Tag Nrho Genome Sequencing Rau Kev Ntsuas Ntawm Neurological Repeat Expansion Disorders hauv UK: Kev Tshawb Fawb Txog Kev Tshawb Fawb Txog Kev Tshawb Fawb Thiab Kev Tshawb Fawb Kev Tshawb Fawb Txog Kev Tshawb Fawb

Yog xav paub ntxiv:ali.ma@wecistanche.com

Cov ntsiab lus

Keeb kwm

Rov ua dua cov kev mob tshwm sim cuam tshuam txog li 1 ntawm 3000 tus neeg thiab yog cov kab mob sib kis los ntawm kev nthuav dav ntawm luv luv tandem DNA rov ua dua. Kev ntsuam xyuas caj ces feem ntau yog qhov tshwj xeeb, ua rau muaj kev kuaj mob tsis zoo ntawm cov neeg uas muaj kev nthuav qhia kho mob atypical, tshwj xeeb tshaj yog nyob rau hauv cov neeg mob menyuam yaus uas tsis muaj keeb kwm yav dhau los hauv tsev neeg zoo. Tag nrho-genome sequencing yog nce siv los ua ib qho kev sim thawj zaug rau lwm yam kab mob uas tsis tshua muaj caj ces, thiab peb tsom mus ntsuam xyuas nws cov kev ua tau zoo hauv kev kuaj mob ntawm cov neeg mob.paj hlwbrov nthuav cov teeb meem.

Cov txheej txheem

Peb rov qab soj ntsuam xyuas qhov tseeb ntawm tag nrho-genome sequencing kom paub txog qhov feem ntau rov nthuav dav loci cuam tshuam nrogpaj hlwbCov txiaj ntsig (AR, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, C9orf72, CACNA1A, DMPK, FMR1, FXN, HTT, thiab TBP) siv cov qauv tau txais hauv National Health Service hauv tebchaws Askiv los ntawm cov neeg mob uas xav tias muajpaj hlwbkev puas tsuaj; Cov txiaj ntsig PCR yav dhau los tau siv los ua tus qauv siv. Qhov tseeb ntawm kev soj ntsuam ntawm tag nrho-genome sequencing los kuaj xyuas qhov kev nthuav dav rov qab mus rau yav dhau los kuaj cov noob caj noob ces thiab cov neeg mob tsis tau kuaj pom hauv 2013-17 mus rau 100 000 Genomes Project hauv UK, uas xav tias muaj caj ces.paj hlwbkev tsis sib haum xeeb (tsev neeg lossis cov ntawv pib ntxov ntawm ataxia, neuropathy, spastic paraplegia, dementia, lub cev muaj zog neuron kab mob,parkinsonian kevkev txav mus losmob, kev tsis taus kev txawj ntse, los yog neuromuscular disorders). Yog hais tias qhov rov nthuav dav hu tau siv tag nrho-genome sequencing, PCR tau siv los lees paub qhov tshwm sim.

Kev tshawb pom

Kev kuaj xyuas qhov tseeb ntawm tag nrho-genome sequencing txhawm rau txheeb xyuas qhov kev nthuav dav rov qab tau soj ntsuam tawm tsam 793 PCR kev sim ua yav dhau los hauv NHS los ntawm 404 tus neeg mob. Tag nrho cov genome sequencing raug cais tawm 215 ntawm 221 nthuav dav alleles thiab 1316 ntawm 1321 non-expanded alleles, qhia 97·3 feem pua ​​​​rhiab heev (95 feem pua ​​​​CI 94·2–99·0) thiab 99·6 feem pua ​​​​txhim kho (99·1–99). · 9) thoob plaws 13 tus kab mob sib txuas nrog loci thaum piv nrog PCR cov txiaj ntsig. Hauv cov qauv los ntawm 11 631 cov neeg mob hauv 100 000 Genomes Project, tag nrho-genome sequencing txheeb xyuas 81 rov nthuav dav, uas kuj tau kuaj los ntawm PCR: 68 tau lees paub tias rov ua dua nyob rau hauv tag nrho cov kab mob pathogenic, 11 tsis yog -pathogenic intermediate expansions los yog permutations, thiab ob yog non-expanded repeats (16 feem pua ​​​​tsis pom tus nqi).

Txhais lus

Hauv peb txoj kev tshawb fawb, tag nrho-genome sequencing rau kev tshawb pom ntawm kev nthuav dav rov qab tau pom tias muaj kev nkag siab zoo thiab tshwj xeeb, thiab nws tau coj mus rau kev txheeb xyuas ntawmpaj hlwbrov nthuav cov teeb meem nyob rau hauv yav tas los undiagnosed cov neeg mob. Cov kev tshawb pom no txhawb nqa kev siv tag nrho-genome sequencing hauv chaw soj ntsuam kuaj mob rau kev kuaj mob ntawm cov neeg mob uas muaj tus kab mob.paj hlwbkev nthuav qhia zoo ib yam nrog kev rov nthuav dav tsis meej.

Nyiaj txiag

Medical Research Council, Department of Health thiab Social Care, National Health Service England, National Institute for Health Research, thiab Illumina.

Taw qhia

Txawm tias tsis ntev los no kev nce qib hauv peb txoj kev nkag siab ntawm cov caj ces ntawm qhov tsis tshua muajpaj hlwbCov kab mob, txog li 70 feem pua ​​​​ntawm cov neeg mob uas muaj cov kab mob zoo li no tseem tsis tau txheeb xyuas cov noob caj noob ces.1–3 Ib feem, qhov no yog vim muaj kev sib tw ntawm kev sim rau txoj kev sib txawv ntawm cov caj ces thiab rov ua dua, suav nrog kev nthuav dav ntxiv; xws li kev nthuav dav tau kwv yees tias yuav cuam tshuam txog 1 ntawm 3000 tus neeg (daim ntawv txuas ntxiv p 1) thiab yog qhov ua rau ntau tshaj 40 cov kab mob neurogenetic, 4 suav nrog Huntington's disease thiab fragile X syndrome. Rov ua dua cov kev mob tshwm sim yog kev kho mob thiab caj ces heterogeneous, thiab rov nthuav dav tuaj yeem cuam tshuam nrog ntau yam kab mob. Piv txwv li, kev nthuav dav hauv C9orf72 tuaj yeem tshwm sim raws li amyotrophic lateral sclerosis lossis frontotemporal dementia.5 Rov ua dua kev nthuav dav hauv cov chaw sib txawv kuj tuaj yeem ua rau muaj cov yam ntxwv zoo sib xws, ua rau lawv nyuaj rau kev paub qhov txawv hauv kev kho mob: rov nthuav dav yam tsawg kawg kaum spinocerebellar ataxia noob nquag tam sim no thaum laus. -onset ataxia,6 thiab cov neeg hauv C9orf72 thiab AR tuaj yeem ua rau lub cev muaj zog neuron kab mob.7,8

