifik\u00e1cia variantu asociovan\u00e9ho s predispoz\u00edciou k\u00a0ur\u010dit\u00e9mu ochoreniu (t.j. vysok\u00fdm rizikom rozvoja ochorenia v bud\u00facnosti), resp. zistenie mut\u00e1cie pre doteraz klinicky sa neprejavuj\u00face ochorenie a n\u00e1lez nosi\u010dstva heterozygotnej mut\u00e1cie pre reces\u00edvne ochorenie. Diagnostick\u00e9 laborat\u00f3rium poskytuj\u00face ES\/WGS anal\u00fdzy by preto malo ma\u0165 vypracovan\u00fd podrobn\u00fd postup oh\u013eadom posudzovania a\u00a0uv\u00e1dzania (reportovania) n\u00e1hodn\u00fdch n\u00e1lezov vo v\u00fdsledkovej spr\u00e1ve.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Diagnostick\u00e9 vyu\u017eitie NGS v\u00a0klinicko-genetickej praxi<\/strong><\/p>\n<p><strong>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/strong>Jednotliv\u00e9 NGS aplik\u00e1cie sa v\u00a0s\u00fa\u010dasnosti efekt\u00edvne vyu\u017e\u00edvaj\u00fa v\u00a0genetickej laborat\u00f3rnej diagnostike zriedkav\u00fdch syndr\u00f3mov, heterog\u00e9nnych ochoren\u00ed a\u00a0pri neinvaz\u00edvnom prenat\u00e1lnom testovan\u00ed plodu. Za zriedkav\u00e9 (raritn\u00e9) ochorenia sa na z\u00e1klade konsenzu pova\u017euj\u00fa choroby s\u00a0individu\u00e1lnou popula\u010dnou incidenciou menej ako 1:2000, \u010di\u017ee vyskytuj\u00face sa menej ako u\u00a0500 jedincov z\u00a0mili\u00f3na. Extr\u00e9mne zriedkav\u00e9 (ultrararitn\u00e9) ochorenia maj\u00fa incidenciu men\u0161iu ako 1:50.000, zis\u0165uj\u00fa sa teda u menej ako 20 jedincov z\u00a0mili\u00f3na. Predpoklad\u00e1 sa, \u017ee kauz\u00e1lne mut\u00e1cie pre raritn\u00e9 ochorenia sa vyskytuj\u00fa so zodpovedaj\u00facou n\u00edzkou frekvenciou, resp. v\u00fdlu\u010dne u\u00a0postihnut\u00fdch jedincov. Tento predpoklad plat\u00ed najm\u00e4 pre vysokopenetrantn\u00e9 mut\u00e1cie, t.j. varianty ve\u013ek\u00e9ho \u00fa\u010dinku asociovan\u00e9 s\u00a0typick\u00fdm fenotypom, ktor\u00e9 sa nevyskytuj\u00fa be\u017ene v\u00a0popul\u00e1cii a\u00a0preto ani v datab\u00e1zach celogen\u00f3mov\u00fdch anal\u00fdz alebo polymorfizmov (napr. dbSNP, HapMap, 1000 Genomes ap.). Nepr\u00edtomnos\u0165 v\u00a0t\u00fdchto datab\u00e1zach predstavuje d\u00f4le\u017eit\u00e9 krit\u00e9rium pri h\u013eadan\u00ed zriedkav\u00e9ho alebo <em>de novo<\/em> variantu pri ES\/WGS anal\u00fdzach. Od uverejnenia pilotnej pr\u00e1ce, ktor\u00e1 pomocou ES identifikovala mut\u00e1ciu v\u00a0g\u00e9ne <em>SLC26A3<\/em> a\u00a0potvrdila p\u00f4vodne klinicky nesuponovan\u00fa diagn\u00f3zu vrodenej chloridovej diarey (11) bolo publikovan\u00fdch viac ako 150 \u010fal\u0161\u00edch \u0161t\u00fadi\u00ed, ktor\u00e9 \u00faspe\u0161ne vyu\u017eili ES\/WGS na detekciu reces\u00edvnych, dominantn\u00fdch aj <em>de novo<\/em> mut\u00e1ci\u00ed.<\/p>\n<p>Geneticky heterog\u00e9nne ochorenia, ktor\u00e9 predstavuj\u00fa jednozna\u010dn\u00fdch kandid\u00e1tov na vyu\u017eitie PS m\u00f4\u017eeme rozdeli\u0165 do nieko\u013ek\u00fdch z\u00e1kladn\u00fdch skup\u00edn: \u0161pecifick\u00e9 genetick\u00e9 syndr\u00f3my s historicky zn\u00e1mou heterogenitou, syndr\u00f3my s ment\u00e1lnou retard\u00e1ciou a\/alebo autizmom, dedi\u010dn\u00e9 metabolick\u00e9 poruchy a ochorenia prim\u00e1rne diagnostikovan\u00e9 in\u00fdmi medic\u00ednskymi odbormi (neurol\u00f3gia, kardiol\u00f3gia, oftalmol\u00f3gia, ORL). Konkr\u00e9tne pr\u00edklady s\u00fa uveden\u00e9 v\u00a0Tab.3.