Allelic dropout in long QT syndrome genetic testing: a possible mechanism underlying false-negative results.
BACKGROUND
Genetic testing for congenital long QT syndrome (LQTS) has been performed in research laboratories for the past decade. Approximately 75% of patients with high clinical probability for LQTS have a mutation in one of five LQTS-causing cardiac channel genes. Possible explanations for the remaining genotype-negative cases include LQTS mimickers, novel LQTS-causing genes, unexplored regions of the known genes, and genetic testing detection failures.


OBJECTIVE
The purpose of this study was to explore the possibility of allelic dropout as a possible mechanism underlying false-negative test results.


METHODS
The published primers currently used by many research laboratories to conduct a comprehensive analysis of the 60 translated exons in the KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6) genes were analyzed for the presence of common intronic single nucleotide polymorphisms (SNPs). Repeat mutational analysis, following primer/amplicon redesign using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, was performed on a cohort of 541 consecutive, unrelated patients referred for LQTS genetic testing.


RESULTS
Common (>1% minor allele frequency) intronic SNPs were discovered within the primer sequences of five of 60 translated exons. Following primer redesign to eliminate the possibility of allelic dropout, four previously genotype-negative index cases were found to possess LQTS-causing mutations: R591H-KCNQ1 and R594Q-KCNQ1 for exon 15 and E229X-KCNH2 in two unrelated cases. Repeat examination of these two amplicons in 400 reference alleles did not identify these or any additional amino acid variants.


CONCLUSIONS
Allelic dropout secondary to intronic SNP-primer mismatch prevented the discovery of LQTS-causing mutations in four cases. Considering that many LQTS genetic testing research laboratories have used these primers, patients who reportedly are genotype negative may benefit from re-examination of those regions susceptible to allelic dropout due to primer-disrupting SNPs, particularly exon 15 in KCNQ1 and exon 4 in KCNH2.
Tester
David J
DJ
Department of Internal Medicine, Divisions of Cardiovascular Diseases, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
Cronk
Lisa B
LB
Carr
Janet L
JL
Schulz
Vincent
V
Salisbury
Benjamin A
BA
Judson
Richard S
RS
Ackerman
Michael J
MJ
D000293:Adolescent; D000328:Adult; D000483:Alleles; D002648:Child; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics*; D005091:Exons; D005188:False Negative Reactions; D005260:Female; D005787:Gene Frequency; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000235:genetics*; D008133:Long QT Syndrome / Q000151:congenital / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D009124:Muscle Proteins / Q000235:genetics; D009154:Mutation; D062554:NAV1.5 Voltage-Gated Sodium Channel; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics; D012189:Retrospective Studies; D015222:Sodium Channels / Q000235:genetics
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C489622:KCNE1 protein, human; C489619:KCNE2 protein, human; C000606913:KCNH2 protein, human; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D024642:Potassium Channels, Voltage-Gated; C568320:SCN5A protein, human; D015222:Sodium Channels; D004247:DNA