Rov ua dua kev nthuav dav tsis zoo yog tshwm sim los ntawm kev nce ntxiv ntawm qhov rov ua dua luv luv tandem DNA ib ntus, thiab cov kab mob pathogenicity pib rau txhua qhov teeb meem yog qhov chaw tshwj xeeb. Qhov loj ntawm kev nthuav dav txawv ntawm tsawg dua 30 rov ua dua (piv txwv li, hauv CACNA1A) mus rau ntau txhiab qhov rov ua dua (piv txwv li, hauv FMR1, DMPK, C9orf72, thiab FXN, uas tuaj yeem txuas ntxiv mus txog 5 kb loj). Rov ua dua qhov nthuav dav pom qhov tsis muaj zog molecular, uas tuaj yeem ua rau muaj kev hloov pauv ntawm qhov rov ua dua tshiab (feem ntau zuj zus ntxiv) nyob rau ntau tiam neeg thiab cov ntaub so ntswg.4 Nyob rau hauv cov xwm txheej no, kev nce ntawm cov rov ua dua feem ntau ua rau muaj qhov tshwm sim ntxov thiab kab mob hnyav dua nyob rau hauv kev ua tiav. generations.4 Pediatric pib ntawm rov nthuav cov teeb meem tuaj yeem tshwm sim raws li cov kab mob ntau yam tsis muaj qhov tshwj xeeb phenotypic kos npe, 9 thiab cov menyuam yaus uas muaj cov kab mob no feem ntau yuav raug kuaj tsis tau thaum tsev neeg keeb kwm ntawm kev rov nthuav dav tsis zoo dua li thaum nws muaj.10– 12

Kev soj ntsuam kev soj ntsuam ntawm kev rov nthuav dav yog feem ntau txwv rau qhov kev soj ntsuam molecular ntawm ib tus neeg qhov chaw coj los ntawm kev kuaj mob uas xav tias siv PCR-based lossis Southern blot txoj kev, 13 uas tuaj yeem raug nqi thiab siv sijhawm. Tsis tas li ntawd, vim muaj qhov sib txawv thiab sib tshooj phenotypic ntawm cov kab mob no, cov kab mob cuam tshuam nrog rov nthuav dav loci tuaj yeem nyob twj ywm untested.14

Tag nrho-genome sequencing tau tshwm sim los ua thawj kab kev tshuaj ntsuam xyuas cov cuab yeej hauv cov neeg mob uas tsis tshua muaj kab mob15 tab sis, txog rau thaum nyuam qhuav, tau xav tias muaj peev xwm ntsuas cov loci uas muaj qhov rov ua dua.16 Kev nce qib hauv bioinformatics, txawm li cas los xij, tau ua kom muaj peev xwm tshawb pom tus kab mob. -ua rau rov nthuav dav ntxiv los ntawm cov ntaub ntawv txuas ntxiv mus rau tiam tom ntej.17–22 Ntawm no, peb tshaj tawm txog kev soj ntsuam kev soj ntsuam ntawm tag nrho-genome sequencing mus kom ze txhawm rau txheeb xyuas qhov kev nthuav dav rov qab siv cov ntaub ntawv PCR rov qab, thiab nws qhov kev kuaj xyuas pom zoo rau cov neeg mob hauv 100000 Genomes Project leej twg. muaj qhov xav tias muaj kab mob hauv lub paj hlwb, tsis tau kuaj pom nrog kev kuaj caj ces yav dhau los.

Cov txheej txheem

Kawm tsim thiab koom nrog

Qhov kev ntsuam xyuas ntawm tag nrho-genome sequencing rau kev tshawb pom ntawm kev nthuav dav ntxiv suav nrog ob qho tib si kuaj qhov tseeb thiab kev soj ntsuam qhov tseeb. Kev kuaj pom tseeb raug soj ntsuam los ntawm kev siv cov ntaub ntawv los ntawm cov neeg mob uas yav dhau los tau sim los ntawm PCR rau kev nthuav dav ntxiv uas paub tias ua rau mob hlwb.4 Cov neeg mob tau txheeb xyuas los ntawm ob qhov chaw: 100 000 Genomes Project thiab Genomic Laboratory raws ntawm Cambridge University Hospitals ( Cambridge, UK). Rau ob qho tib si ntawm cov neeg mob, kev kuaj PCR tau ua rau cov neeg mob kuaj los ntawm cov chaw soj nstuam hauv National Health Service (NHS) ua ib feem ntawm kev soj ntsuam kev soj ntsuam niaj hnub: rau cov qauv hauv 100000 Genomes Project, PCR kuaj tau ua ua ntej nrhiav neeg ua haujlwm los ntawm qhov project. University College London Tsev Kho Mob Neurogenetic Laboratory (London, UK); kuaj nrog PCR-confirmed rov nthuav dav tau txais los ntawm cov neeg mob kuaj los ntawm Genomic Laboratory raws li Cambridge. Cov neeg mob uas muaj PCR-zoo thiab PCR-tsis zoo cov txiaj ntsig rau kev rov nthuav dav ntxiv tau raug txheeb xyuas kom suav nrog hauv peb txoj kev tshawb fawb los ntawm kev kuaj cov ntaub ntawv; txhua tus neeg mob tau sau ntawv tso cai siv lawv cov qauv rau kev lees paub zoo thiab kev tshawb fawb thiab kev cob qhia lub hom phiaj, uas yog ib feem ntawm kev pabcuam kho mob kom zoo thiab siv tau.