<\/p>\n<p>Zavedenie NGS met\u00f3d prinieslo potrebn\u00fa technol\u00f3giu aj na vyrie\u0161enie technick\u00fdch probl\u00e9mov neinvaz\u00edvnej detekcie triz\u00f3mie 21 a \u010fal\u0161\u00edch \u010dast\u00fdch aneuplo\u00eddi\u00ed plodu z vo\u013enej fet\u00e1lnej DNA cirkuluj\u00facej v\u00a0perif\u00e9rnej krvi matky. Po pilotn\u00fdch men\u0161\u00edch pr\u00e1cach principi\u00e1lne testuj\u00facich NGS anal\u00fdzu \u010fal\u0161ie \u0161t\u00fadie potvrdili vysok\u00fa \u0161pecificitu a\u00a0senzitivitu tak\u00e9hoto neinvaz\u00edvneho prenat\u00e1lneho testovania (NIPT) na rozsiahlych s\u00faboroch tehotn\u00fdch \u017eien (12,13). Pri skr\u00edningu naj\u010dastej\u0161\u00edch fet\u00e1lnych aneuplo\u00eddi\u00ed NIPT predstavuje alternat\u00edvu a\u00a0perspekt\u00edvne n\u00e1hradu multimarkerov\u00fdch biochemick\u00fdch testov a\u00a0 invaz\u00edvneho odberu fet\u00e1lnej vzorky. V\u00a0s\u00fa\u010dasnosti je u\u017e v\u00a0ponuke viacero komer\u010dne dostupn\u00fdch NIPT testov a\u00a0ich \u0161irok\u00e9 pou\u017eitie je zatia\u013e limitovan\u00e9 finan\u010dnou n\u00e1ro\u010dnos\u0165ou pre tehotn\u00e9 probandky. Len \u010fal\u0161\u00ed v\u00fdvoj uk\u00e1\u017ee, \u010di tento typ testov \u00faspe\u0161ne nahrad\u00ed doteraj\u0161ie postupy prenat\u00e1lnej genetickej diagnostiky.<\/p>\n<p><strong>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/strong>Postupn\u00e9 roz\u0161\u00edrenie NGS anal\u00fdz v\u00a0klinicko-diagnostickej praxi bude predstavova\u0165 zmenu paradigmy v\u00a0medic\u00edne a\u00a0ve\u013emi pravdepodobne prinesie skuto\u010dne \u201ena mieru \u0161it\u00fa\u201c lek\u00e1rsku starostlivos\u0165 zalo\u017een\u00fa na poznan\u00ed individu\u00e1lneho rizika. U\u017e dnes je zrejm\u00e9, \u017ee aplik\u00e1cie NGS bud\u00fa ma\u0165 ve\u013ek\u00fd v\u00fdznam nielen u\u00a0genetick\u00fdch ochoren\u00ed s\u00a0mendelsk\u00fdm typom dedi\u010dnosti, ale aj u\u00a0polyg\u00e9nov\u00fdch a\u00a0multifaktori\u00e1lnych chor\u00f4b (14). Preto je potrebn\u00e9, aby klinick\u00ed genetici postupne pomohli integr\u00e1cii \u201egenomick\u00e9ho\u201c myslenia aj v\u00a0in\u00fdch medic\u00ednskych \u0161pecializ\u00e1ci\u00e1ch.<\/p>\n<p>&nbsp;<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Literat\u00fara<\/strong><\/p>\n<p>(1) Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. <em>Proc Natl Acad Sci USA<\/em>. 1977;74(12):5463-7.<\/p>\n<p>(2) Metzker ML. Sequencing technologies &#8211; the next generation. <em>Nat Rev Genet<\/em>. 2010;11(1):31-46.<\/p>\n<p>(3) Moorthie S, Hall A, Wright CF. Informatics and clinical genome sequencing: opening the black box. <em>Genet Med<\/em>. 2013;15(3):165-71.<\/p>\n<p>(4) Margulies M, Egholm M, Altman WE, et al. Genome sequencing in microfabricated high-density picolitre reactors. <em>Nature<\/em>. 2005;437(7057):376\u201380.<\/p>\n<p>(5) Shendure J, Porreca GJ, Reppas NB, et al. Accurate multiplex polony sequencing of an evolved bacterial genome. <em>Science<\/em>. 2005;309(5741):1728\u201332<\/p>\n<p>(6) Bentley DR, Balasubramanian S, Swerdlow HP, et al. Accurate whole human genome sequencing using reversible terminator chemistry. <em>Nature<\/em>. 2008; 456(7218):53\u20139.<\/p>\n<p>(7) Rothberg JM, Hinz W, Rearick TM, et al. An integrated semiconductor device enabling nonoptical genome sequencing. <em>Nature<\/em>. 