Original-clinical geneticIdentification of large gene deletions and duplications in KCNQ1 and KCNH2 in patients with long QT syndrome
Introduction
Long QT syndrome (LQTS) is an inherited disorder characterized by abnormal ventricular repolarization leading to a prolonged QT interval on surface ECG. The condition predisposes affected people to ventricular tachyarrhythmias, which may lead to syncope, seizures, or sudden death.1, 2 These events can be triggered by physical or emotional stress, but in some individuals they may occur during periods of sleep or rest.3, 4 Initial speculation that LQTS was an extremely rare disorder has changed with current estimates putting the incidence of LQTS at 1:1,000–5,000.5
Both the phenotypic and genetic heterogeneity of LQTS are widely accepted. The clinical history and ECG phenotype can range from complete absence of symptoms and a normal resting ECG to sudden death in infancy and extreme QT prolongation, respectively.6 Such phenotypic variability can make clinical diagnosis challenging. More than 600 mutations in at least 11 genes have been identified in LQTS patients.5, 7, 8, 9 Most of these genes encode for proteins composing cardiac ion channels, and, of these, more than 90% of mutations are found in five genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2).5, 7, 8, 9
In LQTS, the trigger of a cardiac event, mode of presentation, disease severity (risk of sudden death), and response to therapy can vary considerably according, in part, to the genetic locus at which a mutation is present.4, 10, 11 LQTS mutation carriers, who lack a prolonged QT interval and, therefore, escape clinical diagnosis, have a 10% risk of a major cardiac event by age 40 years if left untreated.11, 12 Therefore, reaching a molecular diagnosis in a proband is valuable in establishing preventative clinical best practice for that patient as well as allowing identification of “at-risk” family members.
Among patients with clinically definite LQTS, approximately 25% remain genotype negative after comprehensive assessment of the three major LQTS genes.5, 8, 13 Current methods for mutation screening of the LQT genes involve denaturing high-performance liquid chromatography (dHPLC) analyses of all intron boundaries and exons, followed by sequence analysis of all those samples/exons with abnormal dHPLC profiles. Alternatively, with the cost of sequencing dropping, the preferential direct sequencing of polymerase chain reaction (PCR)-amplified coding regions is used. These methods do not detect large intragenic deletions or duplications because of the background presence of the remaining normal allele.14 Although a number of other genetic mechanisms exist that might be responsible for the LQTS observed clinically in these patients, including mutations in other yet unidentified genes, a number of cases might be attributable to large genomic rearrangements in these genes. There is a range of disorders in which such deletions and/or duplications of a gene account for a significant proportion of detected mutations, such as Duchenne muscular dystrophy,15 breast cancer,16 and Fanconi anaemia.17 There is no apparent reason why LQTS should be an exception. A tandem duplication of 3.7 kb in KCNH2 has been identified in a Dutch family with LQTS,18 but to our knowledge large gene deletions have not been described in LQTS.
Multiplex ligation-dependent probe amplification (MLPA),14 a quantitative fluorescent approach, was used to determine whether deletions and/or duplications of one or more exons of the main LQTS genes were present in an LQTS mutation-negative cohort. We report the first identification of large multiple exon deletions in the LQTS genes KCNQ1 and KCNH2. We also describe a second, novel large multiple exon duplication in the KCNH2 gene.18
Section snippets
Patients
The study included 26 index cases whose clinical family history and ECG findings supported a definitive clinical diagnosis of LQTS. All subjects included in the study had a Schwartz score of 4 or greater. Clinical details are listed in Table 1. Informed consent for genetic testing was obtained in all cases, following the protocols established in our multicenter ethical approval from the Auckland regional ethics committee. Coding region and splice site mutations in the three major LQTS genes
MLPA analysis
MLPA profiles that reflected altered exon copy numbers were detected in 4 (∼15%) patients among the gene-negative LQTS probands (Figure 1). An apparent deletion of exon 7 of the KCNQ1 gene was not confirmed after sequence analysis. This apparent deletion corresponded to a previously reported missense mutation (c.944A→G, p.Y315C, NM_000218.2)21, 22, 23, 24 in the MLPA probe recognition sequence that escaped initial detection through dHPLC/sequencing screening (Figure 2).13 The remaining two
Discussion
MLPA and real-time PCR were used to identify two large multiple-exon deletions in the KCNQ1 (ex13-14del) and KCNH2 (ex6-14del) genes as well as an intragenic duplication in the KCNH2 gene (ex9-14dup) in 26 mutation-negative LQTS patients. Another patient with an apparent deletion of a single exon was found to carry a disease-associated missense mutation within an MLPA probe recognition sequence. Taken together, the MLPA method detected mutations in 11.5% of LQTS patients who were negative for
Acknowledgments
We thank Jamie Vandenberg (Victor Chang Cardiac Research Institute, NSW) for valuable comments on the effect of our identified mutations on protein and ion channel function.
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This research was supported by the Child Health Research Foundation (Cure Kids) and the Greenlane Research and Education Fund.