Tag nrho-genome sequencing ntawm txhua tus qauv tau ua tiav ntawm ib qho ntawm ob lub chaw soj nstuam: Genomics England (Hinxton, UK) rau 100000 Genomes Project qauv (n=254) thiab Illumina Clinical Services Laboratory (ICSL; San Diego, CA, Tebchaws Asmeskas) rau cov qauv tau txais los ntawm Genomic Laboratory raws li Cambridge (n=150). Zuag qhia tag nrho, cov ntaub ntawv no tau siv rau kev kuaj xyuas qhov tseeb ntawm txoj kev tshawb fawb, thiab muaj PCR thiab tag nrho-genome sequencing cov ntaub ntawv los ntawm 404 cov neeg mob, suav nrog 13 loci uas sawv cev rau feem ntau cov kab mob paj hlwb rov nthuav dav: 11 loci txuam nrog ataxia thiab lig- pib neurodegenerative disorders (HTT, AR, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, CACNA1A, TBP, C9orf72, thiab FXN), ib qho chaw cuam tshuam nrog kev tsis taus kev txawj ntse (FMR1), thiab ib qho chaw cuam tshuam nrog myotonic dystrophy (DMPK). Rau txhua qhov chaw, PCR cov ntaub ntawv xeem tau muaj nyob rau tsawg kawg ib qho kev nthuav dav allele (appendix p 24).

Kev soj ntsuam qhov tseeb tau raug soj ntsuam los ntawm kev tshuaj xyuas qhov sib txuam ntawm kev rov nthuav dav, raws li kuaj pom nrog tag nrho-genome sequencing, nrog kev xav tias kuaj mob tom qab kuaj xyuas PCR hauv cov neeg mob uas xav tias muaj kab mob hauv lub paj hlwb (tsev neeg lossis cov ntawv ntxov ntxov ntawm ataxia, neuropathy, spastic paraplegia, dementia. , motor neuron kab mob,parkinsonian kevkev tsis sib haum xeeb, kev txawj ntse, lossis cov kab mob neuromuscular) tau txais mus rau 100000 Genomes Project hauv 2013-17. 100000 Genomes Project yog UK program los ntsuas tus nqi ntawm tag nrho-genome sequencing nyob rau hauv cov neeg mob uas tsis xav tau kev kuaj mob tsis tshua muaj kab mob thiab mob qog noj ntshav. Ua raws li kev pom zoo rau kev ncaj ncees rau 100000 Genomes Project los ntawm East of England Cambridge South Research Ethics Committee (saib 14/EE/1112), suav nrog rau kev txheeb xyuas cov ntaub ntawv thiab xa rov qab cov kev kuaj pom rau cov neeg mob, cov neeg mob no tau nrhiav los ntawm cov kws kho mob thiab cov kws tshawb fawb los ntawm 13 Genomic Medicine Centers hauv tebchaws Askiv thiab tau tso npe rau hauv qhov project yog tias lawv lossis lawv tus neeg saib xyuas tau sau ntawv tso cai rau lawv cov qauv thiab cov ntaub ntawv los siv rau hauv kev tshawb fawb, suav nrog txoj kev tshawb fawb no. Probands thiab, yog tias ua tau, lwm tus neeg hauv tsev neeg, tau tso npe kawm raws li cov txheej txheem tsim nyog tau teev tseg rau qee yam kab mob tsawg (appendix pp 5–11). Cov neeg mob tau raug xaiv mus rau 100000 Genomes Project tom qab kuaj kab mob raws li kev saib xyuas hauv NHS, raws li tau teev tseg hauv cov qauv tsim nyog. Cov ntaub ntawv kho mob raws li tus qauv tau sau tseg siv Human Phenotyping Ontology (HPO)23 tiv thaiv kab mob cov ntaub ntawv tshwj xeeb.24 Cov xwm txheej kab mob ntawm cov neeg hauv tsev neeg, txheeb ze rau cov khoom siv tshuaj ntsuam xyuas, kuj tau sau tseg.

Txhawm rau txheeb xyuas qhov ua rau rov nthuav dav ntxiv hauv cov neeg mob uas tsis muaj kab mob caj ces, peb tau sim cov neeg mob uas xav tias muaj kab mob caj ces zoo ib yam nrog cov kab mob rov ua dua. Cov neeg mob tau raug xaiv los ntawm kev sib haum xeeb ntawm lawv cov kab mob thiab HPO cov ntsiab lus nrog cov kab mob txuas ntxiv txuas ntxiv. Cov neeg mob 'tag nrho-genome sequencing cov ntaub ntawv raug nug los tshawb nrhiav kev nthuav dav hauv cov txheej txheem ntawm kev rov ua dua siv plaub qhov sib txawv rov ua dua tshiab raws li lawv cov yam ntxwv kho mob (appendix p 5). Qhov rov nthuav dav ntxiv rau kev suav nrog ntawm cov vaj huam sib luag no yog cov kab mob hauv lub paj hlwb uas ua rau rov nthuav dav loci. Cov neeg mob uas muaj cov yam ntxwv kho mob uas muaj feem cuam tshuam nrog ntau dua ib qho kev nthuav dav ntxiv tau raug sim ntawm ntau lub vaj huam sib luag. Yog hais tias qhov kev rov nthuav dav hu tau siv tag nrho-genome sequencing, kev kuaj xyuas los ntawm PCR tau ua.

Rau txhua tus neeg mob uas tau lees paub qhov kev nthuav dav ntxiv, tus kws kho mob hauv zos tau ceeb toom txog qhov muaj peev xwm kuaj tau qhov tshwm sim, thiab kev koom tes ntawm qhov rov nthuav dav rau tus neeg mob cov yam ntxwv kho mob tau soj ntsuam. Rau kev rov nthuav dav ntxiv uas tau piav qhia tag nrho lossis ib nrab piav qhia tus neeg mob cov yam ntxwv kho mob, tau tshaj tawm tsab ntawv ceeb toom kuaj mob raws li cov txheej txheem hauv zos.

Cov txheej txheem

Rau NHS cov qauv keeb kwm siv nyob rau hauv qhov kev kuaj xyuas qhov tseeb ntawm peb txoj kev tshawb fawb, rov nthuav dav dua yav dhau los tau sim siv PCR amplification thiab fragment tsom xam. Yav qab teb blotting tau ua rau loj C9orf72 expansions. Hauv qhov tseeb qhov chaw kho mob ntawm peb txoj kev tshawb fawb, rov nthuav dav ntxiv los ntawm tag nrho-genome sequencing hauv cov neeg mob los ntawm 100 000 Genomes Project tau sim los ntawm PCR hauv cov qauv khaws cia hauv NHS genetic laboratory. Cov ntsiab lus ntxiv, suav nrog cov txheej txheem primer, muaj nyob rau hauv daim ntawv ntxiv (pp 2–3, 25–26).