2011; 475(7356):348\u201352.<\/p>\n<p>(8) Majewski J, Schwartzentruber J, Lalonde E, et al. What can exome sequencing do for you? <em>J Med Genet<\/em>. 2011;48(9):580\u20139.<\/p>\n<p>(9) Rehm HL, Bale SJ, Bayrak-Toydemir P, et al. ACMG clinical laboratory standards for next-generation sequencing. <em>Genet Med<\/em>. 2013;15(9):733-47.<\/p>\n<p>(10) Green RC, Berg JS, Grody WW, et al. ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing. <em>Genet Med<\/em>. 2013;15(7):565-74.<\/p>\n<p>(11) Choi M, Scholl UI, Ji W, et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. <em>Proc Natl Acad Sci U S\u00a0A<\/em>. 2009;106(45):19096\u2013101.<\/p>\n<p>(12) Bianchi DW, Platt LD, Goldberg JD, et al. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. <em>Obstet Gynecol<\/em>. 2012;119(5):890\u2013901.<\/p>\n<p>(13) Palomaki GE, Kloza EM, Lambert-Messerlian GM, et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. <em>Genet Med<\/em>. 2011;13(11):913\u201320.<\/p>\n<p>(14) Thakuria JV, Zaranek AW, Church GM, et al. Back to the future: from genome to metabolome. <em>Hum Mutat<\/em>. 2012;33(5):809-12.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>*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. &nbsp; Sekvena\u010dn\u00e9 technol\u00f3gie prvej a\u00a0druhej gener\u00e1cie Met\u00f3da DNA sekvenovania publikovan\u00e1 F. Sangerom koncom 70. rokov minul\u00e9ho storo\u010dia (1) ozna\u010dovan\u00e1 aj ako dideoxynukleotidov\u00e9 sekvenovanie, priniesla nov\u00e9 a\u00a0v \u010dase svojho vzniku e\u0161te<\/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":[722,721,724,720,725,723],"class_list":["post-1117","post","type-post","status-publish","format-standard","hentry","category-genetics","tag-exome-sequencing-es","tag-gene-panels","tag-genetically-heterogeneous-diseases","tag-next-generation-sequencing-ngs","tag-rare-syndromes","tag-whole-genome-sequencing-wgs","typ_clanku-review-article"],"acf":{"abstrakt":"<p>The revolutionary method of next generation sequencing (NGS) is based on parallel sequencing analysis of a large number of DNA fragments. The chemical principle of currently most widely used NGS technologies includes sequencing during synthesis or ligation followed by the detection of fluorescence, chemiluminescence or changes in pH. Huge amount of data arising from NGS analyses must be effectively processed and correctly evaluated by various bioinformatic algorithms that put high requirements on laboratory\u2019s hardware and software equipment as well as staff trained in bioinformatics. Routine clinical diagnostic implementation of NGS methods requires precise validation<\/p>\n<p>of individual tests and standardization of the entire analytical procedure. Considering the amount of acquired data, genetic testing by NGS yields a lot of results with varying informative value. If a pathological variant (mutation) is found in a known or a gene that is very likely to be associated with the disease, the cause of patient\u2019s phenotype can be diagnostically ascertained. On the other hand, incidental findings identified by exome or whole genome sequencing present us with interpretative and ethical challenges and their reporting must fulfil strict criteria. Various NGS applications are already used in research and clinical diagnosis of many genetically determined diseases. Long-term experience proved clear efficacy of gene-panel and exome sequencing particularly in the diagnosis of rare syndromes and genetically heterogeneous diseases. However, it is apparent that gradual integration of NGS into the clinical practice could lead to the individually tailored patient management even in more common, multifactorial diseases.