Genetics in medicine : official journal of the American College of Medical Genetics · 2007
Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases.
СтатьяПоддержка NIH: внешняя программаПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Miller Todd E, You Lijing, Myerburg Robert J, Benke Paul J, Bishopric Nanette H
Аннотация
PURPOSE: Long QT Syndrome, Marfan Syndrome, hypertrophic and dilated cardiomyopathy are caused by mutations in large, multi-exon genes that are principally expressed in cardiovascular tissues. Genetic testing for these disorders is labor-intensive and expensive. We sought to develop a more rapid, comprehensive, and cost-effective approach.
METHODS: Paired whole blood samples were collected into tubes with or without an RNA-preserving solution, and harvested for whole blood RNA or leukocyte DNA, respectively. Large overlapping cDNA fragments from KCNQ1 and KCNH2 (Long QT Syndrome), MYBPC3 (hypertrophic and dilated cardiomyopathy), or FBN1 (Marfan Syndrome) were amplified from RNA and directly sequenced. Variants were confirmed in leukocyte DNA.
RESULTS: All 4 transcripts were amplified and sequenced from whole blood mRNA. Six known and 2 novel mutations were first identified from RNA of 10 probands, and later confirmed in genomic DNA, at considerable savings in time and cost. In one patient with MFS, RNA sequencing directly identified a splicing mutation. Results from RNA and DNA were concordant for single nucleotide polymorphisms at the same loci.
CONCLUSION: Taking advantage of new whole blood RNA stabilization methods, we have designed a cost-effective, comprehensive method for mutation detection that should significantly facilitate clinical genetic testing in four lethal cardiovascular disorders.
METHODS: Paired whole blood samples were collected into tubes with or without an RNA-preserving solution, and harvested for whole blood RNA or leukocyte DNA, respectively. Large overlapping cDNA fragments from KCNQ1 and KCNH2 (Long QT Syndrome), MYBPC3 (hypertrophic and dilated cardiomyopathy), or FBN1 (Marfan Syndrome) were amplified from RNA and directly sequenced. Variants were confirmed in leukocyte DNA.
RESULTS: All 4 transcripts were amplified and sequenced from whole blood mRNA. Six known and 2 novel mutations were first identified from RNA of 10 probands, and later confirmed in genomic DNA, at considerable savings in time and cost. In one patient with MFS, RNA sequencing directly identified a splicing mutation. Results from RNA and DNA were concordant for single nucleotide polymorphisms at the same loci.
CONCLUSION: Taking advantage of new whole blood RNA stabilization methods, we have designed a cost-effective, comprehensive method for mutation detection that should significantly facilitate clinical genetic testing in four lethal cardiovascular disorders.
Предметные рубрики MeSH
AdolescentAdultCardiomyopathy, DilatedCardiomyopathy, HypertrophicCardiovascular DiseasesCarrier ProteinsChildERG1 Potassium ChannelEther-A-Go-Go Potassium ChannelsExonsFemaleFibrillin-1FibrillinsGenetic TestingGenotypeHumansInfantInfant, NewbornKCNQ1 Potassium ChannelLong QT SyndromeMaleMarfan SyndromeMicrofilament ProteinsMiddle AgedMutationPolymorphism, Single NucleotideProspective StudiesRNAReverse Transcriptase Polymerase Chain ReactionMyosin Binding Protein CAdipokines