DNA tau npaj rau tag nrho genome sequencing siv TruSeq DNA PCR-Dawb tsev qiv ntawv npaj, thiab 150 bp los yog 125 bp khub-kawg sequencing tau ua nyob rau ntawm HiSeq 2000 los yog HiSeq X platforms ntawm high throughput genomes chaw rau Genomics England, thiab ntawm ICSL . Genomes tau ua raws li qhov nruab nrab qhov tob ntawm 35 × (31 × rau 37 ×; appendix p 27). Luv-tandem-repeat genotyping tau ua tiav siv ExpansionHunter software pob version 3.1.2.25,26 Hauv luv luv, ExpansionHunter realigns sequencing nyeem thoob plaws ib txheej predefined ntawm luv luv tandem rov los kwv yees qhov loj ntawm ob qho tag nrho ntawm ib tus neeg (appendix p 3).

ExpansionHunter cov zis suav nrog kev kwv yees ntawm cov lej ntawm cov khoom rov qab, tag nrho qhov loj me, thiab kev ntseeg siab txwv rau txhua qhov chaw soj ntsuam. Cov lus qhia los ntawm Lub Koom Haum rau Kev Kho Mob Pathology thiab Tsev Kawm Qib Siab American Pathologists pom zoo kom pom qhov kev tshuaj xyuas ntawm kev hu xov tooj sib txawv thaum lub sij hawm niaj hnub ntsuam xyuas ntawm cov kev sib tw ua kom sib luag.27

Txawm li cas los xij, luv luv tandem rov sib txawv tsis tuaj yeem pom zoo los ntawm cov cuab yeej pom pom zoo xws li Integrative Genomics Viewer.28 Txhawm rau tshuaj xyuas tag nrho cov genome sequencing cov ntaub ntawv hauv qab txhua genotype hu, siv cov duab kos duab pom, uas ua rau pom kev ncaj qha ntawm haplotypes thiab cov ntawv nyeem sib thooj. ntawm ExpansionHunter genotypes (appendix pp 3, 15). Kev soj ntsuam pom ntawm daim duab pileup tau ua tiav ntawm tag nrho cov genome sequencing luv tandem rov hu xov tooj kom paub meej tias ExpansionHunter kev kwv yees rau alleles tau muaj nyob hauv txhua qhov nyeem (piv txwv li, qhov rov ua ntu ntu me dua li qhov sib txuas nyeem ntev); kom paub meej tias muaj qhov nthuav dav ntawm monoallelic lossis biallelic; txhawm rau txheeb xyuas putative cuav-zoo hu; thiab txhawm rau txheeb xyuas qhov tsis tseeb-tsis zoo alleles hauv biallelic rov nthuav dav, xws li FXN (appendix pp 4, 16).

ExpansionHunter kwv yees qhov loj me los ntawm tag nrho-genome sequencing cov ntaub ntawv los ntawm kev txheeb xyuas cov ntawv nyeem uas tag nrho lossis ib nrab muaj qhov luv luv tandem rov ua dua. Yog hais tias ib tug luv luv tandem rov hais dua allele luv dua qhov ntev nyeem, ExpansionHunter kwv yees qhov loj npaum li cas; Yog hais tias ib tug luv luv tandem rov hais dua allele ntev dua qhov ntev nyeem ntawv, ExpansionHunter kwv yees qhov loj dua nyob rau hauv ib tug CI, nyob ntawm seb qhov chaw nyob ib ntus muaj pes tsawg leeg, qhov tob ntawm sequencing, thiab qhov zoo ntawm sequencing.

Kev txheeb cais

Peb cais cov kev rov ua dua raws li nthuav dav los ntawm tag nrho cov genome sequencing yog tias qhov loj me uas tau kwv yees los ntawm ExpansionHunter yog siab tshaj qhov kev txiav tawm ua ntej, lossis tsis nthuav tawm yog tias qhov kwv yees loj dua qis dua qhov kev txiav tawm (daim ntawv txuas ntxiv p 28).

rhiab heev thiab CIs rau tag nrho-genome sequencing rov nthuav dav nrhiav tau raug xam raws li qhov feem ntawm alleles nrog nthuav rov ua dua ntawm yav tas los PCR-pom zoo alleles nrog nthuav rov ua dua. Qhov tshwj xeeb tau kwv yees raws li kev faib ua feem ntawm cov alleles uas tsis tau nthuav dav ntawm yav dhau los kuaj tsis tau nthuav dav rov qab los ntawm PCR. Cov lus piav qhia tag nrho ntawm cov qauv kev txheeb cais muaj nyob rau hauv daim ntawv ntxiv (p 1).

Txhawm rau sib piv qhov ntau thiab tsawg los ntawm PCR nrog kev kwv yees qhov loj dua los ntawm tag nrho cov genome sequencing, PCR-quantified alleles tau muab piv nrog qhov ntau thiab tsawg tau kwv yees los ntawm ExpansionHunter rau alleles luv dua qhov ntev nyeem ntawv hla tag nrho 13 luv tandem rov loci. Concordance yog xam los ntawm feem pua ​​​​ntawm qhov ntau thiab tsawg tau kwv yees los ntawm ExpansionHunter uas tau pom zoo nrog PCR-quantified loj, coj mus rau hauv tus account PCR yuam kev ntawm ntxiv lossis rho tawm ib zaug. Kev txheeb xyuas txheeb cais tau ua tiav siv R cov ntaub ntawv txheeb xyuas software version 3.6.3.

Lub luag haujlwm ntawm cov peev nyiaj

Txoj kev tshawb fawb tsim, kev sau npe tus neeg mob, sau cov ntaub ntawv, thiab ua raws li cov neeg ua haujlwm ntawm Genomics England thiab cov kws tshawb fawb kev kawm. Cov neeg ua haujlwm ntawm Illumina tau ua qhov sib npaug ntawm 150 tus neeg mob cov qauv ua ib feem ntawm tag nrho cov genome sequencing diagnostic raug kawm thiab tsim ExpansionHunter. Cov neeg ua haujlwm ntawm Genomics England, cov kws tshawb fawb kev kawm, thiab coauthors RTH, ED, thiab MAE tau ua qhov kev tshuaj xyuas thiab kev txhais cov kev nthuav dav ntxiv hauv cov neeg mob uas tau txais los ntawm 100 000 Genomes Project. Cov peev txheej nyiaj txiag tsis muaj lub luag haujlwm hauv kev txhais cov ntaub ntawv lossis sau daim ntawv tshaj tawm.

