Genome-wide diagnostics of Mendelian disorders: from chromosomal microarrays to next-generation sequencing
Date
2017-11-13
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Abstract
Haruldaste pärilike haiguste diagnostika ja ravi keskne paradigma on täpse molekulaarse haiguspõhjuse (mutatsiooni) tuvastamine igal patsiendil. See on aluseks nii patsiendi ravile, perekonna nõustamisele kui ka sünnieelsele diagnostikale. Kui traditsiooniliselt on geneetiliste haiguste diagnostika olnud võimalik vaid üksikute geenide kaupa, siis tänapäevased tehnoloogiad võimaldavad uurida kõiki enam kui 20 000 geeni korraga. Kuigi teaduses on kromosoomiuuringuteks kasutatavad mikrokiibid ja kõigi geenide järjestamist võimaldavad järgmise põlvkonna sekveneerimisanalüüsid juba ennast hästi tõestanud, on kliinilisse kasutusse rakendamisel vajalik neid metoodikaid teaduslikult analüüsida, et leida igale patsiendile parim uuringustrateegia.
Käesoleva uuringuga selgitati nii kromosoomikiipidelt sageli leitud ebaselge tähendusega muutuste, homosügootsete alade, kliinilist tähendust. Näidati, et kolmandik sellistest regioonidest on patsientidel korduvad ja seega tõenäoliselt haigust mittepõhjustavad. Samuti leiti, et üksikud homosügootsed alad sisaldavad harva patsiendi haigusega seostatavat geeni, ent kui selline geen tuvastatakse, on väga tõenäoline leida sealt ka geneetilise haiguse põhjus. Teine osa doktoritööst käsitles suurte geenipaneelide sekveneerimise tulemuslikkust tavapärases kliinilises töös. Töö tulemusena selgus, et ligi 5000 pärilike haigustega seostatud geeni paneeli uuringu tulemuslikkus on võrreldav kõikide geenide ehk eksoomi analüüsiga. Uuringusse kaasatud 501 patsiendist leiti kindel geneetiline haiguspõhjus 132-l (26%). Ligi pooled muutused olid varem kirjeldamata. Doktoritöö viimases osas käsitleti kahte haigusjuhtu. Esiteks kirjeldati maailmas teist korda KPTN geeni mutatsioone intellektipuude põhjusena. Teine haigusjuht, kus lihashaigust põdeva poisi haiguspõhjusena tuvastati uudne MYH7 geeni defekt, laiendas MYH7-seoseliste lihashaiguste teadaolevat kliinilist ja geneetilist spektrit.
The fundamental paradigm of diagnostics and care of patients with rare inherited disorders is the detection of specific molecular causes (mutations) associated with the disorder in every patient. This makes the best treatment, counselling, and prenatal diagnostics possible. Traditionally, genetic diagnostics relied on single gene testing. Modern technologies, however, make simultaneous investigation of more than 20,000 genes possible. Although chromosomal microarrays and next-generation sequencing of entire genomes have already found their place in the research, implementation of these techniques to clinical diagnostics needs further studies before the most optimal testing strategies can be established for patients. This study clarified the clinical role of homozygous chromosomal regions – findings of unclear significance frequently detected by chromosomal microarrays. It was shown that a third of such findings are recurrent between patients, and thus are likely benign. In addition, identification of a candidate gene matching a patient’s symptoms from a homozygous region is very rare; however, if such a gene was found, the mutation would likely be detected. The second part of the study focused on the clinical utility of sequencing a panel of nearly 5,000 disease-associated genes. The results indicated that the large gene panel has a similar diagnostic yield as whole exome sequencing. Out of 501 cases included in this study, a molecular cause of the disorder was detected in 132 cases (26%). Nearly a half of the detected mutations were previously unreported. The last part of the dissertation reported on two patients – the second report in the medical literature on KPTN¬ gene mutations causing intellectual disability, and a boy with muscle disorder, in whom a novel mutation in the MYH7 gene was detected. This MYH7 mutation was confirmed to cause novel molecular effects on the gene transcript, thus expanding the clinical and genetic spectra of MYH7-related disorders.
The fundamental paradigm of diagnostics and care of patients with rare inherited disorders is the detection of specific molecular causes (mutations) associated with the disorder in every patient. This makes the best treatment, counselling, and prenatal diagnostics possible. Traditionally, genetic diagnostics relied on single gene testing. Modern technologies, however, make simultaneous investigation of more than 20,000 genes possible. Although chromosomal microarrays and next-generation sequencing of entire genomes have already found their place in the research, implementation of these techniques to clinical diagnostics needs further studies before the most optimal testing strategies can be established for patients. This study clarified the clinical role of homozygous chromosomal regions – findings of unclear significance frequently detected by chromosomal microarrays. It was shown that a third of such findings are recurrent between patients, and thus are likely benign. In addition, identification of a candidate gene matching a patient’s symptoms from a homozygous region is very rare; however, if such a gene was found, the mutation would likely be detected. The second part of the study focused on the clinical utility of sequencing a panel of nearly 5,000 disease-associated genes. The results indicated that the large gene panel has a similar diagnostic yield as whole exome sequencing. Out of 501 cases included in this study, a molecular cause of the disorder was detected in 132 cases (26%). Nearly a half of the detected mutations were previously unreported. The last part of the dissertation reported on two patients – the second report in the medical literature on KPTN¬ gene mutations causing intellectual disability, and a boy with muscle disorder, in whom a novel mutation in the MYH7 gene was detected. This MYH7 mutation was confirmed to cause novel molecular effects on the gene transcript, thus expanding the clinical and genetic spectra of MYH7-related disorders.
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Keywords
meditsiinigeneetika, pärilikud haigused, mendelism, geeniuuringud, geneetilised assotsiatsiooniuuringud, medical genetics, hereditary diseases, Mendelian genetics, genetic association studies