Identification of large gene deletions and duplications in KCNQ1 and KCNH2 in patients with long QT syndrome

Background

Sequencing or denaturing high-performance liquid chromatography (dHPLC) analysis of the known genes associated with the long QT syndrome (LQTS) fails to identify mutations in approximately 25% of subjects with inherited LQTS. Large gene deletions and duplications can be missed with these methodologies.

Objective

The purpose of this study was 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.

Methods

Multiplex ligation-dependent probe amplification (MLPA), a quantitative fluorescent approach, was used to screen 26 mutation-negative probands with an unequivocal LQTS phenotype (Schwartz score >4). The appropriate MLPA kit contained probes for selected exons in LQTS genes KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2. Real-time polymerase chain reaction was used to validate the MLPA findings.

Results

Altered exon copy number was detected in 3 (11.5%) patients: (1) an ex13-14del of the KCNQ1 gene in an 11-year-old boy with exercise-induced collapse (QTc 580 ms); (2) an ex6-14del of the KCNH2 gene in a 22-year-old woman misdiagnosed with epilepsy since age 9 years (QTc 560 ms) and a sibling with sudden death at age 13 years; and (3) an ex9-14dup of the KCNH2 gene in a 12 year-old boy (QTc 550 ms) following sudden nocturnal death of his 32-year-old mother.

Conclusion

If replicated, this study demonstrates that more than 10% of patients with LQTS and a negative current generation genetic test have large gene deletions or duplications among the major known LQTS susceptibility genes. As such, these findings suggest that sequencing-based mutation detection strategies should be followed by deletion/duplication screening in all LQTS mutation-negative patients.

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