Cov txiaj ntsig

Kev kuaj xyuas qhov tseeb ntawm tag nrho-genome sequencing txhawm rau txheeb xyuas qhov rov nthuav dav tau raug tshuaj xyuas tawm tsam 793 PCR cov kev sim ua yav dhau los hauv NHS los ntawm 404 tus neeg mob (64 tus neeg mob tau sim ntau dua ib zaug; daim duab 1). Ntawm cov kev ntsuam xyuas no, 183 tau muab cais raws li kev nthuav dav ntxiv thiab 610 raws li tsis muaj kev nthuav dav ntxiv los ntawm PCR, ua rau tag nrho 221 nthuav dav thiab 1321 tus neeg tsis tau nthuav dav thoob plaws 13 kab mob loci (appendix pp 24, 28). Tag nrho cov genome sequencing raug cais tawm 215 ntawm 221 nthuav dav alleles thiab 1316 ntawm 1321 tsis nthuav dav alleles piv nrog PCR cov qhabnias (appendix pp 27, 29), uas qhia txog qhov pib rhiab heev ntawm 97·3 feem pua ​​(95 feem pua ​​CI 94·2–99. ·0) thiab qhov tshwj xeeb ntawm 99·6 feem pua ​​(99·1–99·9; table 1). Tom qab kev kho qhov muag pom ntawm txhua qhov kev hu raws li qhov zoo ntawm kev nyeem, rhiab heev rau 99·1 feem pua ​​(96·8–99·9) thiab qhov tshwj xeeb rau 100 feem pua ​​(99·7–100; daim duab 2A, rooj 1). Kev pom kev nthuav dav ntawm alleles tau ua kom pom cov txiaj ntsig tsis tseeb thiab kev faib tawm ntawm txhua qhov tsis tseeb-tsis zoo alleles hauv FXN, ntawm uas tsuas yog ib qho allele tau raug cais raws li nthuav dav hauv cov qauv nrog bialllelic expansions (appendix pp 17, 18).

Rov ua qhov ntev tau txheeb xyuas los ntawm PCR hauv 509 PCR kev kuaj xyuas 945 alleles hla 13 rov nthuav dav loci. Kev sib raug zoo ntawm ExpansionHunter thiab PCR rau qhov ntau thiab tsawg dua luv dua thiab loj dua qhov sib txuas nyeem ntev (piv txwv li, 150 bp) tau qhia hauv daim ntawv ntxiv (appendix p 19). Kev sib raug zoo siab tau pom tias rov ua dua luv dua qhov ntev nyeem, nrog 92·7 feem pua ​​(836 ntawm 902) kev pom zoo ntawm PCR thiab ExpansionHunter. Locus variability tau pom, nrog siab concordance ntawm ExpansionHunter thiab PCR rau ATXN2, ATXN7, CACNA1A, thiab HTT, thiab qis concordance rau DMPK los yog TBP (appendix p 30). Qhov ntev ntawm alleles loj dua qhov ntev nyeem tau underestimated los ntawm ExpansionHunter, uas cuam tshuam qhov tseeb ntawm kev hu hauv DMPK, FMR1, thiab FXN (daim duab 2B, daim ntawv ntxiv pp 19, 31).

Txawm hais tias ExpansionHunter muaj peev xwm txheeb xyuas cov alleles loj loj hauv FMR1, DMPK, C9orf72, thiab FXN (appendix p 29), qhov kev kwv yees qhov loj me me yuav qis dua cov uas tau los ntawm PCR raws li rov loj dua nyob rau hauv cov kab mob pathogenic, uas cuam tshuam cov muaj peev xwm paub qhov txawv ntawm qhov loj thiab me me expansions hauv DMPK, C9orf72, thiab FXN, los yog ntawm tag nrho cov expansions thiab permutations nyob rau hauv FMR1 (appendix p 31). Piv txwv li, loci nrog PCR-kev soj ntsuam qhov ntev dua qhov ntev dua 200 rov ua dua hauv FMR1 thiab cais raws li kev hloov pauv tag nrho muaj qhov rov ua dua qhov loj kwv yees los ntawm ExpansionHunter ntawm 92·6 (SD 17·8; appendix p 31).

Txhawm rau kuaj lub peev xwm ntawm kev tshawb nrhiav rov ua dua los ntawm tag nrho cov genome sequencing los daws qhov kev kuaj mob ntawm cov neeg mob uas tau sim yav dhau los thiab cov neeg mob uas tsis tau kuaj pom, peb tau sim 11 631 cov neeg mob uas xav tias muaj cov kab mob hauv lub paj hlwb tau nrhiav rau 100000 Genomes Project (daim duab 1). Tag nrho-genome sequencing cov ntaub ntawv raug soj ntsuam siv plaub qhov sib txawv rov nthuav dav panels raws li tus neeg mob qhov chaw kho mob. Cov naj npawb ntawm cov neeg mob kuaj nrog txhua ntawm plaub lub vaj huam sib luag tau qhia hauv cov lus 2.

Zuag qhia tag nrho, peb tau kuaj pom thiab pom qhov pom tseeb tias rov nthuav dav hauv cov qauv ntawm 105 tus neeg mob (table 2, appendix pp 20, 33). Ntawm cov no, 81 cov qauv muaj rau kev kuaj xyuas los ntawm PCR, thiab 68 tau lees paub tias muaj qhov rov nthuav dav (0·6 feem pua ​​​​yield): 45 (1·2 feem pua) ntawm 3692 hauv vaj huam sib luag A, yim ({ {18}}·3 feem pua ​​) ntawm 2743 hauv vaj huam sib luag B, tsib (0·6 feem pua) ntawm 860 nyob rau hauv vaj huam sib luag C, thiab kaum (0·1 feem pua) ntawm 6731 nyob rau hauv vaj huam sib luag D. Kaum peb ntawm 81 expansion hu tsis tau lees paub tias yog pathogenic rov nthuav dav ntxiv (16 feem pua ​​​​ntawm kev tshawb pom cuav). Ntawm cov no, ob yog tsis nthuav dav alleles hauv ATXN1 thiab ATXN2, plaub yog FMR1 nruab nrab hu xov tooj (appendix p 21), thiab xya yog FMR1 permutations.