<\/p>\n<p>&nbsp;<\/p>\n<p><strong>Key words:<\/strong> next generation sequencing (NGS), gene panels, exome sequencing (ES), whole genome sequencing (WGS), genetically heterogeneous diseases, rare syndromes<\/p>\n","casopis":[{"ID":1000,"post_author":"7","post_date":"2015-11-21 14:33:24","post_date_gmt":"2015-11-21 13:33:24","post_content":"<h4><strong>GENETICS<\/strong><\/h4>\r\n<ul>\r\n \t<li>The laboratory age<\/li>\r\n \t<li>The Odyssey of DNA reading<\/li>\r\n \t<li>Comparative genomic hybridisation: a methodological introduction<\/li>\r\n \t<li>Next generation sequencing and its application in clinical genetics<\/li>\r\n<\/ul>\r\n<h4><\/h4>\r\n<h4><strong>BIOCHEMISTRY<\/strong><\/h4>\r\n<ul>\r\n \t<li>Determining of the trace elements in blood serum<\/li>\r\n \t<li>Determining of \u03b1-tocopherol (vitamin E) in serum by way of liquid chromatography with tandem mass spectrometry (LC\/MS\/MS)<\/li>\r\n \t<li>Analysis of urinary calculi and its path to Europe<\/li>\r\n<\/ul>\r\n&nbsp;\r\n<h4><strong>IMMUNOLOGY <\/strong><\/h4>\r\n<ul>\r\n \t<li>New autoantibodies in diagnosis of autoimmunity myopathies<\/li>\r\n \t<li>Taking advantage of flow-based cytometry in determining prognostic markers<\/li>\r\n<\/ul>\r\nCD38 and ZAP-70 in patients with B-CLL\r\n\r\n&nbsp;\r\n<h4><strong>CYTOLOGY AND PATHOLOGY <\/strong><\/h4>\r\n<ul>\r\n \t<li>Mucinous ovarian carcinoma \u2013 news in diagnosis from the pathologist\u2019s perspective<\/li>\r\n<\/ul>","post_title":"newsLab","post_excerpt":"","post_status":"publish","comment_status":"closed","ping_status":"closed","post_password":"","post_name":"newslab-12015","to_ping":"","pinged":"","post_modified":"2017-08-16 21:43:10","post_modified_gmt":"2017-08-16 19:43:10","post_content_filtered":"","post_parent":0,"guid":"http:\/\/www.newslab.sk\/casopis\/newslab-12015\/","menu_order":0,"post_type":"casopis","post_mime_type":"","comment_count":"0","filter":"raw"}],"strana":"15","upload_clanok":{"ID":1118,"id":1118,"title":"Sekvenovanie novej gener\u00e1cie a jeho vyu\u017eitie v klinickej genetike - Bujalkova","filename":"MEDIREX_1_2015-\u2013-Bujalkova.pdf","filesize":333064,"url":"https:\/\/www.newslab.sk\/wp-content\/uploads\/2016\/12\/MEDIREX_1_2015-\u2013-Bujalkova.pdf","link":"https:\/\/www.newslab.sk\/en\/next-generation-sequencing-and-its-application-in-clinical-genetics\/medirex_1_2015-bujalkova\/","alt":"","author":"7","description":"","caption":"","name":"medirex_1_2015-bujalkova","status":"inherit","uploaded_to":1117,"date":"2016-12-05 20:23:34","modified":"2016-12-05 20:23:34","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\/1117","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=1117"}],"version-history":[{"count":0,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1117\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/media?parent=1117"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/categories?post=1117"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/tags?post=1117"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}