Whole blood RNA offers a rapid, comprehensive approach to genetic diagnosis of cardiovascular diseases.
OBJECTIVE
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.


CONCLUSIONS
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.
Miller
Todd E
TE
Department of Medicine, Division of Cardiology, Miller School of Medicine, University of Miami, Miami, Florida 33101, USA. Tmillerl@specialtylabs.com
You
Lijing
L
Myerburg
Robert J
RJ
Benke
Paul J
PJ
Bishopric
Nanette H
NH
D000293:Adolescent; D000328:Adult; D002311:Cardiomyopathy, Dilated / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D002312:Cardiomyopathy, Hypertrophic / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D002318:Cardiovascular Diseases / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D002648:Child; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005260:Female; D000071838:Fibrillin-1; D000071837:Fibrillins; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D051657:KCNQ1 Potassium Channel / Q000235:genetics*; D008133:Long QT Syndrome / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D008297:Male; D008382:Marfan Syndrome / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D008840:Microfilament Proteins / Q000235:genetics*; D008875:Middle Aged; D009154:Mutation / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011446:Prospective Studies; D012313:RNA / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000099163:Myosin Binding Protein C; D054392:Adipokines
D002352:Carrier Proteins; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D000071838:Fibrillin-1; D000071837:Fibrillins; D051657:KCNQ1 Potassium Channel; D008840:Microfilament Proteins; D012313:RNA; D000099163:Myosin Binding Protein C; C000605722:FBN1 protein, human; D054392:Adipokines; C000606913:KCNH2 protein, human