Cov ntaub ntawv kho mob ntawm 68 tus neeg mob nrog rov nthuav dav ntxiv tau lees paub los ntawm PCR, suav nrog lawv cov kev nthuav qhia kho mob, qhov rov nthuav dav tau txheeb xyuas, thiab kev koom tes ntawm kev rov nthuav dav rau tus neeg mob cov yam ntxwv kho mob tau muab rau hauv cov lus 3; HPO cov ntsiab lus, rov loj dua kwv yees los ntawm ExpansionHunter, thiab seb daim ntawv qhia txog kev kuaj mob puas tau muab teev tseg rau hauv daim ntawv ntxiv (p 33).

Kev nthuav dav tau pom nyob rau hauv cov neeg mob uas nthuav tawm nrog ntau yam sib tshooj ntawm kev soj ntsuam kev nthuav qhia kuaj nrog vaj huam sib luag A (table 3, appendix p 22), suav nrog ATXN2 rov nthuav dav hauv tus neeg mob nrog levodopa teb thaum ntxov-Parkinson tus kab mob thiab keeb kwm ntawm kev loj hlob cerebellar ataxia. , thiab AR nthuav dav hauv plaub tus neeg mob tau kuaj pom tias muaj tus kab mob Charcot-Marie-Tooth, suav nrog ib qho uas muaj cov tshuaj tiv thaiv kab mob demyelinating neuropathy (ie, Charcot-Marie-Tooth kab mob hom 1, tus neeg mob 42; appendix p 33). Ntau qhov kev kuaj mob yav dhau los tau pom nyob rau hauv cov neeg mob uas muaj kab mob rov ua dua.

Piv txwv li, nyob rau hauv xya tus neeg mob uas muaj amyotrophic lateral sclerosis los yog lwm yam kab mob neuron lub cev muaj zog, kev nthuav dav tau txheeb xyuas hauv AR (n=4) thiab C9orf72 (n=3). Hauv cov neeg mob uas xav tias muaj kab mob ataxia, peb tau txheeb xyuas qhov nthuav dav hauv loci uas tsis tau raug soj ntsuam raws li ib feem ntawm kev ua haujlwm niaj hnub kuaj mob hauv NHS thaum lub sijhawm nrhiav neeg ua haujlwm, suav nrog ATN1, ATXN2, ATXN3, ATXN7, CACNA1A, FXN, TBP, thiab HTT ( tabl 3). Peb kuj tau tshawb pom qhov kev nthuav dav ntxiv hauv cov neeg mob uas muaj cov yam ntxwv kho mob zoo ib yam nrog lwm qhov kev nthuav dav ntxiv, suav nrog kev nthuav dav C9orf72 hauv kev pib ntxov thiab tsev neeg tus kab mob Parkinson (tus neeg mob 24, Table 3) thiab rov nthuav dav hauv qhov txo qis hauv ntau yam hauv HTT (38 rov ua dua) nyob rau hauv ob tug viv ncaus uas muaj kev tsis sib haum xeeb, dementia, kev nyuaj siab, thiab kev hais lus nyuaj (cov neeg mob 44 thiab 45), qhia txog qhov kev sib tw kuaj mob uas tau nthuav tawm los ntawm cov kab mob txuas ntxiv no.

Yim tus menyuam yaus kuaj nrog vaj huam sib luag B tau pom tias muaj CAG loj dua qhov nthuav dav (daim duab 3), xya qhov uas tau piav qhia tag nrho tus neeg mob cov yam ntxwv kho mob. Rau tus neeg mob tsis muaj ntaub ntawv keeb kwm ntawm tsev neeg thiab tsis tau muab kev sim rov nthuav dav ntxiv raws li ib feem ntawm lawv qhov kev soj ntsuam kuaj mob thaum lub sijhawm nrhiav neeg ua haujlwm (cov neeg mob 48–53; cov lus 3, daim ntawv txuas ntxiv p 33). Ob tug ntawm cov menyuam yaus no nqa HTT nthuav dav loj (90–100 CAG rov ua dua). Nco ntsoov, ib tug me nyuam tau txais qhov rov qab los ntawm niam txiv tsis muaj kev cuam tshuam uas tsis muaj tsev neeg keeb kwm ntawm Huntington tus kab mob. Kev ntsuam xyuas tsev neeg tsis tu ncua, tab sis qhov txo qis penetrance allele tau raug txheeb xyuas hauv tsev neeg txuas ntxiv, qhia tias qhov rov ua dua tau nthuav dav dua li 60 rov ua dua hauv ib tiam (tus neeg mob 52). Thaum lub sijhawm sau ntawv, tsis muaj leej twg hauv tsev neeg tau pom muaj cov tsos mob ntawm Huntington tus kab mob, thiab kev sib tham txog caj ces thiab kev kuaj mob txuas ntxiv rau cov niam txiv. Ob tug menyuam yaus hnub nyoog qis dua 5 xyoos tau rov ua qhov kev nthuav dav loj hauv ATXN7 thiab nthuav tawm nrog cov kab mob ntau yam. Rau ib tug ntawm cov menyuam yaus no (tus neeg mob 50), lawv niam lawv txiv tau pom muaj teeb meem taug kev 2 xyoos tom qab tso npe rau hauv 100000 Genomes Project. Ib yam li ntawd, ib tug ntxhais hnub nyoog 10 xyoo uas muaj kev tsis taus kev txawj ntse tau pom tias muaj 99- rov nthuav dav hauv ATXN2, txawm hais tias ob leeg niam txiv tau raug xaiv los tsis muaj kev cuam tshuam, thiab ib tug hluas nkauj hnub nyoog 18 xyoo uas muaj dementia tau pom tias muaj { {19}} rov nthuav dav hauv ATN1 (appendix p 33).

Tsib qhov nthuav dav hauv DMPK ( vaj ​​huam sib luag C) tau kuaj pom, suav nrog hauv tus menyuam yaus thiab leej niam uas muaj kev kuaj mob ntawm cov leeg nqaij dystrophy, hauv ob tug kwv tij uas xav tias muaj kev cuam tshuam myopathy, thiab hauv cov tub ntxhais hluas uas muaj myopathy congenital (cov neeg mob 54-58). FMR1 expansions ( vaj ​​huam sib luag D) tau kuaj pom nyob rau hauv cuaj tus tub thiab ib tug ntxhais, thiab kev kuaj mob ntawm Fragile X syndrome tau piav qhia tag nrho lossis ib nrab piav qhia txog kev kho mob (cov neeg mob 59–68).

Kev sib tham

Kev kuaj mob ntawm kev rov nthuav dav ntxiv yog qhov nyuaj hauv kev saib xyuas kev noj qab haus huv vim yog qhov sib txawv thiab sib tshooj ntawm cov chaw kho mob thiab cov kev tshawb pom tsis tshwj xeeb, uas tuaj yeem nce qhov hnyav nrog lub hnub nyoog thiab hauv txhua tiam tom ntej. Rov ua dua cov kev mob tshwm sim yog ib qho ntawm feem ntau ua rau muaj cov kab mob hauv lub paj hlwb.4 Txawm li cas los xij, cov neeg mob yuav raug kuaj xyuas tsis zoo, vim tias tsis muaj kev ntsuam xyuas caj ces tsis txaus los yog vim tias cov kab mob caj ces tseem tsis tau tshawb pom. Cov kev ntsuam xyuas tam sim no tau tawg, thiab cov neeg mob yuav muaj qhov tsis raug rov nthuav dav qhov chaw kuaj mob29 lossis tau txais kev sim tshuaj molecular rau cov chav sib txawv ntawm qhov sib txawv vim qhov sib tshooj ntawm cov chaw kho mob nrog lwm cov kab mob neurological genetic disorders.30

Tag nrho cov genome sequencing tau siv nyob rau hauv ntau qhov chaw raws li kev kuaj thawj kab rau cov kab mob tsis tshua muaj paj hlwb tab sis yav dhau los tau xav tias muaj peev xwm tsis tshua muaj peev xwm txheeb xyuas qhov rov ua dua.16 Ntau cov cuab yeej tau tsim los txheeb xyuas qhov rov nthuav dav ntawm tag nrho-genome. sequencing nyob rau hauv kev tshawb fawb qhov chaw, 31 tab sis tsis muaj ib qho ntawm cov txheej txheem no tau siv rau tag nrho-genome sequencing cov ntaub ntawv sau los ntawm ib tug loj tus naj npawb ntawm cov neeg mob nyob rau hauv ib qho kev pab kho mob. Peb nthuav qhia cov pov thawj uas ib qho kev tsim los tshawb xyuas qhov kev nthuav dav ntawm tag nrho cov genome sequencing tuaj yeem txheeb xyuas cov kab mob ntau tshaj plaws uas ua rau rov nthuav dav dua thiab daws cov teeb meem uas tsis tau txheeb xyuas yav dhau los hauv pawg loj ntawm cov neeg mob uas muaj kab mob neurological. Peb cov txiaj ntsig qhia tau hais tias tag nrho-genome sequencing tuaj yeem paub qhov txawv ntawm qhov tsis nthuav dav thiab nthuav dav alleles nrog siab rhiab heev thiab tshwj xeeb hla 13 rov nthuav dav loci (uas tuaj yeem txhim kho ntxiv los ntawm kev tshuaj xyuas qhov muag), tuaj yeem xam qhov loj ntawm alleles me dua qhov ntev nyeem. , thiab tej zaum yuav kwv yees qhov loj ntawm kev nthuav dav loj hauv FMR1, DMPK, FXN, thiab C9orf72.

Thaum kuaj pom qhov rov ua dua los ntawm tag nrho cov genome sequencing tau soj ntsuam tawm tsam qhov zoo thiab qhov tsis zoo uas yav dhau los tau los ntawm cov chaw kuaj mob kuaj mob genomic siv cov txheej txheem kub, peb pom qhov tsawg kawg nkaus ntawm 97·3 feem pua ​​​​rhiab thiab 99·6 feem pua ​​​​qhov tseeb. Tsis tas li ntawd, peb tau pom tias ob qho tib si qhov tshwj xeeb thiab qhov rhiab heev tuaj yeem txhim kho los ntawm phau ntawv kho ntawm cov ntawv nyeem, ua kom pom cov txiaj ntsig tsis tseeb thiab rov faib cov khoom tsis tseeb-tsis zoo hauv cov qauv nrog kev nthuav dav biallelic. Ntawm 6731 tus neeg mob kuaj FMR1 (vaj huam sib luag D), 124 hu xov tooj tau kwv yees kom nthuav dav. Peb muaj peev xwm tshem tawm 97 los ntawm kev tshuaj xyuas qhov muag raws li qhov yuav muaj qhov tsis zoo. Qhov no qhia tau hais tias 1 ntawm 54 tag nrho cov kev xeem genome sequencing yuav muaj kev hu FMR1 uas yuav tsum tau kuaj pom qhov muag kom muab pov tseg qhov kev hu tsis tseeb. Kev ua haujlwm tsis tu ncua los txhim kho ExpansionHunter genotyping txoj kev los txo tus naj npawb ntawm kev hu tsis raug rau FMR1.

We show that repeat sizing is accurate for repeats smaller than the sequencing read lengths, and therefore that most non-expanded and premutation CAG repeat expansion disorder alleles can be sized accurately. These results are consistent with other studies showing a strong correlation between whole genome sequencing and PCR quantification of repeat lengths smaller than the sequencing read length.19,25,26 Whole genome sequencing expansion detection is limited in its sizing of alleles considerably larger than the read length, such as in Fragile X syndrome. We note that all FMR1 repeats previously classified by PCR as fully expanded (ie, >200 repeats) were classified by whole genome sequencing as permutation (50–200 repeats) in this study. Repeat size estimation for repeats larger than the read length is particularly important for loci in which the length of the repeat correlates with the disease clinical features. This includes DMPK, for which small expansions (50–150 repeats) cause mild myotonic dystrophy type 1 and large expansions (>1000 rov ua dua) ua rau muaj kab mob hnyav dua, thiab spinocerebellar ataxia hom 36 (NOP56), uas nthuav dav dua 650 rov ua dua yog suav tias yog kab mob thiab rov ua qhov ntau thiab tsawg ntawm 15-650 yog suav tias yog qhov nruab nrab thiab qhov txawv ntawm qhov tsis paub tseeb.

Ntau tshaj 40 rov nthuav dav loci tau raug txheeb xyuas; ntau ntawm cov loci no tsuas yog raug txheeb xyuas tsis ntev los no thiab tam sim no cuam tshuam nrog cov xwm txheej tsis tau piav qhia yav dhau los, suav nrog cerebellar ataxia nrog neuropathy thiab vestibular areflexia syndrome (RFC1) 32 thiab myoclonic epilepsy (SAMD12).33 Cov kab mob ntau tshaj plaws uas ua rau rov nthuav dav loci yog. xaiv rau peb txoj kev tshawb fawb raws li qhov muaj cov qauv tswj kev zoo thiab tsis zoo.

Cov kev tshawb pom tau nthuav tawm ntawm no qhia tias ExpansionHunter yuav tsum muaj peev xwm txheeb xyuas cov uas tsis tau nthuav dav thiab nthuav dav kom raug ntawm txhua qhov chaw nthuav dav yog tias cov alleles tsis nthuav dav me dua qhov ntev nyeem (piv txwv li, 150 bp). Txawm hais tias feem ntau rov nthuav dav loci muaj alleles uas me dua 150 bp thaum tsis nthuav dav, qee qhov loci uas qhov loj ntawm qhov tsis nthuav dav yog ze rau 150 bp (piv txwv li, NOTCH2NLC)34 yuav nyuaj dua rau genotype siv txoj hauv kev no. Rau loci qhov twg qhov kev nthuav dav yog qhov loj dua qhov ntev nyeem, tag nrho-genome sequencing tuaj yeem ntes cov kab mob nthuav dav (piv txwv li, NOP56,35 RFC120,32). Cov technologies uas niaj hnub nyeem ntev ntev tuaj yeem muab cov kev sib txuas ntxiv thaum genotyping loj expansions.36

Kev soj ntsuam ntawm kev rov nthuav dav dua siv tag nrho-genome sequencing hauv 11 631 cov neeg mob uas tsis tau kuaj pom tau raug xaiv los ntawm 100 000 Genomes Project tau muab 68 tus neeg mob nrog kev piav qhia. Cov neeg mob tau raug xaiv los ntawm 100 000 Genomes Project tom qab kuaj kab mob raws li tus qauv; Yog li ntawd, qhov kev faib ua feem ntawm kev nthuav dav ntxiv uas tau txheeb xyuas nyob rau hauv pawg neeg no sawv cev rau kev txhawb nqa ntawm cov txiaj ntsig kev kuaj mob los ntawm cov qauv NHS kuaj, uas suav nrog cov kev sim tshwj xeeb rau kev rov nthuav dav ntxiv xws li FXN lossis DMPK. Nco ntsoov, qee qhov kev kuaj mob tsis tau xav tias yog raws li tus neeg mob qhov chaw kho mob, suav nrog rau rau tus neeg mob menyuam yaus uas tsis paub txog tsev neeg keeb kwm ntawm kev rov ua dua tshiab. Qhov txhais tau tias rov nthuav dav qhov ntau thiab tsawg tau kwv yees los ntawm tag nrho cov genome sequencing hauv cov neeg mob hauv cov menyuam yaus uas tau piav qhia hauv txoj kev tshawb no yog qhov loj dua li qhov nruab nrab ntawm cov neeg laus, ua raws li qhov kev cia siab tias qhov kev nthuav dav loj dua cuam tshuam nrog qhov tshwm sim ntxov thiab hnyav dua, txawm tias hauv cov menyuam yaus. Kev ua haujlwm ntxiv yog xav tau, tab sis qhov kev tshawb pom no qhia tias qhov muaj hnub nyoog raws li qhov muaj hnub nyoog thiab rov ntsuas qhov loj me ntawm cov kab mob tuaj yeem pab txhawb kev kuaj mob menyuam yaus los ntawm kev txo qis kev pheej hmoo ntawm kev txheeb xyuas cov neeg laus-pib qhov pheej hmoo alleles, ua rau tsis muaj kev soj ntsuam kev kwv yees hauv cov menyuam yaus.

Peb qhov kev tshawb pom tau ua kom muaj kev tsim kho kev kuaj mob ua haujlwm rau tag nrho genome sequencing (appendix p 23). Peb tawm tswv yim tias kev tshuaj xyuas qhov muag tau ua tiav rau txhua qhov kev hu xov tooj uas tau nthuav tawm kom pom cov txiaj ntsig tsis tseeb, thiab rau kev nthuav dav biallelic uas tsuas yog ib qho nthuav dav allele tau kuaj pom (xws li FXN). Peb pom zoo kom cov chaw soj nstuam siv ExpansionHunter los ntsuam xyuas qhov muaj qhov nthuav dav yam tsis tau ua raws li kev kwv yees loj thiab ua qhov kev kuaj xyuas PCR uas yog ib qho qauv ntawm kev sim ua haujlwm.

Cov kab mob tsis tshua muaj tshwm sim muaj xws li ntau yam kev kho mob, ua rau qhov chaw tshwj xeeb genomic kuaj tsis muaj txiaj ntsig, nyuaj, thiab kim. Peb nthuav tawm cov pov thawj tias kev soj ntsuam-qib tag nrho cov genome sib txuas nrog lub peev xwm los kuaj xyuas ntau yam kab mob tsis tshua muaj paj hlwb uas feem ntau nthuav tawm nrog ib lub hauv paus, indel, lossis luam cov lej sib txawv tam sim no tuaj yeem txuas ntxiv mus txuas ntxiv. Vim hais tias tag nrho-genome sequencing muab ib qho kev ntsuam xyuas uas tuaj yeem txheeb xyuas qhov kev nthuav dav ntau tshaj plaws, nrog rau kev ua kom muaj kev hloov pauv ntawm cov ntsiab lus hloov pauv thiab luam cov lej sib txawv hauv cov noob uas cuam tshuam nrog cov xwm txheej no ib txhij, nws muaj lub sijhawm los txheeb xyuas cov neeg mob feem ntau nrog cov kab mob sib txawv no. uas tsis tau kuaj pom los ntawm kev kuaj qhov chaw tshwj xeeb. Nyob rau hauv lub sijhawm ntawm cov kev kho mob tshwm sim rau cov teeb meem no, kev tshawb pom ntxov yuav dhau los ua qhov tseem ceeb.37 Cov txiaj ntsig no txhawb nqa kev siv tag nrho-genome sequencing rau kev tshawb nrhiav rov qab nthuav dav hauv cov chaw kuaj mob kuaj mob, ib txoj hauv kev uas twb tau suav nrog hauv NHS England National Genomic. Test Directory, 38 rau kev tshawb nrhiav ntawm cov kab mob tsis tshua muaj paj hlwb.

Cov ntaub ntawv

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